Helicobacter pylori is a Gram-negative spiral shaped microaerophillic bacterium whose ecological niche is the human stomach. It has been
seen that it colonizes over half of the world's population and is the most common bacterial infection worldwide. It is generally acquired during childhood and the infection can persist throughout the life span of the host, if untreated (Evehert JE 2000). In approximately 20% of infected individuals, infection with H. pylori may result in clinical disease that manifests as peptic or duodenal ulcers, gastric adenocarcinoma, or mucosa-associated lymphoid tissue (MALT) lymphoma;(Parsonnet J et al 1991, 1994, Pritchard DM et al 2006, Uemura N et al 2001) these last two manifestations have led H. pylori to be the only bacterium classified as a class I carcinogen by the World Health Organization (IARC 1994). The geographical distribution of Helicobacter pylori differs amongst developing and developed countries that is > 80% in developing countries like India and < 20% in developed countries Mitchell HM 1998, Pounder RR 1995, Parsonnet J 1995).
Indian population is polygenetic and is an amazing amalgamation of diverse races and cultures thus its inhabitants also differ in their genetic traits. The prevlance of gastroduodenal diseases and cancers is high in the North-eastern region of India similar to China and Japan. Till now it was believed that dietry factors are responsible for high prevalence of such gastroduodenal diseases in the North-east region because it consists of a geographically, culturally and ethnically divergent population (Phukan et al 2006).
Since only 20% of the infected individuals develop various gastroduodenal diseases it is possible that the strain specific genetic diversity plays a role in the pathogenesis of H.pylori. It has been suggested that the disease causation by H.pylori is due to the various virulence factors. H.pylori strains vary in the degree of virulence (Atherton JC 1997, Blaser MJ, et al 1995). In particular, the cagA, vacA and iceA genes are considered to be the putative virulence factors of H.pylori (Yamaoka Y et al 1999). Based on the presence and absence of these virulence factors the H.pylori strains are divided into Type I and Type II respectively (Censini S et al 1996, Xiang Z et al 1995). Duodenal ulcer patients are more likely to be infected with the Type I strains (Xiang Z et al 1993, 1995).
CagA protein is one of the best studied virulence factor of H.pylori, the toxin is 120-145 kDa in size and is encoded by the cagA gene which is the part of a 40 kb DNA segment cag pathogenicity island (cagPAI) (Censini S et al 1996). The strains that carry the cagPAI are known to be more virulent than those that do not (Censini S et al 1996). Apart from cagA, the cagPAI encodes several other genes which constitutes the Type IV secretion system(T4SS) which is known to be involved in the translocation of the CagA protein in the host cells (Seagal ED et al 1999, Odenbreit S et al 2000, Stein M et al 2000). The role of CagA is multifactorial as it is required for both acquisition of nutrients and persistence of H. pylori (Tan S et al 2009, Amieva MR et al 2003).
The second toxin in the arsenal of H.pylori is VacA. Unlike cagA, vacA is present in virtually all the H.pylori strains (Blaser MJ et al 2004, Rassow J et al 2012). It is a pore forming toxin. Similar to cagA, vacA also contains allelic diversity that varies across H.pylori strains (Atherton JC et al 1995). There are four identified variable regions in VacA i.e s (signal) region which is typed as s1and s2,m (middle) region which is typed as m1 and m2, i (intermediate) region and d (deletion) region (Atherton JC et al 1995). The vacA can recombine to form many recombinations of which vacA s1/m1 alleles are the most virulent combination, while the s1/m2 and s2/m2 genotypes display virtually no cytotoxicity (Atherton JC et al 1995, Meining A et al 1997, Miehlke S et al 1998, 2000, Letley DP et al 2000, 2003, McClain MS et al 2001).
Another putative virulent factor of H.pylori is iceA gene which is a signalling gene,induced by contact with gastric epithelium. There are two allelic types of the gene that is iceA1 and iceA2.Product of iceA1 is homologous to a restriction endonuclease, NlaIII in Neisseria lactamica (Peek RM et al 1998, Van Doors et al 1998).
