Characterization of Vibrio parahaemolyticus isolated from clinical specimens and oysters in Thailand.

INTRODUCTION: Vibrio parahaemolyticus is a common pathogen that can cause seafood-borne gastroenteritis in humans. We determined the prevalence and characteristics of V. parahaemolyticus isolated from clinical specimens and oysters in Thailand. METHODOLOGY: Isolates of V. parahaemolyticus from clinical specimens (n = 77) and oysters (n = 224) were identified by biochemical testing, polymerase chain reaction (PCR) assays, and serotyping. The toxin genes, antimicrobial resistance, and ß-lactamase production were determined. RESULTS: A total of 301 isolates were confirmed as V. parahaemolyticus by PCR using specific primers for the toxR gene. The majority of clinical isolates carried the tdh+/trh- genotype (82.1%), and one of each isolate was tdh-/trh+ and tdh+/trh+ genotypes. One isolate from oyster contained the tdh gene and another had the trh gene. Twenty-six serotypes were characterized among these isolates, and O3:K6 was the most common (37.7%), followed by OUT:KUT, and O4:K9. In 2010, most clinical and oyster isolates were susceptible to antibiotics, with the exception of ampicillin. In 2012, clinical isolates were not susceptible to cephalothin (52.4%), streptomycin (95.2%), amikacin (66.6%), kanamycin (61.9%), and erythromycin (95.2%), significantly more frequently than in 2010. More than 95% of isolates that were not susceptible to ampicillin produced ß-lactamase enzymes. CONCLUSIONS: We found toxin genes in two oyster isolates, and the clinical isolates that were initially determined to be resistant to several antibiotics. Toxin genes and antimicrobial susceptibility profiles of V. parahaemolyticus from seafood and environment should be continually monitored to determine the spread of toxin and antimicrobial resistance genes.

Prevalence, virulence characteristics, and antimicrobial resistance of Vibrio parahaemolyticus isolates from raw seafood in a province in Northern Thailand.

Vibrio parahaemolyticus (V. parahaemolyticus) is commonly found in seawater and seafood products, but evidence is limited of its presence in seafood marketed in locations very distant from coastal sources. This study determined the prevalence and characterization of V. parahaemolyticus in seafood from markets in landlocked Phayao province, Northern Thailand. Among 120 samples, 26 (21.7%) were positive for V. parahaemolyticus, being highest in shrimp (43.3%), followed by shellfish (36.7%), and squid (6.7%), but was not found in fish. V. parahaemolyticus comprised 33 isolates that were non-pathogenic and non-pandemic. Almost all isolates from shrimp and shellfish samples were positive for T3SS1. Only five isolates (15.2%) showed two antimicrobial resistance patterns, namely, kanamycin-streptomycin (1) carrying sul2 and ampicillin-kanamycin-streptomycin (4) that carried tetA (2), tetA-sul2 (1), as well as one negative. Antimicrobial susceptible V. parahaemolyticus isolates possessing tetA (67.9%) and sul2 (3.5%) were also found. Six isolates positive for integron class 1 and/or class 2 were detected in 4 antimicrobial susceptible and 2 resistant isolates. While pathogenic V. parahaemolyticus was not detected, contamination of antimicrobial resistance V. parahaemolyticus in seafood in locations distant from coastal areas requires ongoing monitoring to improve food safety in the seafood supply chain.

Prevalence, antimicrobial resistance, and enterotoxin gene profiles of Staphylococcus aureus isolated from mobile phones of the food vendors in Phayao province, Thailand.

