Antimicrobial resistance and phylogenetic relatedness of extended-spectrum ß-lactamase (ESBL)-producing Escherichia coli in peridomestic rats (Rattus norvegicus and Rattus tanezumi) linked to city areas and animal farms in Hong Kong.

Extended-spectrum ß-lactamase-producing Escherichia (E.) coli (ESBL-EC) in the clinical setting have emerged as a major threat to public and animal health. Wildlife, including Rattus spp. may serve as reservoirs and spreaders of ESBL-EC in the environment. Peridomestic rats are well adapted to living in proximity to humans and animals in a variety of urban and agricultural environments and may serve as sentinels to identify variations of ESBL-EC within their different habitats. In this study, a set of 221 rats (Rattus norvegicus, R. tanezumi, R. andamanensis, and Niviventer huang) consisting of 104 rats from city areas, 44 from chicken farms, 52 from pig farms, and 21 from stables of horse-riding schools were screened for ESBL-EC. Overall, a total of 134 ESBL-EC were isolated from the caecal samples of 130 (59%) rats. The predominant blaESBL genes were blaCTX-M-14, blaCTX-M-15, blaCTX-M-55, and blaCTX-M-65. Phylogenetic analysis revealed a total of 62 sequence types (STs) and 17 SNP clusters. E. coli ST10 and ST155 were common to ESBL-EC from city areas and chicken farms, and ST44 were found among ESBL-EC from city areas and pig farms. Extra-intestinal pathogenic E. coli (ExPEC) ST69, ST131 and ST1193 were found exclusively among rats from city areas, and avian pathogenic E. coli (APEC) ST177 was restricted to ESBL-EC originating from chicken farms. Phylogenetic analysis showed that the populations of rodent ESBL-EC from city areas, chicken farms and pig farms were genetically different, suggesting a certain degree of partitioning between the human and animal locations. This study contributes to current understanding of ESBL-EC occurring in rats in ecologically diverse locations.

Prevalence and characterization of antimicrobial-resistant Escherichia coli in chicken meat from wet markets in Hong Kong.

Given the close contact between animals, animal products, and consumers in wet markets, fresh meat products are considered a potential source and disseminator of antimicrobial-resistant (AMR) bacteria near the end of the food chain. This cross-sectional study was conducted to estimate the prevalence of select AMR-E. coli in fresh chicken meat collected from wet markets in Hong Kong and to determine target genes associated with the observed resistance phenotypes. Following a stratified random sampling design, 180 fresh half-chickens were purchased from 29 wet markets across Hong Kong in 2022 and immediately processed. After incubation, selective isolation was performed for extended-spectrum ß-lactamase producing (ESBL), carbapenem-resistant (CRE), and colistin-resistant (CSR) E. coli. The bacterial isolates were identified using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). Disc Diffusion was used to determine the susceptibility of ESBL- and CRE-E. coli isolates. The broth microdilution method was used to determine the minimum inhibitory concentration of CSR-E. coli. Targeted resistance genes were then detected by PCR. The prevalence of ESBL-E. coli and CSR-E. coli were estimated at 88.8% (95% CI: 83.4-93.1%) and 6.7% (95% CI: 3.5-11.4%), respectively. No CRE-E. coli isolate was detected. The blaCTX-M-1 gene was the most common ß-lactamase group in isolated E. coli (80%), followed by blaTEM (63.7%); no blaSHV gene was detected. Forty-five percent of the isolates had blaTEM and blaCTX-M-1 simultaneously. The mcr-1 gene was detected in all 12 CSR isolates. Of 180 meat samples, 59 were from Mainland China, and 121 were locally sourced. There was no statistically significant difference in the prevalence of ESBL- and CSR-E. coli between the two sources. Our findings can be used to inform food safety risk assessments and set the stage for adopting targeted control and mitigation measures tailored to the local wet markets.

Serotypes, Antimicrobial Resistance Profiles, and Virulence Factors of Salmonella Isolates in Chinese Edible Frogs (Hoplobatrachus rugulosus) Collected from Wet Markets in Hong Kong.