As virulence markers of H.pylori are not associated with diseases in all the geographical regions, its eradication provides the best and most effective treatment for H.pylori associated diseases (Mukhopadhyay AK et al 2000, Chattopadhyay S et al 2002, Datta S et al 2003, Graham DY et al 1998). Commonly used antimicrobial agents for the eradication of H.pylori are Metronidazole (Mtz), Clarithromycin (Cla), Amoxicillin (Amx), Furazolidone (Fz) and Tetracycline (Tet) etc. Resistance to some of the potentially useful antimicrobial agents, such as metronidazole (Mtz) and Clarithromycin (Cla) is very common. The increased prevalence of antibiotic-resistant H. pylori strains has complicated the efforts to eradicate infection. Therefore, culture and susceptibility tests can prove to be of great value in helping clinicians to choose the most promising therapy for a particular patient or population group (geographical region).
3. REVIEW OF LITERATURE
3.1 Helicobacter pylori
Helicobacter pylori is a species of epsilon proteobacteria which colonizes the harsh environment of the human which colonizes the harsh environment of the human stomach (Chalmers et al 2004). Its name signifies both its spiral shape (Helicobacter) and the area of the lower stomach which it habitually colonizes: the gateway (pylorus) between the stomach and the small intestine (Meyers, 2007). H.pylori is regarded as the most widespread infection in man and has been linked to the development of a number of medical conditions (Chalmers et al 2004). Most infections are believed to be acquired during childhood and can persist for decades (Blaser M J 1995). Its prevalence is closely linked to socioeconomic factors such as low income and poor education and living conditions during childhood such as poor sanitation and overcrowding in developing countries (Webb P M et al 1994, Kurosawa M et al 2000). H pylori is known to be the most important causal agent of many gastroduodenal disease like gastritis, peptic ulcers, gastric cancer etc in humans and thus it is the only bacteria which is classified as the class I carcinogen by IARC (International Agency for Research on Cancer) ( Anonymous 1994).
During the 1970s, there was a sudden increase in the incidences of Peptic Ulcer Disease (PUD) and Gastritis. Throughout most of the 20th century, the conventional thinking was that peptic ulcer disease was caused by gastric juice corroding vulnerable mucosa; the dictum 'no acid- no ulcer' ruled the day and neutralization of the gastric acid was the mainstay of management (Martin B Van Der Wayden et al 2005). The modern era began with the ground breaking research of two Australians, Barry J Marshall and J Robin Warren. The attempts to culture these bacilli were unsuccessful until one lucky accident, in which the plates were unintentionally incubated for 5 days over the long Easter weekend, and the water spray like 1mm transparent colonies were visible in 1982 (Marshall and Warren 1984). They scuttled the prevailing acid-mucosal model by showing that peptic ulcer disease is an infectious disease caused by Campylobacter pylori later named as Helicobacter pylori due to its specific morphologic and structural features. For this breakthrough research, Marshall and Warren were awarded the 2005 Nobel Prize for Physiology or Medicine.
The cultivation of the novel bacterium from gastric mucosa in 1982 marked a turning point in the understanding of gastrointestinal microbial ecology and disease (Warren B J and Marshall). The isolated organism resembled Campylobacter in several aspects including curved morphology, growth on rich media under microaerophilic conditions, failure to ferment glucose, sensitivity to metronidazole, and a G + C content of 34%. It was therefore first referred to as "pyloric Campylobacter" particularly apt; pylorus is Greek for gatekeeper ' one who looks both ways (Owen R J 1998) and was later validated as Campylobacter pyloridis in 1985 (Anonymous 1985). But from its initial cultivation it was suspected that C.pylori is not a true Campylobacter. Early electron micrographs showed multiple sheathed flagella at one pole of the bacterium, in contrast to the single bipolar unsheathed flagellum typical of Campylobacter spp. (Goodwin C S et al 1985). On the basis of the gene sequence analysis of the 16S rRNA, it was renamed as Helicobacter pylori, the first member of the new genus Helicobacter (Goodwin C S et al 1989).
