Role of chitin in the survival of Vibrio parahaemolyticus at different temperatures

Washed cells of Vibrio parahaemolyticus declined in numbers when incubated in phosphate-buffered saline for 6 h at different temperatures. Addition of chitin flakes to phosphate-buffered saline not only helped the organism to survive, but also resulted in an increase in cell numbers, particularly at 10 degrees C. The effect of chitin could not be simulated by N-acetylglucosamine, yeast extract, starch, or casein.     

The aerobic psychrotrophic populations on meat and meat contact surfaces in a meat production system and on meat stored at chill temperatures

At a city abattoir, a wholesaler and 10 different supermarkets, surface microbiological samples were taken of carcasses, hands and apron fronts of members of staff and equipment (mincers and saws). In addition, minced meat, packaged and displayed in chilled cabinets, was also sampled. Carcasses, personnel surfaces and equipment were monitored by a modified agar sausage technique. From each of the highest dilution psychrotrophic plate counts, five colonies were selected randomly, isolated and identified (1265 in total). Microbes developing on chilled meat were also isolated from other surfaces in the production chain. On chilled meat (51%) and at the abattoir (36%) pseudomonads were the predominant organisms followed by the Gram-positive cocci on chilled meat and by Acinetobacter, Moraxella and Alcaligenes spp. at the abattoir. At the wholesaler Gram-positive cocci (32%) predominated, followed by Alcaligenes, Moraxella and Alcaligenes spp. Pseudomonas, Alcaligenes, Neisseriaceae and related genera, Gram-positive cocci, species from the coryneform groups of bacteria and yeasts were identified from all the surfaces monitored. Identification with the API NE20 was unsatisfactory. Enterbacteriaceae, lactobacilli and endospore-forming bacteria were identified occasionally, but their significance as contaminating organisms seems low. No Salmonella spp. were identified. and accepted 25 July 1989     

The aerobic psychotrophic populations on meat and meat contact surfaces in a meat production system and on meat stored at chill temperatures

At a city abattoir, a wholesaler and 10 different supermarkets, surface microbiological samples were taken of carcasses, hands and apron fronts of members of staff and equipment (mincers and saws). In addition, minced meat, packaged and displayed in chilled cabinets, was also sampled. Carcasses, personnel surfaces and equipment were monitored by a modified agar sausage technique. From each of the highest dilution psychotrophic plate counts, five colonies were selected randomly, isolated and identified (1265 in total). Microbes developing on chilled meat were also isolated from other surfaces in the production chain. On chilled meat (51%) and at the abattoir (36%) pseudomonads were the predominant organisms followed by the Gram-positive cocci on chilled meat and by Acinetobacter, Moraxella and Alcaligenes spp. at the abattoir. At the wholesaler Gram-positive cocci (32%) predominated, followed by Alcaligenes, Moraxella and Alcaligenes spp. Pseudomonas, Alcaligenes, Neisseriaceae and related genera, Gram-positive cocci, species from the coryneform groups of bacteria and yeasts were identified from all the surfaces monitored. Identification with the API NE20 was unsatisfactory. Enterbacteriaceae, lactobacilli and endospore-forming bacteria were identified occasionally, but their significance as contaminating organisms seems low. No Salmonella spp. were identified.     

Detection of pathogenic Vibrio parahaemolyticus in oyster enrichments by real time PCR

A real time polymerase chain reaction (PCR) assay was developed and evaluated to detect the presence of the thermostable direct hemolysin gene (tdh), a current marker of pathogenicity in Vibrio parahaemolyticus. The real time PCR fluorogenic probe and primer set was tested against a panel of numerous strains from 13 different bacterial species. Only V. parahaemolyticus strains possessing the tdh gene generated a fluorescent signal, and no cross-reaction was observed with tdh negative Vibrio or non-Vibrio spp. The assay detected a single colony forming unit (CFU) per reaction of a pure culture template. This sensitivity was achieved when the same template amount per reaction was tested in the presence of 2.5 microl of a tdh negative oyster:APW enrichment (oyster homogenate enriched in alkaline peptone water overnight at 35 degrees C). This real time technique was used to test 131 oyster:APW enrichments from an environmental survey of Alabama oysters collected between March 1999 and September 2000. The results were compared to those previously obtained using a streak plate procedure for culture isolation from the oyster:APW enrichment combined with use of a non-radioactive DNA probe for detection of the tdh gene. Real time PCR detected tdh in 61 samples, whereas the streak plate/probe method detected tdh in 15 samples. Only 24 h was required for detection of pathogenic V. parahaemolyticus in oyster:APW enrichments by real time PCR, whereas the streak plate/probe method required 3 days and was more resource intensive. This study demonstrated that real time PCR is a rapid and reliable technique for detecting V. parahaemolyticus possessing the tdh gene in pure cultures and in oyster enrichments.     

