Sucharit Basu Neogi

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.

A highly sensitive and specific multiplex PCR assay for simultaneous detection of Vibrio cholerae, Vibrio parahaemolyticus and Vibrio vulnificus.

Aims:  To develop an effective multiplex PCR for simultaneous and rapid detection of Vibrio cholerae, Vibrio vulnificus and Vibrio parahaemolyticus, the three most important Vibrio species that can cause devastating health hazards among human.

Capsaicin, a potential inhibitor of cholera toxin production in Vibrio cholerae.

The use of natural compounds as inhibitory agents for virulence factor production is a new approach to overcome increased antimicrobial resistance in pathogenic bacteria. In this study, we examined whether red chilli (Capsicum annuum) contains any such compound(s) that can repress the cholera toxin (CT) production in Vibrio cholerae. We found that the methanol extract of red chilli could inhibit CT production in recently emerged V. cholerae O1 El Tor variant strains without affecting their viability. Interestingly, capsaicin, a well-studied active component of red chilli, also drastically inhibited CT production in V. cholerae strains belonging to various serogroups including variants. Real-time quantitative reverse transcription-PCR assay revealed that capsaicin effectively repressed the transcription of ctxA, tcpA and toxT genes, but not of toxR and toxS genes. On the contrary, capsaicin significantly enhanced the transcription of the hns gene, the product of which is known to regulate negatively the transcription of ctxAB, tcpA and toxT genes. These results suggest that capsaicin might act as a potent repressor for CT production possibly by enhancing the transcription of hns.

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 bacteriums 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.

Influence of Catastrophic Climatic Events and Human Waste on Vibrio Distribution in the Karnaphuli Estuary, Bangladesh

Vibrios are bacteria of marine and estuarine origin that can cause human diseases, such as cholera, and also affect aquatic organisms. The impact of storm-driven changes in salinity and suspended particulate matter (SPM) on cultivable Vibrio counts (CVC) and distribution in Karnaphuli estuary, Bangladesh, was compared before and after a strong cyclone in mid May 2007 and after a monsoon landslide a month later. CVC were higher (~103 colony forming units—cfu/ml) at estuary’s mouth (salinity 20–15 parts per thousand, ppt) and steeply declined landwards. CVC and their proportion of total aerobic bacteria were highest after the cyclone and also increased after the landslide, likely due to higher SPM loads. The cyclone did not significantly change previous fecal coliform abundance, contrasting with the ten times increase after the landslide. Sewage input enhanced CVC near the point sources. CVC and salinity correlated highly significantly at salinities <10 ppt; however, at higher values dispersion increased, probably due to the effect of sediment resuspension on CVC. Cyclone or heavy rainfall-mediated turbidity changes jointly with salinity gradients can significantly influence abundance and distribution of estuarine vibrios. Extended salt intrusion and higher turbidities in tropical estuaries by stronger and more frequent storms and deforestation-derived erosion could favor Vibrio growth, with increasing risks for aquatic resources and human health in the coastal zone.

Development of a haemolysin gene-based multiplex PCR for simultaneous detection of Vibrio campbellii, Vibrio harveyi and Vibrio parahaemolyticus.

Aim:  To develop a haemolysin (hly) gene‐based species‐specific multiplex PCR for simple and rapid detection of Vibrio campbellii, V. harveyi and V. parahaemolyticus.

Novel Cholix Toxin Variants, ADP-Ribosylating Toxins in Vibrio cholerae Non-O1/Non-O139 Strains, and Their Pathogenicity

ABSTRACT 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.

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.

Influence of hydrologic and anthropogenic factors on the abundance variability of enteropathogens in the Ganges estuary, a cholera endemic region.

This study deals with the influence of water physico-chemical properties, tides, rainfall and fecal pollution on the abundance of enteropathogens in a main distributary of the Ganges, in the endemic cholera belt of West Bengal. Between January and June 2011, water and sediments were sampled from two sites of the Hooghly River by Kolkata and Diamond Harbour. Counts of cultivable Vibrio (CVC, from~10(2) to~10(5)CFU/L) and total bacteria (TBC, from~10(5) to~10(9)CFU/L) increased with water temperature (17°C to 37°C). A combination of variations in tidal height, salinity and turbidity had a distinct influence on CVC, TBC and coliform counts. At Diamond Harbour, a salinity increase from 0.6 to 7.9 was accompanied by a 1000-fold amplification of initial CVC~10(2)CFU/L, whereas higher prevalence of coliforms in Kolkata was related to greater disposal of untreated sewage into the river. Turbidity-dependent variation of CVC was noteworthy, particularly at Diamond Harbour, where CVC in intertidal surface sediments showed an analogous trend as in surface waters, suggesting bentho-pelagic coupling of Vibrio dynamics. Besides the influence of salinity variation with tidal cycles, sediment re-suspension from tidal flats can play a role on Vibrio abundance in aquatic ecosystems.

Development of simple and rapid PCR-fingerprinting methods for Vibrio cholerae on the basis of genetic diversity of the superintegron

Aims:  To develop simple and rapid PCR‐fingerprinting methods for Vibrio cholerae O1 (El Tor and classical biotypes) and O139 serogroup strains which cause major cholera epidemics, on the basis of the diversity of superintegron (SI) carried by these strains.