Update of Antibiotic Therapy of Brucellosis

*Sara Consuelo Arias Villate and Julio Cesar García Casallas*

#### **Abstract**

Currently, the only option for treating brucellosis is antibiotics especially to prevent complications. In this chapter, we want to talk about the drug therapy in brucellosis and the update of these therapies in the last years. Also, we will expose the principal antibiotics in brucellosis such as doxycycline, rifampin, streptomycin, cotrimoxazole (TMP/SMX), and gentamicin by talking about each one of their mechanism of action, pharmacokinetics, administration, risk assessment, adverse effects, and principal drug interactions. Furthermore, we will add the evidence of efficacy therapy in monotherapy or combinate therapy based on the evidence.

**Keywords:** brucellosis, aminoglycoside, doxycycline, rifampin, treatment

#### **1. Introduction**

Brucellosis is a zoonotic disease that can affect humans around the world, and it can affect any organ system. About the treatment, it is characterized to be prolonged therapy with a concomitant use of at least two or three antibiotics at different administration routes. The antibiotics have some special indications for administration, interactions, and risk assessment to prevent adverse reactions. That is why we will expose the principal antibiotics in brucellosis treatment based on the last evidence.

#### **2. Antibiotic treatment**

The principal objective of the treatment in brucellosis is to control the disease, prevent complications, relapse, and unfavorable outcomes. In the context of a zoonotic infection, the goal of its management is an appropriate antibiotic therapy with a prolonged duration of treatment, nevertheless the most effective antibiotic and treatment durations are unclear. Also, there are some limitations to choose the best treatment because of the need to choose antibiotics that act intracellularly and to prevent relapses with a prolonged therapy that can lead to increase the adverse effects of the drugs [1].

Furthermore, the monotherapy for brucellosis has been considered inadequate due to unacceptably high relapse rates, now we present possible treatment schemes [2, 3].

Uncomplicated brucellosis: (defined by not having focal disease like spondylitis, neurobrucellosis or endocarditis, and adults or > 30 kg):

• Doxycycline 100 mg orally twice daily for 6 weeks, plus streptomycin 1 g intramuscularly one daily for the first 14–21 days (or gentamicin 5 mg/kg for 5–14 days) [1, 2, 4].


Alternative agents: (they may be useful in the setting of drug resistance, allergy, antimicrobial toxicity or relapse in combination with doxycycline or rifampin)


Focal disease: spondylitis, neurobrucellosis, endocarditis, or localized suppurative lesions (it requires longer courses of therapy at least 12 weeks):

	- Doxycycline 100 mg orally twice daily for 12 weeks plus streptomycin 1 g intramuscularly once daily for the first 14–21 days [7].
	- Alternative: doxycycline 100 mg orally twice daily plus rifampin 600–900 mg (15 mg/kg) once daily for 12 weeks.
	- Surgery in the context of spinal instability, persistence or progression of neurological deficit or localizes abscess epidural or paravertebral [8].
	- Doxycycline, rifampin, and ceftriaxone or TMP-SMX.
	- Corticosteroids may be appropriate in the setting of neurobrucellosis complicated by iritis, papilledema, myelopathy, polyneuropathy, or cranial nerve palsies.
	- Doxycycline plus rifampin 300 mg every 12 h and gentamycin 5 mg/kg each day, the duration of therapy is uncertain usually for 6 weeks to 6 months [9].
	- Surgery: valve replacement.

Pregnant women: [2, 10].


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*Update of Antibiotic Therapy of Brucellosis DOI: http://dx.doi.org/10.5772/intechopen.86325*

6 weeks [11].

of therapy.

*2.1.1 Mechanism of action*

*2.1.2 Antimicrobial activity*

*2.1.3 Pharmacokinetics*

See **Table 1**.

*2.1.4 Administration*

**2.1 Doxycycline**

synthesis [13].

kg IV daily for 7 days) for 6 weeks [10–12].

• Osteoarticular disease, neurobrucellosis, or endocarditis

○ (<8 years of age): oral TMP-SMX [10 mg/kg per day TMP (maximum 480 mg/day) and 50 mg/kg per day SMX (maximum 2.4 g/day) by mouth divided into two doses] daily plus rifampin [15–20 mg/kg per day by mouth (maximum 900 mg/day) divided in one or two doses] (or gentamycin 5 mg/

○ (>8 years of age): oral doxycycline [2–4 mg/kg per day by mouth (maximum 200 mg/day) divided into two doses] or tetracycline [30–40 mg/kg per day by mouth (maximum 2 g/day) divided into four doses] plus rifampin for

○ <8 years of age: oral TMP-SMX for at least 6 weeks plus parenteral aminoglycoside [gentamicin (5 mg/kg per day parenterally divided into one to three doses) or streptomycin (20–40 mg/kg per day (maximum dose 1 g/day) parenterally divided in two doses)] for the first 14 days of therapy.

○ >8 years of age: oral doxycycline or tetracycline for at least 6 weeks, plus parenteral aminoglycoside (gentamicin or streptomycin) for the first 14 days

It belongs to the group of tetracyclines that are a series of derivatives of basic four-ring structure. Doxycycline inhibits bacterial protein synthesis by binding to

Doxycycline is a bacteriostatic antibiotic with activity against *Streptococcus pneumoniae* and *H. influenzae* and excellent activity against atypical pathogens such as *Mycoplasma* and *Chlamydophila pneumoniae*, methicillin-resistant *Staphylococcus aureus* and methicillin-susceptible *Staphylococcus aureus*, *Bacillus anthracis*, and *Listeria monocytogenes* and most strains of *Brucella* are susceptible. Some species

• Oral: administer with meals to decrease gastrointestinal (GI) discomfort. Administer capsules and tablets with a considerable amount of water and have patient sit up for at least 30 minutes to reduce esophageal irritation. Oral administration is preferable unless patient has significant nausea and vomiting [13].

• IV: infuse prolonged over 1–4 hours to prevent thrombophlebitis.

the 30S bacterial ribosome and blocking the access of aminoacyl tRNA to the A (acceptor) site on the mRNA-ribosome complex and inhibits protein

such as *Pseudomonas aeruginosa* are resistant [13, 14].

Children

• Uncomplicated brucellosis

*New Insight into* Brucella *Infection and Foodborne Diseases*

for 7 days) to relief symptoms more rapid.

orally one daily for 6 weeks [1, 3].

twice a day.

• Spondylitis

• Neurobrucellosis

• Endocarditis

• Limited data.

Children

6 months [9].

○ Surgery: valve replacement.

Pregnant women: [2, 10].

• Uncomplicated brucellosis

• Doxycycline 100 mg orally twice daily plus rifampin 600–900 mg (15 mg/kg)

• Consider triple therapy with addition of amikacin (intramuscularly twice a day

Alternative agents: (they may be useful in the setting of drug resistance, allergy,

antimicrobial toxicity or relapse in combination with doxycycline or rifampin)

• Ciprofloxacin 500 mg twice daily or ofloxacin 200 mg twice daily [5, 6].

tive lesions (it requires longer courses of therapy at least 12 weeks):

muscularly once daily for the first 14–21 days [7].

(15 mg/kg) once daily for 12 weeks.

• Trimethoprim-sulfamethoxazole (TMP-SMX) one double-strength tablet

Focal disease: spondylitis, neurobrucellosis, endocarditis, or localized suppura-

○ Doxycycline 100 mg orally twice daily for 12 weeks plus streptomycin 1 g intra-

○ Alternative: doxycycline 100 mg orally twice daily plus rifampin 600–900 mg

○ Surgery in the context of spinal instability, persistence or progression of neuro-

○ Corticosteroids may be appropriate in the setting of neurobrucellosis complicated by iritis, papilledema, myelopathy, polyneuropathy, or cranial nerve palsies.

○ Doxycycline plus rifampin 300 mg every 12 h and gentamycin 5 mg/kg each day, the duration of therapy is uncertain usually for 6 weeks to

• Rifampin 900 mg once daily, with or without TMP-SMV (one double-strength

tablet twice a day) for 6 weeks or rifampin with ceftriaxone.

logical deficit or localizes abscess epidural or paravertebral [8].

○ Doxycycline, rifampin, and ceftriaxone or TMP-SMX.

**36**

	- <8 years of age: oral TMP-SMX for at least 6 weeks plus parenteral aminoglycoside [gentamicin (5 mg/kg per day parenterally divided into one to three doses) or streptomycin (20–40 mg/kg per day (maximum dose 1 g/day) parenterally divided in two doses)] for the first 14 days of therapy.
	- >8 years of age: oral doxycycline or tetracycline for at least 6 weeks, plus parenteral aminoglycoside (gentamicin or streptomycin) for the first 14 days of therapy.

#### **2.1 Doxycycline**

#### *2.1.1 Mechanism of action*

It belongs to the group of tetracyclines that are a series of derivatives of basic four-ring structure. Doxycycline inhibits bacterial protein synthesis by binding to the 30S bacterial ribosome and blocking the access of aminoacyl tRNA to the A (acceptor) site on the mRNA-ribosome complex and inhibits protein synthesis [13].

#### *2.1.2 Antimicrobial activity*

Doxycycline is a bacteriostatic antibiotic with activity against *Streptococcus pneumoniae* and *H. influenzae* and excellent activity against atypical pathogens such as *Mycoplasma* and *Chlamydophila pneumoniae*, methicillin-resistant *Staphylococcus aureus* and methicillin-susceptible *Staphylococcus aureus*, *Bacillus anthracis*, and *Listeria monocytogenes* and most strains of *Brucella* are susceptible. Some species such as *Pseudomonas aeruginosa* are resistant [13, 14].

#### *2.1.3 Pharmacokinetics*

#### See **Table 1**.

#### *2.1.4 Administration*



#### **Table 1.**

*Pharmacokinetics parameters of doxycycline [13].*

#### *2.1.5 Risk assessment*

When therapy of doxycycline needs to be used in prolonged therapy, some parameters need to be taken to prevent some of the adverse effects: complete blood count (CBC), renal and liver function tests periodically, during therapy [13].

#### *2.1.6 Adverse effects*


**39**

*2.1.8 Important*

**Table 2.**

**2.2 Streptomycin**

*2.2.1 Mechanism of action*

• Tetracyclines are inexpensive, widely available, and poor associated with side

**Drug Risk rating Interaction Mechanism Management**

Formation of chelates between antibiotic and antacids that reduces absorption from the GI tract [16]

Uncertain. Induction of doxycycline metabolism or excretion by the barbiturates [18]

Buffered aspirin contains antacids that alkaline environment may also reduce absorption [19]

Unknown. Rifampin induction of doxycycline metabolism and/ or excretion [20] Separate administration of both by a few hours when possible Monitor for decreased therapeutic effects of antibiotic [17]

Monitor decreased therapeutic effects of antibiotic if used concurrently with a barbiturate

Administer doxycycline at least 2 hours before or 6 hours after aspirin

ingestion

Monitor closely for reduced doxycycline response in patients receiving rifampin

Antacids may decrease the absorption of doxycycline

Barbiturates may decrease the serum concentration of doxycycline

Aspirin may decrease the serum concentration of doxycycline

Rifampin may decrease the serum concentration of doxycycline

• The doxycycline-streptomycin regimen is considered the first line and has been proven to be more effective than doxycycline-rifampin in some studies [4, 22].

• Do not administer to children <8 years of age due to permanent discoloration of teeth, retardation of skeletal development, and bone growth; more common with long-term use, but may be observed with repeated, short courses [12, 23].

It is an aminoglycoside antibiotic bactericidal. Aminoglycosides diffuse through

aqueous channels formed by porin proteins in the outer membrane of Gramnegative bacteria to enter to the periplasmic space, and its transport across the cytoplasmic membrane depends on an electrical gradient coupled to electron transport to drive permeation of these antibiotics. That is why they are not used in anaerobic environments of abscess. Once streptomycin is inside the cell, it binds to the 30S ribosomal subunit and interferes with protein synthesis by causing misreading and

effects, and also it have proven safe in all age groups [21].

*Update of Antibiotic Therapy of Brucellosis DOI: http://dx.doi.org/10.5772/intechopen.86325*

Barbiturates Consider

Aspirin Monitor

Rifampin Monitor

*Principal drug interactions of doxycycline.*

Consider therapy modification

therapy modification

therapy

therapy

Antacids (aluminum hydroxide, calcium carbonate, magnesium carbonate, sodium bicarbonate)

#### *2.1.7 Principal drug interactions*

#### See **Table 2**.

*Update of Antibiotic Therapy of Brucellosis DOI: http://dx.doi.org/10.5772/intechopen.86325*


#### **Table 2.**

*New Insight into* Brucella *Infection and Foodborne Diseases*

Time to peak serum: oral: 2–4 hours

Poor cerebrospinal fluid penetration

Bioavailability: reduced at high pH

Metabolism Not hepatic, partially inactivated in GI tract by chelate formation Elimination Half-time elimination: 18–22 hours, end-stage renal disease: 18–25 hours

Excretion: feces (30%); urine (23–40%)

Protein binding: >90% Distribution volume

When therapy of doxycycline needs to be used in prolonged therapy, some parameters need to be taken to prevent some of the adverse effects: complete blood count (CBC), renal and liver function tests periodically, during

Absorption Oral: almost completely absorbed from the gastrointestinal tract (GI) (90–100%), plasma

Distribution Widely distributed into tissues and fluids including synovial, pleural, prostatic, seminal

concentration may be reduced 20% by high-fat meal or milk

fluids and bronchial secretions, saliva, aqueous humor

• Gastrointestinal: it can produce GI irritation especially after oral administration (epigastric burning, abdominal discomfort, nausea, vomiting and diarrhea). To prevent this, the patient should take oral formulations with a glass full of water, administration on an empty stomach is generally not

• Photosensitivity: it may produce photosensitivity reactions in treated individuals exposed to sunlight. The patient needs to use skin protection and avoid

• Hepatotoxicity: rarely occurs during the treatment. If patient became symptomatic,

• Hypersensitivity syndromes: severe skin reactions have been reported. Discontinue

• Superinfection: prolonged use may result in fungal or bacterial superinfection

• Tissue hyperpigmentation: may induce hyperpigmentation in many organs: nails, bone, skin, eyes, thyroid, oral cavity (permanent brown discoloration of the teeth in children <8 years or in children from pregnant women in their last half of pregnancy), and sclerae, most dependently of time and

prolonged exposure to sunlight and ultraviolet light [14].

assess liver function tests, and discontinue drug [14].

therapy for serious hypersensitivity reactions.

like pseudomembranous colitis.

chronic use [15].

See **Table 2**.

*2.1.7 Principal drug interactions*

*2.1.5 Risk assessment*

*Pharmacokinetics parameters of doxycycline [13].*

therapy [13].

**Table 1.**

*2.1.6 Adverse effects*

recommended [14].

**38**

*Principal drug interactions of doxycycline.*

#### *2.1.8 Important*


#### **2.2 Streptomycin**

#### *2.2.1 Mechanism of action*

It is an aminoglycoside antibiotic bactericidal. Aminoglycosides diffuse through aqueous channels formed by porin proteins in the outer membrane of Gramnegative bacteria to enter to the periplasmic space, and its transport across the cytoplasmic membrane depends on an electrical gradient coupled to electron transport to drive permeation of these antibiotics. That is why they are not used in anaerobic environments of abscess. Once streptomycin is inside the cell, it binds to the 30S ribosomal subunit and interferes with protein synthesis by causing misreading and

premature termination of mRNA translation, and the resulting aberrant proteins may be inserted into the cell membrane altering permeability [24–26].

#### *2.2.2 Antimicrobial activity*

It is less active than other members of the class against aerobic Gram-negative, and it is used for the treatment of unusual infections and in combination with other antimicrobial agents. The inhibitory activity of aminoglycosides persists after the serum concentration has fallen below de minimum inhibitory concentration (MIC), and it is known as the post antibiotic effect and it improves the efficacy of high-dose extended-interval dosing regimens for aminoglycoside. It is used for the treatment of tuberculosis, tularemia, severe *M. avium* complex, brucellosis, and enterococcal endocarditis in combination with other drugs [24, 27].

#### *2.2.3 Pharmacokinetics*

#### See **Table 3**.


#### **Table 3.**

*Pharmacokinetic parameters of streptomycin [24, 28].*

#### *2.2.4 Administration*

Streptomycin may be administered by deep intramuscular injection into large muscle mass, rotate injection sites (it may be painful with a hot tender mass developing at the site injection) or intravenously (after dilution in admixture, infuse over 30–60 minutes). High-dose, extended-interval administration is the preferred administration of aminoglycosides because of less toxic effect than divided doses [24, 27].

#### *2.2.5 Risk assessment*

It is important to monitor hearing tests (baseline and periodic audiograms), BUN, creatinine, and serum drug concentrations should be monitored in all patients:

• Therapeutic peak: 20–30 mcg/mL [25].

#### *2.2.6 Adverse effects*

• Ototoxicity: aminoglycoside induces ototoxicity irreversible, bilateral, highfrequency hearing loss or vestibular hypofunction. It has been seen degeneration of hair cells and neurons in the cochlea and accumulation in the perilymph and endolymph at high antibiotic concentration in plasma. The initial symptoms such

**41**

species [24].

*Update of Antibiotic Therapy of Brucellosis DOI: http://dx.doi.org/10.5772/intechopen.86325*

*2.2.7 Principal drug interactions*

therapy modification

therapy modification

*Principal drug interactions of streptomycin.*

See **Table 4**.

Colistimethate Consider

Penicillins Consider

*2.2.8 Important*

**Table 4.**

**2.3 Gentamicin**

*2.3.1 Mechanism of action*

*2.3.2 Antimicrobial activity*

as high-pitched tinnitus, nausea, vomiting, and difficulty in equilibrium may be

• Nephrotoxicity: it is because the accumulation and retention of aminoglycoside in the proximal tubular cells and the initial manifestations of damage at this site are mild proteinuria and hyaline and granular casts, and also the glomeru-

• Streptomycin is not available always in some regions and it is administered only

• Gentamicin has replaced streptomycin for some indications because the toxicity of gentamicin is renal and mostly reversible although streptomycin is most vestibular

Gentamicin is a bactericidal aminoglycoside. It binds to the 30S ribosomal subunit and interferes with initiation of protein synthesis causing misreading of mRNA, premature termination of translation, and incomplete synthesized protein,

Gram-negative bacteria such as *Pseudomonas aeruginosa*, *Proteus* species, *Escherichia* 

*coli*, *Klebsiella* species, *Enterobacter* species, *Serratia*, *Citrobacter*, and *Staphylococcus*

intramuscularly or intravenously, so it is a disadvantage [2].

compromise and irreversible [24].

creating nonfunctional proteins [24, 33].

reversible, so it should be monitored carefully for ototoxicity [24].

lar filtration rate is reduced after several additional days [24].

**Drug Risk rating Interaction Mechanism Management**

Additive nephrotoxic

Inactivation of aminoglycosides by extended spectrum penicillins, especially in renal dysfunction

[31, 32]

Alteration in membrane permeability that leads to cellular lysis by colistimethate [29, 30]

This combination should be avoided, if they must be used together to monitor patients' renal and neuromuscular function

Monitor serum aminoglycoside concentration, and do not administer dose together through the same IV line

effects

Aminoglycosides may enhance the nephrotoxic and neuromuscularblocking effect of colistimethate

Penicillins may decrease the serum concentration of aminoglycosides

as high-pitched tinnitus, nausea, vomiting, and difficulty in equilibrium may be reversible, so it should be monitored carefully for ototoxicity [24].

• Nephrotoxicity: it is because the accumulation and retention of aminoglycoside in the proximal tubular cells and the initial manifestations of damage at this site are mild proteinuria and hyaline and granular casts, and also the glomerular filtration rate is reduced after several additional days [24].

#### *2.2.7 Principal drug interactions*

#### See **Table 4**.

*New Insight into* Brucella *Infection and Foodborne Diseases*

*2.2.2 Antimicrobial activity*

*2.2.3 Pharmacokinetics*

See **Table 3**.

*2.2.4 Administration*

**Table 3.**

*2.2.5 Risk assessment*

*2.2.6 Adverse effects*

• Therapeutic peak: 20–30 mcg/mL [25].

premature termination of mRNA translation, and the resulting aberrant proteins

It is less active than other members of the class against aerobic Gram-negative, and it is used for the treatment of unusual infections and in combination with other antimicrobial agents. The inhibitory activity of aminoglycosides persists after the serum concentration has fallen below de minimum inhibitory concentration (MIC), and it is known as the post antibiotic effect and it improves the efficacy of high-dose extended-interval dosing regimens for aminoglycoside. It is used for the treatment of tuberculosis, tularemia, severe *M. avium* complex, brucellosis, and

Distribution Into most body tissues and fluids except the brain and adipose tissue (because of their polar

Streptomycin may be administered by deep intramuscular injection into large muscle mass, rotate injection sites (it may be painful with a hot tender mass developing at the site injection) or intravenously (after dilution in admixture, infuse over 30–60 minutes). High-dose, extended-interval administration is the preferred administration of aminoglycosides because of less toxic effect than divided doses [24, 27].

Excretion Half-time elimination: adults: 2–4, 7 hours, prolonged with renal impairment

It is important to monitor hearing tests (baseline and periodic audiograms), BUN, creatinine, and serum drug concentrations should be monitored in all patients:

• Ototoxicity: aminoglycoside induces ototoxicity irreversible, bilateral, highfrequency hearing loss or vestibular hypofunction. It has been seen degeneration of hair cells and neurons in the cochlea and accumulation in the perilymph and endolymph at high antibiotic concentration in plasma. The initial symptoms such

may be inserted into the cell membrane altering permeability [24–26].

enterococcal endocarditis in combination with other drugs [24, 27].

Absorption Oral: poorly absorbed, IM: well absorbed Time to peak IM: 1–2 hours

Protein binding: 34%

Volume of distribution (Vd): 260 mL/kg

Urine: 29–89% as unchanged drug Bile, saliva, sweat and tears: (1%)

nature)

*Pharmacokinetic parameters of streptomycin [24, 28].*

Metabolism None known

**40**


#### **Table 4.**

*Principal drug interactions of streptomycin.*

#### *2.2.8 Important*


#### **2.3 Gentamicin**

#### *2.3.1 Mechanism of action*

Gentamicin is a bactericidal aminoglycoside. It binds to the 30S ribosomal subunit and interferes with initiation of protein synthesis causing misreading of mRNA, premature termination of translation, and incomplete synthesized protein, creating nonfunctional proteins [24, 33].

#### *2.3.2 Antimicrobial activity*

Gram-negative bacteria such as *Pseudomonas aeruginosa*, *Proteus* species, *Escherichia coli*, *Klebsiella* species, *Enterobacter* species, *Serratia*, *Citrobacter*, and *Staphylococcus* species [24].

#### *2.3.3 Pharmacokinetics*

#### See **Table 5**.


#### **Table 5.**

*Pharmacokinetic parameters of gentamicin [24, 34, 35].*

#### *2.3.4 Administration*


#### *2.3.5 Risk assessment*

During therapy with gentamicin, you should monitor parameters like: urinalysis, urine output, BUN, serum creatinine, plasma gentamicin levels (before and after the third dose), hearing tests before, during and after treatment especially in prolonged therapy [36, 37].

• Therapeutic peak: 5 and 12 μg/mL [36, 37].

#### *2.3.6 Adverse effects*


**43**

**Table 7.**

*Update of Antibiotic Therapy of Brucellosis DOI: http://dx.doi.org/10.5772/intechopen.86325*

**rating**

therapy

Monitor therapy

Monitor therapy

Amphotericin B may enhance the nephrotoxic effect of aminoglycosides

Aminoglycosides may enhance the hypocalcemic effect of bisphosphonate derivates [40]

Diuretics may enhance nephrotoxicity and ototoxicity of aminoglycosides

**Drug Risk** 

Bisphosphonate derivatives

Furosemide, bumetanide, torsemide (loop diuretics)

Amphotericin B Monitor

**2.4 Rifampin**

**Table 6.**

*2.4.1 Mechanism of action*

*Principal drug interactions of gentamicin.*

*2.4.2 Antimicrobial activity*

*tuberculosis* [44].

See **Table 7**.

*2.4.3 Pharmacokinetics*

Rifampin is a bactericidal drug that kills cell growing and it binds to the beta subunit of DNA-dependent RNA polymerase (rpoB) to form a drug-enzyme

**Interaction Mechanism Management**

Probably synergism [39]

Monitor renal function

Monitor serum calcium, serum magnesium and serum creatinine and renal function during concomitant

Monitor toxic effects or avoid concomitant use except in life-threatening situations

use

Unknown.

Association of aminoglycosides with hypocalcemia, probably inhibition of the activity of the parathyroid glands reducing parathyroid hormone production [40, 41]

Uncertain. Damage in proximal tubular cells and decrease glomerular filtration rate.

[42, 43]

It inhibits most Gram-positive bacteria and Gram-negative microorganisms

Absorption Oral: well absorbed (bioavailability 68%). Food may delay or reduce peak by one-third. It

Metabolism Microsomal B-esterases and cholinesterases. Also, 85% liver metabolism (potently induction CYP 1A2, 2C9, 2C19 and 3A4) and enterohepatic recirculation Excretion Half-life elimination: 3–4 hours, prolonged with hepatic impairment feces (60%) and urine

such as *Escherichia coli*, *Pseudomonas*, *Proteus*, and *Klebsiella*, and also it is active again *Neisseria meningitidis*, *Haemophilus influenzae*, and *Mycobacterium* 

complex blocking the chain formation in RNA transcription [44].

should be taken on an empty stomach Time to peak serum: oral: 2–4 hours Distribution Good penetration into many tissues and crosses CSF

> Vd: 53 L/kg Protein binding: 80%

*Pharmacokinetic parameters of rifampin [34, 44–46].*

(30%) as unchanged drug

#### *2.3.7 Principal drug interactions*

#### See **Table 6**.

*Update of Antibiotic Therapy of Brucellosis DOI: http://dx.doi.org/10.5772/intechopen.86325*


#### **Table 6.**

*New Insight into* Brucella *Infection and Foodborne Diseases*

Absorption Intramuscular: rapid and complete Oral: poorly absorbed

(CSF) and ocular tissues

Protein binding: <30%

Metabolism Minimal metabolism

Vd: children: 0.35 L/kg; adults: 0.2–0.3 L/kg

Excretion Half-life elimination: adults 2 hours, renal failure: 41–24 hours Urine: >70% as unchanged drug Clearance is decreased in renal impairment

• IM: it should be administered by deep IM route.

During therapy with gentamicin, you should monitor parameters like: urinalysis, urine output, BUN, serum creatinine, plasma gentamicin levels (before and after the third dose), hearing tests before, during and after treatment especially in

Time to peak: IM 30–90 minutes; IV: 30 minutes after 30-minute infusion Distribution Primarily into extracellular fluid, renal cortex. Poor penetration in cerebrospinal fluid

• Nephrotoxicity: usual risk factors include preexisting renal impairment,

concomitant nephrotoxicity drugs, advanced age, and dehydration. If nephrotoxicity occurs, it is better to discontinue therapy because the renal damage is

• Ototoxicity: use with caution in patients with preexisting vertigo, tinnitus or

• Neuromuscular blockade: aminoglycosides may inhibit prejunctional release of acetylcholine reducing postsynaptic sensitivity to the transmitter, and this reaction can follow intravenous, intramuscular or even oral administration of this antibiotics, especially with concomitant use of anesthesia and other neuromuscular blocking agents. It can be reversed by intravenous administration of calcium salt [24].

• IV: infuse over 30–120 minutes [27].

*Pharmacokinetic parameters of gentamicin [24, 34, 35].*

• Therapeutic peak: 5 and 12 μg/mL [36, 37].

*2.3.3 Pharmacokinetics*

See **Table 5**.

