Label-Free Detection of Waterborne Pathogens Using an All-Solid-State Laser-Induced Graphene Potentiometric Ion Flux Immunosensor.

Waterborne pathogens are harmful microorganisms transmitted through water sources. Early and rapid pathogen detection is important for preventing illnesses and implementing stringent water safety measures to minimize the risk of contamination. This work introduces a miniaturized all-solid-state potentiometric ion flux immunosensor for the rapid and label-free detection of waterborne pathogens. A screen-printed silver/silver chloride electrode coated with a reference electrode membrane and polyurethane as an all-solid-state reference electrode was combined with a solid-state contact ion-selective electrode (ISE). An all-solid-state ISE was constructed on laser-induced graphene by coating it with a cationic marker and a carboxylated poly(vinyl chloride)-based membrane for immobilizing antibodies and controlling ion fluxes through the membrane. Proof-of-concept was achieved by detecting Escherichia coli and Salmonella enterica serovar Typhimurium using the assembled immunosensors within 10 min. The potentiometric response shift attributed to the blocking effect in the ion flux caused by pathogen-antibody interaction corresponded to pathogen concentration, indicating detection limits of 0.1 CFU/mL and working ranges of 0.1-105 CFU/mL. Furthermore, the developed sensors revealed high selectivity and were directly applied in groundwater and tap water without any sample preparation, demonstrating high recovery percentages. The simple operation and elimination of sample preparation are key benefits to further usability of the developed immunosensors for efficient pathogen detection.

Application of machine learning algorithm and modified high resolution DNA melting curve analysis for molecular subtyping of Salmonella isolates from various epidemiological backgrounds in northern Thailand.

Food poisoning from consumption of food contaminated with non-typhoidal Salmonella spp. is a global problem. A modified high resolution DNA melting curve analysis (m-HRMa) was introduced to provide effective discrimination among closely related HRM curves of amplicons generated from selected Salmonella genome sequences enabled Salmonella spp. to be classified into discrete clusters. Combination of m-HRMa with serogroup identification (ms-HRMa) helped improve assignment of Salmonella spp. into clusters. In addition, a machine learning (dynamic time warping) algorithm (DTW) was employed to provide a simple and rapid protocol for clustering analysis as well as to create phylogeny tree of Salmonella strains (n = 40) collected from home, farms and slaughter houses in northern Thailand. Applications of DTW and ms-HRMa clustering analyses were capable of generating molecular signatures of the Salmonella isolates, resulting in 25 ms-HRM and 28 DTW clusters compared to 14 clusters from a standard HRM analysis, and the combination of both analyses permitted molecular subtyping of each Salmonella isolate. Results from DTW and ms-HRMa cluster analyses were in good agreement with that obtained from enterobacterial repetitive intergenic consensus sequence PCR clustering. While conventional serotyping of Clusters 1 and 2 revealed six different Salmonella serotypes, the majority being S. Weltevraden, the new Salmonella subtyping protocol identified five S. Weltevraden subtypes with S.Weltevreden subtype DTW4-M1 being predominant. Based on knowledge of the sources of Salmonella subtypes, transmission of S. Weltevraden in northern Thailand was likely to be farm-to-farm through contaminated chicken stool. In conclusion, the rapid, robust and specific Salmonella subtyping developed in the study can be performed in a local setting, enabling swift control and preventive measures to be initiated against potential epidemics of salmonellosis.

High prevalence of ceftriaxone resistance among invasive Salmonella enterica serotype Choleraesuis isolates in Thailand: The emergence and increase of CTX-M-55 in ciprofloxacin-resistant S. Choleraesuis isolates.

