Exploring the epidemiology of mcr genes, genetic context and plasmids in Enterobacteriaceae originating from pigs and humans on farms in Thailand.

OBJECTIVES: In veterinary medicine, colistin has been widely used as therapeutic and prophylactic agent, and for growth promotion. However, colistin has been re-introduced into treatment of human MDR bacterial infections. We assessed the characteristics and spread of plasmid-borne colistin resistance among healthy pigs, workers with animal-contact and their household members in Thailand. METHODS: WGS and MIC data of 146 mcr-positive isolates from a cross-sectional One Health study were analysed. Long-read sequencing and conjugation were performed for selected isolates. RESULTS: mcr-carrying isolates were detected in 38% of pooled-pig samples and 16% of human faecal samples. Of 143 Escherichia coli and three Escherichia fergusonii, mcr-1, mcr-3, and mcr-9 variants were identified in 96 (65.8%), 61 (41.8%) and one (0.7%) isolate, respectively. Twelve E. coli co-harboured two mcr variants (mcr-1 and mcr-3). Clonal transmission was detected in five out of 164 farms. mcr-1 was mostly harboured by epidemic IncX4 and IncHI1 plasmids (89.9%). Conversely, mcr-3 was harboured by a range of different plasmids. Comparative plasmid studies suggested IncP and IncFII plasmids as possible endemic mcr-3 plasmids in Asian countries. Moreover, mcr-3 was associated with different mobile genetic elements including TnAs2, ISKpn40 and IS26/15DI. Detected genetic signatures (DRs) indicated recent mcr-3 transpositions, underlining the mobilizable nature of the mcr-3 cassette. CONCLUSIONS: The epidemiology of mcr and the possible evolution of successful plasmids and transposition modules should be carefully monitored. Of special concern is the growing number of different horizontal gene transferring pathways encompassing various transposable modules the mcr genes can be shared between bacteria.

Highly conserved composite transposon harbouring aerobactin iuc3 in Klebsiella pneumoniae from pigs.

Klebsiella pneumoniae is an important opportunistic pathogen associated with severe invasive disease in humans. Hypervirulent K. pneumoniae, which are K. pneumoniae with several acquired virulence determinants such as the siderophore aerobactin and others, are more prominent in countries in South and South-East Asia compared to European countries. This Klebsiella pathotype is capable of causing liver abscesses in immunocompetent persons in the community. K. pneumoniae has not been extensively studied in non-human niches. In the present study, K. pneumoniae isolated from caecal samples (n=299) from healthy fattening pigs in Norway were characterized with regard to population structure and virulence determinants. These data were compared to data from a previous study on K. pneumoniae from healthy pigs in Thailand. Lastly, an in-depth plasmid study on K. pneumoniae with aerobactin was performed. Culturing and whole-genome sequencing was applied to detect, confirm and characterize K. pneumoniae isolates. Phylogenetic analysis described the evolutionary relationship and diversity of the isolates, while virulence determinants and sequence types were detected with Kleborate. Long-read sequencing was applied to obtain the complete sequence of virulence plasmids harbouring aerobactin. A total of 48.8 % of the investigated Norwegian pig caecal samples (n=299) were positive for K. pneumoniae. Acquired virulence determinants were detected in 72.6 % of the isolates, the most prominent being aerobactin (69.2 %), all of which were iuc3. In contrast, only 4.6 % of the isolates from Thailand harboured aerobactin. The aerobactin operon was located on potentially conjugative IncFIBK/FIIK plasmids of varying sizes in isolates from both countries. A putative, highly conserved composite transposon with a mean length of 16.2 kb flanked by truncated IS3-family IS407-group insertion sequences was detected on these plasmids, harbouring the aerobactin operon as well as several genes that may confer increased fitness in mammalian hosts. This putative composite transposon was also detected in plasmids harboured by K. pneumoniae from several countries and sources, such as human clinical samples. The high occurrence of K. pneumoniae harbouring aerobactin in Norwegian pigs, taken together with international data, suggest that pigs are a reservoir for K. pneumoniae with iuc3. Truncation of the flanking ISKpn78-element suggest that the putative composite transposon has been permanently integrated into the plasmid, and that it is no longer mobilizable.

