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.

Comparative phenotypic and genotypic characterization of Salmonella spp. in pig farms and slaughterhouses in two provinces in northern Thailand.

Salmonella spp. are an important group of bacterial zoonotic pathogens which can cause acute food-borne diseases in humans. Pork products are the main source of salmonellosis, but the origins and transmission routes of the disease have not been clearly determined. The purpose of this study was to characterize Salmonella spp. isolated in pig production lines both from pig farms and from slaughterhouses in Chiang Mai and Lamphun provinces in northern Thailand. The study focuses on the association among serotypes, antimicrobial resistance patterns and Pulse Field Gel Electrophoresis (PFGE) patterns to investigate possible sources of infection and to provide information which could help strengthen salmonellosis control programs in the region. A total of 86 strains of Salmonella comprising five majority serotypes were identified. Antibiotic resistance to tetracycline was found to be the most prevalent (82.56%) followed by ampicillin (81.40%) and streptomycin (63.95%). Seven clusters and 28 fingerprint-patterns generated by PFGE were identified among strains recovered from various locations and at different times, providing information on associations among the strains as well as evidence of the existence of persistent strains in some areas. Study results suggest that Salmonella control programs should be implemented at slaughterhouse production lines, including surveillance to insure good hygiene practices, in addition to regular monitoring of large populations of farm animals.

Molecular characterization of Thai Salmonella enterica serotype typhimurium and serotype 4,5,12:i:- reveals distinct genetic deletion patterns.

In order to better understand the relationship between Salmonella serotypes Typhimurium and its monophasic variant 4,5,12:i:- found in Thailand, a total of 138 isolates from various sources were characterized using different molecular subtyping methods (i.e., pulsed-field gel electrophoresis [PFGE] and polymerase chain reaction [PCR]) and antibiotic resistance (AbR) patterns. PFGE revealed 52 distinct band patterns among these isolates, 3 of which were shared between the two serotypes. PCR characterization of genomic deletion patterns reveals that Thai S. 4,5,12:i:- isolates contain a distinct deletion pattern in the fljAB region, which can be used as a specific genetic marker for primary identification of S. 4,5,12:i:- sources. AbR study shows that, among 50 representative serotype-confirmed strains, 48.28% (14/29) of Salmonella Typhimurium and 90.48% (19/21) of 4,5,12:i:- isolates are multidrug-resistant Salmonella as they are resistant to at least 3 antimicrobial categories. The AmpST pattern for resistance to ampicillin, streptomycin, and tetracycline was found in high proportions of Salmonella Typhimurium (10 of 29) and S. 4,5,12:i:- (15 of 21) isolates.

Dissemination of clonal Salmonella enterica serovar Typhimurium isolates causing salmonellosis in Mauritius.

Salmonella enterica serotype Typhimurium is one of the leading causes of salmonellosis in Mauritius, where it has also been associated with outbreaks of foodborne illness. However, little is known about its molecular epidemiology in the country. This study was therefore undertaken to investigate the clonality and source of Salmonella Typhimurium in Mauritius by studying human, food, and poultry isolates by pulsed-field gel electrophoresis (PFGE) and antibiotic minimum inhibitory concentration determination. Forty-nine isolates collected between 2008 and 2011 were analyzed, including 25 stool isolates from foodborne illness outbreaks and sporadic gastroenteritis cases, four blood isolates, one postmortem colon isolate, 14 food isolates, and five poultry isolates. All isolates were pansusceptible to the 16 antibiotics tested, except for two isolates that were resistant to sulfamethoxazole and trimethoprim. Overall characterization of the isolates by PFGE digested with XbaI and BlnI resulted in eight different patterns. The largest of the clusters in the composite dataset consisted of 20 isolates, including two raw chicken isolates, four poultry isolates, and nine human stool isolates from two outbreaks. A second cluster consisted of 18 isolates, of which 12 originated from human blood and stool samples from both sporadic and outbreak cases. Six food isolates were also found in this cluster, including isolates from raw and grilled chicken, marlin mousse, and cooked pork. One poultry isolate had a closely related PFGE pattern. The results indicate that one clone of Salmonella Typhimurium found in poultry has been causing outbreaks of foodborne illness in Mauritius and another clone that has caused many cases of gastrointestinal illness and bacteremia in humans could also be linked to poultry. Thus, poultry appears to be a major reservoir for Salmonella Typhimurium in Mauritius. Initiating on-farm control strategies and measures against future dissemination may substantially reduce the number of cases of salmonellosis in the country.

Characterization of Salmonella enterica serovar Enteritidis isolates recovered from blood and stool specimens in Thailand.

