Fast Detection of Pathogenic *Escherichia coli* from Chicken Meats

*Saloua Helali and Adnane Abdelghani*

#### **Abstract**

Food is a means to sustain and enjoy life, but it is also a medium for microbial contamination, causing disease and death. Fruits, vegetables, meat, and water are the common sources of contamination. *Escherichia coli* is one of the most frequent Pathogenic Bacteria responsible for food poisoning and food-related infections. *E. coli* infection causes severe bloody diarrhea, abdominal cramps, and occasional vomiting. In the present study, electrochemical impedance spectroscopy (EIS), surface plasmon resonance (SPR), and physisorption techniques were evaluated to decrease sample preparation time and to improve the sensitivity and specificity for the detection of low levels of pathogenic *Escherichia coli* in frozen chicken meat. The electrical and optical properties of the immobilized anti-*E. coli* antibody were studied. Moreover, the developed biosensor was used for *E. coli* detection in inoculated frozen chicken meat.

**Keywords:** physisorption, impedance spectroscopy, SPR imaging, *E. coli*, frozen chicken meat

#### **1. Introduction**

According to the World Health Organization (WHO), foodborne illnesses are defined as diseases of infectious or toxic nature caused by consumption of contaminated foods or water. The main causes of foodborne illness are viruses, bacteria, parasites, toxins, metals, and prions where bacteria constitute 66% of the problems [1]. *Campylobacter*, *Salmonella*, *Yersinia enterocolitica*, *Clostridium perfringens*, *Listeria monocytogenes*, *Staphylococcus aureus*, and *E. coli O157:H7* are the major Pathogenic Bacteria causing different food illness [2]. The most common clinical symptoms of foodborne illnesses are diarrhea, vomiting, abdominal cramps, headache, and nausea. In many developing countries, the elderly and children under the age of 5 years are at higher risk of food infection. Among the foodborne pathogens, *E. coli* and *Salmonella* are the most common and frequent pathogens responsible for food poisoning and food-related infections. These two Pathogenic Bacteria can be transferred by poultry meat, red meat, desserts, and egg.

Nowadays, *Escherichia coli* and *Salmonella* are the most important and frequent pathogens responsible for food poisoning and food-related infections in chicken meat [2–6]. *E. coli* is a normal inhabitant of intestinal tracts and can be found in chicken feces, litter, dust, and rodent droppings [7]. Preventing food contamination and human infection from *E. coli* requires continuous control measures at all stages of the

food production: from agricultural production to processing, manufacturing, transporting, storing, and preparation of foods in both commercial establishments and the domestic environment. Recently, many analytical applications have been developed for the determination of poultry meat contamination [8–10].

Several microbiological techniques such as conventional culturing, PCR, and ELISA are still considered the oldest and the most accurate approach for bacteria detection. These techniques need traditional sample preparation, though very efficient in extracting the target analyte, which is time-consuming and produces large amount of solvent wastes. Among the various techniques, electrochemical impedance spectroscopy technique and surface plasmon resonance imaging have previously been investigated to study the detection of Pathogenic Bacteria on gold [11]. These two techniques offer several advantages: First, they are label-free and direct detection method for biomolecular interactions because the measurements are based on small electric signal and very large range of frequency (100 mHz–100 kHz) and refractive index changes [12–14]. The analyte does not require any special characteristics (scattering bands) or labels (radioactive or fluorescent) and can be detected directly without the need for multistep detection protocols (sandwich assay). Second, the measurements can be performed in real time, allowing the user to collect kinetic data, as well as thermodynamic data.

In this contribution, an innovative way for sensitive detection of *E.coli* bacteria based on anti-*E. coli* antibody immobilized onto gold surface by physisorption technique is presented. The electrical properties of the immobilization of anti-*E. coli* antibody were studied. Moreover, the developed biosensor was used for *E. coli* detection in inoculated frozen chicken meat.

#### **2. What is** *Escherichia coli* **(***E. coli***)?**


#### **3. History**

In 1982, Riley LW et al. and colleagues were the first to recognize the EHEC serotype O157:H7 as a human pathogen associated with outbreaks of bloody diarrhea in Oregon and Michigan, USA [16]. Since then, *E. coli O157:H7* has become one of the most important foodborne pathogens.

#### **4. Symptoms and mode of transmission**

Virulent strains of *Escherichia coli* are responsible for most diarrheal infections, meningitis, septicemia, and urinary tract infections in children worldwide. A person who is infected with *E. coli O157* can pass it on to other people if there is a situation of insufficient hygiene or handwashing. Small children can still pass the infection on for a couple of weeks after they have recovered from any illness.

**5**

**Figure 2.**

*of IgG anti-*E. coli *antibody followed by* E. coli *detection.*

*Fast Detection of Pathogenic* Escherichia coli *from Chicken Meats*

On the basis of World Health Organization report, the most important symptoms and mode of transmission of *Escherichia coli* are represented in the diagram (**Figure 1**).

Interdigitated microelectrodes were provided by the Microelectronics Institute of Barcelona, National Microelectronics Centre (IMB-CNM), Spain. The different steps of the fabrication of the gold interdigitated electrodes were extensively characterized as described in Ref. [17]. The electrode consists in 3 mm × 3 mm square

*(A) The actual planar interdigital electrode impedance sensors. (B) Schematic representation of physisorption* 

*DOI: http://dx.doi.org/10.5772/intechopen.91437*

**5. Immunosensor conception**

Escherichia coli *symptoms and mode of transmission.*

**5.1 Working electrodes**

**Figure 1.**

*Fast Detection of Pathogenic* Escherichia coli *from Chicken Meats DOI: http://dx.doi.org/10.5772/intechopen.91437*

E. Coli *Infections - Importance of Early Diagnosis and Efficient Treatment*

for the determination of poultry meat contamination [8–10].

