Chromosomal and plasmid localization of ileS2 in high-level mupirocin-resistant Staphylococcus pseudintermedius and Staphylococcus aureus isolated from canine and feline origins.

OBJECTIVES: To characterize the mobile genetic elements and genetic localization of ileS2 in high-level mupirocin-resistant (Hi-MupR) methicillin-resistant Staphylococcus pseudintermedius (MRSP) and MRSA isolates recovered from canine and feline clinical samples. METHODS: The identification of bacterial species and presence of mecA and ileS2 genes in MRSP and MRSA isolates were performed using MALDI-TOF MS and PCR, respectively. Antimicrobial resistance (AMR) phenotypes were determined by broth microdilution assays. The genome characteristics, ileS2-containing elements and staphylococcal cassette chromosome mec (SCCmec) were illustrated using complete circular genomes obtained from hybrid assembly of Illumina short-reads and Oxford Nanopore Technologies long-reads. These were analysed through phylogenetic and bioinformatics approaches. RESULTS: A total of 18 MRSP clinical isolates and four MRSA clinical isolates exhibited the Hi-MupR phenotype and carried multiple AMR genes, including mecA and ileS2 genes. MRSP ST182-SCCmec V (n = 6) and ST282-ΨSCCmec57395-t10 (n = 4) contained the ileS2 transposable unit associated with IS257 on the chromosome. Three MRSA ST398-SCCmec V-t034/t4652 isolates carried ∼42 kb pSK41-like ileS2 plasmids, whereas similar ileS2 plasmids lacking tra genes were found in MRSP ST282-ΨSCCmec57395-t72/t21 isolates. Furthermore, a new group of ileS2 plasmids, carried by MRSP ST45-ΨSCCmec57395, ST433-ΨSCCmecKW21-t05 and ST2165-SCCmec IV-t06, and by one MRSA ST398-SCCmec V-t034 strain, shared the plasmid backbone with the cfr/fexA-carrying plasmid pM084526_1 in MRSA ST398. CONCLUSIONS: This study provides the first evidence of ileS2 integration into the S. pseudintermedius chromosome, which is a rare occurrence in staphylococcal species, and plasmids played a pivotal role in dissemination of ileS2 in both staphylococcal species.

Evaluating Quality Management and Diagnostics Microbiology Performance Within an International External Quality Assessment (EQA) Program Serving National One Health Sector Reference Laboratories Across Asia: Experience Amid the Coronavirus Disease 2019 (COVID-19) Pandemic.

BACKGROUND: Strengthening external quality assessment (EQA) services across the One Health sector supports implementation of effective antimicrobial resistance (AMR) control strategies. Here we describe and compare 2 different approaches for conducting virtual laboratory follow-up assessments within an EQA program to evaluate quality management system (QMS) and procedures for pathogen identification and antimicrobial susceptibility testing (AST). METHODS: During the coronavirus disease 2019 (COVID-19) pandemic in 2021 and 2022, 2 laboratory assessment approaches were introduced: virtual-based and survey-based methodologies. The evaluation of 2 underperforming Animal Health laboratories through a virtual-based approach occurred between May and August 2021. This evaluation encompassed the utilization of 3 online meetings and document reviews, performed subsequent to the execution of EQA procedures. Within a distinct group of laboratories, the survey-based assessment was implemented from December 2021 to February 2022, also following EQA procedures. This phase encompassed the dissemination of an online survey to 31 participating laboratories, alongside a sole online consultation meeting involving 4 specific underperforming laboratories. RESULTS: The virtual-based assessment post-EQA aimed to identify gaps and areas for improvement in the laboratory's practices for pathogen identification and AST. This approach was, however, time-intensive, and, hence, only 2 laboratories were assessed. In addition, limited interactions in virtual platforms compromised the assessment quality. The survey-based post-EQA assessment enabled evaluation of 31 laboratories. Despite limitations for in-depth analysis of each procedure, gaps in QMS across multiple laboratories were identified and tailored laboratory-specific recommendations were provided. CONCLUSIONS: Reliable internet and plans for efficient time management, post-EQA virtual laboratory follow-up assessments are an effective alternative when conducting onsite evaluation is infeasible as observed during the COVID-19 pandemic, although the successful implementation of remediation plans will likely require in person assessments. We advocate application of hybrid approaches (both onsite and virtual) for targeted capacity building of AMR procedures with the ability to implement and oversee the process.

