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1.
Genome analyses of marine microbial communities have revealed the widespread occurrence of genomic islands (GIs), many of which encode for protein secretion machineries described in the context of bacteria-eukaryote interactions. Yet experimental support for the specific roles of such GIs in aquatic community interactions remains scarce. Here, we test for the contribution of type III secretion systems (T3SS) to the environmental fitness of epidemic Vibrio parahaemolyticus. Comparisons of V. parahaemolyticus wild types and T3SS-defective mutants demonstrate that the T3SS encoded on genome island VPaI-7 (T3SS-2) promotes survival of V. parahaemolyticus in the interaction with diverse protist taxa. Enhanced persistence was found to be due to T3SS-2 mediated cytotoxicity and facultative parasitism of V. parahaemolyticus on coexisting protists. Growth in the presence of bacterivorous protists and the T3SS-2 genotype showed a strong correlation across environmental and clinical isolates of V. parahaemolyticus. Short-term microcosm experiments provide evidence that protistan hosts facilitate the invasion of T3SS-2 positive V. parahaemolyticus into a coastal plankton community, and that water temperature and productivity further promote enhanced survival of T3SS-2 positive V. parahaemolyticus. This study is the first to describe the fitness advantage of GI-encoded functions in a microbial food web, which may provide a mechanistic explanation for the global spread and the seasonal dynamics of V. parahaemolyticus pathotypes, including the pandemic serotype cluster O3:K6, in aquatic environments.  相似文献   

2.
Inactivation of mismatch repair (MMR) has been shown to increase the accumulation of spontaneous mutations and frequency of recombination for diverse pathogenic bacteria. Currently, little is known regarding the role of mutator phenotypes for the diversification of natural populations of opportunistic human pathogens in marine environments. In this study, a higher frequency of mutators was detected among V. parahaemolyticus strains obtained from environmental sources compared with clinical sources. Inactivation of the MMR gene mutS caused increased antibiotic resistance and phase variation resulting in translucent colony morphologies. Increased nucleotide diversity in mutS and rpoB alleles from mutator compared with wild-type strains indicated a significant contribution of the mutator phenotype to the evolution of select genes. The results of this study indicate that the inactivation of MMR in V. parahaemolyticus leads to increased genetic and phenotypic diversity. This study is the first to report a higher frequency of natural mutators among Vibrio environmental strains and to provide evidence that inactivation of MMR increases the diversity of V. parahaemolyticus .  相似文献   

3.
4.
AIMS: The objective of this study was to investigate the occurrence and distribution of haemolysin genes, plasmid profile, serogroup analysis and cellular urease activity for Vibrio parahaemolyticus isolates from infected human patients and oysters from the Pacific north-western United States between 1988 and 1997. METHODS AND RESULTS: All of the clinical and environmental isolates tested in this study exhibited the presence of the thermolabile haemolysin gene, tl, confirming that all of the isolates were V. parahaemolyticus. Furthermore, the V. parahaemolyticus isolates that contained either the thermostable direct haemolysin gene, tdh, or the thermostable direct haemolysin-related gene, trh, or both, were also positive for urease. Isolates from infected human patients belong to serogroups O1 and O4, whereas, the isolates from oysters belong to serogroups O1, O4 and O5. These results suggest that the presence of a V. parahaemolyticus serogroup O1 and O4 could indicate the presence of a virulent strain of this pathogen. In this study, the presence of the haemolysin genes, serogroup profiles and urease production in V. parahaemolyticus isolated from human patients correlated with the oysters collected during the outbreaks. However, no significant correlation of the plasmid profiles was detected, based on their distribution and molecular weights, between V. parahaemolyticus isolated from infected human patients and from oysters collected during this outbreak. CONCLUSIONS, SIGNIFICANCE AND IMPACT OF STUDY: It is apparent from this study that the identification of the haemolysin genes by multiplex PCR amplification, in conjunction with serogroup analysis and urease production, can be used to monitor shellfish for the presence of potentially pathogenic strains of V. parahaemolyticus.  相似文献   

