Occurrence and multidrug resistance of Campylobacter spp. at duck farms and associated environmental and anthropogenic risk factors in Bangladesh.

BACKGROUND: The alarming rise in multi-drug resistant (MDR) zoonotic pathogens, including Campylobacter spp., has been threatening the health sector globally. In Bangladesh, despite rapid growth in poultry sector little is known about the potential risks of zoonotic pathogens in homestead duck flocks. The aim of this study was to understand the occurrence, species diversity, and multi-drug resistance in Campylobacter spp., and identify the associated risk factors in duck farms in Bangladesh. METHODS: The study involved 20 duck farms at 6 sub-districts of Mymensingh, Bangladesh. Monthly occurrence of Campylobacter spp. in potential sources at the farms during February-September, 2018, was detected by culture and PCR-based methods. Campylobacter isolates were examined for resistance to different antimicrobials. Risk factors, concerning climatic and environmental disposition, farm management, and anthropogenic practices, of Campylobacter infection were estimated by participatory epidemiological tools. RESULTS: Occurrence of Campylobacter spp. was detected in overall 36.90% (155/420) samples, more frequently in drinking water (60%, 30/50), followed by cloacal swab (37.50%, 75/200), egg surface swab (35%, 35/100) and soil of the duck resting places (30%, 15/50) but was not detected in feed samples (n = 20). PCR assays distinguished the majority (61.30%, 95/155) of the isolates as C. coli, while the rest (38.70%, 60/155) were C. jejuni. Notably, 41.7% (25/60) and 31.6% (30/95) strains of C. jejuni and C. coli, respectively, were observed to be MDR. The dynamics of Campylobacter spp., distinctly showing higher abundance during summer and late-monsoon, correlated significantly with temperature, humidity, and rainfall, while sunshine hours had a negative influence. Anthropogenic management-related factors, including, inadequate hygiene practices, use of untreated river water, wet duck shed, flock age (1-6 months), and unscrupulous use of antimicrobials were identified to enhance the risk of MDR Campylobacter infection. CONCLUSION: The present study clearly demonstrates that duck farms contribute to the enhanced occurrence and spread of potentially pathogenic and MDR C. coli and C. jejuni strains and the bacterial dynamics are governed by a combined interaction of environmental and anthropogenic factors. A long-term holistic research at the environment-animal-human interface would be integral to divulge health risk reduction approaches tackling the spread of Campylobacter spp. from duck farms.

Risk of multi-drug resistant Campylobacter spp. and residual antimicrobials at poultry farms and live bird markets in Bangladesh.

BACKGROUND: Understanding potential risks of multi-drug resistant (MDR) pathogens from the booming poultry sector is a crucial public health concern. Campylobacter spp. are among the most important zoonotic pathogens associated with MDR infections in poultry and human. This study systematically examined potential risks and associated socio-environmental factors of MDR Campylobacter spp. in poultry farms and live bird markets (LBMs) of Bangladesh. METHODS: Microbial culture and PCR-based methods were applied to examine the occurrence and MDR patterns of Campylobacter spp. in potential sources (n = 224) at 7 hatcheries, 9 broiler farms and 4 LBMs in three sub-districts. Antimicrobial residues in broiler meat and liver samples (n = 50) were detected by advanced chromatographic techniques. A questionnaire based cross-sectional survey was conducted on socio-environmental factors. RESULTS: Overall, 32% (71/ 224) samples were found contaminated with Campylobacter spp. In poultry farms, Campylobacter spp. was primarily found in cloacal swab (21/49, 43%), followed by drinking water (8/24, 33%), and meat (8/28, 29%) samples of broilers. Remarkably, at LBMs, Campylobacter spp. was detected in higher prevalence (p < 0.05) in broiler meat (14/26, 54%), which could be related (p < 0.01) to bacterial contamination of drinking water (11/21, 52%) and floor (9/21, 43%). Campylobacter isolates, one from each of 71 positive samples, were differentiated into Campylobacter jejuni (66%) and Campylobacter coli (34%). Alarmingly, 49 and 42% strains of C. jejuni and C. coli, respectively, were observed as MDR, i.e., resistant to three or more antimicrobials, including, tetracycline, amoxicillin, streptomycin, fluoroquinolones, and macrolides. Residual antimicrobials (oxytetracycline, ciprofloxacin and enrofloxacin) were detected in majority of broiler liver (79%) and meat (62%) samples, among which 33 and 19%, respectively, had concentration above acceptable limit. Inadequate personal and environmental hygiene, unscrupulously use of antimicrobials, improper waste disposal, and lack of health surveillance were distinguishable risk factors, with local diversity and compound influences on MDR pathogens. CONCLUSION: Potential contamination sources and anthropogenic factors associated with the alarming occurrence of MDR Campylobacter, noted in this study, would aid in developing interventions to minimize the increasing risks of poultry-associated MDR pathogens under 'One Health' banner that includes poultry, human and environment perspectives.

