Abstract

Helicobacter pylori (H. pylori) is a microaerophilic, Gram-negative, human gastric pathogen found usually in the mucous lining of stomach. It infects more than 50% of the world’s population and leads to gastroduodenal diseases. The outcome of disease depends on mainly three factors: Host genetics, environment and bacterial factors. Among these, bacterial virulence factors such ascagA,vacA are well known for their role in disease outcomes. However, based on the global epidemiological results, none of the bacterial virulence (gene) factors was found to be associated with particular diseases like duodenal ulcer (DU) in all populations. Hence, substantial importance has been provided for research in strain-specific genes outside thecag pathogenicity island, especially genes located within the plasticity regions.dupA found within the plasticity regions was first demonstrated in 2005 and was proposed for duodenal ulcer development and reduced risk of gastric cancer in certain geographical regions. Due to the discrepancies in report from different parts of the world in DU development related toH. pylori virulence factor,dupA became an interesting area of research in elucidating the role of this gene in the disease progression. In this review, we shed light on the detailed information available on the polymorphisms indupA and their clinical relevance. We have critically appraised several pertinent studies ondupA and discussed their merits and shortcomings. This review also highlightsdupA gene as an important biomarker for DU in certain populations.

INTRODUCTION

Helicobacter pylori (H. pylori) is a curved rod-shaped, Gram-negative, microaerophilic bacterium found usually in the mucous lining of the stomach.H. pylori infects more than 50% of the world’s population and 70%-80% of the Indian population[1,2].H. pylori is acquired during childhood and remains in the stomach throughout the life if not treated effectively[3]. Infection withH. pylori causes duodenal ulcer (DU), gastric ulcer (GU), gastric cancer (GC) and gastric mucosa-associated lymphoid tissue lymphoma[4-7]. Considering its clinical importance, the World Health Organization has declaredH. pylori as a class I carcinogen and enlisted GC as the fifth most common cancer and the third most common cause of cancer-related death[8,9]. Infection ofH. pylori is comparatively more prevalent in developing countries than Western countries due to socioeconomic and sanitary conditions[10]. The mode of transmission ofH. pylori is not clearly understood. However, most of the studies suggest thatH. pylori is transmitted from person to personvia oral-oral and fecal-oral route and also through contaminated food and water[11-14].

The enigma ofH. pylori research is that the majority of infected patients remain asymptomatic, whereas around 15%-20% of infected individuals develop symptoms of peptic ulcer (duodenal or gastric) as a long-term consequence of infection. It is not clear what governs the manifestation ofH. pylori infection in some people. This apparent puzzle prompted the proposal that the sheer presence ofH. pylori in the stomach is inadequate to develop acute gastric disease and that other conditions are required. However, it is assumed that the responsible factors inH. pylori-associated diseases are due to its virulence factors, host genetics, immunity and environmental influences. Host factors like polymorphism in the genes (pro-inflammatory cytokine genes) increase the risk of the specific clinical outcome[15]. None of theH. pylori virulence factors such ascagA,vacA, the blood group antigenbabA andoipA have been linked with specific diseases like DU or GC uniformly in all populations[16-20].

Analysis of the full genome sequences of differentH. pylori strains reported specific genetic locus whose G+C content was lower than that of the rest of theH. pylori genome. This indicates the possibility of horizontal deoxyribonucleic acid (DNA) transfer from other species.H. pylori carry an open pan-genome, which maintain a discrete group of strain-specific genes. These strain-specific genes mostly reside in genomic regions that had earlier been coined as plasticity zones. This term was previously used to describe a specific genetic segment with high variation between theH. pylori genome sequences[21,22]. The complete genome sequence ofH. pylori reveals that part of the plasticity zone is normally arranged as genomic islands that may be integrated in the genetic loci. About 50% of the strain-specific genes ofH. pylori are located in the plasticity region. Here, our focus is on the genedupA, which is located within the plasticity region. This gene was first reported in 2005 as an important biomarker for DU[23]. During subsequent years, several investigations were carried out ondupA, and this has become an interesting area of research, as shown in Table1.

METHODOLOGY

To review the importance ofdupA, we have searched the “NCBI-PubMed” using the keywords: “dupA”, “H. pylori” and a total of 80 articles were found, of which 76 were published in English till January 2020. Out of 76, 13 are published as review articles and two as meta-analysis of previous data. The remaining 61 documented as research articles. The research ondupA has spanned 15 years with contradictory findings. In this review, we summarize the result of relevant studies and discuss the pathogenesis ofdupA since its early stage to recent advancements. Finally, this review highlights the significance ofdupA gene ofH. pylori as a virulence factor (virulence marker) and its role in pathogenesis including the progression of DU.