S. Choleraesuis is a highly invasive zoonotic pathogen that causes a serious systemic infection in humans. The emergence and increase of resistance to ceftriaxone and ciprofloxacin among S. Choleraesuis has become a serious therapeutic problem. The present study demonstrated high frequency of antimicrobial resistance in Salmonella Choleraesuis among 414 nontyphoidal Salmonella isolates from bacteremic patients in Thailand. High rates of ceftriaxone (58.3%) and ciprofloxacin (19.6%) resistances were observed in S. Choleraesuis isolates. The dissemination of the self-transferable blaCTX-M-14-carrying IncFIIs, IncFII, and IncI1 plasmids and blaCMY-2-carrying IncA/C plasmid along with the clonal spread of blaCMY-2-harbouring S. Choleraesuis isolates contributed to the high frequency of resistance to extended-spectrum cephalosporins (ESCs; third- and fourth-generation cephalosporins) during 2005-2007. We reported the first occurrence of ceftazidime-hydrolysing CTX-M-55 in S. Choleraesuis isolates which dramatically increased and became the most abundant CTX-M variant among ESC-resistant S. Choleraesuis isolates during 2012-2016. The spread of clone pulsotype B3 was due to the dissemination of IncA/C plasmids carrying both blaCTX-M-55 and qnrS1 among ciprofloxacin-resistant S. Choleraesuis isolates harbouring D87G in GyrA. These isolates were apparently responsible for the high rates of co-resistance to ESCs and ciprofloxacin (51.3%) during 2012-2016. This study emphasizes the importance to have an action plan to control the dissemination of antimicrobial resistance in S. Choleraesuis since this poses a threat to global health due to travel and trade in animal food products.

Genomic dissection of travel-associated extended-spectrum-beta-lactamase-producing Salmonella enterica serovar typhi isolates originating from the Philippines: a one-off occurrence or a threat to effective treatment of typhoid fever?

One unreported case of extended-spectrum-beta-lactamase (ESBL)-producing Salmonella enterica serovar Typhi was identified, whole-genome sequence typed, among other analyses, and compared to other available genomes of S. Typhi. The reported strain was similar to a previously published strain harboring blaSHV-12 from the Philippines and likely part of an undetected outbreak, the first of ESBL-producing S. Typhi.

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 isolates of Salmonella enterica serovar Stanley, a serovar endemic to Asia and associated with travel.

Salmonella enterica serovar Stanley (S. Stanley) is a common serovar in Southeast Asia and was the second most common serovar implicated in human salmonellosis in Thailand in the years 2002 to 2007. In contrast, this serovar is relatively uncommon in Europe. The objective of this study was to characterize a collection of S. Stanley strains isolated from Thai (n = 62), Danish (n = 39), and French (n = 24) patients to gain a broader understanding of the genetic diversity, population dynamics, and susceptibility to antimicrobials. All isolates were characterized by pulsed-field gel electrophoresis and antimicrobial susceptibility testing. The molecular mechanisms of resistance to extended-spectrum cephalosporins and plasmid-mediated resistance to quinolones were characterized by PCR and sequencing. Plasmid profiling, replicon typing, and microarray analysis were used to characterize the genetic mechanisms of antimicrobial resistance in 10 extended-spectrum cephalosporinase-producing isolates. Considerable genetic diversity was observed among the isolates characterized with 91 unique XbaI pulsed-field gel electrophoresis (PFGE) patterns, including 17 distinct clusters consisting of two to seven indistinguishable isolates. We found some of the S. Stanley isolates isolated from patients in Europe were acquired during travel to Southeast Asia, including Thailand. The presence of multiple plasmid lineages carrying the extended-spectrum cephalosporinase-encoding bla(CMY-2) gene in S. Stanley isolates from the central part of Thailand was confirmed. Our results emphasize that Thai authorities, as well as authorities in other countries lacking prudent use of antimicrobials, should improve the ongoing efforts to regulate antimicrobial use in agriculture and in clinical settings to limit the spread of multidrug-resistant Salmonella isolates and plasmids among humans and pigs in Thailand and abroad.

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.