Novel Cholera Toxin Variant and ToxT Regulon in Environmental Vibrio mimicus Isolates: Potential Resources for the Evolution of Vibrio cholerae Hybrid Strains.

Atypical El Tor strains of Vibrio cholerae O1 harboring variant ctxB genes of cholera toxin (CT) have gradually become a major cause of recent cholera epidemics. Vibrio mimicus occasionally produces CT, encoded by ctxAB on CTXФ genome; toxin-coregulated pilus (TCP), a major intestinal colonization factor; and also the CTXФ-specific receptor. This study carried out extensive molecular characterization of CTXФ and ToxT regulon in V. mimicusctx-positive (ctx+) strains (i.e., V. mimicus strains containing ctx) isolated from the Bengal coast. Southern hybridization, PCR, and DNA sequencing of virulence-related genes revealed the presence of an El Tor type CTX prophage (CTXET) carrying a novel ctxAB, tandem copies of environmental type pre-CTX prophage (pre-CTXEnv), and RS1 elements, which were organized as an RS1-CTXET-RS1-pre-CTXEnv-pre-CTXEnv array. Additionally, novel variants of tcpA and toxT, respectively, showing phylogenetic lineage to a clade of V. cholerae non-O1 and to a clade of V. cholerae non-O139, were identified. The V. mimicus strains lacked the RTX (repeat in toxin) and TLC (toxin-linked cryptic) elements and lacked Vibrio seventh-pandemic islands of the El Tor strains but contained five heptamer (TTTTGAT) repeats in ctxAB promoter region similar to those seen with some classical strains of V. cholerae O1. Pulsed-field gel electrophoresis (PFGE) analysis showed that all the ctx+V. mimicus strains were clonally related. However, their in vitro CT production and in vivo toxigenicity characteristics were variable, which could be explainable by differential transcription of virulence genes along with the ToxR regulon. Taken together, our findings strongly suggest that environmental V. mimicus strains act as a potential reservoir of atypical virulence factors, including variant CT and ToxT regulons, and may contribute to the evolution of V. cholerae hybrid strains.IMPORTANCE Natural diversification of CTXФ and ctxAB genes certainly influences disease severity and shifting patterns in major etiological agents of cholera, e.g., the overwhelming emergence of hybrid El Tor variants, replacing the prototype El Tor strains of V. cholerae This report, showing the occurrence of CTXET comprising a novel variant of ctxAB in V. mimicus, points out a previously unnoticed evolutionary event that is independent of the evolutionary event associated with the El Tor strains of V. cholerae Identification and cluster analysis of the newly discovered alleles of tcpA and toxT suggest their horizontal transfer from an uncommon clone of V. cholerae The genomic contents of ToxT regulon and of tandemly arranged multiple pre-CTXФEnv and of a CTXФET in V. mimicus probably act as salient raw materials that induce natural recombination among the hallmark virulence genes of hybrid V. cholerae strains. This report provides valuable information to enrich our knowledge on the evolution of new variant CT and ToxT regulons.

Environmental and hydroclimatic factors influencing Vibrio populations in the estuarine zone of the Bengal delta.