Effect of bile on Vibrio parahaemolyticus.

Many enteric pathogens are thought to enter a viable but nonculturable state when deprived of nutrients. Virulent strains of the enteric pathogen Vibrio parahaemolyticus are rarely isolated from their low-nutrient aquatic environments, possibly due to their nonculturability. Host factors such as bile may trigger release from dormancy and increase virulence in these strains. In this study, the addition of bile or the bile acid deoxycholic acid to estuarine water-cultured bacteria led to an increase in the direct viable count and colony counts among the virulent strains. This effect was not demonstrated in the nonvirulent strains, and it was reversed by extraction of bile acids with cholestyramine. Bile-treated V. parahaemolyticus had lower levels of intracellular calcium than untreated cells, and this effect coincided with an increase in the number of metabolically active cells. Chelation of intracellular calcium with BAPTA/AM (R. Y. Tsien, Biochemistry 19:2396-2402, 1980) produced similar results. Addition of bile to V. parahaemolyticus cultures in laboratory medium enhanced factors associated with virulence such as Congo red binding, bacterial capsule size, and adherence to epithelial cells. These results suggest that a bile acid-containing environment such as that found in the human host favors growth of virulent strains of V. parahaemolyticus and that bile acids enhance the expression of virulence factors. These effects seem to be mediated by a decrease in intracellular calcium.     

Inhibitory Effect of Glycerin on Vibrio parahaemolyticus and Salmonella

In a study of the effect of glycerin in transport media on Vibrio parahaemolyticus and Salmonella, it was found that a concentration of 30% glycerin was highly inhibitory for V. parahaemolyticus and to a lesser degree for Salmonella. The incorporation of peptone or human feces in media did not reduce the inhibitory effect of glycerin. In media with 15% glycerin, viable counts of V. parahaemolyticus and Salmonella increased after 24 hr of incubation both in the presence and absence of feces. Due to the concurrent increase in the total bacterial count in the media containing feces, no enrichment effect was noted.     

Effect of Vibrio parahaemolyticus haemolysin on human erythrocytes

Haemolysin Kanagawa, a toxin from Vibrio parahaemolyticus, is known to trigger haemolysis. Flux studies indicated that haemolysin forms a cation channel. In the present study, channel properties were elucidated by patch clamp and functional significance of ion fluxes by fluorescence-activated cell sorting (FACS) analysis. Treatment of human erythrocytes with 1 U ml-1 haemolysin within minutes induces a non-selective cation permeability. Moreover, haemolysin activates clotrimazole-sensitive K+ channels, pointing to stimulation of Ca2+-sensitive Gardos channels. Haemolysin (1 U ml-1) leads within 5 min to slight cell shrinkage, which is reversed in Ca2+-free saline. Erythrocytes treated with haemolysin (0.1 U ml-1) do not undergo significant haemolysis within the first 60 min. Replacement of extracellular Na+ with NMDG+ leads to slight cell shrinkage, which is potentiated by 0.1 U ml-1 haemolysin. According to annexin binding, treatment of erythrocytes with 0.1 U ml-1 haemolysin leads within 30 min to breakdown of phosphatidylserine asymmetry of the cell membrane, a typical feature of erythrocyte apoptosis. The annexin binding is significantly blunted at increased extracellular K+ concentrations and by K+ channel blocker clotrimazole. In conclusion, haemolysin Kanagawa induces cation permeability and activates endogenous Gardos K+ channels. Consequences include breakdown of phosphatidylserine asymmetry, which depends at least partially on cellular loss of K+.     