DISCREPANCIES OFDUPA WITH CLINICAL OUTCOMES

Studies conducted withH. pylori strains from East Asia and South America identified a novelH. pylori virulence factor encoded in thedupA that was associated with increased risk of DU and decreased risk of GC. However, this perception seems to be region specific. ThisdupA was homologous tovirB4 gene, located in the plasticity zone ofH. pylori. dupA contained two open reading frames (ORFs),jhp0917 andjhp0918, with an overlap of twelve bases and an insertion of either base thymine (T) or cytosine (C) after the position 1385 of thejhp0917 that leads to continuous gene of 1839 bp. Since 2005, several studies have been conducted from different geographical areas to check the association ofdupA with disease outcome considering thedupA has two ORFs (jhp0917/jhp0918) with the insertion of one base (T/C) at position 1385 ofjhp0917. Studies performed in North India during 2007 support the finding of Lu et al[24]. However, studies conducted in different countries (Belgium, South Africa, China, North America and Brazil) found thatdupA is not associated with DU in the respective population[25-27].

Investigations made in Sao Paulo, Brazil showed thatdupA was detected inH. pylori strains of 41.5% patients, which was less from a previous study made by Gomes et al[26] (2008), in whichdupA was present in 89.5% patients[28]. This study showed an association ofdupA withcagA andvacA s1m1 genotypes but without any link to disease outcome. The difference in the results of these two studies from Brazil could be explained by variation in geographic regions, a re-arrangement in the plasticity zone distribution inH. pylori and various methods used for the analysis.

The distribution ofdupA inH. pylori was similar in Iraqi and Iranian population, but there was an association between peptic ulcer anddupA only in the Iraqi population[29]. An independent study by Douraghi et al[30] (2008) reported a non-significant higher distribution ofdupA in DU than non-cardia GC patients in the Iranian population[30]. Another study by Talebi Bezmin Abadi et al[31] (2012) found a positive association between the presence ofdupA and DU along with an inverse association betweendupA and GU in Iranian population[31]. The discrepancy in the finding of Douraghi et al[30] (2008) and Talebi Bezmin Abadi et al[31] (2012) may be due to differences in the study populations. Douraghi et al[30] (2008) focused mainly in Tehran (the densely populated capital of Iran), whereas Talebi Bezmin Abadi et al[31] (2012) collected samples from the extremely rural northern areas of Iran. Recently, Fatahi et al[32] (2019) tested a highly conserved region ofdupA and showed a significant relationship between the occurrence of DU and the presence of an 112 bp segment ofdupA in the Iranian population[32]. Another group from Iran studied the relationship between antibiotic resistance pattern and virulence genotype among 68H. pylori strains and found that metronidazole resistance was significantly associated with the strains harboringcagA, sabA anddupA[33]. One study from Kurdistan region of Northern Iraq reported thatcagA gene was significantly associated with peptic ulcer disease rather thandupA, which contradict the result of Hussein et al[29] (2008). This might be due to the differences in sample size and also in the geographical location of Iraq[34]. In the Shiraz area of Iran, a significant relationship was found between strains withdupA, CagA motif (ABC types) and DU disease, which supports the previous finding in this region[35]. Another study from Western Iran indicated that presence ofdupA gene could be considered as a marker for the onset of severe gastroduodenal diseases[36]. However, there was no association ofdupA with DU in the results obtained from the Turkish population[37].

In China and South Korea, presence ofdupA in clinicalH. pylori isolates is significantly associated with DU and peptic ulcer (DU, benign GU, dysplasia), respectively[38,39]. In the Taiwanese female population, the host factor matrix metalloproteinase-3/tissue inhibitor matrix metalloproteinase-1 genotypes rather thandupA was found to increase the risk of DU inH. pylori infected cases[40]. A case control study conducted in Sweden, Australia, Malaysia and Singapore showed that there was significant variation in the prevalence ofdupA in different locations and among different ethnic groups (Chinese, Indian and Malaya) within a country[41]. Another study in ethnic groups (Indian, Chinese and Malaya) of Malaysia reported that the prevalence ofdupA was 22.9% in patients, which was in line with previous data (21.3%) conducted in Malaysia by Schmidt et al[41] (2009). In the later study, there was no association betweendupA and clinical outcome[42].