Salmonella is an important agent of gastrointestinal disease in humans. While livestock, such as cattle, poultry, and pigs, are well-recognised animal reservoirs of Salmonella, there is a lack of data on Salmonella in edible frogs, even though frog meat is a popular food worldwide. In this study, 103 live edible Chinese frogs (Hoplobatrachus rugulosus) were collected from wet markets throughout Hong Kong. After euthanasia, faeces or cloacal swabs were examined for Salmonella. Overall, Salmonella spp. were isolated from 67 (65%, CI: 0.554-0.736) of the samples. The serotypes included S. Saintpaul (33%), S. Newport (24%), S. Bareilly (7%), S. Braenderup (4%), S. Hvittingfoss (4%), S. Stanley (10%), and S. Wandsworth (16%). Many isolates were phylogenetically related. A high number of genes encoding for resistance to clinically relevant antimicrobials, and a high number of virulence determinants, were identified. Antimicrobial susceptibility testing (AST) identified multidrug resistance (MDR) in 21% of the isolates. Resistance to ampicillin, ciprofloxacin, nalidixic acid, and tetracycline was common. These results demonstrate that a high percentage of live frogs sold for human consumption in wet markets are carriers of multidrug-resistant Salmonella. Public health recommendations for handling edible frogs should be considered, to mitigate the risk of Salmonella transmission to humans.

Phenotypic and Genotypic Characterization and Antifungal Susceptibility of Sporothrix schenckii sensu stricto Isolated from a Feline Sporotrichosis Outbreak in Bangkok, Thailand.

Sporotrichosis, an invasive fungal infection caused by Sporothrix schenckii, has emerged in Southeast Asia, affecting cats and posing a potential zoonotic risk to humans. We evaluated 38 feline sporotrichosis cases in and around Bangkok, Thailand, from 2017 to 2021. The isolates were phenotypically and genotypically characterized. The cats infected with sporotrichosis were mainly young adults, males, and domestic short hairs with uncontrolled outdoor access, and they lived in Bangkok. All isolates showed low thermotolerance and converted to the yeast phase at 35 °C. Based on the internal transcribed spacer region of rDNA sequences, our strains belonged to S. schenckii sensu stricto and clustered with clinical clade D. Based on the concatenated tree of calmodulin and beta-tubulin genes, five groups of S. schenckii were generated, and the monophyletic clade, Group II, of Thai strains was recognized. In vitro antifungal susceptibility testing demonstrated that the MIC50 of our isolates to amphotericin B, itraconazole, and posaconazole were within the limit of the species-specific epidemiological cutoff values, suggesting that the organisms were the wild type. Addressing the outbreak of feline sporotrichosis in Thailand by providing guidelines for diagnosis and effective treatment may help control the spread of disease and reduce the risk of cat-transmitted sporotrichosis to humans.

Complete Genome Sequence of the Extensively Drug-Resistant Extended-Spectrum ß-Lactamase-Producing Proteus mirabilis Isolate HK294, Obtained from Poultry Feces in Hong Kong.

Here, we report the complete genome sequence of Proteus mirabilis isolate HK294, recovered from pooled poultry feces in Hong Kong in 2022. The chromosome contained 32 antimicrobial resistance genes, including the extended-spectrum ß-lactamases blaCTX-M-65 and blaCTX-M-3. Almost all resistance genes were part of either an integrative conjugative element or a Tn7-like transposon.

Serotype Diversity and Antimicrobial Resistance Profile of Salmonella enterica Isolates From Freshwater Turtles Sold for Human Consumption in Wet Markets in Hong Kong.

Chelonians are recognized as a source of human salmonellosis through direct contact or consumption of their meat. Freshwater turtles sold for food are widely available in wet markets in Asia. In this pilot study, 50 turtles belonging to three species were randomly sampled from wet markets throughout Hong Kong. The turtles were humanely euthanised and their feces or the colon were sampled for Salmonella culture. The Salmonella isolates obtained were serotyped and examined for phenotypic antimicrobial resistance and the presence of antimicrobial resistance genes. The study reports a high prevalence (42%, 95% CI: 29.4-55.8) and considerable serotype diversity of Salmonella among turtles sold in wet markets. The most common among the 11 serotypes isolated were S. Oranienburg and S. Thompson, which have been reported in turtles previously. The serotype S. Manhattan is reported in chelonians for the first time. Resistance to streptomycin and chloramphenicol was common, despite the latter being banned from aquaculture in mainland China since 2002. Resistance against fluoroquinolones and third-generation cephalosporins which represent first-line treatment options for salmonellosis was also observed. The multidrug-resistance gene cfr is identified for the first time in Salmonella. This is a worrying finding as it indicates an expansion of the cfr reservoir and potential horizontal spread to other bacteria. The results of this study emphasize the need for close surveillance of Salmonella from turtles sold as food and better regulation of turtle farming to safeguard public health and improve animal welfare.