*2.3.4 Administration*

**Table 5.**

*2.3.5 Risk assessment*

*2.3.6 Adverse effects*

prolonged therapy [36, 37].

usually reversible [24, 38].

hearing loss [24, 38].

*2.3.7 Principal drug interactions*

See **Table 6**.

**42**

*Principal drug interactions of gentamicin.*

#### **2.4 Rifampin**

#### *2.4.1 Mechanism of action*

Rifampin is a bactericidal drug that kills cell growing and it binds to the beta subunit of DNA-dependent RNA polymerase (rpoB) to form a drug-enzyme complex blocking the chain formation in RNA transcription [44].

#### *2.4.2 Antimicrobial activity*

It inhibits most Gram-positive bacteria and Gram-negative microorganisms such as *Escherichia coli*, *Pseudomonas*, *Proteus*, and *Klebsiella*, and also it is active again *Neisseria meningitidis*, *Haemophilus influenzae*, and *Mycobacterium tuberculosis* [44].

#### *2.4.3 Pharmacokinetics*

See **Table 7**.


#### **Table 7.**

*Pharmacokinetic parameters of rifampin [34, 44–46].*

### *2.4.4 Administration*


#### *2.4.5 Risk assessment*

During the therapy with rifampin, it should be monitored with periodical liver function test, CBC, and therapeutic drug monitoring of rifampin [47].

#### *2.4.6 Adverse effects*


#### *2.4.7 Principal drug interactions*

Most of the interactions of rifampin are because it is a strong inducer of CYP3A4 and CYP2C19, moderate inducer of CYP2C8 and CYP2C9, and P-glycoprotein inducer (**Table 8**) [51].

#### *2.4.8 Important*


**45**

**Table 9.**

*Update of Antibiotic Therapy of Brucellosis DOI: http://dx.doi.org/10.5772/intechopen.86325*

combination

combination

therapy modification

Apixaban Avoid

Esomeprazole Avoid

Risperidone Consider

**2.5 Ciprofloxacin**

**Table 8.**

*2.5.1 Mechanism of action*

*Principal drug interactions of rifampin [55].*

*2.5.2 Antimicrobial activity*

*2.5.3 Pharmacokinetics*

See **Table 9**.

*Enterobacter*, and *Campylobacter* [56].

Absorption Oral: well absorbed

tissue

*Pharmacokinetic parameters of ciprofloxacin [55, 56].*

Protein binding: 20–40% Vd: 2.1–2.7 L/kg

and in renal impairment

The fluoroquinolones inhibit two bacterial enzymes: DNA gyrase (in many Gram-negative bacteria) and topoisomerase IV (in many Gram-positive bacteria) blocking the DNA bacterial replication. This action results in damage of bacterial

**Drug Risk rating Interaction Mechanism Management**

Induction of the CYP3A4 mediated metabolism of apixaban [52]

Rifampin induction of CYP3A4- and CYP2C19 mediated esomeprazole metabolism [53]

Unknown. Enzymeinducing drugs may decrease risperidone [54] Avoid concurrent

Avoid concomitant

Consider increasing the dose of oral risperidone (no more than double the original dose) if a CYP3A4 inducer is

initiated

use

use

Rifampin is a strong inducer CYP3A4 and may decrease the serum concentration of apixaban

Rifampin may decrease the serum concentration of esomeprazole

Rifampin is a CYP3A4 inducer that may decrease the serum concentration of risperidone [46]

It is a bactericidal agent against *Proteus*, *E. coli*, *Klebsiella*, *Salmonella*, *Shigella*,

Bioavailability: 70%. Avoid taking with most antacids and milk Distribution Widely distributed in kidneys, gallbladder, liver, lungs, gynecological tissue, and prostatic

Excretion Half-life elimination: children: 4–5 hours and adults: 3–5 hours, prolonged in older adults

Metabolism Poor hepatic metabolism and forms 4 metabolites, inhibitor of CYP1A2

Urine 50% as unchanged drug), feces (15%)

DNA and cell death being bactericidal agents [56, 57].

*Update of Antibiotic Therapy of Brucellosis DOI: http://dx.doi.org/10.5772/intechopen.86325*


#### **Table 8.**

*New Insight into* Brucella *Infection and Foodborne Diseases*

monitor administration to prevent extravasation.

• IV: administer IV preparation by slow infusion rate IV over 30 minutes to 3 hours,

• Oral: administer on an empty stomach with a glass of water to increase

function test, CBC, and therapeutic drug monitoring of rifampin [47].

tinue of therapy and management of the symptoms [48].

During the therapy with rifampin, it should be monitored with periodical liver

• Hypersensitivity reactions: cases of severe cutaneous adverse reactions like Stevens-Johnson syndrome, toxic epidermal necrolysis, and drug reaction with eosinophilia. It is mediated by hypersensitivity type I (IgE). It requires discon-

• Flu-like syndrome: symptoms of fever, chills, headache related with the use of

 weekly, and it resolves spontaneously. Flu-like syndrome is mediated by hypersensitivity type III (antibodies against rifampicin IgM that produce

• Hematologic effects: it may cause thrombocytopenia, leukopenia, or anemia. The platelets are damaged by complement activation following the formation

• Hepatotoxicity: it may cause hepatic dysfunction especially if it is used with

Most of the interactions of rifampin are because it is a strong inducer of CYP3A4

except where tetracyclines are contraindicated or when there are limitations

and CYP2C19, moderate inducer of CYP2C8 and CYP2C9, and P-glycoprotein

• In children, pregnant or lactating women rifampin should not be used

on the use of streptomycin or gentamicin and it should not be used

• It can be an alternative treatment for doxycycline or aminoglycosides, but the use of rifampin should be restricted in endemic areas of tuberculosis because monotherapy with rifampin can lead to the selection of resistant

*Mycobacterium tuberculosis* strains [1, 3, 6, 9].

oral rifampin. It is related with regimens of >600 mg once or twice

*2.4.4 Administration*

• Do not administer IM or SC.

immunocomplex) [48, 49].

of drug-antibody complex [48, 50].

other hepatotoxic agents [44].

*2.4.7 Principal drug interactions*

inducer (**Table 8**) [51].

alone [1, 6, 9].

*2.4.8 Important*

absorption [44].

*2.4.5 Risk assessment*

*2.4.6 Adverse effects*

**44**

*Principal drug interactions of rifampin [55].*

#### **2.5 Ciprofloxacin**

#### *2.5.1 Mechanism of action*

The fluoroquinolones inhibit two bacterial enzymes: DNA gyrase (in many Gram-negative bacteria) and topoisomerase IV (in many Gram-positive bacteria) blocking the DNA bacterial replication. This action results in damage of bacterial DNA and cell death being bactericidal agents [56, 57].

#### *2.5.2 Antimicrobial activity*

It is a bactericidal agent against *Proteus*, *E. coli*, *Klebsiella*, *Salmonella*, *Shigella*, *Enterobacter*, and *Campylobacter* [56].

#### *2.5.3 Pharmacokinetics*

See **Table 9**.


**Table 9.**

*Pharmacokinetic parameters of ciprofloxacin [55, 56].*

#### *2.5.4 Administration*


#### *2.5.5 Risk assessment*

During the treatment with ciprofloxacin parameters like: CBC, renal and hepatic function, signs and symptoms of tendonitis should be monitored [56].

#### *2.5.6 Adverse effects*


#### *2.5.7 Principal drug interactions*

#### See **Table 10**.


**47**

*Update of Antibiotic Therapy of Brucellosis DOI: http://dx.doi.org/10.5772/intechopen.86325*

some cases of relapse.

*2.6.1 Mechanism of action*

dihydrofolic acid [56, 66].

*2.6.2 Antimicrobial activity*

*2.6.3 Pharmacokinetics*

See **Table 11**.

*2.6.4 Administration*

*Pharmacokinetic parameters of TMP/SMX [56].*

**Table 11.**

*2.6.5 Risk assessment*

creatinine, and BUN [56].

**2.6 Trimethoprim-sulfamethoxazole (TMP/SMX)**

*Klebsiella*, *Enterobacter*, also *Brucella abortus* [56].

• It may be useful in the setting of drug resistance, antimicrobial toxicity, and

The combination of trimethoprim with sulfamethoxazole enhances the effectivity and synergist antimicrobial activity. TMP inhibits bacterial dihydrofolate reductase preventing the formation of tetrahydrofolic acid, and SMX is a structural analog of the para-aminobenzoic acid (PABA), and it binds to the dihydropteroate synthetase and competes with PABA to inhibit the synthesis of

The antibacterial spectrum is most *S. pneumoniae*, *S. aureus*, and *Staphylococcus* 

Absorption Oral: rapid 90–100%, TMP is absorbed more rapidly than sulfamethoxazole,

Distribution Good penetration in middle ear fluid, sputum, vaginal fluid, and bronchial

Metabolism Hepatic, SMX via CYP2C9 and also conjugated with glucuronide; TMP to oxide

Excretion Half time elimination: TMP: children 3.7–5.5 hours and adults: 6–11 hours. SMX

Both excreted in urine as metabolites and unchanged drug

Protein binding: SMX 70%, TMP 44%

bioavailability of 85%

and hydroxy derivatives

secretions Vd: adults: 1.3 L/kg

9–12 hours

*epidermidis*, some *E. coli* according to the geographic region, *Proteus mirabilis*,

• Oral: administer without regard to meals and a lot of water.

• IV: infuse over 60–90 minutes, and it is not administered by IM injection [56].

Some monitoring parameters during the treatment are CBC, serum potassium,

*2.5.8 Important*

#### **Table 10.**

*Principal drug interactions of ciprofloxacin.*

*Update of Antibiotic Therapy of Brucellosis DOI: http://dx.doi.org/10.5772/intechopen.86325*

#### *2.5.8 Important*

*New Insight into* Brucella *Infection and Foodborne Diseases*

yogurt or calcium-fortified juices alone.

patients taking corticosteroids [59, 60].

medications that prolong the QT interval [56, 61].

• IV: administer by slow IV infusion over 60 minutes [56].

function, signs and symptoms of tendonitis should be monitored [56].

• Gastrointestinal: nausea, vomiting, and abdominal discomfort [56].

• Neurologic: headache and dizziness, peripheral neuropathy, it can occur at any time during treatment and can last for months to years after finishing the

• Musculoskeletal: tendon rupture or tendinitis usually of the Achilles tendon, arthralgias, and join pain are reported, especially in ancient people and

• QT interval prolongation and arrhythmia: it may be produced by inhibition of potassium channels encoded by the KCNH2 gene (HERG gene). Ciprofloxacin use should be avoided in patients with a history of QT prolongation, torsade de pointes, uncorrected hypokalemia, cardiac disease or concomitant use of other

**Drug Risk rating Interaction Mechanism Management**

The carbonyl functional groups on the antibiotic forms a chelate with the cations of the antacid resulting in inactive antimicrobials [62, 63]

Quinolone inhibition of CYP1A2 and CYP3A4 isoenzymes limiting the metabolism of theophylline [64, 65]

Avoid concurrent administration of quinolones and antacids or quinolones should be administered at least 2 hours before or 2 hours after antacids or 6 hours after multivitamins

Consider a reduction in the dosage of theophylline (25–50%) during the concurrent use to minimize the theophylline toxicity

Antacids may decrease the absorption of quinolones

Quinolones may decrease the metabolism of theophylline

• Oral: administer with food to minimize GI symptoms, avoid antacid use, milk,

During the treatment with ciprofloxacin parameters like: CBC, renal and hepatic

*2.5.4 Administration*

*2.5.5 Risk assessment*

*2.5.6 Adverse effects*

treatment [58].

*2.5.7 Principal drug interactions*

Theophylline Consider

*Principal drug interactions of ciprofloxacin.*

Consider therapy modification

therapy modification

See **Table 10**.

Antacids, multivitamins and minerals like folate and iron

**46**

**Table 10.**

• It may be useful in the setting of drug resistance, antimicrobial toxicity, and some cases of relapse.

#### **2.6 Trimethoprim-sulfamethoxazole (TMP/SMX)**

#### *2.6.1 Mechanism of action*

The combination of trimethoprim with sulfamethoxazole enhances the effectivity and synergist antimicrobial activity. TMP inhibits bacterial dihydrofolate reductase preventing the formation of tetrahydrofolic acid, and SMX is a structural analog of the para-aminobenzoic acid (PABA), and it binds to the dihydropteroate synthetase and competes with PABA to inhibit the synthesis of dihydrofolic acid [56, 66].

#### *2.6.2 Antimicrobial activity*

The antibacterial spectrum is most *S. pneumoniae*, *S. aureus*, and *Staphylococcus epidermidis*, some *E. coli* according to the geographic region, *Proteus mirabilis*, *Klebsiella*, *Enterobacter*, also *Brucella abortus* [56].

#### *2.6.3 Pharmacokinetics*

See **Table 11**.


#### **Table 11.**

*Pharmacokinetic parameters of TMP/SMX [56].*

#### *2.6.4 Administration*


#### *2.6.5 Risk assessment*

Some monitoring parameters during the treatment are CBC, serum potassium, creatinine, and BUN [56].

#### *2.6.6 Adverse effects*


#### *2.6.7 Principal drug interactions*

#### See **Table 12**.


#### **Table 12.**

*Principal drug interactions of TMP/SMX.*

#### *2.6.8 Important*


**49**

*Update of Antibiotic Therapy of Brucellosis DOI: http://dx.doi.org/10.5772/intechopen.86325*

**3. Other considerations about treatment**

with TMP-SMX allergy, they use as an alternative for treatment ciprofloxacin having a good result of the treatment and continue follow up visits, but there are no evidence enough for this treatment, so it is necessary to search for

• Doxycycline is the drug of choice in the treatment of brucellosis, but antibiotic susceptibility patterns of *Brucella* appears to vary geographically, that is why tigecycline can be an option for treatment in brucellosis. Tigecycline is a glycylcycline derivate from tetracycline and minocycline. It has demonstrated activity against *Enterobacteriaceae*, Gram-positives, atypical, and anaerobes. It has the lowest minimal inhibitory concentration on in vitro efficacy models, and also it provided the better synergistic activity compared to doxycycline. Tigecycline can be a therapeutic alternative for brucellosis especially in patients in whom conventional antibiotics is contraindicated or limited because of the presence of severe comorbidities or drug-drug interactions, but

• There are some regional experience and some different treatments that differs

○ The World Health Organization (WHO) recommends the use of doxycycline for 6 weeks combined with rifampicin for 6 weeks, or streptomycin for 2–3 weeks, but this regimen has not been universally used in clinical practice. Even this fact it remains unclear what is the best regimen to be used and

according the regional experiences but here are some considerations:

○ From the comparison of regimens that can be established in randomized clinical trials are: doxycycline and streptomycin vs. doxycycline and rifampicin that favors the first combination in terms of relapse (OR 3.52; CI 95% = 2.14–5.81; p < 0.001); doxycycline and streptomycin vs. doxycycline and gentamicin is not statistically significant as regards either relapses (OR = 1.65; CI 95% = 0.53–5.15; p = 0.386); doxycycline and rifampicin vs. doxycycline and quinolone favors the first one (OR 3.92; CI 95% = 1.35–

○ The most effective regimen is combined doxycycline for 45 days with streptomycin for 14 days, in endemic areas where many patients have a mild form of the disease and diagnosis and prescription can be made in the urgency

○ There are a few studies using doxycycline, rifampicin, and aminoglycosides vs. other regimens in uncomplicated brucellosis with no conclusions on the value of this triple therapy, also some studies were performed only in patients with osteoarticular complications. Another option for triple therapy is doxycycline,

room the used to use gentamycin for the first 5–7 days [4, 73].

○ About the comparison of the efficacy of gentamicin for 5 days plus doxycycline for 8 weeks vs. streptomycin for 2 weeks plus doxycycline for 45 days in human brucellosis, there is a clinical trial that compare the efficacy showing that this treatment is not superior to the standard treat-

alternative treatment for this patient population [12].

it should be supported with more clinical studies [72].

more clinical studies are needed in this regard [2].

11.42; p = 0.01) [73].

ment regimen [74].

*New Insight into* Brucella *Infection and Foodborne Diseases*

related with folate deficient [67].

*2.6.7 Principal drug interactions*

See **Table 12**.

Phenytoin Consider

Warfarin Consider

therapy modification

therapy modification

*Principal drug interactions of TMP/SMX.*

*2.6.8 Important*

**Table 12.**

losis, relapse, or refractory disease [2].

• Blood dyscrasias: agranulocytosis, aplastic anemia, leukopenia, or thrombocytopenia because of the margin between toxicity for bacteria and humans

• Neurologic effects: it is associated with adverse neurologic events like aseptic meningitis, tremor, delirium because TMP/SMX crosses the blood-brain barrier [67].

• Dermatologic reactions: severe reactions including Stevens-Johnson syndrome produced by immune-mediated idiosyncratic reactions associated with reac-

• Hyperkalemia: it is produced because of the TMP similar structure to potassiumsparing diuretics. Potential risk factors include renal impairment, older age, and concomitant use of medications causing or exacerbating hyperkalemia [56].

**Drug Risk rating Interaction Mechanism Management**

TMP inhibition of CYP2C8 and CYP2C9 mediated phenytoin metabolism [68]

Multifactorial. Sulfonamide displacement of warfarin from protein binding sites, reductions in GI flora responsible for production of vitamin

K [69, 70]

Consider alternatives to this combination when possible

Monitor toxic effects of warfarin. Consider reducing warfarin dose by 10–20% prior starting the sulfonamide antibiotic and monitoring INR closely [71]

TMP/SMX may increase the serum concentration of phenytoin

TMP/SMX may enhance the anticoagulant effect of vitamin K antagonists

• TMP-SMX may be used as an additional agent in complex cases of focal brucel-

• TMP-SMZ should not be used in pregnancy, either before 13 weeks because of the risk of teratogenic effects or after 36 weeks because of the risk of kernicterus [21].

• It has been a popular choice and it is included in combination regimens around the world, due to its lower cost compared to other antimicrobials being the most cost-effective drug against brucellosis in developing countries [2].

• No alternative anti-brucellosis therapy for children under 8 years old has been reported, but there is a case that had a 2.5 years old patient with brucellosis

tive metabolite leading to drug-specific antibodies [67].

*2.6.6 Adverse effects*

**48**

with TMP-SMX allergy, they use as an alternative for treatment ciprofloxacin having a good result of the treatment and continue follow up visits, but there are no evidence enough for this treatment, so it is necessary to search for alternative treatment for this patient population [12].

### **3. Other considerations about treatment**

	- The World Health Organization (WHO) recommends the use of doxycycline for 6 weeks combined with rifampicin for 6 weeks, or streptomycin for 2–3 weeks, but this regimen has not been universally used in clinical practice. Even this fact it remains unclear what is the best regimen to be used and more clinical studies are needed in this regard [2].
	- From the comparison of regimens that can be established in randomized clinical trials are: doxycycline and streptomycin vs. doxycycline and rifampicin that favors the first combination in terms of relapse (OR 3.52; CI 95% = 2.14–5.81; p < 0.001); doxycycline and streptomycin vs. doxycycline and gentamicin is not statistically significant as regards either relapses (OR = 1.65; CI 95% = 0.53–5.15; p = 0.386); doxycycline and rifampicin vs. doxycycline and quinolone favors the first one (OR 3.92; CI 95% = 1.35– 11.42; p = 0.01) [73].
	- The most effective regimen is combined doxycycline for 45 days with streptomycin for 14 days, in endemic areas where many patients have a mild form of the disease and diagnosis and prescription can be made in the urgency room the used to use gentamycin for the first 5–7 days [4, 73].
	- About the comparison of the efficacy of gentamicin for 5 days plus doxycycline for 8 weeks vs. streptomycin for 2 weeks plus doxycycline for 45 days in human brucellosis, there is a clinical trial that compare the efficacy showing that this treatment is not superior to the standard treatment regimen [74].
	- There are a few studies using doxycycline, rifampicin, and aminoglycosides vs. other regimens in uncomplicated brucellosis with no conclusions on the value of this triple therapy, also some studies were performed only in patients with osteoarticular complications. Another option for triple therapy is doxycycline,

rifampin, and amikacin (intramuscularly twice a day for 7 days) that have higher efficacy and more rapid action in terms of relief of symptoms, but it has no significant difference in drug side-effects and disease relapse, thus adding amikacin to the standard treatment regimen seems beneficial [6, 75].

#### **4. Conclusion**

In conclusion, there are some antibiotic therapies that are approved for the treatment of brucellosis, and some of them are in prolonged therapy that could affect the adherence of the patient and some of the antibiotics have important recommendations and need to be used in some conditions. Also, they have some parameters that may be monitorable to prevent adverse effects and to improve the outcome of the treatment in all the patients.

**51**

**Author details**

Sabana, Chia, Colombia

provided the original work is properly cited.

Sara Consuelo Arias Villate and Julio Cesar García Casallas\*

\*Address all correspondence to: julio.garcia@unisabana.edu.co

*Update of Antibiotic Therapy of Brucellosis DOI: http://dx.doi.org/10.5772/intechopen.86325*

© 2020 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium,

Evidence-Based Therapeutic Group, Clinical Pharmacology, Universidad de La

#### **Conflict of interest**

The authors declare no conflict of interest.

#### **Notes/thanks/other declarations**

None.

#### **Appendices and nomenclature**


*Update of Antibiotic Therapy of Brucellosis DOI: http://dx.doi.org/10.5772/intechopen.86325*

*New Insight into* Brucella *Infection and Foodborne Diseases*

**4. Conclusion**

the treatment in all the patients.

The authors declare no conflict of interest.

**Notes/thanks/other declarations**

**Appendices and nomenclature**

CBC complete blood count GI gastrointestinal

Vd volume of distribution

MIC minimum Inhibitory concentration TMP/SMX trimethoprim-sulfamethoxazole

**Conflict of interest**

h hours

IM intramuscular IV intravenous kg kilograms mg milligrams

None.

rifampin, and amikacin (intramuscularly twice a day for 7 days) that have higher efficacy and more rapid action in terms of relief of symptoms, but it has no significant difference in drug side-effects and disease relapse, thus adding

In conclusion, there are some antibiotic therapies that are approved for the treatment of brucellosis, and some of them are in prolonged therapy that could affect the adherence of the patient and some of the antibiotics have important recommendations and need to be used in some conditions. Also, they have some parameters that may be monitorable to prevent adverse effects and to improve the outcome of

amikacin to the standard treatment regimen seems beneficial [6, 75].

**50**

#### **Author details**

Sara Consuelo Arias Villate and Julio Cesar García Casallas\* Evidence-Based Therapeutic Group, Clinical Pharmacology, Universidad de La Sabana, Chia, Colombia

\*Address all correspondence to: julio.garcia@unisabana.edu.co

© 2020 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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theophylline interaction using the Food and Drug Administration spontaneous reporting system. Archives of Internal Medicine. 1992;**152**(3):617. DOI: 1001/ archinte.1992.00400150127023

[64] Prince RA, Casabar E, Adair CG, Wexler DB, Lettieri J, Kasik JE. Effect of quinolone antimicrobials on theophylline pharmacokinetics. Journal of Clinical Pharmacology. 1989;**29**(7):650-654. DOI: 10.1002/

Report of 6 cases. The Journal of Rheumatology. 1996;**23**(3):516-520

2001;**59**(1):122-126

in elderly patients taking oral corticosteroids. Archives of Internal Medicine. 2003;**163**(15):1801-1807. DOI:

10.1001/archinte.163.15.1801

**56**

[73] Solís García del Pozo J, Solera J. Systematic review and meta-analysis of randomized clinical trials in the treatment of human brucellosis. PLoS One. 2012;**7**(2):e32090

[74] Roushan MRH, Amiri MJS, Janmohammadi N, Hadad MS, Javanian M, Baiani M, et al. Comparison of the efficacy of gentamicin for 5 days plus doxycycline for 8 weeks versus streptomycin for 2 weeks plus doxycycline for 45 days in the treatment of human brucellosis: A randomized clinical trial. The Journal of Antimicrobial Chemotherapy. 2010;**65**(5):1028-1035

[75] Ranjbar M, Keramat F, Mamani M, Kia AR, Khalilian FO-S, Hashemi SH, et al. Comparison between doxycyclinerifampin-amikacin and doxycyclinerifampin regimens in the treatment of brucellosis. International Journal of Infectious Diseases. 2007;**11**(2):152-156

**59**

**Chapter 6**

**Abstract**

and adaptability.

**1. Introduction**

Salmonellosis

Immunopathogenesis of

*Tufail Banday and Syed Mudasir Ahmad*

*Mashooq Ahmad Dar, Peerzada Tajamul Mumtaz,* 

**Keywords:** *Salmonella*, serovars, adaptability, specificity, invasion, non-typhoidal *Salmonella*, typhoidal *Salmonella*, immune response

*Salmonellae* are facultative anaerobes and gram-negative, non-spore-forming and usually motile bacilli. Two species, namely, *Salmonella enterica and Salmonella bongori*, belong to genus *Salmonella*. *Salmonella enterica* is further subdivided into six subspecies that are distinguished by variations in O (somatic) and H (flagellar) antigens with at least 2500 serotypes. *S. enterica* subsp. *enterica* comprises of more than half of the known serotypes [1]. New serotypes are being discovered increasing the serotype complexity. Approximately 99% of the *Salmonella* serotypes that infect humans and other mammals belong to *S. enterica* subspecies. These serovars are mostly the inhabitants of intestinal tract of humans and other organisms that include reptiles, birds and insects. At farm level, sources of bacterial contamination are faecal matter, litter, feed and soil [2]. *Salmonella* most commonly causes foodborne illnesses worldwide; the two commonly associated foods are eggs and poultry

*Shakil Ahmad Bhat, Qamar Taban, Shabir Ahmad Khan,* 

*Salmonella* is an intracellular pathogenic, gram-negative, facultative anaerobe and non-spore-forming and usually a motile bacillus that leads to salmonellosis in the host. It is a common food-borne disease that ranges from local gastrointestinal inflammation and diarrhoea to life-threatening typhoid fever and presents usually a serious threat to public health due to its socio-economic value. Inadequate sanitation and impure water help in the propagation of this disease. Despite advancement in the sanitation standards, *Salmonella* enters the food chain and affects communities globally. There is an immediate need to develop improved vaccines to minimise *Salmonella*-related illnesses. Some *Salmonella* serovars infect a wide range of hosts, while others are known to be host restricted. Many different factors determine the adaptability and host specificity of *Salmonella*. The host-pathogen interactions play a unique role in *Salmonella* invasion and progression which needs to be studied in detail. This chapter shall focus on our current understanding of *Salmonella* invasion, pathogenesis and interactions with the host, host specificity

#### **Chapter 6**

## Immunopathogenesis of Salmonellosis

*Mashooq Ahmad Dar, Peerzada Tajamul Mumtaz, Shakil Ahmad Bhat, Qamar Taban, Shabir Ahmad Khan, Tufail Banday and Syed Mudasir Ahmad*

#### **Abstract**

*Salmonella* is an intracellular pathogenic, gram-negative, facultative anaerobe and non-spore-forming and usually a motile bacillus that leads to salmonellosis in the host. It is a common food-borne disease that ranges from local gastrointestinal inflammation and diarrhoea to life-threatening typhoid fever and presents usually a serious threat to public health due to its socio-economic value. Inadequate sanitation and impure water help in the propagation of this disease. Despite advancement in the sanitation standards, *Salmonella* enters the food chain and affects communities globally. There is an immediate need to develop improved vaccines to minimise *Salmonella*-related illnesses. Some *Salmonella* serovars infect a wide range of hosts, while others are known to be host restricted. Many different factors determine the adaptability and host specificity of *Salmonella*. The host-pathogen interactions play a unique role in *Salmonella* invasion and progression which needs to be studied in detail. This chapter shall focus on our current understanding of *Salmonella* invasion, pathogenesis and interactions with the host, host specificity and adaptability.