*E. coli* detection in inoculated frozen chicken meat.

**2. What is** *Escherichia coli* **(***E. coli***)?**

a broad spectrum of diseases.

most important foodborne pathogens.

**4. Symptoms and mode of transmission**

food production: from agricultural production to processing, manufacturing, transporting, storing, and preparation of foods in both commercial establishments and the domestic environment. Recently, many analytical applications have been developed

• *Escherichia coli* are gram-negative bacilli of the family *Enterobacteriaceae*.

• *E. coli* is commonly found in the lower intestine of warm-blooded organisms.

• There are many different types (strains) of *E. coli* which cause a number of illnesses. Virulence types of *E. coli* include enterotoxigenic (ETEC), enteroinvasive (EIEC), enteropathogenic (EPEC), and enterohemorrhagic *E. coli* (EHEC) [15].

In 1982, Riley LW et al. and colleagues were the first to recognize the EHEC serotype O157:H7 as a human pathogen associated with outbreaks of bloody diarrhea in Oregon and Michigan, USA [16]. Since then, *E. coli O157:H7* has become one of the

Virulent strains of *Escherichia coli* are responsible for most diarrheal infections,

meningitis, septicemia, and urinary tract infections in children worldwide. A person who is infected with *E. coli O157* can pass it on to other people if there is a situation of insufficient hygiene or handwashing. Small children can still pass the infection on for a couple of weeks after they have recovered from any illness.

• *E. coli* is the most common human and animal pathogens as it is responsible for

Several microbiological techniques such as conventional culturing, PCR, and ELISA are still considered the oldest and the most accurate approach for bacteria detection. These techniques need traditional sample preparation, though very efficient in extracting the target analyte, which is time-consuming and produces large amount of solvent wastes. Among the various techniques, electrochemical impedance spectroscopy technique and surface plasmon resonance imaging have previously been investigated to study the detection of Pathogenic Bacteria on gold [11]. These two techniques offer several advantages: First, they are label-free and direct detection method for biomolecular interactions because the measurements are based on small electric signal and very large range of frequency (100 mHz–100 kHz) and refractive index changes [12–14]. The analyte does not require any special characteristics (scattering bands) or labels (radioactive or fluorescent) and can be detected directly without the need for multistep detection protocols (sandwich assay). Second, the measurements can be performed in real time, allowing the user to collect kinetic data, as well as thermodynamic data. In this contribution, an innovative way for sensitive detection of *E.coli* bacteria based on anti-*E. coli* antibody immobilized onto gold surface by physisorption technique is presented. The electrical properties of the immobilization of anti-*E. coli* antibody were studied. Moreover, the developed biosensor was used for

**4**

**3. History**

**Figure 1.** Escherichia coli *symptoms and mode of transmission.*

On the basis of World Health Organization report, the most important symptoms and mode of transmission of *Escherichia coli* are represented in the diagram (**Figure 1**).

#### **5. Immunosensor conception**

#### **5.1 Working electrodes**

Interdigitated microelectrodes were provided by the Microelectronics Institute of Barcelona, National Microelectronics Centre (IMB-CNM), Spain. The different steps of the fabrication of the gold interdigitated electrodes were extensively characterized as described in Ref. [17]. The electrode consists in 3 mm × 3 mm square

#### **Figure 2.**

*(A) The actual planar interdigital electrode impedance sensors. (B) Schematic representation of physisorption of IgG anti-*E. coli *antibody followed by* E. coli *detection.*

arrays, which consist of 108 fingers 10 μm wide, separated 10 μm from the nearest band (**Figure 2A**). Before modification, the gold microelectrodes were first cleaned in ethanol solution and then electrochemically activated in 0.5 M NaNO3 solution by applying a series of potential pulses from 0 to −2 V vs. Ag/AgCl (3 M KCl). After that, a cyclic voltammetry in 1 mM potassium ferrocyanide [K4Fe(CN)6] was applied to check the degree of activation of the microelectrodes.

The pre-treated working microelectrodes were immersed in 100 μL goat polyclonal IgG anti-*E. coli* antibody solution (5 mg/mL in PBS) for 90 min (**Figure 2B**). The gold substrates were then rinsed with PBS buffer in order to remove the non-bonded antibody; finally the substrate was kept in bovine serum albumin (BSA) 1% for 40 min in order to block any defective areas. The excess of BSA was removed by rinsing with PBS.

#### **5.2 Electrochemical impedance spectroscopy technique**

All electrochemical measurements were performed with a three-electrode configuration using a Pt foil counter electrode, an Ag/AgCl reference electrode, and a modified gold μ-electrodes as a work electrode.

The impedance analysis was performed with a CHI604E Electrochemical Instrumentation (CH Instruments, Inc) in the frequency range of 0.1–100 kHz, using a modulation voltage of 10 mV in sterile PBS buffer.