The objective of this study was to determine environmental parameters driving Vibrio populations in the estuarine zone of the Bengal delta. Spatio-temporal data were collected at river estuary, mangrove, beach, pond, and canal sites. Effects of salinity, tidal amplitude, and a cyclone and tsunami were included in the study. Vibrio population shifts were found to be correlated with tide-driven salinity and suspended particulate matter (SPM). Increased abundance of Vibrio spp. in surface water was observed after a cyclone, attributed to re-suspension of benthic particulate organic carbon (POC), and increased availability of chitin and dissolved organic carbon (DOC). Approximately a two log10 increase in the (p < 0.05) number of Vibrio spp. was observed in < 20 µm particulates, compared with microphytoplankton (20-60 µm) and zooplankton > 60 µm fractions. Benthic and suspended sediment comprised a major reservoir of Vibrio spp. Results of microcosm experiments showed enhanced growth of vibrios was related to concentration of organic matter in SPM. It is concluded that SPM, POC, chitin, and salinity significantly influence abundance and distribution of vibrios in the Bengal delta estuarine zone.

Development and evaluation of a loop-mediated isothermal amplification assay combined with enrichment culture for rapid detection of very low numbers of Vibrio parahaemolyticus in seafood samples.

The aim of this study was to develop and evaluate a rapid and effective method to detect Vibrio parahaemolyticus, a leading pathogen causing seafood-borne gastroenteritis. A newly designed loop-mediated isothermal amplification (LAMP) assay including a short enrichment period was optimized. This assay correctly detected all the target strains (n=61) but none of the non-target strains (n=34). Very low numbers of V. parahaemolyticus (2 colony forming unit (CFU) per gram of seafood) could be detected within 3 h and the minimum time of the whole assay was only 5 h. Comparative screening of various seafood samples (n=70) indicated that the LAMP assay is superior to polymerase chain reaction (PCR) and conventional culture methods because it is more rapid and less complex. This highly sensitive LAMP assay can be applicable as the method of choice in large-scale and rapid screening of seafood and environmental samples to detect V. parahaemolyticus strains.

Seasonal dynamics of Vibrio cholerae and its phages in riverine ecosystem of Gangetic West Bengal: cholera paradigm.

The Gangetic delta is a century-old cholera endemic belt where the role of riverine-estuarine ecosystem in cholera transmission has never been elucidated. Seasonality, distribution, and abundance of environmental Vibrio cholerae O1/O139 and vibriophage in Hooghly riverine-estuarine environment and their correlation with cholera incidence pattern in West Bengal, India, have been analyzed for the first time across summer, monsoon, and winter months. A total of 146 water samples collected from two sites of the Hooghly River (Howrah and Diamond Harbour) were analyzed physicochemically along with cultivable Vibrio count (CVC), V. cholerae O1/O139, and vibriophages. V. cholerae O1 was detected in 56 (38.3%) samples, while 66 (45.2%) were positive for V. cholerae O1 phages. Flood tide, water temperature (31 ± 1.6 °C), and turbidity (≥250 nephelometric turbidity unit (NTU)) significantly stimulated V. cholerae and vibriophage abundance in riverine ecosystem. Solitary existence of V. cholerae O1 and phages (p < 0.0001) in aquatic environment divulges the dominance of either of the entity (V. cholerae O1 or V. cholerae O1 Φ) on the other. Significant association (p < 0.05) between Kolkata cholera cases and V. cholerae O1 in aquatic environment implies the role of riverine-estuarine ecosystem in cholera transmission. A "biomonitoring tool" of physicochemical stimulants, tidal, and climatic variants has been proposed collating V. cholerae and phage dynamics that can forewarn any impending cholera outbreak.

Novel cholix toxin variants, ADP-ribosylating toxins in Vibrio cholerae non-O1/non-O139 strains, and their pathogenicity.

