Human health significance of vibrios from the aquatic environment

For the enrichment and enumeration of Vibrio fluvialis, a broth medium was designed by modifying alkaline peptone (AP) medium. This new V. fluvialis enrichment medium (FEM) was shown to be more effective than AP medium in field samplings where a total of 177 samples (estuarine waters and sediment, sewage, and crabs) were processed over a 14 month period. FEM was particularly superior to AP for water and sewage with low salinities (<6%0). V. vulnificus was shown to have a species-specific antigen by analyzing sonicated whole-cell antigens with two-dimensional immunoelectrophoresis. The antigen (designated as VVA) was purified by various protein chemistry techniques, and specific antiserum to VVA was prepared. Using anti-VVA serum, a simple and rapid microimmunodiffusion method was designed which allowed the specific identification of V. vulnificus in as early as 10 hrs after preparing a bacterial cell lysate from a single colony. suckling mouse assay was utilized to examine enteropathogenicity, i.e., potential to cause diarrhea, of environmental and clinical isolates of 0-1 and non 0-1 V. cholerae, V. mimicus, and V. fluvialis. Cultures from both environmental and clinical origins induced intestinal fluid accumulation (FA) in 3-day-old mice at 4 hr postinoculation followed by diarrheal feces and high mortality. The virulence-associated factor(s) that caused FA were different from cholera toxin (CT) in terms of kinetics of FA and serological behavior, but were correlated with extracellular cytotoxic factors active against Y-1 mouse adrenal tissue cultures. Furthermore, all clinical isolates of non 0-1 V. cholerae, V. mimicus, and V. fluvialis, when grown in brain heart infusion broth supplemented with 0.5% sodium chloride, were found to produce a new extracellular heat-labile enterotoxin which induced significant FA in 3-day-old mice and was distinct from previously reported enterotoxins including CT. Most of the environmental isolates, however, did not produce the newly discovered enterotoxin

Molecular characterization of clinical isolate of Vibrio cholera isolated from outbreaks cases in Malaysia

A total of 32 clinical strains of Vibrio cholerae, including members of the 01 and 0139 serogroup were collected from Klang, Selangor; Penang Island; Samarahan, Sarawak and Miri, Sarawak in Malaysia. In general, all the isolates except the 0139 serotype expressed low resistance to all the antibiotics tested with their Multiple Antibiotic Resistance (MAR) indices ranged from 0.10 to 0.48. The presence of ctx gene that encoded the cholera toxin was confirmed in all these clinical isolates by polymerase chain reaction. The results from the RAPD-PCR were analyzed using the RAPDistance software (Version 1.04). From the dendrogram generated, two main groups were observed which were subdivided into two clusters each. The Selangor's isolates and the 0139 Penang's isolates formed one group whereas the Samarahan, Sarawak isolates and the Miri, Sarawak isolates made up the other group, thus delineating their different sources of origin based on their geographical location

養殖ヨーロッパウナギ(Anguilla anguilla)から分離されたVibrio anguillarum

Vibrio anguillarumはヨーロッパにおいて海水中および汽水中のウナギに鰭赤病を起こすものとして古くから知られている。1969年以来，ニホンウナギ(Anguilla japonica)の種苗不足を補うためヨーロッパウナギ(A. anguilla)のシラスが大量に輸入され，日本各地で養殖されているが，我が国においてはヨーロッパウナギでのV. anguillarum感染症の発生は現在まで報告されていない。 1975年4月末，徳島県下のやや塩分を含む養殖池において，前年から赤点病(Pseudomonas anguilliseptica感染症)が発生していた魚群について検査したところ，P. anguillisepticaとともにV. anguillarumが分離され，ヨーロッパウナギにおける本菌感染症が我が国でも確認された。 しかしながら，その感染率は比較的低く，また本病による大量斃死も認められなかった。これはヨーロッパウナギがニホンウナギと同様，V. anguillarumに対してある程度抵抗性を有しているためと考えられた

Development and Improvement of Methods to Disinfect Raw Beef Using Calcium Hydroxide–Ethanol–Lactate-Based Food Disinfectant for Safe Consumption

The enterohemorrhagic Escherichia coli (EHEC) group is responsible for outbreaks and sporadic cases around the world annually. EHEC produces a potent protein known as Shiga toxin in the human intestine causing mild to bloody diarrhea. Some cases of EHEC infections may develop life-threatening symptoms, which may lead to human death. It also has other virulent factors that enable the EHEC cells to adhere to a target tissue and invade to some extent to crave more nutrition and escape the external extreme conditions, such as disinfection treatment. For those reasons, beef is not permitted for raw consumption unless guaranteed free of harmful bacteria, including EHEC, or the invading bacterial cells are completely removed or reduced to a safe level. A heat treatment that guarantees a sufficiently high temperature to reach inside the tissue of meat through the surface was established in Japan. This treatment may allow the core part of the meat to be consumed raw. However, it seemed to have some limitations. We aimed at developing a disinfection method with, hypothetically, nutrition-preserving property that is equivalent to the heat treatment or even superior. A combination of calcium hydroxide–ethanol–lactate-based food disinfectant and two proposed physical sterilization methods, assisted with microbial detection methods, exerted sufficient bactericidal activities against EHEC cells adhering to and/or invading the beef. These physical methods showed great usefulness in disinfecting fresh full-size boneless Round-beef of around 12 kg including fat on the outside. The first method applied a commercially available wide-drum washing machine (WM method) while the second method applied a specially designed plastic bag and a commercially available vibration machine (VV method). After trimming out the fat and the denatured surface of the beef (1 cm from the surface), the remaining meat mass showed no signs of denaturation and a significant reduction of viable EHEC cells by a factor of &gt;104 CFU/ml. However, in the WM method, the disinfection process required a large amount of the disinfectant (150 L). The improved method, VV method, implemented a system that consumes a smaller amount of the disinfectant (50 L) while ensuring the targeted disinfection power degree