Molecular characterization of extended-spectrum cephalosporin and fluoroquinolone resistance genes in Salmonella and Shigella isolated from clinical specimens in Thailand.

Antimicrobial resistance of Salmonella and Shigella has become a major clinical and public health problem. The incident of co-resistance to third generation cephalosporins and fluoroquinolone is a serious therapeutic issue in Thailand. The present study aimed to investigate the antimicrobial resistance and molecular character of clinical Shigella and Salmonella isolates. A total of 33 Salmonella and 53 Shigella cefotaxime-resistant isolates were collected from human clinical cases in Thailand during the period from 2011-2018. The antimicrobial susceptibility of Salmonella and Shigella was determined by the disk diffusion method, and extended-spectrum beta-lactamase (ESBL) production was characterized by the double-disk synergy test. Genotype characterization was performed by PCR and DNA sequencing. Thirty-two (97.0%) and fifty-two (98.1%) isolates of cefotaxime-resistant Salmonella and Shigella, respectively, were identified as ESBL producers. Shigella sonnei (4 isolates), Salmonella serovar 4,5,12:i:- (6 isolates), Salmonella serovar Agona (2 isolates) and Salmonella serovar Rissen (2 isolates) showed co-resistance to ciprofloxacin and cefotaxime or ceftriaxone. The combination of bla CTX-M-15 plus other ESBL and/or AmpC ß-lactamase genes was the most dominant of the genotype patterns in ESBL-producing isolates. The plasmid harbouring the aac(6')-Ib-cr gene and mutations of gyrA (S83F, D87Y or D87G) and parC (T57S) genes was found in 2 ESBL-producing Salmonella isolates. Three Shigella sonnei isolates harboured mutations in gyrA (S83L, D87Y or D87G), and only one Shigella sonnei phase I isolate showed mutations in both gyrA (S83L and D87G) and parC (S80I) genes. Among these clinical Shigella sonnei isolates, qnrS determinants were identified. Production of ESBLs is an important mechanism for resistance to extended-spectrum cephalosporins in Salmonella and Shigella. The emergence of a decreased susceptibility to extended-spectrum cephalosporins and fluoroquinolone in ESBL-producing isolates has important clinical and therapeutic implications.

Quantitative Risk Assessment of Susceptible and Ciprofloxacin-Resistant Salmonella from Retail Pork in Chiang Mai Province in Northern Thailand.

The adverse human health effects as a result of antimicrobial resistance have been recognized worldwide. Salmonella is a leading cause of foodborne illnesses while antimicrobial resistant (AMR) Salmonella has been isolated from foods of animal origin. The quantitative risk assessment (RA) as part of the guidelines for the risk analysis of foodborne antimicrobial resistance was issued by the Codex Alimentarius Commission more than a decade ago. However, only two risk assessments reported the human health effects of AMR Salmonella in dry-cured pork sausage and pork mince. Therefore, the objective of this study was to quantitatively evaluate the adverse health effects attributable to consuming retail pork contaminated with Salmonella using risk assessment models. The sampling frame covered pork at the fresh market (n = 100) and modern trade where pork is refrigerated (n = 50) in Chiang Mai province in northern Thailand. The predictive microbiology models were used in the steps where data were lacking. Susceptible and quinolone-resistant (QR) Salmonella were determined by antimicrobial susceptibility testing and the presence of AMR genes. The probability of mortality conditional to foodborne illness by susceptible Salmonella was modeled as the hazard characterization of susceptible and QR Salmonella. For QR Salmonella, the probabilistic prevalences from the fresh market and modern trade were 28.4 and 1.9%, respectively; the mean concentrations from the fresh market and modern trade were 346 and 0.02 colony forming units/g, respectively. The probability of illness (PI) and probability of mortality given illness (PMI) from QR Salmonella-contaminated pork at retails in Chiang Mai province were in the range of 2.2 × 10-8-3.1 × 10-4 and 3.9 × 10-10-5.4 × 10-6, respectively, while those from susceptible Salmonella contaminated-pork at retails were in the range 1.8 × 10-4-3.2 × 10-4 and 2.3 × 10-7-4.2 × 10-7, respectively. After 1000 iterations of Monte Carlo simulations of the risk assessment models, the annual mortality rates for QR salmonellosis simulated by the risk assessment models were in the range of 0-32, which is in line with the AMR adverse health effects previously reported. Therefore, the risk assessment models used in both exposure assessment and hazard characterization were applicable to evaluate the adverse health effects of AMR Salmonella spp. in Thailand.

