A decrease in the proportion of infections by pandemic Vibrio parahaemolyticus in Hat Yai Hospital, southern Thailand

Infection by the pandemic clone of Vibrio parahaemolyticus is prevalent in southern Thailand. This study actively surveyed the incidence of V. parahaemolyticus infection in this area. A total of 865 isolates of V. parahaemolyticus was obtained from patients at Hat Yai Hospital, the main public hospital in Songkhla Province, Thailand, from 2000 to 2005. The isolates were examined by group-specific PCR (GS-PCR) specific for the pandemic clone, and for the presence of two major virulence genes, tdh and trh, and the O : K serotype. Representative isolates were also examined by antibiogram pattern and DNA fingerprinting using an arbitrarily primed PCR method to determine the clonal relationships between isolates. The total number of isolates was less in 2000 and more in 2004 and 2005 than in the years 2001–2003. The increase in the numbers of infections in 2004 and 2005 was not due to the emergence of a particular clone having unique characteristics, but was probably due to climate change. From 2000 to 2003, the percentages of pandemic strains of V. parahaemolyticus, defined as GS-PCR-positive tdh + trh −, was stable at 64.1, 67.5, 69.7 and 67.7 % of the total isolates each year, respectively. However, in 2004 and 2005, the percentages decreased to 56.1 and 55.5 %, respectively. The O : K serotypes of the pandemic isolates remained unchanged. The proportional decrease in infections caused by the pandemic strains are probably due to the population in this area gradually developing immunity to the pandemic clone whilst continuing to be susceptible to other strains.

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Prevalence of Pandemic Thermostable Direct Hemolysin-Producing Vibrio parahaemolyticus O3:K6 in Seafood and the Coastal Environment in Japan

Applied and Environmental Microbiology ◽

10.1128/aem.69.7.3883-3891.2003 ◽

2003 ◽

Vol 69 (7) ◽

pp. 3883-3891 ◽

Cited By ~ 101

Author(s):

Yukiko Hara-Kudo ◽

Kanji Sugiyama ◽

Mitsuaki Nishibuchi ◽

Ashrafuzzaman Chowdhury ◽

Jun Yatsuyanagi ◽

...

Keyword(s):

Vibrio Parahaemolyticus ◽

The United States ◽

Most Probable Number ◽

Asian Countries ◽

Probable Number ◽

Specific Pcr ◽

Thermostable Direct Hemolysin ◽

Chromogenic Agar ◽

Pcr Method ◽

Arbitrarily Primed Pcr

ABSTRACT 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.

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Distribution of Virulent and Pandemic Strains of Vibrio parahaemolyticus in Three Molluscan Shellfish Species (Meretrix meretrix, Perna viridis, and Anadara granosa) and Their Association with Foodborne Disease in Southern Thailand

Journal of Food Protection ◽

10.4315/0362-028x-69.11.2615 ◽

2006 ◽

Vol 69 (11) ◽

pp. 2615-2620 ◽

Cited By ~ 21

Author(s):

VARAPORN VUDDHAKUL ◽

SUPATINEE SOBOON ◽

WATTANEE SUNGHIRAN ◽

SUKHON KAEWPIBOON ◽

ASHRAFUZZAMAN CHOWDHURY ◽

...

Keyword(s):