Genomic characterization of antimicrobial resistance in mcr-carrying ESBL-producing Escherichia coli from pigs and humans.

Whole-genome sequencing (WGS) was conducted to characterize mcr-carrying extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli (n=7). These E. coli isolates originated from two pigs (TH2 and TH3) and two humans (TH8 and TH9) from Thailand, and three pigs from Lao PDR (LA1, LA2 and LA3). Four E. coli sequence types/serotypes - ST6833/H20 (TH2 and TH3), ST48/O160:H40 (TH8 and TH9), ST5708/H45 (LA1) and ST10562/O148:H30 (LA2 and LA3) - were identified. The plasmid replicon type IncF was identified in all isolates. The point mutations Ser31Thr in PmrA and His2Arg in PmrB were found concurrently in all isolates (colistin MIC=4-8 µg ml-1). LA1 contained up to five point mutations in PmrB, and the colistin MIC was not significantly different from that for the other isolates. All mcr-1.1 was located in the ISApl1-mcr-1-pap2 element, while all mcr-3.1 was located in the TnAs2-mcr-3.1-dgkA-ISKpn40 element. The mcr-3.1 and blaCTX-M-55 genes were co-localized on the same plasmid, which concurrently contained cml, qnrS1 and tmrB. The blaCTX-M-55 and mcr-3.1 genes were located on conjugative plasmids and could be transferred horizontally under selective pressure from ampicillin or colistin. In conclusion, comprehensive insights into the genomic information of ESBL-producing E. coli harbouring mcr were obtained. As mcr-carrying ESBL-producing E. coli were detected in pigs and humans, a holistic and multisectoral One Health approach is required to contain antimicrobial resistance (AMR).

Plasmid profile analysis of Escherichia coli and Salmonella enterica isolated from pigs, pork and humans.

This study aimed to determine the epidemiology and association of antimicrobial resistance (AMR) among Escherichia coli and Salmonella in Thailand. The E. coli (n = 1047) and Salmonella (n = 816) isolates from pigs, pork and humans were screened for 18 replicons including HI1, HI2, I1-γ, X, L/M, N, FIA, FIB, W, Y, P, FIC, A/C, T, FIIAs, F, K and B/O using polymerase chain reaction-based replicon typing. The E. coli (n = 26) and Salmonella (n = 3) isolates carrying IncF family replicons, ESBL and/or mcr genes were determined for FAB formula. IncF represented the major type of plasmids. Sixteen and eleven Inc groups were identified in E. coli (85.3%) and Salmonella (25.7%), respectively. The predominant replicon patterns between E. coli and Salmonella were IncK-F (23.7%) and IncF (46.2%). Significant correlations (P < 0.05) were observed between plasmid-replicon type and resistance phenotype. Plasmid replicon types were significantly different among sources of isolates and sampling periods. The most common FAB types between E. coli and Salmonella were F2:A-:B- (30.8%) and S1:A-:B- (66.7%), respectively. In conclusion, various plasmids present in E. coli and Salmonella. Responsible and prudent use of antimicrobials is suggested to reduce the selective pressures that favour the spread of AMR determinants. Further studies to understand the evolution of R plasmids and their contribution to the dissemination of AMR genes are warranted.

Metagenomics of antimicrobial and heavy metal resistance in the cecal microbiome of fattening pigs raised without antibiotics.