#### **5.3 SPR imaging technique**

John Mitchell [18] has been successfully explaining the physical principles of surface plasmon resonance. The SPR is an optoelectronic phenomenon that occurs when a photon of light is incident upon a noble metal surface such as gold or silver. When the wavelength of the photon equals the resonance wavelength of the metal, then the photon couples with the surface and induces the electrons in the metal surface to move as a single electrical entity called a plasmon. This oscillation of electrons sets up an electromagnetic field that exponentially decays out from the metal surface, with significant electrical field strength typically occurring within 300 nm of the surface. When molecules with sufficient mass bind to the surface within the range of the electric field, they perturb the plasmon and change the resonance wavelength. When dealing with a fixed planar surface, this is seen as a shift in the resonance angle of the incoming photons.

In this work, the surface plasmon resonance imaging system was from GWC technologies (USA). The system is based on Charge-Couple Device (CCD) camera which can simultaneously capture all data for all the gold spots and converts the reflectivity changes to pixels data. The sensor surface was an array format with 16 gold spots (each gold spot has a surface of 0.004 cm<sup>2</sup> ) deposited on glass substrate. An incident beam of excitation wavelength of 850 nm was used. At resonance condition, the variation of the reflected light was due to the refractive index variation of the external dielectric medium or immobilized thin layer. The noise of such system is equal to 0.5 pixel (**Figure 3**).

#### **5.4 Physisorption of polyclonal antibody on interdigitated gold microelectrodes**

Physisorption is defined as weak electrostatic interactions including Van Der Waals interactions, dipole-dipole, and London forces. This physical interaction resulting from nonspecific was forming on substrate have energy range from 0.2 to 4 kJ/mol. The binding energy depends on the polarizability and on the number of atoms involved of the molecules. It takes place on all surfaces provided that temperature and pressure conditions are favorable.

Random physisorption is the easiest and fastest strategy for biomolecule immobilization onto substrates. Mainly, physisorption does not depend on multistep, long

**7**

**Figure 3.**

*Fast Detection of Pathogenic* Escherichia coli *from Chicken Meats*

experimental procedures and is easily reversed [19]. In addition, physisorbed phages have been described to promote bacteria-specific capture, infection, and lysis, when monitored by SPR [20, 21]. This work was carried using physisorption functionalization based on its simplicity. First, the gold microelectrodes were modificated with anti-*E. coli* antibody, followed by washing with PBS then physical blocking with BSA. Blocking prevented nonspecific adsorption of unwanted nontarget components during subsequent incubations. Then, in this work, a fast and suitable immunosensor for *E. coli* bacteria detection, using physically adsorbed antibodies, SPR and EIS, is developed.

The rapid and specific detection of Pathogenic Bacteria has become an increasingly demanding field in recent years for ensuring the safety of human health. EIS is a sensitive technique, which monitors the electrical response of the system studied after the application of a periodic small amplitude AC signal [22]. With this aim, the gold microelectrode surface and antibody coverage are of high importance for

The typical response of electrochemical impedance spectra of gold, "gold/ Antibody /BSA" interfaces was illustrated in **Figure 4**(I). The curve shows the typical Nyquist plots presented as a combination of the real, Zre, and imaginary, Zim, components originating mainly from the resistance and capacitance of the cell, respectively. The impedance spectra corresponding to each step were fitted with computer-simulated spectra using Randles circuit in **Figure 4**(II) by Zview model-

**6. Electrochemical impedance measurement**

*Schematic representation of the SPR imaging system.*

ensuring high reactivity and stability of the immunosensor.

ing program (Scriber and Associates, Charlottesville, VA).

*DOI: http://dx.doi.org/10.5772/intechopen.91437*

*Fast Detection of Pathogenic* Escherichia coli *from Chicken Meats DOI: http://dx.doi.org/10.5772/intechopen.91437*

E. Coli *Infections - Importance of Early Diagnosis and Efficient Treatment*

applied to check the degree of activation of the microelectrodes.

**5.2 Electrochemical impedance spectroscopy technique**

using a modulation voltage of 10 mV in sterile PBS buffer.

gold spots (each gold spot has a surface of 0.004 cm<sup>2</sup>

system is equal to 0.5 pixel (**Figure 3**).

perature and pressure conditions are favorable.

a modified gold μ-electrodes as a work electrode.

**5.3 SPR imaging technique**

arrays, which consist of 108 fingers 10 μm wide, separated 10 μm from the nearest band (**Figure 2A**). Before modification, the gold microelectrodes were first cleaned in ethanol solution and then electrochemically activated in 0.5 M NaNO3 solution by applying a series of potential pulses from 0 to −2 V vs. Ag/AgCl (3 M KCl). After that, a cyclic voltammetry in 1 mM potassium ferrocyanide [K4Fe(CN)6] was

The pre-treated working microelectrodes were immersed in 100 μL goat polyclonal IgG anti-*E. coli* antibody solution (5 mg/mL in PBS) for 90 min (**Figure 2B**). The gold substrates were then rinsed with PBS buffer in order to remove the non-bonded antibody; finally the substrate was kept in bovine serum albumin (BSA) 1% for 40 min in order to block any defective areas. The excess of BSA was removed by rinsing with PBS.

