Vibrio parahaemolyticus and Vibrio vulnificus in South America: water,seafood and human infections

The bacterial species, Vibrio parahaemolyticus and Vibrio vulnificus, are ubiquitous in estuaries and coastal waters throughout the world, but they also happen to be important human pathogens. They are concentrated by filter‐feeding shellfish which are often consumed raw or undercooked, providing an important potential route of entry for an infective dose of these bacteria. Vibrio parahaemolyticus can cause abdominal cramping, nausea, diarrhoea, vomiting, chills and fever. Vibrio vulnificus can cause similar gastrointestinal‐related symptoms, but can also spread to the bloodstream, resulting in primary septicaemia, and it can also cause disease via wound infections. The objective of this article is to summarize, for the first time, the incidence and importance of V. parahaemolyticus and V. vulnificus in South America, in environmental waters and seafood, especifically molluscan shellfish, as well as human infection cases and outbreaks. It appears that infections from V. parahaemolyticus have been more strongly related to shellfish ingestion and have been more frequently reported on the Pacific coast of South America. Conversely, V. vulnificus has been more frequently acquired by water contact with open wounds and its presence has been more heavily reported along the Atlantic coast of South America, and while documented to cause serious mortality, have been relatively few in number. The impacts of El Nino Southern Oscillation (ENSO) have been observed to cause an increase in V. parahaemolyticus outbreaks on the Pacific coast of South America. The implementation of a regulated monitoring approach, along with the use of faster, more accurate and virulence‐specific detection approaches, such as PCR confirmation, should be considered to detect the presence of pathogenic Vibrio strains in environmental and seafood samples for protection of public health. Furthermore, improved clinical surveillance with suspected cases should be implemented. This review highlights the need for more research and monitoring of vibrios in South America, in water, shellfish and clinical samples.     

Differences in the API 20E biochemical patterns of clinical and environmental Vibrio parahaemolyticus isolates

Genetic differences in clinical and environmental strains of Vibrio parahaemolyticus have been widely used as criteria in identifying pathogenic isolates. However, few studies have been carried out to assess the differences in biochemical characteristics of V. parahaemolyticus isolates from human and environmental sources. We compared the biochemical profiles obtained by the characterization of V. parahaemolyticus isolates from human infections and the marine environment using the API 20E system. Environmental and clinical isolates showed significant differences in the gelatin and arabinose tests. Additionally, clinical isolates were correctly identified according to the API 20E profile using 0.85% NaCl diluent, but they presented nonspecific profiles with 2% NaCl diluent. In contrast, use of 2% NaCl diluent facilitated correct identification of the environmental isolates. Clinical isolates showed significant differences in up to five biochemical tests with respect to the API 20E database. The API 20E system is widely used in routine identification of bacteria in clinical laboratories, and this discrepancy in an important number of biochemical tests may lead to misidentification of V. parahaemolyticus infection.     

Effects of organic matter on acidic electrolysed water for reduction of foodborne pathogens on lettuce and spinach

Aims: To evaluate the efficacy of acidic electrolysed water (EW) in the presence of organic matter (bovine serum) on the inoculated surfaces of lettuce and spinach. Materials and Results: Lettuce and spinach leaves were inoculated with a cocktail of three strains each of Escherichia coli O157:H7, Salmonella Typhimurium and Listeria monocytogenes and treated with deionized water, acidic EW and acidic EW containing bovine serum (5, 10, 15 and 20 ml l^?1) for 15 s, 30 s, 1 min, 3 min and 5 min at room temperature (22 ± 2°C). In the absence of bovine serum, acidic EW treatment reduced levels of cells below the detection limit (0·7 log) in 5 min. In the presence of bovine serum, bactericidal activity of acidic EW decreased with increasing serum concentration. Conclusions: Organic matter reduces the effectiveness of acidic EW for reducing pathogens on the surfaces of lettuce and spinach. Significance and Impact of the Study: From a practical standpoint, organic matter reduces the efficacy of acidic EW. This study was conducted to confirm the effect of organic matter on the properties of acidic EW in the inactivation of foodborne pathogens on the surface of vegetables.     