Longitudinal Monitoring Reveals Persistence of Colistin-Resistant Escherichia coli on a Pig Farm Following Cessation of Colistin Use.

Colistin-resistant bacteria harboring plasmid-mediated mcr genes are of concern as they may be a cause of serious nosocomial infections. It is hypothesized that cessation of colistin use as a feed additive for pigs will reduce the occurrence and distribution of mcr genes in farms. The aim of this study was to investigate this hypothesis by longitudinal monitoring and characterizing of mcr positive Escherichia coli (MCRPE) isolates after colistin was withdrawn on a central Thailand pig farm that previously had a high frequency of MCRPE. Colistin use ceased at the beginning of 2017, and subsequently 170 samples were collected from farrowing sows and suckling piglets (n = 70), wastewater (n = 50) and farm workers (n = 50) over a 3.5-year period. Bacteria were identified by MALDI-TOF mass spectrometry and minimal inhibitory concentrations were determined by broth microdilution. The antibiogram of mcr positive E. coli isolates was determined using the Vitek2 automated susceptibility machine, and multiplex and simplex PCRs were performed for mcr-1-8 genes. MCRPE containing either mcr-1 or mcr-3 were isolated from pigs throughout the investigation period, but with a declining trend, whereas MCRPE isolates were recovered from humans only in 2017. MCRPE were still being recovered from wastewater in 2020. Most MCRPE isolates possessed the virulence genes Stap, Stb, or Stx2e, reflecting pathogenic potential in pigs, and showed high rates of resistance to ampicillin, gentamicin and tetracycline. Pulsed-field gel electrophoresis and multi-locus sequence typing showed that diverse MCRPE clones were distributed on the farm. The study identified a decline of pathogenic MCRPE following withdrawal of colistin, with pigs being the primary source, followed by wastewater. However, short-term therapeutic usage of other antibiotics could enhance the re-occurrence of mcr-carrying bacteria. Factors including the environment, management, and gene adaptations that allow maintenance of colistin resistance require further investigation, and longer-term studies are needed.

Reducing the Risk of Transmission of Critical Antimicrobial Resistance Determinants From Contaminated Pork Products to Humans in South-East Asia.

Antimicrobial resistance (AMR) is a critical challenge worldwide as it impacts public health, especially via contamination in the food chain and in healthcare-associated infections. In relation to farming, the systems used, waste management on farms, and the production line process are all determinants reflecting the risk of AMR emergence and rate of contamination of foodstuffs. This review focuses on South East Asia (SEA), which contains diverse regions covering 11 countries, each having different levels of development, customs, laws, and regulations. Routinely, here as elsewhere antimicrobials are still used for three indications: therapy, prevention, and growth promotion, and these are the fundamental drivers of AMR development and persistence. The accuracy of detection of antibiotic resistant bacteria (ARB) and antibiotic resistance genes (ARG) depends on the laboratory standards applicable in the various institutes and countries, and this affects the consistency of regional data. Enterobacteriaceae such as Escherichia coli and Klebsiella pneumoniae are the standard proxy species used for indicating AMR-associated nosocomial infections and healthcare-associated infections. Pig feces and wastewater have been suspected as one of the hotspots for spread and circulation of ARB and ARG. As part of AMR surveillance in a One Health approach, clonal typing is used to identify bacterial clonal transmission from the production process to consumers and patients - although to date there have been few published definitive studies about this in SEA. Various alternatives to antibiotics are available to reduce antibiotic use on farms. Certain of these alternatives together with improved disease prevention methods are essential tools to reduce antimicrobial usage in swine farms and to support global policy. This review highlights evidence for potential transfer of resistant bacteria from food animals to humans, and awareness and understanding of AMR through a description of the occurrence of AMR in pig farm food chains under SEA management systems. The latter includes a description of standard pig farming practices, detection of AMR and clonal analysis of bacteria, and AMR in the food chain and associated environments. Finally, the possibility of using alternatives to antibiotics and improving policies for future strategies in combating AMR in a SEA context are outlined.

