**6. Functional characteristics of the** *jhp0940***,** *jhp094***5,** *jhp0947* **and**  *jhp0949* **genes**

Among the *jhp0940*, *jhp0945*, *jhp0947* and jhp0949 genes, *jhp0940* is the best characterised. Rizwan et al. [25] purified recombinant JHP0940 protein, which, when incubated with macrophage cells (TPH-1), was able to induce the synthesis of TNF-α, IL-6 and IL-8 via translocation of NF-κB. This gene is currently known as *ctkA* (cell translocation kinase A), and the encoded protein has autophosphorylation activity [43]. In addition to being a serine/threonine kinase, CtkA can increase the phosphorylation of NF-κB by inducing TNF-α in a dose-dependent manner in HeLa cells [40]. However, Tenguria et al. [44] observed that *H. pylori* can secrete the CtkA and induce the expression of caspase-1 in macrophages (RAW264.7), generating an increase in the transcription of IL-1β and promoting the recruitment of infiltrated immune cells in the gastric mucosa. In addition, CtkA may be able to decrease cell viability through the Fas receptor. Recently, a study showed that CtkA is expressed from its native host and can induce stimulation of a pro-inflammatory response from gastric epithelial cells. This interaction is dependent upon a complement of the tfs3 T4SS genes but independent of the T4SS proteins encoded by either tfs4 or the *cag* PAI [45].

The function of the genes *jhp0945*-*jhp0947-jhp0949* is not yet well understood. However, it has been proposed that since they are consecutive genes and are oriented in the same direction, it is possible that they are expressed as an operon [30]. A study of the Dutch population revealed that the presence of these three genes in *H. pylori* strains induced higher amounts of IL-12 than in strain 26695, which does not possess these genes. However, the disruption of this locus reduces the production of IL-12 in THP-1 monocytic cells [32]. The presence of these three genes in strains from India induces a greater amount of IL-8 and induction of cell death by apoptosis (caspase-3 activity) in AGS cells than in strains that lack these genes [22]. *jhp0947* shows homology with *jhp0938* (*hp0990*) and *jhp0253* (*hp1333*), but their functions are still unknown. This gene also shows homology in the 5′ region with *jhp0477* (*hp0528*), which encodes a *virB9* homologue, an important component of the T4SS encoded by the *cag* PAI [46].

#### **7. Conclusion**

was assessed in 17 strains from patients with gastric cancer and 26 strains from patients with gastritis. The results showed a high prevalence of *jhp0940* and *jhp0947* in patients with gastric cancer [30]. Later, in a study conducted in Brazil that included 200 *H. pylori* isolates from patients with duodenal ulcers, gastric cancer or gastritis, only *jhp0947* continued to show an association with gastric cancer and duodenal ulcers [23]. However, in another study of strains from Brazil, an association between *jhp0947* and peptic ulcers was not observed [31]. A different study assessed the prevalence of the *jhp0945-jhp947-jhp0949* locus in a Dutch population with gastritis and duodenal ulcers. In addition to *jhp0947*, the presence of *jhp0949* was associated with duodenal ulcers, whereas *jhp0945* was not associated with this disease [32]. Another cluster of genes for which the prevalence was examined consisted of *jhp0926*, *jhp0931*, *jhp0933*, *jhp0944* and *jhp0945* in isolates from Turkish patients with gastritis and peptic ulcers. Among

Yakoob et al. [27] observed that the *jhp0947* gene is more frequently present than *jhp0940* in strains associated with duodenal ulcers and gastric cancer. This association was determined to be independent of the presence of the virulence factor *cagA* in *H. pylori* strains. A study that included 296 Western isolates (from the United States and Colombia) and 217 East Asian isolates (from Korea and Japan) reported that the prevalence of *jhp0945*, *jhp0947* and *jhp0949* differs significantly between the two geographical regions. In the Western isolates, the presence of *jhp0945* was higher in isolates obtained from individuals with gastric ulcers, duodenal