Class: Epsilon Proteobacteria
3.4 Microbial Characteristics
Helicobacter pylori is a non spore forming gram negative bacterium which is enclosed within two membranes (Curry and Jones, 1990). It is generally of spiral shape, 2.5 to 5 ??m long with four to six unipolar sheathed flagella which allows it to move with a corkscrew motility (Goodwin et al 1985). The combination of its curved shape and unipolar flagella allows H pylori to move easily through the thick mucus layer of the stomach (Salyers and Whitt 2002). Under stress it loses its spiral morphology and eventually become coccoid are not culturable (Bode et al, 1993, Nilius et al 1993). It is theorized that it is the coccal forms that are involved in the transmission of H. pylori. H.pylori possesses five major outer membrane protein (OMP) families. The largest family is of adhesins. The outer membrane of H pylori is made up of phospholipids and lipopolysaccharides (LPS). The O- specific chain of H. pylori LPS mimics the Lewis blood group antigens in structure (Appelmelk et al, 1996, Aspinall et al, 1996, Sherburne et al, 1995, Wirth et al, 1996), which is thought to contribute in pathogenesis. The expression of Lewis antigen on the surface of H pylori helps them to camouflage and thus aid in their survival (Wirth et al, 1997). It tests positive for oxidase and catalase.
Fig : The spiral morphology of Helicobacter pylori ?? 2008 Michelle Wiepjes
Helicobacter pylori is a microaerophillic organism that is, it thrives best in low oxygen environment (Helicobacter foundation 2006). Analysis of the H pylori genome, sequenced by Tomb et al (1997) indicated that it utilizes glucose as its primary carbon source for energy production.
Another interesting feature of H.pylori physiology is that it thrives at a neutral pH of 7.0. In order to protect itself from the acidic environment of the stomach, H.pylori penetrates the mucus lining of the stomach. H.pylori has a large urease enzyme protein that produces urease which breaks down the urea in the stomach into ammonia and bicarbonate both of which are strong bases which counteracts the acid in the stomach, thus maintaining a favourable environment for the survival of H.pylori (Chalmers et al 2004).
H.pylori obtains its nutrients by taking the advantage of the human inflammatory response. As a matter of fact the human body sends extra nutrients to the areas where there is an inflammation in order to help white blood cells attack the pathogen which in this case is the H.pylori. Since H.pylori is inaccessible to these cells because of their location within the mucus and are therefore able to utilize the nutrients without any threat to themselves.
Fig : Helicobacter pylori cells under the stomach mucus layer. ?? Michelle Wiepjes
3.4.3 Nutritional Requirements
A key feature of H.pylori is its microaerophilicity and capnophilicity with optimal growth at O 2 levels of 2-5% and an additional need of 5-10% CO 2 and high humidity. The standard microaerophillic condition for H.pylori is 85% N 2, 10% CO 2, 5% O 2. H.pylori are considered to be neutrophilic. The range of pH required for its growth ranges from 6.0 to 8.0; however, the ideal pH is around 7.0 (Scott D et al 1998). H.pylori is a fastidious microorganism and requires a complex basal media (either solid or liquid) with some form of supplementation such as whole blood, heme, serum, charcoal, cornstarch, egg yolk emulsion (Hachem et al, 1995, Henriksen et al, 1995), whose key function in addition to providing nutritional substrates may be detoxification of the medium and protection of the organism against the long chain fatty acids (Hazell et al 1990). For primary isolation and also for routine culture, selective antibiotics like Vancomycin, Polymixin B, Amphotericin B and Trimethoprim are used. In the absence of serum H.pylori becomes sensitive to Polymixin B and/or Trimethoprim (Testerman et al 2006).
A shift in the paradigm of bacteriology came in 1995 when the first complete genomic sequence of a free-living bacterium, Haemophilus influenza was released (Fleischmann R. D et al 1995). There has been an explosion of microbial genomic sequence data generated from a wide range of organisms, in the last 5 years, with almost 30 completed microbial genomes, including three strains of the gastric pathogen Helicobacter pylori namely 26695, J99 and 908 (last two are the Indian strains) (Alm et al 1999, Tomb et al 1997). The advent of these sequences has afforded novel insights into the molecular mechanisms of genetic change and adaptive mutations of the bacteria.
