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Emergence and spread of antimicrobial-resistant bacteria, including extended-spectrum β-lactamase (ESBL)-producing Escherichia coli, have become serious problems worldwide. Recent studies conducted in Vietnam revealed that ESBL-producing E. coli are widely distributed in food animals and people. CTX-M-9 and CTX-M-1 are the most prevalent β-lactamases among the identified ESBLs. Furthermore, most of the ESBL-producing E. coli isolates were multi-drug resistant. Residual antimicrobials such as sulfamethoxazole, trimethoprim, sulfadimidine, cephalexin, and sulfadiazine were also detected at a high level in both animal meats and environmental water collected from several cities, including Ho Chi Minh city and Can Tho city. These recent studies indicated that improper use of antimicrobials in animal-originated food production might contribute to the emergence and high prevalence of ESBL-producing E. coli in Vietnam. Although clonal ESBL-producing E. coli was not identified, CTX-M-55 gene-carrying plasmids with similar sizes (105–139 kb) have been commonly detected in the ESBL-producing E. coli strains isolated from various food animals and human beings. This finding strongly suggests that horizontal transfer of the CTX-M plasmid among various E. coli strains played a critical role in the emergence and high prevalence of ESBL-producing E. coli in Vietnam. [ABSTRACT FROM AUTHOR]

Abstract: An extracellular lipase (EC 3.1.1.3), SAL-PP1, from Staphylococcus aureus isolated from Arachis hypogaea rhizosphere was purified and characterized. The enzyme was purified using PALL''S Microsep centrifugal device (10kD cut off), hydrophobic interaction (phenyl sepharose CL-4B column) and Superose-12 gel filtration chromatography and found to have a molecular mass of around 49kDa. The gene fragment encoding the part of the catalytic site of the SAL-PP1 lipase was sequenced and the deduced amino acid sequence shows 93% identity with that of SEL3. SAL-PP1 showed activity against long acyl-chain triglycerides, various p-nitrophenyl esters and phospholipids. The enzyme shows high stability and activity after incubation with various metal ions (retained >90% activity in presence of Ca2+, Na+, Cu2+, Mg2+, Fe2+, or Hg2+ at 10mM), organic solvents (retained >80% activity in presence of acetonitrile, ethanol, DMSO, methanol, isopropanol, toluene, or ethylene glycol at 10mM), detergents (retained >70% activity in Triton X-100, Tween 80, or sodium deoxycholate at 10mM) and irreversible inhibitors (retained >77% activity in presence of PMSF, leupetin, or β-mercaptoethanol, at 1mM). Thermal inactivation studies revealed a temperature dependent unfolding of secondary structure of protein. SAL-PP1 showed maximal activity and stability at pH 8.0 and pH 9.0, respectively. The alkali-thermostability, organic solvent-tolerance and broad substrate specificity of this enzyme may have potential implications in detergent formulations, biotransformation, industries, and medicine. [Copyright &y& Elsevier]

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. [ABSTRACT FROM AUTHOR]

Abstract: Effect of silicon dioxide (silica) addition into titanium oxide (titania) films on crystallinity and photo-induced hydrophilicity was investigated. Titania films and titania–silica mixed films with 20% or 50% silica were prepared by spin-coating deposition. For the titania films, photo-induced hydrophilicity is only observed in the films with anatase polycrystalline structure. For the titania–silica mixed films, however, despite these films showing no obvious anatase polycrystalline structure, very strong photo-induced hydrophilicity is observed. Particularly, even super-hydrophilicity where the contact angle shows 0° is easily observed for the titania–silica mixed films with 20% silica. The hydrophilicity of the titania films correlates closely with anatase crystalline structure. In the titania–silica mixed films, the synergistic effects of anatase polycrystalline structure of titania and water-trapping effect of silica are important for their strong photo-induced hydrophilicity. [Copyright &y& Elsevier]

Vibrio cholerae O139, the new serogroup associated with epidemic cholera, came into being in the second half of the year 1992 in an explosive fashion and was responsible for several outbreaks in India and other neighbouring countries. This was an unprecedented event in the history of cholera and the genesis of the O139 serogroup was, at that time, thought to be the beginning of the next or the eighth pandemic of cholera. However, with the passage of time, the O1 serogroup of the El Tor biotype again reappeared and displaced the O139 serogroup on the Indian subcontinent, and there was a feeling among cholera workers that the appearance of this new serogroup may have been a one-time event. The resurgence of the O139 serogroup in September 1996 in Calcutta and the coexistence of both the O1 and O139 serogroups in much of the cholera endemic areas in India and elsewhere, suggested that the O139 serogroup has come to stay and is a permanent entity to contend with in the coming years. During the past 10 years, intensive work on all aspects of the O139 serogroup was carried out by cholera researchers around the world. The salient findings on this serogroup over the past 10 years pertinent to its prevalence, clinico-epidemiological features, virulence-associated genes, rapid screening and identification, molecular epidemiology, and vaccine developments have been highlighted. [Copyright &y& Elsevier]

Deals with a study which investigated the prevalance of Shiga toxin-producing Escherichia coli in hospitalized diarrhea patients in Calcutta, India, as well as in healthy domestic cattle and raw beef samples collected from the city's abattoir. Background to the study; Materials and methods; Results.

An unusually high number of sporadic cholera outbreaks have occurred in various parts of Kenya since January 2009. Clinical symptoms of cholera play an important role in the diagnosis and management of the disease, especially in resource-poor settings in most developing countries. We describe a case report of a patient who was treated for cholera according to symptoms at presentation to hospital. Non-typhoid Salmonella was later isolated and the patient's condition improved after administration of ciprofloxacin. [ABSTRACT FROM AUTHOR]

• SARS-CoV-2 epitope dominance patterns are either NTD- or RBD-dominant. • NTD-dominant sera neutralise the B.1.617.2 variant less than RBD-dominant sera. • Infectivity-enhancing antibodies are unlikely to impair cross-variant neutralisation. SARS-CoV-2 serological studies suggest that individual serum antibody repertoires can affect neutralisation breadth. Herein, we asked whether a BNT162b2 vaccine-induced epitope dominance pattern (i.e., predominant viral structural domain targeted by serum antibodies for virus neutralisation) affects cross-variant neutralisation. When a neutralisation assay against the ancestral strain was carried out using 16 vaccine sera preabsorbed with a recombinant receptor-binding domain (RBD) or an N-terminal domain (NTD) protein, three and 13 sera, respectively, showed lower neutralisation under NTD and RBD protein-preabsorbed conditions than under the other protein-preabsorbed conditions. This suggests that the NTD was responsible for virus neutralisation in three sera, whereas the other 13 sera elicited RBD-dominant neutralisation. The results also suggest the presence of infectivity-enhancing antibodies in four out of the 13 RBD-dominant sera. A neutralisation assay using SARS-CoV-2 variants revealed that NTD-dominant sera showed significantly reduced neutralising activity against the B.1.617.2 variant, whereas RBD-dominant sera retained neutralising activity even in the presence of infectivity-enhancing antibodies. Taken together, these results suggest the followings: (i) epitope dominance patterns are divided into at least two types: NTD-dominant and RBD-dominant; (ii) NTD-dominant sera have less potential to neutralise the B.1.617.2 variant than RBD-dominant sera; and (iii) infectivity-enhancing antibodies play a limited role in cross-variant neutralisation against the five variants tested. [ABSTRACT FROM AUTHOR]