This study aimed to detect the cecal microbiome, antimicrobial resistance (AMR) and heavy metal resistance genes (MRGs) in fattening pigs raised under antibiotic-free (ABF) conditions compared with ordinary industrial pigs (control, C) using whole-genome shotgun sequencing. ABF pigs showed the enrichment of Prevotella (33%) and Lactobacillus (13%), whereas Escherichia coli (40%), Fusobacterium and Bacteroides (each at 4%) were notably observed in the C group. Distinct clusters of cecal microbiota of ABF and C pigs were revealed; however, microbiota of some C pigs (C1) appeared in the same cluster as ABF and were totally separated from the remaining C pigs (C2). For AMR genes, the highest abundance tet(Q) (35.7%) and mef(A) (12.7%) were markedly observed in the ABF group whereas tet(Q) (26.2%) and tet(W) (10.4%) were shown in the C group. tet(Q) was positively correlated to Prevotella in ABF and C1 samples. In the C2 group, the prominent tet(W) was positively correlated to Fusobacterium and Bacteroides Pigs have never received tetracycline but pregnant sows used chlortetracycline once 7 d before parturition. Chromosomal Cu and Zn resistance genes were also shown in both groups regardless the received Cu and Zn feed additives. A higher abundance of multi-metal resistance genes was observed in the C group (44%) compared with the ABF group (41%). In conclusion, the microbiome clusters in some C pigs were similar to that in ABF pigs. High abundant tetracycline resistance genes interrelated to major bacteria were observed in both ABF and C pigs. MRGs were also observed.IMPORTANCE: Owing to the increased problem of AMR in farm animals, raising farm animals without antibiotics is one method that could solve this problem. Our study showed that only some tetracycline and macrolide resistance genes, tet(Q), tet(W) and mef(A), were markedly abundant in ABF and C groups. The tet(Q) and tet(W) genes interrelated to different predominant bacteria in each group, showing the potential role of major bacteria as reservoirs of AMR genes. In addition, chromosomal Cu and Zn resistance genes were also observed in both pig groups, not depending on the use of Cu and Zn additives in both farms. The association of MRGs and AMR genotypes and phenotypes together with the method to re-sensitize bacteria to antibiotics should be studied further to unveil the cause of high resistance genes and solve the problems.

Novel dual regulators of Pseudomonas aeruginosa essential for productive biofilms and virulence.

Gene regulation network in Pseudomonas aeruginosa is complex. With a relatively large genome (6.2 Mb), there is a significant portion of genes that are proven or predicted to be transcriptional regulators. Many of these regulators have been shown to play important roles in biofilm formation and maintenance. In this study, we present a novel transcriptional regulator, PA1226, which modulates biofilm formation and virulence in P. aeruginosa. Mutation in the gene encoding this regulator abolished the ability of P. aeruginosa to produce biofilms in vitro, without any effect on the planktonic growth. This regulator is also essential for the in vivo fitness and pathogenesis in both Drosophila melanogaster and BALB/c mouse lung infection models. Transcriptome analysis revealed that PA1226 regulates many essential virulence genes/pathways, including those involved in alginate, pili, and LPS biosynthesis. Genes/operons directly regulated by PA1226 and potential binding sequences were identified via ChIP-seq. Attempts to confirm the binding sequences by electrophoretic mobility shift assay led to the discovery of a co-regulator, PA1413, via co-immunoprecipitation assay. PA1226 and PA1413 were shown to bind collaboratively to the promoter regions of their regulons. A model is proposed, summarizing our finding on this novel dual-regulation system.

Two Regulators, PA3898 and PA2100, Modulate the Pseudomonas aeruginosa Multidrug Resistance MexAB-OprM and EmrAB Efflux Pumps and Biofilm Formation.