3.5 Role of H.pylori in human disease development
By 1984, it became almost clear that H.pylori infections are strongly associated with the presence of inflammation in the gastric mucosa ( chronic superficial gastritis), and specially with polymorphonuclear cell infilteration (chronic active gastritis)(Blaser 1990). Once established H.pylori infection persist, usually for life, unless eradicated by antimicrobial therapy (Blaser 1990). Marshall and Warren noted that H.pylori infection was associated with duodenal ulceration (Marshall and Warren 1984), and this observation too was rapidly confirmed and extended to include gastric ulceration (Blaser 1990). In 1994, a consensus conference convened by The National Institute of Health concluded that H.pylori was a major cause of peptic ulcer disease and recommended that infected individuals with ulcers should be treated to eradicate the organism. In 1991, four reports showed an association between H.pylori infection and the establishment of gastric cancer (Talley et al 1991). Thus, in total, H.pylori a previously obscure organism has now been associated with many of the important diseases involving gastrointestinal tract.
3.6.1 Geographical Distribution
Helicobacter pylori is one of the human pathogens with 50% prevalence around the world; yet, its principal mode of transmission still remains unknown. The epidemiology of H. pylori infection is characterized by marked differences between developing and developed countries, that is > 80% in developing countries like India and Algeria and < 20% in developed countries, (Mitchell HM 1998, Pounder RR 1995, Parsonnet J 1995) notably among children (Mohammed M K et al 2010). H. pylori prevalence markedly differ amongst different ethnic, social strata that one belongs to, and is also different amongst different age groups within the same country or region, but is separated based on the sanitation facilities, overall hygiene, and standardss of living (Malaty HM. et al 1999, 1992, Mitchell H M et al 2001).
The mode of transmission of H. pylori is one of the most controversial areas in the study of this pathogen. H.pylori infections are thought to occur as a consequence of direct human to human transmission, via either an oral-oral or oral-faecal route or both. Also H.pylori has been detected and cultured from saliva, vomitus, faeces and gastric refluxate (Parsonnet 1999, Allaker 2002, Ferguson 1993, Kabir S 2004, Leung 1999), but there is no conclusive evidence for predominant
transmission by any of these products.
H. pylori has adapted to the inhospitable conditions found at the gastric mucosal surface. Urease production and high motility are very vital for its survival in this environment (Tanih et al., 2008). Adherence of H. pylori to epithelial cells is a relevant step in the development of gastro duodenal pathologies. Although, it has been seen that the primary disorder, which occurs after the colonization of H. pylori, is chronic active gastritis. The intra gastric distribution and severity of this chronic inflammatory process depends upon a variety of factors, such as virulence factors of the colonizing strain (CagA, VacA and other membrane proteins), host genetic factors and immune response, diet, and the levels of acid production (Kusters et al 2006).
Some strains are more virulent than others particularly those expressing the highly immunogenic cytotoxin associated gene A protein (cag A) which is present in approximately 50 to 70% of these organisms (El-Omar, 2006; Epplein et al., 2008; Lee et al., 2008; Tanih et al., 2010a) and VacA toxin. Patients infected with CagA+ strains usually have a higher inflammatory response and are significantly more at risk of developing a symptomatic outcome (Torres et al., 2009). The vacuolating cytotoxin (VacA) is a highly immunogenic 95-kDa protein which has been reported to induce massive vacuolization in epithelial cells in-vitro (El-Omar, 2006). This toxin producing gene is found in 50% of the H. pylori strains (Epplein et al., 2008).
Complications like H. pylori-induced ulcer disease, gastric cancer, and lymphoma are all a result of this chronic inflammation; out of this, ulcer disease and gastric cancer, particularly occurs at those sites where there is a severe inflammation (El Omar 2006). Understanding of these factors (Virulence factors) is thus very crucial for the recognition of the role of H. pylori in the etiology of the upper gastrointestinal pathology.
3.8 H.pylori virulence factors
Virulence or pathogenicity can be defined as the ability of a microbe to induce disease. It has been suggested that the disease causation by H.pylori is due to the various factors including host factors, environment, and diet and also the virulence factors. H.pylori strains vary in the degree of virulence (Atherton JC 1997, Blaser MJ, et al 1995). In particular, the cagA, vacA, iceA, OipA and DupA genes are considered to be the putative virulence factors of H.pylori (Yamaoka Y et al 1999).