Only a small number of islands were colonized by Homo sapiens during the Pleistocene. One such island group is the Ryukyu Archipelago located between Kyushu and Taiwan. The Minotogawa Fissure site, located on the island of Okinawa in the Ryukyu Archipelago, is known for well-preserved late Pleistocene Homo sapiens fossil remains that were unearthed in the 1960s. In addition, eight Paleolithic sites and four other possible ones have been identified in the Central Ryukyus (Amami and Okinawa Archipelagos) in the last four decades. Were these Paleolithic groups direct ancestors of modern peoples who currently live in the region? Recent studies in archaeology and other related fields have shown that colonization processes in the Central Ryukyus were complex. It has been suggested that the islands' initial successful colonizers were people who migrated there during the later part of the Early 3 to Early 4 Periods; however, recent studies and findings do not support this hypothesis. Currently, the Early 1, pre-Early 1, and Paleolithic hypotheses seem to be the most acceptable regarding the timing of initial colonization of the islands. Whatever the case, there appears to have been a second major population influx at the end of the Shellmidden or initial Gusuku Period. [ABSTRACT FROM AUTHOR]

Aim The aim of this study was to develop a selective enrichment broth for efficient isolation of Escherichia albertii. Methods and Results A total of 412 raccoon rectal swabs suspended in PBS (phosphate-buffered saline) were tested by a real-time PCR to quantify the number of E. albertii followed by its isolation. The number of E. albertii in the PBS suspension strongly affected the isolation rate (1.2%–89%), which notably dropped (≤33%) when the number was <4 log10 CFU ml−1. However, enrichment of PBS suspension containing raccoon feces in tryptic soy broth containing cefixime, tellurite, and deoxycholate (CTD-TSB), the selective medium developed in this study, remarkably improved the isolation efficiency (up to 48%) of E. albertii. Conclusions CTD-TSB is a useful enrichment culture medium for E. albertii and contributes to increase its isolation rate. [ABSTRACT FROM AUTHOR]

Aim: A novel coronavirus, termed severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) suddenly appeared in Wuhan, China, and has caused pandemic. In this study, we evaluated antiviral activity of purified hypochlorous acid (HClO) against coronaviruses such as SARS‐CoV‐2 and transmissible gastroenteritis virus (TGEV) responsible for pig diseases. Materials and Results: In a suspension test, 28.1 ppm HClO solution inactivated SARS‐CoV‐2 in phosphate‐buffered saline with the reduction of 104 of 50% tissue culture infectious dose per ml (TCID50 per ml) within 10 s. When its concentration increased to 59.4 ppm, the virus titre decreased to below the detection limit (reduction of 5 logs TCID50) within 10 s even in the presence of 0.1% foetal bovine serum. In a carrier test, incubation with 125 ppm HClO solution for 10 min or 250 ppm for 5 min inactivated SARS‐CoV‐2 by more than 4 logs TCID50 per ml or below the detection limit. Because the titre of TGEV was 10‐fold higher, TGEV was used for SARS‐CoV‐2 in a suspension test. As expected, 56.3 ppm HClO solution inactivated TGEV by 6 logs TCID50 within 30 s. Conclusions: In a carrier test, 125 ppm HClO solution for 10 min incubation is adequate to inactivate 4 logs TCID50 per ml of SARS‐CoV‐2 or more while in a suspension test 56.3 ppm HClO is adequate to inactivate 5 logs TCID50 per ml of SARS‐CoV‐2 when incubated for only 10 s regardless of presence or absence of organic matter. Significance and Impact of the Study: Effectiveness of HClO solution against SARS‐CoV‐2 was demonstrated by both suspension and carrier tests. HClO solution inactivated SARS‐CoV‐2 by 5 logs TCID50 within 10 s. HClO solution has several advantages such as none toxicity, none irritation to skin and none flammable. Thus, HClO solution can be used as a disinfectant for SARS‐CoV‐2. [ABSTRACT FROM AUTHOR]

We applied a new geoarchaeological method with two carbonate archives, which are fossil snails from Sakitari Cave and stalagmites from Gyokusen Cave, on Okinawa Island, Japan, to reconstruct surface air temperature changes over the northwestern Pacific since the last glacial period. Oxygen isotope ratios (δ18O) of modern and fossil freshwater snail shells were determined to infer seasonal temperature variations. The observational and analytical data confirm that δ18O values of fluid inclusion waters in the stalagmite can be regarded as those of spring waters at the sites where snails lived. Our results indicate that the annual mean, summer, and winter air temperatures were lower by 6–7 °C at ca. 23 thousand years ago (ka) and 4–5 °C at ca. 16–13 ka than those of the present day. Our reconstruction implies that surface air cooling was possibly two times greater than that of seawater around the Ryukyu Islands during the Last Glacial Maximum, which potentially enhanced the development of the East Asian summer monsoon during the last deglaciation. Considering the potential uncertainties in the temperature estimations, the climatic interpretations of this study are not necessarily definitive due to the limited number of samples. Nevertheless, our new geoarchaeological approach using coupled δ18O determinations of fossil snails and stalagmite fluid inclusion waters will be useful for reconstructing snapshots of seasonally resolved time series of air temperatures during the Quaternary. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Campylobacter enteritis is the leading cause of gastroenteritis in humans worldwide including Bangladesh. The objectives of this study were to estimate the prevalence and antimicrobial-resistance status of Campylobacter spp. in human diarrheal samples collected from Surya Kanta Hospital, Mymensingh, Bangladesh. In this study, we evaluated a total of 330 clinical samples for the presence Campylobacter spp. via cultural and biochemical tests and molecular assays. Furthermore, antimicrobial susceptibility testing for Campylobacter species was accomplished by the standard agar disc diffusion technique against eight commercially available antimicrobial agents. A pretested semistructured questionnaire was used to capture the data on socioanthropological factors from the diarrheal patients. Pearson's chi-square test was performed, and a p value of <0.05 was considered for the level of significance. Nearly one in three diarrheal patients admitted in this hospital were infected with Campylobacter spp. Overall prevalence of Campylobacter spp. was estimated to be 31.5% (104/330) that comprised the prevalence of C. jejuni, 21.8% (n = 72), and C. coli, 9.6% (n = 32). Among the positive cases, the prevalence of Campylobacter was higher in the age group 0-5 years (52%) followed by 6-18 years (42.7%), 19-40 years (34.0%), 41-60 years (25.4%), and >60 years (10.5%). Age, family level's personal hygiene, and involvement with animal husbandry were captured as potential determinants to be associated with the Campylobacter positive status. Among the isolates, 27.3% (n = 20) of C. jejuni and 31.2% (n = 10) of C. coli demonstrated as multidrug-resistant (MDR) to three or more antimicrobial agents. The present study shows that Campylobacter spp. is most prevalent among the hospital-admitted diarrheal patients, and proper measures should be taken to reduce the burden focusing on the potential determinants. [ABSTRACT FROM AUTHOR]

