Orasa Suthienkul

Evidence for genetic linkage between the ure and trh genes in Vibrio parahaemolyticus.

Although V. parahaemolyticus does not generally produce urease, several studies have reported urease-positive V. parahaemolyticus isolates from clinical sources. Recently, studies have shown a complete coincidence between the urease-producing phenotype of V. parahaemolyticus strains and the possession of the thermostable direct haemolysin (TDH)-related haemolysin (TRH) gene (trh). TRH, like TDH, is considered to be an important virulence factor in the pathogenesis of V. parahaemolyticus gastroenteritis. The present study attempted to identify the gene ure encoding urease in V. parahaemolyticus to clarify the relationship between urease production and possession of trh. The polymerase chain reaction with mixed oligonucleotide primers targeted for conserved sequences of reported ure genes from other species was used to prepare a DNA probe to detect the V. parahaemolyticus ure gene. Colony hybridisation with this ure probe demonstrated that all the ure-positive strains produced urease. Considering the coincidence between production of urease and possession of trh in V. parahaemolyticus, it was concluded that the presence or absence of the ure gene is completely coincident with that of the trh gene in V. parahaemolyticus strains. Furthermore, the relative location of ure and trh on V. parahaemolyticus chromosomal DNA was analysed by pulsed-field gel electrophoresis. The results showed that, in all the strains examined, ure and trh were detected on the same NotI fragment, showing that the two genes localise within a relatively small portion of the chromosome DNA. These results suggest that the ure and trh genes are genetically linked in V. parahaemolyticus strains.

Unstable Lysogeny and Pseudolysogeny in Vibrio harveyi Siphovirus-Like Phage 1†

ABSTRACT Exposure of Vibrio harveyi (strain VH1114) to V. harveyi siphovirus-like phage 1 (VHS1) resulted in the production of a low percentage of lysogenized clones of variable stability. These were retrieved most easily as small colonies within dot plaques. Analysis revealed that VHS1 prophage was most likely carried by VH1114 as an episome rather than integrated into the host chromosome. In the late exponential growth phase, lysogenized VH1114 continuously produced VHS1 but also gave rise to a large number of cured progeny. The absence of phage DNA in the cured progeny was confirmed by the absence of VHS1 DNA in Southern blot and PCR assays. Curiously, these very stable, cured subclones did not show the parental phenotype of clear plaques with VHS1 but instead showed turbid plaques, both in overlaid lawns and in dot plaque assays. This phenotypic difference from the original parental isolate suggested that transient lysogeny by VHS1 had resulted in a stable genetic change in the cured clones. Such clones may be called pseudolysogens (i.e., false lysogens), since they have undergone transient lysogeny and have retained some resistance to full lytic phage development, despite the loss of viable or detectable prophage.

Genetic Relationships of Vibrio parahaemolyticus Isolates from Clinical, Human Carrier, and Environmental Sources in Thailand, Determined by Multilocus Sequence Analysis

ABSTRACT Vibrio parahaemolyticus is a seafood-borne pathogenic bacterium that is a major cause of gastroenteritis worldwide. We investigated the genetic and evolutionary relationships of 101 V. parahaemolyticus isolates originating from clinical, human carrier, and various environmental and seafood production sources in Thailand using multilocus sequence analysis. The isolates were recovered from clinical samples (n = 15), healthy human carriers (n = 18), various types of fresh seafood (n = 18), frozen shrimp (n = 16), fresh-farmed shrimp tissue (n = 18), and shrimp farm water (n = 16). Phylogenetic analysis revealed a high degree of genetic diversity within the V. parahaemolyticus population, although isolates recovered from clinical samples and from farmed shrimp and water samples represented distinct clusters. The tight clustering of the clinical isolates suggests that disease-causing isolates are not a random sample of the environmental reservoir, although the source of infection remains unclear. Extensive serotypic diversity occurred among isolates representing the same sequence types and recovered from the same source at the same time. These findings suggest that the O- and K-antigen-encoding loci are subject to exceptionally high rates of recombination. There was also strong evidence of interspecies horizontal gene transfer and intragenic recombination involving the recA locus in a large proportion of isolates. As the majority of the intragenic recombinational exchanges involving recA occurred among clinical and carrier isolates, it is possible that the human intestinal tract serves as a potential reservoir of donor and recipient strains that is promoting horizontal DNA transfer, driving evolutionary change, and leading to the emergence of new, potentially pathogenic strains.