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.

Molecular Characterization of Cephalosporin and Fluoroquinolone Resistant Salmonella Choleraesuis Isolated from Patients with Systemic Salmonellosis in Thailand.

The antimicrobial resistance of nontyphoidal Salmonella has become a major clinical and public health problem. Southeast Asia has a high level of multidrug-resistant Salmonella and isolates resistant to both fluoroquinolone and third-generation cephalosporins. The incidence of co-resistance to both drug classes is a serious therapeutic problem in Thailand. The aim of this study was to determine the antimicrobial resistance patterns, antimicrobial resistance genes and genotypic relatedness of third-generation cephalosporins and/or fluoroquinolone-resistant Salmonella Choleraesuis isolated from patients with systemic salmonellosis in Thailand. Antimicrobial susceptibility testing was performed using the agar disk diffusion method, and ESBL production was detected by the combination disc method. A molecular evaluation of S. Choleraesuis isolates was performed using PCR and DNA sequencing. Then, a genotypic relatedness study of S. Choleraesuis was performed by pulse field gel electrophoresis. All 62 cefotaxime-resistant S. Choleraesuis isolates obtained from 61 clinical specimens were multidrug resistant. Forty-four isolates (44/62, 71.0%) were positive for ESBL phenotypes. Based on the PCR sequencing, 21, 1, 13, 23, 20 and 6 ESBL-producing isolates harboured the ESBL genes blaCTX-M-14, blaCTX-M-15, blaCTX-M-55, blaCMY-2, blaACC-1 and blaTEM-1, respectively. This study also found that nine (9/62, 14.5%) isolates exhibited co-resistance to ciprofloxacin and cefotaxime. All of the co-resistant isolates harboured at least one PMQR gene. The qnr genes and the aac(6')-Ib-cr gene were the most prevalent genes detected. The QRDR mutation, including the gyrA (D87Y and D87G) and parC (T57S) genes, was also detected. PFGE patterns revealed a high degree of clonal diversity among the ESBL-producing isolates.

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.

Prevalence and antimicrobial susceptibility of Campylobacter isolated from retail chickens in Thailand.

Campylobacter is an important foodborne pathogen causing bacterial gastroenteritis worldwide; however, there has been a lack of information over the past decade on its occurrence, antibiotic susceptibility and genetic diversity in Thailand. Poultry meat is considered as a reservoir for transmission of Campylobacter to humans. This study determines the prevalence and antimicrobial resistance patterns of Campylobacter spp. on chicken samples purchased from 50 local wet markets and supermarkets in central Thailand. Of the 296 samples, 99 (33.5%) were contaminated with C. jejuni, 54 (18.2%) were C. coli and 15 (5.1%) were contaminated with both species. Antibiotic resistance rate is higher among C. coli isolates; 100%, 76.8%, 37.7%, 36.2% and 13.0% were resistant to quinolones, cyclines, macrolides, clindamycin and gentamicin, respectively. Most of the C. jejuni isolates were resistant to quinolones (79.8%) and cyclines (38.6%) whereas resistance to macrolides, clindamycin and gentamicin was found to be 1.8%. Multi-drug resistance (i.e. to three or more unrelated antimicrobials) was detected in 37.7% of C. coli and 1.8% of C. jejuni isolates. This study has revealed high contamination rates and alarming levels of antimicrobial resistance in Campylobacter spp. isolated from retail chicken samples in Thailand, suggesting the necessity of implementing interventions to reduce its prevalence from farm to table in the country.