Variability in biofilm formation correlates with hydrophobicity and quorum sensing among Vibrio parahaemolyticus isolates from food contact surfaces and the distribution of the genes involved in biofilm formation

Vibrio parahaemolyticus is one of the leading foodborne pathogens causing seafood contamination. Here, 22 V. parahaemolyticus strains were analyzed for biofilm formation to determine whether there is a correlation between biofilm formation and quorum sensing (QS), swimming motility, or hydrophobicity. The results indicate that the biofilm formation ability of V. parahaemolyticus is positively correlated with cell surface hydrophobicity, autoinducer (AI-2) production, and protease activity. Field emission scanning electron microscopy (FESEM) showed that strong-biofilm-forming strains established thick 3-D structures, whereas poor-biofilm-forming strains produced thin inconsistent biofilms. In addition, the distribution of the genes encoding pandemic clone factors, type VI secretion systems (T6SS), biofilm functions, and the type I pilus in the V. parahaemolyticus seafood isolates were examined. Biofilm-associated genes were present in almost all the strains, irrespective of other phenotypes. These results indicate that biofilm formation on/in seafood may constitute a major factor in the dissemination of V. parahaemolyticus and the ensuing diseases.     

Fate of foodborne pathogens on green onions and tomatoes by electrolysed water

Aims: To investigate the efficacy of electrolysed water (EW) in killing Escherichia coli O157:H7, Salmonella typhimurium and Listeria monocytogenes on the surfaces of spot‐inoculated green onions and tomatoes. Methods and Results: Green onions and tomatoes were inoculated with a cocktail of three strains each of E. coli O157:H7, Salm. typhimurium and L. monocytogenes and treated with acidic electrolysed water (AC‐EW), alkaline electrolysed water (AK‐EW), alkaline electrolysed water followed by acidic electrolysed water (AK‐EW + AC‐EW), deionized water followed by acidic electrolysed water (DW + AC‐EW) and deionized water (control, DW) for 15 s, 30 s, 1 min, 3 min and 5 min at room temperature (22 ± 2°C). The relative efficacy of reduction was AC‐EW > DW + AC‐EW ≈ AK‐EW + AC‐EW > AK‐EW > DW. Conclusions: Acidic EW treatment was able to significantly reduce populations of the three tested pathogens from the surfaces of green onions and tomatoes with increasing exposure time. Significance and Impact of the Study: Rinsing in acidic EW reveals an effective method to control the presence of E. coli O157:H7, Salm. typhimurium and L. monocytogenes on the surfaces of fresh green onions and tomatoes, without affecting their organoleptic characteristics. This indicates its potential application for the decontamination of fresh produce surfaces.     

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.     

副溶血弧菌LacZ报告基因融合实验方法的建立与应用

目的:建立副溶血弧菌(Vibrio parahaemolyticus,VP)的LacZ报告基因融合实验方法。方法:PCR扩增靶基因的整个启动子区序列,并将其直接克隆入pHRP309质粒中,构建重组质粒;将重组质粒VPA1513转入VP野生株(WT)和opaR突变株(ΔopaR)中,而后通过比较两者β半乳糖苷酶活性的差异,确定OpaR对VPA1513的调控关系,以检验实验的稳定性。结果:构建出9个VP生物膜或毒力相关基因的LacZ重组质粒;OpaR对VPA1513的转录具有抑制作用。结论:建立了VP的LacZ报告基因融合实验方法,为后续转录调控机制的研究奠定了基础。     

Demonstration of the presence of alditols and galacturonic acid in Vibrio parahaemolyticus O10 lipopolysaccharide

Abstract Identification of 4 unidentified neutral substances (X₁, X₂, X₃ and X₄) in lipopolysaccharides of Vibrio parahaemolyticus (Miyano et al. (1980) FEMS Microbiol. Lett. 8, 23–28, and 14, 145–148) was attempted. X₁ (1,4-anhydroribitol) was found to be formed from ribitol-5-phosphate during hydrolysis. X₂ was identified to be 2- O -methylribitol. X₃ and X₄ were found to be formed during hydrolysis of galacturonic acid and D-glycero-L-mannoheptose (or L-glycero-D-mannoheptose), respectively. The chemical structures of X₃ and X₄ remain to be determined.     