In *H. pylori* isolates of Chinese origin, the prevalence of *jhp0945*, *jhp0947* and *jhp0949* was significantly higher in individuals with duodenal ulcers and gastric cancer than in individuals with chronic gastritis [24]. Similarly, in isolates from India, the presence of *jhp0945*, *jhp0947* and *jhp0949* in *H. pylori* isolates was associated with disease [22]. The prevalence of *jhp0945*, *jhp0947* and *jhp0949* is associated with a greater risk of serious diseases in India [28]. PZ-associated genes (outside of locus *jhp0945*-*jhp0947*-*jhp0949*) that also have exhibited an association with disease include *jhp0950* and *jhp0917*-*jhp0918*. The *jhp0950* gene was associated with marginal zone B cell lymphomas (MZBL) and mucosa-associated lymphoid tissue (MALT) when its prevalence was examined in patients with gastritis, duodenal ulcers and gastric cancer [34]. Another gene considered a risk factor for the development of duodenal ulcers is the *dupA* gene (duodenal ulcer-promoting gene, *jhp0917*-*jhp0918*); its presence in strains from patients

these genes, *jhp0931* was the most prevalent in patients with peptic ulcers [33].

82 Helicobacter Pylori - New Approaches of an Old Human Microorganism

ulcers or gastric cancer than in those obtained from individuals with gastritis [21].

with this gastroduodenal pathology resulted in its association with this condition.

countries and 64% of strains in Western countries [36–40].

Predominant inflammation in the antrum region of the stomach as well as the infiltration of polymorphonuclear leukocytes can lead to the appearance of a duodenal ulcer [35]. The prevalence of the *dupA* gene among *H. pylori* strains varies according to the geographic region and duodenal pathology. The *dupA* gene is present in approximately 31% of strains in Asian

However, two genotypes were observed for this gene, including strains with and without an extra 600 bp in the gene sequence [41]; another important characteristic is that it has high homology with VirB4, a component of the type IV secretion system (T4SS) of *H. pylori*, and recent studies suggest that *dupA* and the six homologues of adjacent vir genes (virB8-virB11, virD4 and virD2) in the PZ could form the third T4SS [42]. Many unanswered questions still The PZ of the *H. pylori* genome contains several genes that have not been fully explored but could be important for understanding the pathogenesis of *H. pylori* due to their location in an area of the genome associated with genetic exchange. Among the most studied genes are *jhp0945*, *jhp0947* and *jhp0949*, which have been found to be associated with gastroduodenal disease, although their mechanism is still not clearly defined. However, the prevalence and association of *jhp0940* with ulcer or gastric cancer is still not entirely clear, although progress has been made in the characterisation of the function of this gene. It is important to continue exploring the presence and *in vivo* expression of these genes in strains isolated from children and adults from different geographical regions to elucidate their potential role in the pathogenesis of *H. pylori* infection.

[6] Gressmann H, Linz B, Ghai R, Pleissner KP, Schlapbach R, Yamaoka Y, et al. Gain and loss of multiple genes during the evolution of *Helicobacter pylori*. PLoS Genetics.

*Helicobacter pylori* Genes *jhp0940*, *jhp0945*, *jhp0947* and *jhp0949* are Associated with Gastroduodenal Disease

http://dx.doi.org/10.5772/intechopen.81290

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#### **Acknowledgements**

We thank Mariana Espinosa and Miguel Angel Rojas for the help in generating the figures. The publication of this chapter was supported by funds from the National Institute of pediatrics, Mexico. No conflicts of interest exist for this paper.

#### **Author details**

Romo-González Carolina\* and Coria-Jiménez Rafael

\*Address all correspondence to: crgaro\_06@yahoo.com.mx

Laboratory of Experimental Bacteriology, National Institute of Pediatrics, Mexico City, Mexico

#### **References**


[6] Gressmann H, Linz B, Ghai R, Pleissner KP, Schlapbach R, Yamaoka Y, et al. Gain and loss of multiple genes during the evolution of *Helicobacter pylori*. PLoS Genetics. 2005;**1**:e43. DOI: 10.1371/journal.pgen.0010043

characterisation of the function of this gene. It is important to continue exploring the presence and *in vivo* expression of these genes in strains isolated from children and adults from different geographical regions to elucidate their potential role in the pathogenesis of *H. pylori* infection.