BACKGROUND: Bacteremia due to Salmonella spp. is a life-threatening condition and is commonly associated with immune compromise. A 2009 observational study estimated risk factors for the ten most common non-typhoidal Salmonella (NTS) serovars isolated from Thai patients between 2002-2007. In this study, 60.8% of Salmonella enterica serovar Enteritidis isolates (n = 1517) were recovered from blood specimens and infection with Salmonella serovar Enteritidis was a statistically significant risk factor for bacteremia when compared to other NTS serovars. Based on this information, we characterized a subset of isolates collected in 2008 to determine if specific clones were recovered from blood or stool specimens at a higher rate. Twenty blood isolates and 20 stool isolates were selected for antimicrobial resistance testing (MIC), phage typing, PFGE, and MLVA. RESULT: Eight antibiogrammes, seven MLVA types, 14 XbaI/BlnI PFGE pattern combinations, and 11 phage types were observed indicating considerable diversity among the 40 isolates characterized. Composite analysis based on PFGE and MLVA data revealed 22 genotypes. Seven of the genotypes containing two or more isolates were from both stool and blood specimens originating from various months and zones. Additionally, those genotypes were all further discriminated by phage type and/or antibiogramme. Ninety percent of the isolates were ciprofloxacin resistant. CONCLUSIONS: The increased percentage of bloodstream infections as described in the 2009 observational study could not be attributed to a single clone. Future efforts should focus on assessing the immune status of bacteriaemic patients and identifying prevention and control measures, including attribution studies characterizing non-clinical (animal, food, and environmental) isolates.

Prevalence and characterization of human Shigella infections in Henan Province, China, in 2006.

In 2006, 3,531 fecal samples were collected from patients with diarrhea in Henan Province, China. A total of 467 (13.2%) Shigella strains were isolated and serotyped. Seventy-one Shigella flexneri strains were characterized by MIC determination, pulsed-field gel electrophoresis (PFGE), and detection of genes encoding cephalosporin resistance. Most infections were caused by S. flexneri variant X [IV:(7),8] (27.6%), S. sonnei (24.2%), and S. flexneri 2a (20.8%). However, large regional differences were observed. Significantly higher odds (2.0) of females compared to males were infected with S. flexneri 2a. Untypeable S. flexneri (-:6) isolates were absent among males, as were untypeable S. flexneri [I:(7),8] isolates among females. Patient ages ranged from 2 months to 82 years, with 231 subjects (49.7%) <5 years of age. Most of the patients were male (62.1% [n = 290]). Infections peaked in July; week 27 with 38 cases (8.1%). All of the 71 S. flexneri conferred resistance to nalidixic acid; in addition, 21% (n = 15) and 79% (n = 56) were high- and low-level resistant to ciprofloxacin, respectively. Six S. flexneri isolates {serotype 2b [II:7,(8)] and 2b [II:(3),4;7,(8)]} harbored the bla(CTX-M-14) or bla(CTX-M-15) gene. A total of 52 unique XbaI PFGE patterns were observed among the 71 S. flexneri isolates with 11 distinct PFGE clusters. This study revealed a high prevalence of shigellosis with geographical differences in the distribution of serotypes in the distribution of serotypes and also differences in comparisons by gender. A high frequency of resistance, including 100% resistance to ciprofloxacin and resistance to extended-spectrum cephalosporins, was observed. We detected several isolates exhibiting the same PFGE type and MIC profile, indicating multiple undetected outbreaks.

Epidemiological investigation of Salmonella enterica serovar Kedougou in Thailand.

OBJECTIVE: Salmonella enterica serovar Kedougou is among the top 10 serovars reported in northern Thailand. The objective of this study was to identify risk factors associated with Salmonella Kedougou infection in Thailand and to compare the molecular types and antimicrobial resistance with Salmonella Kedougou isolates of human origin from United States and of animal origin from the United Kingdom. METHODS: Data from 13,976 Salmonella infections of which 253 were Salmonella Kedougou collected in Thailand between 2002 and 2008 were analyzed by logistic regression. Antimicrobial susceptibility testing and pulsed-field gel electrophoresis (PFGE) were performed on selected Salmonella Kedougou strains causing infections in Thailand (n = 66), and compared to isolates from the United States (n = 5) and the United Kingdom (n = 20). RESULTS: Logistic analysis revealed season (hot/dry; p = 0.023), region (northern Thailand; p < 0.001), and specimen (stool; p < 0.001) as significant risk factors associated with Salmonella Kedougou infection compared to other nontyphoid Salmonella. Of the Salmonella Kedougou isolates of human origin, 84% exhibited resistance to at least three antimicrobial classes. Three strains recovered from human stool in Thailand were resistant to third-generation cephalosporins: two harbored bla(CTX-M-63) and one bla(CMY-2). PFGE revealed 45 unique clusters. Isolates obtained from humans in Thailand and the United States presented identical PFGE profiles suggesting a travel association, whereas the majority of the animal isolates from United Kingdom clustered separately. CONCLUSIONS: This study reveals Salmonella Kedougou as a major cause of human infections in northern Thailand especially during the hot period and suggests a global spread probably due to travel. The clonal types causing infections in humans differed from those observed in animals in United Kingdom, which suggests the absence of an epidemiological link and could suggest differences in virulence. The high frequency of antimicrobial resistance, including emergence of resistance to fluoroquinolones and third-generation cephalosporins, might pose problems for treatment of infections.

