Multidrug-resistance and mobile colistin resistance (mcr) genes of Salmonella isolates from pork in Thailand during 2014-2017: comparison between two different types of slaughterhouses and retails

Food-producing animals are the major reservoir for Salmonella infections in humans. Salmonella contamination and spread of antimicrobial resistance genes can occur during the production chain of animal products. The aims of this study were to investigate antimicrobial resistance patterns and compare the proportions of multidrug resistance and the presence of mobile colistin resistance (mcr) genes, mcr-1, mcr-2 and mcr-3, among Salmonella isolates which were recovered from pork at two different standard practice slaughterhouses and retails during 2014-2017 in Thailand. Salmonella isolates recovered from good standard practice slaughterhouses (GSH, n=75), below standard practice slaughterhouses (BSH, n=75), good standard practice retails (GRT, n=75) and below standard practice retails (BRT, n=75) were examined for their antimicrobial resistance patterns and the existence of mcr-1 to mcr-3 genes. Salmonella strains of the 4 origins showed similar resistance rates to almost all antimicrobial agents tested. BRT origin (33/75, 44%) had slightly higher proportion of MDR Salmonella than the others group with no statistical difference. Five MDR Salmonella isolates carrying the mcr-3 gene were detected among isolates of all origins, while only 4 isolates (1.33%) displayed colistin resistance phenotype (MIC 4-8 ug/mL). This study revealed that MDR Salmonella isolates have widely spread in both standard and low hygiene practice slaughterhouses and retails. This is the first report of mcr-3 positive MDR Salmonella isolates from pork in Thailand. Effective monitoring program in slaughterhouses and retails should be continually implemented to reduce the contamination of MDR Salmonella carrying the mcr gene to consumers

OXA-48-positive carbapenem-resistant Enterobacteriaceae in a farrow-to-finish pig farm: First report in Thailand

Carbapenem-resistant Enterobacteriaceae (CRE) have emerged as an urgent threat to public health. This study aimed to determine the occurrence of CRE and the carbapenemase genes in a farrow-to-finish pig farm, and to investigate carriage proportion and maintenance of CRE during the pig production cycle. We conducted a cross-sectional study by collecting 200 rectal swabs from healthy pigs of 5 groups: gilts, sows, piglets, weaners, and fatteners. In the longitudinal study, 20 healthy pigs were followed from 2 to 26 weeks old, and rectal swabs were collected from each pig for 5 times. Samples were screened for CRE using MacConkey agar supplemented with meropenem at 0.5 µg/mL. Identification and antimicrobial susceptibility pattern of the recovered isolates were determined using an automated system. PCR was used to detect carbapenemase genes. The occurrence of Enterobacteriaceae isolates with the carbapenem resistant phenotype and/or harboring the blaOXA-48 gene was 3% (6/200) in the cross-sectional study. Groups of sows and piglets had the same occurrence rate at 5% (2/40), while weaner and fattener groups had 2.5% (1/40). In the longitudinal study, CRE were not detected in pigs at an early age; however, two isolates were detected at the age of finishing. This study is the first report of Enterobacteriaceae with a carbapenem resistant phenotype and/or carrying blaOXA-48 gene in pigs in Thailand. Finding CRE in pigs at all age categories including finisher in the study farm underscores the need for active monitoring and surveillance studies to determine the occurrence of CRE in pig farms in Thailand

Epidemiology and Antimicrobial Resistance of Salmonella Isolated from Racehorses and Horsemen in Northeastern Thailand

Salmonella is one of the major causes of foodborne diseases in humans. These bacteria can colonize within the gastrointestinal tracts of both humans and animals, and there have been reports of incidences of Salmonella in horses. This study aimed to investigate the prevalence, serotypes, and antimicrobial resistance of Salmonella isolates from racehorses and horsemen, as well as to explore the possible transmission between horses and humans. Fecal samples from racehorses (247 samples) and horsemen (33 samples) were collected from horse farms located in 3 provinces of upper Northeastern Thailand between March and August 2019. Salmonella was isolated and identified. Broth microdilution was used to determine the minimal inhibitory concentrations (MICs) of the antimicrobial agents for antimicrobial. Salmonella isolates were detected in 4.86% (12/247) of racehorses and 3.03% (1/33) of horsemen. The most commonly found serotypes in the isolates obtained from the racehorses were Abony (25%) and Iganda (16.67%). Only the Tumodi II serotype was found in one horseman. Salmonella isolates collected from the racehorses had been the most resistant to streptomycin (66.67%), while the isolate from a horsemen had been resistant to ampicillin, streptomycin, oxytetracycline, and to tetracycline. Although Salmonella transmission between racehorses and horsemen was not found to be obviously present in this study, the appropriate use of antimicrobials and hygienic procedures are still necessary in order to prevent antimicrobial resistance and the transmission of drug-resistant Salmonella between horses and humans