Escherichia albertii, often misidentified as Escherichia coli, has become an emerging foodborne human enteric pathogen. However, the prevalence and major animal reservoirs of this significant pathogen are still not clear. Here, we performed comprehensive microbiological, molecular, comparative genomics and animal studies to understand the status and features of E. albertii in the US domestic and food animals. Although no E. albertii was identified in a total of 1,022 diverse E. coli strains isolated from pets and food animals in a retrospective screening, in a pilot study, E. albertii was successfully isolated from a broiler farm (6 out of 20 chickens). The chicken E. albertii isolates showed clonal relationship as indicated by both pulsed‐field gel electrophoresis (PFGE) and whole‐genome sequence analysis. The isolated chicken E. albertii displayed multidrug resistance; all the resistance determinants including the extended‐spectrum beta‐lactamase gene, carried by plasmids, could be conjugatively transferred to E. coli, which was further confirmed by S1‐PFGE and Southern hybridization. Whole‐genome sequence‐based phylogenetic analysis showed the chicken E. albertii strains were phylogenetically close to those of human origins. Challenge experiment demonstrated that the E. albertii strains isolated from human and wild bird could successfully colonize in the chicken intestine. Together, this study, for the first time, reported the isolation of E. albertii in poultry at the pre‐hrvest level. The findings from multi‐tier characterization of the chicken E. albertii strains indicated the importance of chickens as a reservoir for E. albertii. A large scale of E. albertii survey in poultry production at the pre‐harvest level is highly warranted in the future. [ABSTRACT FROM AUTHOR]

Ready-to-eat (RTE) foods have been considered to be reservoirs of antibiotic resistance bacteria, which constitute direct threat to human health, but the potential microbiological risks of RTE foods remain largely unexplored. In this study, the metagenomic approach was employed to characterize the comprehensive profiles of bacterial community and antibiotic resistance gene (ARG) in 18 RTE food samples (8 RTE meat, 7 RTE vegetables and 3 RTE fruit) in southern China. In total, the most abundant phyla in RTE foods were Proteobacteria, Firmicutes, Cyanobacteria, Bacteroidetes and Actinobacteria. 204 ARG subtypes belonging to 18 ARG types were detected with an abundance range between 2.81 × 10−5 and 7.7 × 10−1 copy of ARG per copy of 16S rRNA gene. Multidrug-resistant genes were the most predominant ARG type in the RTE foods. Chloramphenicol, macrolide-lincosamide-streptogramin, multidrug resistance, aminoglycoside, bacitracin, tetracycline and β-lactam resistance genes were dominant, which were also associated with antibiotics used extensively in human medicine or veterinary medicine/promoters. Variation partitioning analysis indicated that the join effect of bacterial community and mobile genetic elements (MGEs) played an important role in the resistome alteration. This study further deepens the comprehensive understanding of antibiotic resistome and the correlations among the antibiotic resistome, microbiota, and MGEs in the RTE foods. [ABSTRACT FROM AUTHOR]

Natural reservoirs of Escherichia albertii remain unclear. In this study, we detected E. albertii by PCR in 248 (57.7%) of 430 raccoons from Osaka, Japan, and isolated 143 E. albertii strains from the 62 PCR-positive samples. These data indicate that raccoons could be a natural reservoir of E. albertii in Japan. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Many Escherichia albertii isolates, an emerging pathogen of human and birds, might have been misidentified due to the difficulty of differentiating this bacterium from Escherichia coli and Shigella spp. by routine biochemical tests, resulting in underestimation of E. albertii infections. We have developed a polymerase chain reaction (PCR) assay that targets E. albertii cytolethal distending toxin (Eacdt) genes, which include the genes previously identified as Escherichia coli cdt-II. This assay could generate a single 449-bp PCR product in each of 67 confirmed E. albertii strains but failed to produce PCR product from any of the tested non– E. albertii enteric strains belonging to 37 different species, indicating 100% sensitivity and specificity of the PCR assay. The detection limit was 10 CFU per PCR tube and could detect 105 CFU E. albertii per gram of spiked healthy human stool. The Eacdt gene-based PCR could be useful for simple, rapid, and accurate detection and identification of E. albertii. [ABSTRACT FROM AUTHOR]

Background: Toxigenic Vibrio cholerae resides in aquatic reservoirs of cholera-endemic areas mostly in a dormant form known as conditionally viable environmental cells (CVEC) in which the bacteria remain embedded in an exopolysaccharide matrix, and fail to grow in routine bacteriological culture. The CVEC can be resuscitated by supplementing culture media with either of two autoinducers CAI-1 and AI-2, which are signal molecules controlling quorum sensing, a regulatory network of bacterial gene expression dependent on cell density. This study investigated possible existence of variant strains that overproduce AIs, sufficient to resuscitate CVEC in environmental waters. Methods: Environmental V. cholerae isolates and Tn insertion mutants of a V. cholerae strain C6706 were screened for production of AIs using bioluminescent reporter strains. Relevant mutations in environmental strains which overproduced AI-2 were characterized by nucleotide sequencing and genetic complementation studies. Effect of AIs produced in culture supernatants of relevant strains on reactivation of CVEC in water was determined by resuscitation assays. Results: Two of 54 environmental V. cholerae isolates were found to overproduce AI-2. Screening of a Tn-insertion library of V. cholerae strain C6706, identified a mutant which overproduced AI-2, and carried Tn insertion in the cqsS gene. Nucleotide sequencing also revealed mutations inactivating the cqsS gene in environmental isolates which overproduced AI-2, and this property was reversed when complemented with a wild type cqsS gene. Culture of river water samples supplemented with spent medium of these mutants resuscitated dormant V. cholerae cells in water. Significance: V. cholerae strains with inactivated cqsS gene may offer a convenient source of AI-2 in enhanced assays for monitoring bacteriological quality of water. The results also suggest a potential role of naturally occurring cqsS mutants in the environmental biology of V. cholerae. Furthermore, similar phenomenon may have relevance in the ecology of other waterborne bacterial pathogens beyond V. cholerae. [ABSTRACT FROM AUTHOR]