Vibrio Parahaemolyticus ◽

Immunomagnetic Separation ◽

Hard Clam ◽

Perna Viridis ◽

Clinical Strains ◽

Specific Pcr ◽

Alkaline Peptone Water ◽

Southern Thailand ◽

Peptone Water ◽

Pandemic Strains

Distribution of pandemic strains of Vibrio parahaemolyticus in seafood, particularly in molluscan shellfish, and their serological and molecular relationships to clinical strains were examined from Hat Yai City in southern Thailand. During 2000 to 2002, virulent strains (tdh+ or trh+) were isolated from 13 of 230 molluscan shellfish samples using alkaline peptone water enrichment followed by immunomagnetic separation. The isolates included 12 pandemic strains (tdh+, trh−, group-specific PCR positive) from five Oriental hard clam samples, five green mussel samples, and one bloody clam sample. Among the pandemic strains, eight belonged to serogroup O3:K6, three belonged to O1:K25, and one was O1:K untypeable. One hundred eighty-seven strains of V. parahaemolyticus were isolated from clinical specimens obtained from a hospital in this city during 2000 to 2001. The pandemic strains comprised 64 and 68% of the isolates in 2000 and 2001, respectively. Among the serotypes of the pandemic strains, O3:K6 was dominant at 73% in 2000 and 76% in 2001 followed by O1:K25 at 20% in 2000 and 13% in 2001. Pulsed-field gel electrophoresis profiles of the pandemic strains from molluscan shellfish were indistinguishable or very similar to those of patient isolates. Similarity of the serotype distribution and DNA fingerprints occurring between the molluscan shellfish strains and clinical strains suggests that molluscan shellfish may be an important source of pandemic V. parahaemolyticus infection in southern Thailand. For public health, proper cooking of molluscan shellfish in this area is strongly recommended.

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Isolation of a Pandemic O3:K6 Clone of a Vibrio parahaemolyticus Strain from Environmental and Clinical Sources in Thailand

Applied and Environmental Microbiology ◽

10.1128/aem.66.6.2685-2689.2000 ◽

2000 ◽

Vol 66 (6) ◽

pp. 2685-2689 ◽

Cited By ~ 49

Author(s):

Varaporn Vuddhakul ◽

Ashrafazzuman Chowdhury ◽

Varaporn Laohaprertthisan ◽

Punnee Pungrasamee ◽

Nuanjira Patararungrong ◽

...

Keyword(s):

Vibrio Parahaemolyticus ◽

Clinical Case ◽

Parahaemolyticus Strain ◽

Enrichment Method ◽

Southern Thailand ◽

Immunomagnetic Enrichment ◽

Arbitrarily Primed Pcr

ABSTRACT Application of an immunomagnetic enrichment method selective forVibrio parahaemolyticus serovar K6 allowed isolation of a strain belonging to the pandemic O3:K6 clone of V. parahaemolyticus from fresh shellfish not implicated in a clinical case in southern Thailand. Arbitrarily primed PCR profiles of this strain, clinical O3:K6 strains isolated from sporadic diarrhea cases in the same area, and a standard pandemic O3:K6 strain were indistinguishable.

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Pandemic strains of O3:K6Vibrio parahaemolyticusin the aquatic environment of Bangladesh

Canadian Journal of Microbiology ◽

10.1139/w04-072 ◽

2004 ◽

Vol 50 (10) ◽

pp. 827-834 ◽

Cited By ~ 19

Author(s):

M Sirajul Islam ◽

Rizwana Tasmin ◽

Sirajul Islam Khan ◽

Habibul Bari Mahmud Bakht ◽

Zahid Hayat Mahmood ◽

...

Keyword(s):