Genome-associations of extended-spectrum ß-lactamase producing (ESBL) or AmpC producing E. coli in small and medium pig farms from Khon Kaen province, Thailand.

Thailand is undergoing rapid intensification of livestock production where small subsistence farms and medium sized commercial farms coexist. In medium farms, antimicrobials are prescribed by a veterinarian, whereas in small farms antimicrobial use remains largely unsupervised. The impact of these differences as well as other farming practices on the emergence and composition of antimicrobial resistance genes (ARGs) remains largely unknown. We analyzed 363 genomes of extended-spectrum ß-lactamase producing (ESBL) and/or AmpC producing Escherichia coli recovered from humans and pigs at small and medium farms from the Khon Kaen province, Thailand. We tested for genome-wide associations to identify links between ARGs, host, and farm size. Pig isolates from small farms were associated with mcr and qnr genes conferring resistance to colistin and fluoroquinolones, respectively. In contrast, pig isolates from medium farms were associated with ARGs conferring resistance to drugs commonly used on medium farms (i.e., streptomycin). ESBL plasmids from small farms co-carried ARGs conferring resistance to critically important antimicrobials more frequently compared to plasmid from medium farms. Frequent ARG combinations included blaCTX-M-55 + qnrS1 (29.8% vs 17.5% in small and medium farms, respectively), blaCTX-M-55 + qnrS1 + mcr-3.19 (5% vs 0%), blaCTX-M-14 + qnrS1 (9.3% vs 6.2%), and blaCTX-M-14 + qnrS1 + mcr-1.1 (3.1% vs 0%). The co-location on plasmids of ARGs conferring resistance to critically important antimicrobials as defined by the World Health Organization is concerning, and actions to curb their spread are urgently needed. Legislation on limiting antimicrobial sales and initiatives to better inform farmers and veterinarians on appropriate antimicrobial usage and farm biosecurity could help reduce antimicrobial use on farms.

Expression of IL-23 in gilt endometrium and oviduct after insemination with seminal plasma, spermatozoa or semen extender.

OBJECTIVE: Insemination with spermatozoa, seminal plasma and extender, cause a rapid inflammatory response in pig endometrium, characterized by an influx of neutrophils into the uterus. The transient inflammatory response to semen involves cytokine induction. Potential functions for Interleukin-23 (IL-23) in the inflammatory response to different insemination treatments were examined by studying mRNA expression and immunostaining in gilt oviduct and endometrium 35-40 h after insemination. Insemination was performed with seminal plasma (SP), spermatozoa (SPZ) without SP in the extender Beltsville thawing solution (BTS), or BTS alone. In control gilts an insemination catheter was inserted without anything being inseminated. RESULTS: Results showed that IL-23 mRNA was expressed in oviduct and endometrium after insemination regardless of treatment. There was an approximate two- to fourfold increase in expression of IL-23 mRNA in catheter-insertion control compared with SPZ, SP and BTS treatment groups. IL-23 immunolabelling was detected in a small number of separate cells and in the sub-epithelial connective tissue of the endometrium, the endosalpinx of isthmus and infundibulum. CONCLUSION: In conclusion, insemination with SP, SPZ in BTS, and BTS alone decreased the expression of IL-23 mRNA in the endometrium compared to catheter-insertion control, indicating a possible role for IL-23 in the inflammatory response after insemination in gilts.

Exploring the Antibiotic Resistance Burden in Livestock, Livestock Handlers and Their Non-Livestock Handling Contacts: A One Health Perspective.

Antibiotics are freqeuently used in the livestock sector in low- and middle-income countries for treatment, prophylaxis, and growth promotion. However, there is limited information into the zoonotic prevalence and dissemination patterns of antimicrobial resistance (AMR) within these environments. In this study we used pig farming in Thailand as a model to explore AMR; 156 pig farms were included, comprising of small-sized (<50 sows) and medium-sized (≥100 sows) farms, where bacterial isolates were selectively cultured from animal rectal and human fecal samples. Bacterial isolates were subjected to antimicrobial susceptibility testing (AST), and whole-genome sequencing. Our results indicate extensive zoonotic sharing of antibiotic resistance genes (ARGs) by horizontal gene transfer. Resistance to multiple antibiotics was observed with higher prevalence in medium-scale farms. Zoonotic transmission of colistin resistance in small-scale farms had a dissemination gradient from pigs to handlers to non-livestock contacts. We highly recommend reducing the antimicrobial use in animals' feeds and medications, especially the last resort drug colistin.