We developed a new GeneFields® EHEC/SS PCR dipstick DNA chromatography kit for the simultaneously detection of invA , ipaH , and stx genes in Salmonella enterica (56 strains), Shigella spp. (44), and enterohemorrhagic Escherichia coli (EHEC) (28), respectively, and evaluated the sensitivity and specificity with other bacteria (57) by this kit. The sensitivity and specificity were 100%, respectively. The detection limit of various methods was determined using 5% (w / v) stool suspensions spiked with each bacterium. The detection limit of the GeneFields® EHEC/SS kit ranged from approximately 102–103 CFU/g. Additionally, the relative sensitivities and specificities of the GeneFields® EHEC/SS kit vs two commercially available real-time PCR kits were >85.0% and >90.0%, respectively. These results indicate that the GeneFields® EHEC/SS kit can be used for genetic screening of S. enterica , Shigella spp., and EHEC in human stool specimens with sensitivities and specificities similar to those of the commercially available real-time PCR kits. • GeneFields® EHEC/SS detected S. enterica , Shigella spp., and EHEC in stool specimens. • The detection limit of this kit is comparable to that of the real-time PCR kits. • This kit can be employed in a routine surveillance test of 5,000 stool specimens. [ABSTRACT FROM AUTHOR]

Abstract 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. Highlights • A multiplex PCR for detection of clinically important virulence genes in V. cholerae is described • Virulence genes not only from V. cholerae O1 but also non-O1/non-O139 are included • The multiplex PCR targeted genes for cholera toxin, cholix toxin, NAG-ST and T3SS • This assay can also detect virulence gene homologues in other vibrio species • This assay is simple, rapid, highly specific and sensitive to detect virulence genes in vibrios [ABSTRACT FROM AUTHOR]

Food poisonings caused by Clostridium perfringens and Shiga toxin (Stx)-producing Escherichia coli (STEC) occur frequently worldwide; however, no vaccine is currently available. Therefore, we aimed to develop a bivalent vaccine against C. perfringens and STEC infections. Although it has been considered that the C-terminal region of C. perfringens enterotoxin (C-CPE) could be a good vaccine antigen to block the binding to its receptor, it was insufficient for induction of a protective immune response because of the low antigenicity. However, the fusion of C-CPE with Stx2 B subunit (Stx2B) augmented the antigenicity of C-CPE without affecting the antigenicity of Stx2B. Indeed, high levels of C-CPE-specific neutralizing IgG were found in the serum of mice immunized with the fusion protein Stx2B–C-CPE. Additionally, comparable and substantial levels of Stx2B-specific neutralizing IgG were induced in mice receiving Stx2B–C-CPE or Stx2B alone. These antibody responses against C-CPE and Stx2B lasted for at least 48 weeks, which were sufficient for protective immunity in vitro and in vivo, indicating that Stx2B–C-CPE could induce long-term protective immunity. As an underlying mechanism, ex vivo stimulation with Stx2B, but not with C-CPE, induced cytokine production from splenic T cells collected from mice immunized with Stx2B–C-CPE, suggesting that Stx2B-specific, but not C-CPE-specific, T cells were induced by the immunization with Stx2B–C-CPE and plausibly promoted immunoglobulin class switching of both Stx2B- and C-CPE-specific B cells from IgM to IgG. These findings collectively indicate that Stx2B–C-CPE is a T-cell-antigen-supplement-type bivalent vaccine, which could be an efficient against C. perfringens and STEC infections. [ABSTRACT FROM AUTHOR]

The ecology of the most relevant Vibrio species for human health and their relation to water quality and biogeochemistry were studied in two estuaries in Argentinian Patagonia. Vibrio cholerae and Vibrio parahaemolyticus were reported in > 29% of cases at the Río Colorado and Río Negro estuaries. Neither the pandemic serogroups of Vibrio cholerae O1, Vibrio cholerae O139 nor the cholera toxin gene were detected in this study. However, several strains of V. cholerae (not O1 or O139) are able to cause human disease or acquire pathogenic genes by horizontal transfer. Vibrio vulnificus was detected only in three instances in the microplankton fraction of the Río Negro estuary. The higher salinity in the Río Colorado estuary and in marine stations at both estuaries favours an abundance of culturable Vibrio . The extreme peaks for ammonium, heterotrophic bacteria and faecal coliforms in the Río Negro estuary supported a marked impact on sewage discharge. Generally, the more pathogenic strains of Vibrio have a faecal origin. Salinity, pH, ammonium, chlorophyll a, silicate and carbon/nitrogen ratio of suspended organic particulates were the primary factors explaining the distribution of culturable bacteria after distance-based linear models. Several effects of dissolved organic carbon on bacterial distribution are inferred. Global change is expected to increase the trophic state and the salinisation of Patagonian estuaries. Consequently, the distribution and abundance of Vibrio species is projected to increase under future changing baselines. Adaptation strategies should contribute to sustaining good water quality to buffer climate- and anthropogenic- driven impacts. [ABSTRACT FROM AUTHOR]

Escherichia albertii is an emerging enteric bacterial pathogen causing watery diarrhea, abdominal distension, vomiting and fever in humans. E. albertii has caused many foodborne outbreaks in Japan and was also reported in other countries worldwide. However, the important animal reservoirs of this pathogen are still largely unknown, impeding us to combat this emerging pathogen. Recently, we reported that wild raccoons (Procyon lotor) and broiler chickens are significant reservoirs of E. albertii in Japan and the U.S., respectively. Here, we performed a longitudinal surveillance to monitor prevalence of E. albertii in wild raccoons in the U.S. and conducted comprehensive comparative analyses of the E. albertii of different origins. A total of 289 fecal swab samples were collected from wild raccoons in Tennessee and Kentucky in the U.S. (2018–2020). Approximately 26% (74/289) of the raccoons examined were PCR-positive for E. albertii and eventually 22 E. albertii isolates were obtained. PFGE analysis showed the U.S. raccoon E. albertii were phylogenetically distant even though the corresponding raccoons were captured from a small area. Unlike the high prevalence of multidrug resistance (83%) observed in previous chicken E. albertii survey, antibiotic resistance was rarely observed in all the U.S. raccoon and 22 Japan raccoon strains with only one Japan strain displaying multidrug resistance (2%). Whole genome sequencing of 54 diverse E. albertii strains and subsequent comparative genomics analysis revealed unique clusters that displayed close evolutionary relationships and similar virulence gene profiles among the strains of different origins in terms of geographical locations (e.g., U.S. and Japan) and hosts (raccoon, chicken, swine, and human). Challenge experiment demonstrated raccoon E. albertii strains could successfully colonize in the chicken intestine at 3 and 8 days postinfection. A pilot environmental survey further showed all the four tested water samples from Tennessee river were E. albertii -positive; two different E. albertii strains, isolated from a single water sample, showed close relationships to those of human origin. Together, the findings from this study provide new insights into the ecology, evolution, and pathobiology of E. albertii, and underscore the need to control the emerging E. albertii in a complex ecosystem using One Health approach. [ABSTRACT FROM AUTHOR]