We thank Mariana Espinosa and Miguel Angel Rojas for the help in generating the figures. The publication of this chapter was supported by funds from the National Institute of pediat-

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**Section 3**

**Helicobacter Pylori and Eradication Therapies**


**Helicobacter Pylori and Eradication Therapies**

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88 Helicobacter Pylori - New Approaches of an Old Human Microorganism

DOI: 10.1073/pnas.1010153107

DOI: 10.1046/j.1462-5822.2003.00286.x

**Chapter 7**

**Provisional chapter**

**Gastric Microbiota and Resistance to Antibiotics**

**Gastric Microbiota and Resistance to Antibiotics**

DOI: 10.5772/intechopen.80662

Studies on gastric microbiota find several bacterial families and species in the stomach using molecular-based techniques. When biopsies are cultured, there may be growth of bacteria, pure culture of *Helicobacter pylori*, or no growth. When looking at the histological sections of corresponding biopsies no bacteria may be seen, except curved rods (*H. pylori*) adherent to the gastric epithelial cells. In a number of biopsies, several different bacteria are cultured with or without *H. pylori*. The non–*H. pylori* bacteria cultured are like the normal oral flora and may be contamination of the samples during endoscopy. In histological sections, these bacteria are seen above the mucin layer and not adherent to the epithelial cells confirming that it is contamination of the samples and can thus not be regarded as gastric microbiota. Therefore, the susceptibility of *H. pylori* to antibiotics is independent of coexisting bacterial flora. A review of *H. pylori* susceptibility to antibiotics in untreated and previous treated patients will be given including meta-analyses of *H. pylori* susceptibility to metronidazole (MTZ), clarithromycin, and levofloxacin. These data indicate that these antibiotics become more doubtful to use for primary therapy and

should be banned for secondary therapy without susceptibility testing.

**Keywords:** gastric microbiota, *H. pylori*, histology, susceptibility testing, resistant rates

Microbiota and microbiome are not always clearly defined or distinguished. The human microbiota comprises the population of microbial species that live on or in the human body. This is the resident flora of the body and does not include the transient flora (sampling contamination, etc.).

> © 2016 The Author(s). Licensee InTech. 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.

© 2018 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.

Agnes Tving Stauning,

**Abstract**

**1. Introduction**

Agnes Tving Stauning,

Rie Louise Møller Nordestgaard,

Rie Louise Møller Nordestgaard,

http://dx.doi.org/10.5772/intechopen.80662

Tove Havnhøj Frandsen and Leif Percival Andersen

Tove Havnhøj Frandsen and Leif Percival Andersen

Additional information is available at the end of the chapter

Additional information is available at the end of the chapter

#### **Gastric Microbiota and Resistance to Antibiotics Gastric Microbiota and Resistance to Antibiotics**

DOI: 10.5772/intechopen.80662

Agnes Tving Stauning, Rie Louise Møller Nordestgaard, Tove Havnhøj Frandsen and Leif Percival Andersen Agnes Tving Stauning, Rie Louise Møller Nordestgaard, Tove Havnhøj Frandsen and Leif Percival Andersen

Additional information is available at the end of the chapter Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/intechopen.80662

#### **Abstract**

Studies on gastric microbiota find several bacterial families and species in the stomach using molecular-based techniques. When biopsies are cultured, there may be growth of bacteria, pure culture of *Helicobacter pylori*, or no growth. When looking at the histological sections of corresponding biopsies no bacteria may be seen, except curved rods (*H. pylori*) adherent to the gastric epithelial cells. In a number of biopsies, several different bacteria are cultured with or without *H. pylori*. The non–*H. pylori* bacteria cultured are like the normal oral flora and may be contamination of the samples during endoscopy. In histological sections, these bacteria are seen above the mucin layer and not adherent to the epithelial cells confirming that it is contamination of the samples and can thus not be regarded as gastric microbiota. Therefore, the susceptibility of *H. pylori* to antibiotics is independent of coexisting bacterial flora. A review of *H. pylori* susceptibility to antibiotics in untreated and previous treated patients will be given including meta-analyses of *H. pylori* susceptibility to metronidazole (MTZ), clarithromycin, and levofloxacin. These data indicate that these antibiotics become more doubtful to use for primary therapy and should be banned for secondary therapy without susceptibility testing.

**Keywords:** gastric microbiota, *H. pylori*, histology, susceptibility testing, resistant rates

#### **1. Introduction**

Microbiota and microbiome are not always clearly defined or distinguished. The human microbiota comprises the population of microbial species that live on or in the human body. This is the resident flora of the body and does not include the transient flora (sampling contamination, etc.).

© 2016 The Author(s). Licensee InTech. 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. © 2018 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.