Anticonjugation and Antibiofilm Evaluation of Probiotic Strains Lactobacillus plantarum 22F, 25F, and Pediococcus acidilactici 72N Against Escherichia coli Harboring mcr-1 Gene.

Several species of lactic acid bacteria (LAB) are commonly used as probiotics and as an alternative to antibiotics in various industries, especially in the livestock industry. This study aimed to investigate the anticonjugation and antibiofilm activity of cell-free supernatant (CFS) of Thai LAB strains (Lactobacillus plantarum 22F, 25F, and Pediococcus acidilactici 72N) against colistin-resistant Escherichia coli isolates. A total of six colistin-resistant E. coli strains were isolated from different sources, including pigs, farmers, and farmhouse environments. The E. coli were characterized by plasmid profiling, PCR detection of mcr-1 gene, and antibiotic susceptibility patterns. The CFS at dilutions ≥1:16 was chosen as the proper dilution for anticonjugation assay. Besides, it could significantly reduce the transfer frequencies of resistance gene mcr-1 up to 100 times compared to the neutralizing CFS (pH 6.5). The biofilm production in the planktonic stage was reduced by non-neutralizing and neutralizing CFS determining with crystal violet staining assay up to 82 and 60%, respectively. Moreover, the non-neutralizing CFS also inhibited the biofilm formation in the sessile stage up to 52%. The biofilm illustration was confirmed by scanning electron microscopy (SEM). These results agreed with the findings of the crystal violet technique, which showed a significant reduction in cell density, aggregation, and extracellular polysaccharide (EPS) matrix. The application of Thai LAB may serve as an attractive alternative to antibiotics for reducing biofilm formation and limiting the proliferation of antibiotic-resistant genes.

Multidrug Resistance and Virulence Factors of Escherichia coli Harboring Plasmid-Mediated Colistin Resistance: mcr-1 and mcr-3 Genes in Contracted Pig Farms in Thailand.

The presence of the plasmid-mediated colistin resistance encoding mcr gene family in the Enterobacteriaceae is one of the crucial global concerns. The use of colistin in livestock rearing is believed to be the cause of mcr gene spreading and is of impact to public health. The objective of this research was to detect the frequency and virulent genes of mcr-positive Escherichia coli (MCRPE) in fecal samples from healthy pigs in a contract farming system across Thailand. A total of 696 pooled samples were derived from 80 farms, located in 49 provinces across six regions of Thailand. The colistin-resistant E. coli were identified by MALDI-TOF mass spectrometry and antimicrobial susceptibility testing by broth microdilution. The antibiogram was determined using an automated susceptibility machine, and the genetic characteristics were investigated for mcr-1-5 genes, phylogenetic group, replicon types, and virulent genes. In total, 31 of 696 samples were positive, with E. coli containing mcr-1 or combination of mcr-1 and mcr-3 with incidence of 4.45 and 0.43%. Phylogenetic groups A and B1 and the IncF and IncFIB replicon types were predominantly found in the MCRPE located in the central area, with multidrug-resistant traits against 3-14 types of antimicrobials. Additionally, 19 of 31 isolates identified as enterotoxigenic E. coli were with the stap and stb (enterotoxin-encoding genes). In conclusion, a low carriage rate of mcr-positive E. coli was detected in the large-scale farming of healthy pigs. The association between multidrug-resistant MCRPE and their pathogenic potential should be of concern.

First identification and characterisation of Brachyspira hyodysenteriae in pigs in Hong Kong.