Induction of viable but nonculturable state in Vibrio parahaemolyticus and its susceptibility to environmental stresses

AIMS: This work analysed factors that influence the induction of viable but nonculturable (VBNC) state in the common enteric pathogen, Vibrio parahaemolyticus. The susceptibility of the VBNC cells to environmental stresses was investigated. METHODS AND RESULTS: Bacterium was cultured in tryptic soy broth-3% NaCl medium, shifted to a nutrient-free Morita mineral salt-0.5% NaCl medium (pH 7.8) and further incubated at 4 degrees C in a static state to induce the VBNC state in 28-35 days. The culturability and viability of the cells were monitored by the plate count method and the Bac Light viable count method, respectively. Cells grown at the optimum growth temperature and in the exponential phase better induced the VBNC state than those grown at low temperature and in the stationary phase. Low salinity of the medium crucially and markedly shortened the induction period. The VBNC cells were highly resistant to thermal (42, 47 degrees C), low salinity (0% NaCl), or acid (pH 4.0) inactivation. CONCLUSIONS: Optimal conditions for inducing VBNC V. parahaemolyticus were reported. The increase in resistance of VBNC V. parahaemolyticus to thermal, low salinity and acidic inactivation verified that this state is entered as part of a survival strategy in an adverse environment. SIGNIFICANCE AND IMPACT OF THE STUDY: The methods for inducing VBNC V. parahaemolyticus in a markedly short time will facilitate further physiological and pathological study. The enhanced stress resistance of the VBNC cells should attract attention to the increased risk presented by this pathogen in food.     

Identification and characterization of a type III secretion-associated chaperone in the type III secretion system 1 of Vibrio parahaemolyticus

Vibrio parahaemolyticus causes human gastroenteritis. Genomic sequencing of this organism has revealed that it has two sets of type III secretion systems, T3SS1 and T3SS2, both of which are important for its pathogenicity. However, the mechanism of protein secretion via T3SSs is unknown. A characteristic of many effectors is that they require specific chaperones for efficient delivery via T3SSs; however, no chaperone has been experimentally identified in the T3SSs of V. parahaemolyticus . In this study, we identified candidate T3SS1-associated chaperones from genomic sequence data and examined their roles in effector secretion/translocation and binding to their cognate substrates. From these experiments, we concluded that there is a T3S-associated chaperone, VecA, for a cytotoxic T3SS1-dependent effector, VepA. Further analysis using pulldown and secretion assays characterized the chaperone-binding domain encompassing the first 30–100 amino acids and an amino terminal secretion signal encompassing the first 5–20 amino acids on VepA. These findings will provide a strategy to clarify how the T3SS1 of V. parahaemolyticus secretes its specific effectors.     

酒、盐度及酸碱度对醉泥螺中副溶血性弧菌生长的影响

醉泥螺中的副溶血性弧菌对人有较高的致病风险,为了提高醉泥螺的食用安全性,本文对其腌制工艺进行了优化,探讨了酒、盐度和酸碱度三个因素对醉泥螺中副溶血性弧菌生长状况的影响,并用描述性检验对盐度不同的醉泥螺进行了感官评价。研究结果表明,52%vol白酒,6. 6%盐度,酸碱度p H 5. 5的工艺条件能够最高效地降低醉泥螺腌制过程中副溶血性弧菌引起的食用安全风险,副溶血性弧菌的生长最受抑制。但兼顾不同盐度醉泥螺的感官评价,本研究认为52%vol白酒,4. 4%盐度,酸碱度pH 5. 5是最适合腌制醉泥螺的加工条件。     

Inactivation of Vibrio parahaemolyticus in pure culture, whole live and half shell oysters (Crassostrea virginica) by X-ray