Prevalence, genetic characterization, and antimicrobial resistance of Salmonella isolated from meat goats in the Northeastern region of Thailand

This study aimed to determine the prevalence, genotypic diversity, and antimicrobial resistance pattern of Salmonella isolated from meat goats in the Northeastern region of Thailand. A total of 1,014 rectal swabs were collected from 30 meat goat farms during April to November, 2018. Salmonella was isolated and identified according to the International Organization for Standardization protocol (ISO-6579:2002/AMD:2017) and serotyped using a slide agglutination test following the Kauffmann-White scheme. An antimicrobial susceptibility test to determine minimal inhibitory concentration (MIC) of 12 antimicrobial agents was performed using a broth microdilution method following the CLSI protocol (2017). Pulsed-field gel electrophoresis (PFGE) of XbaI digested chromosomal DNA was used to determine genotypic diversity of the isolates. The overall prevalence of Salmonella in the meat goats was 1.28%. A total of 13 Salmonella isolates recovered from the meat goats belonged to 4 serovars includings. Weltevreden (n=4), S. Bovismorbificans (n=4), S. Paratyphi B (n=4), and S. Stanley (n=1). Antimicrobial susceptibility testing revealed 2 antibiogram patternS. Eleven Salmonella isolates were susceptible to all antimicrobial agents tested, except sulfamethoxazole, and the other 2 isolates were susceptible to all antimicrobials. Genetic characterization of 13 Salmonella isolates by PFGE revealed 9 PFGE patterns that were grouped into 4 major clusters, A, B, C and D, with an 80% similarity value. This study revealed a low prevalence of Salmonella in meat goats in the Northeastern region of Thailand. Salmonella isolates were susceptible to most antimicrobials tested, with a very high proportion of resistance to sulfamethoxazole being observed

Mapping genetic determinants of the cell-culture growth phenotype of enterovirus 71

Enterovirus 71 (EV71) is a member of the species Human enterovirus A within the family Picornaviridae and is a major causative agent of epidemics of hand, foot and mouth disease associated with severe neurological disease. Three EV71 genogroups, designated A, B and C, have been identified, with 75–84 % nucleotide sequence similarity between them. Two strains, EV71-26M (genogroup B) and EV71-6F (genogroup C), were found to have distinct cell-culture growth (26M, rapid; 6F, slow) and plaque-formation (26M, large; 6F, small) phenotypes. To identify the genome regions responsible for the growth phenotypes of the two strains, a series of chimeric viruses was constructed by exchanging the 5′ untranslated region (UTR), P1 structural protein or P2/P3 non-structural protein gene regions plus the 3′UTR using infectious cDNA clones of both virus strains. Analysis of reciprocal virus chimeras revealed that the 5′UTRs of both strains were compatible, but not responsible for the observed phenotypes. Introduction of the EV71-6F P1 region into the EV71-26M clone resulted in a small-plaque and slow-growth phenotype similar to that of EV71-6F, whereas the reciprocal chimera displayed intermediate-growth and intermediate-sized plaque phenotypes. Introduction of the EV71-26M P2–P3–3′UTR regions into the EV71-6F clone resulted in a large-plaque and rapid-growth phenotype identical to that of strain EV71-26M, whereas the reciprocal chimera retained the background strain large-plaque phenotype. These results indicate that, although both the P1 and P2–P3–3′UTR genome regions influence the EV71 growth phenotype in cell culture, phenotype expression is dependent on specific genome-segment combinations and is not reciprocal