Occurrence of mcr-mediated colistin resistance in Salmonella clinical isolates in Thailand.

Nontyphoidal Salmonella, an important zoonotic pathogen and a major cause of foodborne illnesses, could be a potential reservoir of plasmids harbouring mobile colistin resistance gene (mcr). This study reported, for the first time, a high rate of mcr-carrying Salmonella clinical isolates (3.3%, 24/724) in Thailand, associated with mcr-3 gene (3.0%, 22/724) in S. 4,[5],12:i:-(15.4%, 4/26), S. Typhimurium (8.8%, 5/57), and S. Choleraesuis (5.6%, 13/231). Remarkably, the increasing trends of colistin and extended-spectrum cephalosporin resistances have displayed a high agreement over the years, with a dramatic rise in the mcr-carrying Salmonella from 1.1% (6/563) during 2005-2007 to 11.2% (18/161) during 2014-2018 when CTX-M-55 became abundant. Clonal and plasmid analysis revealed that the self-transferable IncA/C and a novel hybrid IncA/C-FIIs MDR plasmids were the major vehicles to disseminate both mcr-3 and blaCTX-M55 genes among diverse Salmonella strains, from as early as 2007. To our knowledge the occurrence of mcr-3 and the co-existence of it with blaCTX-M-55 in S. Choleraesuis are reported here for the first time, leading to clinical concern over the treatment of the invasive salmonellosis. This study provides evidence of the potential reservoirs and vectors in the dissemination of the mcr and highlights the co-selection by colistin and/or cephalosporins.

Molecular characterization of extended-spectrum cephalosporinase-producing Salmonella enterica serovar Choleraesuis isolates from patients in Thailand and Denmark.

The objective of this study was to characterize extended-spectrum cephalosporinase (ESC)-producing isolates of Salmonella enterica serovar Choleraesuis recovered from patients in Thailand and Denmark. Twenty-four blood culture isolates from 22 patients were included in the study, of which 23 isolates were recovered from 21 Thai patients during 2003, 2007, or 2008 and one isolate was recovered from a Danish traveler to Thailand. ESC production was confirmed in 13 out of the 24 isolates by MIC testing. Microarray and plasmid profiling (replicon typing and restriction fragment length polymorphism [RFLP]) were used to characterize the genetic mechanisms of antimicrobial resistance in the 13 ESC-producing isolates. Pulsed-field gel electrophoresis (PFGE) and MIC testing were used to compare the clonality between the 13 ESC-producing isolates and the 11 non-ESC-producing isolates. Based on susceptibility patterns, the ESC-producing isolates were more closely related than non-ESC-producing isolates. Microarray, PCR, plasmid profiling, and replicon typing revealed that the 13 ESC-producing isolates harbored either bla(CMY-2) containing incA/C or bla(CTX-M-14) containing incFIIA, incFrepB, and an unknown replicon located on plasmids ranging in size from 75 to 200 kb. The RFLP and replicon typing clustered the isolates into four distinct groups. PFGE revealed 16 unique patterns and five clusters; each cluster contained two or three of the 24 isolates. The isolate from the Danish patient was indistinguishable from two Thai clinical isolates by PFGE. This study revealed the emergence of the bla(CTX-M-14) gene among several clones of Salmonella serovar Choleraesuis. Numerous plasmids were identified containing up to two different ESC genes and four distinct replicons. A "travel-associated" spread was confirmed. Overall, a high degree of clonal diversity between isolates resistant and susceptible to cephalosporins was observed. The findings represent a serious threat to public health for the Thai people and tourists.

CRISPR 2 PCR and high resolution melting profiling for identification and characterization of clinically-relevant Salmonella enterica subsp. enterica.