3.8.1 Cag Pathogenicity Island (PAI) and CagA
The cag pathogenicity island qualifies as an important virulence factor because its presence is associated with an increased risk of peptic ulcer and gastric carcinoma. This island is a 40kb DNA segment that contains 31 open reading frames out of which 6 genes code for a putative Type IV secreation system (T4SS) that is associated with the translocation of the CagA protein into the extracellular compartment of the host cells (Censini et al 1996). At one end of this cag PAI is the cagA gene which codes for CagA protein. CagA is one of the best studied virulence markers of H.pylori, the toxin is 120-145 kDa in size and is highly immunogenic (Censini et al 1996). The strains of H.pylori that carry the cagPAI are known to be more virulent than those that do not. The exact role of CagA is multifactorial as it is required for both acquisition of nutrients and persistence of H. pylori in the host ( Tan S et al 2009, Amieva MR 2003). The structure of the cagA gene revealed a 5' highly conserved region. Variation in the size of the protein is correlated with the presence of a variable number of repeat sequences that are located in the 3' region of the gene (Covacci A 1993, Maeda S 1997). The 3' repeat regions of the cagA contain EPIYA motifs (consisting of Glutamine-Proline-Isoleucine-Tyrosine-Alanine), and the number of these motifs determines the level of CagA phosphorylation (Argent R H 2004). Once delivered inside the cell, the CagA protein is phosphorylated at the tyrosine residues by kinases, which results in morphological changes in the epithelial cells commonly termed as the 'Hummingbird Phenotype' (El-Omar, 2003; 2006).
Analysis of the primary sequence and structural organization of the 3' region of the cagA gene showed that there are four subtypes of the gene namely cagA type A-D (Azuma T 2004). Types A and C can be distinguished by their PCR product size ( 642-651 bp and 810-813 bp respectively) but Types B and D have the same PCR product length and can be distinguished only by sequencing. It was observed that cagA type A is associated with duodenal ulcer and cagA type C is associated with high rates of gastric cancer in China and Japan.
Fig : Primary structure variants of the 3' region of the cagA gene (Yamaoka Y et al 1998)
3.8.2 Vacuolating Cytotoxin Gene (VacA)
The second most important and well-studied toxin in the array of H.pylori's virulence factors is VacA. Unlike cagA, it is present essentially in all the H.pylori strains (Blaser M J et al 2004, Rassow J et al 2012). It is a highly immunogenic 95-kDa protein which has been reported to induce massive vacuole formation in epithelial cells in-vitro (El-Omar, 2006). VacA is a pore forming toxin which is expressed as a 140 kDa prototoxin initially, which after secreation is cleaved to form a mature 88-95 kDa toxin (Lenuk R D 1988). This mature toxin is further cleaved into two fragments each of 33 kDa and 55 kDa respectively (Rassow J et al 2012, Boquet P et al 2012, Kim I J et al 2012). The 33 kDa domain of the vacA inserts itself in the outer membrane and forms a channel through which the mature VacA toxin is secreted (Cover T L et al 2005) (Fig 4(a)).
Similar to cagA, vacA also contains allelic diversity that varies amongst H.pylori strains worldwide (Atherton J C et al 2005). Allelic diversity of vacA is striking particularly at the 5' terminus (Cover T L 2005). VacA contains four identified variable regions each of which are subdivided into subtypes. The best characterized regions are, s (signal) region which is typed as s1and s2, m (middle) region which is typed as m1 and m2, i (intermediate) region and d (deletion) region (Atherton JC et al 1995). The i region and the d region are discovered recently therefore not much is known about their role in the disease progression (Rhead J L et al 2007, Chung C et al 2010). The i region lies between the s and m regions and are typed as i1, i2 or i3 (Rhead J L et al 2007, Chung C et al 2010). The d region is located between the i and m region and is typed as d1 if there is no deletion or as d2 if a 69-89 bp deletion is present (Ogiwara H et al 2009). The vacA can recombine to form many recombinations of which vacA s1/m1 alleles are the most virulent combination, while the s1/m2 and s2/m2 genotypes display virtually no cytotoxicity (Atherton JC et al 1995, Meining A et al 1997, Miehlke S et al 1998, 2000, Letley DP et al 2000, 2003, McClain MS et al 2001). Some studies show that the i1 polymorphism is a significant and independent risk factor for the gastric malignancies (Rhead J L et al 2007), and is also associated with the s1 polymorphism (Yordanov D et al 2012).