Prevalence of contagious and environmental mastitis-causing bacteria in bulk tank milk and its relationships with milking practices of dairy cattle herds in São Miguel Island (Azores)

This study aimed to assess the degree of contamination of bulk tank milk (BTM) by Staphylococcus spp. and coliform bacteria and to identify major milking practices that help perpetuate them in dairy cattle herds in São Miguel Island. In July 2014, BTM was sampled and a survey concerning local milking practices was conducted on 100 herds. Semi quantitative multiplex polymerase chain reaction detected coagulase-negative staphylococci, Escherichia coli, Staphylococcus aureus, and other coliform bacteria (Klebsiella oxytoca, Klebsiella pneumoniae, andSerratia marcescens) in 100, 75, 59, and 35 % of BTM, respectively. According to multivariable univariate models, on herds not using hot water for cleaning the milking machine and teat liners, there was at least 3.4 more odds (P<0.01) to have S. aureus or coliform bacteria contamination in BTM. The likelihoodoffinding S.aureus inBTMwas higher(P<0.001)on herds without high hygiene during milking, when milking mastitic cows at the end, on abrupt cessation of milking at dry-off, and official milk control implementation. The glove use also favored (odds ratio (OR) 5.8; P<0.01)thedetection ofcoliformbacteriainBTM.Poormilkingpracticesidentified in this study should be avoided in order to decrease S. aureus and coliform bacteria contamination of BTM. Other factors associated with milk quality in São Miguel Island also should be further investigated

Mastitis diagnostics and performance monitoring: a practical approach

In this paper a review is given of frequently used mastitis diagnostic methods in modern dairy practice. Methods used at the quarter, cow, herd and regional or national level are discussed, including their usability for performance monitoring in udder health. Future developments, such as systems in which milk-derived parameters are combined with modern analytical techniques, are discussed. It is concluded that, although much knowledge is available and science is still developing and much knowledge is available, it is not always fully exploited in practice

Development of a reverse genetic system for human enterovirus 71 (HEV71) and the molecular basis of its growth phenotype and adaptation to mice

Human enterovirus 71 (HEV71) is a member of the Human Enterovirus A species within the Family Picornaviridae. Since 1997, HEV71 has emerged as a major cause of epidemics of hand, foot and mouth disease (HFMD) associated with severe neurological disease in the Asia-Pacific region. At the present time, little is known about the pathogenesis of acute neurological disease caused by HEV71. The major aim of this study was to generate infectious cDNA clones of HEV71 and use them as tools for investigating the biology of HEV71 and molecular genetics of HEV71 virulence and pathogenesis. Two infectious cDNA clones of HEV71 clinical isolates, 26M (genotype B3) and 6F (genotype C2) were successfully constructed using a low copy number plasmid vector and an appropriate bacterial host. Transfection of cDNA clones or RNA transcripts derived from these clones produced infectious viruses. Phenotypic characterisation of clone-derived viruses (CDV-26M and CDV-6F) was performed, and CDV-26M and CDV-6F were found to have indistinguishable phenotypes compared to their wild type viruses. Strains HEV71-26M and HEV71-6F were found to have distinct cell culture growth phenotypes. To identify the genome regions responsible for the growth phenotypes of the two strains a series of chimeric viruses were constructed by exchanging the 5„S untranslated region (5„S UTR), structural protein (P1), and nonstructural protein (P2 and P3) gene regions using infectious cDNA clones of both virus strains. Analysis of reciprocal virus chimeras revealed that the 5„S UTR of both strains were compatible but not responsible for the observed phenotypes. Both the P1 and P2-P3 genome regions influence the HEV71 growth phenotype in cell culture, phenotype expression is dependent on specific P1/P2-P3 combinations and is not reciprocal. In the previous study, in order to investigate the pathogenesis of HEV71 infection, a mouse HEV71 model was developed using a mouse-adapted variant of HEV71-26M. Mouse-adapted strain MP-26M caused fore- and/or hindlimb paralysis in mice, whereas HEV71-26M-infected mice did not develop clinical signs of infection at any virus dose or route of inoculation tested. In this study, the molecular basis of mouse adaptation by HEV71 was identified. Nucleotide sequence analysis of HEV71-26M and MP-26M revealed three point mutations in the open reading frame, each resulting in an amino acid substitution in the VP1, VP2 and 2C proteins; no mutations were identified in the untranslated regions of the genome. To determine which of the three amino acid mutations were responsible for the adaptation and virulence of HEV71-26M in mice, recombinant cDNA clones containing one, or a combination of two or three mutations, were constructed. Mouse virulence assays of the mutated viruses clearly demonstrated that a non-conservative amino acid substitution (G710„_E) in the capsid protein VP1 alone was sufficient to confer the mouse virulence phenotype on HEV71. In addition, a mouse oral infection model was established in this study. Oral inoculation with the mouse-adapted HEV71 virus, MP-26M, induced fore-or hindlimb paralysis in newborn mice in an age- and dose-dependent manner. As oral transmission is the natural route of HEV71 infection, this murine HEV71 oral infection model will provide a suitable tool for studying HEV71 pathogenesis, for defining neurological determinants, and for testing vaccine efficacy and immunogenicity in the future