The microbiome is constituted by all the genes inside these microbial cells and is thus restricted to detection by molecular methods (sequencing, polymerase chain reactions [PCR]) [1].

up free DNA with resistance genes from the environment (transcription) or DNA can be transferred by bacteriophages (transduction). Furthermore, mutations can occur in the bacterial genome which may result in resistance if the mutation occurs in the part of the genome that codes for a structure on which the antibiotics act; this action may be interfered, and the bacteria becomes resistant to the antibiotic [10–12]. The conjugation of plasmids increases with the number of different bacteria in the microbiota and depends on a close contact between the bacteria. Uptake of free DNA does not demand a direct contact with other bacteria, but bacteria should probably be present in the close environment [3]. Mutations occur in all bacteria with a certain time because of natural replication errors [12]. Some bacteria mutate more often than others; but because of the short generation time for bacteria, each bacterial clone will have several mutations. If the mutation occurs in a part of the genome, which is target for the antibiotics, resistance to

Gastric Microbiota and Resistance to Antibiotics http://dx.doi.org/10.5772/intechopen.80662

In a previous unpublished study that included 411 biopsies from patients undergoing upper gastrointestinal endoscopy were investigated both by microaerobic culture and by histology (**Table 1**). From 249 (60%) biopsies other bacteria than *H. pylori* were cultured. These bacteria were oral flora, that is, *Streptococcus* spp., *Staphylococcus* spp., *Corynebacterium* spp., *Neisseria* spp., etc., which may indicate contamination of both the endoscope and the biopsies during the procedure. In histological sections, very few bacteria except *H. pylori* were seen in 20 (5%) of the biopsies. In all cases, the bacteria were located superficial to the mucus layer and not in relation to the epithelial cells and *H. pylori*, which confirm that it is contamination from the oral cavity. The discrepancy in the number of biopsies with other bacteria than *H. pylori* between culture and histology may be because very few bacteria (less than 5 colonies) are cultured and the preparation of histological sections may remove much of the mucin and the contaminating bacteria. *H. pylori* was found alone without contamination in 60 biopsies by culture and in 83 biopsies by histology which indicate that *H. pylori* is a true gastric micro-

All known mechanisms for *H. pylori* resistance to all antibiotics are point mutations located on the chromosome (**Table 2**), indicating no uptake of plasmids or free DNA, which support that *H. pylori* is the only bacteria in the true gastric microbiota and everything else is transient

**Table 1.** Comparison of culture and histological finding of *H. pylori* and other bacteria (oral flora) in gastric biopsies.

*H. pylori* **Other bacteria** *H. pylori* **Other bacteria**

**No. of biopsies Culture Histology**

411 106 249 83 20

the antibiotic may occur.

biota (**Figure 2**).

contaminating flora [13].

**2. Study on gastric microbiota**

By molecular methods, bacteria are usually identified to family and genera level [2]. Bacterial families and genera may include species and types of bacteria that may have completely opposite actions in the human body [3]. It is, therefore, doubtful if molecular methods alone are sensitive enough to predict the effect of the composition of microbiota. The limited original literature on gastric microbiota has mainly focused on gastric cancer and contains conflicting results [4–7]. There are many difficulties in investigating the gastric microbiota. One thing many authors are not aware of is the difficulty of getting samples without contaminating bacterial flora (**Figure 1**) [8]. In animal models, the whole stomach can be removed, and contamination of the stomach can be avoided, but in most animal species, physiology, acidity, etc. of the stomach are very different from the human stomach. Samples from the human stomach are usually taken as biopsies during gastroscopy. Even though the endoscope and the forceps are sterilized or decontaminated, it will be contaminated with oral bacterial flora during gastroscopy and thereby will the samples be contaminated by oral flora mainly of the phyla *Firmicutes* [8, 9].

Bacterial resistance to antibiotics can occur either if the bacteria obtain plasmids containing resistance genes from other bacteria in the microbiota (conjugation); they can take

**Figure 1.** Schematic illustration of the gastric mucosa with the main cell types of oxyntic and pyloric glands in the gastric epithelium. Gastric stem cells reside in the isthmus zone of the gland and differentiate into precursors of the different cell lineages, which migrate either apically toward the gastric lumen or downwards to the base. The superficial epithelium and the gastric glands are covered by a viscous mucus layer mainly composed of MUC5AC, secreted by the SMCs, and MUC6, secreted mainly by MNCs and antral gland cells. The mucus layer consists of an inner layer, which is firmly attached to the epithelium, and an outer loose layer. The gastric pathogen *Helicobacter pylori* has been shown to use the transmucus pH gradient between the acidic gastric lumen and the near-neutral epithelial surface for spatial orientation to reach its niche at the juxtamucosal epithelium. The precise location of non–*H. pylori* microbiota is still hypothetical. [8].