BACKGROUND: Mobile phones are widely used and may cause bacterial pathogens to spread among various professionals. Staphylococcus aureus from the mobile phones can contaminate the hands of food vendors and food during the cooking or packaging process. This research aimed to determine the prevalence, enterotoxin genes, and antimicrobial resistance (AMR) profiles of S. aureus contaminating the vendors' mobile phones. METHODS: In this study, 266 mobile phone samples were randomly collected from food vendors selling food on walking streets (n = 139) and in food centers (n = 127) in Phayao province. All samples were identified as S. aureus by the conventional culture method and confirmed species-specific gene by polymerase chain reaction (PCR). Then, all identified S. aureus isolates were tested for antimicrobial susceptibility by broth microdilution method and for the presence of staphylococcal enterotoxin (SE) genes by PCR. RESULTS: The results showed that 12.8% of the mobile phones collected were contaminated with S. aureus. Of 49 S. aureus isolates obtained, 30 (61.2%) were positive for SE genes. The most common SE gene was sea followed by sec, seb, sem, seq, and sel. Moreover, S. aureus was most frequently resistant to penicillin, followed by chloramphenicol and tetracycline, erythromycin, clindamycin, and gentamicin. Methicillin-resistant S. aureus (MRSA), vancomycin-resistant S. aureus (VRSA), and multidrug-resistant (MDR) strains were also detected. CONCLUSIONS: This study showed that mobile phones were an intermediate surface for the transmission of S. aureus, including MDR variants. It indicates that hand hygiene and the decontamination of mobile phones are essential to prevent cross-contamination of S. aureus in food settings.

First study on virulence genes, antimicrobial resistance, and integrons in Escherichia coli isolated from cage, free-range, and organic commercial eggs in Phayao Province, Thailand.

Background and Aim: Antimicrobial resistance (AMR) is a global problem that affects human and animal health, and eggs can act as a vehicle for pathogenic and non-pathogenic resistant bacteria in the food chain. Escherichia coli is an indicator of food contamination with fecal materials as well as the occurrence and levels of AMR. This study aimed to investigate the presence of AMR, integrons, and virulence genes in E. coli isolated from eggshell samples of three egg production systems, from supermarkets in Thailand. Materials and Methods: A total of 750 hen's egg samples were purchased from supermarkets in Phayao Province: Cage eggs (250), free-range eggs (250), and organic eggs (250). Each sample was soaked in buffered peptone water (BPW), and the BPW samples were incubated at 37°C for 18-24 h. All samples were tested for E. coli by the standard conventional culture method. Then, all identified E. coli were tested for antimicrobial susceptibility to 15 antimicrobial agents by the agar disk diffusion method. All E. coli strains were subsequently found to have virulence genes and Classes 1 and 2 integrons by polymerase chain reaction. Results: Among the eggshell samples, 91 samples were identified as having E. coli (cage eggs, 24 strains; free-range eggs, 27 strains; and organic eggs, 40 strains). Then, among the E. coli strains, 47 (51.6%) were positive for at least one virulence gene. The proportion of AMR in the eggshell samples was 91.2% (83/91), and streptomycin (STR), ampicillin (AMP), and tetracycline (TET) had a high degree of resistance. Among the E. coli strains, 27 (29.7%) strains were positive for class 1 or 2 integrons, and integron-positive strains were commonly found in STR-, AMP-, and TET-resistant strains. Multidrug resistance (MDR) was detected in 57.1% (52/91) of the E. coli strains, with STR-AMP-TET (5.5%) as the most frequent pattern. The proportion of MDR in cage eggs was 75.0% (18/24), which was higher than in both free-range and organic eggs. On the other hand, 53.2% (25/47) of E. coli carrying virulence genes had MDR, distributed across the production systems as follows: Cage eggs, 76.9% (10/13); free-range eggs, 63.6% (7/11); and organic eggs, 34.8% (8/23). Conclusion: Escherichia coli was detected in eggshell samples from all three egg production systems. The high level of virulence genes, AMR, and integrons indicated the possibility of dissemination of AMR among pathogenic and commensal E. coli through eggshells. These findings could be a major concern to farmers, food handlers, and consumers, especially regarding raw egg consumption.

Multilocus Sequence Typing and Virulence Potential of Vibrio parahaemolyticus Strains Isolated from Aquatic Bird Feces.