**Keywords:** *Salmonella*, serovars, adaptability, specificity, invasion, non-typhoidal *Salmonella*, typhoidal *Salmonella*, immune response

#### **1. Introduction**

*Salmonellae* are facultative anaerobes and gram-negative, non-spore-forming and usually motile bacilli. Two species, namely, *Salmonella enterica and Salmonella bongori*, belong to genus *Salmonella*. *Salmonella enterica* is further subdivided into six subspecies that are distinguished by variations in O (somatic) and H (flagellar) antigens with at least 2500 serotypes. *S. enterica* subsp. *enterica* comprises of more than half of the known serotypes [1]. New serotypes are being discovered increasing the serotype complexity. Approximately 99% of the *Salmonella* serotypes that infect humans and other mammals belong to *S. enterica* subspecies. These serovars are mostly the inhabitants of intestinal tract of humans and other organisms that include reptiles, birds and insects. At farm level, sources of bacterial contamination are faecal matter, litter, feed and soil [2]. *Salmonella* most commonly causes foodborne illnesses worldwide; the two commonly associated foods are eggs and poultry meat [3]. Serovars *S.* Enteritidis, *S.* Typhimurium, *S*. Heidelberg and *S.* Newport are linked to such food-borne diseases, with farm animals being reservoirs for these serotypes [4, 5]. Salmonellosis is a big socio-economic threat worldwide that causes considerable mortality and morbidity in both humans and animals [6]. Most of the human-related diseases are food-borne, and exposure to these bacteria at different places has also been linked to human salmonellosis. The most orthodox mode of bacterial transmission is the faecal-oral route. Once the bacteria are transmitted, the initial site for bacterial infection is the small intestine. Following infection, different manifestations that arise range from gastroenteritis to enteric fever [7].

#### **2. Epidemiology**

The extensive investigation of the associated epidemiological risk factors that make an organism a persistent *Salmonella* carrier needs to be carried out. Nontyphoidal *Salmonella* (NTS) infections that cause self-limiting manifestations are the most common to occur globally. In comparison, typhoidal *Salmonella* (TS) causing enteric fever leads to a high rate of mortality and morbidity that predominantly affects the underdeveloped countries [8]. Recent studies conclude that *Salmonella* Paratyphi A incidences have risen especially in South East Asia where approximately half of the TS-infected enteric fever patients are reported to be infected with *S*. Paratyphi A [9]. The food chain can get contaminated at any stage, and most of the transmission can occur by contaminated foods like poultry and dairy-related products. Apart from contaminated food products, NTS transmission can also result from person-to-person contact or from contact with other bacterial reservoirs. After gaining entry into the host, both TS and NTS serovars initially invade the intestinal epithelium of the small intestine.

#### **3. Diseases caused by** *Salmonella* **infection**

*Salmonella* species cause a varying number of clinical manifestations in the host that can range from self-limiting gastroenteritis typically associated with nontyphoidal *Salmonella* (NTS) to typhoidal or paratyphoidal fevers, which can be life-threatening [6].

#### **3.1 Typhoidal** *Salmonella* **(TS)**

Humans are exclusive hosts for serovars such as *S*. Typhi, *S*. Sendai and *S*. Paratyphi A, B and C. These serovars are known as typhoidal serovars (TS) that can cause enteric fever/typhoidal/paratyphoidal fevers. Enteric fever is a systemic disease that is highly invasive and life threatening and is endemic in the developing world. Within an incubation period of around 2 weeks, bacteraemia occurs, which is marked by fever and malaise. The symptoms that start to appear after a week include fever, malaise, nausea, dry cough and abdominal discomfort. Common symptoms include tender abdomen, coated tongue, splenomegaly and hepatomegaly [10]. As the lack of adequate water and sanitation facilities in the developing countries help in the spread of *Salmonella* through faecal-oral route [11], so to prevent typhoidal *Salmonella* transmission, societal standards need to be improved. The development of improved societal infrastructures is generally cost prohibitive in developing countries, hence, having little significance in reducing the disease frequency. In comparison, the development of effective and safe typhoidal vaccines can have a significant effect on reducing typhoidal cases.

**61**

*Immunopathogenesis of Salmonellosis*

*DOI: http://dx.doi.org/10.5772/intechopen.85371*

*Salmonella* serovars that shall be discussed in detail.

**4. Host specificity and adaptation**

Many industrialised and underdeveloped countries across the globe face a significant threat of non-typhoidal *Salmonella* (NTS). Worldwide, about 93.8 million gastroenteritis cases arise from *Salmonella* infections leading to 1.5 million deaths annually [7]. In infants, young, aged and immunologically compromised subjects NTS cause invasive bacterial infection [12, 13]. After post infection with NTS, symptoms that arise last for about 10 days that triggers a massive inflammatory response involving the release of pro-inflammatory cytokines and chemokines. The human NTS patients have higher serum levels of different interleukins and cytokines like IL-18, IL-12, IL-10, IL-15, TNF- α and IFN γ [14]. Non-typhoidal *Salmonella* serovars can cause severe extra-intestinal disease in patients with deficiencies in type 1 cytokine pathways such as IFN-γ/IL-12/IL-23 especially IL-12 abnormalities. Effective vaccination against NTS is lacking as there is a greater variance among different serovars. So, for generating effective vaccines against NTS, knowledge regarding different target antigens needs to be studied in detail. To disrupt the bacterial transmission and the incidence, effective preventive measures such as improving sanitation, hygiene and drinking clean water must be taken into consideration. Different host specificities and adaptability are shown by different

Salmonellosis susceptibly ranges from organism to organism and can occur in almost all animal species, but the clinical severity of this disease varies among the hosts. There are only specific serovars that cause severe clinical manifestations in their specific hosts [15]. Although most of the serovars of *S. enterica* subspecies cause infections which give rise to gastroenteritis that lasts for short durations, some serovars lead to severe systemic illness in humans and animals accompanied by septicaemia, fever and in some cases abortion. Based on the host specificity, these serovars can be grouped into two categories: the first category consists of serovars that are single-host restricted and the second category infecting a broad range of hosts. Different factors can be considered for grouping different serovars under the above-mentioned categories. Also, serovar pathogenicity and host epidemiology define host specificity. Keeping the above factors into account, different serovars have been grouped into three major groups. The first group comprises of serovars that mainly infect cattle and pigs but can also infect other animals including humans in some accidental cases. In this group, *S. choleraesuis* and dublin have been included that cause systemic diseases in the above-mentioned hosts. In humans and other animals, clinical symptoms may not be visible, thus making them asymptomatic. *Salmonella* carriers that can shed the bacteria in the surroundings thus leading to increased risk for susceptible hosts are also known as host-adapted serovars (HA) [16]. The second group infects specific hosts and is collectively known as host-restricted (HR) serovars. The HR serovars cause systemic diseases and can sometimes prove lethal in their hosts that include poultry, humans, sheep, equine and pigs. This group includes *S. gallinarum*, *S*. typhi, *S.* abortus and *S.* abortusequi. They interfere with the environment of their hosts in a way that paves their way for invading the host. This ability to cause mammalian abortions and loss in poultry egg production is due to their remarkable ability to multiply in the foetal tissues [16–18]. The serovars of the third group are known as unrestricted serovars that are of zoonotic, epidemiological importance and impose a great threat to many animals and humans. The serovars of this group that are of much clinical

**3.2 Non-typhoidal** *Salmonella* **(NTS)**

#### **3.2 Non-typhoidal** *Salmonella* **(NTS)**

*New Insight into* Brucella *Infection and Foodborne Diseases*

invade the intestinal epithelium of the small intestine.

can have a significant effect on reducing typhoidal cases.

**3. Diseases caused by** *Salmonella* **infection**

**2. Epidemiology**

life-threatening [6].

**3.1 Typhoidal** *Salmonella* **(TS)**

meat [3]. Serovars *S.* Enteritidis, *S.* Typhimurium, *S*. Heidelberg and *S.* Newport are linked to such food-borne diseases, with farm animals being reservoirs for these serotypes [4, 5]. Salmonellosis is a big socio-economic threat worldwide that causes considerable mortality and morbidity in both humans and animals [6]. Most of the human-related diseases are food-borne, and exposure to these bacteria at different places has also been linked to human salmonellosis. The most orthodox mode of bacterial transmission is the faecal-oral route. Once the bacteria are transmitted, the initial site for bacterial infection is the small intestine. Following infection, different manifestations that arise range from gastroenteritis to enteric fever [7].

The extensive investigation of the associated epidemiological risk factors that make an organism a persistent *Salmonella* carrier needs to be carried out. Nontyphoidal *Salmonella* (NTS) infections that cause self-limiting manifestations are the most common to occur globally. In comparison, typhoidal *Salmonella* (TS) causing enteric fever leads to a high rate of mortality and morbidity that predominantly affects the underdeveloped countries [8]. Recent studies conclude that *Salmonella* Paratyphi A incidences have risen especially in South East Asia where approximately half of the TS-infected enteric fever patients are reported to be infected with *S*. Paratyphi A [9]. The food chain can get contaminated at any stage, and most of the transmission can occur by contaminated foods like poultry and dairy-related products. Apart from contaminated food products, NTS transmission can also result from person-to-person contact or from contact with other bacterial reservoirs. After gaining entry into the host, both TS and NTS serovars initially

*Salmonella* species cause a varying number of clinical manifestations in the host

that can range from self-limiting gastroenteritis typically associated with nontyphoidal *Salmonella* (NTS) to typhoidal or paratyphoidal fevers, which can be

Humans are exclusive hosts for serovars such as *S*. Typhi, *S*. Sendai and *S*. Paratyphi A, B and C. These serovars are known as typhoidal serovars (TS) that can cause enteric fever/typhoidal/paratyphoidal fevers. Enteric fever is a systemic disease that is highly invasive and life threatening and is endemic in the developing world. Within an incubation period of around 2 weeks, bacteraemia occurs, which is marked by fever and malaise. The symptoms that start to appear after a week include fever, malaise, nausea, dry cough and abdominal discomfort. Common symptoms include tender abdomen, coated tongue, splenomegaly and hepatomegaly [10]. As the lack of adequate water and sanitation facilities in the developing countries help in the spread of *Salmonella* through faecal-oral route [11], so to prevent typhoidal *Salmonella* transmission, societal standards need to be improved. The development of improved societal infrastructures is generally cost prohibitive in developing countries, hence, having little significance in reducing the disease frequency. In comparison, the development of effective and safe typhoidal vaccines

**60**

Many industrialised and underdeveloped countries across the globe face a significant threat of non-typhoidal *Salmonella* (NTS). Worldwide, about 93.8 million gastroenteritis cases arise from *Salmonella* infections leading to 1.5 million deaths annually [7]. In infants, young, aged and immunologically compromised subjects NTS cause invasive bacterial infection [12, 13]. After post infection with NTS, symptoms that arise last for about 10 days that triggers a massive inflammatory response involving the release of pro-inflammatory cytokines and chemokines. The human NTS patients have higher serum levels of different interleukins and cytokines like IL-18, IL-12, IL-10, IL-15, TNF- α and IFN γ [14]. Non-typhoidal *Salmonella* serovars can cause severe extra-intestinal disease in patients with deficiencies in type 1 cytokine pathways such as IFN-γ/IL-12/IL-23 especially IL-12 abnormalities. Effective vaccination against NTS is lacking as there is a greater variance among different serovars. So, for generating effective vaccines against NTS, knowledge regarding different target antigens needs to be studied in detail. To disrupt the bacterial transmission and the incidence, effective preventive measures such as improving sanitation, hygiene and drinking clean water must be taken into consideration. Different host specificities and adaptability are shown by different *Salmonella* serovars that shall be discussed in detail.

#### **4. Host specificity and adaptation**

Salmonellosis susceptibly ranges from organism to organism and can occur in almost all animal species, but the clinical severity of this disease varies among the hosts. There are only specific serovars that cause severe clinical manifestations in their specific hosts [15]. Although most of the serovars of *S. enterica* subspecies cause infections which give rise to gastroenteritis that lasts for short durations, some serovars lead to severe systemic illness in humans and animals accompanied by septicaemia, fever and in some cases abortion. Based on the host specificity, these serovars can be grouped into two categories: the first category consists of serovars that are single-host restricted and the second category infecting a broad range of hosts. Different factors can be considered for grouping different serovars under the above-mentioned categories. Also, serovar pathogenicity and host epidemiology define host specificity. Keeping the above factors into account, different serovars have been grouped into three major groups. The first group comprises of serovars that mainly infect cattle and pigs but can also infect other animals including humans in some accidental cases. In this group, *S. choleraesuis* and dublin have been included that cause systemic diseases in the above-mentioned hosts. In humans and other animals, clinical symptoms may not be visible, thus making them asymptomatic. *Salmonella* carriers that can shed the bacteria in the surroundings thus leading to increased risk for susceptible hosts are also known as host-adapted serovars (HA) [16]. The second group infects specific hosts and is collectively known as host-restricted (HR) serovars. The HR serovars cause systemic diseases and can sometimes prove lethal in their hosts that include poultry, humans, sheep, equine and pigs. This group includes *S. gallinarum*, *S*. typhi, *S.* abortus and *S.* abortusequi. They interfere with the environment of their hosts in a way that paves their way for invading the host. This ability to cause mammalian abortions and loss in poultry egg production is due to their remarkable ability to multiply in the foetal tissues [16–18]. The serovars of the third group are known as unrestricted serovars that are of zoonotic, epidemiological importance and impose a great threat to many animals and humans. The serovars of this group that are of much clinical

importance are *S. typhimurium* and *S. enteritidis* [18]. These cause mild symptoms in the adult host, and sometimes the host does not show any visible clinical symptoms despite infection. They severely invade young hosts as compared to adult hosts because the adult hosts have a well-built immune system that hinders the invasion by these serovars [16]. The host specificity and adaptability of different serovars are a complex process and involve many molecular mechanisms. The exact mechanisms are poorly studied, but certain factors have been found to be responsible for determining host specificity and adaptability.

#### **4.1 Factors determining** *Salmonella* **host specificity and adaptation**

Although the exact mechanisms to host specificity have not been fully deciphered, the existing evidence shows that serovars act independently of each other at the various phases of infection. The expression of serovar's pathogenicity is affected by the environmental and genetic factors influencing each host during adaptation [19]. Each HA/HR serovar must overcome the encountered specific and nonspecific immune mechanisms. Thus, pathogenicity of HA serovars results from the development of ways helping their survival in a host. Examples of this are serovars of *S*. *enterica* subsp. *enterica*, which have developed the ability to evade the immune mechanisms of warm-blooded animals. They have, during their evolution, acquired the ability to modify to their favour the physiological functions of their host, such as intracellular engulfment, apoptosis, transfer of antigens by M (microfold) cells, migration of macrophages and lymphocytes in the reticuloendothelial system and others [20]. A well-known serovar *S*. *typhi* has evolved to survive in human macrophages making it pathogenic to man, but not to mice [21]. Serovars such as the HR *S*. *typhi*, *S. gallinarum* and *S. abortusovis* show high tropism for the lymphatic organs of their hosts, thereby regulating their natural host's biological environment in their favour [19]. By anchoring to the cells of the bone marrow, PPs and bursa of Fabricius in the development of B cells, thus immune response is affected. The result of such interactions, particularly in adult animals, helps in the establishment of chronic or subclinical infection and thus prolonged subclinical excretion, but they do not help in the development of severe gastroenteritis [22]. Serovars not fully adapted to evade the mature immune system lack specificity, causing deadly systemic disease in adult animals by invading their defence mechanisms compared to HR serovars [20]. HR serovars are mildly enteropathogenic compared to the unrestricted serovars; thus, they do not cause intestinal inflammation [23]. It has also been shown that the ability of a serovar to metabolise a wide range of amino acids adds to its virulence and is thought to be closely related [24, 25]; however, HR or HA serovars have most likely evolved independently. On the other hand, the heterogenicity of serovars in relation to different metabolic profiles facilitates either the completion of the pathway to infection [26] or, when lacking specificity, it is favouring host adaptation [19]. The process of *Salmonella* host adaptation is believed to be involving either the loss of genes or the acquisition of novel genetic elements that encode specific virulence factors, and thus an inconvenience is observed frequently in the pathogenic strains. Best examples of host specificity dependent on gene deletions are, perhaps, of *S. enteritidis*, Typhimurium, Choleraesuis, Gallinarum, Abortusovis, Pullorum and Paratyphi C [27]. Genome sequencing of HA/HR serovars, such as Typhi, Gallinarum, Choleraesuis and the newly emerging in sub-Saharan Africa invasive strains of *S*. Typhi, has divulged that these have encountered extensive gene deletions and truncation [28, 29]. In systemically noninvasive *Salmonella*, the majority of lost genes have functional orthologues, which play a key role in intestinal colonisation, thus resulting in the loss of an intestinal multiplication cycle for narrowly host-adapted *Salmonellae*

**63**

*Immunopathogenesis of Salmonellosis*

*DOI: http://dx.doi.org/10.5772/intechopen.85371*

followed by a concurrent acquisition of mechanisms helping the microorganism to survive in a systemic niche [30]. Point mutations, horizontal gene transfer, positive selection and genome degradation could be responsible for a differential pathoadaptive evolution of some *Salmonella* serovars [31]. It appears from the analysis of the mannose-sensitive fimbrial adhesin FimH that even single amino acid replacement, resulting in specific structural mutations in FimH variants of HA serovars, plays an important role in the differential adaptive evolution of *Salmonella* spp. Thus, activation or inactivation of mannose-specific adhesive properties in different systemically invasive serovars reflects the dynamic trajectories of adaptation to the biological environment of a specific host. Furthermore, phylogenetic analysis has indicated that these mutations are, most likely, of a convergent nature (common pathogenic traits incorporated into different genetic backgrounds) and occur under strong positive selection, illustrative of the role of point amino acid changes for HA *Salmonella*. Certainly, deep study for the molecular composition of flagella, chemotaxis genes [32], fimbriae and bacteriophages and the presence of virulence plasmids and subtypes of each specific serovar is needed, to understand mechanisms of pathogenicity and host specificity [19]. Correlation between some phage types of *S*. Typhi with their hosts has shown considerable host specificity [33]. However, the majority of phage types studied had a broad host range, perhaps, suggesting a phage transfer of virulent genes between hosts, leading eventually to host specificity. The unrestricted serovar Typhimurium may comprise a spectrum of variants differing in regard to virulence, reflecting a summation of the spatial and/or temporal selective pressures within a particular host [34]. *Salmonella* Typhimurium strains derived from animal cases were also virulent in mice, whereas many strains derived from a clinically ill man lacked this ability. Of interest was that many derived from human gastroenteritis lacked the *Salmonella* virulence plasmid, present in all animal strains and strains isolated from human bacteraemia. Furthermore, some strains harbouring the virulence plasmid isolated from the man were avirulent in mice, and the opposite was observed with those derived from animals. Altogether, isolates of a specific bacterial serovar obtained from human salmonellosis are different from those isolated from animals. This means that selective pressure within a specific host gives rise to bacterial strain variants that exhibit different pathogenicity determinants, thus varying degree of pathogenicity. Similarly, serovars of *S. enterica* subsp. *enterica*, associated with disease in mammals and birds, show different degrees of adaptability. Pathogenicity determinants, such as the FimH adhesins, play an important role. Type 1 FimH adhesins are expressed by serovars of *S*. *enterica* isolated from mammalian and avian hosts, while type 2 FimH is expressed exclusively by the avian-adapted serovar Gallinarum. Allelic variation of the *S*. *enterica* FimH adhesin directs host-cell-specific recognition, thus selectively binding to mammalian or avian receptors [35]. The distribution of SPIs, fimbriae operons and virulence plasmids has shown that various combinations of virulent determinants formed during the evolution of the microorganism are needed for a variant to become pathogenic in a particular range of host species. Mutations horizontally transmitted could have helped the development of host specificity by helping *Salmonella* serovars to harbour unique virulence factors [36]. Molecular and phylogenetic analyses of the SPI genes showed that these encode for translocon proteins (SipD, SseC and SseD) present on both *Salmonella* pathogenicity islands SPI-1 and SPI-2 and also encode an effector protein that inhibits the MAPK pathway of the host cells [37]. In addition, they encode effector proteins (SseF and SifA) important in placing the *Salmonella*-containing vacuole (SCV) in a juxtanuclear position. The products of SPI genes interact directly with the host and modulate its functions, thus favouring host specificity. Another study of the SPI genes has shown the close evolutionary relatedness between serovars Gallinarum

#### *Immunopathogenesis of Salmonellosis DOI: http://dx.doi.org/10.5772/intechopen.85371*

*New Insight into* Brucella *Infection and Foodborne Diseases*

mining host specificity and adaptability.

importance are *S. typhimurium* and *S. enteritidis* [18]. These cause mild symptoms in the adult host, and sometimes the host does not show any visible clinical symptoms despite infection. They severely invade young hosts as compared to adult hosts because the adult hosts have a well-built immune system that hinders the invasion by these serovars [16]. The host specificity and adaptability of different serovars are a complex process and involve many molecular mechanisms. The exact mechanisms are poorly studied, but certain factors have been found to be responsible for deter-

**4.1 Factors determining** *Salmonella* **host specificity and adaptation**

Although the exact mechanisms to host specificity have not been fully deciphered, the existing evidence shows that serovars act independently of each other at the various phases of infection. The expression of serovar's pathogenicity is affected by the environmental and genetic factors influencing each host during adaptation [19]. Each HA/HR serovar must overcome the encountered specific and nonspecific immune mechanisms. Thus, pathogenicity of HA serovars results from the development of ways helping their survival in a host. Examples of this are serovars of *S*. *enterica* subsp. *enterica*, which have developed the ability to evade the immune mechanisms of warm-blooded animals. They have, during their evolution, acquired the ability to modify to their favour the physiological functions of their host, such as intracellular engulfment, apoptosis, transfer of antigens by M (microfold) cells, migration of macrophages and lymphocytes in the reticuloendothelial system and others [20]. A well-known serovar *S*. *typhi* has evolved to survive in human macrophages making it pathogenic to man, but not to mice [21]. Serovars such as the HR *S*. *typhi*, *S. gallinarum* and *S. abortusovis* show high tropism for the lymphatic organs of their hosts, thereby regulating their natural host's biological environment in their

favour [19]. By anchoring to the cells of the bone marrow, PPs and bursa of Fabricius in the development of B cells, thus immune response is affected. The result of such interactions, particularly in adult animals, helps in the establishment of chronic or subclinical infection and thus prolonged subclinical excretion, but they do not help in the development of severe gastroenteritis [22]. Serovars not fully

adapted to evade the mature immune system lack specificity, causing deadly systemic disease in adult animals by invading their defence mechanisms compared to HR serovars [20]. HR serovars are mildly enteropathogenic compared to the unrestricted serovars; thus, they do not cause intestinal inflammation [23]. It has also been shown that the ability of a serovar to metabolise a wide range of amino acids adds to its virulence and is thought to be closely related [24, 25]; however, HR or HA serovars have most likely evolved independently. On the other hand, the heterogenicity of serovars in relation to different metabolic profiles facilitates either the completion of the pathway to infection [26] or, when lacking specificity, it is favouring host adaptation [19]. The process of *Salmonella* host adaptation is believed to be involving either the loss of genes or the acquisition of novel genetic elements that encode specific virulence factors, and thus an inconvenience is observed frequently in the pathogenic strains. Best examples of host specificity dependent on gene deletions are, perhaps, of *S. enteritidis*, Typhimurium,

Choleraesuis, Gallinarum, Abortusovis, Pullorum and Paratyphi C [27]. Genome sequencing of HA/HR serovars, such as Typhi, Gallinarum, Choleraesuis and the newly emerging in sub-Saharan Africa invasive strains of *S*. Typhi, has divulged that these have encountered extensive gene deletions and truncation [28, 29]. In systemically noninvasive *Salmonella*, the majority of lost genes have functional orthologues, which play a key role in intestinal colonisation, thus resulting in the loss of an intestinal multiplication cycle for narrowly host-adapted *Salmonellae*

**62**

followed by a concurrent acquisition of mechanisms helping the microorganism to survive in a systemic niche [30]. Point mutations, horizontal gene transfer, positive selection and genome degradation could be responsible for a differential pathoadaptive evolution of some *Salmonella* serovars [31]. It appears from the analysis of the mannose-sensitive fimbrial adhesin FimH that even single amino acid replacement, resulting in specific structural mutations in FimH variants of HA serovars, plays an important role in the differential adaptive evolution of *Salmonella* spp. Thus, activation or inactivation of mannose-specific adhesive properties in different systemically invasive serovars reflects the dynamic trajectories of adaptation to the biological environment of a specific host. Furthermore, phylogenetic analysis has indicated that these mutations are, most likely, of a convergent nature (common pathogenic traits incorporated into different genetic backgrounds) and occur under strong positive selection, illustrative of the role of point amino acid changes for HA *Salmonella*. Certainly, deep study for the molecular composition of flagella, chemotaxis genes [32], fimbriae and bacteriophages and the presence of virulence plasmids and subtypes of each specific serovar is needed, to understand mechanisms of pathogenicity and host specificity [19]. Correlation between some phage types of *S*. Typhi with their hosts has shown considerable host specificity [33]. However, the majority of phage types studied had a broad host range, perhaps, suggesting a phage transfer of virulent genes between hosts, leading eventually to host specificity. The unrestricted serovar Typhimurium may comprise a spectrum of variants differing in regard to virulence, reflecting a summation of the spatial and/or temporal selective pressures within a particular host [34]. *Salmonella* Typhimurium strains derived from animal cases were also virulent in mice, whereas many strains derived from a clinically ill man lacked this ability. Of interest was that many derived from human gastroenteritis lacked the *Salmonella* virulence plasmid, present in all animal strains and strains isolated from human bacteraemia. Furthermore, some strains harbouring the virulence plasmid isolated from the man were avirulent in mice, and the opposite was observed with those derived from animals. Altogether, isolates of a specific bacterial serovar obtained from human salmonellosis are different from those isolated from animals. This means that selective pressure within a specific host gives rise to bacterial strain variants that exhibit different pathogenicity determinants, thus varying degree of pathogenicity. Similarly, serovars of *S. enterica* subsp. *enterica*, associated with disease in mammals and birds, show different degrees of adaptability. Pathogenicity determinants, such as the FimH adhesins, play an important role. Type 1 FimH adhesins are expressed by serovars of *S*. *enterica* isolated from mammalian and avian hosts, while type 2 FimH is expressed exclusively by the avian-adapted serovar Gallinarum. Allelic variation of the *S*. *enterica* FimH adhesin directs host-cell-specific recognition, thus selectively binding to mammalian or avian receptors [35]. The distribution of SPIs, fimbriae operons and virulence plasmids has shown that various combinations of virulent determinants formed during the evolution of the microorganism are needed for a variant to become pathogenic in a particular range of host species. Mutations horizontally transmitted could have helped the development of host specificity by helping *Salmonella* serovars to harbour unique virulence factors [36]. Molecular and phylogenetic analyses of the SPI genes showed that these encode for translocon proteins (SipD, SseC and SseD) present on both *Salmonella* pathogenicity islands SPI-1 and SPI-2 and also encode an effector protein that inhibits the MAPK pathway of the host cells [37]. In addition, they encode effector proteins (SseF and SifA) important in placing the *Salmonella*-containing vacuole (SCV) in a juxtanuclear position. The products of SPI genes interact directly with the host and modulate its functions, thus favouring host specificity. Another study of the SPI genes has shown the close evolutionary relatedness between serovars Gallinarum

and Enteritidis [38], although the former is highly adapted (restricted) to poultry and is the only known non-motile serovar, while serovar Enteritidis is unrestricted. Analysis of the functions of genes associated to SPI-1 showed that virulence genes might have evolved under positive selection imposed by a serovar's respective host(s) contributing to the different host specificity observed between different serovars. This has displayed that a close similarity of core regions exists within as well as among different serovar genomes [39]. In particular, genomic comparisons of HR and HA serovars show that genomic degradation is a common evolutionary mechanism for host adaptation and increased pathogenicity [39, 40]. Others have shown that host restriction and change of ecological niche are associated with the accumulation of pseudogenes and an overall reduction in genome size [28]. For example, *S*. Typhi and Paratyphi A are restricted to the man and cause a similar systemic disease. Genome sequence similarity between Typhi and Paratyphi A serovars and their different pathogenicity when compared to the unrestricted serovars of *S. enterica* have been attributed to a relatively recent recombination of a quarter of their genomes, making the aggregation of pseudogenes a key feature of convergent evolution for these and other HA pathogens [31]. Another example supporting the role of convergent evolution is serovar Paratyphi C, which has diverged from the same ancestor as serovar Choleraesuis, by accumulating genomic novelty during its adaptation process to man. The genomic analysis of these two *Salmonella* serovars has revealed a highly similar genomic construction between the two and their distinct pathogenic features, making them excellent models for studying *Salmonella*'*s* host adaptation and pathogenic divergence [39]. Hence, *Salmonella* adaptation to a particular host species is a complex phenomenon, which depends, apparently, on a large number of gene products. The prowess of understanding host-pathogen interactions requires analysis of the physiological associations between various animal species and genetic composition.