All electrochemical measurements were performed with a three-electrode configuration using a Pt foil counter electrode, an Ag/AgCl reference electrode, and

The impedance analysis was performed with a CHI604E Electrochemical Instrumentation (CH Instruments, Inc) in the frequency range of 0.1–100 kHz,

plasmon resonance. The SPR is an optoelectronic phenomenon that occurs when a photon of light is incident upon a noble metal surface such as gold or silver. When the wavelength of the photon equals the resonance wavelength of the metal, then the photon couples with the surface and induces the electrons in the metal surface to move as a single electrical entity called a plasmon. This oscillation of electrons sets up an electromagnetic field that exponentially decays out from the metal surface, with significant electrical field strength typically occurring within 300 nm of the surface. When molecules with sufficient mass bind to the surface within the range of the electric field, they perturb the plasmon and change the resonance wavelength. When dealing with a fixed planar surface, this is seen as a shift in the resonance angle of the incoming photons. In this work, the surface plasmon resonance imaging system was from GWC technologies (USA). The system is based on Charge-Couple Device (CCD) camera which can simultaneously capture all data for all the gold spots and converts the reflectivity changes to pixels data. The sensor surface was an array format with 16

An incident beam of excitation wavelength of 850 nm was used. At resonance condition, the variation of the reflected light was due to the refractive index variation of the external dielectric medium or immobilized thin layer. The noise of such

**5.4 Physisorption of polyclonal antibody on interdigitated gold microelectrodes**

Physisorption is defined as weak electrostatic interactions including Van Der Waals interactions, dipole-dipole, and London forces. This physical interaction resulting from nonspecific was forming on substrate have energy range from 0.2 to 4 kJ/mol. The binding energy depends on the polarizability and on the number of atoms involved of the molecules. It takes place on all surfaces provided that tem-

Random physisorption is the easiest and fastest strategy for biomolecule immobilization onto substrates. Mainly, physisorption does not depend on multistep, long

John Mitchell [18] has been successfully explaining the physical principles of surface

) deposited on glass substrate.

**6**

**Figure 3.** *Schematic representation of the SPR imaging system.*

experimental procedures and is easily reversed [19]. In addition, physisorbed phages have been described to promote bacteria-specific capture, infection, and lysis, when monitored by SPR [20, 21]. This work was carried using physisorption functionalization based on its simplicity. First, the gold microelectrodes were modificated with anti-*E. coli* antibody, followed by washing with PBS then physical blocking with BSA. Blocking prevented nonspecific adsorption of unwanted nontarget components during subsequent incubations. Then, in this work, a fast and suitable immunosensor for *E. coli* bacteria detection, using physically adsorbed antibodies, SPR and EIS, is developed.

#### **6. Electrochemical impedance measurement**

The rapid and specific detection of Pathogenic Bacteria has become an increasingly demanding field in recent years for ensuring the safety of human health. EIS is a sensitive technique, which monitors the electrical response of the system studied after the application of a periodic small amplitude AC signal [22]. With this aim, the gold microelectrode surface and antibody coverage are of high importance for ensuring high reactivity and stability of the immunosensor.

The typical response of electrochemical impedance spectra of gold, "gold/ Antibody /BSA" interfaces was illustrated in **Figure 4**(I). The curve shows the typical Nyquist plots presented as a combination of the real, Zre, and imaginary, Zim, components originating mainly from the resistance and capacitance of the cell, respectively. The impedance spectra corresponding to each step were fitted with computer-simulated spectra using Randles circuit in **Figure 4**(II) by Zview modeling program (Scriber and Associates, Charlottesville, VA).

#### **Figure 4.**

*(I) [A] Nyquist impedance plots for gold microelectrode and [B] Nyquist impedance plots after physisorption of anti-*E. coli *and BSA on gold microelectrodes. (II) equivalent circuit used to model impedance data.*

This equivalent circuit includes the ohmic resistance of the electrolyte solution, Rs, at 100 kHz; the Warburg impedance, Zw, from the diffusion; the constant phase element, CPE, which was introduced into the circuit instead of a capacitance in order to depict the nonhomogeneous quality of the deposited layer, respectively [23, 24]; and the charge transfer resistance, Rct. The constant phase element impedance (CPE) was introduced into the circuit instead of a capacitance: ZCPE = \_1

$$\mathbf{Z\_{CPE}} = \frac{1}{K \, (j\omega)^{\*}} \tag{1}$$

**9**

tration of 105

**Figure 5.**

*Fast Detection of Pathogenic* Escherichia coli *from Chicken Meats*

anti-*E. coli* antibody solution. The real-time sensorgram showed a gradual increase in the response with antibody immobilization onto the sensor chip. The highest

**8. Detection of** *E. coli* **bacteria in inoculated frozen chicken meat sample**

important sources of good-quality proteins, it is highly susceptible to microbial contamination and often implicated in foodborne disease. Epidemiological reports suggest that poultry meat is still the primary cause of human food poisoning [25]. According to Osman Albarri (2017) [26], in turkey the highest percentage (93.75%) of *E. coli* was isolated from chicken, while the lowest percentage (56.25%) was isolated from meat. Therefore, ensuring the microbial safety of chicken meat products is an important issue in the context of increasing consumption and production [24]. This moved us to develop rapid, easy, simple, sensitive, and non-time-consuming physical adsorption methods for the detection of *E. coli* bacteria in frozen chicken meat.