It is generally believed that the Pseudomonas aeruginosa biofilm matrix itself acts as a molecular sieve or sink that contributes to significant levels of drug resistance, but it is becoming more apparent that multidrug efflux pumps induced during biofilm growth significantly enhance resistance levels. We present here a novel transcriptional regulator, PA3898, which controls biofilm formation and multidrug efflux pumps in P. aeruginosa A mutant of this regulator significantly reduced the ability of P. aeruginosa to produce biofilm in vitro and affected its in vivo fitness and pathogenesis in Drosophila melanogaster and BALB/c mouse lung infection models. Transcriptome analysis revealed that PA3898 modulates essential virulence genes/pathways, including multidrug efflux pumps and phenazine biosynthesis. Chromatin immunoprecipitation sequencing (ChIP-seq) identified its DNA binding sequences and confirmed that PA3898 directly interacts with promoter regions of four genes/operons, two of which are mexAB-oprM and phz2 Coimmunoprecipitation revealed a regulatory partner of PA3898 as PA2100, and both are required for binding to DNA in electrophoretic mobility shift assays. PA3898 and PA2100 were given the names MdrR1 and MdrR2, respectively, as novel repressors of the mexAB-oprM multidrug efflux operon and activators for another multidrug efflux pump, EmrAB. The interaction between MdrR1 and MdrR2 at the promoter regions of their regulons was further characterized via localized surface plasmon resonance and DNA footprinting. These regulators directly repress the mexAB-oprM operon, independent of its well-established MexR regulator. Mutants of mdrR1 and mdrR2 caused increased resistance to multiple antibiotics in P. aeruginosa, validating the significance of these newly discovered regulators.

Prevalence and antimicrobial resistance in Salmonella enterica isolated from broiler chickens, pigs and meat products in Thailand-Cambodia border provinces.

This study aimed to examine the prevalence and antimicrobial resistance (AMR) of Salmonella isolates from broiler chickens, pigs and their associated meat products in the Thailand-Cambodia border provinces. A total of 941 samples were collected from pigs and broiler chickens at slaughter houses and from carcasses at local fresh markets in Sa Kaeo, Thailand (n = 554) and Banteay Meanchey, Cambodia (n = 387) in 2014 and 2015. From these samples, 345 Salmonella isolates were collected from Sa Keao (n = 145; 23%) and Banteay Meanchey (n = 200; 47%) and assayed for antimicrobial susceptibility, class 1 integrons and extended-spectrum ß-lactamase (ESBL) genes. Serovars Typhimurium (29%) and Rissen (29%) were the most common serotypes found in Thai and Cambodian isolates, respectively. Multidrug resistance was detected in 34% and 52% of isolates from Sa Keao and Banteay Meanchey, respectively. The majority of the Thai isolates were resistant to ampicillin (72.4%), whereas most Cambodian isolates were resistant to sulfamethoxazole (71%). Eleven isolates from Sa Keao and 44 from Banteay Meanchey carried class 1 integrons comprising resistance gene cassettes. The most common gene cassette array was dfrA12-aadA2 (61.1%). Six isolates were ESBL producers. The ß-lactamase genes found included blaTEM-1 , blaCTX-M-55 and blaCMY-2 . Some of these class 1 integrons and ESBL genes were located on conjugative plasmid. In conclusion, multidrug-resistant Salmonella are common in pigs, chickens and their products in the Thailand-Cambodia border provinces. Our findings indicate that class 1 integrons play a role in spread of AMR in the strains in this study.

Occurrence and molecular characteristics of antimicrobial resistance of Escherichia coli from broilers, pigs and meat products in Thailand and Cambodia provinces.

Nine hundred and forty-one samples were collected in Sa Keao, Thailand (n = 554) and Banteay Meanchey, Cambodia (n = 387) from July 2014 to January 2015. A total of 667 Escherichia coli isolates (381 isolates from Sa Keao and 286 isolates from Banteay Meanchey) were obtained and examined for antimicrobial susceptibility, class 1 integrons, ESBL genes and horizontal transfer of resistance determinants. Prevalence of E. coli in pig and broiler carcass samples from slaughterhouses and fresh markets was 36-85% in Sa Keao and 11-69% in Banteay Meanchey. The majority of these isolates were multidrug resistant (75.3%). Class 1 integrons were common in both Thai (47%) and Cambodian (62%) isolates, of which four resistance gene cassette arrays including aadA1, dfrA1-aadA1, dfrA12-aadA2 and aadA2-linF were identified. Class 1 integrons in two broiler isolates from Sa Keao (dfrA12-aadA2) and one broiler isolate from Banteay Meanchey (dfrA1-aadA1) were horizontally transferable. Sixteen isolates were confirmed to be ESBL-producing strains with ESBL gene blaCTX-M-15 , broad spectrum ß-lactamase gene blaTEM-1 and the AmpC gene blaCMY-2 being detected. The blaTEM-1 gene was most prevalent and located on a conjugative plasmid.