#### **5.** *Salmonella* **invasion**

After ingestion of S*almonella* by the host organism, it travels from the stomach and invades intestinal epithelial cells. Bacterial recognition generates an inflammatory response following the recruitment of a variety of bone-marrow-derived phagocytes [41]. The ability of *Salmonella* to access intestinal epithelium (M cells) is due to the presence of virulence genes encoded by *Salmonella* pathogenicity island 1 (SPI-1). Proteins that are encoded by SPI-1 form a needle-like Type III secretion system which allows the transport of several bacterial proteins into the host cell cytosol. These proteins induce changes in the host cells such as the rearrangement of the cytoskeleton and cell membrane and disconnection of epithelial cell junctions, facilitating bacterial invasion [42]. The primary site of *Salmonella* infection occurs at specialised microfold, or M cells, that are dispersed among the enterocytes, covering the follicle-associated epithelium (FAE) of the Peyer's patch (PP) [43] (**Figure 1**). *Salmonella* is considered to preferentially invade PPs in the distal ileum, but in practice, all intestinal PPs will harbour bacteria after moderate-to-high-dose infection. Once *Salmonella* is penetrated, it initiates destruction of M cell which disrupts the mucosal barrier and allows additional entry of bacteria through neighbouring enterocytes [43]. This process is extremely efficient, with M-cell penetration followed by M-cell destruction. Once access to PP via FAE is gained, invading bacteria enter the lymphatic system where they interact with professional killing cells (phagocytes) that ultimately determine the fate of the infection. Phagocytes are involved in both oxygen-dependent and oxygen-independent killing of the engulfed bacteria. During intestinal NTS infection, the release of reactive oxygen

**65**

**Figure 1.**

*Immunopathogenesis of Salmonellosis*

*DOI: http://dx.doi.org/10.5772/intechopen.85371*

and nitrogen species creates a highly oxidative environment which is not permissive for the growth of bacteria. The subepithelial dome also contains dendritic cell (DC) subsets apart from macrophage populations, each of which can phagocytise the bacteria and then undergo apoptosis through a caspase-1-dependent mechanism [44]. Consequently, during *Salmonella* infection, the number of obligate anaerobes decline in the gut. Also, host-signalling environment is highly crucial for the disease development initiated by contact between microbe and host cells in various tissues, largely mediated by cytokine signalling. These cytokines aid in initiating and regulating the innate and adaptive branches of the immune response against *Salmonella*. In order to avoid damage to the host, the release of pro- and anti-inflammatory cytokines must be balanced [45]. M cells are not only abundant in PPs; they also predominate in other intestinal locations and so can, therefore, mediate infection of non-PP intestinal tissues (**Figure 1**). The most likely non-PP entry route is through the bacterial invasion of solitary intestinal lymphoid tissues (SILTs), which are heterogeneous intestinal lymphoid aggregates found in mice and humans that contain

*Salmonella entry in intestinal epithelial cells. SPI-1 facilitates uptake and destruction of M cells, SILTs. After invasion of under tissues, Salmonella is taken by phagocytes and transported to mesenteric lymph nodes.*

*New Insight into* Brucella *Infection and Foodborne Diseases*

tions between various animal species and genetic composition.

After ingestion of S*almonella* by the host organism, it travels from the stomach and invades intestinal epithelial cells. Bacterial recognition generates an inflammatory response following the recruitment of a variety of bone-marrow-derived phagocytes [41]. The ability of *Salmonella* to access intestinal epithelium (M cells) is due to the presence of virulence genes encoded by *Salmonella* pathogenicity island 1 (SPI-1). Proteins that are encoded by SPI-1 form a needle-like Type III secretion system which allows the transport of several bacterial proteins into the host cell cytosol. These proteins induce changes in the host cells such as the rearrangement of the cytoskeleton and cell membrane and disconnection of epithelial cell junctions, facilitating bacterial invasion [42]. The primary site of *Salmonella* infection occurs at specialised microfold, or M cells, that are dispersed among the enterocytes, covering the follicle-associated epithelium (FAE) of the Peyer's patch (PP) [43] (**Figure 1**). *Salmonella* is considered to preferentially invade PPs in the distal ileum, but in practice, all intestinal PPs will harbour bacteria after moderate-to-high-dose infection. Once *Salmonella* is penetrated, it initiates destruction of M cell which disrupts the mucosal barrier and allows additional entry of bacteria through neighbouring enterocytes [43]. This process is extremely efficient, with M-cell penetration followed by M-cell destruction. Once access to PP via FAE is gained, invading bacteria enter the lymphatic system where they interact with professional killing cells (phagocytes) that ultimately determine the fate of the infection. Phagocytes are involved in both oxygen-dependent and oxygen-independent killing of the engulfed bacteria. During intestinal NTS infection, the release of reactive oxygen

**5.** *Salmonella* **invasion**

and Enteritidis [38], although the former is highly adapted (restricted) to poultry and is the only known non-motile serovar, while serovar Enteritidis is unrestricted. Analysis of the functions of genes associated to SPI-1 showed that virulence genes might have evolved under positive selection imposed by a serovar's respective host(s) contributing to the different host specificity observed between different serovars. This has displayed that a close similarity of core regions exists within as well as among different serovar genomes [39]. In particular, genomic comparisons of HR and HA serovars show that genomic degradation is a common evolutionary mechanism for host adaptation and increased pathogenicity [39, 40]. Others have shown that host restriction and change of ecological niche are associated with the accumulation of pseudogenes and an overall reduction in genome size [28]. For example, *S*. Typhi and Paratyphi A are restricted to the man and cause a similar systemic disease. Genome sequence similarity between Typhi and Paratyphi A serovars and their different pathogenicity when compared to the unrestricted serovars of *S. enterica* have been attributed to a relatively recent recombination of a quarter of their genomes, making the aggregation of pseudogenes a key feature of convergent evolution for these and other HA pathogens [31]. Another example supporting the role of convergent evolution is serovar Paratyphi C, which has diverged from the same ancestor as serovar Choleraesuis, by accumulating genomic novelty during its adaptation process to man. The genomic analysis of these two *Salmonella* serovars has revealed a highly similar genomic construction between the two and their distinct pathogenic features, making them excellent models for studying *Salmonella*'*s* host adaptation and pathogenic divergence [39]. Hence, *Salmonella* adaptation to a particular host species is a complex phenomenon, which depends, apparently, on a large number of gene products. The prowess of understanding host-pathogen interactions requires analysis of the physiological associa-

**64**

#### **Figure 1.**

*Salmonella entry in intestinal epithelial cells. SPI-1 facilitates uptake and destruction of M cells, SILTs. After invasion of under tissues, Salmonella is taken by phagocytes and transported to mesenteric lymph nodes.*

and nitrogen species creates a highly oxidative environment which is not permissive for the growth of bacteria. The subepithelial dome also contains dendritic cell (DC) subsets apart from macrophage populations, each of which can phagocytise the bacteria and then undergo apoptosis through a caspase-1-dependent mechanism [44]. Consequently, during *Salmonella* infection, the number of obligate anaerobes decline in the gut. Also, host-signalling environment is highly crucial for the disease development initiated by contact between microbe and host cells in various tissues, largely mediated by cytokine signalling. These cytokines aid in initiating and regulating the innate and adaptive branches of the immune response against *Salmonella*. In order to avoid damage to the host, the release of pro- and anti-inflammatory cytokines must be balanced [45]. M cells are not only abundant in PPs; they also predominate in other intestinal locations and so can, therefore, mediate infection of non-PP intestinal tissues (**Figure 1**). The most likely non-PP entry route is through the bacterial invasion of solitary intestinal lymphoid tissues (SILTs), which are heterogeneous intestinal lymphoid aggregates found in mice and humans that contain

certain features of PPs, including the presence of FAE-containing M cells [46, 47]. These SILTs are invaded by bacteria in a much similar manner as described above for PPs [48]. SILTs can be important in humans since in a study of typhoid patients, both PPs and SILTs showed inflammation. It has also been reported that intravillous M cells, which are sparsely located along the intestinal tract, may serve as a portal of entry for invasive *Salmonella* bacteria [49, 50] (**Figure 1**).

#### **5.1 Alternative route for invasion**

The main entry route described above involve, bacterial interactions with M cells, the possibility is that it can invade the host by an alternative route that does not involve M cells. A population of phagocytes in the lamina propria capture bacteria directly from luminal contents which also allow bacterial entry [51, 52]. This is for those bacteria that lack SPI-1 genes as this route does not involve M cell-mediated uptake. These cells might have been referred to as DCs, but as this is not clear [53, 54], they will be referred to as lamina propria phagocytes in this chapter. Although this pathway has now become an alternative to our general understanding of bacterial entry through M cells, the physiological importance of this route to systemic salmonellosis is poorly defined. The compelling evidence for a non-M-cell pathway is largely derived from microbiological and immunological investigations. Recent interest was stimulated by demonstrating that strains lacking SPI-1 and the fimbrial lpfC gene that did not normally infect mice retained the ability to infect mice in a CD18-dependent manner and were rapidly detected in the blood after oral inoculation [55, 56]. This extremely rapid dissemination to the blood and lack of serovar specificity might be due to bacterial entry in the bloodstream of the host through abrasions caused during gavage. Many cervical lymph node infection cases that attributed to the entry through mucosal abrasions during gavage were revealed through bacterial imaging system [57]. Expression of the SPI-2 type-III secretion system effector protein (SrfH) of bacteria was required for very early dissemination of bacteria to the blood and spleen [58]. This finding supports the idea that rapid entry through an alternative pathway involves active processes, so, therefore, it is important to examine this route from a microbiological perspective. In vitro studies demonstrated that DCs could capture bacteria by extending processes between the tight junctions of a monolayer and the apical surface of epithelial cells [59]. Subsequently, a similar process was directly visualised in vivo when CX3CR1-expressing phagocytes were detected extending transepithelial dendrites in the lamina propria, and the number of dendrites increased in the terminal ileum after infection [60]. So, these studies suggested an alternative entry model, whereby *Salmonella* might commonly access the intestinal lamina propria by cell sampling, as large numbers of bacteria were detected within the lamina propria [60]. However, *Salmonella* is not normally recoverable in large numbers from the lamina propria unless the bacterial flora is first depleted before infection [48]. Also, the formation of transepithelial dendrites is dispensable for the uptake of other pathogenic microorganisms [61]. More importantly, it has been demonstrated that CX3CR1+ lamina propria cells are unlikely to migrate to the mesenteric lymph nodes (MLN) and have poor immunostimulatory capacity [53]. Thus, CX3CR1+ cells most likely represent a population of non-migrating phagocytes that provide innate immune defence against infection within the lamina propria. Surprisingly, the role of cell-mediated uptake has not been examined carefully in PPs or in SILTs, but still, phagocytic cells are often found in association with the epithelium of tissues [48, 62]. In summary, a prominent role for M cell-mediated intestinal entry by *Salmonella* is played both in the PPs and SILTs, whereas *Salmonella* entry of the lamina propria and the mechanisms like immune activation and bacterial dissemination associated with this pathway of entry remain largely speculative.

**67**

*Immunopathogenesis of Salmonellosis*

**tissues**

*DOI: http://dx.doi.org/10.5772/intechopen.85371*

**6.** *Salmonella* **infection of mesenteric lymph nodes (MLNs) and systemic** 

After initial invasion through PPs, the ultimate fate of infection is decided in the lymphatic system. The indication for the bacterial migration is based on our understanding of the lymph and the conjectural finding that bacteria are detected initially in PPs, followed by the MLN and finally the liver and spleen [63, 64]. *Salmonella* after getting access to efferent lymphatics reaches the systemic tissues via the thoracic duct and blood after reaching the MLN [65, 66]. The immune cell population that aids in the transport of bacteria to the blood and other tissues is not well known; however, intestinal DCs are usually considered as a possibility. The majority of bacteria were found free in the lymph or were associated with non-DC phagocytes [67], but it is not clear whether this also occurs during exit from the MLN. Disseminated bacteria show a tropism of tissues that contain a high number of phagocytic cells, and in most circumstances, this involves the spleen, liver, and bone marrow [65, 68]. Disruption of erythropoiesis and splenomegaly by *Salmonella* can be explained majorly by the expansion of immature erythrocytes in the spleen in an erythropoietin-dependent manner. Cancer studies have demonstrated that bacteria preferentially accumulate in primary and metastatic tumours [69, 70], suggesting that it does not have a precise organ tropism but finds tissues that contain a sufficient number of cells that support bacterial replication. The large size of the spleen, liver, and bone marrow means that these tissues gradually comprise the major sites of bacterial replication [71, 72]. Thus, *Salmonella* causes systemic infection that uses intestinal lymphoid tissues as a portal of entry. Also that bacteria clearance from the host and resistance to secondary infection requires

the coordinated action of both systemic and mucosal immunity.

After phagocytosis by macrophages, *Salmonella* can survive and replicate within modified intracellular vesicles, termed as *Salmonella*-containing vacuoles (SCV) [73, 74]. The ability of *Salmonella* to survive within the phagosome is mediated by SPI-2, which prevents movement of RNS and ROS into the phagosome where the bacteria reside [75, 76]. In addition, *Salmonella* phoP/phoQ regulon inhibits fusion of the SCV with toxic lysosomes and endosomes [77]. The natural resistanceassociated macrophage protein encoding gene, which enables macrophages to transport ions into the SCV, provides resistance/susceptibility to infection [78]. Survival of bacteria intracellularly within tissue phagocytes is a prerequisite to the bacterial virulence, and bacterial mutants that cannot survive and replicate within macrophages are attenuated for virulence [79]. The initial invasion induces a massive inflammatory response, characterised by recruitment of neutrophils, DCs, inflammatory monocytes and macrophages [48, 80]. Neutrophils follow the chemokine gradient to the gut and extravagate into the mucosa. As they encounter and eliminate the bacteria by mechanisms that are not yet fully elucidated [81], neutrophils are believed to be important in preventing dissemination of the bacteria from the intestine to systemic tissues, so the patients with low neutrophil levels have a high risk of bacteraemia during infection with NTS strains [82]. Also, that depletion of neutrophils allows extracellular growth of bacteria, suggesting that neutrophils confine and reduce bacterial replication immediately after entry. Inflammatory monocytes are an important source of antimicrobial factors, such as TNFs and inducible NO synthase, during the initial stages of infection [80]. Myd88 dependent chemokine production within the PPs drives the recruitment of these

**7. Host innate immune response to** *Salmonella*

*New Insight into* Brucella *Infection and Foodborne Diseases*

**5.1 Alternative route for invasion**

of entry for invasive *Salmonella* bacteria [49, 50] (**Figure 1**).

certain features of PPs, including the presence of FAE-containing M cells [46, 47]. These SILTs are invaded by bacteria in a much similar manner as described above for PPs [48]. SILTs can be important in humans since in a study of typhoid patients, both PPs and SILTs showed inflammation. It has also been reported that intravillous M cells, which are sparsely located along the intestinal tract, may serve as a portal

The main entry route described above involve, bacterial interactions with M cells, the possibility is that it can invade the host by an alternative route that does not involve M cells. A population of phagocytes in the lamina propria capture bacteria directly from luminal contents which also allow bacterial entry [51, 52]. This is for those bacteria that lack SPI-1 genes as this route does not involve M cell-mediated uptake. These cells might have been referred to as DCs, but as this is not clear [53, 54], they will be referred to as lamina propria phagocytes in this chapter. Although this pathway has now become an alternative to our general understanding of bacterial entry through M cells, the physiological importance of this route to systemic salmonellosis is poorly defined. The compelling evidence for a non-M-cell pathway is largely derived from microbiological and immunological investigations. Recent interest was stimulated by demonstrating that strains lacking SPI-1 and the fimbrial lpfC gene that did not normally infect mice retained the ability to infect mice in a CD18-dependent manner and were rapidly detected in the blood after oral inoculation [55, 56]. This extremely rapid dissemination to the blood and lack of serovar specificity might be due to bacterial entry in the bloodstream of the host through abrasions caused during gavage. Many cervical lymph node infection cases that attributed to the entry through mucosal abrasions during gavage were revealed through bacterial imaging system [57]. Expression of the SPI-2 type-III secretion system effector protein (SrfH) of bacteria was required for very early dissemination of bacteria to the blood and spleen [58]. This finding supports the idea that rapid entry through an alternative pathway involves active processes, so, therefore, it is important to examine this route from a microbiological perspective. In vitro studies demonstrated that DCs could capture bacteria by extending processes between the tight junctions of a monolayer and the apical surface of epithelial cells [59]. Subsequently, a similar process was directly visualised in vivo when CX3CR1-expressing phagocytes were detected extending transepithelial dendrites in the lamina propria, and the number of dendrites increased in the terminal ileum after infection [60]. So, these studies suggested an alternative entry model, whereby *Salmonella* might commonly access the intestinal lamina propria by cell sampling, as large numbers of bacteria were detected within the lamina propria [60]. However, *Salmonella* is not normally recoverable in large numbers from the lamina propria unless the bacterial flora is first depleted before infection [48]. Also, the formation of transepithelial dendrites is dispensable for the uptake of other pathogenic microorganisms [61]. More importantly, it has been demonstrated that CX3CR1+ lamina propria cells are unlikely to migrate to the mesenteric lymph nodes (MLN) and have poor immunostimulatory capacity [53]. Thus, CX3CR1+ cells most likely represent a population of non-migrating phagocytes that provide innate immune defence against infection within the lamina propria. Surprisingly, the role of cell-mediated uptake has not been examined carefully in PPs or in SILTs, but still, phagocytic cells are often found in association with the epithelium of tissues [48, 62]. In summary, a prominent role for M cell-mediated intestinal entry by *Salmonella* is played both in the PPs and SILTs, whereas *Salmonella* entry of the lamina propria and the mechanisms like immune activation and bacterial dissemination associated with

**66**

this pathway of entry remain largely speculative.

#### **6.** *Salmonella* **infection of mesenteric lymph nodes (MLNs) and systemic tissues**

After initial invasion through PPs, the ultimate fate of infection is decided in the lymphatic system. The indication for the bacterial migration is based on our understanding of the lymph and the conjectural finding that bacteria are detected initially in PPs, followed by the MLN and finally the liver and spleen [63, 64]. *Salmonella* after getting access to efferent lymphatics reaches the systemic tissues via the thoracic duct and blood after reaching the MLN [65, 66]. The immune cell population that aids in the transport of bacteria to the blood and other tissues is not well known; however, intestinal DCs are usually considered as a possibility. The majority of bacteria were found free in the lymph or were associated with non-DC phagocytes [67], but it is not clear whether this also occurs during exit from the MLN. Disseminated bacteria show a tropism of tissues that contain a high number of phagocytic cells, and in most circumstances, this involves the spleen, liver, and bone marrow [65, 68]. Disruption of erythropoiesis and splenomegaly by *Salmonella* can be explained majorly by the expansion of immature erythrocytes in the spleen in an erythropoietin-dependent manner. Cancer studies have demonstrated that bacteria preferentially accumulate in primary and metastatic tumours [69, 70], suggesting that it does not have a precise organ tropism but finds tissues that contain a sufficient number of cells that support bacterial replication. The large size of the spleen, liver, and bone marrow means that these tissues gradually comprise the major sites of bacterial replication [71, 72]. Thus, *Salmonella* causes systemic infection that uses intestinal lymphoid tissues as a portal of entry. Also that bacteria clearance from the host and resistance to secondary infection requires the coordinated action of both systemic and mucosal immunity.

#### **7. Host innate immune response to** *Salmonella*

After phagocytosis by macrophages, *Salmonella* can survive and replicate within modified intracellular vesicles, termed as *Salmonella*-containing vacuoles (SCV) [73, 74]. The ability of *Salmonella* to survive within the phagosome is mediated by SPI-2, which prevents movement of RNS and ROS into the phagosome where the bacteria reside [75, 76]. In addition, *Salmonella* phoP/phoQ regulon inhibits fusion of the SCV with toxic lysosomes and endosomes [77]. The natural resistanceassociated macrophage protein encoding gene, which enables macrophages to transport ions into the SCV, provides resistance/susceptibility to infection [78]. Survival of bacteria intracellularly within tissue phagocytes is a prerequisite to the bacterial virulence, and bacterial mutants that cannot survive and replicate within macrophages are attenuated for virulence [79]. The initial invasion induces a massive inflammatory response, characterised by recruitment of neutrophils, DCs, inflammatory monocytes and macrophages [48, 80]. Neutrophils follow the chemokine gradient to the gut and extravagate into the mucosa. As they encounter and eliminate the bacteria by mechanisms that are not yet fully elucidated [81], neutrophils are believed to be important in preventing dissemination of the bacteria from the intestine to systemic tissues, so the patients with low neutrophil levels have a high risk of bacteraemia during infection with NTS strains [82]. Also, that depletion of neutrophils allows extracellular growth of bacteria, suggesting that neutrophils confine and reduce bacterial replication immediately after entry. Inflammatory monocytes are an important source of antimicrobial factors, such as TNFs and inducible NO synthase, during the initial stages of infection [80]. Myd88 dependent chemokine production within the PPs drives the recruitment of these

inflammatory cells [81]. Indeed, *Salmonella* expresses several pathogen-associated molecular patterns (PAMPs), including lipopolysaccharide (LPS) and flagellin, which can be detected by Toll-like receptors (TLRs) expressed by enterocytes and phagocytes [83]. Also, macrophages after sensing cytosolic flagellin through NLRC4 (also known as Ipaf) activate caspase-1 and induce the production of IL-18 (pro-inflammatory) [84, 85]. Dendritic cells are professional antigen-presenting cells and increase the expression of MHC class II and the co-stimulatory molecules CD86, CD80 and CD40 by responding to the recognition of *Salmonella* LPS or flagellin [86, 87]. DCs present antigen to naive CD4<sup>+</sup> T cells, thus providing a vital link between innate immune responses and the induction of adaptive immunity. In the PPs, flagellin also induces the secretion of the inflammatory chemokine CCL20, which is an important ligand for CCR6 [88]. This response activates an early process whereby CCR6-expressing DCs are recruited to the FAE, for efficient activation of CD4+ T cells [89].

#### **8. Host-adaptive immune response to** *Salmonella*

Adaptive immune response to *Salmonella* can be mediated via early CD4+ T-cell activation. Due to the small size of intestinal lymphoid tissues and low frequency of naive CD4+ T cells specific for any given antigen [90], detecting initial bacterial specific T-cell activation in these tissues is challenging. However, studies with T-cell receptor transgenic mice visualised the processes of bacterial specific CD4+ T cells responding to oral infection [64, 91]. An artificially elevated naive precursor frequency of CD4+ T cells at a high dose of infection provides the most accurate assessment of *Salmonella*-specific CD4+ T-cell activation [92]. The earliest *Salmonella*-specific CD4<sup>+</sup> T-cell activation occurs within the MLN after oral infection but usually peaks few hours after that in the PPs [91]. At very early time points, CD4+ T cells were not found to be activated in any other secondary lymphoid tissues [89], suggesting that whatever the explanation for early bacterial dissemination to blood as discussed above, no early adaptive immune response is initiated outside the gut-associated lymphoid tissue. Interestingly, the T-cell receptor transgenic model used recognises a bacterial peptide from the carboxy-terminal region of flagellin [93] which is also a ligand for TLR5 [94]. Generally, the early activation of flagellin-specific CD4+ T cells in the PPs is representative of the naive CD4+ response to other bacterial antigens [95, 96]. However, this is difficult to demonstrate conclusively as very few, naturally occurring bacterial specific MHC class-II peptides are known [97]. Interestingly, activation of CD4+ T cells in the MLN is also dependent on CD11c<sup>+</sup> DCs and CCR6, indicating that T-cell activation in the MLN and PP has similar requirements. The evidence clearly suggests that the MLNs are an important site for immune protection during the course of *Salmonella* infection. Indeed, after MLNs were surgically removed, there was an elevated bacterial load and severe immunopathology in the liver [98]. The importance was also highlighted using a relapsing model of murine typhoid in which primary infection returns after apparent antibiotic clearance [57]. Although MLN is often considered a potential site of bacterial accumulation [99, 100], it acts as a protective firewall, preventing bacterial dissemination and relapsing *Salmonella* infection.

#### **9. Host effector responses against** *Salmonella*

The development of robust protective immunity against *Salmonella* infection requires the coordination of B and T cells. One hundred and sixteen CD4+ T

**69**

**Figure 2.**

*macrophages.*

*Immunopathogenesis of Salmonellosis*

tempo of CD4+

*DOI: http://dx.doi.org/10.5772/intechopen.85371*

cells eventually comprises ~50% of all CD4+

infection. Thus, it was found that when CD4+

*Induction of IFN γ production by Salmonella-specific CD4+*

expansion [109]. In contrast, the gradual loss of effector CD4+

cells have a critical role in clearing the primary infection and are also required for acquired resistance to secondary infection [100]. In contrast, B cells are dispensable for resolving primary infection but are required for protection against secondary challenge [101]. There is a massive expansion of *Salmonella*-specific CD4+

and rapid acquisition of Th1 effector functions, i.e. the enhanced ability to secrete INF-γ, TNF α and IL-2 upon restimulation [102] (**Figure 2**). These activated Th1 cells can be clearly detected a week after infection, which is consistent with the rapid

require expression of both programmed death ligand-1 (PD-L1) and the TNF receptor family members, OX40 and CD30. Appropriately, expansion of activated Th1

Furthermore, Th1 cells are capable of responding to innate signals such as *Salmonella* LPS by secreting cytokines [104]. This innate response is unexpected as effector Th1 cells are normally stimulated only after recognition of cognate peptide and MHCs [105]. This innate immune responsiveness suggests a means by which the host can rapidly produce INF-γ to activate macrophages within an infected tissue, even if bacteria are capable of inhibiting antigen presentation by infected phagocytes [106]. Despite the rapid and efficient development of Th1 effector cells, there is actually little evidence that suggests their contribution to bacterial clearance during primary

absent, bacterial growth enhanced a few weeks after infection which indicates that Th1 cells contribute little to regulate bacterial growth before this point [107]. Many in vitro studies point to an inhibitory effect of *Salmonella* on antigen presentation to naive T cells in vitro [108], but in vivo, there is no effect on *Salmonella*-specific CD4+

process of *Salmonella* infection that required the presence of live bacteria and the expression of SPI-2 genes indicated that the effector function of cells is specifically

*secondary lymphoid tissues which in turn produces IFNγ at infection sites. Production of IFNγ finally activates* 

T-cell activation. Optimal expansion of Th1 cells have been shown to

or CD4+

T cells few weeks after infection [103].