Swine dysentery (SD) is an important endemic disease of pigs throughout the world. The most common aetiological agent is the anaerobic intestinal spirochaete Brachyspira hyodysenteriae. The related spirochaete Brachyspira pilosicoli causes a milder form of colitis. We report the first isolation of B. hyodysenteriae and B. pilosicoli from a pig farm in Hong Kong. Faecal samples containing mucus or fresh blood were collected from the ground where finisher pigs had just been loaded into a truck for transport to the abattoir. The samples were subjected to selective anaerobic culture and PCR for B. hyodysenteriae and B. pilosicoli, and two isolates of both species were obtained. The B. hyodysenteriae isolates showed clinical resistance to tylosin and lincomycin, whilst the B. pilosicoli isolates were resistant to tylosin and showed intermediate susceptibility to lincomycin. The B. hyodysenteriae isolates were subjected to multilocus sequence typing and a single previously undescribed sequence type (ST250) was identified. Disease was not recorded in other pigs on the farm, but it may have been masked by the use of antimicrobials. Further work is required to examine the distribution of these two pathogens in this and other farms in Hong Kong and in adjoining mainland China.

Molecular Characterization and Antimicrobial Resistance of Livestock-Associated Methicillin-Resistant Staphylococcus aureus Isolates from Pigs and Swine Workers in Central Thailand.

This study presents molecular characteristics of livestock-associated methicillin-resistant Staphylococcus aureus (LA-MRSA) from pigs and swine workers in central Thailand. Sixty-three MRSA isolates were recovered from pigs (n = 60) and humans (n = 3). Two major LA-MRSA lineages, including sequence type (ST) 398 and clonal complex 9 (ST9 and ST4576, a novel single-locus variant of ST9), were identified. ST398 had spa type t034 (n = 55). ST9 and ST4576 had t337 (n = 8) and carried staphylococcal cassette chromosome mec (SCCmec) IX only. MRSA-ST398-t034 contained various SCCmec, including SCCmec V (n = 42), a novel SCCmec composite island (n = 12), and a nontypeable SCCmec (n = 1). All isolates were multidrug resistant and carried common resistance genes found in LA-MRSA. This is the first report of the presence of swine MRSA ST398 and multidrug resistance gene cfr in MRSA ST9 in Thailand. With identical molecular characteristics, pigs could be a source of MRSA ST398 spread to humans. A minor variation of genetic features and resistance gene carriage in both lineages represented a heterogeneous population and evolution of the endemic clones. A monitoring program and farm management, with prudent antimicrobial uses, should be implemented to reduce spreading. Strict hygiene and personal protection are also necessary to prevent transfer of LA-MRSA to humans.

Antimicrobial resistance in Brachyspira - An increasing problem for disease control.

Across all bacterial species the continuing reduction in susceptibility to antimicrobial agents is a critical and increasing threat for disease control. This mini-review outlines the extent of this problem amongst anaerobic intestinal spirochaetes of the genus Brachyspira, of which there are currently nine officially recognised species. These include some important pathogens that may cause colitis with diarrhoea and/or dysentery in various mammalian and avian species, but most notably in pigs and in adult chickens. The most economically significant pathogen is Brachyspira hyodysenteriae, the spirochaete which causes swine dysentery in countries throughout the world. Control of infections with Brachyspira species has long relied on the prophylactic or therapeutic use of antimicrobials, but increasingly strains with reduced susceptibility and sometimes multidrug resistance to previously effective antimicrobial agents are being encountered. In this mini-review we outline these problems and explain the extent and molecular basis of the emerging resistance. Future control will rely on developing and applying standardised methods for measuring antimicrobial susceptibility; improving surveillance of resistance using traditional phenotypic as well as genomic analysis of known resistance determinants; improving understanding of the molecular basis of resistance to different drug classes; improving farmer and veterinarian education about prudent antimicrobial use so as to reduce selective pressure on the emergence of resistance; and developing alternatives to antimicrobials as a means to control these infections.

Examination of Australian Streptococcus suis isolates from clinically affected pigs in a global context and the genomic characterisation of ST1 as a predictor of virulence.