The objectives of this study were to investigate prevalence and pathogenic potential of Escherichia coli contaminating raw milk and its products in Egypt. Out of 187 dairy products including 72 raw milk samples, 55 Karish cheese and 60 Ras cheese, 222 E. coli isolates including 111, 89 and 22 were obtained from 55 raw milk samples (76.4%), 41 Karish cheese (74.5%), and 13 Ras cheese (21.7%), respectively. Isolated E. coli strains were examined for 24 representative virulence genes present in diarrheagenic E. coli (DEC) and extraintestinal pathogenic E. coli (ExPEC). Among DEC and ExPEC virulence factors, genes for enteropathogenic E. coli ( eaeA , bfpA , EAF), enterohemorrhagic E. coli ( stx1 , stx2 , eaeA ), enterotoxigenic E. coli ( elt , est ), enteroinvasive E. coli ( invE ), enteroaggregative E. coli ( Eagg , astA ), diffusely adherent E. coli ( daaD ), ExPEC ( cdt-I to cdt - V , cnf1 , cnf2 , hlyA ) and putative adhesins ( efa1 , iha , ehaA , saa , and lpfA O113 ) were screened by colony hybridization assay. Out of 222 E. coli strains, 104 (46.8%) isolated from 69 (36.9%) samples carried one or more virulence genes. The most prevalent gene detected was lpfA O113 (40.5%), followed by ehaA (32.4% , ), astA (3.15% , ), iha (1.80%), hlyA (1.35%), stx1 (0.90%), stx2 (0.90%), eaeA (0.45%), cdt-III (0.45%) and cnf2 (0.45%). Two strains isolated from Karish cheese harbored 5 virulence genes ( stx1 , stx2 , iha , ehaA , lpfA O113 ). Stx subtype was determined to be stx1 (not stx1c or stx1d ) and stx2d . Indeed, expression of hemolysin A, CDT-III, CNF-II, Stx1 and Stx2d was confirmed by blood agar plate, cytotoxicity assay and Western blotting, respectively. Among the 222 E. coli strains, 54 (48.6%), 38 (42.6%) and 12 (54.7%) isolated from raw milk, Karish cheese and Ras cheese were potentially virulent, respectively. O-genotyping indicated that most of the potentially virulent E. coli isolates did not belong to clinically important O serogroups except O75, O91 and O166, which have been associated with human diseases. Phylogenetic grouping revealed that 150 (67.6%), 67 (30.2%) and 5 (2.30%) strains were clustered into A, B1 and D groups, respectively, which are considered to be associated with intestinal infection, indicating that these E. coli strains might have a potential to cause gastroenteritis. To the best of our knowledge, this is the first comprehensive study regarding prevalence and pathogenic potential of E. coli in dairy products in Egypt. Raw milk, Karish cheese and Ras cheese in Egypt are highly contaminated with E. coli including potentially pathogenic strains, which may impose a public health threat. [ABSTRACT FROM AUTHOR]

Use of natural compounds as antivirulence drugs could be an alternative therapeutic approach to modify the outcome of bacterial infections, particularly in view of growing resistance to available antimicrobials. Here, we show that sub-bactericidal concentration of anethole, a component of sweet fennel seed, could suppress virulence potential in O1 El Tor biotype strains of toxigenic Vibrio cholerae, the causative agent of the ongoing 7th cholera pandemic. The expression of cholera toxin (CT) and toxin coregulated pilus (TCP), the major virulence factors of V. cholerae, is controlled through a regulatory cascade involving activation of ToxT with synergistic coupling interaction of ToxR/ToxS with TcpP/TcpH. We present evidence that anethole inhibits in vitro expression of CT and TCP in a toxT-dependent but toxR/toxS-independent manner and through repression of tcpP/tcpH, by using bead-ELISA, western blotting and quantitative real-time RT-PCR assays. The cyclic AMP (cAMP)-cAMP receptor protein (CRP) is a well-studied global signaling system in bacterial pathogens, and this complex is known to suppress expression of tcpP/tcpH in V. cholerae. We find that anethole influences the virulence regulatory cascade by over-expressing cyaA and crp genes. Moreover, suppression of toxigenic V. cholerae-mediated fluid accumulation in ligated ileum of rabbit by anethole demonstrates its potentiality as an antivirulence drug candidate against the diseases caused by toxigenic V. cholerae. Taken altogether, these results revealing a mechanism of virulence inhibition in V. cholerae by the natural compound anethole, may have relevance in designing antivirulence compounds, particularly against multiple antibiotic resistant bacterial pathogens. [ABSTRACT FROM AUTHOR]

Phenotypic and genotypic tolerance in 71 Listeria monocytogenes isolates from different varieties of foods to benzalkonium chloride (BC) and cadmium were investigated by susceptibility test and molecular methods. To investigate the role of efflux pumps in BC tolerance, reserpine, an efflux pump inhibitor, was added to the BC tolerant strains. Tolerance to BC and cadmium were 26.8% (19/71) and 49.3% (35/71) respectively. Strains with BC tolerance were significantly more frequent among those of serotype 4b (100%, 6/6) than among those of serotype 1/2a (or 3a) (13.5%, 5/37), which represent the predominant number of strains (52.1%, 37/71). Tolerance to cadmium was encountered among 62.2% (23/37) and 50.0% (3/6) of the serotype 1/2a (or 3a) and 4b strains, respectively, and among 19.0% (4/21) of the strains of the serotype 1/2c. All of the 10 (14.1%) isolates found to be BC and cadmium co-tolerance were isolated from raw meat or quick-frozen food made of wheat flour and rice. Five multi-drug resistant strains were tolerant to cadmium as well. Among 71 isolates examined, one contained qacA and three contained qacEΔ1-sul . To the best of our knowledge, this is the first detection of qacA and qacEΔ1-sul in L. monocytogenes , an indication of the possible horizontal transfer of the two genes. Addition of reserpine to the tolerant strains resulted in the loss of tolerance among seven out of 19 BC strains, suggesting a certain role the efflux pump played in mediating BC tolerance. Of the three distinct cadA types known to date in L. monocytogenes , the cadA1 and cadA2 genes were detected among 24 (33.8%) and three (4.2%) isolates respectively. The presence of cadA1 and cadA2 largely corresponded to the susceptibility phenotype. A subset (9/35 [25.7%]) of the cadmium-tolerant isolates lacked the known cadmium resistance determinants. These findings suggest that food products could act as a reservoir for L. monocytogenes harboring tolerance to BC and cadmium and will further our understanding of the adaptations of this organism to these two compounds. [ABSTRACT FROM AUTHOR]