Antimicrobial Resistance, Extended-Spectrum β-Lactamase Productivity, and Class 1 Integrons in Escherichia coli from Healthy Swine

Administration of antimicrobials to food-producing animals increases the risk of higher antimicrobial resistance in the normal intestinal flora of these animals. The present cross-sectional study was conducted to investigate antimicrobial susceptibility and extended-spectrum β-lactamase (ESBL)-producing strains and to characterize class 1 integrons in Escherichia coli in healthy swine in Thailand. All 122 of the tested isolates had drug-resistant phenotypes. High resistance was found to ampicillin (98.4% of isolates), chloramphenicol (95.9%), gentamicin (78.7%), streptomycin (77.9%), tetracycline (74.6%), and cefotaxime (72.1%). Fifty-four (44.3%) of the E. coli isolates were confirmed as ESBL-producing strains. Among them, blaCTX-M (45 isolates) and blaTEM (41 isolates) were detected. Of the blaCTX-M-positive E. coli isolates, 37 carried the blaCTX-M-1 cluster, 12 carried the blaCTX-M-9 cluster, and 5 carried both clusters. Sequence analysis revealed blaTEM-1, blaTEM-135, and blaTEM-175 in 38, 2, and 1 isolate, respectively. Eighty-seven (71%) of the 122isolates carried class 1 integrons, and eight distinct drug-resistance gene cassettes with seven different integron profiles were identified in 43 of these isolates. Gene cassettes were associated with resistance to aminoglycosides (aadA1, aadA2, aadA22, or aadA23), trimethoprim (dfrA5, dfrA12, or dfrA17), and lincosamide (linF). Genes encoding β-lactamases were not found in class 1 integrons. This study is the first to report ESBL-producing E. coli with a class 1 integron carrying the linF gene cassette in swine in Thailand. Our findings confirm that swine can be a reservoir of ESBL-producing E. coli harboring class 1 integrons, which may become a potential health risk if these integrons are transmitted to humans. Intensive analyses of animal, human, and environmental isolates are needed to control the spread of ESBL-producing E. coli strains.

Genetic diversity of Vibrio parahaemolyticus strains isolated from farmed Pacific white shrimp and ambient pond water affected by acute hepatopancreatic necrosis disease outbreak in Thailand

Acute hepatopancreatic necrosis disease (AHPND) is an emerging shrimp disease that causes massive die-offs in farmed shrimps. Recent outbreaks of AHPND in Asia have been causing great losses for shrimp culture and have become a serious socioeconomic problem. The causative agent of AHPND is Vibrio parahaemolyticus, which is typically known to cause food-borne gastroenteritis in humans. However, there have been few reports of the epidemiology of V. parahaemolyticus AHPND strains, and the genetic relationship among AHPND strains is unclear. Here, we report the genetic characterization of V. parahaemolyticus strains isolated from AHPND outbreaks in Thailand. We found eight isolates from AHPND-suspected shrimps and pond water that were positive for AHPND markers AP1 and AP2. PCR analysis confirmed that none of these eight AP-positive AHPND strains possesses the genes for the conventional virulence factors affecting to humans, such as thermostable direct hemolysin (TDH), TDH-related hemolysin (TRH) and type III secretion system 2. Phylogenetic analysis by multilocus sequence typing showed that the AHPND strains are genetically diverse, suggesting that AHPND strains were not derived from a single genetic lineage. Our study represents the first report of molecular epidemiology of AHPND-causing V. parahaemolyticus strains using multilocus sequence typing, and provides an insight into their evolutionary mechanisms.