Th1 cells were completely

*T cells. Expansion of activated CD4+*

*T cells in* 

T cells detected in the

T cells

#### *Immunopathogenesis of Salmonellosis DOI: http://dx.doi.org/10.5772/intechopen.85371*

*New Insight into* Brucella *Infection and Foodborne Diseases*

flagellin [86, 87]. DCs present antigen to naive CD4<sup>+</sup>

**8. Host-adaptive immune response to** *Salmonella*

accurate assessment of *Salmonella*-specific CD4+

peptides are known [97]. Interestingly, activation of CD4+

bacterial dissemination and relapsing *Salmonella* infection.

**9. Host effector responses against** *Salmonella*

inflammatory cells [81]. Indeed, *Salmonella* expresses several pathogen-associated molecular patterns (PAMPs), including lipopolysaccharide (LPS) and flagellin, which can be detected by Toll-like receptors (TLRs) expressed by enterocytes and phagocytes [83]. Also, macrophages after sensing cytosolic flagellin through NLRC4 (also known as Ipaf) activate caspase-1 and induce the production of IL-18 (pro-inflammatory) [84, 85]. Dendritic cells are professional antigen-presenting cells and increase the expression of MHC class II and the co-stimulatory molecules CD86, CD80 and CD40 by responding to the recognition of *Salmonella* LPS or

link between innate immune responses and the induction of adaptive immunity. In the PPs, flagellin also induces the secretion of the inflammatory chemokine CCL20, which is an important ligand for CCR6 [88]. This response activates an early process whereby CCR6-expressing DCs are recruited to the FAE, for efficient activation of

Adaptive immune response to *Salmonella* can be mediated via early CD4+

rial specific T-cell activation in these tissues is challenging. However, studies with T-cell receptor transgenic mice visualised the processes of bacterial specific

activation. Due to the small size of intestinal lymphoid tissues and low frequency

T cells responding to oral infection [64, 91]. An artificially elevated naive

tion but usually peaks few hours after that in the PPs [91]. At very early time points,

response to other bacterial antigens [95, 96]. However, this is difficult to demonstrate conclusively as very few, naturally occurring bacterial specific MHC class-II

and PP has similar requirements. The evidence clearly suggests that the MLNs are an important site for immune protection during the course of *Salmonella* infection. Indeed, after MLNs were surgically removed, there was an elevated bacterial load and severe immunopathology in the liver [98]. The importance was also highlighted using a relapsing model of murine typhoid in which primary infection returns after apparent antibiotic clearance [57]. Although MLN is often considered a potential site of bacterial accumulation [99, 100], it acts as a protective firewall, preventing

The development of robust protective immunity against *Salmonella* infection requires the coordination of B and T cells. One hundred and sixteen CD4+

 T cells were not found to be activated in any other secondary lymphoid tissues [89], suggesting that whatever the explanation for early bacterial dissemination to blood as discussed above, no early adaptive immune response is initiated outside the gut-associated lymphoid tissue. Interestingly, the T-cell receptor transgenic model used recognises a bacterial peptide from the carboxy-terminal region of flagellin [93] which is also a ligand for TLR5 [94]. Generally, the early activation

T cells specific for any given antigen [90], detecting initial bacte-

T cells at a high dose of infection provides the most

T-cell activation occurs within the MLN after oral infec-

T cells in the PPs is representative of the naive CD4+

DCs and CCR6, indicating that T-cell activation in the MLN

T cells, thus providing a vital

T-cell activation [92]. The earliest

T cells in the MLN is also

T

T-cell

**68**

CD4+

CD4+

CD4+

of naive CD4+

T cells [89].

precursor frequency of CD4+

*Salmonella*-specific CD4<sup>+</sup>

of flagellin-specific CD4+

dependent on CD11c<sup>+</sup>

cells have a critical role in clearing the primary infection and are also required for acquired resistance to secondary infection [100]. In contrast, B cells are dispensable for resolving primary infection but are required for protection against secondary challenge [101]. There is a massive expansion of *Salmonella*-specific CD4+ T cells and rapid acquisition of Th1 effector functions, i.e. the enhanced ability to secrete INF-γ, TNF α and IL-2 upon restimulation [102] (**Figure 2**). These activated Th1 cells can be clearly detected a week after infection, which is consistent with the rapid tempo of CD4+ T-cell activation. Optimal expansion of Th1 cells have been shown to require expression of both programmed death ligand-1 (PD-L1) and the TNF receptor family members, OX40 and CD30. Appropriately, expansion of activated Th1 cells eventually comprises ~50% of all CD4+ T cells few weeks after infection [103]. Furthermore, Th1 cells are capable of responding to innate signals such as *Salmonella* LPS by secreting cytokines [104]. This innate response is unexpected as effector Th1 cells are normally stimulated only after recognition of cognate peptide and MHCs [105]. This innate immune responsiveness suggests a means by which the host can rapidly produce INF-γ to activate macrophages within an infected tissue, even if bacteria are capable of inhibiting antigen presentation by infected phagocytes [106]. Despite the rapid and efficient development of Th1 effector cells, there is actually little evidence that suggests their contribution to bacterial clearance during primary infection. Thus, it was found that when CD4+ or CD4+ Th1 cells were completely absent, bacterial growth enhanced a few weeks after infection which indicates that Th1 cells contribute little to regulate bacterial growth before this point [107]. Many in vitro studies point to an inhibitory effect of *Salmonella* on antigen presentation to naive T cells in vitro [108], but in vivo, there is no effect on *Salmonella*-specific CD4+ expansion [109]. In contrast, the gradual loss of effector CD4+ T cells detected in the process of *Salmonella* infection that required the presence of live bacteria and the expression of SPI-2 genes indicated that the effector function of cells is specifically

#### **Figure 2.**

*Induction of IFN γ production by Salmonella-specific CD4+ T cells. Expansion of activated CD4+ T cells in secondary lymphoid tissues which in turn produces IFNγ at infection sites. Production of IFNγ finally activates macrophages.*

inhibited by actively replicating bacteria [110]. Effector Th1 cells are effective in providing immunity to salmonellosis [111]; however, effector CD4+ subsets including regulatory T cells (Tregs) and Th17 cells are also known to contribute. Tregs arise from the thymus or develop after naive T-cell activation in the presence of TNF β which suppresses effector T-cell responses [112]. In contrast, Th17 cells arise from naive CD4+ T-cell stimulation in the presence of IL-6 and TNF-β and are important in mediating immunity against extracellular bacterial infections [113, 114]. During the development of Th1 cells and Tregs after infection, it was found that changes in the cogency of Tregs reduced the efficacy of Th1 responses and increased bacterial growth [102]. After oral infection with *Salmonella*, cytokines associated with Th17 cells, IL-17 and IL-22 are rapidly produced within the intestinal mucosa [115], and the production is induced by innate responses to infection rather than Th17 cells, however, still indicating the potential for Th17 cytokines to participate in intestinal defence against bacteria. In vivo, production of IL-22 dependent on IL12B, rather than IL-17, contributed to bacterial clearance [116]. Taken together, it is suggested that Th17 cells contribute additionally to protection against *Salmonella* infection by not only initiating or enhancing neutrophil infiltration to intestinal tissues but by the production of antimicrobial peptides by the epithelium which is effective against luminal bacteria as well [117]. In summary, it has been suggested that Th17 cells have an additional role in defence against *Salmonella* in the intestine and a role for Tregs in modulating the potency of *Salmonella*-specific Th1 cells in vivo.

#### **10. Host antibody (Ab) response against** *Salmonella*

*Salmonella*-specific B-cell responses contribute to bacterial clearance in the hosts [39, 120, 121]. Although the bacteria are generally found within SCV in phagocytic cells, there is a short period during the infection cycle when bacteria are expected to be extracellular. *Salmonella* is not only tightly associated with mononuclear phagocytes in vivo [118] but also induces these infected cells to undergo apoptosis [44]. After cell death, bacteria are presumably found in the extracellular compartment before infecting a neighbouring phagocyte. Thus, antibody might have direct access to the bacteria during this short period of time and prevent cell-to-cell transmission [92]. Bacterial colonisation obstructs the bacterial opsonization with *Salmonella*-specific Ab [119]. The Ab also plays a role in amplifying the processing and presentation of antigens to CD4+ T cells, thus affecting the Th1 response [120]. B-cell innate immune response to TLR-specific ligands is necessary for the development of Th1 responses in vivo [121]. New findings also suggest the suppression of protective immunity through B-cell MyD88 pathway during infection [122]. Therefore, innate immune signalling in B cells contributes to an important regulatory function but requires further analysis. The presence of *Salmonella*-specific Ab IgA in the intestinal mucosa may also prevent or reduce bacterial penetration of the intestinal barrier [123]. However, which of these mechanisms makes the greatest contribution to protective immunity is yet to be deciphered, but an important role for Ab is also suggested from human studies [124]. Although the specificity of Ab responses is undefined, Abs specific for the LPS O-antigen, flagellin, Vi capsular polysaccharide (ViCPS) antigen and outer membrane porin protein (OmpD) are all believed to be protective [125].

#### **11. Conclusion**

Members belonging to genus *Salmonella* are the major intestinal pathogens of human beings and animals. The increased food production and growth in human

**71**

*Immunopathogenesis of Salmonellosis*

are required.

**Abbreviations**

*DOI: http://dx.doi.org/10.5772/intechopen.85371*

NTS non-typhoidal *Salmonella* TS typhoidal *Salmonella* HA host-adapted serovars

FAE follicle-associated epithelium

MHC major histocompatibility complex SPI *Salmonella* pathogenicity island SCV *Salmonella*-containing vacuoles MLN mesenteric lymph nodes

PAMPs pathogen-associated molecular patterns

Myd myeloid differentiation primary response

HR host restricted

PP Peyer's patch DC dendritic cells

LPS lipopolysaccharide Tregs regulatory T cells

IL interleukin IFN interferon DC dendritic cell

Fim fimbrin

PDL programmed death ligand

TNF tumour necrosis factor CD cluster of differentiation

ViCPS Vi capsular polysaccharide

OmpD outer membrane porin protein

TLRs Toll-like receptors

MAPK mitogen-activated protein kinase SILTs solitary intestinal lymphoid tissues

populations have led to the increase in dissemination potential of these ubiquitous microorganisms. Due to the systemic nature of some infections, where many tissues get involved to display immunity to specific infection, salmonellosis and the immune response that results are pliable. Deciphering the pathogenesis of invasive salmonellosis may hopefully lead to potential therapeutic treatment strategies that are urgently required in light of propagating antimicrobial resistance. Future studies must focus on the identification of molecular targets of *Salmonella* virulence factors during intracellular life in immune cells and designate the molecular mechanisms of interference. This would impart a novel perception into the cell biology of DCs and other immune cells. Furthermore, understanding the intracellular life of *Salmonella* may lead to new advancement in generating reliable vaccines against infections, to wield *Salmonella* strains as live carriers for recombinant vaccines and to evolve novel forms of treatment that target the function of specific virulence factors. Further explorations to clarify the contribution of genes differently represented/expressed in the genomes of various *Salmonella* serotypes during infection

#### *Immunopathogenesis of Salmonellosis DOI: http://dx.doi.org/10.5772/intechopen.85371*

populations have led to the increase in dissemination potential of these ubiquitous microorganisms. Due to the systemic nature of some infections, where many tissues get involved to display immunity to specific infection, salmonellosis and the immune response that results are pliable. Deciphering the pathogenesis of invasive salmonellosis may hopefully lead to potential therapeutic treatment strategies that are urgently required in light of propagating antimicrobial resistance. Future studies must focus on the identification of molecular targets of *Salmonella* virulence factors during intracellular life in immune cells and designate the molecular mechanisms of interference. This would impart a novel perception into the cell biology of DCs and other immune cells. Furthermore, understanding the intracellular life of *Salmonella* may lead to new advancement in generating reliable vaccines against infections, to wield *Salmonella* strains as live carriers for recombinant vaccines and to evolve novel forms of treatment that target the function of specific virulence factors. Further explorations to clarify the contribution of genes differently represented/expressed in the genomes of various *Salmonella* serotypes during infection are required.

### **Abbreviations**

*New Insight into* Brucella *Infection and Foodborne Diseases*

naive CD4+

inhibited by actively replicating bacteria [110]. Effector Th1 cells are effective in

ing regulatory T cells (Tregs) and Th17 cells are also known to contribute. Tregs arise from the thymus or develop after naive T-cell activation in the presence of TNF β which suppresses effector T-cell responses [112]. In contrast, Th17 cells arise from

in mediating immunity against extracellular bacterial infections [113, 114]. During the development of Th1 cells and Tregs after infection, it was found that changes in the cogency of Tregs reduced the efficacy of Th1 responses and increased bacterial growth [102]. After oral infection with *Salmonella*, cytokines associated with Th17 cells, IL-17 and IL-22 are rapidly produced within the intestinal mucosa [115], and the production is induced by innate responses to infection rather than Th17 cells, however, still indicating the potential for Th17 cytokines to participate in intestinal defence against bacteria. In vivo, production of IL-22 dependent on IL12B, rather than IL-17, contributed to bacterial clearance [116]. Taken together, it is suggested that Th17 cells contribute additionally to protection against *Salmonella* infection by not only initiating or enhancing neutrophil infiltration to intestinal tissues but by the production of antimicrobial peptides by the epithelium which is effective against luminal bacteria as well [117]. In summary, it has been suggested that Th17 cells have an additional role in defence against *Salmonella* in the intestine and a role for Tregs in

*Salmonella*-specific B-cell responses contribute to bacterial clearance in the hosts [39, 120, 121]. Although the bacteria are generally found within SCV in phagocytic cells, there is a short period during the infection cycle when bacteria are expected to be extracellular. *Salmonella* is not only tightly associated with mononuclear phagocytes in vivo [118] but also induces these infected cells to undergo apoptosis [44]. After cell death, bacteria are presumably found in the extracellular compartment before infecting a neighbouring phagocyte. Thus, antibody might have direct access to the bacteria during this short period of time and prevent cell-to-cell transmission [92]. Bacterial colonisation obstructs the bacterial opsonization with *Salmonella*-specific Ab [119]. The Ab also plays a role in amplifying the processing and presentation of

response to TLR-specific ligands is necessary for the development of Th1 responses in vivo [121]. New findings also suggest the suppression of protective immunity through B-cell MyD88 pathway during infection [122]. Therefore, innate immune signalling in B cells contributes to an important regulatory function but requires further analysis. The presence of *Salmonella*-specific Ab IgA in the intestinal mucosa may also prevent or reduce bacterial penetration of the intestinal barrier [123]. However, which of these mechanisms makes the greatest contribution to protective immunity is yet to be deciphered, but an important role for Ab is also suggested from human studies [124]. Although the specificity of Ab responses is undefined, Abs specific for the LPS O-antigen, flagellin, Vi capsular polysaccharide (ViCPS) antigen and outer membrane

Members belonging to genus *Salmonella* are the major intestinal pathogens of human beings and animals. The increased food production and growth in human

T cells, thus affecting the Th1 response [120]. B-cell innate immune

T-cell stimulation in the presence of IL-6 and TNF-β and are important

subsets includ-

providing immunity to salmonellosis [111]; however, effector CD4+

modulating the potency of *Salmonella*-specific Th1 cells in vivo.

**10. Host antibody (Ab) response against** *Salmonella*

porin protein (OmpD) are all believed to be protective [125].

**70**

antigens to CD4+

**11. Conclusion**


#### **Author details**

Mashooq Ahmad Dar1,2, Peerzada Tajamul Mumtaz1 , Shakil Ahmad Bhat1 , Qamar Taban1 , Shabir Ahmad Khan1 , Tufail Banday3 and Syed Mudasir Ahmad1 \*

1 Division of Biotechnology, Faculty of Veterinary Sciences and Animal Husbandry, Shere Kashmir University of Agricultural Sciences and Technology, Kashmir, India

2 Department of Biochemistry, University of Kashmir, Kashmir, India

3 Division of Livestock Production and Management Faculty of Veterinary Sciences and Animal Husbandry, Shere Kashmir University of Agricultural Sciences and Technology, Kashmir, India

\*Address all correspondence to: mudasirbio@gmail.com

© 2019 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

**73**

*Immunopathogenesis of Salmonellosis*

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[2] Andino A, Hanning I. *Salmonella enterica*: Survival, colonization, and virulence differences among serovars. The Scientific World Journal. 2015. DOI: 10.1155/2015/520179 (review article)

[3] Dar MA, Ahmad SM, Bhat SA, Ahmad R, Urwat U, Mumtaz PT, et al. Salmonella typhimurium in poultry: A review. World's Poultry Science Journal.

[4] Rabsch W, Tschäpe H, Baumler AJ. Non-typhoidal salmonellosis: Emerging problems. Microbes and

[5] Rodriguez A, Pangloli P, Richards HA, Mount JR, Draughon FA. Prevalence of Salmonella in diverse environmental farm samples. Journal of Food Protection. 2006;**69**:2576-2580

[6] Smith SI, Seriki A, Ajayi A. Typhoidal and non-typhoidal Salmonella infections in Africa. European Journal of Clinical

2016;**35**:1913-1922

pgmj.2003.016584

Microbiology & Infectious Diseases.

[7] Majowicz SE, Musto J, Scallan E, Angulo FJ, Kirk M, O'Brien SJ, et al. The global burden of nontyphoidal *Salmonella gastroenteritis*. Clinical Infectious Diseases. 2010;**50**:882-889

[8] Hardy A. Salmonella: A continuing problem. Postgraduate Medical Journal.

2004;**80**:541-545. DOI: 10.1136/

[9] Meltzer E, Schwartz E. Enteric fever: A travel medicine oriented view.

Infection. 2001;**3**:237-247

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#### **References**

*New Insight into* Brucella *Infection and Foodborne Diseases*

**72**

**Author details**

Qamar Taban1

Technology, Kashmir, India

Mashooq Ahmad Dar1,2, Peerzada Tajamul Mumtaz1

, Shabir Ahmad Khan1

\*Address all correspondence to: mudasirbio@gmail.com

provided the original work is properly cited.

, Shakil Ahmad Bhat1

and Syed Mudasir Ahmad1

, Tufail Banday3

1 Division of Biotechnology, Faculty of Veterinary Sciences and Animal Husbandry, Shere Kashmir University of Agricultural Sciences and Technology, Kashmir, India

3 Division of Livestock Production and Management Faculty of Veterinary Sciences and Animal Husbandry, Shere Kashmir University of Agricultural Sciences and

© 2019 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium,

2 Department of Biochemistry, University of Kashmir, Kashmir, India

,

\*

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[109] Yrlid U, Wick MJ. Antigen presentation capacity and cytokine production by murine splenic dendritic cell subsets upon Salmonella encounter. Journal of Immunology. 2002;**169**:108-116

[110] Srinivasan A, Nanton M, Griffin A, McSorley SJ. Culling of activated CD4 T cells during typhoid is driven by Salmonella virulence genes. Journal of Immunology. 2009;**182**:7838-7845

[111] VanCott JL, Chatfield SN, Roberts M, Hone DM, Hohmann EL, Pascual DW, et al. Regulation of host immune responses by modification of Salmonella virulence genes. Nature Medicine. 1998;**4**:1247-1252

[112] Xu L, Kitani A, Strober W. Molecular mechanisms regulating TGF beta-induced Foxp3 expression. Mucosal Immunology. 2010;**3**:230-238

[113] Weaver CT, Hatton RD, Mangan PR, Harrington LE. IL-17 family cytokines and the expanding diversity of effector T cell lineages. Annual Review of Immunology. 2007;**25**:821-852

[114] Curtis MM, Way SS. Interleukin-17 in host defence against bacterial, mycobacterial and fungal pathogens. Immunology. 2009;**126**:177-185

[115] Raffatellu M, Santos RL, Verhoeven DE, George MD, Wilson RP, Winter SE, et al. Simian immunodeficiency virus-induced mucosal interleukin-17 deficiency promotes Salmonella dissemination from the gut. Nature Medicine. 2008;**14**:421-428

[116] Schulz SM, Kohler G, Holscher C, Iwakura Y, Alber G. IL-17A is

produced by Th17, gamma delta T cells and other CD4- lymphocytes during infection with *Salmonella enterica* serovar enteritidis and has a mild effect in bacterial clearance. International Immunology. 2008;**20**:1129-1138

[117] Santos RL, Raffatellu M, Bevins CL, Adams LG, Tükel C, Tsolis RM, et al. Life in the inflamed intestine, Salmonella style. Trends in Microbiology. 2009;**17**:498-506

[118] MacLennan CA, Esther NG, Chisomo LM, Robert AK, Nicholas R, Thomson SA. The neglected role of antibody in protection against bacteremia caused by nontyphoidal strains of Salmonella in African children. The Journal of Clinical Investigation. 2008;**118**:1553-1562

[119] Cunningham AF, Gaspal F, Serre K, Mohr E, Henderson IR, Scott-Tucker A, et al. Salmonella induces a switched antibody response without germinal centers that impedes the extracellular spread of infection. Journal of Immunology. 2007;**178**:6200-6207

[120] Bueno SM, Gonzalez PA, Schwebach JR, Kalergis AM. T cell immunity evasion by virulent *Salmonella enterica*. Immunology Letters. 2007;**111**:14-20

[121] Barr TA, Brown S, Mastroeni P, Gray D. TLR and B cell receptor signals to B cells differentially program primary and memory Th1 responses to *Salmonella enterica*. Journal of Immunology. 2010;**185**:2783-2789

[122] Neves P, Lampropoulou V, Calderon-Gomez E, Roch T, Stervbo U, Shen P, et al. Signaling via the MyD88 adaptor protein in B cells suppresses protective immunity during *Salmonella typhimurium* infection. Immunity. 2010;**33**:777-790

**81**

*Immunopathogenesis of Salmonellosis*

Medicine. 2006;**203**:21-26

2006;**24**:3804-3811

[124] Guzman CA, Borsutzky S, Griot-Wenk M, Metcalfe IC, Pearman J, Collioud A, et al. Vaccines against typhoid fever. Vaccine.

Brandtzaeg P, Strugnell RA. Innate secretory antibodies protect against natural *Salmonella typhimurium*

*DOI: http://dx.doi.org/10.5772/intechopen.85371*

infection. The Journal of Experimental

[125] Gil-Cruz C, Bobat S, Jennifer L, Marshall RA, Kingsley EA, Ross IR. The porin OmpD from nontyphoidal Salmonella is a key target for a

protective B1b cell antibody response. Proceedings of the National Academy of Sciences of the United States of America. 2009;**106**:9803-9808

[123] Wijburg OL, Uren TK, Simpfendorfer K, Johansen FE, *Immunopathogenesis of Salmonellosis DOI: http://dx.doi.org/10.5772/intechopen.85371*

*New Insight into* Brucella *Infection and Foodborne Diseases*

produced by Th17, gamma delta T cells and other CD4- lymphocytes during infection with *Salmonella enterica* serovar enteritidis and has a mild effect in bacterial clearance. International Immunology. 2008;**20**:1129-1138

[117] Santos RL, Raffatellu M, Bevins CL, Adams LG, Tükel C, Tsolis RM, et al. Life in the inflamed intestine, Salmonella style. Trends in Microbiology. 2009;**17**:498-506

[118] MacLennan CA, Esther NG, Chisomo LM, Robert AK, Nicholas R, Thomson SA. The neglected role of antibody in protection against bacteremia caused by nontyphoidal strains of Salmonella in African children. The Journal of Clinical Investigation. 2008;**118**:1553-1562

[119] Cunningham AF, Gaspal F, Serre K, Mohr E, Henderson IR, Scott-Tucker A, et al. Salmonella induces a switched antibody response without germinal centers that impedes the extracellular spread of infection. Journal of Immunology. 2007;**178**:6200-6207

[120] Bueno SM, Gonzalez PA, Schwebach JR, Kalergis AM. T cell immunity evasion by virulent *Salmonella enterica*. Immunology

[121] Barr TA, Brown S, Mastroeni P, Gray D. TLR and B cell receptor signals to B cells differentially program primary and memory Th1 responses to *Salmonella enterica*. Journal of Immunology. 2010;**185**:2783-2789

[122] Neves P, Lampropoulou V,

[123] Wijburg OL, Uren TK, Simpfendorfer K, Johansen FE,

2010;**33**:777-790

Calderon-Gomez E, Roch T, Stervbo U, Shen P, et al. Signaling via the MyD88 adaptor protein in B cells suppresses protective immunity during *Salmonella typhimurium* infection. Immunity.

Letters. 2007;**111**:14-20

[108] Halici S, Zenk SF, Jantsch J, Hensel M. Functional analysis of the Salmonella

pathogenicity island 2-mediated inhibition of antigen presentation in dendritic cells. Infection and Immunity.

[109] Yrlid U, Wick MJ. Antigen presentation capacity and cytokine production by murine splenic

dendritic cell subsets upon Salmonella encounter. Journal of Immunology.

[110] Srinivasan A, Nanton M, Griffin A, McSorley SJ. Culling of activated CD4 T cells during typhoid is driven by Salmonella virulence genes. Journal of Immunology. 2009;**182**:7838-7845

[111] VanCott JL, Chatfield SN, Roberts M, Hone DM, Hohmann EL, Pascual DW, et al. Regulation of host immune responses by modification of Salmonella virulence genes. Nature Medicine.

[112] Xu L, Kitani A, Strober W. Molecular mechanisms regulating TGF beta-induced Foxp3 expression. Mucosal Immunology. 2010;**3**:230-238

[114] Curtis MM, Way SS. Interleukin-17 in host defence against bacterial, mycobacterial and fungal pathogens. Immunology.

Medicine. 2008;**14**:421-428

[116] Schulz SM, Kohler G, Holscher C, Iwakura Y, Alber G. IL-17A is

2009;**126**:177-185

[113] Weaver CT, Hatton RD, Mangan PR, Harrington LE. IL-17 family cytokines and the expanding diversity of effector T cell lineages. Annual Review of Immunology. 2007;**25**:821-852

[115] Raffatellu M, Santos RL, Verhoeven DE, George MD, Wilson RP, Winter SE, et al. Simian immunodeficiency virus-induced mucosal interleukin-17 deficiency promotes Salmonella dissemination from the gut. Nature

2008;**76**:4924-4933

2002;**169**:108-116

1998;**4**:1247-1252

**80**

Brandtzaeg P, Strugnell RA. Innate secretory antibodies protect against natural *Salmonella typhimurium* infection. The Journal of Experimental Medicine. 2006;**203**:21-26

[124] Guzman CA, Borsutzky S, Griot-Wenk M, Metcalfe IC, Pearman J, Collioud A, et al. Vaccines against typhoid fever. Vaccine. 2006;**24**:3804-3811

[125] Gil-Cruz C, Bobat S, Jennifer L, Marshall RA, Kingsley EA, Ross IR. The porin OmpD from nontyphoidal Salmonella is a key target for a protective B1b cell antibody response. Proceedings of the National Academy of Sciences of the United States of America. 2009;**106**:9803-9808

**83**

**Chapter 7**

**Abstract**

**1. Introduction**

Application of Artificial Barrier

through Riverbank Filtration

*Nur Aziemah Abd Rashid and Ismail Abustan*

as Mitigation of *E. coli* Which Pass

Water security in the water treatment plant has been doubted, and the treatment

process may have given unreliable and unsafe water to the public. A newspaper reported on November 19, 2011, that laboratory tests on water samples in Kelantan for each year by the Ministry of Health have found harmful bacteria including *Escherichia coli* (*E. coli*) in the water samples. More worryingly, it was stated in a study that chlorine in water treated with high chlorine can be harmful to human health. In 2010, Malaysia has begun to approach a natural treatment technique, namely, riverbank filtration (RBF), and firstly used it at the Water Treatment Plant in Jeli, Kelantan, and Kuala Kangsar, Perak. RBF limitation is the invisible groundwater flow that makes it difficult to predict the transport of contaminants. Managing groundwater is important to ensure that water is aligned in compliance with government legislation and environmental protection. Due to that, this study suggests an implementation of an artificial barrier for microorganism in RBF to sustain the good water quality abstracted from the abstraction well. This pretreatment or purifying method is to improve the effectiveness of RBF in removing pollutants during shock loads and reduce the load placed in the water treatment process.