5.
Abstract An environmental survey was done to study the ecology and distribution of Vibrio parahaemolyticus in 5 selected stations in Okayama Prefecture, which included fresh, brackish, and marine aquatic environments. Water and plankton samples were collected monthly for quantitative and qualitative analyses during the period October, 1987 to October, 1989 for V. parahaemolyticus . The pathogen was not detected from fresh water environments. A seasonality of the organism was observed in brackish and marine environments where average salinity ranged between 0.39 and 1.28%.Plankton samples yielded higher densities of V. parahaemolyticus compared with water samples. By applying several enrichment techniques, the pathogen was detected quite frequently during the winter months in the environments with temperatures ranging between 10 and 14°C. The identification following conventional tests, by the API 20E system and by serological methods reveal that the API 20E system is satisfactory to identify V. parahaemolyticus and further confirms that the serological method could be a simpler and more rapid procedure for V. parahaemolyticus identification.  相似文献   

6.
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.  相似文献   

7.
Repeated subculturing of Kanagawa-negative strains of Vibrio parahaemolyticus on Wagatsuma agar induced the production of a hemolysin which was not the thermostable direct hemolysin. Crude hemolysin exhibited a 30 to 40% lethal toxicity in mice after intraperitoneal injection. A 21-kilodalton protein band was observed with all the environmental isolates in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Results suggested that a certain percentage of environmental strains of V. parahaemolyticus is responsible for pathogenesis.  相似文献   

8.
B L Sarkar  R Kumar  S P De    S C Pal 《Applied microbiology》1987,53(11):2696-2698
Repeated subculturing of Kanagawa-negative strains of Vibrio parahaemolyticus on Wagatsuma agar induced the production of a hemolysin which was not the thermostable direct hemolysin. Crude hemolysin exhibited a 30 to 40% lethal toxicity in mice after intraperitoneal injection. A 21-kilodalton protein band was observed with all the environmental isolates in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Results suggested that a certain percentage of environmental strains of V. parahaemolyticus is responsible for pathogenesis.  相似文献   

9.
Vibrio parahaemolyticus is an estuarine bacterium that is the leading cause of shellfish-associated cases of bacterial gastroenteritis in the United States. Our laboratory developed a real-time multiplex PCR assay for the simultaneous detection of the thermolabile hemolysin (tlh), thermostable direct hemolysin (tdh), and thermostable-related hemolysin (trh) genes of V. parahaemolyticus. The tlh gene is a species-specific marker, while the tdh and trh genes are pathogenicity markers. An internal amplification control (IAC) was incorporated to ensure PCR integrity and eliminate false-negative reporting. The assay was tested for specificity against >150 strains representing eight bacterial species. Only V. parahaemolyticus strains possessing the appropriate target genes generated a fluorescent signal, except for a late tdh signal generated by three strains of V. hollisae. The multiplex assay detected <10 CFU/reaction of pathogenic V. parahaemolyticus in the presence of >10(4) CFU/reaction of total V. parahaemolyticus bacteria. The real-time PCR assay was utilized with a most-probable-number format, and its results were compared to standard V. parahaemolyticus isolation methodology during an environmental survey of Alaskan oysters. The IAC was occasionally inhibited by the oyster matrix, and this usually corresponded to negative results for V. parahaemolyticus targets. V. parahaemolyticus tlh, tdh, and trh were detected in 44, 44, and 52% of the oyster samples, respectively. V. parahaemolyticus was isolated from 33% of the samples, and tdh(+) and trh(+) strains were isolated from 19 and 26%, respectively. These results demonstrate the utility of the real-time PCR assay in environmental surveys and its possible application to outbreak investigations for the detection of total and pathogenic V. parahaemolyticus.  相似文献   