Aquatic Environment ◽

Vibrio Parahaemolyticus ◽

Blot Hybridization ◽

Virulence Gene ◽

Gene Probe ◽

Chromosomal Dna ◽

Specific Pcr ◽

Pandemic Strains ◽

Positive Results ◽

Tdh Gene

A total of 1500 environmental strains of Vibrio parahaemolyticus, isolated from the aquatic environment of Bangladesh, were screened for the presence of a major V. parahaemolyticus virulence factor, the thermostable direct haemolysin (tdh) gene, by the colony blot hybridization method using a digoxigenin-labeled tdh gene probe. Of 1500 strains, 5 carried the tdh sequence, which was further confirmed by PCR using primers specific for the tdh gene. Examination by PCR confirmed that the 5 strains were V. parahamolyticus and lacked the thermostable direct haemolysin-related haemolysin (trh) gene, the alternative major virulence gene known to be absent in pandemic strains. All 5 strains gave positive Kanagawa phenomenon reaction with characteristic β-haemolysis on Wagatsuma agar medium. Southern blot analysis of the HindIII-digested chromosomal DNA demonstrated, in all 5 strains, the presence of 2 tdh genes common to strains positive for Kanagawa phenomenon. However, the 5 strains were found to belong to 3 different serotypes (O3:K29, O4:K37, and O3:K6). The 2 with pandemic serotype O3:K6 gave positive results in group-specific PCR and ORF8 PCR assays, characteristics unique to the pandemic clone. Clonal variations among the 5 isolates were analyzed by comparing RAPD and ribotyping patterns. Results showed different patterns for the 3 serotypes, but the pattern was identical among the O3:K6 strains. This is the first report on the isolation of pandemic O3:K6 strains of V. parahaemolyticus from the aquatic environment of Bangladesh.Key words: pandemic strains, Vibrio parahaemolyticus, aquatic environment.

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Characteristics of a pandemic clone of O3 : K6 and O4 : K68 Vibrio parahaemolyticus isolated in Beira, Mozambique

Journal of Medical Microbiology ◽

10.1099/jmm.0.2008/004275-0 ◽

2008 ◽

Vol 57 (12) ◽

pp. 1502-1507 ◽

Cited By ~ 15

Author(s):

M. Ansaruzzaman ◽

Ashrafuzzaman Chowdhury ◽

Nurul A. Bhuiyan ◽

Marzia Sultana ◽

Ashrafus Safa ◽

...

Keyword(s):

Multilocus Sequence Typing ◽

Vibrio Parahaemolyticus ◽

Common Ancestor ◽

Filamentous Phage ◽

Genetic Characteristics ◽

Clonal Group ◽

Specific Pcr ◽

Arbitrarily Primed Pcr ◽

Single Cluster ◽

Reference Strains

The genetic characteristics of Vibrio parahaemolyticus strains isolated in 2004 and 2005 in Mozambique were assessed in this study to determine whether the pandemic clone of V. parahaemolyticus O3 : K6 and O4 : K68 serotypes has spread to Mozambique. Fifty-eight V. parahaemolyticus strains isolated from hospitalized diarrhoea patients in Beira, Mozambique, were serotyped for O : K antigens and genotyped for toxR, tdh and trh genes. A group-specific PCR, a PCR that detects the presence of ORF8 of the filamentous phage f237, arbitrarily primed PCR, PFGE and multilocus sequence typing were performed to determine the pandemic status of the strains and their ancestry. All strains of serovars O3 : K6 (n=38) and O4 : K68 (n=4) were identified as a pandemic clonal group by these analyses. These strains are closely related to the pandemic reference strains of O3 : K6 and O4 : K68, which emerged in Asia in 1996 and were later found globally. The pandemic serotypes O3 : K6 and O4 : K68 including reference strains grouped into a single cluster indicating emergence from a common ancestor. The O3 : K58 (n=8), O4 : K13 (n=6), O3 : KUT (n=1) and O8 : K41 (n=1) strains showed unique characteristics different from the pandemic clone.

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Molecular Characterization of Vibrio parahaemolyticus Strains Associated with Foodborne Illness in Florida

Journal of Food Protection ◽

10.4315/0362-028x-70.10.2396 ◽

2007 ◽

Vol 70 (10) ◽

pp. 2396-2401 ◽

Cited By ~ 9

Author(s):

CARISA R. DAVIS ◽

DAVID L. WINGFIELD ◽

K. KEALY PEAK ◽

WILLIAM VEGUILLA ◽

PHILIP T. AMUSO ◽

...