**Keywords:** artificial barrier, riverbank filtration, *E. coli*, groundwater, water security

Potable water access globally is now under crisis, which leads to poor human health issue, affecting Malaysia as one of the countries facing this problem. The main reasons why this happens are due to climate change, deterioration of river water quality, unreliable water treatment system, and increase of population, which, at the same time, causes water shortage to occur. During dry weather conditions, further depletion of water occurs. Pertinently, climate changes make the drought season becomes longer and hotter than usual. The dam water becomes low and the river water dries up. The deterioration of river water quality in Malaysia has brought an impact to the water treatment plant due to the increase of treatment cost and maintenance. Chemicals such as PACI, alum, and others will also be increased to treat the polluted river. In the year of 2011, it was stated in a study that chlorine in water treated with high chlorine can be harmful to human health [1]. Thus, water security in the water treatment plant has been doubted, and the treatment process may have given unreliable and unsafe water to the public. Recently, *Utusan Malaysia*

#### **Chapter 7**

## Application of Artificial Barrier as Mitigation of *E. coli* Which Pass through Riverbank Filtration

*Nur Aziemah Abd Rashid and Ismail Abustan*

#### **Abstract**

Water security in the water treatment plant has been doubted, and the treatment process may have given unreliable and unsafe water to the public. A newspaper reported on November 19, 2011, that laboratory tests on water samples in Kelantan for each year by the Ministry of Health have found harmful bacteria including *Escherichia coli* (*E. coli*) in the water samples. More worryingly, it was stated in a study that chlorine in water treated with high chlorine can be harmful to human health. In 2010, Malaysia has begun to approach a natural treatment technique, namely, riverbank filtration (RBF), and firstly used it at the Water Treatment Plant in Jeli, Kelantan, and Kuala Kangsar, Perak. RBF limitation is the invisible groundwater flow that makes it difficult to predict the transport of contaminants. Managing groundwater is important to ensure that water is aligned in compliance with government legislation and environmental protection. Due to that, this study suggests an implementation of an artificial barrier for microorganism in RBF to sustain the good water quality abstracted from the abstraction well. This pretreatment or purifying method is to improve the effectiveness of RBF in removing pollutants during shock loads and reduce the load placed in the water treatment process.

**Keywords:** artificial barrier, riverbank filtration, *E. coli*, groundwater, water security

#### **1. Introduction**

Potable water access globally is now under crisis, which leads to poor human health issue, affecting Malaysia as one of the countries facing this problem. The main reasons why this happens are due to climate change, deterioration of river water quality, unreliable water treatment system, and increase of population, which, at the same time, causes water shortage to occur. During dry weather conditions, further depletion of water occurs. Pertinently, climate changes make the drought season becomes longer and hotter than usual. The dam water becomes low and the river water dries up. The deterioration of river water quality in Malaysia has brought an impact to the water treatment plant due to the increase of treatment cost and maintenance. Chemicals such as PACI, alum, and others will also be increased to treat the polluted river. In the year of 2011, it was stated in a study that chlorine in water treated with high chlorine can be harmful to human health [1]. Thus, water security in the water treatment plant has been doubted, and the treatment process may have given unreliable and unsafe water to the public. Recently, *Utusan Malaysia*

newspaper reported on November 19, 2011, that laboratory tests on water samples in Kelantan for each year by the Ministry of Health have found heavy metals and harmful bacteria including *Escherichia coli* (*E. coli*) in the water samples. More worryingly, *E. coli* was also found in water supplied to homes by Air Kelantan Sdn. Bhd. (AKSB). The discovery of *E. coli* in water samples in Kelantan detected by the ministry was then carried out from 2008 to 2010.

Providing reliable and safe potable water has become a human right for us. Therefore, finding a solution to these issues is highly desirable to improve the safety and reliability of potable water. In 2010, Malaysia has begun to approach a new treatment technique, namely, riverbank filtration (RBF). RBF is a method using groundwater that is expected to provide a new way to increase water intake and untapped resources in Malaysia, firstly used at the Water Treatment Plant in Jeli, Kelantan, and Kuala Kangsar, Perak. RBF is a natural system in which it involves the entry of river water into underground aquifers and is caused by hydraulic gradients, whereby water retrieval is from collector wells located at banks, at a certain distance from the river [2]. Although it is still less than 10 years in Malaysia, RBF method shows good results to reduce the use of chemicals and produces biologically stable water; the system also improves water quality by removing particles (turbidity and suspended solids), organic pollutants, microorganisms, heavy metals, and nitrogen. One previous experience in Germany shows that RBF provides a strong barrier for various pollutants and can help to ease the temperature fluctuations and concentration peaks when it is associated with spills into rivers. It also replaces and supports other treatment processes and reduces the overall costs of water treatment plant [3]. The removal of sediment, organic and inorganic compounds, and pathogens takes place during the first meters from the river in what is known as the hyporheic zone, which usually presents reducing conditions, due to high microbial activity that consumes oxygen in the water. Within this zone, there are important biochemical processes and redox reactions that affect groundwater quality [4]. In general, every stage of RBF has an environmental influence that is from the river until abstraction well.

Safe potable water is one of the implicit requisites for a healthy human population. In the existence of RBF, artificial barrier is a new efficient purifying method to maintain safer water abstraction. This study demonstrates the potential of a new application of artificial barrier to filtrate *E. coli* in water in RBF system. The artificial barrier efficiency was examined for different media ratio. Artificial barrier is a man-made vertical barrier to pretreat water abstraction intake. It is a mixture of sand (local soil), granular activated carbon (GAC), and zeolite. Generally, the individual application of coconut shell GAC and zeolite has shown great advantages in terms of characteristics, adsorption capacities, as well as their physicochemical versatility. For that reason, the idea of combining the precursors in order to make an effective filter-based adsorbent for RBF purifying process is highly recommended. Besides that, the inherited limitation of an individual precursor in water treatment process could be minimized by combining them in layered filter adsorbent as first and second barriers in RBF aquifer due to low turbidity. GAC and zeolite have high permeability which make them suitable to be applied in RBF aquifer, which requires high permeability condition as for the RBF site. However, studies on the removal of *E. coli* from actual river water using artificial barrier (GAC and zeolite) in RBF as the pretreatment or purifying process are still limited until now. Similarly, studies concerning the optimization of adsorption treatment for the studied parameter removal from river water are inadequate. Due to that, this research study is mainly focused on the treatment of actual river water from Sungai Kerian, Lubok Buntar, Kedah, via artificial barrier fixed-bed flow studies.

**85**

**Figure 1.**

*Riverbank filtration system.*

*Application of Artificial Barrier as Mitigation of* E. coli *Which Pass through Riverbank Filtration*

RBF has begun to be widely used in Malaysia as to optimize the water supply. The introduction of RBF in Malaysia is started in 2010 at Jeli, Kelantan. The plants' operation has demonstrated the success of the combination of RBF (as pretreatment) and water treatment plant (as posttreatment). Most RBF in Malaysia have been applied in Kelantan areas. After calculating all the costs (not including the

0.04, which is considered to be a competitive price for the Malaysian. The combined method has therefore proved to be both technologically and financially viable. These findings should pave the way for other municipal authorities to follow suit by

RBF post water treatment has been employed dating back to the nineteenth century. During RBF, river or lake water is extracted indirectly by drawing it through the subsurface prior to use as in **Figure 1**. The extraction is accomplished by an infiltration line of well either vertical or horizontal. The well is located at a short (below 30 m) to intermediate (up to 60 m) distance from the riverbank or lake. During extraction of water, the groundwater that discharges into the river decreases, and the groundwater table near the waterline may decrease below the river water level. To ensure a satisfactory purification, the distance between the river and the extrac-

During infiltration and travel through the soil and aquifer sediments, surface water is subjected to a combination of physical and chemical and biological processes of filtration. The top few centimeters of the riverbank materials formed are a screen or filter medium that removes the suspended solids present in the water. Heavy metal, phosphorous, and hydrophobic organic compounds present in the water are removed by adsorption onto certain aquifer materials. In the presence of biomass, the organic matter is further biodegraded (initially under oxic conditions and later under anoxic conditions). The water quality in most cases is improved by dilution of the surface water source with native groundwater [6]. When a particle becomes attached to the biofilm on the sand grain, microorganism may degrade that particle. There is an interception when particles are carried by one of the streamlines closest to the sand grain and a brushing effect occurs. There is general agreement that straining, adhesion, attachment, chemical adsorption, sedimentation,

of drinking water costs approximately USD

*DOI: http://dx.doi.org/10.5772/intechopen.86079*

cost of pumps, pipes, valves, etc.), 1 m3

introducing their own combined RBF with ultrafiltration.

tion well should such that the travel time exceeds 30–60 days [5].

and biological growth all operate to some extent.

**2. Riverbank filtration**

**2.1 Principle and treatment**

*Application of Artificial Barrier as Mitigation of* E. coli *Which Pass through Riverbank Filtration DOI: http://dx.doi.org/10.5772/intechopen.86079*

#### **2. Riverbank filtration**

*New Insight into* Brucella *Infection and Foodborne Diseases*

ministry was then carried out from 2008 to 2010.

newspaper reported on November 19, 2011, that laboratory tests on water samples in Kelantan for each year by the Ministry of Health have found heavy metals and harmful bacteria including *Escherichia coli* (*E. coli*) in the water samples. More worryingly, *E. coli* was also found in water supplied to homes by Air Kelantan Sdn. Bhd. (AKSB). The discovery of *E. coli* in water samples in Kelantan detected by the

Providing reliable and safe potable water has become a human right for us. Therefore, finding a solution to these issues is highly desirable to improve the safety and reliability of potable water. In 2010, Malaysia has begun to approach a new treatment technique, namely, riverbank filtration (RBF). RBF is a method using groundwater that is expected to provide a new way to increase water intake and untapped resources in Malaysia, firstly used at the Water Treatment Plant in Jeli, Kelantan, and Kuala Kangsar, Perak. RBF is a natural system in which it involves the entry of river water into underground aquifers and is caused by hydraulic gradients, whereby water retrieval is from collector wells located at banks, at a certain distance from the river [2]. Although it is still less than 10 years in Malaysia, RBF method shows good results to reduce the use of chemicals and produces biologically stable water; the system also improves water quality by removing particles (turbidity and suspended solids), organic pollutants, microorganisms, heavy metals, and nitrogen. One previous experience in Germany shows that RBF provides a strong barrier for various pollutants and can help to ease the temperature fluctuations and concentration peaks when it is associated with spills into rivers. It also replaces and supports other treatment processes and reduces the overall costs of water treatment plant [3]. The removal of sediment, organic and inorganic compounds, and pathogens takes place during the first meters from the river in what is known as the hyporheic zone, which usually presents reducing conditions, due to high microbial activity that consumes oxygen in the water. Within this zone, there are important biochemical processes and redox reactions that affect groundwater quality [4]. In general, every stage of RBF has an environmental influence that is from the river

Safe potable water is one of the implicit requisites for a healthy human population. In the existence of RBF, artificial barrier is a new efficient purifying method to maintain safer water abstraction. This study demonstrates the potential of a new application of artificial barrier to filtrate *E. coli* in water in RBF system. The artificial barrier efficiency was examined for different media ratio. Artificial barrier is a man-made vertical barrier to pretreat water abstraction intake. It is a mixture of sand (local soil), granular activated carbon (GAC), and zeolite. Generally, the individual application of coconut shell GAC and zeolite has shown great advantages in terms of characteristics, adsorption capacities, as well as their physicochemical versatility. For that reason, the idea of combining the precursors in order to make an effective filter-based adsorbent for RBF purifying process is highly recommended. Besides that, the inherited limitation of an individual precursor in water treatment process could be minimized by combining them in layered filter adsorbent as first and second barriers in RBF aquifer due to low turbidity. GAC and zeolite have high permeability which make them suitable to be applied in RBF aquifer, which requires high permeability condition as for the RBF site. However, studies on the removal of *E. coli* from actual river water using artificial barrier (GAC and zeolite) in RBF as the pretreatment or purifying process are still limited until now. Similarly, studies concerning the optimization of adsorption treatment for the studied parameter removal from river water are inadequate. Due to that, this research study is mainly focused on the treatment of actual river water from Sungai Kerian, Lubok Buntar, Kedah, via artificial barrier

**84**

fixed-bed flow studies.

until abstraction well.

#### **2.1 Principle and treatment**

RBF has begun to be widely used in Malaysia as to optimize the water supply. The introduction of RBF in Malaysia is started in 2010 at Jeli, Kelantan. The plants' operation has demonstrated the success of the combination of RBF (as pretreatment) and water treatment plant (as posttreatment). Most RBF in Malaysia have been applied in Kelantan areas. After calculating all the costs (not including the cost of pumps, pipes, valves, etc.), 1 m3 of drinking water costs approximately USD 0.04, which is considered to be a competitive price for the Malaysian. The combined method has therefore proved to be both technologically and financially viable. These findings should pave the way for other municipal authorities to follow suit by introducing their own combined RBF with ultrafiltration.

RBF post water treatment has been employed dating back to the nineteenth century. During RBF, river or lake water is extracted indirectly by drawing it through the subsurface prior to use as in **Figure 1**. The extraction is accomplished by an infiltration line of well either vertical or horizontal. The well is located at a short (below 30 m) to intermediate (up to 60 m) distance from the riverbank or lake. During extraction of water, the groundwater that discharges into the river decreases, and the groundwater table near the waterline may decrease below the river water level. To ensure a satisfactory purification, the distance between the river and the extraction well should such that the travel time exceeds 30–60 days [5].

During infiltration and travel through the soil and aquifer sediments, surface water is subjected to a combination of physical and chemical and biological processes of filtration. The top few centimeters of the riverbank materials formed are a screen or filter medium that removes the suspended solids present in the water. Heavy metal, phosphorous, and hydrophobic organic compounds present in the water are removed by adsorption onto certain aquifer materials. In the presence of biomass, the organic matter is further biodegraded (initially under oxic conditions and later under anoxic conditions). The water quality in most cases is improved by dilution of the surface water source with native groundwater [6]. When a particle becomes attached to the biofilm on the sand grain, microorganism may degrade that particle. There is an interception when particles are carried by one of the streamlines closest to the sand grain and a brushing effect occurs. There is general agreement that straining, adhesion, attachment, chemical adsorption, sedimentation, and biological growth all operate to some extent.

**Figure 1.** *Riverbank filtration system.*

The conventional treatment commonly involves screening, aeration, coagulation, flocculation, sedimentation, slow sand filtration, and chlorination. The chemical treatment and waste product will increase if the pollutants in surface water increased. The RBF reduces the posttreatment step from six to only two steps which is removal of heavy metals (usually iron and manganese) by either aeration, activated carbon filter, or ultrafiltration and chlorination for taste and odor. This RBF system as a pretreatment technique applied in countries like the Netherlands, Germany, China, Korea, India, Egypt, and others has already succeeded in optimizing the potable water supply. The underground passage ensures the high quality of drinking water, which does not need any further treatment or disinfection before supply [7].

The posttreatment after RBF depends on the water abstraction water quality. Each RBF site has a different technique step for posttreatment. Previous study shows the most common pollutants that occur in RBF sites are iron and manganese. The treatments used to remove these contaminants in water are aeration, activated carbon filter, and ultrafiltration method. The second contaminant that occurs is taste and odor which are usually removed using chlorination. The third contaminant was microbiology which is solved by using ozonation and UV disinfection. This all posttreatment technique is commonly used at RBF site and summarized in **Table 1**. Meanwhile, there are RBF sites which are not using a posttreatment as a means for direct usage such as in China. However, in several years there will be oocyst problems.

#### **2.2 Benefits and limitation**

The RBF is a sustainable natural treatment process which avoids or reduces the use of chemicals and produces biologically stable water. The system improves water quality by removing particles (turbidity and suspended solid), organic pollutants,


**87**

*Application of Artificial Barrier as Mitigation of* E. coli *Which Pass through Riverbank Filtration*

microorganism, heavy metals, and nitrogen. The RBF also helps to dampen the temperature fluctuations and concentration peaks when it is associated with spills into a river or lake. This treatment process also replaces and supports the other treatment processes by providing a robust barrier for multiple contaminants and reduces the

RBF limitation is the invisible groundwater flow that makes it difficult to predict the transport of contaminants. A specific concern of the RBF limitation is due to hydrology and dynamics of the river and groundwater, which have different climate variations (drought and rainy seasons), and thus, the groundwater level patterns result in significant fluctuation of contaminants in well stream loads. In rainy season, the rate of groundwater flow increases to a maximum level and causes small particles and pollutants to absorb into the soil where it encloses the flow along the groundwater flow, which initiates pollutants to enter the borehole. On the other hand, in dry season, minimum and ideal flow rates for pollutants are attached to the local soil. Moreover, since maximum groundwater flow rate occurs frequently in Malaysia, this incident is predicted to often result in significant fluctuations of underground hydraulic conductivity of groundwater and shock load of pollutants. Significant amount of pollutants may exist in borehole water due to high hydraulic conductivity and soil feature, which concludes that RBF is a natural treatment method that depends on natural behavior. In general, the quality of RBF water is influenced by the environmental conditions, where managing groundwater is important to ensure that water is aligned in compliance with government legislation

The posttreatment step in most RBF sites is usually focused on iron and manganese treatment which result in the usage of aeration, activated carbon filter, and ultrafiltration treatment process. The weakness of this treatment which cannot be ignored has been discussed in the above section. The occurrence of the pollutants can be worse during shock load and clogging. Due to that, artificial barrier seems important which can increase the hydraulic conductivity of the underground water flow, reduce the pressure load to the aquifer during clogging, and enhance the pollutants adsorption during shock load. This can reduce the consumption of chemical

There are four basic important criteria affecting the performance of RBF which

groundwater, distance of the well from riverbank and spacing of wells and pumping rates, and sediment permeability. The effectiveness of RBF for removing surface water contaminants depends largely on hydrogeological conditions. It is about the soil microbiology, characteristic of the bank materials and streambed, and scouring characteristic [13]. In many countries, the alluvial soil aquifers hydraulically connected to a water course would be preferred sites for drinking water production [14]. The actual biochemical interactions that sustain the quality of the pumped bank filtration depend on numerous factors, including aquifer mineralogy and the

The RBF shows a decreasing RBF water level with an increasing distance of the well apart from the riverbank. In addition to the decreasing RBF water level due to increasing distance, there is no cross flow of natural groundwater that the well could abstract river water [12]. Pumping test result shows that the water in well (below 60 m) comes from river water. However, the low-lying coastal aquifer is generally fragile and easily depleted due to anthropogenic activities and overexploitation of groundwater and agriculture. To manage and protect precious groundwater

are hydrogeological conditions, source water quality and mixing with native

*DOI: http://dx.doi.org/10.5772/intechopen.86079*

overall cost of water treatment [3].

and environmental protection.

extent of the aquifer [15].

treatment and strengthen the RBF barrier.

**2.3 Factors influencing optimization of RBF**

**Table 1.** *Summary of RBF post treatment from other country and its limitations.* *Application of Artificial Barrier as Mitigation of* E. coli *Which Pass through Riverbank Filtration DOI: http://dx.doi.org/10.5772/intechopen.86079*

microorganism, heavy metals, and nitrogen. The RBF also helps to dampen the temperature fluctuations and concentration peaks when it is associated with spills into a river or lake. This treatment process also replaces and supports the other treatment processes by providing a robust barrier for multiple contaminants and reduces the overall cost of water treatment [3].

RBF limitation is the invisible groundwater flow that makes it difficult to predict the transport of contaminants. A specific concern of the RBF limitation is due to hydrology and dynamics of the river and groundwater, which have different climate variations (drought and rainy seasons), and thus, the groundwater level patterns result in significant fluctuation of contaminants in well stream loads. In rainy season, the rate of groundwater flow increases to a maximum level and causes small particles and pollutants to absorb into the soil where it encloses the flow along the groundwater flow, which initiates pollutants to enter the borehole. On the other hand, in dry season, minimum and ideal flow rates for pollutants are attached to the local soil. Moreover, since maximum groundwater flow rate occurs frequently in Malaysia, this incident is predicted to often result in significant fluctuations of underground hydraulic conductivity of groundwater and shock load of pollutants. Significant amount of pollutants may exist in borehole water due to high hydraulic conductivity and soil feature, which concludes that RBF is a natural treatment method that depends on natural behavior. In general, the quality of RBF water is influenced by the environmental conditions, where managing groundwater is important to ensure that water is aligned in compliance with government legislation and environmental protection.

The posttreatment step in most RBF sites is usually focused on iron and manganese treatment which result in the usage of aeration, activated carbon filter, and ultrafiltration treatment process. The weakness of this treatment which cannot be ignored has been discussed in the above section. The occurrence of the pollutants can be worse during shock load and clogging. Due to that, artificial barrier seems important which can increase the hydraulic conductivity of the underground water flow, reduce the pressure load to the aquifer during clogging, and enhance the pollutants adsorption during shock load. This can reduce the consumption of chemical treatment and strengthen the RBF barrier.

#### **2.3 Factors influencing optimization of RBF**

There are four basic important criteria affecting the performance of RBF which are hydrogeological conditions, source water quality and mixing with native groundwater, distance of the well from riverbank and spacing of wells and pumping rates, and sediment permeability. The effectiveness of RBF for removing surface water contaminants depends largely on hydrogeological conditions. It is about the soil microbiology, characteristic of the bank materials and streambed, and scouring characteristic [13]. In many countries, the alluvial soil aquifers hydraulically connected to a water course would be preferred sites for drinking water production [14]. The actual biochemical interactions that sustain the quality of the pumped bank filtration depend on numerous factors, including aquifer mineralogy and the extent of the aquifer [15].

The RBF shows a decreasing RBF water level with an increasing distance of the well apart from the riverbank. In addition to the decreasing RBF water level due to increasing distance, there is no cross flow of natural groundwater that the well could abstract river water [12]. Pumping test result shows that the water in well (below 60 m) comes from river water. However, the low-lying coastal aquifer is generally fragile and easily depleted due to anthropogenic activities and overexploitation of groundwater and agriculture. To manage and protect precious groundwater

*New Insight into* Brucella *Infection and Foodborne Diseases*

The conventional treatment commonly involves screening, aeration, coagulation, flocculation, sedimentation, slow sand filtration, and chlorination. The chemical treatment and waste product will increase if the pollutants in surface water increased. The RBF reduces the posttreatment step from six to only two steps which is removal of heavy metals (usually iron and manganese) by either aeration, activated carbon filter, or ultrafiltration and chlorination for taste and odor. This RBF system as a pretreatment technique applied in countries like the Netherlands, Germany, China, Korea, India, Egypt, and others has already succeeded in optimizing the potable water supply. The underground passage ensures the high quality of drinking water, which does not need any further treatment or disinfection before supply [7].

The posttreatment after RBF depends on the water abstraction water quality. Each RBF site has a different technique step for posttreatment. Previous study shows the most common pollutants that occur in RBF sites are iron and manganese. The treatments used to remove these contaminants in water are aeration, activated carbon filter, and ultrafiltration method. The second contaminant that occurs is taste and odor which are usually removed using chlorination. The third contaminant was microbiology which is solved by using ozonation and UV disinfection. This all posttreatment technique is commonly used at RBF site and summarized in **Table 1**. Meanwhile, there are RBF sites which are not using a posttreatment as a means for direct usage such as in China. However, in several years there will be

The RBF is a sustainable natural treatment process which avoids or reduces the use of chemicals and produces biologically stable water. The system improves water quality by removing particles (turbidity and suspended solid), organic pollutants,

**86**

**Table 1.**

*Summary of RBF post treatment from other country and its limitations.*

oocyst problems.

**2.2 Benefits and limitation**

resources in a sustainable manner, the characterization and understanding of the natural evolution of groundwater chemistry are crucial to elucidate their geochemical nature and its relation.

The collector well can be far from the river if the soil type is sand and gravel such as RBF at Yellow River, China. The combination of vertical and horizontal collector well can maximize the water capacity such as RBF at Elbe River, Germany. However, clayey alluvial soil will limit the water capacity as RBF site at Lek River, Netherlands, shows the water capacity is only 0.01 MLD, compared to clayey alluvial soil at Nakdong River, Korea, which can be abstracted to 10 MLD water capacity. This shows clayey alluvial soil type needs deeper built collector well near the riverbank. The nearer to riverbank, the more water capacity can be abstracted than collector well at Nakdong River, Korea, which is only 10 MLD with 150 m distance from river, and collector well at Nile River, Egypt, with 22 MLD. Some sites do not contain gravelly sand alluvial soil type but can apply RBF such as Kali River, India. The highly pollutant river demands to use RBF methods; however, it only can abstract 0.8 MLD water capacity because the transmissivity of brownish red silty loam alluvial soil is low. Sites with clayey alluvial soil can apply limestone to increase the transmissivity of water such RBF sites at Ohio River, Kentucky, and Great Miami River, USA. Malaysia RBF sites at Sungai Semerak contain gravelly sand and shallow vertical well collector type. The shallow collector well nearer to riverbank helps RBF to avoid problem with iron and manganese. Thus, the RBF site that can supply huge water capacity is 25 MLD.

#### **3.** *Escherichia coli* **in riverbank filtration**

The abstracted water from RBF is very clear which has less contaminants than river water. According to previous study from other RBF sites, the contaminants that are below drinking water standard are turbidity, color, pH, TDS, chloride, ammonia, COD, BOD5, sulfate, iron, manganese, total coliform, and *E. coli*. RBF sites show great anthropogenic activity with the absence of total coliform and *E. coli* because the schmutzdecke (biofilm) layer exists at the bottom of the streamline [16] which can reduce the disinfection treatment. According to data obtained from the monitoring wells, the shallow geology of the RBF area is related to the alluvial deposition at the bottom of the streamline by the river which usually consists of upper fine, medium, and lower fine sand layers [17]. The quality of the ambient groundwater of the previous RBF sites at Louisville also shows that distance and location of the RBF wells from river are the key parameters of the RBF performance. If the RBF wells are very close to the river, then the problems of *E. coli* will be detected [18]. The existence of these enteropathogenic bacteria in abstracted well can be high in the range of 1–140 MPN/100 mL, respectively, as in **Table 2**.

Several of *E. coli* infection issues related to groundwater as drinking water were detected [19, 20] which the source of the infection was positively identified


**89**

system pipeline.

*Application of Artificial Barrier as Mitigation of* E. coli *Which Pass through Riverbank Filtration*

This study is focusing mainly on *E. coli* removal from groundwater. Typically the amount of *E. coli* depends on the aquifer types, distance of abstracted well to river, and climates. The removal of these parameters is crucial to ensure the treated groundwater can safely deliver to water treatment plant or directly distribute to consumer. *E. coli* is a Gram-negative, facultative anaerobic bacterium that belongs to the family of *Enterobacteriaceae*. *E. coli* is recognized as the most important parameter of fecal contaminants by microbiology and public health experts [26]. Depending on environmental conditions, *E. coli* can survive for 4–12 weeks [27]. There are various factors affecting the survival of *E. coli* in environment such as protozoa, antagonists, temperature, light, soil, pH, toxic substances, and oxygen [28]. The survival periods of *E. coli* in various surroundings were reported: in the groundwater at 10°C, recharged well and river water at 9–16°C, *E. coli* survived for 100 days, 63 days, and 55 days, respectively [29, 30]. Due to its strong relevance with the fecal contamination and relatively easy quantification methods, *E. coli* has been employed in a wide range of investigation including water treatment [31–33]. In natural conditions at RBF sites, water percolates through the organic soil where dissolved oxygen (DO) is consumed by the decomposition of organic matter and microbes in the soil. The decomposition process reduces the pH due to microbial action. When groundwater is pumped up to the surface, it gets into contact with air (O2) which enters the solutions and starts the oxidation process that releases

as a contaminated well or runoff from cow manure after torrential rain was thought to have been responsible for contamination [21]. As a safety precaution against *E. coli* infection in the body, the WHO fixed a 0.0 MPN/100 ml of *E. coli*

carbon dioxide (CO2) from the groundwater to the atmosphere.