Distribution and expression of the Ade multidrug efflux systems in Acinetobacter baumannii clinical isolates.

One hundred Acinetobacter baumannii clinical isolates were examined for inhibitory effect of reserpine and carbonyl cyanide m-chlorophenylhydrazone (CCCP) on the antimicrobial susceptibility and expression of 4 resistant-nodulation-cell division (RND)-type multidrug efflux systems, including AdeABC, AdeDE, AdeIJK, and AdeFGH, using RT-PCR. Ten A. baumannii isolates expressing AdeABC, AdeIJK, or AdeFGH were randomly selected for determination of transcription level and regulatory mutations. While all the isolates were resistant to multiple drugs, the reserpine and CCCP experiment showed that the multidrug resistance phenotype in most A. baumannii isolates was associated with efflux pumps. Most isolates expressed at least one of the RND-type efflux pumps tested (97%). AdeIJK expression was most common (97%), but none of the isolates produced AdeDE. Fifty-two percent of the A. baumannii isolates simultaneously produced up to 3 RND-type efflux systems (i.e., AdeABC, AdeFGH, and AdeIJK). No good correlation between the expression of RND-type efflux pumps and the type of antimicrobial resistance was observed. Overexpression of AdeABC, AdeIJK, and AdeFGH was not always related to the presence of mutations in their corresponding regulatory genes. This study highlights (i) the universal presence of the RND-type efflux pumps with variable levels of expression level among the A. baumannii in this collection and (ii) the complexity of their regulation of expression.

Characterization of Antimicrobial Resistance in Salmonella enterica Isolated from Pork, Chicken Meat, and Humans in Northeastern Thailand.

A total of 221 Salmonella enterica from raw pork (n=64), raw chicken (n=80), and humans (n=77) were characterized for antimicrobial resistance phenotypes and genotypes and virulence plasmid-associated genes. Most Salmonella isolates (95.9%) were multidrug resistant and exhibited high resistance to sulfamethoxazole (96.4%), streptomycin (93.2%), spectinomycin (76.5%), tetracycline (73.3%), ampicillin (70.1%), and trimethoprim (60.2%). Forty-one percent of all isolates were intI1-positive, of which 60% carried class 1 integrons with variable region ranging in size from 0.2 to 2.0 kb. Six integron profiles (IP-I to IP-VI) were defined. The dfrA12-aadA2 cassette was most prevalent (66.7%). Class 1 integrons with the dfrA12-aadA2 cassette in five pork isolates could be horizontally transferred. Three pork isolates carried Salmonella genomic island 1 (SGI1), of which a serovar Anatum harbored SGI1 gene cluster located between thdF and int2. Two single-point mutations (i.e., G-259-T and C-248-T) in gyrA leading to Asp-87-Tyr and Ser-83-Phe substitutions in GyrA, respectively, were detected. Of all plasmid-mediated quinolone resistance genes tested, only qnrS (4.1%) and qnrB (1.8%) were found. The virulence plasmid-associated genes including spvC, pefA, and rck were identified in 8.1%, 1.8%, and 1.4% of all Salmonella isolates, respectively.

Simultaneous overexpression of multidrug efflux pumps in Pseudomonas aeruginosa non-cystic fibrosis clinical isolates.

The purpose of this study was to examine expression and regulation of 6 multidrug efflux systems, including MexAB-OprM, MexCD-OprJ, MexEF-OprN, MexXY, MexJK, and MexVW, in 13 non-cystic fibrosis (CF) clinical isolates of Pseudomonas aeruginosa. These isolates displayed a high level of resistance to many clinically important antibiotics. Some isolates simultaneously overexpressed up to 4 different Mex systems, as determined by quantitative real-time reverse transcription PCR. None of the isolates overexpressed MexCD-OprJ, and only 1 isolate overproduced MexJK. All the isolates overexpressed MexXY, while overexpression of MexEF-OprN and MexVW was common. DNA sequencing analysis of regulatory genes showed that no clear correlation could be established among (i) the presence of mutations, (ii) the type of mutations, (iii) the expression level of the Mex systems, and (iv) resistance to antibiotic substrates. The results suggest that the concomitant overexpression of some Mex systems may superimpose their antimicrobial drug efflux capabilities, contributing to the multidrug resistance phenotype in the P. aeruginosa non-CF clinical isolates. The existence of uncharacterized regulators for the Mex systems was signified.