Risk factors and epidemiology of the ten most common Salmonella serovars from patients in Thailand: 2002-2007.

We conducted a retrospective observational study to assess epidemiological trends and risk factors associated with the 10 most common Salmonella serovars isolated from humans in Thailand between 2002 and 2007. A total of 11,656 Salmonella isolates covering all 6 years were included in the study. The top 10 Salmonella serovars identified during the course of this study were Enteritidis, Stanley, Weltevreden, Rissen, I [1],4,[5],12:i:-, Choleraesuis, Anatum, Typhimurium, Corvallis, and Panama, which accounted for 8108 (69.6%) of the isolates. Most isolates were from patients <5 years (33%), were isolated during June (13%), and were recovered from stool (82%) and from patients in Bangkok (27%). Statistical analysis revealed that S. Enteritidis and S. Choleraesuis were recovered from blood with a higher frequency than other nontyphoidal serovars. While both serovars tended to be isolated from patients >5 years; S. Choleraesuis was recovered with a higher frequency from patients in Bangkok and the central region, whereas S. Enteritidis was recovered predominantly from patients in the southern region. This study also indicates a shift in prevalence of the most common Salmonella serovars responsible for human infections in Thailand compared to previous studies. Notably, there was an increase in human infections with S. Stanley, S. Corvallis, and S. Choleraesuis, three serovars that have previously been associated with swine, and a decrease in infections due to S. Weltevreden and S. Anatum. The study also revealed differences in the epidemiology among the different serovars, suggesting that serovar-specific interventions are needed. We recommend initiating targeted interventions for the two serovars associated with a high odds ratio for submitted blood samples, S. Enteritidis and S. Choleraesuis. The authors also recommend additional epidemiologic studies to investigate the observed increase in swine associated serovars (S. Stanley, S. Corvallis, and S. Choleraesuis) and determine interventions to reduce the burden of disease from these serovars.

Shigella species and serotypes among clinical isolates in Thailand from 2001 to 2005.

OBJECTIVE: To study the species and the serotypes of the clinical isolates of Shigella obtained from patients in Thailand. MATERIAL AND METHOD: The World Health Organization National Salmonella and Shigella Center Thailand, had confirmed the species and performed serotype identification of 1,913 clinical isolates of Shigella collected from the laboratory network of Department of Medical Sciences and the collaborated hospitals across Thailand from 2001 to 2005. RESULTS: Between the year 2001 and 2005, 728, 481, 160, 247, 297 clinical isolates were tested, respectively. There were 5 isolates of S. dysenteriae (group A), 416 isolates of S. flexneri (group B), 4 isolates of S. boydii (group C) and 1,488 isolates of S. sonnei (group D). A total of 21 Shigella serotypes were identified and there were 3 serotypes in group A, 11 serotypes in group B, 4 serotypes in group C, and 3 serotypes in group D. Throughout these five years, the five common serotypes were S. sonnei Phases I and II, 28.6% (548 isolates); S. sonnei Phase I, 24.6% (470 isolates); S. sonnei Phase II, 24.6% (470 isolates); S. flexneri Type 2a, 10.9% (208 isolates), and S. flexneri Type 3a, 6.3% (121 isolates), respectively. CONCLUSION: At the national scale in Thailand from 2001 to 2005, S. sonnei was the most frequent Shigella spp. isolated from patients in Thailand. In addition, S. dysenteriae and S. boydii were extremely uncommon. These findings are important in future vaccine development.

The epidemiological relationship between Salmonella enterica serovar typhimurium and Salmonella enterica serovar 4,[5],12:i:- isolates from humans and swine in Thailand.

A total of 138 isolates of S. Typhimurium and S. 4,[5],12:i:- from humans and swine in Thailand during 2003-2006, were evaluated for antimicrobial sensitivity by the disk diffusion method against 10 antimicrobial drugs and pulsed-field gel electrophoresis (PFGE) with endonuclease Xbal to investigate the epidemiological relationship among isolates. It was found that all isolates were classified into 27 antimicrobial resistance patterns, and 80% of S. Typhimurium and 95.4% of S. 4,[5],12:i:- isolates were resistant to three or more antimicrobial agents. By PFGE testing, the 84 PFGE patterns were categorized into A to Z patterns. Eighty percent of S. Typhimurium and 71.3% of S. 4,[5],12:i:- isolates in 7 major PFGE patterns had close clonal relationships (_85% similarity). Our studies indicate the spread of genetically identical clones of S. Typhimurium and S. 4,[5],12:i:- in humans and swine in Thailand.