Vibrio parahaemolyticus is a Gram-negative, foodborne pathogenic bacterium that causes human gastroenteritis. This organism is ubiquitously present in the marine environment. Detection of V. parahaemolyticus in aquatic birds has been previously reported; however, the characterization of isolates of this bacterium recovered from these birds remains limited. The present study isolated and characterized V. parahaemolyticus from aquatic bird feces at the Bangpu Recreation Center (Samut Prakan province, Thailand) from 2016 to 2017, using multilocus sequence typing (MLST) and genome analysis. The results showed that V. parahaemolyticus was present in 34.9% (76/218) of the collected bird fecal samples. Among the ldh-positive V. parahaemolyticus isolates (n = 308), 1% (3/308) were positive for tdh, 1.3% (4/308) were positive for trh, and 0.3% (1/308) were positive for both tdh and trh. In turn, the MLST analysis revealed that 49 selected V. parahaemolyticus isolates resolved to 36 STs, 26 of which were novel (72.2%). Moreover, a total of 10 identified STs were identical to globally reported pathogenic strains (ST1309, ST1919, ST491, ST799, and ST2516) and environmental strains (ST1879, ST985, ST288, ST1925, and ST260). The genome analysis of isolates possessing tdh and/or trh (ST985, ST1923, ST1924, ST1929 and ST2516) demonstrated that the organization of the T3SS2α and T3SS2ß genes in bird fecal isolates were almost identical to those of human clinical strains posing public health concerns of pathogen dissemination in the recreational area. The results of this study suggest that aquatic birds are natural reservoirs of new strains with high genetic diversity and are alternative sources of potentially pathogenic V. parahaemolyticus in the marine environment. IMPORTANCE To our knowledge, infection of foodborne bacterium V. parahamolyticus occurs via the consumption of undercooked seafood contaminated with pathogenic strains. Aquatic bird is a neglectable source that can transmit V. parahaemolyticus along coastal areas. This study reported the detection of potentially pathogenic V. parahamolyticus harboring virulence genes from aquatic bird feces at the recreational center situated near the Gulf of Thailand. These strains shared identical genetic profile to the clinical isolates that previously reported in many countries. Furthermore, the strains from aquatic birds showed extremely high genetic diversity. Our research pointed out that the aquatic bird is possibly involved in the evolution of novel strains of V. parahaemolyticus and play a role in dissimilation of the potentially pathogenic strains across geographical distance.

Prevalence and Multidrug Resistance of Salmonella in Swine Production Chain in a Central Province, Thailand.

ABSTRACT: Salmonella causes foodborne disease outbreaks worldwide and raises concerns about public health and economic losses. To determine prevalence, serovar, antimicrobial resistance patterns, and the presence of extended-spectrum ß-lactamase (ESBL) genes in a cross-sectional study, 418 total samples from feces and carcasses (from three slaughterhouses) and pork and cutting boards (from four markets) were collected in a central Thailand province in 2017 and 2018. Of the 418 samples, 272 (65.1%) were positive for Salmonella. The prevalence of Salmonella-positive samples from markets (158 of 178; 88.8%) was significantly higher than that among samples from slaughterhouses (114 of 240; 47.5%) (P < 0.05). A total of 1,030 isolates were identified; 409 were assigned to 45 serovars, with Salmonella Rissen the most common (82 of 409; 20%). Two serovars, Salmonella Cannstatt and Salmonella Braubach, were identified for the first time in Thailand in market and slaughterhouse samples, respectively. Among 180 isolates representing 19 serovars, 133 (73.9%) exhibited multidrug resistance. Screening for ESBL production revealed that 41 (10.3%) of 399 isolates were ESBL positive. The prevalence of ESBL-producing Salmonella isolates was significantly higher among the market isolates (31 of 41; 75.6%) than among the slaughterhouse isolates in (10 of 41; 24.4%) (P < 0.05). In market samples, 24 (77.4%) of 31 isolates were recovered from pork and 7 (22.6%) were recovered from cutting boards. Nine ESBL-producing isolates carried single ESBL genes, either blaTEM (4 of 41 isolates; 9.8%) or blaCTX-M (5 of 41 isolates; 12.2%), whereas 11 (26.8%) carried both blaTEM and blaCTX-M. No ESBL-producing Salmonella isolate carried the blaSHV gene. These results suggest that pigs, their flesh, and cutting boards used for processing pork could be reservoirs for widespread ESBL-producing Salmonella isolates with multidrug resistance and outbreak potential across the food chain.