Geographic Drivers of Antimicrobial Use and Resistance in Pigs in Khon Kaen Province, Thailand.

In Thailand, pig production has increased considerably in the last decades to meet a growing demand for pork. Antimicrobials are used routinely in intensive pig production to treat infections and increase productivity. However, the use of antimicrobials also contributes to the rise of antimicrobial resistance with potential consequences for animal and human health. Here, we quantify the association between antimicrobial use and resistance rates in extensive and intensive farms with a focus on geographic proximity between farm and drugstores. Of the 164 enrolled farms, 79% reported using antimicrobials for disease prevention, treatment, or as a feed additive. Antimicrobial-resistant E. coli were present in 63% of farms. These drugs included critically important antimicrobials, such as quinolones and penicillins. Medium-scale farms with intensive animal production practices showed higher resistance rates than small-scale farms with extensive practices. Farms with drug-resistant Escherichia coli were located closer to drugstores and a had a higher proportion of disease than farms without drug-resistant E. coli. We found no association between the presence of resistance in humans and antimicrobial use in pigs. Our findings call for actions to improve herd health to reduce the need for antimicrobials and systematic training of veterinarians and drugstore owners on judicious use of antimicrobials in animals to mitigate resistance.

Characterization of Klebsiella pneumoniae complex isolates from pigs and humans in farms in Thailand: population genomic structure, antibiotic resistance and virulence genes.

OBJECTIVES: To define characteristics of Klebsiella pneumoniae complex (hereafter KP) isolates from healthy pigs, farm workers and their household members in Thailand. METHODS: A total of 839 individual rectal swabs from pigs on 164 farms and 271 faecal samples of humans working on pig farms and persons living in the same household in Khon Kaen, Thailand were screened for gut colonization by KP. Genomic sequences were investigated for antibiotic resistance and virulence genes. Phylogenetic analyses were performed in addition to comparison with isolates from previous studies from Thailand. RESULTS: KP was detected in approximately 50% of pig and human samples. In total, 253 KP isolates were obtained: 39% from pigs, 34% from farmers and 26% from individuals living on the same farm but without animal contact. MLST revealed high genetic diversity with 196 different STs distributed over four phylogroups (Kp1 to Kp4). Low prevalence of ESBL-KP (7.5%) and colistin-resistant KP (3.2%) was observed among pigs and humans. Remarkably, four convergent MDR and hypervirulent strains were observed: one from pigs (ST290) and three from humans [ST35, ST3415 (strain 90CP1), ST17 (strain 90CM2)]. Sharing of KP clones among pigs and humans was identified for some STs including ST4788, ST661, ST3541 and ST29. CONCLUSIONS: The study indicated a low prevalence of ESBL and mcr genes among KP isolated from pigs and healthy humans in Thailand and suggested the possibility of zoonotic transmission for a subset of circulating KP clones.

Antibiotic use in pig farms at different levels of intensification-Farmers' practices in northeastern Thailand.

Understanding the patterns and drivers of antibiotic use in livestock is crucial for tailoring efficient incentives for responsible use of antibiotics. Here we compared routines for antibiotic use between pig farms of two different levels of intensification in Khon Kaen province in Thailand. Among the 113 family-owned small-scale farms (up to 50 sows) interviewed did 76% get advice from the pharmacy about how to use the antibiotics and 84% used it primarily for treating disease. Among the 51 medium-scale-farms (100-500 sows) belonging to two companies did 100% get advice about antibiotic use from the company's veterinarian (P<0.0001) and 94% used antibiotics mostly as disease preventive measure (P<0.0001). In 2 small scale farms 3rd generation cephalosporins, tylosin or colistin were used; antibiotics belonging to the group of highest priority critically important antimicrobials for human medicine. Enrofloxacin, belonging to the same group of antimicrobials, was used in 33% of the small-scale and 41% of the medium-scale farms. In the latter farms, the companies supplied 3-4 antibiotics belonging to different classes and those were the only antibiotics used in the farms. The median and mean estimated expenditure on antibiotics per sow was 4.8 USD (IQR = 5.8) for small-scale farms and 7 USD and 3.4 USD for the medium-scale farms belonging to the two respective companies. Our observations suggest to target the following areas when pig farming transitions from small-scale to medium-scale: (i) strengthening access to professional animal health services for all farmers, (ii) review of the competence and role of veterinary pharmacies in selling antibiotics and (iii) adjustment of farming company animal health protocols towards more medically rational use of antibiotics.