Campylobacter jejuni causes foodborne disease associated with abdominal pain, gastroenteritis, and diarrhea. These symptoms are induced by bacterial adherence and invasion of host epithelial cells. C. jejuni infection can occur with a low infective dose, suggesting that C. jejuni may have evolved strategies to cope with the bacterial clearance system in the gastrointestinal tract. The mucosa layer is the first line of defense against bacteria. Mucus conditions are maintained by water and anion (especially Cl − ) movement. Cystic fibrosis transmembrane conductance regulator (CFTR) is the main Cl − channel transporting Cl − to the lumen. Mutations in CFTR result in dehydrated secreted mucus and bacterial accumulation in the lungs, and recent studies suggest that closely related pathogenic bacteria also may survive in the intestine. However, the relationship between C. jejuni infection and CFTR has been little studied. Here, we used an 125 I − efflux assay and measurement of short-circuit current to measure Cl − secretion in C. jejuni -infected T-84 human intestinal epithelial cells. The basic state of Cl − secretion was unchanged by C. jejuni infection, but CFTR activator was observed to induce Cl − secretion suppressed in C. jejuni -infected T-84 cells. The suppression of activated Cl − secretion was bacterial dose-dependent and duration-dependent. A similar result was observed during infection with other C. jejuni strains. The mechanism of suppression may occur by affecting water movement or mucus condition in the intestinal tract. A failure of mucus barrier function may promote bacterial adhesion or invasion of host intestinal epithelial cells, thereby causing bacterial preservation in the host intestinal tract. [ABSTRACT FROM AUTHOR]

Abstract: We evaluated the usefulness of the loop-mediated isothermal amplification (LAMP) using a portable ESE Quant tube scanner as a rapid and simple method for the detection of Vibrio parahaemolyticus, an important pathogen causing seafood-borne gastroenteritis. The real time LAMP (RT-LAMP) assay using a hemolysin gene (tlh/ldh)-specific primers was verified using V. parahaemolyticus strains (n = 91) from different countries and other non-target strains to check the utility of this method. Both the sensitivity and specificity of the RT-LAMP using 3 pairs of tlh/ldh-specific primers developed in this study were excellent (100%). The detection limit of the RT-LAMP was as low as 7 Colony forming unit per reaction and detection time was only 20 min. Comparative evaluation of the target bacterial strains with the RT-LAMP using ESE Quant tube scanner, API 20E system and conventional RT-PCR method revealed that the RT-LAMP assay developed in this study is simpler and more rapid than the latter two methods. Therefore, the RT-LAMP method using the easily portable ESE Quant tube scanner can be considered as an effective tool for the rapid screening of V. parahaemolyticus strains in environmental and clinical samples, especially, in remote areas of developing countries during epidemic periods. [Copyright &y& Elsevier]

We identified 131 strains of Vibrio fluvialis among 400 nonagglutinating Vibrio spp. isolated from patients with diarrhea in Kolkata, India. For 43 patients, V. fluvialis was the sole pathogen identified. Most strains harbored genes encoding hemolysin and metalloprotease; this finding may contribute to understanding of the pathogenicity of V. fluvialis. [ABSTRACT FROM AUTHOR]

Abstract: Laribacter hongkongensis is a novel emerging pathogen associated with human gastroenteritis. We aimed to investigate the prevalence, antimicrobial resistance and genotypic relationship of 199 L. hongkongensis isolates from 690 intestinal samples of fish and frogs. These samples were collected from retail markets in the city of Guangzhou in southern China from October 2008 to September 2009. L. hongkongensis was detected in from 80 (16.3%) out of 490 freshwater fish, and this number included 76 (32.3%) out of 235 grass carp and 4 (14.8%) out of 27 bighead carp. A higher isolation rate of 59.5% (119 out of 200) was observed in edible frogs. The isolation rate was highest in the spring in comparison with other seasons. Notably, 63.8% of the isolates were resistant to at least one class of antimicrobial agents. Analysis by pulsed-field gel electrophoresis (PFGE) revealed that the isolates could be grouped into three clusters. Isolates from fish intestines were grouped into two clusters: cluster I and II. Isolates of frog-origin and several fish-origin isolates were grouped into cluster III. Two patient-derived strains could be classed into cluster III. Extensive genetic heterogeneity among the isolates was observed. The results indicate that L. hongkongensis isolates exhibits host tropism, extensive resistance to widely used antimicrobials and diverse biological evolution in an aquatic environment. The frog is more likely than the freshwater fish to be the potential source for human infection with L. hongkongensis. [Copyright &y& Elsevier]

Changes of structure and thermal stability of soy protein isolate after pulsed electric field treatment were analyzed by Fourier transform infrared spectroscopy and differential scanning calorimetry (DSC). When the applied pulsed electric field (PEF) treatment intensity was over 35 kV/cm, the amino acid side chain, anti-parallel β-sheets, β-turn as well as β-sheets in soy protein isolate (SPI) secondary structure were significantly changed, which suggested that PEF treatment might be a new processing method for SPI; the dipole moments of some bonds, such as C=O, C-O, and C-O-C were partially polarized, accompanying with complete denaturation of β-conglycinin and glycinin obtained from DSC. Furthermore, self-reassembly from β-turn to α-helix in SPI structure abruptly happened under intense PEF treatment condition, which suggested that the PEF treatment had induced change of the orientation of α-helix dipole moment to stabilize α-helix. This observation implies that PEF treatment technique may be a novel method for preparation of protein nanotube. [ABSTRACT FROM AUTHOR]

Cholix toxin (Cholix) is a novel ADP-ribosylating cytotoxin produced by Vibrio cholerae, which utilizes eukaryotic elongation factor 2 as a substrate and acts by a mechanism similar to that of diphtheria toxin and Pseudomonas exotoxin A. First it was found that Cholix-treated HeLa cells exhibited caspase-dependent apoptosis, whereas intestinal cells such as Caco-2, HCT116, and RKO did not. Here we investigated Cholix-induced cell death signaling pathways in HeLa cells. Cholix-induced cytochrome c release into cytosol was initiated by specific conformational changes of pro-apoptotic Bak associated with Bax. Silencing of bak/bax genes or bak gene alone using siRNA significantly suppressed cytochrome c release and caspase-7 activation, but not activation of caspases-3 and -9. Although pretreatment with a caspase-8 inhibitor (Z-IETD-FMK) reduced Cholix-induced cytochrome c release and activation of caspases-3, -7, and -9, cytotoxicity was not decreased. Pretreatment with Z-YVAD-FMK, which inhibits caspase-1, -4, and -5, suppressed not only cytochrome c release, activation of caspase-3, -7, -8, or -9, and PARP cleavage, but also cytotoxicity, indicating that caspase-1, -4, and -5 activation is initiated at an early stage of Cholix-induced apoptosis and promotes caspase-8 activation. These results show that the inflammatory caspases (caspase-1, -4, and -5) and caspase-8 are responsible for both mitochondrial signals and other caspase activation. In conclusion, we showed that Cholix-induced caspase activation plays an essential role in generation of apoptotic signals, which are mediated by both mitochondria-dependent and -independent pathways. [ABSTRACT FROM AUTHOR]