Cholix toxin (ChxA) is a recently discovered exotoxin in Vibrio cholerae which has been characterized as a third member of the eukaryotic elongation factor 2-specific ADP-ribosyltransferase toxins, in addition to exotoxin A of Pseudomonas aeruginosa and diphtheria toxin of Corynebacterium diphtheriae. These toxins consist of three characteristic domains for receptor binding, translocation, and catalysis. However, there is little information about the prevalence of chxA and its genetic variations and pathogenic mechanisms. In this study, we screened the chxA gene in a large number (n = 765) of V. cholerae strains and observed its presence exclusively in non-O1/non-O139 strains (27.0%; 53 of 196) and not in O1 (n = 485) or O139 (n = 84). Sequencing of these 53 chxA genes generated 29 subtypes which were grouped into three clusters designated chxA I, chxA II, and chxA III. chxA I belongs to the prototype, while chxA II and chxA III are newly discovered variants. ChxA II and ChxA III had unique receptor binding and catalytic domains, respectively, in comparison to ChxA I. Recombinant ChxA I (rChxA I) and rChxA II but not rChxA III showed variable cytotoxic effects on different eukaryotic cells. Although rChxA II was more lethal to mice than rChxA I when injected intravenously, no enterotoxicity of any rChxA was observed in a rabbit ileal loop test. Hepatocytes showed coagulation necrosis in rChxA I- or rChxA II-treated mice, seemingly the major target for ChxA. The present study illustrates the potential of ChxA as an important virulence factor in non-O1/non-O139 V. cholerae, which may be associated with extraintestinal infections rather than enterotoxicity.

Inhibition of virulence potential of Vibrio cholerae by natural compounds.

The rise in multi-drug resistant Vibrio cholerae strains is a big problem in treatment of patients suffering from severe cholera. Only a few studies have evaluated the potential of natural compounds against V. cholerae. Extracts from plants like 'neem', 'guazuma', 'daio', apple, hop, green tea and elephant garlic have been shown to inhibit bacterial growth or the secreted cholera toxin (CT). However, inhibiting bacterial growth like common antimicrobial agents may also impose selective pressure facilitating development of resistant strains. A natural compound that can inhibit virulence in V. cholerae is an alternative choice for remedy. Recently, some common spices were examined to check their inhibitory capacity against virulence expression of V. cholerae. Among them methanol extracts of red chili, sweet fennel and white pepper could substantially inhibit CT production. Fractionation of red chili methanol extracts indicated a hydrophobic nature of the inhibitory compound(s), and the n-hexane and 90 per cent methanol fractions could inhibit >90 per cent of CT production. Purification and further fractionation revealed that capsaicin is one of the major components among these red chili fractions. Indeed, capsaicin inhibited the production of CT in various V. cholerae strains regardless of serogroups and biotypes. The quantitative reverse transcription real-time PCR assay revealed that capsaicin dramatically reduced the expression of major virulence-related genes such as ctxA, tcpA and toxT but enhanced the expression of hns gene that transcribes a global prokaryotic gene regulator (H-NS). This indicates that the repression of CT production by capsaicin or red chili might be due to the repression of virulence genes transcription by H-NS. Regular intake of spices like red chili might be a good approach to fight against devastating cholera.

Prevalence, Risk Factors, and Molecular Detection of Campylobacter in Farmed Cattle of Selected Districts in Bangladesh.

A cross-sectional survey was conducted in selected districts of Bangladesh to estimate prevalence, risk factors, and molecular detection of Campylobacter isolates from 540 farmed cattle of 90 herds. As an individual sample, 540 feces, and as a pooled sample, 180 milk samples, 90 feed samples, 90 water samples, 90 manure samples, and 90 animal attendants' hand-rinse water were collected and tested via culture, biochemical, and molecular assays. A pretested semi-structured questionnaire was used to collect herd-level data on risk factors with the herd owners. The herd-level data on risk factors were analyzed through univariate and multivariate analyses, and a p-value <0.05 was considered statistically significant for all analyses. Overall, farm-level prevalence of bovine Campylobacter was enumerated to be 53.3% (95% confidence interval [CI]: 42.5-63.9%). The feces sample was found to be a high level of contamination of 30.9% (95% CI: 27-35%) followed by the manure swab (pooled) at 15.6% (95% CI: 8.8-24.7%). Campylobacter jejuni was documented as an abundant species (12.6%), followed by Campylobacter coli (5.1%), and Campylobacter fetus (0.3%). Older farms (>5 years of age), no/minimum cleaning and disinfection practices, along with animal roaming outside of the farm, were documented as significant risk factors for farm-level Campylobacter occurrence. Evidence-based control measures need to be taken through stringent biosecurity and hygienic measurement to lessen the load of the Campylobacter pathogen in the farm environment and prevent further transmission to animals and humans.

