Sunpetch Angkititrakul

Serogroup, virulence, and molecular traits of Vibrio parahaemolyticus isolated from clinical and cockle sources in northeastern Thailand.

Vibrio parahaemolyticus is responsible for seafood-borne gastroenteritis worldwide. Isolates of V. parahaemolyticus from clinical samples (n=74) and cockles (Anadara granosa) (n=74) in Thailand were analyzed by serotyping, determination of virulence and related marker genes present, response to antimicrobial agents, and genetic relatedness. Serological analysis revealed 31 different serotypes, 10 of which occurred among both clinical and cockle samples. The clinical isolates commonly included the pandemic serogroup O3:K6, while a few of the cockle isolates exhibited likely pandemic serovariants such as O3:KUT and O4:KUT, but not O3:K6. The pandemic (orf8 gene-positive) strains were more frequently found among clinical isolates (78.4%) than cockle isolates (28.4%) (p<0.001). Likewise, the virulence and related marker genes were more commonly detected among clinical than cockle isolates; i.e., tdh gene (93.2% versus 29.7%), vcrD2 (97.3% versus 23.0%), vopB2 (89.2% versus 13.5%), vopT (98.6% versus 36.5%) (all p<0.001) and trh (10.8% versus 1.4%) (p<0.05). Pulsed-field gel electrophoresis of NotI-digested genomic DNA of 41 randomly selected V. parahaemolyticus isolates representing different serotypes produced 33 pulsotypes that formed 5 different clusters (clonal complexes) (A-E) in a dendrogram. Vibrio parahaemolyticus O3:K6 and likely related pandemic serotypes were especially common among the numerous clinical isolates in cluster C, suggesting a close clonal link among many of these isolates. Most clinical and cockle isolates were resistant to ampicillin. This study indicates that O3:K6 and its likely serovariants based on the PFGE clusters, are causative agents. Seafoods such as cockles potentially serve as a source of virulent V. parahaemolyticus, but further work is required to identify possible additional sources.

SCCmec IX in meticillin-resistant Staphylococcus aureus and meticillin-resistant coagulase-negative staphylococci from pigs and workers at pig farms in Khon Kaen, Thailand

Livestock-associated meticillin-resistant Staphylococcus aureus, clonal complex (CC) 398, has been reported in Europe, whereas CC9 MRSA has mostly been found in Asia. Therefore, we aimed to detect MRSA on pig farms in north-eastern Thailand. A total of 257 nasal swabs (159 samples from pigs and 98 from pig-farm workers) were collected from three pig farms in north-eastern Thailand from 2010 to 2011. MRSA isolates were confirmed for femA and mecA genes by PCR. The MICs of eight antimicrobials, namely vancomycin (VA), cefazolin (CZ), ofloxacin (OF), tetracycline (TET), erythromycin (ER), oxacillin (OX), cefoxitin (FOX) and gentamicin (GN), were tested by agar dilution method. The virulence genes for Panton-Valentine leukocidin toxin (lukSF-PV), toxic shock syndrome toxin-1 (tst) and α-haemolysin (hla) were detected by PCR. Strain typing was performed by staphylococcal cassette chromosome (SCC) mec, agr, spa and multilocus sequence typing. Four MRSA were isolated: three from workers and one from a pig. All the MRSA isolates were resistant to OX, GN, ER, TET and CZ, and they all carried hla only. Two MRSA from humans carried SCCmec II-sequence type (ST)764-agrII, whereas the two remaining MRSA (one each from a human and a pig) contained SCCmec IX-ST9-agrII. Interestingly, meticillin-resistant coagulase-negative Staphylococcus isolates carrying SCCmec IX were also obtained from five workers and three pigs. This study suggests that the SCCmec IX element is distributed among the Staphylococcus found in pigs and pig-farm workers, and pigs may be a reservoir for MRSA in the community.

Prevalence and antimicrobial resistance in Salmonella enterica isolated from broiler chickens, pigs and meat products in Thailand-Cambodia border provinces

This study aimed to examine the prevalence and antimicrobial resistance (AMR) of Salmonella isolates from broiler chickens, pigs and their associated meat products in the Thailand–Cambodia border provinces. A total of 941 samples were collected from pigs and broiler chickens at slaughter houses and from carcasses at local fresh markets in Sa Kaeo, Thailand (n = 554) and Banteay Meanchey, Cambodia (n = 387) in 2014 and 2015. From these samples, 345 Salmonella isolates were collected from Sa Keao (n = 145; 23%) and Banteay Meanchey (n = 200; 47%) and assayed for antimicrobial susceptibility, class 1 integrons and extended‐spectrum β‐lactamase (ESBL) genes. Serovars Typhimurium (29%) and Rissen (29%) were the most common serotypes found in Thai and Cambodian isolates, respectively. Multidrug resistance was detected in 34% and 52% of isolates from Sa Keao and Banteay Meanchey, respectively. The majority of the Thai isolates were resistant to ampicillin (72.4%), whereas most Cambodian isolates were resistant to sulfamethoxazole (71%). Eleven isolates from Sa Keao and 44 from Banteay Meanchey carried class 1 integrons comprising resistance gene cassettes. The most common gene cassette array was dfrA12‐aadA2 (61.1%). Six isolates were ESBL producers. The β‐lactamase genes found included blaTEM‐1, blaCTX‐M‐55 and blaCMY‐2. Some of these class 1 integrons and ESBL genes were located on conjugative plasmid. In conclusion, multidrug‐resistant Salmonella are common in pigs, chickens and their products in the Thailand–Cambodia border provinces. Our findings indicate that class 1 integrons play a role in spread of AMR in the strains in this study.