The reason for choosing *E. coli* as the main parameter is because it is a model for waterborne bacteria and reduces chemical usage in posttreatment. The *Escherichia coli* which is easily called as *E. coli* is a group of bacteria that are commonly found in food and water. Most of the *E. coli* is harmless, but some can cause sickness to human. These bacteria will lead to stomach and intestinal problems such as diarrhea and vomiting. The disease-causing *E. coli* strains live in the intestinal tracts of animals that ruminate, such as cows, deer, and goats. Bacteria early pretreatment seems important since it avoids to stimulate the bacterial growth in distribution

**4. The possibility of** *Escherichia coli* **infection in riverbank filtration**

The site was located at coordinates 5° 07′38.61" N and 100° 35'44.24", Lubok Buntar, Kedah. The examined site was influenced by the water from the Kerian River which was also influenced by the discharge of the wastewater from palm oil, mining industry, and poultry farming area at Sungai Mahang (upstream). The river water and borehole water samples were taken for laboratory (characteristics) test. **Figure 2** shows concentration plots of *E. coli* against height of water in tube well. It can be observed that the increase of height of water in tube well was caused by *E. coli* existence. The existing of *E. coli* was changed from absent to <200 MPN.

The depth of borehole was 30 m signifying that this borehole was under uncon-

fined aquifer. The unconfined aquifer is recharged more rapidly when raining and groundwater hydraulic conductivity at maximum due to infiltration and runoff [34]. The increase of solute concentration during rainy season due to the groundwater flow exceeded the permeability of alluvial soil. Groundwater flow was maximized when raining which creates pressure to the alluvial soil. This leads small particle to flow together into abstraction well which in turn increases contaminant

*DOI: http://dx.doi.org/10.5772/intechopen.86079*

for drinking water standard.

**Table 2.** *E. coli concentration during treated with RBF.* *Application of Artificial Barrier as Mitigation of* E. coli *Which Pass through Riverbank Filtration DOI: http://dx.doi.org/10.5772/intechopen.86079*

as a contaminated well or runoff from cow manure after torrential rain was thought to have been responsible for contamination [21]. As a safety precaution against *E. coli* infection in the body, the WHO fixed a 0.0 MPN/100 ml of *E. coli* for drinking water standard.

This study is focusing mainly on *E. coli* removal from groundwater. Typically the amount of *E. coli* depends on the aquifer types, distance of abstracted well to river, and climates. The removal of these parameters is crucial to ensure the treated groundwater can safely deliver to water treatment plant or directly distribute to consumer. *E. coli* is a Gram-negative, facultative anaerobic bacterium that belongs to the family of *Enterobacteriaceae*. *E. coli* is recognized as the most important parameter of fecal contaminants by microbiology and public health experts [26]. Depending on environmental conditions, *E. coli* can survive for 4–12 weeks [27]. There are various factors affecting the survival of *E. coli* in environment such as protozoa, antagonists, temperature, light, soil, pH, toxic substances, and oxygen [28]. The survival periods of *E. coli* in various surroundings were reported: in the groundwater at 10°C, recharged well and river water at 9–16°C, *E. coli* survived for 100 days, 63 days, and 55 days, respectively [29, 30]. Due to its strong relevance with the fecal contamination and relatively easy quantification methods, *E. coli* has been employed in a wide range of investigation including water treatment [31–33].

In natural conditions at RBF sites, water percolates through the organic soil where dissolved oxygen (DO) is consumed by the decomposition of organic matter and microbes in the soil. The decomposition process reduces the pH due to microbial action. When groundwater is pumped up to the surface, it gets into contact with air (O2) which enters the solutions and starts the oxidation process that releases carbon dioxide (CO2) from the groundwater to the atmosphere.

The reason for choosing *E. coli* as the main parameter is because it is a model for waterborne bacteria and reduces chemical usage in posttreatment. The *Escherichia coli* which is easily called as *E. coli* is a group of bacteria that are commonly found in food and water. Most of the *E. coli* is harmless, but some can cause sickness to human. These bacteria will lead to stomach and intestinal problems such as diarrhea and vomiting. The disease-causing *E. coli* strains live in the intestinal tracts of animals that ruminate, such as cows, deer, and goats. Bacteria early pretreatment seems important since it avoids to stimulate the bacterial growth in distribution system pipeline.

#### **4. The possibility of** *Escherichia coli* **infection in riverbank filtration**

The site was located at coordinates 5° 07′38.61" N and 100° 35'44.24", Lubok Buntar, Kedah. The examined site was influenced by the water from the Kerian River which was also influenced by the discharge of the wastewater from palm oil, mining industry, and poultry farming area at Sungai Mahang (upstream). The river water and borehole water samples were taken for laboratory (characteristics) test. **Figure 2** shows concentration plots of *E. coli* against height of water in tube well. It can be observed that the increase of height of water in tube well was caused by *E. coli* existence. The existing of *E. coli* was changed from absent to <200 MPN.

The depth of borehole was 30 m signifying that this borehole was under unconfined aquifer. The unconfined aquifer is recharged more rapidly when raining and groundwater hydraulic conductivity at maximum due to infiltration and runoff [34]. The increase of solute concentration during rainy season due to the groundwater flow exceeded the permeability of alluvial soil. Groundwater flow was maximized when raining which creates pressure to the alluvial soil. This leads small particle to flow together into abstraction well which in turn increases contaminant

*New Insight into* Brucella *Infection and Foodborne Diseases*

that can supply huge water capacity is 25 MLD.

**3.** *Escherichia coli* **in riverbank filtration**

cal nature and its relation.

resources in a sustainable manner, the characterization and understanding of the natural evolution of groundwater chemistry are crucial to elucidate their geochemi-

The collector well can be far from the river if the soil type is sand and gravel such as RBF at Yellow River, China. The combination of vertical and horizontal collector well can maximize the water capacity such as RBF at Elbe River, Germany. However, clayey alluvial soil will limit the water capacity as RBF site at Lek River, Netherlands, shows the water capacity is only 0.01 MLD, compared to clayey alluvial soil at Nakdong River, Korea, which can be abstracted to 10 MLD water capacity. This shows clayey alluvial soil type needs deeper built collector well near the riverbank. The nearer to riverbank, the more water capacity can be abstracted than collector well at Nakdong River, Korea, which is only 10 MLD with 150 m distance from river, and collector well at Nile River, Egypt, with 22 MLD. Some sites do not contain gravelly sand alluvial soil type but can apply RBF such as Kali River, India. The highly pollutant river demands to use RBF methods; however, it only can abstract 0.8 MLD water capacity because the transmissivity of brownish red silty loam alluvial soil is low. Sites with clayey alluvial soil can apply limestone to increase the transmissivity of water such RBF sites at Ohio River, Kentucky, and Great Miami River, USA. Malaysia RBF sites at Sungai Semerak contain gravelly sand and shallow vertical well collector type. The shallow collector well nearer to riverbank helps RBF to avoid problem with iron and manganese. Thus, the RBF site

The abstracted water from RBF is very clear which has less contaminants than river water. According to previous study from other RBF sites, the contaminants that are below drinking water standard are turbidity, color, pH, TDS, chloride, ammonia, COD, BOD5, sulfate, iron, manganese, total coliform, and *E. coli*. RBF sites show great anthropogenic activity with the absence of total coliform and *E. coli* because the schmutzdecke (biofilm) layer exists at the bottom of the streamline [16] which can reduce the disinfection treatment. According to data obtained from the monitoring wells, the shallow geology of the RBF area is related to the alluvial deposition at the bottom of the streamline by the river which usually consists of upper fine, medium, and lower fine sand layers [17]. The quality of the ambient groundwater of the previous RBF sites at Louisville also shows that distance and location of the RBF wells from river are the key parameters of the RBF performance. If the RBF wells are very close to the river, then the problems of *E. coli* will be detected [18]. The existence of these enteropathogenic bacteria in abstracted well can be high in the range of 1–140 MPN/100 mL, respectively, as in **Table 2**.

Several of *E. coli* infection issues related to groundwater as drinking water were detected [19, 20] which the source of the infection was positively identified

**88**

**Table 2.**

*E. coli concentration during treated with RBF.*

**Figure 2.** *The monitoring of E. coli concentration and height of borehole water for duration 2015–2017.*

concentrations in abstraction water. For that reason, the application of artificial barrier seemed beneficial since it will increase the permeability of aquifer near the river avoiding small particles to flow together to abstraction well during rainy season. Besides raining, *E. coli* can penetrate into abstracted well due to pollution in streamline, abstracted well is near the riverbank, and sources of pollution such as poultry field and sanitary tank are close to abstracted well.

The experiment shows that the application of artificial barrier as RBF water purification method seems important to avoid the possibility of *E. coli* infection. Smith et al. [35] and Uhlmann et al. [36] previously identified exposure to drinking water from private underground water supply as a significant risk factor in human pathogen infections in the UK and Canada, respectively. In addition, O'Sullivan et al. [37] and Garvey et al. [38] have proposed that increases in *E. coli* infection in Ireland may be associated with water consumption from untreated water wells in rural areas, particularly following periods of excessive rainfall.

#### **5. Artificial barrier for riverbank filtration**

#### **5.1 Methodology**

The fixed-bed flow studies were carried out to evaluate their ability to remove *E. coli* during filtration process. The column was made from Perspex glass with inner diameter 8.5 cm. **Figure 3** shows a schematic diagram of the column setup used in this study. The pretreated media were filled in the column. To avoid channeling, the river water was pumped upward through the column at flow rate 50 mL/min. The flow rate was controlled by a peristaltic pump.

The water samples used in the column were taken from the Kerian River at coordinates 5° 07′38.61" N and 100° 35'44.24" E. The sand, GAC, and zeolite were oven dried for 24 hours at 105°C. Before placing the sand, GAC, and zeolite in the column, the column was washed with a solution of 3% acid nitric. The removal of *E. coli* in column test was observed in close exposure to light. This is due to the real condition in the aquifer which is close to sunlight exposure.

The *E. coli* was measured according to Method 9223B. The sample was transferred into the sterile vessel, and the water sample bottle is vigorously shaked 25 times within 7 seconds. The interval between shaking and measuring the test portion does not exceed 3 minutes. Aseptically the lid was removed, and the sample volume was adjusted to the calibrated 100 ml line of the sample container. Aseptically one packet of Colilert reagent was added to the 100 ml test bottle.

**91**

**Figure 3.**

**5.2 Result and discussion**

*Application of Artificial Barrier as Mitigation of* E. coli *Which Pass through Riverbank Filtration*

Aseptic technique refers to a procedure that is performed under sterile conditions. The bottle was recapped and shaked until reagent was mostly dissolved. One hand was used to hold open the Quanti-Tray 2000. Well side was facing the palm of the hand. The upper part of the tray was squeezed so it bent toward the palm and gently pulled the foil tab to open the tray. Avoid touching inside of the tray or foil tab. The 100-ml sample was poured into the tray, and small wells are tapped two to three times to release air bubbles. The tray was placed with the sample into rubber insert so that the wells sat within the cutouts and rubber insert slided with tray into the sealer. The Quanti-Tray once sealed was incubated for 24 hours at 35 +/− 0.5°C. After 24 hours, the fluorescence light under UV light

The measured *E. coli* using IDEXX was also validated with modified mTEC agar plates. Modified mTEC agar plates are prepaid powder by Arachem, BCBS2082V number. The powder was suspended in 1000 mL of distilled water for 45.6 g. The suspended powder was autoclaved and sterilized at 15 lbs. pressure (121°C) for 15 minutes. After that, the suspended powder was cooled to 45–50°C and poured into sterile petri plates. The filtered sample is placed at the top of agar and incubated at 35°C for 2 hours followed by incubation at 44.5°C for 22 hours. The modified mTEC agar contains selective and differential agents. Sodium lauryl sulfate and sodium desoxycholate are selective agents that inhibit Gram + cocci and endospore-forming bacteria. The modified mTEC agar contains the differential agent, 5-bromo-6-chloro-3-indolyl-β-D-glucuronide, which is catabolized to glucuronidase. Unlike the original mTEC method, the modified mTEC does not require the transfer of the membrane filter to another substrate. The positive colony was in magenta color. The analysis on surface morphology of the raw material was carried out using scanning electron microscope (Leo Supra 50 VP Field Emission, UK).

In this study, 15 mixture components that are represented by soil, GAC, and zeolite bed height (in real site of RBF equal to distance of abstracted well water to river) were chosen for the optimization studies since they influenced the presence of *E. coli* in RBF abstracted water as well as volume of abstracted water. In addition, since the absence of *E. coli* and volume of abstracted water was concomitant, the experiments were done using high flow rate. This study determined the optimum

*DOI: http://dx.doi.org/10.5772/intechopen.86079*

*Laboratory fixed bed column experimental setup.*

was counted which indicated as positive *E. coli*.

*Application of Artificial Barrier as Mitigation of* E. coli *Which Pass through Riverbank Filtration DOI: http://dx.doi.org/10.5772/intechopen.86079*

**Figure 3.** *Laboratory fixed bed column experimental setup.*

*New Insight into* Brucella *Infection and Foodborne Diseases*

concentrations in abstraction water. For that reason, the application of artificial barrier seemed beneficial since it will increase the permeability of aquifer near the river avoiding small particles to flow together to abstraction well during rainy season. Besides raining, *E. coli* can penetrate into abstracted well due to pollution in streamline, abstracted well is near the riverbank, and sources of pollution such as

*The monitoring of E. coli concentration and height of borehole water for duration 2015–2017.*

The experiment shows that the application of artificial barrier as RBF water purification method seems important to avoid the possibility of *E. coli* infection. Smith et al. [35] and Uhlmann et al. [36] previously identified exposure to drinking water from private underground water supply as a significant risk factor in human pathogen infections in the UK and Canada, respectively. In addition, O'Sullivan et al. [37] and Garvey et al. [38] have proposed that increases in *E. coli* infection in Ireland may be associated with water consumption from untreated water wells in

The fixed-bed flow studies were carried out to evaluate their ability to remove *E. coli* during filtration process. The column was made from Perspex glass with inner diameter 8.5 cm. **Figure 3** shows a schematic diagram of the column setup used in this study. The pretreated media were filled in the column. To avoid channeling, the river water was pumped upward through the column at flow rate

The water samples used in the column were taken from the Kerian River at coordinates 5° 07′38.61" N and 100° 35'44.24" E. The sand, GAC, and zeolite were oven dried for 24 hours at 105°C. Before placing the sand, GAC, and zeolite in the column, the column was washed with a solution of 3% acid nitric. The removal of *E. coli* in column test was observed in close exposure to light. This is due to the real

The *E. coli* was measured according to Method 9223B. The sample was transferred into the sterile vessel, and the water sample bottle is vigorously shaked 25 times within 7 seconds. The interval between shaking and measuring the test portion does not exceed 3 minutes. Aseptically the lid was removed, and the sample volume was adjusted to the calibrated 100 ml line of the sample container. Aseptically one packet of Colilert reagent was added to the 100 ml test bottle.

poultry field and sanitary tank are close to abstracted well.

rural areas, particularly following periods of excessive rainfall.

50 mL/min. The flow rate was controlled by a peristaltic pump.

condition in the aquifer which is close to sunlight exposure.

**5. Artificial barrier for riverbank filtration**

**5.1 Methodology**

**Figure 2.**

**90**

Aseptic technique refers to a procedure that is performed under sterile conditions. The bottle was recapped and shaked until reagent was mostly dissolved. One hand was used to hold open the Quanti-Tray 2000. Well side was facing the palm of the hand. The upper part of the tray was squeezed so it bent toward the palm and gently pulled the foil tab to open the tray. Avoid touching inside of the tray or foil tab. The 100-ml sample was poured into the tray, and small wells are tapped two to three times to release air bubbles. The tray was placed with the sample into rubber insert so that the wells sat within the cutouts and rubber insert slided with tray into the sealer. The Quanti-Tray once sealed was incubated for 24 hours at 35 +/− 0.5°C. After 24 hours, the fluorescence light under UV light was counted which indicated as positive *E. coli*.

The measured *E. coli* using IDEXX was also validated with modified mTEC agar plates. Modified mTEC agar plates are prepaid powder by Arachem, BCBS2082V number. The powder was suspended in 1000 mL of distilled water for 45.6 g. The suspended powder was autoclaved and sterilized at 15 lbs. pressure (121°C) for 15 minutes. After that, the suspended powder was cooled to 45–50°C and poured into sterile petri plates. The filtered sample is placed at the top of agar and incubated at 35°C for 2 hours followed by incubation at 44.5°C for 22 hours. The modified mTEC agar contains selective and differential agents. Sodium lauryl sulfate and sodium desoxycholate are selective agents that inhibit Gram + cocci and endospore-forming bacteria. The modified mTEC agar contains the differential agent, 5-bromo-6-chloro-3-indolyl-β-D-glucuronide, which is catabolized to glucuronidase. Unlike the original mTEC method, the modified mTEC does not require the transfer of the membrane filter to another substrate. The positive colony was in magenta color. The analysis on surface morphology of the raw material was carried out using scanning electron microscope (Leo Supra 50 VP Field Emission, UK).

#### **5.2 Result and discussion**

In this study, 15 mixture components that are represented by soil, GAC, and zeolite bed height (in real site of RBF equal to distance of abstracted well water to river) were chosen for the optimization studies since they influenced the presence of *E. coli* in RBF abstracted water as well as volume of abstracted water. In addition, since the absence of *E. coli* and volume of abstracted water was concomitant, the experiments were done using high flow rate. This study determined the optimum

ratio for combination of soil with GAC and zeolite that would support and improve the capability of *E. coli* removal compared to alluvial soil in RBF with a constant 50 mL/min flow rate. The removal of *E. coli* was less than 85% for soil with 81 and 82% removal as in **Figure 4**. In comparison with 70% soil combined with 15% GAC and 15% zeolite, the removal of *E. coli* was increased to 89%. Meanwhile, with 50% soil combined with 15% zeolite, the removal of *E. coli* was increased higher up to 90%. However, the combination of GAC and zeolite showed the lowest removal of *E. coli* compared to soil only by less than 50%. The honeycomb structure in GAC created the strongest biofilm layer which assisted the trap of microbe during high flow rate. Effective microbial adhesion and immobilization are essential for biofilm activities [39].

The GAC morphology (**Figure 5(a)**) showed that the surface structure and pore were well developed similar to honeycomb structure. The surface morphology of the GAC was also comparable to the analysis done by Hameed and Ahmad [40]. However, the adsorption of *E. coli* to GAC surfaces occurred on the outside of the

**Figure 4.** *Laboratory fixed bed column experimental setup.*

**Figure 5.** *The morphology of GAC for (a) before and (b) after adsorption with images of E. coli cells attach to surface.*

**93**

adsorption method.

*Application of Artificial Barrier as Mitigation of* E. coli *Which Pass through Riverbank Filtration*

pore (honeycomb structure) as depicted in **Figure 5(b)**. *E. coli* adhesion to media surface is the initial step to schmutzdecke (biofilm) layer formation which later will create sticky surface and help in more adsorption of *E. coli*. The honeycomb structure provides strong physical confinement for the bacterial cells' adhesion and

The enumeration of *E. coli* throughout optimization using Colilert. However,

(MPN acceptable values +20%). This means the results measured using mTEC agar are quite close to the mean value of MPN and in 95% of confidence limit for MPN

Until now, the health effects endemic to human for groundwater supply in Malaysia are not investigated. Casemore [42] notes that the occurrence of sporadic or pseudo-sporadic infection is particularly important in the context of groundwater-related infection. This is because the groundwater is often seen as pure quality and therefore not examined as potential sources of enteric infections that occur,

The performance of RBF depended on alluvial soil particles' size distribution,

soil gradation, and soil structure. From the monitoring, results show that the possibility of *E. coli* infection may happen. Thus, the purification method using artificial recharge seems important. In this study, the adsorption of *E. coli* by soil becomes higher in combination with GAC and zeolite. It was the honeycomb morphology of GAC that assists the attachment of *E. coli*. The schmutzdecke (biofilm) layer formation helps to enhance the *E. coli* adhesion to media surface which later will create sticky surface and help more adsorption of *E. coli*. The zeolite has higher CaO than other adsorbents; the attachment of *E. coli* in zeolite is based on mineral content. The aquifer is advisable but should not have too high or too low permeability for RBF because majority of removal mechanism was assisted by medium filter media permeability. The chemical usage technique in controlling *E. coli* in water treatment may not be a suitable method, whereby in a certain time, *E. coli* may resist to that chemical. Thus, from that reasoning, it's better to use the

of 0.92 which was acceptable

due to sensitivity and verification of the result, the mTEC agar enumeration method was used to increase the reliability of the result. **Figure 6** shows that the

*DOI: http://dx.doi.org/10.5772/intechopen.86079*

subsequently resists biofilm formation [41].

measurement.

**Figure 6.**

**6. Conclusion**

thus leading to important effect.

*E. coli* enumeration can be trusted due to linear R2

*Validation of measurement E. coli with using colilert and mTEC agar.*

*Application of Artificial Barrier as Mitigation of* E. coli *Which Pass through Riverbank Filtration DOI: http://dx.doi.org/10.5772/intechopen.86079*

**Figure 6.** *Validation of measurement E. coli with using colilert and mTEC agar.*

pore (honeycomb structure) as depicted in **Figure 5(b)**. *E. coli* adhesion to media surface is the initial step to schmutzdecke (biofilm) layer formation which later will create sticky surface and help in more adsorption of *E. coli*. The honeycomb structure provides strong physical confinement for the bacterial cells' adhesion and subsequently resists biofilm formation [41].

The enumeration of *E. coli* throughout optimization using Colilert. However, due to sensitivity and verification of the result, the mTEC agar enumeration method was used to increase the reliability of the result. **Figure 6** shows that the *E. coli* enumeration can be trusted due to linear R2 of 0.92 which was acceptable (MPN acceptable values +20%). This means the results measured using mTEC agar are quite close to the mean value of MPN and in 95% of confidence limit for MPN measurement.

Until now, the health effects endemic to human for groundwater supply in Malaysia are not investigated. Casemore [42] notes that the occurrence of sporadic or pseudo-sporadic infection is particularly important in the context of groundwater-related infection. This is because the groundwater is often seen as pure quality and therefore not examined as potential sources of enteric infections that occur, thus leading to important effect.

#### **6. Conclusion**

*New Insight into* Brucella *Infection and Foodborne Diseases*

activities [39].

ratio for combination of soil with GAC and zeolite that would support and improve the capability of *E. coli* removal compared to alluvial soil in RBF with a constant 50 mL/min flow rate. The removal of *E. coli* was less than 85% for soil with 81 and 82% removal as in **Figure 4**. In comparison with 70% soil combined with 15% GAC and 15% zeolite, the removal of *E. coli* was increased to 89%. Meanwhile, with 50% soil combined with 15% zeolite, the removal of *E. coli* was increased higher up to 90%. However, the combination of GAC and zeolite showed the lowest removal of *E. coli* compared to soil only by less than 50%. The honeycomb structure in GAC created the strongest biofilm layer which assisted the trap of microbe during high flow rate. Effective microbial adhesion and immobilization are essential for biofilm

The GAC morphology (**Figure 5(a)**) showed that the surface structure and pore were well developed similar to honeycomb structure. The surface morphology of the GAC was also comparable to the analysis done by Hameed and Ahmad [40]. However, the adsorption of *E. coli* to GAC surfaces occurred on the outside of the

*The morphology of GAC for (a) before and (b) after adsorption with images of E. coli cells attach to surface.*

**92**

**Figure 5.**

**Figure 4.**

*Laboratory fixed bed column experimental setup.*

The performance of RBF depended on alluvial soil particles' size distribution, soil gradation, and soil structure. From the monitoring, results show that the possibility of *E. coli* infection may happen. Thus, the purification method using artificial recharge seems important. In this study, the adsorption of *E. coli* by soil becomes higher in combination with GAC and zeolite. It was the honeycomb morphology of GAC that assists the attachment of *E. coli*. The schmutzdecke (biofilm) layer formation helps to enhance the *E. coli* adhesion to media surface which later will create sticky surface and help more adsorption of *E. coli*. The zeolite has higher CaO than other adsorbents; the attachment of *E. coli* in zeolite is based on mineral content. The aquifer is advisable but should not have too high or too low permeability for RBF because majority of removal mechanism was assisted by medium filter media permeability. The chemical usage technique in controlling *E. coli* in water treatment may not be a suitable method, whereby in a certain time, *E. coli* may resist to that chemical. Thus, from that reasoning, it's better to use the adsorption method.

### **Acknowledgements**

The authors would like to acknowledge the Ministry of Education Malaysia for providing LRGS Grant on Water Security entitled Protection of Drinking Water: Source Abstraction and Treatment (203/PKT/6720006).

#### **Author details**

Nur Aziemah Abd Rashid\* and Ismail Abustan Universiti Sains Malaysia, Nibong Tebal, Malaysia

\*Address all correspondence to: nuraziemahabdrashid@gmail.com

© 2020 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

**95**

*Application of Artificial Barrier as Mitigation of* E. coli *Which Pass through Riverbank Filtration*

Groundwater. The Netherland: Balkema,

[9] Chew CM, David Ng KM, Richard Ooi HH, Ismail WMZW. Malaysia's largest river bank filtration and ultrafiltration systems for municipal drinking water production. Journal of Water Practice and Technology. 2015;**10**:59-65. DOI: 10.2166/

[10] Nodler K, Hillebrand O, Idzik K, Strathmann M, Schiperski F, Zirlewagen J, et al. Occurrence and fate of the angiotensin II receptor antagonist transformation product valsartan acid in water cycle—A comparative study with selected β-blockers and the persistent anthropogenic wastewater indicators carbamazepine and

acesulfame. Journal of Water Research. 2013;**47**:6650-6659. DOI: 10.1016/j.

[11] Singh P, Kumar P, Mehrotra I, Grischek T. Impact of riverbank filtration on treatment of polluted river water. Journal of Environmental Management. 2009;**91**:1055-1062. DOI:

10.1016/j.jenvman.2009.11.013

[12] Shamrukh M, Ahmed AW. Water pollution and riverbank filtration for water supply along river Nile, Egypt. Riverbank Filtration for Water Security in Desert Countries. 2011;**233**:1824-1835. DOI: 10.1007/978-94-007-0026-0\_2

[13] Sahoo GB, Ray C, Wanf JZ, Hubbs SA, Song R, Jasperse J, et al. Use of artificial neural networks to evaluate the effectiveness of riverbank filtration. Water Research. 2005;**39**:2505-2516. DOI: 10.1016/j.watres.2005.04.020

[14] Doussan C, Poitevin G, Ledoux E, Detay M. River bank filtration: modeling of the changes in water chemistry with emphasis on nitrogen species. Journal of Contaminant

Rotterdam; 1998

wpt.2015.008

watres.2013.08.034

*DOI: http://dx.doi.org/10.5772/intechopen.86079*

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[4] Lewandowski JA, Putschew A,Schwesig D, Neumann C, Radke M. Fate of organic micropollutants in the hyporheic zone of a eutrophic

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scitotenv.2011.01.028

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*Application of Artificial Barrier as Mitigation of* E. coli *Which Pass through Riverbank Filtration DOI: http://dx.doi.org/10.5772/intechopen.86079*

#### **References**

*New Insight into* Brucella *Infection and Foodborne Diseases*

Source Abstraction and Treatment (203/PKT/6720006).