Antimicrobial Resistance in Fecal Escherichia coli from Humans and Pigs at Farms at Different Levels of Intensification.

The overall aim of the current study was to test the hypotheses that (i) antibiotic resistance in bacteria were more frequent in clinically health pigs in intensified company owned, medium-scale farms (MSFs) (100-500 sows) than in pigs in family-owned, small-scale farms (SSFs) (1-50 sows) and (ii) that farmers working at the MSFs were more prone to attain antibiotic resistant bacteria than farmers working at SSFs. The study was conducted in North-Eastern Thailand, comprising fecal Escherichia coli isolates from pigs, farmers working with the pigs (contact humans) and persons living in the same household as the farmer (non-contact humans) at 51 MSFs and 113 SSFs. Samples from all farms were also screened for methicillin-resistant staphylococcus aureus (MRSA), which was not detected in pig samples, but was found in one human sample. Susceptibility was tested by disc-diffusion for seven antibiotics commonly used in the study area. Resistance in pig isolates from MSFs were more frequent for chloramphenicol which (P < 0.001), trimethoprim/sulfamethoxazole (P < 0.001) and gentamicin (P < 0.05) compared with isolates from SSFs, whereas the opposite was true for tetracycline (P < 0.01). Resistance in the human isolates was lower than those in the isolates from pigs for tetracycline, trimethoprim/sulfamethoxazole and chloramphenicol (P < 0.001). The frequency of resistance in the contact human samples from SSFs and MSFs did not differ. There was no difference between isolates from contact and non-contact humans for any of the tested antibiotics. Multidrug resistance in isolates from pigs was 26%, significantly higher (P < 0.01) than the 13% from humans. The data indicate that (i) resistance to antibiotics, including those critical and highly important for human medicine, were more common in fecal E. coli from pigs at the MSFs than at the SSFs, whereas (ii) the resistance in fecal E. coli from pig farmers seemed not to be influenced by the level of intensification of the farm they were working at.

A bread-based lyophilized C-strain CSF virus vaccine as an oral vaccine in pigs.

A live attenuated cereal-based classical swine fever (CSF) vaccine for oral application, a form of vaccine that farmers can use themselves, demonstrated the improvement of CSF control in backyard production systems in endemic areas. Due to the dependency on very low storage temperature (-20°C) of the cereal-based oral CSF vaccine, a lyophilized cereal-based oral vaccine has been developed and tested. Although some studies showed total protection against a virulent virus strain, the production procedure is still considered complex. In this study, the lyophilized oral CSF virus, which is easy to produce and could be kept in the form of a bread-based vaccine at 4°C for an extended time, was tested for the ability to induce a humoral immune response in pigs. Among the materials tested as a base for the CSF virus vaccine, plain sliced bread was considered the most appropriate base because of its absorbing ability and virus titre maintenance. Titres of bread-based lyophilized CSF virus vaccine were stable at around 3.67 log10 TCID50 per ml for 7 months at 4°C. Pigs aged 5 and 8 weeks that orally received five bread-based lyophilized CSF virus vaccine showed seroconversion of over 90% at 14 dpv. At 28 dpv, both age groups showed 100% seroconversion. In conclusion, the bread-based lyophilized CSF virus vaccine can be an alternative choice for oral vaccination of pigs. Due to the simple process of production and the need for less virus titre, vaccine prices could be lowered. However, vaccine thermostability has to be improved to allow the vaccine delivery to be less dependent on functioning cool chains.