Aims:  To study the inactivation effect of different doses of X-ray on Vibrio parahaemolyticus in pure culture, inoculated whole live and half shell oysters and to evaluate the efficacy of X-ray doses on reduction of inherent microflora on oysters.
Methods and Results:  X-ray was produced using RS 2400 generator system (Rad Source Technologies Inc.). Pure culture of V. parahaemolyticus , inoculated half and whole shell oysters with V. parahaemolyticus were treated with 0·0, 0·1, 0·5, 0·75, 1·0, 1·5, 2·0, 3·0 and 5·0 kGy X-ray. Surviving bacteria in the pure culture and inoculated oysters, before and after treatment, were enumerated using overlay plating (in TSA then TCBS) and most probable number (MPN) methods. A greater than 6·0 log reduction of V. parahaemolyticus was observed with 0·75, 2·0 and 5·0 kGy X-ray for pure culture, half shell and whole shell oysters, respectively. Treatment with 0·75, 2·0 and 5·0 kGy X-ray reduced the MPN to <3 for pure culture, half and whole shell oysters, respectively. Treatment with 1·0 kGy X-ray significantly ( P <  0·05) reduced the inherent micro-organisms on whole shell oysters from 4·7 ± 0·1 to less than the detectable limit (<1·0 log CFU g⁻¹).
Conclusions:  X-ray (1–5 kGy) significantly ( P <  0·05) reduced V . parahaemolyticus and inherent microflora on oysters to less than detectable limit (<1·0 log CFU g⁻¹).
Significance and Impact of the Study:  Treatment with X-ray could control pathogenic bacteria and extend the shelf life of oysters.     

Inactivation of Escherichia coli, Listeria monocytogenes, Pseudomonas aeruginosa and Staphylococcus aureus on stainless steel and glass surfaces by neutral electrolysed water

AIM: To ascertain the efficacy of neutral electrolysed water (NEW) in reducing Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus and Listeria monocytogenes on glass and stainless steel surfaces. Its effectiveness for that purpose is compared with that of a sodium hypochlorite (NaClO) solution with similar pH, oxidation-reduction potential (ORP) and active chlorine content. METHODS AND RESULTS: First, the bactericidal activity of NEW was evaluated over pure cultures (8.5 log CFU ml-1) of the abovementioned strains: all of them were reduced by more than 7 log CFU ml-1 within 5 min of exposure either to NEW (63 mg l-1 active chlorine) or to NaClO solution (62 mg l-1 active chlorine). Then, stainless steel and glass surfaces were inoculated with the same strains and rinsed for 1 min in either NEW, NaClO solution or deionized water (control). In the first two cases, the populations of all the strains decreased by more than 6 log CFU 50 cm-2. No significant difference (P相似文献   

Cleaning and decontamination efficacy of wiping cloths and silver dihydrogen citrate on food contact surfaces

Aims: To test the efficacy of four wipe cloth types (cotton bar towel, nonwoven, microfibre and blended cellulose/cotton) with either quaternary ammonia cleaning solution or silver dihydrogen citrate (SDC) in cleaning food contact surfaces. Methods: Swab samples collected from untreated, cloth‐treated and cloth disinfectant‐treated surfaces were subjected to hygiene monitoring using adenosine triphosphate (ATP) bioluminescence and aerobic total plate counting (TPC) assays. Results: Adenosine triphosphate measurements taken after wiping the surfaces showed poor cleaning by nonwoven cloths (2·89 RLU 100 cm^?2) than the microfibre (2·30 RLU 100 cm^?2), cotton terry bar (2·26 RLU 100 cm^?2) and blended cellulose/cotton cloth types (2·20 RLU 100 cm^?2). The cellulose/cotton cloth showed highest log reduction in ATP‐B RLU values (95%) and CFU values (98·03%) when used in combination with SDC disinfectant. Conclusions: Cleaning effect of wiping cloths on food contact surfaces can be enhanced by dipping them in SDC disinfectant. ATP‐B measurements can be used for real‐time hygiene monitoring in public sector, and testing microbial contamination provides more reliable measure of cleanliness. Significance and Impact of the Study: Contaminated food contact surfaces need regular hygiene monitoring. This study could help to estimate and establish contamination thresholds for surfaces at public sector facilities and to base the effectiveness of cleaning methods.     