Effectiveness of Fluoroquinolones with Difluoropyridine Derivatives as R1 Groups on the Salmonella DNA Gyrase in the Presence and Absence of Plasmid-Encoded Quinolone Resistance Protein QnrB19.

Aims: WQ-3810 has strong inhibitory activity against Salmonella and other fluoroquinolone-resistant pathogens. The unique potentiality of this is attributed to 6-amino-3,5-difluoropyridine-2-yl at R1 group. The aim of this study was to examine WQ-3810 and its derivatives WQ-3334 and WQ-4065 as the new drug candidate for wild-type Salmonella and that carrying QnrB19. Materials and Methods: The half maximal inhibitory concentrations (IC50s) of WQ-3810, WQ-3334 (Br atom in place of methyl group at R8), and WQ-4065 (6-ethylamino-3,5-difluoropyridine-2-yl in place of 6-amino-3,5-difluoropyridine-2-yl group at R1) in the presence or absence of QnrB19 were assessed by in vitro DNA supercoiling assay utilizing recombinant DNA gyrase and QnrB19. Results: IC50s of WQ-3810, WQ-3334, and WQ-4065 against Salmonella DNA gyrase were 0.031 ± 0.003, 0.068 ± 0.016, and 0.72 ± 0.39 µg/mL, respectively, while QnrB19 increased IC50s of WQ-3810, WQ-3334, and WQ-4065 to 0.44 ± 0.05, 0.92 ± 0.34, and 9.16 ± 2.21 µg/mL, respectively. Conclusion: WQ-3810 and WQ-3334 showed stronger inhibitory activity against Salmonella Typhimurium DNA gyrases than WQ-4065 even in the presence of QnrB19. The results suggest that 6-amino-3,5-difluoropyridine-2-yl group at R1 is playing an important role and WQ-3810 and WQ-3334 to be good candidates for Salmonella carrying QnrB19.

Antimicrobial Resistance and Molecular Characterization of Methicillin-Resistant Staphylococcus aureus Isolated from Slaughtered Pigs and Pork in the Central Region of Thailand.

Methicillin-resistant Staphylococcus aureus (MRSA) have been a major public health concern in humans. Among MRSA, livestock-associated (LA)-MRSA strains have always been associated with exposure to livestock or their products and have emerged in different countries globally. Although studies have identified LA-MRSA from healthy pigs and pork in Thailand, prevalence in slaughtered pigs is still unknown. In addition, there are few reports on the epidemiology and molecular characteristics of LA-MRSA in Thailand. Hence, this is the first report investigating the epidemiology and molecular characteristics of MRSA in individual slaughtered pigs and pork in Thailand. A total of 204 nasal swab and 116 retailed pork samples were collected from three slaughterhouses and four fresh markets, respectively. Individual samples were used for screening for MRSA and obtained isolates were examined for drug- resistance profiling for 12 antimicrobial agents of 10 drug classes. In addition, SCCmec typing and multi-locus sequence typing were conducted to obtain genotype profiles. MRSA were isolated from 11 and 52 nasal swab and pork samples, respectively. The prevalence was significantly higher in the pork than in the nasal swab samples (p-value < 0.05). A high prevalence of ST9-SCCmecIX and ST398-SCCmecV with high-level antimicrobial resistance from markets and slaughterhouses indicated the spreading of MRSA with these genotypes in the Thai swine processing chains and suggested the need for further investigation to determine a control.

Characterization of Arcobacter spp. Isolated from human diarrheal, non-diarrheal and food samples in Thailand.