Two independent systematic review and meta-analyses showed thatdupA is more associated with DU in some Asian populations than in Western populations[43,44]. Between 2005 and 2009, almost all the studies used polymerase chain reaction (PCR) of two ORFsjhp0917, jhp0918 and sequencing to identify thedupA. Functional analysis ofdupA in the Japanese population showed no association with DU but another study from different parts of Japan showed thatdupA is inversely related to GC[45,46].

Results from a study using different molecular methods [PCR, dot-blot hybridization, sequencing and reverse transcription PCR (RT-PCR)] indicated thatdupA gene was prevalent more than six times in DU than in non-ulcer dyspepsia patients, indicating its significant association in India[47]. This result also corroborated the finding of Arachchi et al[24] (2007) from North India. The RT-PCR analysis of South and East Indian population revealed that all PCR positive strains were not able to producedupA transcripts, which was inconsistent with the finding of Nguyen et al[45] (2009) where all thedupA positive strains showed the expression of the gene[47]. Further, the real-time PCR analysis revealed that the expression level of thedupA transcripts varied from strain to strain in this study.

Studies conducted in Chile supported a significant association ofdupA gene with non-severe clinical outcome like DU and also played a role in protecting severe diseases like GC[48]. The Costa Rica study with 151 dyspeptic patients showed that presence ofdupA was significantly associated with decreased risk of DU[49].

Some of the above-mentioned studies verified the finding of Lu et al[23] (2005), but others could not find an association betweendupA and disease outcome in their study populations. The differences in the results could be explained due to variation in the distribution of plasticity region genes and differences in the study population and techniques chosen for detection ofdupA gene. Several studies ondupA were restricted to PCR ofjhp0917 andjhp0918 along with sequencing of only the 3' region ofjhp0917 to find the insertion of T/C at 1385 position ofjhp0917. Numerous studies have shown the presence of frame shift mutation withindupA gene leading to the formation of truncated non-functional DupA. These findings provide evidence that only PCR based analysis ofdupA may yield erroneous interpretation. Studies conducted by Queiroz et al[50] (2011) and Moura et al[51] (2012) from Brazil showed the presence of a mutation indupA that results in a stop codon, making the gene truncated or non-functional. In addition, these studies revealed the importance of sequence analysis ofdupA amplicons[50,51]. TruncateddupA might not be involved in the pathogenesis ofH. pylori.

Hussein et al[52] (2010) coined the term “dupA1”. ThedupA positiveH. pylori strains were categorized into two alleles based on the sequence;dupA1 (intact 1884 bp) anddupA2 (truncated). It was shown that the intactdupA1 positive strains induced the production of interleukin (IL)-12 subunit p40 (IL-12p40) and IL-12p70 from CD14 (+) mononuclear cells and IL-8 expression in the human stomach, respectively[52]. Takahashi et al[53] (2012) first reported the presence of an additional 615 bp in the 5' region of ORFjhp0917 (absent in strain J99) and 45 bp in the 3' ofjhp0918 (consist of 37 bp of intergenic region ofjhp0918-jhp0919 and 8 bp of 5' region ofjhp0919 in J99) to make 2499 bp ofdupA in the Japanese population (Figure1). This variation formed the basis for classification ofdupA into two types; “long and short types”. The long type of intact 2499 bp (with an additional 615 bp at 5' region ofjhp0917) has been considered as an actual virulence factor, and the absence of the additional segment should be interpreted with caution[53].

None of theH. pylori strains from Iraq carried the completedupA cluster containingvirB8,virB9,virB10,virB11,virD4 andvirD2, but there was a significant association betweendupA1 and DU. Moreover, higher levels of gastric mucosa IL-8 production were documented indupA1 than indupA2 ordupA negative strains[54]. Further studies withH. pylori infected patients showed thatcagA, completedupA cluster and smoking habit were associated with increased levels of IL-8 production from gastric mucosa[55]. It was also shown in another study that the high IL-8 level in gastric mucosa was neither significantly associated withdupA1 positive strains nor withdupA negative strains[56]. A significant association has also been found betweendupA1 and A2147G clarithromycin resistance mutation. However, the result ofdupA1 and IL-8 association in the Iraqi population was not well elucidated. In BrazilianH. pylori strains, it was found thatH. pylori strains had the 45 base at the 3' end ofdupA, similar to that ofdupA1[57].