Although chicken is the most consumed meat in the world and is one of the most

With this aim, two samples of fresh chicken meat were kept in freezers at −18°C during 45 days [27]. The first sample (S1) was inoculated with *E. coli* with a concen-

**Figure 6** shows Nyquist plots for gold microelectrode (curve A), gold electrode with immobilized anti-*E. coli* antibody with BSA blocking layer (curve B), gold electrode with immobilized anti-*E. coli* antibody with BSA blocking layer without inoculated bacteria (sample S2, curve C), and gold electrode with immobilized anti-*E. coli* antibody with BSA blocking layer with inoculated bacteria (sample S1, curve D). Using the same equivalent circuit model described in **Figure 4 (II)**, the best-fit equivalent circuit parameters for each step are given in **Table 1**. The injection of sample S1 and S2 on the microelectrode induce an increase in the charge transfer resistance proving the detection of *E. coli* bacteria in the chicken meat sample. It is obvious that the chicken was initially contaminated by the *E. coli* bacteria

(reference). These samples were characterized by EIS as previously described.

CFU/ML in PBS, and the second sample (S2) was kept in PBS buffer

CFU/ML.

*DOI: http://dx.doi.org/10.5772/intechopen.91437*

response obtained was equal to 87 pixels.

*SPRi signal versus time for anti-*E. coli*.*

and the concentration of the bacteria is about 105

where *ω* = 2πf is the angular frequency and *K* and *n* are the experimental parameters. When *n* approaches 1, the CPE acts as an ideal capacitor. The CPE can be viewed as a heuristic method to incorporate the effects of surface heterogeneity both along and through the electrochemical interface. Data was fitted to the Randles circuit shown in **Figure 4**(II). The plot shows the expected increases in the charge transfer resistance after immobilization of anti-*E. coli* antibody from 0.14 to 1.22 MΩ. This increase could be attributed to a rearrangement in the structure of the antibody and showed that the grafted layer becomes more insulating, whereas the gradual decrease in capacitance was related to the positive change in thickness after immobilization of anti-*E. coli* antibody and BSA. This behavior is consistent with the successful immobilization of anti-*E. coli* antibody and BSA molecules.

#### **7. Surface plasmon resonance measurement**

In the last few years, surface plasmon resonance was used as a sensitive method and as a label-free detection method for biomolecular interactions.

The immunosensing protocols exposing sensor surface to the PBS buffer as the baseline, followed by injecting anti-*E. coli* antibody, which is allowed to flow over the sensor surface, leading to binding. This binding alters the mass of the surface layer which translates into refractive index variation and resonance angle shift. **Figure 5** shows the resulting response obtained from the injection of 5 mg/mL

*Fast Detection of Pathogenic* Escherichia coli *from Chicken Meats DOI: http://dx.doi.org/10.5772/intechopen.91437*

**Figure 5.** *SPRi signal versus time for anti-*E. coli*.*

E. Coli *Infections - Importance of Early Diagnosis and Efficient Treatment*

This equivalent circuit includes the ohmic resistance of the electrolyte solution, Rs, at 100 kHz; the Warburg impedance, Zw, from the diffusion; the constant phase element, CPE, which was introduced into the circuit instead of a capacitance in order to depict the nonhomogeneous quality of the deposited layer, respectively [23, 24]; and the charge transfer resistance, Rct. The constant phase element imped-

*(I) [A] Nyquist impedance plots for gold microelectrode and [B] Nyquist impedance plots after physisorption of anti-*E. coli *and BSA on gold microelectrodes. (II) equivalent circuit used to model impedance data.*

ZCPE = \_1

where *ω* = 2πf is the angular frequency and *K* and *n* are the experimental parameters. When *n* approaches 1, the CPE acts as an ideal capacitor. The CPE can be viewed as a heuristic method to incorporate the effects of surface heterogeneity both along and through the electrochemical interface. Data was fitted to the Randles circuit shown in **Figure 4**(II). The plot shows the expected increases in the charge transfer resistance after immobilization of anti-*E. coli* antibody from 0.14 to 1.22 MΩ. This increase could be attributed to a rearrangement in the structure of the antibody and showed that the grafted layer becomes more insulating, whereas the gradual decrease in capacitance was related to the positive change in thickness after immobilization of anti-*E. coli* antibody and BSA. This behavior is consistent with the successful immobilization of anti-*E. coli* antibody and BSA

*K* ( *j*)

In the last few years, surface plasmon resonance was used as a sensitive method

The immunosensing protocols exposing sensor surface to the PBS buffer as the baseline, followed by injecting anti-*E. coli* antibody, which is allowed to flow over the sensor surface, leading to binding. This binding alters the mass of the surface layer which translates into refractive index variation and resonance angle shift. **Figure 5** shows the resulting response obtained from the injection of 5 mg/mL

*<sup>n</sup>* (1)

ance (CPE) was introduced into the circuit instead of a capacitance:

**7. Surface plasmon resonance measurement**

and as a label-free detection method for biomolecular interactions.

**8**

molecules.

**Figure 4.**

anti-*E. coli* antibody solution. The real-time sensorgram showed a gradual increase in the response with antibody immobilization onto the sensor chip. The highest response obtained was equal to 87 pixels.

#### **8. Detection of** *E. coli* **bacteria in inoculated frozen chicken meat sample**

Although chicken is the most consumed meat in the world and is one of the most important sources of good-quality proteins, it is highly susceptible to microbial contamination and often implicated in foodborne disease. Epidemiological reports suggest that poultry meat is still the primary cause of human food poisoning [25]. According to Osman Albarri (2017) [26], in turkey the highest percentage (93.75%) of *E. coli* was isolated from chicken, while the lowest percentage (56.25%) was isolated from meat. Therefore, ensuring the microbial safety of chicken meat products is an important issue in the context of increasing consumption and production [24]. This moved us to develop rapid, easy, simple, sensitive, and non-time-consuming physical adsorption methods for the detection of *E. coli* bacteria in frozen chicken meat.