Dynamics, Diversity, and Virulence of Aeromonas spp. in Homestead Pond Water in Coastal Bangladesh.

Aeromonads are aquatic bacteria associated with frequent outbreaks of diarrhea in coastal Bangladesh, but their potential risks from environmental sources have remained largely unexplored. This study, over 2 years, examined homestead pond waters in the region for monthly dynamics and diversity of Aeromonas spp. The bacterial counts showed bi-modal annual growth peak, pre- and post-monsoon, strongly correlating (p < 0.0005) with temperature. Of 200 isolates characterized, Aeromonas veronii bv. sobria (27%) was predominant among co-existent Aeromonas schubertii (20%), Aeromonas hydrophila (17%), Aeromonas caviae (13%), and three more. PCR screening of virulence-related genes identified 15 genotypes (I to XV), however, enterotoxigenicity in animal model was observed for five genotypes, ca. 18% (nine of 50) strains, prevalent in A. veronii bv. sobria, A. hydrophila, and A. caviae. Pathogenic strains were distinguishable by possessing at least three of the major virulence genes: ascV, hlyA, ela, ast, and alt, together with accessory virulence factors. PFGE of XbaI-digested genomic DNA revealed high genetic diversity and distant lineage of potentially toxigenic clones. Therefore, along with increased global warming, Aeromonas spp. having multi-factorial virulence potential in coastal ponds that serve as drinking water sources pose a potential health risk, and underscores the need for routine monitoring.

Salmonella Gallinarum in Small-Scale Commercial Layer Flocks: Occurrence, Molecular Diversity and Antibiogram.

Salmonella Gallinarum is one of the most important bacterial pathogens associated with diminished egg production in poultry. The aim of this study was to understand the occurrence, molecular traits and antimicrobial resistance patterns of Salmonella Gallinarum strains isolated from small-scale commercial layer flocks with low level biosecurity standards in Bangladesh. A total of 765 samples, including cloacal swabs (535), visceral organs (50), and droppings (180), were collected from chickens of 12 layer flocks in 11 districts. Salmonella Gallinarum was isolated and characterized through culture-based method, followed by biochemical tests, sero-grouping, PCR assays, sequencing, and antibiogram. The identity of biochemically detected isolates of Salmonella Gallinarum was confirmed via genus-specific 16S rRNA gene based PCR, followed by invA and spvC genes based PCR assays. Occurrence of Salmonella Gallinarum was detected in overall 25.75% (197/765) samples, with a significantly (p < 0.05) higher incidence in visceral organs (42%) in comparison to cloacal swab (24%) and droppings (26%). Sequencing and subsequent phylogenetic analysis of invA and spvC genes in representative strains of Salmonella Gallinarum revealed a close genetic lineage, with a sequence similarity of 98.05-99.21% and 97.51-99.45%, respectively, to previously published sequences of the corresponding genes from the same serogroup strains. Remarkably, 66.5% (131/197) of the isolated strains of Salmonella Gallinarum were found to be resistant to 3 to 6 antimicrobial agents, and interpreted as multidrug resistant (MDR). The findings of this study underscore an inherent need of appropriate control measures to curb the widespread incidence of MDR Salmonella Gallinarum in small-scale commercial layer flocks, thereby, facilitating enhanced egg production and further support to the food security and safety in low resource settings.

Comparison of Established PCR Assays for Accurate Identification of Campylobacter jejuni and Campylobacter coli.

Proper surveillance of Campylobacter jejuni and Campylobacter coli, major pathogens associated with human gastroenteritis, is necessary to tackle the increasing disease burden. To detect these pathogenic species, a variety of PCR assays have been developed. This study examined the sensitivity and specificity of 12 PCR assays targeting 23S rRNA, ceuE, lpxA, hipO, mapA, ask, and cdt genes of C. jejuni and C. coli. The sensitivities of PCR assays were 85.2-100%, and 97-100%, and the specificities were 90.5-100%, and 94.3-100% for the tested C. jejuni (n = 61) and C. coli (n = 33) strains, respectively. Two PCR assays, targeting cdtC and hipO genes, were found to be 100% sensitive and/or specific for all C. jejuni strains, while 3 assays, targeting cdtB, cdtA, and ask genes, were 100% sensitive and/or specific for C. coli strains. However, PCR assays for hipO and ask genes are problematic to conduct simultaneously due to the differences in PCR conditions. Overall, multiplex PCR assays targeting cdtC and cdtB genes, encoding 2 subunits of the same toxin, were concluded to be the most reliable. The results of this study would aid in proper surveillance of C. jejuni and C. coli and adopting intervention strategies in the near future.