Persistence of Campylobacter jejuni on surfaces in a processing environment and on cutting boards

AIMS: The objectives of the study were to determine the spread and persistence of Campylobacter in a poultry processing plant and to provide a quantitative estimate of the survival of Campylobacter jejuni on the surface of a cutting board. METHODS AND RESULTS: Several contact surfaces in a poultry processing plant were sampled before the start of processing, after 30 min and after 120 min. Next, the survival of four C. jejuni strains was studied on a beech and polypropylene cutting board during 120 min. CONCLUSIONS: A rapid introduction and spread of Campylobacter in a well cleaned processing plant as well as a significant survival in time on the example of a cutting board is shown. SIGNIFICANCE AND IMPACT OF THE STUDY: The need to prevent cross-contamination in the food processing and preparation area and the importance of an integrated approach throughout the whole food chain to control transmission of Campylobacter is highlighted.     

Effect of Mg(2+) ion in protein secretion by magnesium-resistant strains of Pseudomonas aeruginosa and Vibrio parahaemolyticus isolated from the coastal water of Haldia port

A rapidly growing industrial complex including oil refineries and chemical industries has developed around the coastal area of Haldia port in the district of Midnapore, West Bengal, India. The coastal water is highly polluted with industrial wastes along with petroleum hydrocarbons. The bacteria isolated from the different sites of the coastal waters were Escherichia coli, Alcaligenes, Acinetobacter, Klebsiella spp., Micrococcus spp., Vibrio spp., Pseudomonas aeruginosa and Vibrio parahaemolyticus. The salinity of the water during the time of collection of samples around the port area was 8. 2 ppt. Among the isolated organisms, only two isolates, P. aeruginosa and V. parahaemolyticus, showed growth at 300 mM Mg(2+) ion concentration. However, a 3 mM Mg(2+) concentration was detected in the coastal water whereas other metal ion concentrations were less than 3x10(-5) mM. Resistance to Mg(2+) (300 mM) was determined by a 5.5-kb plasmid. A large amount of a 40-kDa outer membrane protein, which was highly soluble in 1 M MgCl(2), was isolated from both V. parahaemolyticus and P. aeruginosa. The secretion of proteins in the culture supernatant of V. parahaemolyticus was highly increased when the cells were grown in the presence of 300 mM Mg(2+), whereas very low secretion was observed in the same concentration of Mg(2+) in the case of P. aeruginosa. Mg(2+) may act as a specific release factor in protein secretion by V. parahaemolyticus strains.     

Detection of the tdh and trh genes in Vibrio parahaemolyticus isolates in fish and mussels from Middle Black Sea Coast of Turkey

Aim:  The aim of this study was to demonstrate the occurrence of potential pathogenic Vibrio parahaemolyticus in seafoods using DNA-based techniques in comparison with bacteriological methods.
Methods and Results:  From 120 fresh and processed fish and mussel samples collected from Middle Black Sea, 32 isolates were identified as V. parahaemolyticus by bacteriological methods and confirmed by tl gene-based conventional PCR. Of them, 13 isolates were found positive for only tdh gene, six isolates for only trh gene and 13 isolates for both genes by multiplex PCR.
Conclusions:  It is the first report demonstrating the presence of potential pathogenic V. parahaemolyticus isolates from the Black Sea seafoods by PCR detection of tl , trh and tdh genes that was found more rapid than bacteriological methods.
Significance and Impact of the Study:  This study confirmed the previous reports that characterization of potential pathogenic V. parahaemolyticus isolates based on the PCR techniques was reliable and cost-effective. These results suggest that molecular detection methods should be included in Turkish Standards of seafood control in addition to bacteriological methods.