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.

Characterization of Campylobacter jejuni DNA gyrase as the target of quinolones.

Quinolones have long been used as the first-line treatment for Campylobacter infections. However, an increased resistance to quinolones has raised public health concerns. The development of new quinolone-based antibiotics with high activity is critical for effective, as DNA gyrase, the target of quinolones, is an essential enzyme for bacterial growth in several mechanisms. The evaluation of antibiotic activity against Campylobacter jejuni largely relies on drug susceptibility tests, which require at least 2 days to produce results. Thus, an in vitro method for studying the activity of quinolones against the C. jejuni DNA gyrase is preferred. To identify potent quinolones, we investigated the interaction of C. jejuni DNA gyrase with a number of quinolones using recombinant subunits. The combination of purified subunits exhibited DNA supercoiling activity in an ATP dependent manner. Drug concentrations that inhibit DNA supercoiling by 50% (IC50s) of 10 different quinolones were estimated to range from 0.4 (sitafloxacin) to >100 µg/mL (nalidixic acid). Sitafloxacin showed the highest inhibitory activity, and the analysis of the quinolone structure-activity relationship demonstrated that a fluorine atom at R-6 might play the important role in the inhibitory activity against C. jejuni gyrase. Measured quinolone IC50s correlated well with minimum inhibitory concentrations (R = 0.9943). These suggest that the in vitro supercoiling inhibition assay on purified recombinant C. jejuni DNA gyrase is a useful and predictive technique to monitor the antibacterial potency of quinolones. And furthermore, these data suggested that sitafloxacin might be a good candidate for clinical trials on campylobacteriosis.

Draft genome sequence data of the multidrug-resistant bacterium Staphylococcus haemolyticus 010503B isolated from an aerosol sample in a hospital waiting area in Thailand.

Staphylococcus haemolyticus 010503B is a multidrug-resistant bacterium isolated from an outpatient clinic in a hospital waiting area in Thailand. Here we present the draft genome sequence of S. haemolyticus 010503B. The paired-end reads were generated on the Illumina NextSeq 550 sequencer using genomic DNA from the pure culture of S. haemolyticus 010503B. The draft genome consisted of 114 contigs with a total size of 2,457,654 base pairs, an N50 of 57,312 base pairs and a GC content of 32.60%. The dDDH between 010503B and Staphylococcus haemolyticus SM 131T was 91.9%, identifying the strain as Staphylococcus haemolyticus. The data presented holds promise for bacterial classification, comparative genomics, analysing antimicrobial resistance comprehensively, and assessing bacterial virulence factors of S. haemolyticus. The draft genome sequence data has been deposited at NCBI under Bioproject accession number PRJNA550309.

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.

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.

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.

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.

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.

Antimicrobial Resistance, Extended-Spectrum ß-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 ß-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 ß-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.

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.