Development of a multiplex PCR assay for the detection of major virulence genes in Vibrio cholerae including non-O1 and non-O139 serogroups.

Vibrio cholerae strains producing cholera toxin (CT) and toxin co-regulated pilus (TCP) and belonging to O1 and O139 serogroups are responsible for cholera. However, non-CT producing V. cholerae from non-O1/non-O139 serogroups have been increasingly isolated from diarrheal stools and extra-intestinal infections. In this study, we have developed a multiplex PCR for the simultaneous detection of heat-stable enterotoxin (stn), type three-secretion system (vopF), and cholix toxin (chxA), along with CT (ctx) in V. cholerae strains. As other species from genus Vibrio carries homologous virulence genes, V. cholerae specific ompW was also included to differentiate V. cholerae from other vibrios. This assay was 100% specific and sensitive, and could detect homologous virulence genes like ctxA in V. mimicus and vopF in V. parahaemolyticus. This multiplex PCR assay, which can detect four major virulence genes in V. cholerae, is novel and important for epidemiologic and environmental surveillance of pathogenic V. cholerae.

Occurrence, seasonality and genetic diversity of Vibrio vulnificus in coastal seaweeds and water along the Kii Channel, Japan.

Vibrio vulnificus is a ubiquitous toxigenic bacterium found in a coastal environment but little is known about its occurrence and seasonality among seaweeds, which are widely consumed as seafood in Japan. Therefore, we have observed the bacterium's abundance in seawater and seaweed samples from three areas of the Kii Channel, Japan, during June 2003 to May 2004. A total of 192 samples were collected: 24 from each source in summer, autumn, winter and spring. The samples were selectively cultivated following the most probable number (MPN) technique. Vibrio vulnificus population ranged from 0 to 10(3) MPN 100 mL(-1) seawater or 10 g seaweeds; higher counts were observed during summer. The optimum temperature, salinity and pH for the bacterium were 20-24 degrees C, 24-28 p.p.t. and 7.95-8.15, respectively. However, seaweeds always contained higher V. vulnificus than seawater. Among 280 V. vulnificus strains, detected by species-specific colony hybridization and PCR, 78, 74, 11 and 16 were from seaweeds and 46, 42, 2 and 11 were from seawater during summer, autumn, winter and spring, respectively. Ribotyping of 160 selected strains revealed a higher genotypic diversity (18 patterns) among strains from seaweeds than from seawater (10 patterns). Seaweeds can thus act as a potential habitat for V. vulnificus and are more unsafe for consumption during summer.

Seaweeds as a reservoir for diverse Vibrio parahaemolyticus populations in Japan.

Gastroenteritis caused by Vibrio parahaemolyticus has recently been associated with foods prepared with seaweeds, but little is known about the bacterium's abundance and diversity among seaweeds in coastal environment. Therefore, we determined its phenotypic and genotypic diversity in relation to its seasonal abundance in seawater and seaweed samples from three areas of Kii Channel, Japan during June 2003 to May 2004. Isolates were obtained by selective enrichment of samples and detection of V. parahaemolyticus by colony hybridization with a species-specific probe. A total of 128 isolates comprising 16 from each source in each season were characterized by serotyping and ribotyping. V. parahaemolyticus was more abundant in seaweeds (3,762 isolates) than in water samples (2,238 isolates). Twenty and 17 serotypes were found among the selected seaweed and seawater isolates, respectively. Cluster analysis revealed 19, 11, 7 and 9 ribotypes during summer, autumn, winter and spring, respectively. Seaweeds supported a diverse V. parahaemolyticus population throughout the year and thus seaweeds are a reservoir for the organism. However, V. parahaemolyticus occurrence had positive correlation with water temperature and its abundance in seaweeds was at least 50 times higher during summer than in winter.