Abstract: In situ loop-mediated isothermal amplification (in situ LAMP) as a novel technique for detection of food-borne pathogens, has been successfully applied to detect Salmonella in artificially contaminated eggshells. Escherichia coli C600 was used as a negative control in specificity experiment. The sensitivity of the in situ LAMP assay was confirmed by the test in serial 10-fold dilutions of Salmonella cells, and the detection limit of this method may reach 1 CFU/cm2 on eggshells. Compared with traditional culture methods and PCR-based methods, the in situ LAMP assay is advantageous on rapidity, high specificity, less time consumption and ease in operation. This is the first study carried out to apply the in situ LAMP method in practical food-borne pathogens detection. The results indicate that this method has great potential for use in food safety laboratories. [ABSTRACT FROM AUTHOR]

Abstract: 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. [ABSTRACT FROM AUTHOR]

Abstract: 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. [Copyright &y& Elsevier]

We show here that oral immunization with purified outer membrane vesicles (OMVs) of Vibrio cholerae induces a prolonged high rise in the protective antibody titre. Rabbit immune sera were vibriocidal against the homologous and against several heterologous V. cholerae strains in vitro. In addition, OMV immunization conferred significant protective immunity against subsequent bacterial challenges. Thirty OMV-immunized rabbits were challenged with different V. cholerae strains; each challenged group contained five immunized and three unimmunized animals. All the immunized rabbits survived bacterial challenges and were healthy after 24 h, except the two from each group that received the SG24 and SG06 strains, respectively, which developed watery diarrhoea. In contrast, all the unimmunized animals developed cholera-like symptoms, with a death toll of eight within 24 h of challenge. This is the first report of the induction of protective immunity by V. cholerae OMVs in a rabbit model (removable intestinal tie-adult rabbit diarrhoea) that mimics the human disease. Finally, OMVs were found to be significantly less reactogenic than the live and the heat-killed bacteria. Our studies show that oral immunization with OMVs of V. cholerae may induce long-term immunity and may be useful as a ‘nonliving’ vaccine candidate for the future. [ABSTRACT FROM AUTHOR]

Abstract: A novel hot spring bacterial strain was isolated and characterized from Bakreshwar, West Bengal, India. Phylogenetic analysis of partial 16 S-rRNA gene sequence (697 nucleotides) suggested that the isolate was affiliated with the family ‘Firmicutes’ and 99.8% homologous with Thermoanaerobactor thermohydrosulfuricus. A unique thermostable and high pH tolerant protein (28kDa) secreted by the bacterium was isolated whose N-terminal partial sequence (25 AA) suggested that the protein was similar to mammalian carbonic anhydrase-II. This bacterium or its unique protein both increase the compressive (>25%) and tensile (>20%) strengths, hardness and elastic modulus of cementitious material when incorporated to the cement/cement–sand mixture. The protein (named as bioremediase) possesses silica leaching activity, which is increased by calcium ions. No esterase and/or proteolytic activities are present in the protein. The use of such isolated bacterium or its protein in concrete technology develops new construction material that may be used for self-healing concrete in future. [Copyright &y& Elsevier]

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. [ABSTRACT FROM AUTHOR]

Abstract: In the present study we purified 34kDa major outer membrane protein (MOMP) of Shigella flexneri 2a for the first time, which was cross-reactive and antigenically conserved among Shigella spp. and the epitope was surface exposed on the intact bacterium. The purified antigen was found to be glycosylated, which aids in binding to macrophages and up-regulated the production of nitric oxide, granulocyte-colony stimulating factor and IL-12p70, indicating that the MOMP is immunogenic and has the ability to commence protective immune responses against intracellular pathogens, thereby it may be considered as a potential vaccine candidate. [Copyright &y& Elsevier]

Six methicillin-resistant Staphylococcus aureus MRSA strains from two nosocomial infection cases described in a previous study [], of which two ocurred in March and the other four in May, 2005, were found to possess one copy of class 1 integron with aadA2 gene cassette located on chromosomes by Southern hybridization. Polymerase chain reaction (PCR) detection of mecA and pvl, SCC mec typing, multilocus sequence typing (MLST), spaA typing and coa typing were also performed. The results revealed 6 MRSA fell into the ST239-MRSA-III group (clonal complex 8), with the spaA type GKAOMQ and coa type HIJKL, whereas the pvl locus was not detected. DNA fingerprinting analysis by random amplified polymorphic DNA–PCR using three different assays were also performed, and all strains exhibited identical patterns, indicating that they were clonally related and might be mainly due to a specific clone in the hospital. This was the first time, to our knowledge, that class 1 integron-bearing MRSA (I-MRSA), simultaneously carrying two mobile genetic elements was confirmed: class 1 integron and SCC mec. [ABSTRACT FROM AUTHOR]

The aim of the present study was to genotypically characterize Vibrio cholerae strains isolated from cholera patients in various provinces of Thailand. Two hundred and forty V. cholerae O1 strains, isolated from patients with cholera during two outbreaks, i.e. March 1999--April 2000 and December 2001--February 2002, in Thailand, were genotypically characterized by NotI digestion and pulsed-field gel electrophoresis (PFGE). In total, 17 PFGE banding patterns were found and grouped into four Dice-coefficient clusters (PF-I to PF-IV). The patterns of V. cholerae O1, El Tor reference strains from Australia, Peru, Romania, and the United States were different from the patterns of reference isolates from Asian countries, such as Bangladesh, India, and Thailand, indicating a close genetic relationship or clonal origin of the isolates in the same geographical region. The Asian reference strains, regardless of their biotypes and serogroups (classical O1, El Tor O1, O139, or O151), showed a genetic resemblance, but had different patterns from the strains collected during the two outbreaks in Thailand. Of 200 Ogawa strains collected during the first outbreak in Thailand, two patterns (clones)--PF-I and PF-II--predominated, while other isolates caused sporadic cases and were grouped together as pattern PF-III. PF-II also predominated during the second outbreak, but none of the 40 isolates (39 Inaba and 1 Ogawa) of the second outbreak had the pattern PF-I; a minority showed a new pattern--PF-IV, and others caused single cases, but were not groupable. In summary, this study documented the sustained appearance of the pathogenic V. cholerae O1 clone PF-II, the disappearance of clones PF-I and PF-III, and the emergence of new pathogenic clones during the two outbreaks of cholera. Data of the study on molecular characteristics of indigenous V. cholerae clinical isolates have public-health implications, not only for epidemic tracing of existing strains but also for the recognition of strains with new genotypes that may emerge in the future. [ABSTRACT FROM AUTHOR]