up free DNA with resistance genes from the environment (transcription) or DNA can be transferred by bacteriophages (transduction). Furthermore, mutations can occur in the bacterial genome which may result in resistance if the mutation occurs in the part of the genome that codes for a structure on which the antibiotics act; this action may be interfered, and the bacteria becomes resistant to the antibiotic [10–12]. The conjugation of plasmids increases with the number of different bacteria in the microbiota and depends on a close contact between the bacteria. Uptake of free DNA does not demand a direct contact with other bacteria, but bacteria should probably be present in the close environment [3]. Mutations occur in all bacteria with a certain time because of natural replication errors [12]. Some bacteria mutate more often than others; but because of the short generation time for bacteria, each bacterial clone will have several mutations. If the mutation occurs in a part of the genome, which is target for the antibiotics, resistance to the antibiotic may occur.

#### **2. Study on gastric microbiota**

The microbiome is constituted by all the genes inside these microbial cells and is thus restricted

By molecular methods, bacteria are usually identified to family and genera level [2]. Bacterial families and genera may include species and types of bacteria that may have completely opposite actions in the human body [3]. It is, therefore, doubtful if molecular methods alone are sensitive enough to predict the effect of the composition of microbiota. The limited original literature on gastric microbiota has mainly focused on gastric cancer and contains conflicting results [4–7]. There are many difficulties in investigating the gastric microbiota. One thing many authors are not aware of is the difficulty of getting samples without contaminating bacterial flora (**Figure 1**) [8]. In animal models, the whole stomach can be removed, and contamination of the stomach can be avoided, but in most animal species, physiology, acidity, etc. of the stomach are very different from the human stomach. Samples from the human stomach are usually taken as biopsies during gastroscopy. Even though the endoscope and the forceps are sterilized or decontaminated, it will be contaminated with oral bacterial flora during gastroscopy and thereby will the samples be contaminated by oral flora mainly of the

Bacterial resistance to antibiotics can occur either if the bacteria obtain plasmids containing resistance genes from other bacteria in the microbiota (conjugation); they can take

**Figure 1.** Schematic illustration of the gastric mucosa with the main cell types of oxyntic and pyloric glands in the gastric epithelium. Gastric stem cells reside in the isthmus zone of the gland and differentiate into precursors of the different cell lineages, which migrate either apically toward the gastric lumen or downwards to the base. The superficial epithelium and the gastric glands are covered by a viscous mucus layer mainly composed of MUC5AC, secreted by the SMCs, and MUC6, secreted mainly by MNCs and antral gland cells. The mucus layer consists of an inner layer, which is firmly attached to the epithelium, and an outer loose layer. The gastric pathogen *Helicobacter pylori* has been shown to use the transmucus pH gradient between the acidic gastric lumen and the near-neutral epithelial surface for spatial orientation to reach its niche at the juxtamucosal epithelium. The precise location of non–*H. pylori* microbiota is still hypothetical. [8].

to detection by molecular methods (sequencing, polymerase chain reactions [PCR]) [1].

92 Helicobacter Pylori - New Approaches of an Old Human Microorganism

phyla *Firmicutes* [8, 9].

In a previous unpublished study that included 411 biopsies from patients undergoing upper gastrointestinal endoscopy were investigated both by microaerobic culture and by histology (**Table 1**). From 249 (60%) biopsies other bacteria than *H. pylori* were cultured. These bacteria were oral flora, that is, *Streptococcus* spp., *Staphylococcus* spp., *Corynebacterium* spp., *Neisseria* spp., etc., which may indicate contamination of both the endoscope and the biopsies during the procedure. In histological sections, very few bacteria except *H. pylori* were seen in 20 (5%) of the biopsies. In all cases, the bacteria were located superficial to the mucus layer and not in relation to the epithelial cells and *H. pylori*, which confirm that it is contamination from the oral cavity. The discrepancy in the number of biopsies with other bacteria than *H. pylori* between culture and histology may be because very few bacteria (less than 5 colonies) are cultured and the preparation of histological sections may remove much of the mucin and the contaminating bacteria. *H. pylori* was found alone without contamination in 60 biopsies by culture and in 83 biopsies by histology which indicate that *H. pylori* is a true gastric microbiota (**Figure 2**).