Major tdh(+)Vibrio parahaemolyticus serotype changes temporally in the Bay of Bengal estuary of Bangladesh.

Vibrio parahaemolyticus is responsible for seafood-related gastroenteritis worldwide. In Bangladesh, diarrhea is endemic and diarrheagenic V. parahaemolyticus serotypes occur naturally in the coastal and estuarine aquatic environment. V. parahaemolyticus strains, isolated from estuarine surface water of the Bay of Bengal villages of Bangladesh during 2006-2008, were tested for the presence of virulence and pandemic-marker genes, serodiversity, and phylogenetic relatedness. PCR analysis of V. parahaemolyticus (n=175) showed 53 (30.3%) strains to possess tdh, the major virulence gene encoding thermostable direct hemolysin. Serotyping results revealed the tdh(+)V. parahaemolyticus strains to belong to 10 different serotypes, of which the O8:K21 (30.2%) and O3:K6 (24.5%) were predominantly non-pandemic and pandemic serotypes, respectively; while O5:K30 and O9:KUT were new. The pandemic markers, orf8 and toxRS(variant), were present only in the pandemic serotype O3:K6 (n=13) and its serovariant O4:K68 (n=2). Temporal distribution of the tdh(+) serotypes revealed the O8:K21 to be predominant in 2006 and 2007, while O3:K6 was the predominant tdh(+) serotype in 2008. Pulsed-field gel electrophoresis (PFGE) of SfiI-digested genomic DNA revealed high genetic diversity among the V. parahaemolyticus strains, while dendrogram constructed with the PFGE patterns formed two major clusters separating the tdh(+) O3:K6 and its pandemic serovariants from the tdh(+) non-pandemic (O8:K21) strains, suggesting different lineages for them. The potential health risk related to the prevalent tdh(+) strains, including the observed temporal change of the predominant tdh(+) serotype, from O8:K21 to the pandemic serotype O3:K6 in estuarine surface waters serving as the major source of drinking water suggests the need for routine environmental monitoring to prevent V. parahaemolyticus infection in Bangladesh.

Development of a PCR-restriction fragment length polymorphism assay for detection and subtyping of cholix toxin variant genes of Vibrio cholerae.

Cholix toxin (ChxA) is an exotoxin reported in Vibrio cholerae non-O1/non-O139. Apart from its prototype (ChxA I) we have recently identified two novel variants of this toxin, ChxA II and ChxA III. Our previous investigations indicated that the first two variants may instigate extra-intestinal infections and ChxA II can be more lethal than ChxA I in mice. However, all three cholix toxins (ChxA I to III) failed to show any enterotoxicity in rabbit ileal loops. In this study we developed a PCR-restriction fragment length polymorphism (RFLP) assay to differentiate all three chxA variants to further understand the importance of each subtype. By using 53 V. cholerae non-O1/non-O139 strains harbouring chxA genes, which were previously categorized by sequencing, and various other strains as negative controls, the PCR-RFLP assay showed 100 % typability and specificity. Furthermore, when applied to differentiate additional V. cholerae strains, which were also screened for the chxA gene by colony hybridization, this assay identified chxA I and chxA II genes among 18.5 % and 4.5 % of non-O1/non-O139 strains (n = 178), respectively. One non-O1/non-O139 strain was untypable due to the insertion of an IS911-like element. Interestingly, the chxA I gene was detected in 10 out of 137 cholera toxin gene-negative V. cholerae O1 strains. These results suggest that the PCR-RFLP assay developed in this study can be a rapid and simple method to differentiate the chxA subtypes.

Role of polyphosphate kinase gene (ppk) for survival of Vibrio cholerae O1 in surface water of Bangladesh.