Molecular characterisation of Vibrio parahaemolyticus carrying tdh and trh genes using ERIC-, RAPD- and BOX-PCR on local Malaysia bloody clam and Lala

Molecular typing methods have been widely applied for many purposes. In this study, such methods were adopted as DNA fingerprinting tools to determine the origin and divergence of virulent Vibrio parahaemolyticus strains found in local seafood. Although not all strain carry virulent tdh and trh gene, increasing prevalence demands an effective fingerprinting scheme which can constantly monitor and trace the sources of such emerging food pathogens. By using ERIC-, RAPD-, and BOX-PCR methods, 33 Vibrio parahaemolyticus isolates from local Malaysia bloody clam (Anadara granosa) and Lala (Orbicularia orbiculata) with confirmed presence of tdh and trh gene were characterised, followed by determination of clonal relatedness among virulent strains using cluster analysis and discriminatory index. This study also involved application of Immunomagnetic Separation (IMS) Method which significantly improved the specificity of strain isolation. Cluster analysis using Unweighted Pair Group Mathematical Averaging (UPGMA) and Dice Coefficient shown clustering according to isolation food source, IMS level and haemolysin gene possessed. Nevertheless, different DNA fingerprinting methods generated different clustering at different similarity cut-off percentage, regardless as individual or as composite dendrograms. ERIC- and RAPD-PCR composite fingerprinting relatively shown the highest discriminatory index at following similarity cut- off percentage: 0.68 at 50%; 0.83 at 65%; and 0.93 at 75%. Discriminatory power increased with similarity cut-off percentage. However, result also suggested that BOX-PCR might be an effective fingerprinting tool, as it generated three clusters with no single-colony isolate at 70% similarity cut-off. This study not only achieved its objective to determine clonal relatedness among virulent strains from local seafood via characterisation, but also speculated the best possible combination of molecular typing methods to effectively do s

Efficiency of four Malaysian commercial disinfectants on removing Listeria monocytogenes biofilm

Listeria monocytogenes(L. monocytogenes) is a gram positive food-borne pathogen that is able to form biofilm on food factory surfaces. Formation of biofilm makes the bacteria much more resistance to environmental stresses such as disinfectant. The extracellular polymeric matrix (biofilm structure) which is mostly comprised of sticky extracellular polysaccharides (EPS) and proteins can protect bacteria in a harsh condition. The efficiency of four disinfectants on removing L. monocytogenes biofilm was investigated. Five concentration levels (100, 50,25, 12.5, and 6.25%) of disinfectants were tested. In the microtitre assay, the optical density at 595 nm CV-OD 595 value, was used to measure the amount of remained biofilm after 24 h. Results showed that disinfectants did not have significant effect on removing L. monocytogenes biofilm. Formation of L. monocytogenes biofilm significantly decreased the efficiency of disinfectants. Biofilm produced by strain number 9 showed higher resistance to disinfectant. Low concentrations (<50%) of disinfectants did not show significant effect on removing L. monocytogenes biofilm

Comparison of thermophilic Campylobacter spp. occurrence in two types of retail chicken samples

The aim of this study is to compare the occurrence of thermophilic Campylobacter spp. in chicken retail at wet markets and hypermarkets. Campylobacter contaminations in chicken samples from wet market (70.7%) were comparatively lower than chicken samples sold in hypermarket (91.4%). Of the 77 Campylobacter isolates, 59 (76.6%) were identified as Campylobacter jejuni and 18 (23.4%) isolates were identified as C. coli. All Campylobacter isolates are multi-resistant to the antimicrobial agents. Most of the isolates were resistant to tetracycline (92.2%) and erythromycin (98.7%). This study concluded that chicken samples from both wet market and hypermarket were contaminated with Campylobacter, most of which are antimicrobial-resistant strains

Molecular characterization of Escherichia coli isolated from different food sources

Escherichia coli and Escherichia coli O157 were identified from "selom" (Oenanthe stolonifera), "pegaga" (Centella asiatica), beef, chicken, lamb, buffalo, "ulam Raja" (Cosmos caudatus) and "tenggek burung" (Euodia redlevi). The bacteria were recovered using chromagenic agar. Isolated Escherichia coli and Escherichia coli 0157 were further characterized by plasmid profiling and enterobacterial repetitive intergenic consensus-polymerase chain reaction (ERIC-PCR). The virulence genes of the isolates (VT1, VT2, LT, ST, eaeA, inV) that produces pathogenic Escherichia coli and 16S rRNA gene were screened by a multiplex PCR assay. The plasmid profiling analysis showed that out of 176 isolates, only 103 isolates contained plasmids. ERIC-PCR analysis generated amplified products in the range of ~150 bp to > 1000 bp categorizing isolates into a total of 52 different profiles. Multiplex PCR showed that 20 (32.3%) of the isolates carried eaeA gene, 6 (9.7%) isolates possessed inV genes, only 1 (1.6%) have VT2 genes and 1 (1.6%) as well carried VT1 genes, 2 (3.2%) of the isolates harboured LT genes, and only 1 (1.6%) isolate possessed ST genes. There were no correlation between plasmid, ERIC-PCR and virulence genes profiles