Keyword(s):

Multilocus Sequence Typing ◽

Vibrio Parahaemolyticus ◽

The United States ◽

Pcr Markers ◽

Specific Pcr ◽

Foodborne Outbreaks ◽

Pcr Ribotyping ◽

Typing Methods ◽

Pandemic Strains ◽

Novel Alleles

Vibrio parahaemolyticus is the leading cause of bacterial seafood-based illness in the United States. Real-time PCR, pandemic group–specific PCR, ribotyping, and multilocus sequence typing were used to characterize 30 strains of V. parahaemolyticus including 11 strains associated with foodborne outbreaks in Florida and 6 known pandemic strains. Thirteen strains were positive for four pandemic group–specific PCR markers, including 5 strains associated with outbreaks in Florida. Molecular typing methods were used to further define the pandemic status of these strains because the current PCR markers are not sufficient to identify pandemic isolates. Nine of the Florida strains clustered with a majority of the known pandemic strains, based on ribotyping patterns using PvuII, but no isolated pandemic branch was formed. Using multilocus sequence typing, it was determined that 14 strains possess a previously determined pandemic sequence type. This study identified 13 novel sequence types and seven to nine novel alleles for each locus. Furthermore, the results indicate that seven of the strains from recent foodborne outbreaks in Florida are pandemic strains, and that multilocus sequence typing was the most accurate molecular method to identify these strains.

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Pandemic Spread of an O3:K6 Clone of Vibrio parahaemolyticus and Emergence of Related Strains Evidenced by Arbitrarily Primed PCR and toxRS Sequence Analyses

Journal of Clinical Microbiology ◽

10.1128/jcm.38.2.578-585.2000 ◽

2000 ◽

Vol 38 (2) ◽

pp. 578-585 ◽

Cited By ~ 245

Author(s):

Chiho Matsumoto ◽

Jun Okuda ◽

Masanori Ishibashi ◽

Masaaki Iwanaga ◽

Pallavi Garg ◽

...

Keyword(s):

Phylogenetic Analysis ◽

Vibrio Parahaemolyticus ◽

The United States ◽

Epidemiological Investigation ◽

Sequence Analyses ◽

History Of ◽

Pcr Method ◽

Arbitrarily Primed Pcr ◽

Positive Results ◽

Unique Clone

Vibrio parahaemolyticus O3:K6 strains responsible for the increase in the number of cases of diarrhea in Calcutta, India, beginning in February 1996 and those isolated from Southeast Asian travelers beginning in 1995 were shown to belong to a unique clone characterized by possession of the tdh gene but not thetrh gene and by unique arbitrarily primed PCR (AP-PCR) profiles (J. Okuda, M. Ishibashi, E. Hayakawa, T. Nishino, Y. Takeda, A. K. Mukhopadhyay, S. Garg, S. K. Bhattacharya, G. B. Nair, and M. Nishibuchi, J. Clin. Microbiol. 35:3150–3155, 1997). Evidence supporting a hypothesis that this clone emerged only recently and is spreading to many countries was obtained in this study. Of 227 strains isolated in a hospital in Bangladesh between 1977 and 1998, only 22 strains isolated between 1996 and 1998 belonged to the new O3:K6 clone (defined by the serovar, the tdh andtrh typing, and AP-PCR profiles). The O3:K6 strains isolated from clinical sources in Taiwan, Laos, Japan, Thailand, Korea, and the United States between 1997 and 1998 were also shown to belong to the new O3:K6 clone. The clonality of the new O3:K6 strains was also confirmed by analysis of the toxRS sequence, which has been shown to be useful for phylogenetic analysis of the members of the genus Vibrio. The toxRS sequences of the representative strains of the new O3:K6 clone differed from those of the O3:K6 strains isolated before 1995 at least at 7 base positions within a 1,346-bp region. A new PCR method targeted to 2 of the base positions unique to the new O3:K6 clone was developed. This PCR method could clearly differentiate all 172 strains belonging to the new O3:K6 clone from other O3:K6 strains isolated earlier. One hundred sixty-six strains belonging to 28 serovars other than O3:K6 were also examined by the new PCR method. The tdh-positive andtrh-lacking strains that belonged to the O4:K68 and O1:K untypeable serovars and were isolated in three countries and from international travelers beginning in 1997 gave positive results. The AP-PCR profiles of these strains were nearly identical to those of the new O3:K6 clone, and their toxRS sequences were 100% identical to that of the new O3:K6 clone. The results suggest that these strains may have diverged from the new O3:K6 clone by alteration of the O:K antigens. In conclusion, this study presents strong evidence for the first pandemicity in the history of V. parahaemolyticus and reports a novel toxRS-targeted PCR method that will be useful in epidemiological investigation of the cases associated with the current pandemic spread.