10.
Total Vibrio parahaemolyticus densities and the occurrence of pathogenic strains in shellfish were determined following outbreaks in Washington, Texas, and New York. Recently developed nonradioactive DNA probes were utilized for the first time for direct enumeration of V. parahaemolyticus in environmental shellfish samples. V. parahaemolyticus was prevalent in oysters from Puget Sound, Wash.; Galveston Bay, Tex.; and Long Island Sound, N.Y., in the weeks following shellfish-associated outbreaks linked to these areas. However, only two samples (one each from Washington and Texas) were found to harbor total V. parahaemolyticus densities exceeding the level of concern of 10,000 g(-1). Pathogenic strains, defined as those hybridizing with tdh and/or trh probes, were detected in a few samples, mostly Puget Sound oysters, and at low densities (usually <10 g(-1)). Intensive sampling in Galveston Bay demonstrated relatively constant water temperature (27.8 to 31.7 degrees C) and V. parahaemolyticus levels (100 to 1,000 g(-1)) during the summer. Salinity varied from 14.9 to 29.3 ppt. A slight but significant (P < 0.05) negative correlation (-0.25) was observed between V. parahaemolyticus density and salinity. Based on our data, findings of more than 10,000 g(-1) total V. parahaemolyticus or >10 g(-1) tdh- and/or trh-positive V. parahaemolyticus in environmental oysters should be considered extraordinary.  相似文献   

11.
We investigated the recovery of dormant and injured cells along with the normally culturable cells of Vibrio species with special emphasis on V. parahaemolyticus using both selective and non-selective media at moderate (20 C) and standard (37 C) culture temperatures from a bay water environment. Culture temperatures (20 or 37 C) did not affect the recovery of V. parahaemolyticus but did for other vibrios. We observed similar seasonality of V parahaemolyticus as in most other environmental studies. V. parahaemolyticus and other Vibrio species were recovered in higher numbers by a replica plating method compared to most probable number (MPN) and direct TCBS (thiosulfate citrate bile-salt sucrose) agar counts. Even with the replica plating method, however, vibrios number goes down to a minimum level and V. parahaemolyticus was undetectable during the cool temperature period of the year, although total bacterial cells and CFU on nutrient agar (with 2% NaCl) did not vary so much during the study period.  相似文献   

12.
Although thermostable direct hemolysin (TDH)-producing Vibrio parahaemolyticus has caused many infections in Asian countries, the United States, and other countries, it has been difficult to detect the same pathogen in seafoods and other environmental samples. In this study, we detected and enumerated tdh gene-positive V. parahaemolyticus in Japanese seafoods with a tdh-specific PCR method, a chromogenic agar medium, and a most-probable-number method. The tdh gene was detected in 33 of 329 seafood samples (10.0%). The number of tdh-positive V. parahaemolyticus ranged from <3 to 93/10 g. The incidence of tdh-positive V. parahaemolyticus tended to be high in samples contaminated with relatively high levels of total V. parahaemolyticus. TDH-producing strains of V. parahaemolyticus were isolated from 11 of 33 tdh-positive samples (short-necked clam, hen clam, and rock oyster). TDH-producing strains of V. parahaemolyticus were also isolated from the sediments of rivers near the coast in Japan. Representative strains of the seafood and sediment isolates were examined for the O:K serovar and by the PCR method specific to the pandemic clone and arbitrarily primed PCR and pulsed-field gel electrophoresis techniques. The results indicated that most O3:K6 tdh-positive strains belonged to the pandemic O3:K6 clone and suggested that serovariation took place in the Japanese environment.  相似文献   

13.
目的 了解宁波地区环境来源海产品中副溶血弧菌血清学特点及毒力相关基因分布。方法 采集并分离2013年6‒10月宁波地区海产品中副溶血弧菌,对其进行O、K抗原血清学分型;并采用PCR或多重PCR的方法来检测溶血素基因(tlh、tdh、trh)、大流行群遗传标志基因(toxRS/new、orf8)和Ⅲ型分泌系统(T3SS1、T3SS2α、T3SS2β)基因。结果 从海产品样本中分离鉴定到44株副溶血弧菌的菌株,分属于20种血清型,型别多样,未见优势血清型;溶血素基因检测发现3株tdh+trh-致病性菌株,遗传标志基因检测发现1株tdh+trh-toxRS/new+大流行株,其血清型为O3:K6型;Ⅲ型分泌系统基因检测发现T3SS1基因存在于所有的副溶血弧菌菌株中,而T3SS2α基因则主要分布在tdh+的菌株中。结论 宁波地区环境中副溶血弧菌致病性菌株和大流行株的检出,说明该地区具有潜在的食源性疾病爆发的风险。  相似文献   