Molecular basis of the persistence of chloramphenicol resistance among Escherichia coli and Salmonella spp. from pigs, pork and humans in Thailand.

This study aimed to investigate the potential mechanisms associated with the persistence of chloramphenicol (CHP) resistance in Escherichia coli and Salmonella enterica isolated from pigs, pork, and humans in Thailand. The CHP-resistant E. coli (n = 106) and Salmonella (n = 57) isolates were tested for their CHP susceptibility in the presence and absence of phenylalanine arginine ß-naphthylamide (PAßN). The potential co-selection of CHP resistance was investigated through conjugation experiments. Whole genome sequencing (WGS) was performed to analyze the E. coli (E329, E333, and E290) and Salmonella (SA448, SA461, and SA515) isolates with high CHP MIC (32-256 µg/mL) and predominant plasmid replicon types. The presence of PAßN significantly reduced the CHP MICs (≥4-fold) in most E. coli (67.9%) and Salmonella (64.9%). Ampicillin, tetracycline, and streptomycin co-selected for CHP-resistant Salmonella and E. coli-transconjugants carrying cmlA. IncF plasmids were mostly detected in cmlA carrying Salmonella (IncFIIAs) and E. coli (IncFIB and IncF) transconjugants. The WGS analysis revealed that class1 integrons with cmlA1 gene cassette flanked by IS26 and TnAs1 were located on IncX1 plasmid, IncFIA(HI1)/HI1B plasmids and IncFII/FIB plasmids. IncFIA(HI1)/HI1B/Q1in SA448 contained catA flanked by IS1B and TnAs3. In conclusion, cross resistance through proton motive force-dependent mechanisms and co-selection by other antimicrobial agents involved the persistence of CHP-resistance in E. coli in this collection. Dissemination of CHP-resistance genes was potentially facilitated by mobilization via mobile genetic elements.

Microbiological Quality and Antimicrobial Resistance of Commercial Probiotic Products for Food-Producing Animals.

Probiotics have been popularly used in livestock production as an alternative to antibiotics. This study aimed to investigate the microbiological quality and phenotypic and genotypic antimicrobial resistance of bacteria in probiotic products sold for food animals. A total of 45 probiotic products were examined for the number of viable cells, species, and antimicrobial susceptibility; the contamination of Escherichia coli and Salmonella; and the presence of 112 genes encoding resistance to clinically important antimicrobials and transferability of AMR determinants. The results showed that 29 of 45 products (64.4%) were incorrectly labeled in either number of viable cells or bacterial species. None of the tested products were contaminated with E. coli and Salmonella. A total of 33 out of 64 bacterial isolates (51.6%) exhibited resistance to at least one antimicrobial agent. Of the 45 products tested, 16 (35.5%) carried AMR genes. Almost all AMR genes detected in probiotic products were not correlated to the AMR phenotype of probiotic strains formulated in the products. Three streptomycin-resistant Lactobacillus isolates could horizontally transfer their AMR determinants. The findings demonstrated that the probiotic products could serve as reservoirs for the spread of AMR genes and may not yield benefits to animals as claimed. The need for the adequate quality control of probiotic products is highlighted.

Metagenomic insights into isolable bacterial communities and antimicrobial resistance in airborne dust from pig farms.