Polyphosphate provides a substitute for ATP and energy source when phosphorus is a limiting resource in nature. The present study focuses on the role ofpolyphosphate for the survival of Vibrio cholerae in the aquatic habitats as an autochthonous bacterium. The survival advantages of polyphosphate of V. cholerae O1 having (wild type) and lacking (mutant) polyphosphate kinase (ppk) gene in surface water and with Anabaena variabilis were compared by cultural, Direct Fluorescent Antibody (DFA) and polymerase chain reaction methods in natural water microcosms. The microcosm's water was prepared by filtering and physicochemical parameters were also investigated by standard methods. The results revealed that both fresh and saline water, the wild type strain enhanced survival in cultural conditioned than ppk mutant strain. However, Fluorescent Antibody Direct Viable Counts (FADVC) and Polymerase Chain Reaction (PCR) results noted both strains have the equal survival strategy in viable but nonculturable state (VNC). In conclusion, it could be hypothesized that the polyphosphate inclusion body might keep cultivable and survivable at low phosphate natural environment of the aquatic bacterium.

Identification and characterization of integron-associated antibiotic resistant Laribacter hongkongensis isolated from aquatic products in China.

Laribacter hongkongensis is a recently discovered bacterium associated with gastroenteritis. In this study, a total of 199 isolates of this species obtained from aquatic products (n=462) in Guangzhou City, China, were examined for their susceptibility to 19 antimicrobial agents and the presence of antimicrobial resistance integrons. The genetic relatedness of the isolates with integrons was also evaluated. A PCR-based method was used to screen integrons and found that 13 (6.5%) of the isolates harbored class 1 integrons. The antimicrobial resistance rates of integron-positive isolates were significantly higher than integron-negative ones for cefepime, ciprofloxacin, gentamicin, tetracycline, trimethoprim-sulfamethoxazole and rifampicin. Genetic sequence analysis revealed that these integrons contained various antimicrobial-resistance genes (dfrA1, dfrA14, dfrA17, dfrA32, aadA1, aadA2, aadA5, cmlA5, arr2, ereA and orfC) organized into different gene cassettes arrangements including a novel array of dfrA14-arr2-cmlA5. Pulsed-field gel electrophoresis (PFGE) yielded 13 different patterns among 13 integron-positive isolates, which could be grouped into four clusters. These indicate the dispersal of multi-resistant integrons among different molecular types. To the best of our knowledge, this is the first report showing distribution and characterization of class 1 integrons among L. hongkongensis isolates.

Prevalence and characterization of antimicrobial resistance of foodborne Listeria monocytogenes isolates in Hebei province of Northern China, 2005-2007.

A total of 2177 food samples collected from nine cities in northern China during 2005 to 2007 were screened for the presence of Listeria monocytogenes. All L. monocytogenes isolates were subjected to serotyping, antimicrobial susceptibility, pulsed-field gel electrophoresis (PFGE), as well as PCR screening to identify genes responsible for tetracycline resistance [tet(L), tet(M), tet(K), tet(S) and tet(B)], transposon Tn916, and class 1 integron. Contamination with L. monocytogenes was detected in 4.13% (90/2177) of the total samples representing various food products. The pathogen was mainly isolated from frozen food made of wheat flour or rice products (26/252, 10.32%) and raw meat products (46/733, 6.28%). Besides, 3.31% (10/302) of cooked meat, 1.17% (4/343) of seafood, 0.98% (2/204) of non-fermented bean products and 0.62% (2/323) of vegetables samples were contaminated by this bacterium. The L. monocytogenes isolates belonged to five serotypes (1/2a, 1/2b, 1/2c, 4b, and 3a), with serotype 1/2a being dominant (48.88%). Antimicrobial resistance was most frequently observed for ciprofloxacin (17.8%), tetracycline (15.6%) and streptomycin (12.2%). Overall, resistance was observed against 14 out of 18 antimicrobials tested while multiple resistances occurred among 18.9% (17/90) isolates. Interestingly, two isolates were resistant to more than five antimicrobials. Among 14 tetracycline-resistant isolates, 13 carried tet(M) gene including nine possessing Tn916, and one harbored tet(S) gene. PFGE analysis revealed genetic heterogeneity among individual serotypes as well as scattered occurrence of some genotypes without any clear-cut correlation to source or food type. The widespread distribution of epidemiologically important serotypes (1/2a, 1/2b and 4b) of L. monocytogenes, and their resistance to commonly used antibiotics indicate a potential public health risk. Our data also indicate that L. monocytogenes could act as a reservoir of mobile tet genes along the food chain.