The authors would like to acknowledge the Ministry of Education Malaysia for providing LRGS Grant on Water Security entitled Protection of Drinking Water:

**Acknowledgements**

**94**

**Author details**

provided the original work is properly cited.

Nur Aziemah Abd Rashid\* and Ismail Abustan Universiti Sains Malaysia, Nibong Tebal, Malaysia

© 2020 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium,

\*Address all correspondence to: nuraziemahabdrashid@gmail.com

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[12] Shamrukh M, Ahmed AW. Water pollution and riverbank filtration for water supply along river Nile, Egypt. Riverbank Filtration for Water Security in Desert Countries. 2011;**233**:1824-1835. DOI: 10.1007/978-94-007-0026-0\_2

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*New Insight into* Brucella *Infection and Foodborne Diseases*

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[23] Hu B, Teng Y, Zhai Y, Zuo R, Li J, Chen H. Riverbank filtration in China: A review and perspective. Journal of Hydrology. 2016;**541**:914-927. DOI: 10.1016/j.jhydrol.2016.08.004

[24] Abdalla F, Shamrukh M. Riverbank filtration: Developing countries choice for water supply treatment, Egypt case. In: The 1st IWA Malaysia Young Water Professionals Conference (IWAYP2010), 1-4 March. Kuala Lumpur, Malaysia;

[25] Enrico H, Pieter JS, Janek G, Harrie T, Gudrun M. The fate of organic micropollutants during long-term/ long-distance river bank filtration. Journal of Science of the Total

Environment. 2016;545-546, 629-640. DOI: 10.1016/j.scitotenv.2015.12.057

[26] Bartram J, Cotruvo J, Exner M, Fricker C, Glasmacher A. Heterotrophic plate count measurement in drinking water safety management—Report of an Expert Meeting Geneva. International

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Applied Microbiology. 2000

[28] Foppen JWA, Schijven JF.

conditions. Water Research.

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2010. p. 2010

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[15] Hiscock KM, Grischeck T.

2002;**266**:139-144. DOI: 10.1016/

S0022-1694(02)00158-0

10.2166/aqua.2012.097

jhydrol.2009.07.030

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et al. Escherichia coli O157:H7 in drinking water from private water supplies in the Netherlands. Water Research. 2005;**39**(18):4485-4493

et al. Massive microbiological

with a waterborne outbreak in Lake Erie, South Bass Island, Ohio. Environmental Health Perspectives.

2007;**115**(6):856-864

1999;**319**:873

10.1007/0-306-48154-5\_8

Technology. 2002;**43**(3):117-145. DOI:

[19] Schets FM, During M, Italiaander R,

[20] Fong TT, Mansfield LS, Wilson DL,

groundwater contamination associated

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Attenuation of groundwater pollution by bank filtration. Journal of Hydrology.

[16] Freitas DA, Cabral JJSP, Paiva ALR, Molica RJR. Application of bank filtration technology for water quality improvement in a warm climate: A case study at Beberibe River in Brazil. Journal of Water Supply: Research and Technology. 2012;**61**:319-330. DOI:

[17] Lee JH, Hamm SY, Cheong JY, Kim HS, Ko EJ, Lee KS, et al. Characterizing riverbank-filtered water and river water qualities at a site in the lower Nakdong River basin, Republic of Korea. Journal of Hydrology. 2009;**376**:209-220. DOI: 10.1016/j.

**96**

[29] Goldshmi MG, Pantsili VD. Device for carving out spacing nets as series of circles. Zavodskaya Laboratoriya. 1972;(1):117

[30] Grabow WOK, Prozesky OW, Burger JS. Behavior in a river and dam of coliform bacteria with transferable or non-transferable drug-resistance. Water Research. 1975;**9**(9):777-782

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[32] Lutterodt G, Foppen JWA, Maksoud A, Uhlenbrook S. Transport of Escherichia coli in 25 m quartz sand column. Journal of Contaminant Hydrology. 2011;**119**(1-4):80-88

[33] Sinton LW, Mackenzie ML, Karki N, Dann RL, Pang L, Close ME. Transport of Escherichia coli and F-RNA bacteriophages in a 5-M column of saturated heterogenous gravel. Water, Air, & Soil Pollution. 2012;**223**:2347-2360. DOI: 10.1007/s11270-011-1029-9

[34] Zhang G, Feng G, Li X, Xie C, Pi X. Flood effect on groundwater recharge on a typical silt loam soil. Water. 2017;**9**:1-15. DOI: 10.3390/w9070523

[35] Smith A, Reacher M, Smerdon W, et al. Outbreaks of waterborne infectious intestinal disease in England and Wales, 1992-2003. Epidemiology and Infection. 2006;**134**(6):1141-1149

[36] Uhlmann S, Galanis E, Takaro T, et al. Where's the pump? Associating sporadic enteric disease with drinking water using a geographic information system, in British Columbia, Canada, 1996-2005. Journal of Water and Health. 2009;**07**(4):692-698

[37] O'Sullivan MB, Garvey P, O'Riordan M, et al. Increase in VTEC cases in the south of Ireland: Link to private wells? Euro Surveillance. 2008;**13**(39):1-2

[38] Garvey P, McKeown P, Carroll A, et al. Epidemiology of verotoxigenic *E. coli* in Ireland, 2008. Epi Insight. 2009;**10**(9):1-10

[39] Zhu IX, Brian JB. Conventional media filtration with biological activities. In: Walid E, Rezaul K, editors. Water Treatment. Chowdhury: IntechOpen; 2013. DOI: org/10.5772/50481

[40] Hameed BH, Ahmad AA. Batch adsorption of methylene blue from aqueous solution by garlic peel, an agricultural waste biomass. Journal of Hazardous Materials. 2009;**164**(2-3):870-875. DOI: 10.1016/j. jhazmat.2008.08.084

[41] Meng Y, Yonghui D, Xiang G, Yang L. Control of bacterial adhesion and growth on honeycomb-like patterned surfaces. Colloids and Surfaces B. 2015;**135**(2015):549-555

[42] Casemore D. Towards a US national estimate of the risk of endemic waterborne disease—Seroepidemiologic studies. Journal of Water and Health. 2006;**4**(Suppl. 2):121-164

**99**

**Chapter 8**

**1. Introduction**

**2.** *E. coli*

Scientific classification Domain: Prokaryota Kingdom: Bacteria Phylum: Proteobacteria Class: Gamma Proteobacteria Order: Enterobacteriales Family: Enterobacteriaceae

Species: *E. coli*

ferment glucose and lactose.

mon in children and in elderly people [1].

and *Salmonella*

*Maria Teresa Mascellino*

Prologue: *Escherichia coli, Listeria*,

The present book deals with the following microorganisms: *E. coli*, *Salmonella*, and *Listeria*. The first two are Gram-negative bacteria belonging to the group of Enterobacteriaceae with the characteristic of becoming resistant to the most common antibiotics; whereas, the last one is a Gram-positive bacterium belonging to *Corynebacterium*, *Erysipelothrix*, and other Gram-positive microorganisms showing an involvement in pathologies as newborn meningitis and gynecological infection which may interfere with the pregnancy outcome. The peculiarity of all these

The bacteria, in fact, can be found in the gastrointestinal tract (GI) of humans

This bacterium includes a single species (*E. coli*) and is divided into 171 serotypes, aerobic-anaerobic Gram-negative rods with flagella fimbriae, and able to

The most important serotype is *Escherichia coli* O157:H7 or enterohemorrhagic *Escherichia coli* (EHEC), which often leads to enterohemorrhagic diarrhea and is also able to induce hemolytic uremic syndrome (HUS) which is characterized by acute renal failure, hemolytic anemia, and thrombocytopenia that are more com-

Serotype O157-H7 causes numerous outbreaks and sporadic cases of bloody diarrhea. Foodborne pathogenic E. coli contamination, such as that with *E. coli* O157 and O104, is very common even in developed countries. Bacterial contamination may occur from environmental, animal, or human sources and cause foodborne illness [2]. The three main diseases, depending on each particular serotype involved, are

Many different mechanisms of action are reported regarding the virulence of *E. coli.* Although most strains are saprophytic colonizing the large bowel, some types of them are involved in different pathologies such as traveler's and childhood

urinary tract infections, intestinal diseases, and neonatal meningitis [3].

and animals, but they are mainly considered as ubiquitous microorganisms.

bacteria is that they can be transmitted by contaminated food.

#### **Chapter 8**

## Prologue: *Escherichia coli, Listeria*, and *Salmonella*

*Maria Teresa Mascellino*

#### **1. Introduction**

The present book deals with the following microorganisms: *E. coli*, *Salmonella*, and *Listeria*. The first two are Gram-negative bacteria belonging to the group of Enterobacteriaceae with the characteristic of becoming resistant to the most common antibiotics; whereas, the last one is a Gram-positive bacterium belonging to *Corynebacterium*, *Erysipelothrix*, and other Gram-positive microorganisms showing an involvement in pathologies as newborn meningitis and gynecological infection which may interfere with the pregnancy outcome. The peculiarity of all these bacteria is that they can be transmitted by contaminated food.

#### **2.** *E. coli*

Scientific classification Domain: Prokaryota Kingdom: Bacteria Phylum: Proteobacteria Class: Gamma Proteobacteria Order: Enterobacteriales Family: Enterobacteriaceae Species: *E. coli*

The bacteria, in fact, can be found in the gastrointestinal tract (GI) of humans and animals, but they are mainly considered as ubiquitous microorganisms.

This bacterium includes a single species (*E. coli*) and is divided into 171 serotypes, aerobic-anaerobic Gram-negative rods with flagella fimbriae, and able to ferment glucose and lactose.

The most important serotype is *Escherichia coli* O157:H7 or enterohemorrhagic *Escherichia coli* (EHEC), which often leads to enterohemorrhagic diarrhea and is also able to induce hemolytic uremic syndrome (HUS) which is characterized by acute renal failure, hemolytic anemia, and thrombocytopenia that are more common in children and in elderly people [1].

Serotype O157-H7 causes numerous outbreaks and sporadic cases of bloody diarrhea. Foodborne pathogenic E. coli contamination, such as that with *E. coli* O157 and O104, is very common even in developed countries. Bacterial contamination may occur from environmental, animal, or human sources and cause foodborne illness [2].

The three main diseases, depending on each particular serotype involved, are urinary tract infections, intestinal diseases, and neonatal meningitis [3].

Many different mechanisms of action are reported regarding the virulence of *E. coli.* Although most strains are saprophytic colonizing the large bowel, some types of them are involved in different pathologies such as traveler's and childhood diarrhea (ETEC and EPEC also in Mexico and North Africa EAEC), hemorrhagic colitis (EHEH), and a Shiga-like disease (EIEC). As far as this last point is concerned, it is reported that the differentiation between *Shigella* and *E. coli* is quite more complicated when we consider enteroinvasive *E. coli* (EIEC). In fact, EIEC are strains that are similar to *E. coli* but are able to cause dysentery using the same method of invasion as *Shigella*. In fact, in this specific situation, EIEC is more related to *Shigella* than to non-invasive *E. coli* [4]. This strain is among the most common cause of foodborne diseases other than of neurological and renal complications, especially in children.

*Escherichia coli* K1 strains are major causative agents of invasive disease of newborn infants. Colonization of the small intestine following oral administration of K1 bacteria leads rapidly to blood stream infections (BSI). Indeed, these microorganisms are the cause of life-threatening infections that are acquired from the mother at birth thus colonizing the small intestine, from where they invade the blood and central nervous system.

*E. coli* is increasingly present as a MDR (multi-drug resistant) bacterium, in fact its genomic outfit has acquired various antibiotic resistances through the production of ESBL [5] and carbapenemases as well as metallo-beta lactamases (NDM = New Delhi metallo-beta lactamases) making the infections of this bacterium extremely worrying [6] (**Figures 1** and **2**).

#### **3.** *Listeria monocytogenes*

Class: Bacilli Kingdom: Bacteria Family: Listeriaceae Classification: *Listeria*

*Listeria monocytogenes* is a Gram-positive, mobile, rod-shaped bacterium that is ubiquitous in the environment. It can be isolated in soil and wood and decays in the natural environment; however, the principal acquisition of *Listeria* is through the ingestion of contaminated food products. *Listeria* is a foodborne pathogen that contaminates food-processing environments and persists within biofilms in the surroundings. The peculiar characteristic of this microorganism is its ability to grow even in extreme situations, such as under high salt conditions and refrigeration temperatures, maintaining its vitality in various food products [7]. Even though the incidence of listeriosis is lower than other enteric illnesses, infections caused by *L. monocytogenes* are more serious and may lead to hospitalizations and fatalities. These infections mainly affect women and children who acquire the disease

*Morphology of* E. coli*. http://www.lacolonscopia.it/colonscopia/escherichia-coli-come-prevenirlo-e-curarlo/*

**101**

**Figure 3.**

*(Hpt)) [10].*

**Figure 2.**

*Prologue:* Escherichia coli, Listeria, *and* Salmonella *DOI: http://dx.doi.org/10.5772/intechopen.89654*

through vertical transmission from mother to infant during pregnancy or childbirth. Nosocomial infections between children are rare but anyhow they were reported. The most important disease for the newborns is the neonatal meningitis, which shows a high degree of mortality (higher in the developing countries which can reach 40–58% of cases). Listeriosis requires rapid treatment with antibiotics and most drugs suitable for Gram-positive bacteria are effective against *L. monocytogenes*. Generally, the *Listeria* clinical strains are susceptible to the common antibiotics because only a minority results as being resistant to antimicrobial agents. In the same way, several

*Phagocytosis of Listeria. Legenda: (internalins InlA and InlB), phagosome lysis (listeriolysin O (LLO)), phosphatidylinositol-specific phospholipase C (PI-PLC) and phosphatidylcholine ((PC)-PLC), cell-to-cell spread (actin assembly-inducing protein (ActA)), intracellular growth (hexose-6-phosphate transporter* 

*Slide from "New Delhi metallo-beta-lactamase (NDM-1). Facts, controversies, solutions. An update" T.V. Rao* 

*(Powerpoint 2016). https://www.slideshare.net/doctorrao/new-delhi-metallobetalactamse*

#### **Figure 2.**

*New Insight into* Brucella *Infection and Foodborne Diseases*

rium extremely worrying [6] (**Figures 1** and **2**).

central nervous system.

**3.** *Listeria monocytogenes*

Class: Bacilli Kingdom: Bacteria Family: Listeriaceae Classification: *Listeria*

diarrhea (ETEC and EPEC also in Mexico and North Africa EAEC), hemorrhagic colitis (EHEH), and a Shiga-like disease (EIEC). As far as this last point is concerned, it is reported that the differentiation between *Shigella* and *E. coli* is quite more complicated when we consider enteroinvasive *E. coli* (EIEC). In fact, EIEC are strains that are similar to *E. coli* but are able to cause dysentery using the same method of invasion as *Shigella*. In fact, in this specific situation, EIEC is more related to *Shigella* than to non-invasive *E. coli* [4]. This strain is among the most common cause of foodborne diseases other than of neurological and renal complications, especially in children. *Escherichia coli* K1 strains are major causative agents of invasive disease of newborn infants. Colonization of the small intestine following oral administration of K1 bacteria leads rapidly to blood stream infections (BSI). Indeed, these microorganisms are the cause of life-threatening infections that are acquired from the mother at birth thus colonizing the small intestine, from where they invade the blood and

*E. coli* is increasingly present as a MDR (multi-drug resistant) bacterium, in fact its genomic outfit has acquired various antibiotic resistances through the production of ESBL [5] and carbapenemases as well as metallo-beta lactamases (NDM = New Delhi metallo-beta lactamases) making the infections of this bacte-

*Listeria monocytogenes* is a Gram-positive, mobile, rod-shaped bacterium that is ubiquitous in the environment. It can be isolated in soil and wood and decays in the natural environment; however, the principal acquisition of *Listeria* is through the ingestion of contaminated food products. *Listeria* is a foodborne pathogen that contaminates food-processing environments and persists within biofilms in the surroundings. The peculiar characteristic of this microorganism is its ability to grow even in extreme situations, such as under high salt conditions and refrigeration temperatures, maintaining its vitality in various food products [7]. Even though the incidence of

listeriosis is lower than other enteric illnesses, infections caused by

*L. monocytogenes* are more serious and may lead to hospitalizations and fatalities. These infections mainly affect women and children who acquire the disease

*Morphology of* E. coli*. http://www.lacolonscopia.it/colonscopia/escherichia-coli-come-prevenirlo-e-curarlo/*

**100**

**Figure 1.**

*Slide from "New Delhi metallo-beta-lactamase (NDM-1). Facts, controversies, solutions. An update" T.V. Rao (Powerpoint 2016). https://www.slideshare.net/doctorrao/new-delhi-metallobetalactamse*

through vertical transmission from mother to infant during pregnancy or childbirth. Nosocomial infections between children are rare but anyhow they were reported. The most important disease for the newborns is the neonatal meningitis, which shows a high degree of mortality (higher in the developing countries which can reach 40–58% of cases). Listeriosis requires rapid treatment with antibiotics and most drugs suitable for Gram-positive bacteria are effective against *L. monocytogenes*. Generally, the *Listeria* clinical strains are susceptible to the common antibiotics because only a minority results as being resistant to antimicrobial agents. In the same way, several

#### **Figure 3.**

*Phagocytosis of Listeria. Legenda: (internalins InlA and InlB), phagosome lysis (listeriolysin O (LLO)), phosphatidylinositol-specific phospholipase C (PI-PLC) and phosphatidylcholine ((PC)-PLC), cell-to-cell spread (actin assembly-inducing protein (ActA)), intracellular growth (hexose-6-phosphate transporter (Hpt)) [10].*

strains detected from food exhibited resistance to antimicrobials not suitable against listeriosis [8]. Pregnant women can carry *Listeria* asymptomatically in their gastrointestinal tract or vagina and the risk of transmitting this infection to their babies is high. The consequence of listeriosis to human health is a very important issue due to its virulence mainly in children with an underlying immunodeficiency. Symptoms include fever, headache, abdominal pain, diarrhea, vomiting, and convulsions. The complications can be appendicitis and Meckel's diverticulitis [9].

*Listeria* which is saprophyte in the environments such as water, soil, and food, once internalizes into the mammalian host, shows its virulence through the expression of many gene products reported in **Figure 3** [10].

#### **4.** *Salmonella*

Domain: Prokaryota Kingdom: Bacteria Phylum: Proteobacteria Class: Gammaproteobacteria Order: Enterobacteriales Family: Enterobacteriaceae Genre: *Salmonella*

*Salmonella* is the most commonly isolated bacterial agent of foodborne and epidemic infections. It was reported for the first time in 1886, in a case of swine fever by the American doctor Daniel Elmer Salmon.

The genus *Salmonella* is characterized by Gram-negative facultative anaerobic bacilli without spores. They are mobile through peritrichous flagella with the exception of *S. gallinarum* and *S. pullorum*. The serotypes are diversified according to the somatic antigen "O," the flagellar antigen "H" and the surface antigen "Vi." The Vi antigen is exclusively expressed by *S. typhi* and is able to circumvent the innate immune response by repressing flagellin and LPS expression [11]. The "O" antigens are distinguished in the serogroups A, B, C1, C2, D, and E.

*Salmonella* is present in the environment and can be either commensal or pathogen for men and various animals; some serotypes are exclusively pathogen for humans (i.e., *S. typhi* and *S. paratyphi* A and C), others infect both humans and animals such as *S. typhimurium* [12].

In humans, there are two kind of infectious diseases:

1.typhoid and paratyphoid fever [13]

2.minor salmonellosis [14]

*Salmonella* infection is transmitted through fecal route by the ingestion of contaminated food and drink. *Salmonella typhi* is responsible for typhoid fever, and its transmission can occur, especially in developing countries, by water and food infected or with direct contact among people, especially in poor hygienic conditions. The minimum infectious dose can be less than 15–20 cells. Individual sensitivity depends on the patients' age and on the nature of *Salmonella* strains.

In most cases, *Salmonella* infection occurs in mild form and resolves on its own within a few days. In these situations, the advice is not to consider the diarrheal phenomenon, since it is the natural defense mechanism used by the organism to expel germs. Normally, for S*almonella*, it should be enough to adopt a supportive therapy: administration of oral rehydration solutions (which are used to compensate for water and salts lost with vomiting and diarrhea), lactic ferments, and probiotics.

**103**

**Author details**

**Figure 4.**

*rise to healthy carriers [18].*

Maria Teresa Mascellino

Sapienza University of Rome, Italy

provided the original work is properly cited.

\*Address all correspondence to: mariateresa.mascellino@uniroma1.it

© 2020 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium,

*Prologue:* Escherichia coli, Listeria, *and* Salmonella *DOI: http://dx.doi.org/10.5772/intechopen.89654*

3 months and in subjects with chronic-degenerative diseases.

Although salmonellosis is a bacterial infection, the use of antibiotics is not recommended as it could lengthen the persistence time of *Salmonella* in feces or induce antimicrobials resistance [15]. Hospitalization and the use of antibiotics are indicated only in severe cases (with extra-intestinal symptoms), in infants under

In recent times, *Salmonella* has changed its characteristics worldwide, becoming the etiologic agent of many peculiar pathological processes such as cancer development, inflammatory process, and immune-pathogenesis [16, 17] (**Figure 4**).

Salmonella *infection pathogenesis. The ingestion of contaminated food or water begins the infective processes (gastroenteritis or systemic infection) depending on the species of* Salmonella *involved (minor and major Salmonellae). The microorganisms reach the intestinal epithelial cells and migrate to the lamina propria invading the liver from where* Salmonella *reaches the gall bladder and can cause chronic carriage which gives*  *Prologue:* Escherichia coli, Listeria, *and* Salmonella *DOI: http://dx.doi.org/10.5772/intechopen.89654*

Although salmonellosis is a bacterial infection, the use of antibiotics is not recommended as it could lengthen the persistence time of *Salmonella* in feces or induce antimicrobials resistance [15]. Hospitalization and the use of antibiotics are indicated only in severe cases (with extra-intestinal symptoms), in infants under 3 months and in subjects with chronic-degenerative diseases.

In recent times, *Salmonella* has changed its characteristics worldwide, becoming the etiologic agent of many peculiar pathological processes such as cancer development, inflammatory process, and immune-pathogenesis [16, 17] (**Figure 4**).

#### **Figure 4.**

*New Insight into* Brucella *Infection and Foodborne Diseases*

complications can be appendicitis and Meckel's diverticulitis [9].

sion of many gene products reported in **Figure 3** [10].

fever by the American doctor Daniel Elmer Salmon.

are distinguished in the serogroups A, B, C1, C2, D, and E.

In humans, there are two kind of infectious diseases:

animals such as *S. typhimurium* [12].

2.minor salmonellosis [14]

1.typhoid and paratyphoid fever [13]

**4.** *Salmonella*

Domain: Prokaryota Kingdom: Bacteria Phylum: Proteobacteria Class: Gammaproteobacteria Order: Enterobacteriales Family: Enterobacteriaceae

Genre: *Salmonella*

strains detected from food exhibited resistance to antimicrobials not suitable against listeriosis [8]. Pregnant women can carry *Listeria* asymptomatically in their gastrointestinal tract or vagina and the risk of transmitting this infection to their babies is high. The consequence of listeriosis to human health is a very important issue due to its virulence mainly in children with an underlying immunodeficiency. Symptoms include fever, headache, abdominal pain, diarrhea, vomiting, and convulsions. The

*Listeria* which is saprophyte in the environments such as water, soil, and food, once internalizes into the mammalian host, shows its virulence through the expres-

*Salmonella* is the most commonly isolated bacterial agent of foodborne and epidemic infections. It was reported for the first time in 1886, in a case of swine

The genus *Salmonella* is characterized by Gram-negative facultative anaerobic bacilli without spores. They are mobile through peritrichous flagella with the exception of *S. gallinarum* and *S. pullorum*. The serotypes are diversified according to the somatic antigen "O," the flagellar antigen "H" and the surface antigen "Vi." The Vi antigen is exclusively expressed by *S. typhi* and is able to circumvent the innate immune response by repressing flagellin and LPS expression [11]. The "O" antigens

*Salmonella* is present in the environment and can be either commensal or pathogen for men and various animals; some serotypes are exclusively pathogen for humans (i.e., *S. typhi* and *S. paratyphi* A and C), others infect both humans and

*Salmonella* infection is transmitted through fecal route by the ingestion of contaminated food and drink. *Salmonella typhi* is responsible for typhoid fever, and its transmission can occur, especially in developing countries, by water and food infected or with direct contact among people, especially in poor hygienic conditions. The minimum infectious dose can be less than 15–20 cells. Individual sensitivity depends on the patients' age and on the nature of *Salmonella* strains.

In most cases, *Salmonella* infection occurs in mild form and resolves on its own within a few days. In these situations, the advice is not to consider the diarrheal phenomenon, since it is the natural defense mechanism used by the organism to expel germs. Normally, for S*almonella*, it should be enough to adopt a supportive therapy: administration of oral rehydration solutions (which are used to compensate for water and salts lost with vomiting and diarrhea), lactic ferments, and probiotics.

**102**

Salmonella *infection pathogenesis. The ingestion of contaminated food or water begins the infective processes (gastroenteritis or systemic infection) depending on the species of* Salmonella *involved (minor and major Salmonellae). The microorganisms reach the intestinal epithelial cells and migrate to the lamina propria invading the liver from where* Salmonella *reaches the gall bladder and can cause chronic carriage which gives rise to healthy carriers [18].*

#### **Author details**

Maria Teresa Mascellino Sapienza University of Rome, Italy

\*Address all correspondence to: mariateresa.mascellino@uniroma1.it

© 2020 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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AAC.00642-11

*New Insight into* Brucella *Infection and Foodborne Diseases*

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**107**

**Chapter 9**

*Dilek ÇAM*

**Abstract**

methods.

rapid detection

**1. Introduction**

Lateral Flow Assay for *Salmonella*

Detection and Potential Reagents

*Salmonella* is among the very important pathogens threating human and animal health. It is a common food pathogen transmitted from animals to humans via contaminated food, drinking water, and air. It invades the intestinal tract of hosts and causes salmonellosis leading to death. *S. enteritidis* was the most common species accounted for all salmonellosis cases. *S. typhimurium* is also another significant species causing the serious cases worldwide. To ensure public health, early detection of pathogens is crucial. Lateral flow assay (LFA), immunochromatographic assay, is a simple and rapid diagnostic test kits used in various fields and can be developed by, aptamers, antibodies (Abs), and nucleic acids. They are also being continued to develop different capture reagents coming from the recombinant technology. It has many advantages such as having mature technology, market presence, low cost, easy to use for end users without education, and stable shelf life. Gold nanoparticles (GNPs) are the most commonly used labels in the LFAs for the naked-eye analysis. Therefore, *Salmonella* detection by LFA based on GNPs in a rapid and simple way is always open to be developed by new reagents and

**Keywords:** *Salmonella*, gold nanoparticles, lateral flow, food pathogens,

selectivity of detection with low cost as rapid tests.

Most of *Salmonella* infections are typically food-borne illness. It was reported that around 15% of salmonellosis cases is caused by pork [1], turkey products, and meat [2]. Early detection of pathogens which contaminated the foods or consumption products is a crucial issue especially for the government authorities to ensure public health. Thus, many kinds of identification methods are in use, and new detection platforms are also being tried to develop for improving the sensitivity and

Traditionally, the *Salmonella* diagnosis in the laboratory is based on common cultural techniques [3], biochemical and serological confirmation tests. Along with immunomagnetic nanospheres as immunological tools [4], multiplex PCR [5] and real-time multiplex PCR [6–9] are other detection methods of *Salmonella* in chicken samples or other sources. However, some of those techniques require 5 or 7 days, skilled personnel, sterile working conditions, and sensitive and costly equipment, and they are inconvenient for food sector or industrial applications [10] and not

#### **Chapter 9**