Phenotypic and genotypic antimicrobial resistance and virulence genes of Salmonella enterica isolated from pet dogs and cats

Salmonella enterica isolates (n = 122), including 32 serotypes from 113 dogs and 9 cats, were obtained from household dogs (n = 250) and cats (n = 50) during 2012–2015. The isolates were characterized by serotyping, antimicrobial resistance phenotyping and genotyping, and virulence gene screening. Serovars Weltevreden (15.6%) and Typhimurium (13.9%) were the most common. The majority (43%) of the isolates were multidrug resistant. The dog isolates (12.3%) harbored class 1 integrons, of which the dfrA12-aadA2 cassette was most frequent (66.7%). The only class integron in serovar Albany was located on a conjugative plasmid. Two ESBL-producing isolates (i.e., a serovar Krefeld and a serovar Enteritridis) carried blaTEM and blaCTX-M, and the blaTEM gene in both was horizontally transferred. Of the plasmid-mediated quinolone resistance genes tested, only qnrS (4.9%) was detected. Most Salmonella isolates harbored invA (100%), prgH (91.8%), and sipB (91%). Positive associations between resistance and virulence genes were observed for blaPSE-1/orgA, cmlA/spaN, tolC, and sul1/tolC (p < 0.05). The results suggest that companion dogs and cats are potential sources of S. enterica strains that carry resistance and virulence genes and that antimicrobial use in companion animals may select for the examined Salmonella virulence factors.

Genotypic and phenotypic characterization of Salmonella enterica subsp. enterica serovar Typhimurium monophasic variants isolated in Thailand and Japan

Monophasic variants of Salmonella enterica serovar Typhimurium isolated in Thailand and Japan were characterized to elucidate the genetic basis of the monophasic phenotype, genetic relatedness, and antimicrobial resistance. A total of 20 Salmonella isolates agglutinated with anti-O4 and anti-H:i serum and not agglutinated with either anti-H:1 or anti-H:2 serum were identified as monophasic variants of Salmonella serovar Typhimurium because they harbored IS200, specific to this serovar, and lacked the fljB gene. An allele-specific PCR-based genotyping method that detects a clade-specific single nucleotide polymorphism indicated that seven swine isolates and one human isolate from Thailand were grouped into clade 1; five isolates from layer chicken houses and layer chicken feces from Japan were grouped into clade 8, together with two Salmonella serovar Typhimurium isolates from chicken houses in Japan; and five isolates from swine feces from Thailand and two isolates from layer chicken feces from Japan were grouped into clade 9. Multilocus sequencing typing demonstrated that sequence type (ST) 34 isolates were solely grouped into clade 9. Clade 1 and 8 isolates were assigned as ST19. Pulsed-field gel electrophoresis revealed multiple types within each of the clades. The presence of antimicrobial resistance genes and plasmid replicon type, of the clade 1 and 9 isolates were comparable to those reported for epidemic strains of monophasic variants. Our results suggest that monitoring monophasic variants of serovar Typhimurium is important for understanding of the spread of these variants in Thailand and Japan.

Molecular Characterization of Carbapenemase-Nonproducing Clinical Isolates of Escherichia coli (from a Thai University Hospital) with Reduced Carbapenem Susceptibility

Twelve nonreplicate carbapenemase-negative ertapenem (ETP)-nonsusceptible (CNENS) Escherichia coli isolates obtained at a Thai university hospital between 2010 and 2014 were characterized and compared with 2 carbapenemase-producing E. coli isolates from the same hospital. Eight unique pulsed-field gel electrophoresis patterns were obtained. All the isolates produced CTX-M-15 β-lactamase and 2 either coexpressed CMY-2 cephalosporinase or showed increased efflux pump activity. Amino acid sequence analysis revealed that an OmpF defect (in 7 isolates) due to mutations generating truncated proteins or an IS1 insertion was more prevalent than a defect in OmpC was (no truncated proteins detected). Seven out of 10 isolates possessing OmpC variants with any OmpF defect were weakly ETP-resistant (minimum inhibitory concentrations [MICs] of 1-4 μg/mL) and imipenem (IPM)- and meropenem (MEM)-susceptible (MICs 0.125-0.5 μg/mL). Two isolates with ompC PCR-negative results and an OmpF defect showed higher carbapenem MICs (8-32, 1-8, and 1-4 μg/mL for ETP, IPM, and MEM, respectively) with the highest MICs associated with the additional efflux pump activity. Both carbapenemase producers possessing CTX-M-15 and a porin background identical to that in the CNENS isolates showed ETP, IPM, and MEM MICs of 128-256, 8, and 2-32 μg/mL, respectively. These findings suggest that a porin defect combined with CTX-M-15 production is the major mechanism of low carbapenem susceptibility among our CNENS isolates, which have potential to become strongly carbapenem-resistant because of additional carbapenemase or efflux pump activities.