With this aim, two samples of fresh chicken meat were kept in freezers at −18°C during 45 days [27]. The first sample (S1) was inoculated with *E. coli* with a concentration of 105 CFU/ML in PBS, and the second sample (S2) was kept in PBS buffer (reference). These samples were characterized by EIS as previously described.

**Figure 6** shows Nyquist plots for gold microelectrode (curve A), gold electrode with immobilized anti-*E. coli* antibody with BSA blocking layer (curve B), gold electrode with immobilized anti-*E. coli* antibody with BSA blocking layer without inoculated bacteria (sample S2, curve C), and gold electrode with immobilized anti-*E. coli* antibody with BSA blocking layer with inoculated bacteria (sample S1, curve D). Using the same equivalent circuit model described in **Figure 4 (II)**, the best-fit equivalent circuit parameters for each step are given in **Table 1**. The injection of sample S1 and S2 on the microelectrode induce an increase in the charge transfer resistance proving the detection of *E. coli* bacteria in the chicken meat sample.

It is obvious that the chicken was initially contaminated by the *E. coli* bacteria and the concentration of the bacteria is about 105 CFU/ML.

#### **Figure 6.**

*[A] Nyquist impedance plots for gold microelectrode, [B] nyquist impedance plots for gold microelectrode with anti-*E. coli *and BSA, and [C and D] nyquist impedance plots for gold microelectrode with anti-*E. coli *and BSA after injection of sample S2 and S1, respectively.*


#### **Table 1.**

*The electrical parameters of Randle's circuit.*

#### **9. Conclusion**

In this work, we describe an approach of detecting of *Escherichia coli* bacteria by Electrochemical Impedance Spectroscopy technique and surface plasmon resonance imaging technique. The physisorption method used for immobilization of anti-*E. coli* into interdigitated microelectrode is rapid and easy. Furthermore, *Escherichia coli* bacteria detection was also possible in frozen chicken meat. This method can be used for real-time detection of meat contamination.

#### **Acknowledgements**

This work is funded by the Science for Peace and Security Program of the North Atlantic Treaty Organization (NATO) under grant no. SFP G5571. The authors dedicate this work to the memory of Mr. Naoufel Gaouar, Associate Professor at the National Institute of Applied Science and Technology (INSAT, Tunisia) who died on March 8, 2020.

**11**

Tunisia

**Author details**

Kingdom of Saudi Arabia

Saloua Helali1,2\* and Adnane Abdelghani3

1 Faculty of Science, Department of Physics, University of Tabuk, Tabuk,

3 Carthage University, National Institute of Applied Science and Technology,

© 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,

2 Centre de Recherche et Technologies de L'Energie, Tunisia

\*Address all correspondence to: s.helali@ut.edu.sa

provided the original work is properly cited.

*Fast Detection of Pathogenic* Escherichia coli *from Chicken Meats*

*DOI: http://dx.doi.org/10.5772/intechopen.91437*

*Fast Detection of Pathogenic* Escherichia coli *from Chicken Meats DOI: http://dx.doi.org/10.5772/intechopen.91437*

E. Coli *Infections - Importance of Early Diagnosis and Efficient Treatment*

In this work, we describe an approach of detecting of *Escherichia coli* bacteria by Electrochemical Impedance Spectroscopy technique and surface plasmon resonance imaging technique. The physisorption method used for immobilization of anti-*E. coli* into interdigitated microelectrode is rapid and easy. Furthermore, *Escherichia coli* bacteria detection was also possible in frozen chicken meat. This method can be

**BSA/anti-***E. coli***/ gold**

*[A] Nyquist impedance plots for gold microelectrode, [B] nyquist impedance plots for gold microelectrode with anti-*E. coli *and BSA, and [C and D] nyquist impedance plots for gold microelectrode with anti-*E. coli *and* 

> 7.464E-7 0.86 74.74 1.225E6 1452

**Sample S2 Sample S1**

5.128E-7 0.9 71 5.01E6 120

5.76E-7 0.88 70.7 4.68E6 132

This work is funded by the Science for Peace and Security Program of the North

Atlantic Treaty Organization (NATO) under grant no. SFP G5571. The authors dedicate this work to the memory of Mr. Naoufel Gaouar, Associate Professor at the National Institute of Applied Science and Technology (INSAT, Tunisia) who died on

used for real-time detection of meat contamination.

**Parameters Gold** 

*BSA after injection of sample S2 and S1, respectively.*

*The electrical parameters of Randle's circuit.*

**microelectrode**

8.34E-7 0.93 110 1.43E5 45,364

**10**

**9. Conclusion**

Capacitance, CPE (F)

Resistance, Rs (Ω) Resistance, Rct (Ω) Warburg, ZW (Ω)

n

**Figure 6.**

**Table 1.**

**Acknowledgements**

March 8, 2020.