All known mechanisms for *H. pylori* resistance to all antibiotics are point mutations located on the chromosome (**Table 2**), indicating no uptake of plasmids or free DNA, which support that *H. pylori* is the only bacteria in the true gastric microbiota and everything else is transient contaminating flora [13].


**Table 1.** Comparison of culture and histological finding of *H. pylori* and other bacteria (oral flora) in gastric biopsies.

Histology is an invasive method which requires a least one antral biopsy and preferably two antral and two corpus biopsies. The biopsy is stained with hematoxylin and eosin, Giemsa, or silver staining. *H. pylori* is identified by the color, shape, and close relation to the mucosa and can be confirmed by immunohistochemistry using *H. pylori–*specific antibodies. The histology has shown to have a sensitivity at the same level as culture but is influenced by the size of the biopsy [14]. The number of biopsies and the location in the stomach also modify the sensitivity. The specificity of histology is lower than the specificity of the culture as histology cannot distinguish *H. pylori* from non–pylori *Helicobacter* species. The detection rates in cultures and histology varies with varying expertise of examiners. If the patient is taking proton pump inhibitor (PPI), bismuth, or antibiotics prior to gastroscopy, it might change the shape of *H. pylori* from curved rod to a coccoid form. This form is undetectable in the routine microscopy technique and requires fluorescent *in situ* hybridization, immunohistochemistry with specific antibodies to *H. pylori,* or confirmation by the 16s rRNA and 23rRAN sequencing, which are irrespective of the shape of the bacteria [16]. *H. pylori* urease breaks down urea to ammonia and carbon dioxide. This feature is used in the diagnostic methods "rapid urease test" (RUT) and "urea breath test" (UBT). RUT is an invasive method that preferably needs two biopsies. If the biopsy contains *H. pylori,* the release of ammonia increases the pH of the test medium, which is seen by a color change due to a pH indicator. The result of the test is fast and takes approximately ½ hour. UBT is a noninvasive method where the patient ingests 13C-labeled urea. If the patient is infected with *H. pylori,* orally ingested 13C-urea is broken down to 13C-labeled carbon dioxide, which is then exhaled. The sensitivity of the two tests is 75–85% for RUT and >95% for UBT. Likewise, the UBT has a higher specificity (<95%) when compared to RUT (85–95%). For both RUT and UBT, PPI and antibiotics can give false negative results. Furthermore, coccoid forms of *H.* 

Gastric Microbiota and Resistance to Antibiotics http://dx.doi.org/10.5772/intechopen.80662

*pylori* would not produce urease and would therefore give a false negative result [17].

to false negative results.

Stool antigen test is another noninvasive method. It was first successfully described in 1997 using polyclonal antibodies [18]. Today monoclonal antibodies are used, and the sensitivity and the specificity are at the same levels as for UBT, but are preferred in special patients like children and patients with bleeding ulcers. This test can be done within ½ hour and is good for screening a patient for an infection with *H. pylori*. Despite this, antigen excretion may vary over time, and antigens may degrade while passing through the intestines, which may lead

The humoral antibody response to *H. pylori* can be measured by either serum IgG antibodies to *H. pylori*, which shows an ongoing or a previous infection, or by serum IgM antibodies, which shows an ongoing acute infection. *H. pylori* IgG antibodies can be detected in sputum or urine but have a much lover sensitivity and specificity than serum antibodies. Antibodies to *H. pylori* in serum can be tested by ELISA or "near patient test (NPT)." NPT uses immunechromatography or passive agglutination. A 2013 study compared the NPT and the ELISA test. The study showed that the NPT never reach 90% in sensitivity, and the frequency of false negatives and false positives were high [19]. Several tested ELISA kits showed a high specificity and sensitivity above 90%. However, the serological kits may differ considerably depending on the antigens that are included in the kit as antibodies to low-molecular-weight antigens (outer membrane antigens) decline significantly within 3 months, whereas antibodies to high-molecular-weight antigens (CagA, VacA, etc.) may stay potent for years [20]. CagA antibodies remain stable for a long period of time and can probably be useful for the detection

**Figure 2.** Imprint cytology showing the presence of *H. pylori* (Giemsa stain, ×400) Rahbar [84].


**Table 2.** Examples of mutations in *H. pylori* causing resistance to antibiotics.

#### **3. Diagnosis of** *H. pylori*

The detection of *H. pylori* can be done by invasive and noninvasive methods. The invasive methods require a biopsy, whereas the noninvasive methods are gentler for the patient.