Fig : VacA toxin ( Cover T et al 2005)
3.8.3 iceA, Induced upon contact with epithelium
Another putative virulent factor of H.pylori is iceA gene which is a signalling gene, induced by contact with gastric epithelium. An initial series of studies showed that iceA has two allelic variants, iceA1 and iceA2. iceA1 shows sequence homology with a gene from Neisseria lactamica, NlaIIIR which encodes for a CTAG specific restriction endonuclease (Van Doorn L et al 1998, Peek R M et al 1998). On the other hand iceA2 shows no homology to any known genes and its function remains unclear. It was reported in one study that the iceA allelic types are independent of the cagA and vacA status and there is a significant association between the presence of the iceA1 and peptic ulcer disease (van Doorn L et al 1998). The expression of iceA1 is upregulated on contact of H.pylori with the epithelial cells, which results in the enhanced mucosal interleukin (IL)-8 expression and acute antral inflammation (Peek R M et al 1998, Xu Q et al 2002). The overall prevalence of iceA1is 64.6% in Asian countries and 42.1% in western countries. On the other hand prevalence of iceA2 is 25.8% in Asian countries and 45.1% in western countries (Shiota S et al 2012).
3.8.4 Interleukin (IL)-8 Production
Infection of the gastric mucosa by H. pylori is characterized by the production of proinflammatory cytokines, especially interleukin (IL)-8, a potent neutrophil-activating chemokine (Yamaoka Y et al 1996, 1998, 1999, 2001). Levels of IL-8 are directly related to the severity of gastritis (Peek R M et al 1995). Compared with cag' strains, cag+ strains induce an enhanced IL-8 and inflammatory response in human tissue (Crabtree J E 1993, Peek R M et al 1995, Yamaoka Y et al 1996). It has been reported that the IL-8-251 A/T polymorphism is associated with higher expression of IL-8 protein, severe neutrophil infiltration and increased risk of atrophic gastritis and gastric cancer (Taguchi A et al 2005).
3.8.5 Oip A (Outer inflammatory protein)
Approximately 4% of the H. pylori genome is predicted to encode outer membrane proteins (OMPs), some of which may function as adhesins (Figueiredo C et al 2005, Fujimoto S et al 2007, Lu H et al 2005, Yamaoka Y 2006, 2008). One such OMP is OipA (Outer inflammatory protein). Its function has been suggested to amplify IL-8 secretion via interferon-stimulated responsive element (ISRE) (Yamaoka Y et al 2000, 2004). Yamaoka and co-workers have reported that the expression of functional OipA in H.pylori is phase-variable, and can be switched 'on' or 'off' by a slipped strand mispairing mechanism during chromosomal replication (Yamaoka Y et al 2000, Saunders N J et al 1998,de Vries L et al 2002). The OipA expression status is often observed being associated with the presence of cagPAI and VacAs1/m1, allelic variants (Markovska R et al 2011). Some animal studies demonstrate that OipA alone plays an important role in the development of gastric cancer.
3.8.6 DupA (Duodenal ulcer promoting gene A)
Till 2004 it was thought that CagA, VacA and OipA are all involved in the development of both gastric cancer and duodenal ulcer, which are at the opposite ends of the disease spectrum. But in 2005, the first disease-specific H. pylori virulence factor that induced duodenal ulcer and had a suppressive action on gastric cancer was identified, and was named duodenal ulcer promoting gene A (DupA) (Lu H et al 2005). Some of the in vitro studies using DupA deleted and DupA complemented suggests that virulent H. pylori strains cause inflammation by stimulating epithelial cells via CagA, OipA and/or VacA-related proteins and mononuclear inflammatory cells through DupA (Yamaoka Y 2010). Analyzing 500
H. pylori strains isolated from patients in Colombia, South Korea, and Japan, Lu et al reported that infection with dupA-positive strains is significantly associated with duodenal ulceration but negatively associated with gastric cancer (Lu H et al 2005).