#### **Author details**

Saloua Helali1,2\* and Adnane Abdelghani3

1 Faculty of Science, Department of Physics, University of Tabuk, Tabuk, Kingdom of Saudi Arabia

2 Centre de Recherche et Technologies de L'Energie, Tunisia

3 Carthage University, National Institute of Applied Science and Technology, Tunisia

\*Address all correspondence to: s.helali@ut.edu.sa

© 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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[13] Huber W, Mueller F. Biomolecular interaction analysis in drug discovery using surface plasmon resonance technology. Current Pharmaceutical Design. 2006;**12**:3999-4021. DOI: 10.2174/138161206778743600

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*Fast Detection of Pathogenic* Escherichia coli *from Chicken Meats*

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[26] Albarri O, Var I, Meral M, Bedir B, Heshmati B, Köksal F. Prevalence of Escherichia coli isolated from meat, chicken and vegetable samples in Turkey. Journal of Biotechnology Science Research. 2017;**4**(3):214-2022

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immunosensing using surface plasmon resonance. Sensors. 2010;**10**:7323-7346.

[20] Mejri MB, Baccar H, Baldrich E, Del Campo FJ, Helali S, Ktari T, et al. Impedance biosensing using phages for bacteria detection: Generation of dual signals as the clue for in-chip assay confirmation. Biosensors and Bioelectronics. 2010;**26**:1261-1267. DOI:

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Nanobiotechnology. 2014;**5**:153-158. DOI: 10.4236/jbnb.2014.53018

[22] Hleli S, Martelet C, Abdelghani A, Burais N, Jaffrezic-Renault N. Atrazine

analysis using an impedimetric

10.1016/j.bios.2010.06.054

Journal of Biomaterials and

2010;**20**:5-14

2199-1

sl.2011.1800

*Fast Detection of Pathogenic* Escherichia coli *from Chicken Meats DOI: http://dx.doi.org/10.5772/intechopen.91437*

Environmental Research and Public Health. 2013;**10**:6235-6254. DOI: 10.3390/ijerph10126235

[16] Lim JY, Yoon JW, Hovde CJ. A brief overview of Escherichia coli O157:H7 and its plasmid O157. Journal of Microbiology and Biotechnology. 2010;**20**:5-14

[17] Laczka O, Baldrich E, Javier del Campo F, Muñoz FX. Immunofunctionalisation of gold transducers for bacterial detection by physisorption. Analytical and Bioanalytical Chemistry. 2008;**391**: 2825-2835. DOI: 10.1007/s00216-008- 2199-1

[18] Mitchell J. Small molecule immunosensing using surface plasmon resonance. Sensors. 2010;**10**:7323-7346. DOI: 10.3390/s100807323

[19] Marrakchi M, Campos Sánchez I, Helali S, Mejri N, Soto Camino J, Gonzalez-Martinez MA, et al. A label-free interdigitated microelectrodes immunosensor for pesticide detection. Sensor Letters. 2011;**9**:2203-2206. DOI: 10.1166/ sl.2011.1800

[20] Mejri MB, Baccar H, Baldrich E, Del Campo FJ, Helali S, Ktari T, et al. Impedance biosensing using phages for bacteria detection: Generation of dual signals as the clue for in-chip assay confirmation. Biosensors and Bioelectronics. 2010;**26**:1261-1267. DOI: 10.1016/j.bios.2010.06.054

[21] Chammem H, Hafaid I, Meilhac O, Menaa F, Mora L, Abdelghani A. Surface plasmon resonance for C-reactive protein detection in human plasma. Journal of Biomaterials and Nanobiotechnology. 2014;**5**:153-158. DOI: 10.4236/jbnb.2014.53018

[22] Hleli S, Martelet C, Abdelghani A, Burais N, Jaffrezic-Renault N. Atrazine analysis using an impedimetric

immunosensor based on mixed biotinylated self-assembled monolayer. Sensors and Actuators B. 2006;**113**:711-717

[23] Prodromidis MI. Impedimetric immunosensors - a review. Electrochimica Acta. 2010;**55**:4227-4233. DOI: 10.1016/j.electacta.2009.01.081

[24] Nahar MK, Zakaria Z, Hashim U. Protein solubility behaviour of fresh and frozen chicken meat in slaughtering and non-slaughtering condition. International Food Research Journal. 2014;**21**:135-137

[25] Kozačinski L, Hadžiosmanović M, Zdolec N. Microbiological quality of poultry meat on the Croatian market. Veterinarski Arhiv. 2006;**76**(4):305-313

[26] Albarri O, Var I, Meral M, Bedir B, Heshmati B, Köksal F. Prevalence of Escherichia coli isolated from meat, chicken and vegetable samples in Turkey. Journal of Biotechnology Science Research. 2017;**4**(3):214-2022

[27] Helali S, Alatawi ASE, Abdelghani A. Pathogenic Escherichia coli biosensor detection on chicken food samples. Journal of Food Safety. 2018;**38**:1-6. DOI: 10.1111/jfs.12510

**12**

E. Coli *Infections - Importance of Early Diagnosis and Efficient Treatment*

[9] Skóra J, Matusiak K, Wojewódzki P, Nowak A, Sulyok M, Ligocka A, et al. Evaluation of microbiological and chemical contaminants in poultry farms. International Journal of Environmental Research and Public Health. 2016;**13**:1-16. DOI: 10.3390/

[10] Feng YZ, Elmasry G, Sun DW, Scannell AG, Walsh D, Morcy N. Nearinfrared hyperspectral imaging and partial least squares regression for rapid and reagentless determination of *Enterobacteriaceae* on chicken fillets. Food Chemistry. 2013;**138**:1829-1836. DOI: 10.1016/j.foodchem.2012.11.040

[11] Moghtader F, Congur G, Zareie HM, Erdem A, Piskin E. Impedimetric detection of Pathogenic Bacteria with bacteriophages using gold nanorod deposited graphite electrodes. RSC

[12] Boozer C, Kim G, Cong S, Guan HW, Londergan T. Looking towards labelfree biomolecular interaction analysis in a high-throughput format: A review of new surface plasmon resonance technologies. Current Opinion in Biotechnology. 2006;**17**:400-405. DOI:

Advances. 2016;**6**:97832

10.1016/j.copbio.2006.06.012

[14] Yang L, Bashir R. Electrical/ electrochemical impedance for rapid detection of foodborne Pathogenic Bacteria. Biotechnology Advances. 2008;**26**:135-150. DOI: 10.1016/j.