Culture of *H. pylori* may be difficult and the sensitivity may be rather low (50–85%) [14]. The sensitivity of the culture depends on transport time to the lab and the culture method used [15]. Different agar plates or incubation time can also give different results on the same biopsy. Two biopsies from the antrum and two biopsies from the fundus are preferred when making a culture as *H. pylori* is unevenly distributed in the stomach. Culture is the only method by which it is possible to make a full susceptibility test.

Histology is an invasive method which requires a least one antral biopsy and preferably two antral and two corpus biopsies. The biopsy is stained with hematoxylin and eosin, Giemsa, or silver staining. *H. pylori* is identified by the color, shape, and close relation to the mucosa and can be confirmed by immunohistochemistry using *H. pylori–*specific antibodies. The histology has shown to have a sensitivity at the same level as culture but is influenced by the size of the biopsy [14]. The number of biopsies and the location in the stomach also modify the sensitivity. The specificity of histology is lower than the specificity of the culture as histology cannot distinguish *H. pylori* from non–pylori *Helicobacter* species. The detection rates in cultures and histology varies with varying expertise of examiners. If the patient is taking proton pump inhibitor (PPI), bismuth, or antibiotics prior to gastroscopy, it might change the shape of *H. pylori* from curved rod to a coccoid form. This form is undetectable in the routine microscopy technique and requires fluorescent *in situ* hybridization, immunohistochemistry with specific antibodies to *H. pylori,* or confirmation by the 16s rRNA and 23rRAN sequencing, which are irrespective of the shape of the bacteria [16].

*H. pylori* urease breaks down urea to ammonia and carbon dioxide. This feature is used in the diagnostic methods "rapid urease test" (RUT) and "urea breath test" (UBT). RUT is an invasive method that preferably needs two biopsies. If the biopsy contains *H. pylori,* the release of ammonia increases the pH of the test medium, which is seen by a color change due to a pH indicator. The result of the test is fast and takes approximately ½ hour. UBT is a noninvasive method where the patient ingests 13C-labeled urea. If the patient is infected with *H. pylori,* orally ingested 13C-urea is broken down to 13C-labeled carbon dioxide, which is then exhaled. The sensitivity of the two tests is 75–85% for RUT and >95% for UBT. Likewise, the UBT has a higher specificity (<95%) when compared to RUT (85–95%). For both RUT and UBT, PPI and antibiotics can give false negative results. Furthermore, coccoid forms of *H. pylori* would not produce urease and would therefore give a false negative result [17].

Stool antigen test is another noninvasive method. It was first successfully described in 1997 using polyclonal antibodies [18]. Today monoclonal antibodies are used, and the sensitivity and the specificity are at the same levels as for UBT, but are preferred in special patients like children and patients with bleeding ulcers. This test can be done within ½ hour and is good for screening a patient for an infection with *H. pylori*. Despite this, antigen excretion may vary over time, and antigens may degrade while passing through the intestines, which may lead to false negative results.

The humoral antibody response to *H. pylori* can be measured by either serum IgG antibodies to *H. pylori*, which shows an ongoing or a previous infection, or by serum IgM antibodies, which shows an ongoing acute infection. *H. pylori* IgG antibodies can be detected in sputum or urine but have a much lover sensitivity and specificity than serum antibodies. Antibodies to *H. pylori* in serum can be tested by ELISA or "near patient test (NPT)." NPT uses immunechromatography or passive agglutination. A 2013 study compared the NPT and the ELISA test. The study showed that the NPT never reach 90% in sensitivity, and the frequency of false negatives and false positives were high [19]. Several tested ELISA kits showed a high specificity and sensitivity above 90%. However, the serological kits may differ considerably depending on the antigens that are included in the kit as antibodies to low-molecular-weight antigens (outer membrane antigens) decline significantly within 3 months, whereas antibodies to high-molecular-weight antigens (CagA, VacA, etc.) may stay potent for years [20]. CagA antibodies remain stable for a long period of time and can probably be useful for the detection

**3. Diagnosis of** *H. pylori*

which it is possible to make a full susceptibility test.

The detection of *H. pylori* can be done by invasive and noninvasive methods. The invasive methods require a biopsy, whereas the noninvasive methods are gentler for the patient.