3.9 Antibiotic Susceptibility of H.pylori: Prevalence
As virulence markers of H.pylori are not associated with diseases in all the geographical regions, its eradication provides the best and most effective treatment for H.pylori associated diseases (Mukhopadhyay AK et al 2000, Chattopadhyay S et al 2002, Datta S et al 2003, Graham DY et al 1998). Commonly used antimicrobial agents for the eradication of H.pylori are Metronidazole (Mtz), Clarithromycin (Cla), Amoxicillin (Amx), Furazolidone (Fz) and Tetracycline (Tet) etc. Resistance to some of the potentially useful antimicrobial agents, such as metronidazole (Mtz) and Clarithromycin (Cla) is unfortunately very common. The successful treatment of H.pylori infection entails use of combination of two antibiotics and a proton pump inhibitor (Wong BCY et al 2000, Chu KM et al 2004). Reports have indicated that the prevalence of resistance amongst H.pylori varies geographically, ranging from 10 to 90% for Mtz and from 0 to 15% for Clarithromycin (Cla) (Nahar S et al 2004). In one multicentric study in India reported, 80% resistance against Amoxicillin (Amx) and 96% resistance against Clarithromycin (Cla) amongst H.pylori isolates from Hyderabad (Thyagarajan S P et al 2003).
The increased prevalence of antibiotic-resistant H. pylori strains has complicated the efforts to eradicate the infection. Therefore, culture and susceptibility testing can prove to be of great value in helping clinicians to choose the most promising therapy for a particular patient or population group (geographical region).
As mentioned above commonly used antibiotics in the treatment regimen of H.pylori and their mechanism of action are as follows:
1. Amoxicillin (Amx)
H.pylori is very sensitive in vitro to this antibiotic. Resistance to this drug is very rare. It acts by interfering with the peptidoglycan synthesis, especially by blocking transporters named penicillin binding proteins (PBP). The rare amoxicillin-resistant H. pylori strains harbor mutations on the pbp-1a gene. The amino acid substitution Ser-414'Arg is involved in conferring the ressistance to this drug (Gerrits et al 2002), which leads to the blockage of penicillin transport.
2. Tetracycline (Tet)
Tetracycline is a very effective drug against H.pylori but is quite underused in the anti-H.pylori therapies. It works best at the low pH. Resistance to this drug is very rare. It works by interfering with the protein synthesis at the ribosome level by binding to the 30S subunit (Gerrits M et al 2002).
3. Metronidazole (Mtz)
Metronidazole is a nitroimidazole which is commonly used in the H.pylori eradication regimen. H.pylori, ordinarily is highly sensitive to this drug. Metronidazole otherwise is a harmless prodrug which is converted to hydroxylamine like compounds which is both bactericidal and mutagenic by two nitroreductase genes namely rdxA and frxA (flavin oxidoreductase). Inactivation due to mutations in these chromosomal genes is implicated in conferring resistance in H.pylori to metronidazole (Debetes- Ossenkopp Y J et al 1999, Goodwin A et al 1998).
4. Clarithromycin (Cla)
This antibiotic belongs to the macrolide family. This drug acts by binding to the ribosomes at the level of the peptidyl transferase loop of the 23S rRNA gene. Resistance to this drug is a consequence of point mutations at two nucleotide positions, 2142 (A2142G and A2142C) and 2143 (A2143G), which leads to a conformational change and a decrease in macrolide binding (Occialini A et al 1997, Versalovic J et al 1996) but in Indian strains mutation is observed at 1825 (A 1825 G), 1834 (T 1834 C) and 2147 (A 2147 G) which is quite different from the other data (Rajashree Das et al 2013). The success rate of H.pylori treatment with this drug is up to 54%.
5. Furazolidone (Fz)
This antibiotic is a nitrofuran antibiotic and is a monoamine oxidase inhibitor and has a broad spectrum antibacterial activity. It has been empirically used in the treatment of peptic ulcer disease. A furazolidone- based regimen is given to those patients who do not achieve cure of the H.pylori infection with the metronidazole- based therapy. It is not considered as a first line treatment though it has reported good results.
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