[15] Allocati N, Masulli M, Alexeyev MF, Di Ilio C. Escherichia coli in Europe: An overview. International Journal of

biotechadv.2007.10.003

[13] Huber W, Mueller F. Biomolecular interaction analysis in drug discovery using surface plasmon resonance technology. Current Pharmaceutical Design. 2006;**12**:3999-4021. DOI: 10.2174/138161206778743600

ijerph13020192

[1] Addis M, Sisay D. A review on major food borne bacterial illnesses. Journal of Tropical Diseases. 2015;**3**:1-7. DOI:

[2] Adu-Gyamfi A, Torgby-Tetteh W, Appiah V. Microbiological quality of chicken sold in accra and determination of D10-value of *E. coli.* Food and Nutrition Sciences. 2012;**3**:693-698

[3] Borch E, Arinder P. Bacteriological safety issues in beef and readyto-eat meat products, as well as control measures. Meat Science. 2002;**62**:381-390. DOI: 10.1016/ S0309-1740(02)00125-0

[4] Alvarez-Astorga M, Capita R,

[5] Akbar A, Sitara U, Khan SA, Ali I, Khan MI, Phadungchob T, et al. Presence of Escherichia coli in poultry meat: A potential food safety threat. International Food Research Journal.

[6] Rouger A, Tresse O, Zagorec M. Bacterial contaminants of poultry meat: Sources, species, and dynamics. Microorganisms. 2017;**5**:1-16. DOI: 10.3390/microorganisms5030050

[7] Kozačinski L, Hadžiosmanović M, Zdolec N. Microbiological quality of poultry meat on the Croatian market. Veterinarski Arhiv. 2006;**76**:305-313.

[8] Kamruzzaman M, Makino Y, Oshita S. Non-invasive analytical technology for the detection of contamination, adulteration, and authenticity of meat, poultry, and fish: A review. Analytica Chimica Acta. 2015;**853**:19-29. DOI:

DOI: hrcak.srce.hr/5139

10.1016/j.aca.2014.08.043

2014;**21**:941-945

Alonso-Callega C, Moreno B, Del Camoni Garcia-Fernandez M. Microbiological quality of retail chicken in by-products in Spain. Meat Science. 2002;**62**:45-50. DOI: 10.1016/S0309-1740(01)00225-X

10.4176/2329-891X.1000176

**References**

**15**

**Chapter 2**

**Abstract**

health status of pigs.

**1. Introduction**

infection caused by ETEC.

Young Pigs

*Peng Ji, Xunde Li and Yanhong Liu*

Dietary Intervention to Reduce

Postweaning piglets are immediately imposed to remarkable environmental and psychosocial stressors, which adversely affect their intestinal development and health and predispose them to diarrhea. The ratio of postweaning mortality is 6–10% and may rise up to 20% with poor management strategies. Diarrhea per se accounts for 20–30% of cases of mortality in weanling pigs. *E. coli* postweaning diarrhea is one of the most important causes of postweaning diarrhea in pigs. This diarrhea is responsible for huge economic losses due to high mortality and morbidity, weight loss, and cost of medication. Burgeoning evidence suggested feed-based intervention are one of the promising measures to prevent postweaning diarrhea and to enhance overall health of weaned pigs. Although the exact protective mechanisms may vary and are still not completely understood, a number of feed ingredients or feed additives are marketed to assist in boosting intestinal immunity and regulating gut microbiota. The promising results have been demonstrated in several nutrients (i.e., functional amino acids, organic acids, micro minerals, nondigestible carbohydrates, and antimicrobial peptides), non-nutrients (i.e., phytochemicals and probiotics), and many other feed additives. The efficiencies of each candidate may differ based on their exact modes of action, the basal diet formulation, and the

**Keywords:** dietary intervention, *E. coli* infectious diarrhea, ETEC, young pigs

*Escherichia coli* (*E. coli*), a Gram-negative rod-shaped bacterium, was first discovered in 1885 by Theodor Escherich, who noted that *E. coli* are highly prevalent in the intestinal microflora of healthy individuals and have potential to cause disease when directly inoculated into extraintestinal sites. Diarrheagenic *E. coli* can be further divided into six groups: enterotoxigenic *E. coli* (ETEC), enteropathogenic *E. coli,* enterohemorrhagic *E. coli*, enteroinvasive *E. coli*, diffusely adhering *E. coli*, and enteroaggregative *E. coli* [1]. Different groups of diarrheagenic *E. coli* express different virulence genes, exhibit different adhesion characteristics, and therefore have different mechanisms of pathogenicity. This book chapter only covers the

ETEC is the major etiological agent causing acute watery diarrhea in postweaning piglets. The duration of diarrheal symptom may be shortened by

*E. coli* Infectious Diarrhea in

#### **Chapter 2**