14.
Screening for pathogenic Vibrio parahaemolyticus has become routine in certain areas associated with food-borne outbreaks. This study is an evaluation of the CHROMagar Vibrio (CV) medium-PCR protocol and the conventional method (TCBS (thiosulfate-citrate-bile salts-sucrose) agar plus biochemical and Wagatsuma agar tests) for detection of V. parahaemolyticus in shrimp, water, sediment, and stool samples collected for biosurveillance in an endemic area of northwestern Mexico. A total of 131 environmental and clinical samples were evaluated. The CV medium-PCR protocol showed a significantly improved ability (P < 0.05) to isolate and detect V. parahaemolyticus, identifying isolates of this bacteria missed by the conventional method. Although some other bacteria, distinct from pathogenic V. parahaemolyticus, produced violet colonies similar to that of V. parahaemolyticus on CV medium, we were able to detect a superior number of samples of V. parahaemolyticus with the CV medium-PCR protocol than with the conventional method. The Kanagawa phenomenon is routinely determined on Wagatsuma agar for the diagnosis of V. parahaemolyticus (pathogenic) positive for thermostable direct hemolysin (TDH) in developing countries. In our results, Wagatsuma agar showed low sensitivity (65.4% at 24 h and 75.6% at 48 h) and specificity (52.4% at 48 h) for identifying V. parahaemolyticus positive for TDH. Overall, our data support the use of the CV medium-PCR protocol in place of the conventional method (TCBS-biochemical tests-Wagatsuma agar) for detection of pathogenic V. parahaemolyticus, both in terms of effectiveness and cost efficiency.  相似文献   

15.
目的建立用于评价副溶血弧菌毒力的小鼠模型,为研究副溶血弧菌的致病机制奠定基础。方法将适宜浓度的菌液经腹腔感染4~5周龄雌性BALB/c小鼠,观察小鼠的症状及死亡数。结果高盐(2%NaCl)条件下培养的强毒株RIMD2210633,经腹腔感染107CFU的菌量,小鼠存活率为20%~30%,而环境无毒株S251的小鼠存活率为100%。结论建立了评价副溶血弧菌毒力的实验小鼠模型,并应用于不同盐分浓度培养的强毒株与环境无毒株的毒力比较实验。  相似文献   

16.
Vibrio parahaemolyticus is an important human pathogen whose transmission is associated with the consumption of contaminated seafood. There is a growing public health concern due to the emergence of a pandemic strain causing severe outbreaks worldwide. Many questions remain unanswered regarding the evolution and population structure of V. parahaemolyticus. In this work, we describe a multilocus sequence typing (MLST) scheme for V. parahaemolyticus based on the internal fragment sequences of seven housekeeping genes. This MLST scheme was applied to 100 V. parahaemolyticus strains isolated from geographically diverse clinical (n = 37) and environmental (n = 63) sources. The sequences obtained from this work were deposited and are available in a public database (http://pubmlst.org/vparahaemolyticus). Sixty-two unique sequence types were identified, and most (50) were represented by a single isolate, suggesting a high level of genetic diversity. Three major clonal complexes were identified by eBURST analysis. Separate clonal complexes were observed for V. parahaemolyticus isolates originating from the Pacific and Gulf coasts of the United States, while a third clonal complex consisted of strains belonging to the pandemic clonal complex with worldwide distribution. The data reported in this study indicate that V. parahaemolyticus is genetically diverse with a semiclonal population structure and an epidemic structure similar to that of Vibrio cholerae. Genetic diversity in V. parahaemolyticus appears to be driven primarily by frequent recombination rather than mutation, with recombination ratios estimated at 2.5:1 and 8.8:1 by allele and site, respectively. Application of this MLST scheme to more V. parahaemolyticus strains and by different laboratories will facilitate production of a global picture of the epidemiology and evolution of this pathogen.  相似文献   