Arcobacter butzleri is an emerging zoonotic food-borne and water-borne pathogen that can cause diarrhea in humans. The global prevalence of A. butzleri infection is underestimated, and little is known about their phenotypic and genotypic characterization. The aim of this study was to determine antimicrobial susceptibility (AST) profiles, detect related virulence genes, and classify sequence type (ST) of A. butzleri isolates obtained from human stool and food samples. A total of 84 A. butzleri isolates were obtained from human diarrheal (n = 25), non-diarrheal (n = 24) stool, and food (n = 35) samples in Thailand. They were evaluated for phenotypic identification by conventional microbiological procedures and AST by Kirby-Bauer disc diffusion method as well as virulence genes detection. Representative isolates from each origin were selected based on the presence of virulence genes and AST profiles to analyze genetic diversity by multilocus sequence typing (MLST). All isolates showed resistance to nalidixic acid 40.5% (34/84), ciprofloxacin 11.9% (10/84), azithromycin 8.3% (7/84), and erythromycin 3.6% (3/84). Regarding the ten virulence genes detected, cj1349, mviN and pldA had the highest prevalence 100% (84/84), followed by tlyA 98.8% (83/84), cadF 97.6% (82/84), ciaB 71.4% (60/84), hecA and hecB 22.6% (19/84), iroE 15.5% (13/84) and irgA 10.7% (9/84), respectively. Three virulence genes were present among A. butzleri isolates of human diarrheal stool and food samples, with a significant difference observed among isolates; hecB [36% (9/25) and 8.6% (3/35)], hecA [36% (9/25) and 5.7% (2/35)], and irgA [24% (6/25) and 2.9% (1/35)] (p < 0.05), respectively. The hecA and hecB virulence genes functions are related to the mechanism of hemolysis, while irgA supports a bacterial nutritional requirement. MLST analysis of 26 A. butzleri isolates revealed that 16 novel STs exhibited high genetic diversity. The results of this study is useful for understanding potentially pathogenic and antimicrobial-resistant A. butzleri in Thailand. The pathogenic virulence markers hecB, hecA, and irgA have the potential to be developed for rapid diagnostic detection in human diarrheal stool. No significant relationships among STs and sources of origin were observed. Little is known about A. butzleri, the mechanism of action of these virulence genes, is a topic that needs further investigation.

Characterization of Vibrio parahaemolyticus strains isolated from clinically asymptomatic seafood workers.

Vibrio parahaemolyticus (VP) is a major cause of gastroenteritis outbreaks in Thailand and other countries due to the consumption of contaminated and undercooked seafood. However, there have been few reports of the molecular epidemiology of VP isolates from asymptomatic seafood handlers. Here, we report the phenotypic and genetic characterization of 61 VP isolates obtained from asymptomatic workers in two seafood-processing plants. We found 24 O:K serotypes, of which O11:KUT, O1:KUT and O3:KUT were the dominant serotypes. Analysis by PCR showed that 12 isolates harbored either tdh or trh genes with the potential to be pathogenic VP strains. The presence of T3SS2α and T3SS2ß genes was correlated with the presence of tdh and trh, respectively. Four tdh+ isolates were positive for pandemic marker. In this study, VP isolates were commonly resistant to ampicillin, cephazolin, fosfomycin and novobiocin. Phylogenetic analysis of VP1680 loci in 35 isolates from 17 asymptomatic workers, 6 gastroenteritis patients, 7 environmental samples and 5 genomes from a database showed 22 different alleles. Gene VP1680 was conserved in tdh+ isolates and pandemic strains, while that of trh + isolates was diverse. Asymptomatic workers carrying VP were the most likely source of contamination, which raises concerns over food safety in seafood-processing plants.

Interaction of the plasmid-encoded quinolone resistance protein QnrB19 with Salmonella Typhimurium DNA gyrase.

BACKGROUND: Plasmid-encoded quinolone resistance protein Qnr is an important factor in bacterial resistance to quinolones. Qnr interacts with DNA gyrase and reduces susceptibility to quinolones. The gene qnr likely spreads rapidly among Enterobacteriaceae via horizontal gene transfer. Though the vast amounts of epidemiological data are available, molecular details of the contribution of QnrB19, the predominant Qnr in Salmonella spp., to the acquisition of quinolone resistance has not yet been understood well. OBJECTIVE: We aimed to examine the role of QnrB19 in quinolone resistance acquisition using recombinant Salmonella Typhimurium DNA gyrases and QnrB19. MATERIALS AND METHODS: Recombinant QnrB19 was expressed in E. coli and purified by Ni-NTA agarose column chromatography. DNA supercoiling activities of recombinant Salmonella Typhimurium DNA gyrase were assessed with or without QnrB19 under the existence of three quinolones to measure IC50s, the concentration of each quinolone required for 50% inhibition in vitro. RESULTS: The IC50s of norfloxacin, ciprofloxacin and nalidixic acid against DNA gyrases were measured to be 0.30, 0.16 and 17.7 µg/mL, respectively. The addition of QnrB19 increased the IC50s of norfloxacin and ciprofloxacin to be 0.81 and 0.48 µg/mL, respectively, where no effect of QnrB19 was observed on the IC50 of nalidixic acid. CONCLUSION: QnrB19 was shown for the first time in vitro to have ability to grant non-classical quinolone resistance to S. Typhimurium DNA gyrase. Structural insight on quinolones in this study may contribute to investigate drugs useful for preventing the spread of plasmid carrying PMQR along with other factors associating with antimicrobial resistance in S. Typhimurium and other bacteria.