dupA gene of IndianH. pylori strains has been classified into two forms based on the presence of additional 615 bp at the 5' region ofdupA followedby a stop codon. This includesdupA1 without any frameshift mutation (either long type or short type) anddupA2 with the truncated version having frameshift mutation[58]. Among these,dupA1 (intactdupA) was significantly associated with DU. Phylogenetic analysis of completedupA gene sequencing revealed that IndianH. pylori strains intermingled with the East Asian strains, but differed from European strains[58].dupA is the first known genetic element of IndianH. pylori strains, which phylogenetically formed the same cluster with the East Asian strains.In vitro study showed that IL-8 production was significantly associated with DU in intactdupA1 rather than truncateddupA2 ordupA negative strains[58]. In Chinese strains, the prevalence of long typedupA (2499 bp) was significantly higher in patients with GU, GC and DU than in those with gastritis[59].

In the Japanese population, prevalence ofdupA was higher in the group whereH. pylori cannot be eradicated, indicating thatdupA may be an associated risk factor in the eradication failure[60]. A study from Pakistan on the influence ofdupA in the eradication failure showed thatH. pylori strains harboringdupA andcagA were multidrug (metronidazole, clarithromycin and amoxicillin) resistant as compared to strains having other virulence factors. This finding was similar to the observation made in the Japanese population[61]. In the northern part of Spain,dupA was more prevalent in mild diseases (peptic ulcer) than severe diseases (GC)[62]. In Switzerland and South Africa,dupA ofH. pylori was not associated with severe gastritis or DU[63,64].

DUPA CLUSTER: THIRD TYPE IV SECRETION SYSTEM (T4SS) OFH. PYLORI

The T4SS is an important bacterial transport system, and it is involved in the transport of large molecules (e.g., DNA, protein,etc). across the bacterial cell envelope[65,66]. Till now, three types of T4SS have been identified inH. pylori, of which much work has been done for the first two categories (cagPAI and ComB) and little is known about the third T4SS termeddupA cluster ortfs3 (Figure2)[67,68]. The third putative type IV secretion system (tfs3) is a 16 kb gene fragment present in the plasticity zone ofH. pylori, whose seven ORFs (viB4,virB8, virB9, virB10, virB11, virD4 andvirD2) were homologous to virB4/D ofAgrobacterium tumefecians (A. tumefecians). The function of thetfs3 elements is not yet clear as there is no direct evidence to show its role in transformation, conjugation or mouse colonization[69,70]. Some researchers divided thetfs3 intotfs3a (all sixvirB homologues withdupA) andtfs3b (all sixvirB homologues withvirB4), whereas others named all sixvirB homologues with virB4 astfs3 and all sixvirB homologues withdupA astfs4[71-73]. In order to avoid confusion, we will use the termtfs3a ordupA cluster (all sixvirB homologues withdupA). VirB8, VirB9 and VirB10 are expected to form the core complex that bridges cytoplasm and the outer membrane. The VirB4, VirB11, VirD4 may be localized to the inner bacterial membrane and recognize the substrate and energize translocation and assembly of T4SS[74]. Further, the novel putative T4SS (tfs3a) ordupA cluster has been divided into three groups: Viz, a completedupA cluster (dupA-positive and all sixvirB genes-positive), an incompletedupA cluster (dupA-positive but one/more than onevirB genes negative) anddupA-negative group (dupA negative andvirB gene positive/negative).

The study ofdupA cluster from the United States population showed that the completedupA cluster (dupA with sixvirB homologues) was associated with DU rather thandupA gene only[75]. Another report from the northeast part of China showed a significant association of completedupA cluster with IL-8 production (P < 0.01), but it did not show any correlation betweendupA cluster and disease outcome[76]. The studies from United States and China were conducted to check the prevalence oftfs3a ordupA cluster in their population by PCR only. However, the mere presence of the gene does not express functional protein and there is no direct evidence that showstfs3a ordupA cluster forming a functional T4SS. The earlier studies ontfs3adid not find a direct pathogenic role oftfs3a inH. pylori, but found increased colonization fitness and up-regulation of pro-inflammatory signaling from cultured cells. A novel pathogenicity island (PAI) calledtfs3-PAI was identified in China that had 17 ORFs, of which six are functionally homologues of T4SS and coordinate with the well-studiedcag-PAI[77]. The completetfs plasticity cluster was associated with IL-8 induction. The expression of some of the genes oftfs3a/tfs4 (virB2,virB4,virB6,virB8,virB10) inH. pylori is up-regulated in low pH and enhances bacterial adhesion that support the role oftfs3a/tfs4 in the colonization and virulence[78]. It is not known whether thevirB genes ofdupA cluster work independently or in a coordinated manner by interacting among themselves or complementing each other’s function. We checked the interaction ofdupA with sixvirB genes oftfs3a to identify the assembly and function of completetfs3 usingin vivo studies (yeast two-hybrid system) and found thatdupA gene did not interact directly with anyvirB gene. It seems thatdupA may interact with some intermediates or work independently (unpublished data). This interpretation supports our earlier finding thattfs3 is not significantly associated with DU in Indian population. More studies are required to know the structure, assembly and functions of the VirB proteins inH. pylori.