17.
This study describes the optimization of PCR parameters and testing of a wide number of microbial species to establish a highly specific and sensitive PCR-based method of detection of a newly emerged pandemic Vibrio parahaemolyticus O3:K6 strain in pure cultures and seeded waters from the Gulf of Mexico (gulf water). The selected open reading frame 8 (ORF8) DNA-specific oligonucleotide primers tested were found to specifically amplify all 35 pathogenic V. parahaemolyticus O3:K6 pandemic isolates, whereas these primers were not found to detectably amplify two strains of V. parahaemolyticus O3:K6 that were isolated prior to the 1996 outbreaks, 122 non-O3:K6 strains of V. parahaemolyticus, 198 non-V. parahaemolyticus spp., or 16 non-Vibrio bacterial spp. The minimum level of detection by the PCR method was 1 pg of purified genomic DNA or 10(2) ORF8-positive V. parahaemolyticus O3:K6 cells in 100 ml of water. The effectiveness of this method for the detection of ORF8-positive isolates in environmental samples was tested in gulf water seeded with 10-fold serial dilutions of this pathogen. A detection level of 10(3) cells per 100 ml of gulf water was achieved. Also, the applicability of this methodology was tested by the detection of this pathogen in gulf water incubated at various temperatures for 28 days. This PCR approach can potentially be used to monitor with high specificity and well within the required range of sensitivity the occurrence and distribution of this newly emerged pathogenic V. parahaemolyticus O3:K6 strain in coastal, marine, and ship ballast waters. Early detection of V. parahaemolyticus O3:K6 will help increase seafood safety and decrease the risk of infectious outbreaks caused by this pathogen.  相似文献   

18.
The thermostable direct hemolysin (TDH) and TDH-related hemolysin (TRH) are the main virulence factors of Vibrio parahaemolyticus. We isolated V. parahaemolyticus from seawater, fish, and oysters obtained from the Pueblo Viejo Lagoon in Veracruz, determined the serogroups, phenotypically and genotypically characterized TDH and TRH, and investigated the presence of the toxR gene. A total of 46 V. parahaemolyticus strains were isolated, and all of them amplified the 368-bp toxR gene fragment. The trh gene was not identified in any of the strains; 4 of the 46 strains were Kanagawa phenomenon (KP) positive and amplified the 251-bp tdh gene fragment. The most frequent serogroup was serogroup O3. This is the first report of the presence of KP-positive tdh-positive environmental V. parahaemolyticus strains in Mexico.  相似文献   

19.
Vibrio parahaemolyticus is known to exist in a viable but nonculturable state when incubated at low temperature under starvation. It has long been debated whether the culturable cells which appear after temperature upshift are the result of true resuscitation or regrowth of a few residual culturable cells. Starved V. parahaemolyticus cells at 4 degrees C reached the nonculturable stage in about 12 days. The true resuscitation of nonculturable cells of V. parahaemolyticus occurred after spreading them onto an agar medium supplemented with H(2)O(2)-degrading compounds such as catalase or sodium pyruvate. The proposed method may be applicable to detecting the enteropathogen from environmental samples.  相似文献   

20.
Culturable vibrios were isolated from water and plankton fractions collected during an 18-month sampling study performed along the north-central coast of the Adriatic Sea (Italy). Unculturable Vibrio vulnificus and V. parahaemolyticus were detected in plankton fractions by polymerase chain reaction amplification of DNA sequences for cytotoxin-haemolysin and thermolabile haemolysin respectively. The presence of V. parahaemolyticus, V. vulnificus and V. cholerae virulence genes and the expression of pathogenicity-associated traits were analysed in all isolates. The results showed the spreading of these properties among the environmental isolates and confirm the need of both monitoring the presence of vibrios in coastal areas and studying their pathogenicity potential in order to properly protect human health.  相似文献   

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