**Figure 2.** Imprint cytology showing the presence of *H. pylori* (Giemsa stain, ×400) Rahbar [84].

rp1V A2142C A2142G A2143G

frxA fdxB

gyrB

**Resistance to Mutation** Amoxicillin PBP1 Clarithromycin InfB

94 Helicobacter Pylori - New Approaches of an Old Human Microorganism

Metronidazole rdxA

Fluoroquinolones gyrA

Tetracycline AGA925-967TTC

**Table 2.** Examples of mutations in *H. pylori* causing resistance to antibiotics.

Rifampicin RNA polymerase subunit beta/beta

Culture of *H. pylori* may be difficult and the sensitivity may be rather low (50–85%) [14]. The sensitivity of the culture depends on transport time to the lab and the culture method used [15]. Different agar plates or incubation time can also give different results on the same biopsy. Two biopsies from the antrum and two biopsies from the fundus are preferred when making a culture as *H. pylori* is unevenly distributed in the stomach. Culture is the only method by of *H. pylori* infections in patients with gastric cancer when other tests are negative [21]. Due to local strain distribution of *H. pylori,* the serology kits should be made by using local *H. pylori* strains, and the kits should be locally validated [21].

Gastrin and pepsinogen are compounds produced in the stomach that depend on the changes in the gastric mucosa, and the serum levels of pepsinogens are a marker of atrophic gastritis [22]. This can be combined with the *H. pylori* antibody test to predict the risk of developing gastric cancer.

Molecular methods have been of increasing interest in the field of microbiology and for detection of *H. pylori*. Polymerase chain reaction (PCR) seems to be more sensitive than any other method to detect *H. pylori* [23]. The main problem is that the method does not distinguish between live bacteria and DNA from dead bacteria. Real-time PCR (RT-PCR), which is a fast and quantitative PCR, seems to be more sensitive than classical PCR [24]. By sequencing the 16S RNA or 23S RNA region, it is possible to detect *Helicobacter* species and susceptibility to clarithromycin and tetracycline [25–27]. However, it is a more expensive and time-consuming method. A commercial kit has combined detection of *H. pylori* and susceptibility to clarithromycin in a classical PCR. However, culture is still needed for a full susceptibility testing. There are so many point mutations causing resistance to antibiotics in *H. pylori* that a full susceptibility analysis can only be detected by whole genome sequencing [28].

growth of *H. pylori*. This is the inhibition zone, and the diameter of the zone can be translated to an MIC value, which shows whether or not the bacteria are resistant to the antibiotic. To make the susceptibility testing of *H. pylori*, a McFarland 3.0 dilution of *H. pylori* and Mueller-Hinton agar plates with 10% blood or chocolate ager plates should be used and incubated in

**Figure 3.** Reading guide for E tests. (A) Colonies of a metronidazole-resistant subpopulation in the ellipse minimum inhibitory concentration (MIC) >32; (B) trailing of microcolonies at the end point MIC 0.5 μg/ml. Warburton-Timms and

Gastric Microbiota and Resistance to Antibiotics http://dx.doi.org/10.5772/intechopen.80662

The E-test is a stripe with a concentration gradient of an antibiotic. The stripe is placed on the agar plate and is incubated for 3 days. After 3 days, there will be a droplet shape around the stripe with no growth of *H. pylori* (**Figure 3**). That concentration where *H. pylori* grows close to

*H. pylori* infections are usually treated with a combination of antibiotics and nonantibiotics (proton pump inhibitor [PPI] or bismuth salts). Usually, a combination of two or three antibiotics is used, as the effect of monotherapy has been found insufficient. The most commonly used antibiotics are amoxicillin, clarithromycin, metronidazole, fluoroquinolones, tetracy-

*H. pylori* is found in very different environments such as the gastric lumen with a relatively low pH, in between the epithelial cells and on the basement membrane with a neutral pH but protected as intracellular microorganisms. When choosing antibiotics, it is important to select antibiotic to which *H. pylori* is sensitive and is active in all the environmental niches where *H. pylori* occurs. It is also important to look at the duration of the efficacy of antibiotics to keep

PPI in standard doses do not have antibacterial effect on *H. pylori*, but 5–10 times higher doses have a direct effect on *H. pylori*. Bismuth salts binds to the surface of *H. pylori* but have

microaerobic conditions at 37°

McNulty [85].

stripe is the MIC value [33].

cline, and rifampicin (**Table 3**).

**5. Treatment of** *H. pylori* **infection**

C.

stable levels above the minimal inhibitory concentrations.