THE PROSPECTIVE FUNCTIONS OF DUPA

The bioinformatics analysis (PDB search tool, UniProt database) showed that thedupA gene is homologous to VirB4 adenosine triphosphate (ATP)ase of virB/virD ofA. tumefecians and is predicted to be involved in DNA/protein transfer. The N-terminal of long type DupA has no homologous motif. Only the middle portion (jhp0917) and C-terminal (part ofjhp0918) showed homologous motifs suggesting that the N-terminal region might act as signal sequence. The amino acid sequence (210-406 AA) ofjhp0917 gene protein was homologous to CagE_TrbE_VirB family, a component of type IV transporter secretion system. The first middle region of the DupA protein (430-500 AA) is homologous to FtsK/SpoIIIE family, which contains ATP binding P-loop motif. This was found in the Ftsk protein ofEscherichia coli involved in peptidoglycan synthesis and spoIIIE ofBacillus subtilis, facilitating in the intercellular chromosomal DNA transfer.

The second middle region (464-503aa) is homologous to TrwB, which has an ATP binding domain, and a part of T4SS may be responsible for the DNA binding and horizontal DNA transfer. The C-terminal region (668-738aa) is homologous to TraG_C_D, which is involved in the interaction of DNA-processing (Dtr) and mating pair formation (Mpf) system, leading to DNA transfer in bacterial conjugation. Many reports have shown that the growth rate ofdupA positive strains is higher in low pH as compared todupA deleted/negative strains. This phenomenon indicates that DupA protein acts as an interactive protein and hence regulates urease secretion inH. pylori[79].

Thein vitro andin vivo studies showed the role ofdupA gene in the activation of transcription factors nuclear factor kappa light chain enhancer of activated B cells and activator protein-1, which leads to IL-8 production. DupA protein act as an ATPase associated efflux pump, which probably confers its virulence. Evidence suggests thatDupA is involved in the pathogenesis ofH. pylori by activating the mitochondria dependent apoptotic pathway of the host’s cell, which ultimately inhibits gastric cell growth.

Studies to understand the apoptotic effect ofdupA on human gastric adenocarcinoma epithelial cell line (commonly known as AGS) by propidium iodide staining and fragmentation assay determined thatdupA gene can induce apoptosis in AGS cells during an early stage of infection (unpublished data). This finding supports the results of Wang et al[79] (2015) and finds thatdupA may act as a pathogenic factor ofH. pylori to cause gastroduodenal diseases. Further studies are required to confirm the pathogenic effect ofdupA in anin vivo model.

The growth kinetics between wild typedupA positive strains and its isogenic mutant strain showed that exponential phase was retarded indupA mutant cells as compared to the wild type strain. Our growth curve results, supported by the microarray data, showed that cell division gene in the mutantH. pylori was downregulated (unpublished data). It has also been suggested that motility is an essential feature in the colonization and therefore the pathogenicity ofH. pylori. The decrease in motility indupA mutant strain as compared to wild type inferred the role ofdupA gene in the motility. This motility result was further confirmed by the gene expression profile ofdupA mutant strain whose flagella proteins (FlgE, FliD and FliG) were found to be down-regulated (unpublished data). It might be possible thatdupA gene is directly or indirectly involved in negatively affecting the expression of cell division and flagellar genes ofH. pylori.