This study aims to investigate bacterial communities and antimicrobial resistance (AMR) in airborne dust from pig farms. Airborne dust, pig feces and feed were collected from nine pig farms in Thailand. Airborne dust samples were collected from upwind and downwind (25 meters from pig house), and inside (in the middle of the pig house) of the selected pig house. Pig feces and feed samples were individually collected from the pen floor and feed trough from the same pig house where airborne dust was collected. A direct total bacteria count on each sampling plate was conducted and averaged. The ESKAPE pathogens together with Escherichia coli, Salmonella, and Streptococcus were examined. A total of 163 bacterial isolates were collected and tested for MICs. Pooled bacteria from the inside airborne dust samples were analyzed using Metagenomic Sequencing. The highest bacterial concentration (1.9-11.2 × 103 CFU/m3) was found inside pig houses. Staphylococcus (n = 37) and Enterococcus (n = 36) were most frequent bacterial species. Salmonella (n = 3) were exclusively isolated from feed and feces. Target bacteria showed a variety of resistance phenotypes, and the same bacterial species with the same resistance phenotype were found in airborne dust, feed and fecal from each farm. Metagenomic Sequencing analysis revealed 1,652 bacterial species across all pig farms, of which the predominant bacterial phylum was Bacillota. One hundred fifty-nine AMR genes of 12 different antibiotic classes were identified, with aminoglycoside resistance genes (24%) being the most prevalent. A total of 251 different plasmids were discovered, and the same plasmid was detected in multiple farms. In conclusion, the phenotypic and metagenomic results demonstrated that airborne dust from pig farms contained a diverse array of bacterial species and genes encoding resistance to a range of clinically important antimicrobial agents, indicating the significant role in the spread of AMR bacterial pathogens with potential hazards to human health. Policy measurements to address AMR in airborne dust from livestock farms are mandatory.

Aminoglycoside resistance mechanisms in Pseudomonas aeruginosa isolates from non-cystic fibrosis patients in Thailand.

This study aimed to examine aminoglycosides (AMGs) resistance mechanisms, including the AMG-modifying enzyme genes, mexXY, rplY, nuoG, and galU, in the Pseudomonas aeruginosa non-cystic fibrosis (CF) isolates in Thailand. One hundred P. aeruginosa isolates from non-CF patients were examined for susceptibility to AMGs and for the presence of 10 AMG-modifying enzyme genes. Thirty randomly selected isolates were tested for transcription of mexXY and nuoG and mutations in rplY and galU. All the P. aeruginosa isolates exhibited simultaneous resistance to at least 4 AMGs. High resistance rates to amikacin (92%), gentamicin (95%), streptomycin (99%), and tobramycin (96%) were observed, and all isolates were resistant to kanamycin, neomycin, and spectinomycin. Nine AMG-modifying enzyme genes were detected, including aadA1 (84%), aadB (84%), aadA2 (67%), ant(2″)-Ia (72%), strA-strB (70%), aph(3')-IIb (57%), aac(3')-Ia (40%), and aac(6')-IIa (27%). None of the isolates harbored aac(6')-IIb. Of 30 isolates tested, all but 1 isolate expressed MexXY. Two isolates did not express nuoG. Six isolates carried an amino acid change in RplY, but none of the isolates harbored mutation in galU. The results indicated that the AMG-modifying enzyme genes were widespread among the P. aeruginosa non-CF isolates. The MexXY efflux pump and inactivation for rplY played a role in AMG resistance but disruption of nuoG or galU did not.

Antimicrobial resistance, Extended-Spectrum ß-Lactamase production and virulence genes in Salmonella enterica and Escherichia coli isolates from estuarine environment.