Comparison of Database Search Methods for the Detection of Legionella pneumophila in Water Samples Using Metagenomic Analysis.

Metagenomic analysis has become a powerful tool to analyze bacterial communities in environmental samples. However, the detection of a specific bacterial species using metagenomic analysis remains difficult due to false positive detections of sequences shared between different bacterial species. In this study, 16S rRNA amplicon and shotgun metagenomic analyses were conducted on samples collected along a stream and ponds in the campus of Hokkaido University. We compared different database search methods for bacterial detection by focusing on Legionella pneumophila. In this study, we used L. pneumophila-specific nested PCR as a gold standard to evaluate the results of the metagenomic analysis. Comparison with the results from L. pneumophila-specific nested PCR indicated that a blastn search of shotgun reads against the NCBI-NT database led to false positive results and had problems with specificity. We also found that a blastn search of shotgun reads against a database of the catalase-peroxidase (katB) gene detected L. pneumophila with the highest area under the receiver operating characteristic curve among the tested search methods; indicating that a blastn search against the katB gene database had better diagnostic ability than searches against other databases. Our results suggest that sequence searches targeting long genes specifically associated with the bacterial species of interest is a prerequisite to detecting the bacterial species in environmental samples using metagenomic analyses.

Quinolone Resistance Determinants of Clinical Salmonella Enteritidis in Thailand.

Salmonella Enteritidis has emerged as a global concern regarding quinolone resistance and invasive potential. Although quinolone-resistant S. Enteritidis has been observed with high frequency in Thailand, information on the mechanism of resistance acquisition is limited. To elucidate the mechanism, a total of 158 clinical isolates of nalidixic acid (NAL)-resistant S. Enteritidis were collected throughout Thailand, and the quinolone resistance determinants were investigated in the context of resistance levels to NAL, norfloxacin (NOR), and ciprofloxacin (CIP). The analysis of point mutations in type II topoisomerase genes and the detection of plasmid-mediated quinolone resistance genes showed that all but two harbored a gyrA mutation, the qnrS1 gene, or both. The most commonly affected codon in mutant gyrA was 87, followed by 83. Double codon mutation in gyrA was found in an isolate with high-level resistance to NAL, NOR, and CIP. A new mutation causing serine to isoleucine substitution at codon 83 was identified in eight isolates. In addition to eighteen qnrS1-carrying isolates showing nontypical quinolone resistance, one carrying both the qnrS1 gene and a gyrA mutation also showed a high level of resistance. Genotyping by multilocus variable number of tandem repeat analysis suggested a possible clonal expansion of NAL-resistant strains nationwide. Our data suggested that NAL-resistant isolates with single quinolone resistance determinant may potentially become fluoroquinolone resistant by acquiring secondary determinants. Restricted therapeutic and farming usage of quinolones is strongly recommended to prevent the emergence of fluoroquinolone-resistant isolates.

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.

Impact of mutations in DNA gyrase genes on quinolone resistance in Campylobacter jejuni.