The molecular basis of mouse adaptation by human enterovirus 71

A mouse-adapted strain of human enterovirus 71 (HEV71) was selected by serial passage of a HEV71 clinical isolate (HEV71-26M) in Chinese hamster ovary (CHO) cells (CHO-26M) and in newborn BALB/c mice (MP-26M). Despite improved growth in CHO cells, CHO-26M did not show increased vilrulence in newborn BALB/c mice compared with HEV71-26M. By contrast, infection of newborn mice with MP-26M resulted in severe disease of high mortality. Skeletal muscle was the primary site of replication in mice for both viruses. However, MP-26M infection induced severe necrotizing myositis, whereas CHO-26M infection caused only mild inflammation. MP-26M was also isolated from whole blood, heart, liver, spleen and brain of infected mice. CHO-26M harboured a single mutation within the open reading frame (ORF), resulting in an amino acid substitution of K149-I in the VP2 capsid protein; two further ORF mutations that resulted in amino acid substitutions were identified in MP-26M, located within the VP1 capsid protein (G145-E) and the 2C protein (K216-R). Infectious cDNA clone-derived mutant virus populations containing the mutations identified in CHO-26M and MP-26M were generated in order to study the molecular basis of CHO cell and mouse adaptation. The VP2 (K149-I) change was responsible only for improved growth in CHO cells and did not lead to increased virulence in mice. Of the two amino acid substitutions identified in MP-26M, the VP1 (G145-E) mutation alone was sufficient to increase virulence in mice to the level observed in MP-26M-infected mice

Occurrence of ciprofloxacin resistance, plasmid-mediated quinolone resistance genes and virulence factors in Salmonella enterica serovar Enteritidis isolated from broilers in Thailand isolated from broilers in Thailand

Salmonella Enteritidis is one of the most common serovars associated with gastroenteritis in humans. Fluoroquinolone resistance in non-typhoidal Salmonella including S. Enteritidis has increased globally and is considered as a threat to public health. In this study, we aimed to investigate the occurrence of ciprofloxacin resistance and plasmid-mediated quinolone resistance (PMQR) genes, and examine virulence gene profiles of 69 S. Enteritidis isolates recovered from 46 boot swab and 23 intestinal samples collected from 69 commercial intensive broiler farms in Thailand. Ciprofloxacin susceptibility of these isolates was determined using microbroth dilution method. PCR was used to detect 5 common PMQR genes (qnrA, qnrB, qnrS, aac(6')-Ib-cr and qepA) and 12 important virulence genes (agfA, invA, spaN, prgH, sitC, ssaQ, mgtC, sopB, sifA, tolC, cdtB and spvC). All S. Enteritidis showed reduced susceptibility to ciprofloxacin, with the MIC values of 0.125-0.50 µg/mL. However, these isolates did not carry PMQR genes investigated. The same virulence profile was observed among 69 S. Enteritidis isolates in which 11 virulence genes, except cdtB, were detected. The presence of virulence genes identified in invasive salmonellosis in the S. Enteritidis isolates with reduced susceptibility to ciprofloxacin could be of public health concerns. Our findings underline the need for constant monitoring of ciprofloxacin-resistant S. Enteritidis in poultry production chain to reduce public health risk