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Real-Time PCR Assay for Detection and Enumeration of Dekkera bruxellensis in Wine

Applied and Environmental Microbiology ◽

10.1128/aem.69.12.7430-7434.2003 ◽

2003 ◽

Vol 69 (12) ◽

pp. 7430-7434 ◽

Cited By ~ 77

Author(s):

Trevor G. Phister ◽

David A. Mills

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Pcr Primers ◽

Rrna Gene ◽

Dekkera Bruxellensis ◽

Pcr Assay ◽

Specific Pcr ◽

Spoilage Yeast ◽

Pcr Method ◽

26S Rrna

ABSTRACT Traditional methods to detect the spoilage yeast Dekkera bruxellensis from wine involve lengthy enrichments. To overcome this difficulty, we developed a quantitative real-time PCR method to directly detect and enumerate D. bruxellensis in wine. Specific PCR primers to D. bruxellensis were designed to the 26S rRNA gene, and nontarget yeast and bacteria common to the winery environment were not amplified. The assay was linear over a range of cell concentrations (6 log units) and could detect as little as 1 cell per ml in wine. The addition of large amounts of nontarget yeasts did not impact the efficiency of the assay. This method will be helpful to identify possible routes of D. bruxellensis infection in winery environments. Moreover, the time involved in performing the assay (3 h) should enable winemakers to more quickly make wine processing decisions in order to reduce the threat of spoilage by D. bruxellensis.

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Prevalence and Serotype Determination of Streptococcus agalactiae Isolated From Non-Pregnant Women in Tehran, Iran

ACTA MEDICA IRANICA ◽

10.18502/acta.v57i9.2641 ◽

2020 ◽

Author(s):

Leila Goudarzi ◽

Mohammad Bagher Khalili ◽

Mahmood Vakili ◽

Maryam Sadeh

Keyword(s):

Pregnant Women ◽

Streptococcus Agalactiae ◽

Group B Streptococcus ◽

Cross Sectional Study ◽

Chi Square ◽

Cross Sectional ◽

Specific Pcr ◽

Pcr Method ◽

Group B ◽

Tehran Area

Consequence of Streptococcus agalactiae, Group B Streptococcus (GBS) relating infant’s diseases are well documented. Although many women carry this bacterium in their vagina, they may transfer to their infant during delivery and may result in different neonatal invasive diseases. The aim of this study was to determine the prevalence of GBS and serotyping the isolated species among un-selective non-pregnant women who attended two gynecology clinics in Tehran. In this cross-sectional study, a total of 560 vaginal samples collected from non-pregnant women. Following inoculation of the specimen on Blood Agar, the standard technology was applied for the final identification of GBS. Detected GBS species were further confirmed using specific PCR directed on dlts gene. Capsular serotyping was done by using the multiplex PCR method. The chi-square method was used for statistical analysis. Fifty (8.9%) out of 560 non-pregnant women were carriers of GBS. The most common types were III (36%), followed by type II (32%), Ia (26%), and Ib (6%), respectively. Results represent that the prevalence rate of GBS in non-pregnant women was reliable and similar to what obtained from pregnant women. In addition, the serotype III was found the most dominant types, as well as other investigations in the Tehran area. Therefore, vaccine designation based on type III is recommended.

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