Possible horizontal transmission of Salmonella via reusable egg trays in Thailand

Salmonella contamination of eggshells, egg contents, reusable egg trays, and various environmental samples was assessed. Although the overall Salmonella contamination rate from egg farms was low (3.2%), over a quarter (26.7%) of egg trays from farms and more than one third (36.7%) of trays from the market were contaminated. Salmonella strains isolated from reusable egg trays were analyzed by serotyping, antimicrobial susceptibility test and XbaI pulsed-field gel electrophoresis (PFGE) typing. Five serovars (S. Braenderup, S. Emek, S. Weltevreden, S. Stanley, and S. Derby) were isolated, and half of the strains assessed were found to be resistant to one or more of the six antimicrobial agents examined. The overall resistance rates to nalidixic acid, trimethoprim-sulfamethoxazole, tetracycline, and ampicillin were 40.7%, 36.0%, 26.7% and 3.5%, respectively. The PFGE types were matched against sample location and drug resistance. S. Braenderup PFGE type A2 (susceptible to all tested drugs) was isolated from all sample sites; PFGE type A2 (resistant to nalidixic acid) was isolated from Farm C and the market. S. Braenderup PFGE type A1 (resistant to four drugs) was isolated from Farms A and C. S. Weltevreden PFGE type C3 (susceptible to all tested drugs) was isolated from Farms A and B and type C4 (susceptible to all tested drugs) was isolated from Farm A and the market. The distribution of the related genotypes and resistance patterns of Salmonella in egg farms and the market indicate drug-resistant strains of Salmonella may be spread on reusable egg trays.

Plasmid dynamics in Vibrio parahaemolyticus strains related to shrimp Acute Hepatopancreatic Necrosis Syndrome (AHPNS)

Vibrio parahaemolyticus is a causative agent of acute hapatopancreatic necrosis syndrome (AHPNS) which causes early mortality in white shrimp. Emergence of AHPNS has caused tremendous economic loss for aquaculture industry particularly in Asia since 2010. Previous studies reported that strains causing AHPNS harbor a 69-kb plasmid with possession of virulence genes, pirA and pirB. However, genetic variation of the 69-kb plasmid among AHPNS related strains has not been investigated. This study aimed to analyze genetic composition and diversity of the 69-kb plasmid in strains isolated from shrimps affected by AHPNS. Plasmids recovered from V. parahaemolyticus strain VPE61 which represented typical AHPNS pathogenicity, strain VP2HP which did not represent AHPNS pathogenicity but was isolated from AHPNS affected shrimp and other AHPNS V. parahaemolyticus isolates in Genbank were investigated. Protein coding genes of the 69-kb plasmid from the strain VPE61 were identical to that of AHPNS strain from Vietnam except the inverted complement 3.4-kb transposon covering pirA and pirB. The strain VP2HP possessed remarkable large 183-kb plasmid which shared similar protein coding genes to those of the 69-kb plasmid from strain VPE61. However, the 3.4-kb transposon covering pirA and pirB was absent from the 183-kb plasmid in strain VP2HP. A number of protein coding genes from the 183-kb plasmid were also detected in other AHPNS strains. In summary, this study identified a novel 183-kb plasmid that is related to AHPNS causing strains. Homologous recombination of the 69-kb AHPNS plasmid and other naturally occurring plasmids together with loss and gain of AHPNS virulence genes in V. parahaemolyticus were observed. The outcome of this research enables understanding of plasmid dynamics that possibly affect variable degrees of AHPNS pathogenicity.

Characterization of antibiotic resistance in Escherichia coli isolated from shrimps and their environment.