As predicted from the bioinformatics analysis, our experimental data (unpublished data) have shown that natural transformation ability indupA mutant strains has been totally inhibited in comparison to their wild type counterparts. There is a need for more studies on the heat-shock transformation efficiency, which will confirm the natural transformation assay, if any. Resistance to antimicrobials is of serious concern inH. pylori infection, as this may be the basis for eradication failure. It is important to use therapeutic regimens based on the results of antibiotic susceptibility testing. Metronidazole is considered a key drug in several therapies againstH. pylori infection. The results of the metronidazole susceptibility test showed that inactivation ofdupA gene transforms theH. pylori strains to resistance phenotype. This phenomenon has not been explained very well. It is possible that thedupA gene might help in the DNA/protein/drug import (unpublished data).

ThedupA ordupA cluster may have an intermediate function to linkcagPAI andcomB system, asdupA gene shows homology withcagE ofcagPAI andcomB4 ofcomB system. So, there is a need of anin vivo study to establish the precise function ofdupA. It is assumed that thedupA in combination with other sixvir genes form a novel third T4SS calledtfs3a ordupA cluster that might play a pathogenic role in gastroduodenal diseases.

CONCLUSION

H. pylori is one of the most diverse bacterial species.H. pylori demonstrate panmictic population structure. DNA-fingerprint of two strains isolated from two different persons generally displays a non-identical pattern, which suggests genetic exchange along with co-evolution of this gastric pathogen with its host. One study from the Indian population demonstrated that all the tested patients carried multipleH. pylori strains in their gastric mucosa[80]. Analyses of certain genetic loci showed the micro diversity among the colonies from a single patient, which may be due to the recombination events during long-term carriage of the pathogen. From the results of this study, researchers predicted that many patients from the developing world acquired infections ofH. pylori due to repeated exposure to this pathogen with different genetic make-up[80]. This may enhance the probability of super infections, which favor genetic exchanges among these unrelatedH. pylori strains. As a result, this led to the genesis of certainH. pylori variants with different genetic makeup than the parental strain, which in turn increases the chance of the severe infection. Therefore, the exploration of appropriate biomarker(s) that envisage the clinical condition inH. pylori-infected patient is a challenging area of research.

There is a lack of relevant biomarker(s) capable of predicting important digestive diseases in clinical settings. Even though there is ample information regarding thedupA ofH. pylori, many unanswered questions still exist, especially regarding the specificity of thedupA proposed for clinical manifestation.dupA was categorized as long and short types in one study, but in another study, this gene was typed asdupA1 (intactdupA1 may be long type or short type) anddupA2 (truncated version). This gene classification should be resolved for international use to avoid any misperception. We propose the longdupA asdupA1 and short typedupA asdupA2, and the truncated version ofdupA has to be disregarded, as it has no role in pathogenesis.dupA should be screened by PCR, sequencing of the full-length gene (1884 and 2499 nt) and western blotting. Nevertheless, the discrepancy prevails between the association ofdupA (short type or long type) ordupA cluster and the disease outcome. Currently, the prevalence of intactdupA in East Asian countries is lower than Western countries. DupA with another six Vir proteins (VirB8, VirB9, VirB10, VirB11, VirD4 and VirD2) predicted to form novel third type-IV secretion system (tfs3a), which may be involved in transformation/conjugation or injection of DNA/new effector molecules in gastric epithelial cells. However, the function of specific Vir protein of completedupA cluster (tfs3a) is not well characterized. Recent reports and other unpublished data showed that DupA has multifunctional biological activities, and it can be considered as an important biomarker for DU. It is also not clear whether the DupA works alone or in combination with other VirB proteins. There is an urgent need for reliablein vitro and animal models from diverse geographical areas of the world to elucidate further the pathogenic role ofdupA anddupA cluster in gastroduodenal diseases, particularly the DU and GC.

Footnotes

Contributor Information

Jawed Alam, Division of Infectious Diseases, Institute of Life Science, Bhubaneswar 751023, India.

Avijit Sarkar, Division of Bacteriology, ICMR-National Institute of Cholera and Enteric Diseases, Kolkata 700010, India.

Bipul Chandra Karmakar, Division of Bacteriology, ICMR-National Institute of Cholera and Enteric Diseases, Kolkata 700010, India.

Mou Ganguly, Division of Bacteriology, ICMR-National Institute of Cholera and Enteric Diseases, Kolkata 700010, India.

Sangita Paul, Division of Bacteriology, ICMR-National Institute of Cholera and Enteric Diseases, Kolkata 700010, India.

Asish K Mukhopadhyay, Division of Bacteriology, ICMR-National Institute of Cholera and Enteric Diseases, Kolkata 700010, India. asish1967@gmail.com.

References