The development of rapid real-time PCR detection system for Vibrio parahaemolyticus in raw oyster

Aims:  To develop a new rapid real-time polymerase chain reaction (PCR) based detection system for Vibrio parahaemolyticus ( V. parahaemolyticus ) applicable to raw oyster samples.
Methods and Results:  V. parahaemolyticus cells were artificially inoculated to oysters. Samples were homogenized in 100 ml of sterile saline water and serially diluted to 1·5 CFU ml⁻¹ level. One millilitre of diluents was centrifuged and the pellet was resuspended with 100  μ l of de-ionized water. DNA was extracted by boiling for 20 min, and 0·5  μ l was used as a template for PCR reaction. Real-time PCR was performed with TMC-1000 system (1  μ l PCR system). The detection system was found to achieve detection limit of 1·5 CFU g⁻¹ for V. parahaemolyticus . Furthermore, the specificities of these assay systems were confirmed with more than 20 bacterial strains, including various Vibrio species.
Conclusions:  Rapid and sensitive food-borne pathogen detection techniques for V. parahaemolyticus is important to the food industry and consumers. The direct detection of V. parahaemolyticus from food is possible with micro real-time PCR system.
Significance and Impact of the Study:  This study shows that oyster samples can be tested for V. parahaemolyticus with a rapid, specific and simple procedure.     

Spoilage of vacuum-packaged dark, firm, dry meat at chill temperatures.

The flora of vacuum-packaged dark, firm, dry meat included thred organisms not usually found on vacuum-packaged meat, Yersinia enterocolitica, Enterobacter liquefaciens, and Alteromonas putrefaciens. Y. enterocolitica did not affect the meat quality. Production of spoilage odors by E. liquefaciens could be prevented by addition of glucose or citrate to the meat. Greening of meat could be prevented by addition of glucose or citrate to the meat. Greening of meat by A. putrefaciens was not prevented by addition of glucose, as the organism degraded cysteine with the release of H2S even when glucose was present. To prevent greening, growth of A. putrefaciens must be inhibited by reducing the meat pH to less than 6.0.     

Spoilage of vacuum-packaged dark, firm, dry meat at chill temperatures.

The flora of vacuum-packaged dark, firm, dry meat included thred organisms not usually found on vacuum-packaged meat, Yersinia enterocolitica, Enterobacter liquefaciens, and Alteromonas putrefaciens. Y. enterocolitica did not affect the meat quality. Production of spoilage odors by E. liquefaciens could be prevented by addition of glucose or citrate to the meat. Greening of meat could be prevented by addition of glucose or citrate to the meat. Greening of meat by A. putrefaciens was not prevented by addition of glucose, as the organism degraded cysteine with the release of H2S even when glucose was present. To prevent greening, growth of A. putrefaciens must be inhibited by reducing the meat pH to less than 6.0.     

Identification of tdh-positive Vibrio parahaemolyticus from an outbreak associated with raw oyster consumption in Spain

Between August and September 1999, a total of 64 cases of illness were identified in three episodes of acute gastroenteritis associated with the consumption of live oysters from a typical outdoor street market in Galicia (northwest Spain). Nine case patients were hospitalized and analysis of their stool samples revealed the presence of Vibrio parahaemolyticus. The strains isolated from two stool samples were studied for antibiotic susceptibility, biochemical characteristics and presence of virulence factors. Both isolates were Kanagawa phenomenon positive and produced thermostable direct hemolysin, which is related to pathogenicity in humans. These results show the presence of pathogenic V. parahaemolyticus in mollusks harvested in Europe and reveal the risk of illness associated with their consumption, suggesting the revision of V. parahaemolyticus risk assessment associated with consumption of raw live shellfish.     

Effect of Hydrostatic Pressure on Growth and Viability of Vibrio parahaemolyticus

Six strains of marine bacteria, including three strains of Vibrio parahaemolyticus, two Vibrio spp isolated from coastal regions, and the deep ocean isolate Pseudomonas bathycetes, were examined for ability to survive and grow at deep ocean hydrostatic pressures. V. parahaemolyticus and the coastal Vibrio spp. were unable to survive or grow at 200, 400, 600, 800, or 1,000 atm of pressure. In contrast, the deep ocean isolate P. bathycetes was capable of survival and growth at these pressures. The evidence strongly supports the neritic or estuarine origin and habitat for V. parahaemolyticus.     