Amino acid substitutions providing quinolone resistance to Campyloabcter jejuni have been found in the quinolone resistance-determining region of protein DNA gyrase subunit A (GyrA), with the highest frequency at position 86 followed by position 90. In this study, wild-type and mutant recombinant DNA gyrase subunits were expressed in Escherichia coli and purified using Ni-NTA agarose column chromatography. Soluble 97 kDa GyrA and 87 kDa DNA gyrase subunit B were shown to reconstitute ATP-dependent DNA supercoiling activity. A quinolone-inhibited supercoiling assay demonstrated the roles of Thr86Ile, Thr86Ala, Thr86Lys, Asp90Asn, and Asp90Tyr amino acid substitutions in reducing sensitivity to quinolones. The marked effect of Thr86Ile on all examined quinolones suggested the advantage of this substitution in concordance with recurring isolation of quinolone-resistant C. jejuni. An analysis of the structure-activity relationship showed the importance of the substituent at position 8 in quinolones to overcome the effect of Thr86Ile. Sitafloxacin (SIT), which has a fluorinate cyclopropyl ring at R-1 and a chloride substituent at R-8, a characteristic not found in other quinolones, showed the highest inhibitory activity against all mutant C. jejuni gyrases including ciprofloxacin-resistant mutants. The results suggest SIT as a promising drug for the treatment of campylobacteriosis caused by CIP-resistant C. jejuni. Copyright © 2016 John Wiley & Sons, Ltd.

Genetic diversity and antimicrobial resistance pattern of Salmonella enterica serovar Enteritidis clinical isolates in Thailand.

OBJECTIVE: To trace the history of antimicrobial resistance in Salmonella enterica serovar Enteritidis (S. Enteritidis, SE) circulating in Thailand, we characterised clinical isolates obtained during 2004-2007. METHODS: Antimicrobial resistance profiles, multi-locus variable number tandem repeat analysis (MLVA) types and 3 representative virulence determinants (spvA, sodCI and sopE) were established from SE isolates (n = 192) collected from stool and blood of patients throughout Thailand during the period 2004-2007. RESULTS: Resistance was found in SE against 10 out of 11 antimicrobials studied. The highest resistance ratios were observed for nalidixic acid (83.2%), ciprofloxacin (51.1%) and ampicillin (50.5%), and 25.5% were multidrug resistant. Based on five polymorphic tandem repeat loci analysis, MLVA identified 20 distinct types with three closely related predominant types. A significant increase of AMP resistance from 2004 to 2006 was strongly correlated with that of a MLVA type, 5-5-11-7-3. CONCLUSION: The usage of antimicrobials in human medicine or farm settings might act as selective pressures and cause the spread of resistant strains. Hence, a strict policy on antimicrobial usage needs to be implemented to achieve the control of resistant SE in Thailand.

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.

Amino acid substitutions in GyrA affect quinolone susceptibility in Salmonella typhimurium.

The prevalence of quinolone-resistant Salmonella has become a public health concern. Amino acid substitutions have generally been found within the quinolone resistance-determining region in subunit A of DNA gyrase (GyrA) of Salmonella Typhimurium. However, direct evidence of the contribution of these substitutions to quinolone resistance remains to be shown. To investigate the significance of amino acid substitutions in S. Typhimurium GyrA to quinolone resistance, we expressed recombinant wild-type (WT) and five mutant DNA gyrases in Escherichia coli and characterized them in vitro. WT and mutant DNA gyrases were reconstituted in vitro by mixing recombinant subunits A and B of DNA gyrase. The correlation between the amino acid substitutions and resistance to quinolones ciprofloxacin, levofloxacin, nalidixic acid, and sitafloxacin was assessed by quinolone-inhibited supercoiling assays. All mutant DNA gyrases showed reduced susceptibility to all quinolones when compared with WT DNA gyrases. DNA gyrase with a double amino acid substitution in GyrA, serine to phenylalanine at codon 83 and aspartic acid to asparagine at 87 (GyrA-S83F-D87N), exhibited the lowest quinolone susceptibility amongst all mutant DNA gyrases. The effectiveness of sitafloxacin was shown by the low inhibitory concentration required for mutant DNA gyrases, including the DNA gyrase with GyrA-S83F-D87N. We suggest sitafloxacin as a candidate drug for the treatment of salmonellosis caused by ciprofloxacin-resistant S. Typhimurium. Copyright © 2015 John Wiley & Sons, Ltd.

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.