BACKGROUND: Nontyphoidal Salmonella spp. constitute a major bacterial cause of food poisoning. Each Salmonella serotype causes distinct virulence to humans. METHOD: A small cohort study was conducted to characterize several aspects of Salmonella isolates obtained from stool of diarrheal patients (n = 26) admitted to Phayao Ram Hospital, Phayao province, Thailand. A simple CRISPR 2 molecular analysis was developed to rapidly type Salmonella isolates employing both uniplex and high resolution melting (HRM) curve analysis. RESULTS: CRISPR 2 monoplex PCR generated a single Salmonella serotype-specific amplicon, showing S. 4,[5],12:i:- with highest frequency (42%), S. Enteritidis (15%) and S. Stanley (11%); S. Typhimurium was not detected. CRISPR 2 HRM-PCR allowed further classification of S. 4,[5],12:i:- isolates based on their specific CRISPR 2 signature sequences. The highest prevalence of Salmonella infection was during the summer season (April to August). Additional studies were conducted using standard multiplex HRM-PCR typing, which confirmed CRISPR 2 PCR results and, using a machine-learning algorithm, clustered the majority of Salmonella serotypes into six clades; repetitive element-based (ERIC) PCR, which clustered the serotypes into three clades only; antibiogram profiling, which revealed the majority resistant to ampicillin (69%); and test for extended spectrum ß-lactamase production (two isolates) and PCR-based detection of bla alleles. CONCLUSION: CRISPR 2 PCR provided a simple assay for detection and identification of clinically-relevant Salmonella serotypes. In conjunction with antibiogram profiling and rapid assay for ß-lactamase producers, this approach should facilitate detection and appropriate treatment of Salmonellosis in a local hospital setting. In addition, CRISPR 2 HRM-PCR profiling enabled clustering of S. 4,[5],12:i:-isolates according to CRISPR 2 locus signature sequences, indicative of their different evolutionary trajectories, thereby providing a powerful tool for future epidemiological studies of virulent Salmonella serotypes.

Risk of Antimicrobial Resistant Non-Typhoidal Salmonella during Asymptomatic Infection Passage between Pet Dogs and Their Human Caregivers in Khon Kaen, Thailand.

To explore the risk of antimicrobial resistant (AMR) non-typhoidal Salmonella during asymptomatic infection passage between pet dogs and human caregivers in Khon Kaen, Thailand, one hundred forty paired fecal samples (n = 280) were obtained from companion dogs and their human caregivers, interviewed from 140 households during 2019-2020. The purified Salmonella isolates were serotype-identified and tested for antimicrobial resistance against ampicillin, ciprofloxacin, chloramphenicol, nalidixic acid, streptomycin, sulfamethoxazole-trimethoprim, and tetracycline. The homologous Salmonella isolate pairs (suggesting Salmonella infections may have been due to passage between each one of the pair, or derived from the same source) were subsequently characterized by serotype screening, pulsed field gel electrophoresis (PFGE), and Synchrotron Fourier transform infrared spectroscopy (SR-FTIR). The Salmonella prevalence observed in dogs, 12.86% (18/140), was not significantly different from that observed in humans, 17.86% (25/140) using McNemar's test. The AMR patterns (the patterns among the isolates of pet dogs and caregivers) and the serotypes (thirteen serotypes with 18 isolates from pet dogs plus thirteen serotypes with 25 isolates from humans) between pet dogs and humans were not significantly different using Pearson's chi-squared test. The homologous Salmonella isolates from the Salmonella-present households was 5.13% (2/39). This study demonstrated that the hypothesis that there is a high risk of Salmonella infection passage between dogs and humans with close contact in Khon Kaen is doubtful. Only 5.13% of homologous Salmonella isolates from Salmonella-present households were found in Khon Kaen, Thailand, although the prevalence of Salmonella-positive samples, serotypes, and antimicrobial resistance patterns were quite similar among the two populations.

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.