Effect of essential oils on pathogenic and biofilm-forming Vibrio parahaemolyticus strains

Abstract

In this study, the effect of three essential oils (EOs) – clove oil (CO), thyme oil (TO), and garlic oil (GO), which are generally recognized as safe – on the planktonic growth, minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC), motility, biofilm formation, and quorum sensing (QS) of Vibrio parahaemolyticus was investigated. All three EOs showed bacteriostatic activity, with MICs in the range 0.02%–0.09% (v/v). CO and TO completely controlled planktonic growth at 0.28% and 0.08% (v/v), which is four times their MIC (4?×?MIC), after 10?min, whereas GO completely controlled growth at 0.36% (v/v) (4?×?MIC) after treatment for 20?min. V. parahaemolyticus motility was significantly reduced by all three EOs at 4?×?MIC (0.28% for CO, 0.08% for TO, and 0.36% for GO), whereas QS was controlled and biofilm formation reduced by all three EOs at 8?×?MIC (0.56% for CO, 0.16% for TO, and 0.72% for GO) after 30?min of treatment. These results suggest that CO, TO, and GO have a significant inhibitory effect on V. parahaemolyticus cells in biofilm sand thus represent a promising strategy for improving food safety. These results provide the evidence required to encourage further research into the practical use of the proposed EOs in food preparation processes.     

外源化学物质对副溶血性弧菌药物敏感性的影响

【目的】细菌对抗生素的耐药性已成为全球公共卫生问题关注的热点。有研究表明外源添加化学物质可以增强耐药细菌对抗生素的敏感性。本研究比较了3种化学物质葡萄糖、丙氨酸、甘油对增强副溶血性弧菌抗生素敏感性的作用效果。【方法】在亚抑菌浓度抗生素胁迫条件下,通过比较副溶血性弧菌在添加终浓度为10 mmol/L葡萄糖、丙氨酸、甘油后细菌存活率随时间的变化水平,来观察弧菌对亚抑菌浓度抗生素敏感性作用效果的改变,并采用氧化磷酸化解偶联剂CCCP对实验结果进行验证。【结果】发现3种外源化学物质均能增强亚抑菌浓度氨基糖苷类抗生素对副溶血性弧菌的杀菌能力,其中外源添加葡萄糖对增强亚抑菌浓度卡那霉素的杀菌能力最为显著,而对其他种类抗生素的杀菌能力则无明显增强作用。加入氧化磷酸化解偶联剂CCCP后可消除由外源化学物质引发的弧菌抗生素敏感性作用增强的现象。【结论】通过调节细菌细胞代谢水平可提高耐药副溶血性弧菌对氨基糖苷类抗生素的敏感性,对多重耐药副溶血性弧菌的防控具有一定的实际应用价值。     

不同温度条件下副溶血性弧菌tdh基因表达及其代谢组响应

【目的】采用RT-PCR的方法分析致病性副溶血性弧菌毒力基因表达,并应用代谢组学的方法研究毒力基因不同表达水平下致病性副溶血性弧菌代谢组的响应。【方法】本文以致病性副溶血性弧菌(Vibrio parahaemolyticus)ATCC33846为材料,分别提取不同温度(4、25和37℃)下菌体总RNA和代谢组。采用相对定量的方法检测副溶血性弧菌tdh基因在不同温度条件下的表达差异,同时应用超高压液相色谱-四级杆飞行时间质谱联用仪(UPLC/Q-TOF-MS)系统为工作平台检测其代谢组。采用主成分分析法(principal component analysis,PCA)比较副溶血性弧菌代谢组轮廓差异,并通过皮尔森和斯皮尔曼相关性分析法分析代谢组与tdh基因表达之间相关性。【结果】结果表明,不同温度条件下tdh基因表达强弱的排列顺序25℃4℃37℃;在tdh基因不同表达水平下发生显著性(P0.05)变化的主要代谢物是有机酸、氨基酸、醇、酮、酯;共得到11种代谢物与tdh基因表达高度相关(相关性系数︱r︱=1,P0.05),其中3种为负相关,8种为正相关,且醇类代谢物与tdh基因表达的正相关性最显著。【结论】本研究发现副溶血性弧菌代谢组与毒力基因表达存在一定的相关性,有望为副溶血性弧菌致病机理的深入探究提供一定的理论支持。