The aim of the present study was to genotypically characterize Vibrio cholerae strains isolated from cholera patients in various provinces of Thailand. Two hundred and forty V. cholerae O1 strains, isolated from patients with cholera during two outbreaks, i.e. March 1999--April 2000 and December 2001--February 2002, in Thailand, were genotypically characterized by NotI digestion and pulsed-field gel electrophoresis (PFGE). In total, 17 PFGE banding patterns were found and grouped into four Dice-coefficient clusters (PF-I to PF-IV). The patterns of V. cholerae O1, El Tor reference strains from Australia, Peru, Romania, and the United States were different from the patterns of reference isolates from Asian countries, such as Bangladesh, India, and Thailand, indicating a close genetic relationship or clonal origin of the isolates in the same geographical region. The Asian reference strains, regardless of their biotypes and serogroups (classical O1, El Tor O1, O139, or O151), showed a genetic resemblance, but had different patterns from the strains collected during the two outbreaks in Thailand. Of 200 Ogawa strains collected during the first outbreak in Thailand, two patterns (clones)--PF-I and PF-II--predominated, while other isolates caused sporadic cases and were grouped together as pattern PF-III. PF-II also predominated during the second outbreak, but none of the 40 isolates (39 Inaba and 1 Ogawa) of the second outbreak had the pattern PF-I; a minority showed a new pattern--PF-IV, and others caused single cases, but were not groupable. In summary, this study documented the sustained appearance of the pathogenic V. cholerae O1 clone PF-II, the disappearance of clones PF-I and PF-III, and the emergence of new pathogenic clones during the two outbreaks of cholera. Data of the study on molecular characteristics of indigenous V. cholerae clinical isolates have public-health implications, not only for epidemic tracing of existing strains but also for the recognition of strains with new genotypes that may emerge in the future. [ABSTRACT FROM AUTHOR]

The aim of the present study was to genotypically characterize Vibrio cholerae strains isolated from cholera patients in various provinces of Thailand. Two hundred and forty V. cholerae O1 strains, isolated from patients with cholera during two outbreaks, i.e. March 1999--April 2000 and December 2001--February 2002, in Thailand, were genotypically characterized by NotI digestion and pulsed-field gel electrophoresis (PFGE). In total, 17 PFGE banding patterns were found and grouped into four Dice-coefficient clusters (PF-I to PF-IV). The patterns of V. cholerae O1, El Tor reference strains from Australia, Peru, Romania, and the United States were different from the patterns of reference isolates from Asian countries, such as Bangladesh, India, and Thailand, indicating a close genetic relationship or clonal origin of the isolates in the same geographical region. The Asian reference strains, regardless of their biotypes and serogroups (classical O1, El Tor O1, O139, or O151), showed a genetic resemblance, but had different patterns from the strains collected during the two outbreaks in Thailand. Of 200 Ogawa strains collected during the first outbreak in Thailand, two patterns (clones)--PF-I and PF-II--predominated, while other isolates caused sporadic cases and were grouped together as pattern PF-III. PF-II also predominated during the second outbreak, but none of the 40 isolates (39 Inaba and 1 Ogawa) of the second outbreak had the pattern PF-I; a minority showed a new pattern--PF-IV, and others caused single cases, but were not groupable. In summary, this study documented the sustained appearance of the pathogenic V. cholerae O1 clone PF-II, the disappearance of clones PF-I and PF-III, and the emergence of new pathogenic clones during the two outbreaks of cholera. Data of the study on molecular characteristics of indigenous V. cholerae clinical isolates have public-health implications, not only for epidemic tracing of existing strains but also for the recognition of strains with new genotypes that may emerge in the future. [ABSTRACT FROM AUTHOR]

A cytolethal distending toxin ( cdt) gene-based species-specific multiplex PCR assay for the detection of cdtA, cdtB or cdtC gene of Campylobacter jejuni, Campylobacter coli or Campylobacter fetus, respectively, was developed and evaluated with 76 Campylobacter strains belonging to seven different species and 131 other bacterial strains of eight different genera. The cdtA, cdtB or cdtC gene of C. jejuni, C. coli or C. fetus, respectively, could be successfully amplified using the corresponding set of primers in a highly species-specific manner. Furthermore, the specific primer set for the cdtA, cdtB or cdtC gene of a particular species could amplify the desired gene from a mixture of DNA templates of any of two or all three species. The detection limit of C. jejuni, C. coli or C. fetus was 10–100 CFU tube−1 by the multiplex PCR assay on the basis of the presence of the cdtA, cdtB or cdtC gene. These data indicate that the cdt gene-based multiplex PCR assay may be useful for rapid and accurate detection as well as identification of Campylobacter strains in a species-specific manner. [ABSTRACT FROM AUTHOR]

A total of 53 methicillin-resistant coagulase-negative staphylococci strains isolated in a hospital in Guangzhou, China, were analyzed to detect class 1 integrons and SCC mec typing. Thirty strains had the class 1 integrase ( intI1) gene and 26 strains possessed the 3′ conserved region of qacEΔ 1- sul1. Four different types of gene cassette arrays were found and a highly prevalent array of dfrA12-orfF-aadA2 gene cassettes was observed. Thirty class 1 integron-positive coagulase-negative staphylococci strains were subjected to Southern hybridization analysis; the result showed that class 1 integrons were located on chromosome, not plasmid. According to the results of SCC mec typing for 30 integron-bearing MRCNS strains, five, 15 and five strains belonged to type I, II and III SCC mec, respectively, and five strains were untypeable. For 23 non-integron-bearing methicillin-resistant coagulase-negative staphylococci strains, four, nine and seven strains belonged to type I, II and III SCC mec, respectively, and three strains were untypeable. None of the strains belonged to type IV or V. Twenty-three coagulase-negative staphylococci isolates of three Staphylococcal species that contained the dfrA12-orfF-aadA2 gene cassette array were phylogenetically unrelated to each other by randomly amplified polymorphic DNA, indicating that the gene cassettes might be disseminated in the clinical strains by a horizontal gene transfer. [ABSTRACT FROM AUTHOR]

Cytolethal distending toxins (CDTs) are inhibitory cyclomodulins, which block eukaryotic cell proliferation and are produced by a diverse group of Gram-negative bacteria, including Escherichia coli strains associated with intestinal and extraintestinal infections. However, the mode of transmission of the toxin gene clusters among diverse bacterial pathogens is unclear. We found that Cdt-1 produced by enteropathogenic E. coli strains associated with diarrhea is encoded by a lambdoid prophage, which is inducible and infectious. The genome of Cdt-1 converting phage (CDT-1(b) comprises 47,021 nucleotides with 60 predicted ORFs organized into six genomic regions encoding the head and tail, virulence, integrase, unknown functions, regulation, and lysis. The genomic organization of CDT-1Φ is similar to those of SfV, a serotype-converting phage of Shigella flexneri, and UTI89, a prophage identified in uropathogenic E. coll. Besides the cdt1 gene cluster, the virulence region of CDT-1Φ genome contains sequences homologous to a truncated cycle inhibiting factor and a type 3 effector protein. Mutation analysis of susceptible E. coli strain C600 suggested that the outer membrane protein OmpC is a putative receptor for CDT-1Φ. CDT-1Φ genome was also found to integrate into the host bacterial chromosome forming lysogens, which produced biologically active Cdt-1. Furthermore, phage induction appeared to cause enhanced toxigenicity of the E. coli strains carrying lysogenic CDT-1Φ. Our results suggest that CDT-1Φ is the latest member of a growing family of lambdoid phages encoding bacterial cyclomodulins and that the phage may have a role in horizontal transfer of these virulence genes. [ABSTRACT FROM AUTHOR]