Streptococcus suis is a major zoonotic pathogen that causes severe disease in both humans and pigs. Australia's pig herd has been quarantined for over 30 years, however S. suis remains a significant cause of disease. In this study, we investigated S. suis from 148 cases of clinical disease in pigs from 46 pig herds over a period of seven years, to determine the level of genetic difference from international isolates that may have arisen over the 30 years of separation. Isolates underwent whole genome sequencing, genome analysis and antimicrobial susceptibility testing. Data was compared at the core genome level to clinical isolates from overseas. Results demonstrated five predominant multi-locus sequence types and two major cps gene types (cps2 and 3). At the core genome level Australian isolates clustered predominantly within one large clade consisting of isolates from the UK, Canada and North America. A small proportion of Australian swine isolates (5%) were phylogenetically associated with south-east Asian and UK isolates, many of which were classified as causing systemic disease, and derived from cases of human and swine disease. Based on this dataset we provide a comprehensive outline of the current S. suis clones associated with disease in Australian pigs and their global context, with the main finding being that, despite three decades of separation, Australian S. suis are genomically similar to overseas strains. In addition, we show that ST1 clones carry a constellation of putative virulence genes not present in other Australian STs.

Antimicrobial Resistance in Commensal Escherichia coli Isolated from Pigs and Pork Derived from Farms Either Routinely Using or Not Using In-Feed Antimicrobials.

The aims of this study were (i) to evaluate whether routine in-feed antimicrobial use in pigs or not resulted in differences in antimicrobial resistance (AMR) E. coli at different pig producing stages, and (ii) to determine whether resistant strains were presented in pig meat postslaughter. A total of 300 commensal E. coli isolates were obtained and examined for antibiograms, AMR genes, plasmid replicons, and molecular types. The isolates were from two farms either using (A) or not using in-feed antimicrobials (NA), sampled four times during the production cycle and once postslaughter. E. coli resistant to aminoglycosides containing aadA1, aadA2, and aadB and extended-spectrum beta-lactamase-producing (ESBLP) E. coli containing blaCTX-M-1 were significantly increased in the nursery and growing periods in farm A compared to farm NA. IncI1-Iγ and IncHI2 were common in the nursery period and were shown to transfer blaCTX-M genes by conjugation. ST10 was the most common type only found in live pigs. ST604, ST877, ST1209, and ST2798 ESBLP were found only in live pigs, whereas ST72, ST302, and ST402 ESBLP were found in pig meat.

Routine Prophylactic Antimicrobial Use Is Associated with Increased Phenotypic and Genotypic Resistance in Commensal Escherichia coli Isolates Recovered from Healthy Fattening Pigs on Farms in Thailand.

This study examined antimicrobial resistance (AMR) profiles in commensal Escherichia coli derived from healthy fattening pigs in Thai farms that used prophylactic antimicrobials (in-feed tiamulin fumarate and amoxicillin) [PAs], therapeutic antimicrobials (injectable enrofloxacin or gentamicin) [TAs], or no antimicrobials [NAs]. Commensal E. coli were used as a proxy for overall AMR on the farms. There was a high level of multidrug resistance in all three categories of farm, with isolates showing resistance to ß-lactams (amoxicillin, ampicillin, and piperacillin) and tetracyclines (tetracycline), and commonly possessing tetA, blaTEM, and plasmid replicons FIB and F. On the other hand, isolates with an extended-spectrum beta-lactamase phenotype (ESBLP) and with resistance to aminoglycosides, chloramphenicol, fluoroquinolones, nitrofurantoin, tiamulin, and trimethoprim/sulfamethoxazole were significantly more common among the PA farms (p < 0.05) than in the other two farm categories. In the PA farms, ESBLP E. coli commonly contained the blaCTX-M-1 group, blaCTX-M-9 group, or both gene groups, and were shown to transfer blaCTX-M genes in a conjugation experiment. E. coli containing N, FIC and A/C replicons were found only in PA farms. In summary, although E. coli isolates from all farms contained a core set of resistance to ß-lactams and tetracyclines, the routine use of PA increased resistance rates to other important antimicrobials.

Molecular detection and isolation of pathogenic Leptospira from asymptomatic humans, domestic animals and water sources in Nan province, a rural area of Thailand.