Antimicrobial resistance in bacteria associated with food and water is a global concern. To survey the risk, 312 Escherichia coli isolates from shrimp farms and markets in Thailand were examined for susceptibility to 10 antimicrobials. The results showed that 17.6% of isolates (55 of 312) were resistant to at least one of the tested drugs, and high resistance rates were observed to tetracycline (14.4%; 45 of 312), ampicillin (8.0%; 25 of 312), and trimethroprim (6.7%; 21 of 312); 29.1% (16 of 55) were multidrug resistant. PCR assay of the tet (A), tet (B), tet (C), tet (D), tet (E), and tet (G) genes detected one or more of these genes in 47 of the 55 resistant isolates. Among these genes, tet (A) (69.1%; 38 of 55) was the most common followed by tet (B) (56.4%; 31 of 55) and tet (C) (3.6%; 2 of 55). The resistant isolates were further investigated for class 1 integrons. Of the 55 resistant isolates, 16 carried class 1 integrons and 7 carried gene cassettes encoding trimethoprim resistance (dfrA12 or dfrA17) and aminoglycosides resistance (aadA2 or aadA5). Two class 1 integrons, In54 (dfrA17-aadA5) and In27 (dfrA12-orfF-aadA2), were found in four and three isolates, respectively. These results indicate a risk of drug-resistant E. coli contamination in shrimp farms and selling places. The occurrence of multidrug-resistant E. coli carrying tet genes and class 1 integrons indicates an urgent need to monitor the emergence of drug-resistant E. coli to control the dissemination of drug-resistant strains and the further spread of resistance genes to other pathogenic bacteria.

Enhancement of Legionella pneumophila Culture Isolation from Microenvironments by Macrophage Infectivity Potentiator (mip) Gene‐Specific Nested Polymerase Chain Reaction

The combination of a Legionella pneumophila culture isolation technique and macrophage infectivity potentiator (mip) gene‐specific nested polymerase chain reaction (PCR) is pivotal for effective routine use in an environmental water system laboratory. Detection of Legionella organisms in 169 environmental samples was performed by using modified buffered charcoal yeast extract (MBCYE) agar for conventional culture. Nested PCR specific for L. pneumophila was performed using boiled genomic DNA extracts from filtered and Chelex 100‐treated water samples, or by using silica‐gel membrane spin column‐eluted DNA from concentrated pond, canal and river samples. Overall, the nested PCR was twelvefold more sensitive than the culture method. The target amplicons (471 basepairs) of all 4 biochemically characterized L. pneumophila isolates were sequenced. They had homology at the DNA and protein levels to 3′ proximity of the mip‐coding gene of L. pneumophila deposited in genome databases. EcoRI‐ or KpnI‐digested PCR fragments with expected sizes were also confirmed in all 52 PCR‐positive samples that were isolated from cooling towers and condenser drains. Viable but nonculturable L. pneumophila might have been present in 48 PCR‐positive samples. This study demonstrates that detection of the genetically stable mip gene by nested PCR with a modified process of water sample preparation can be rapidly and effectively used to enhance isolation of the L. pneumophila taxon from microenvironments.

Multidrug resistance in Clostridium perfringens isolated from diarrheal neonatal piglets in Thailand.

Clostridium perfringens causes diarrhea in neonatal piglets, thereby affecting commercial swine farming. The objective of this study was to determine the prevalence and characterize antimicrobial resistance in C. perfringens isolated from diarrheal neonatal piglets in Thailand. A total of 260 rectal swab samples were collected from 13 farms and were subjected to C. perfringens isolation. A total of 148 samples were PCR-positive for C. perfringens toxin genes, from which 122 were recovered. All isolates were cpb2-encoding C. perfringens type A and enterotoxin gene negative. Most of the isolates were susceptible to ampicillin, bacitracin, chlorotetracycline, doxycycline, and oxytetracycline with MIC50 values ranging from 0.32 to 8 μg/ml. The high resistance rates were observed for ceftiofur, enrofloxacin, erythromycin, lincomycin, and tylosin. Among resistant isolates, 82% were resistant to more than one type of antibiotics. The distinct pattern of multiple drug resistance in C. perfringens was observed in different regions, potentially reflecting the farm specific usage of these agents.