Leptospirosis is an important zoonotic disease that is often associated with animal carriers and contamination of the environment via infected urine. This study aimed to assess pathogenic leptospiral carriage in Nan province, a rural area of Thailand where leptospirosis is endemic. Samples from 20 villages were obtained during the period 2013 to 2016, comprising urine samples collected from asymptomatic people (n=37) and domestic animals (n=342), and environmental water samples (n=14). Leptospira were cultured in Ellinghauson McCullough Johnson and Harris (EMJH) media. An rrs nested PCR identified 9.92% (95% confidence interval (CI) 6.96-12.88) of the urine and water samples as being positive for Leptospira spp., and phylogenetic analysis was conducted on the 443bp amplicons. Leptospira weilii, which has not previously been identified in Thailand, was recovered from 13 cattle, 9 pigs, 2 dogs, 2 water samples and 1 goat. L. interrogans was found in 4 dogs, 3 pigs, 3 cattle, 1 human and 1 water sample. Four leptospiral strains were isolated and multilocus sequence typing (MLST) analysis was performed on these. Three novel sequence types were identified, including two singletons of L. interrogans in ST26 and ST33, and one of L. weilii in ST94, with this having a close relationship to previous isolates from cases of human leptospirosis in Laos and China. Our results revealed that pathogenic Leptospira occur commonly in asymptomatic domestic animals, humans and environmental water samples in Nan Province, and emphasize the high potential for zoonotic transmission in the province.

Salmonella serovar distribution in cobras (Naja kaouthia), snake-food species, and farm workers at Queen Saovabha Snake Park, Thailand.

The purpose of the current study was to investigate the prevalence and serovar distribution of Salmonella isolates in cobras and their environment at a snake park. A total of 166 fecal or intestinal samples were examined, comprising 39 samples from captive cobras (Naja kaouthia), 70 from recently wild-caught cobras, 19 from wild-caught cobras that had been kept on the farm for over 3 months, 18 from mice (Mus musculus), 12 from frogs (Hoplobatrachus rugulosus), and 8 from farm workers. Specific serological identification was performed, and pulsed-field gel electrophoresis (PFGE) was utilized for DNA analysis. Out of all snakes (n = 128), 20 of the 30 animals used for snake food and 3 of the 8 samples from personnel were positive for Salmonella spp. There were 228 Salmonella isolates, with a total of 29 serovars from subspecies I and IIIb, composed of 24 serovars from cobras and 5 from the other sources. Salmonella Amsterdam was predominant in captive-born and captive cobras, followed by S. Poona and S. Bareilly, respectively (P < 0.05). Salmonella I 4,[5],12:i:- was the sole serovar detected from the mice, while 3 serovars including Ramatgan, I 4,[5],12:e,h:-, and rough strain were detected only from frogs (P < 0.001). Salmonella Derby was only detected in workers. On the basis of the PFGE results, evidence of movement of isolates between human beings and snakes, and between snakes and frogs, was found for S. Poona and S. Wandsworth, respectively. The study suggests that Salmonella spp. act as true residents in the intestinal tract of cobras with high risk of environmental contamination through fecal shedding.

Faecal excretion of intestinal spirochaetes by urban dogs, and their pathogenicity in a chick model of intestinal spirochaetosis.

This study aimed to obtain information about the types of spirochaetes colonising urban dogs in Thailand, and to investigate their pathogenic potential in a day-old chick model of intestinal spirochaetosis. Spirochaetes were isolated from the faeces of six of 47 (12.8%) healthy dogs and 11 of 104 (10.6%) dogs with diarrhoea. Their biochemical properties and 16S ribosomal DNA sequences were analysed. Four isolates were identified as Brachyspira pilosicoli, three resembled "Brachyspira pulli", nine clustered with "Brachyspira canis" and one was similar to Brachyspira intermedia. Canine isolates of B. pilosicoli, "B. canis" and "B. pulli", and control strains of Brachyspira hyodysenteriae, B. pilosicoli and Brachyspira innocens colonised experimentally infected day-old chicks. The chicks did not develop diarrhoea, but were significantly lighter than the non-infected group and those infected with B. innocens after 21 days (P<0.05). Using immunohistochemistry, spirochaetes were observed covering the surface epithelium and in the crypts of chicks in all three groups challenged with the canine isolates. Variable histopathological changes were seen, with the greatest inflammatory cell infiltration into the lamina propria occurring in the group infected with "B. pulli". Canine "B. canis", "B. pulli" and B. pilosicoli isolates may have pathogenic potential.