The impact of antimicrobial resistance (AMR) on global public health has been widely documented. AMR in the environment poses a serious threat to both human and animal health but is frequently overlooked. This study aimed to characterize the association between phenotype and genotype of AMR, virulence genes and Extended-Spectrum ß-Lactamase (ESBL) production from estuarine environment. The Salmonella (n = 126) and E. coli (n = 409) were isolated from oysters and estuarine water in Thailand. The isolates of Salmonella (96.9%) and E. coli (91.4%) showed resistance to at least one antimicrobial agent. Multidrug resistance (MDR) was 40.1% of Salmonella and 23.0% of E. coli. Resistance to sulfamethoxazole was most common in Salmonella (95.2%) and E. coli (77.8%). The common resistance genes found in Salmonella were sul3 (14.3%), followed by blaTEM (11.9%), and cmlA (11.9%), while most E. coli were blaTEM (31.5%) and tetA (25.4%). The ESBL production was detected in Salmonella (1.6%, n = 2) of which one isolate was positive to blaTEM-1. Eight E. coli isolates (2.0%) were ESBL producers, of which three isolates carried blaCTX-M-55 and one isolate was blaTEM-1. Predominant virulence genes identified in Salmonella were invA (77.0%), stn (77.0%), and fimA (69.0%), while those in E. coli isolates were stx1 (17.8%), lt (11.7%), and stx2 (1.2%). Logistic regression models showed the statistical association between resistance phenotype, virulence genes and ESBL production (p < 0.05). The findings highlighted that estuarine environment were potential hotspots of resistance. One Health should be implemented to prevent AMR bacteria spreading.

Antimicrobial resistance, virulence, and phylogenetic characteristics of Escherichia coli isolates from clinically healthy swine.

A total of 344 commensal Escherichia coli isolates from clinically healthy pigs were examined for antimicrobial resistance phenotypes, class 1 integrons, resistance genes, virulence gene profile, and phylogenetic groups. The majority of E. coli isolates were resistant to tetracycline (96.2%) and ampicillin (91.6%). Up to 98% were multidrug resistant. Seventy-three percent of the isolates carried class 1 integrons. Inserted-gene cassette arrays in variable regions included incomplete sat, aadA22, aadA1, dfrA12-aadA2, and sat-psp-aadA2, of which the aadA2 gene cassette was most prevalent (42.9%). Horizontal transfer was detected in eight E. coli isolates carrying class 1 integrons with dfrA12-aadA2 gene cassette array. Sixteen resistance genes were identified among the E. coli isolates with corresponding resistance phenotype. Ten virulence genes (including elt, estA, estB, astA, faeG, fasA, fedA, eaeA, paa, and sepA) were detected, of which fasA was most commonly found (98.3%). Most of the E. coli isolates belonged to phylogenetic group B1. Significantly positive associations were observed between some virulence genes and some resistance phenotypes and genotypes (p < 0.05). The results support a finding that commensal E. coli have a role as reservoirs for antimicrobial resistance-encoding genes and virulence determinants.

Class 1 integrons in Pseudomonas aeruginosa and Acinetobacter baumannii isolated from clinical isolates.

Resistance to various antimicrobial agents is an increasing problem in Pseudomonas aeruginosa and Acinetobacter baumannii infections. In this study, the roles of integrons were examined in 101 P. aeruginosa isolates and 176 A. baumannii isolates from patients. The frequencies and characteristics of class 1, 2 and 3 integrons were investigated and the horizontal transfer of integrons was assessed. Among these isolates, class 1 integrons with a resistance gene cassette were detected in 69.3% of P. aeruginosa and 31.8% of A. baumannii isolates, but class 2 and 3 integrons were not found. Five novel gene cassette arrays were identified in P. aeruginosa: aacA7-cmlA, aadB-blaOXA,-o-aadA1, aadB-arr-2-cmlA-blaOXA,-o-aadA1, aadB-cmlA-aadA1 and aadB-cmlA-blaOXA-10-aadA15. The integrons found in A. baumannii isolates in this study were previously reported. Horizontal transfer of some class 1 integrons was detected in both P. aeruginosa (2/70) and A. baumannii (5/57). These data confirm the high prevalence of class 1 integrons with a variety of gene cassette combinations among multidrug-resistant P. aeruginosa and A. baumannii clinical isolates.

Clinically healthy household dogs and cats as carriers of multidrug-resistant Salmonella enterica with variable R plasmids.

Introduction. Antimicrobial resistance (AMR) is a One Health issue concerning humans, animals and the environment and a unified One Health approach is required to contain this problematic issue. Dogs and cats are popular pet animals and are known to carry many bacterial pathogens that are of public health importance, including Salmonella. However, data on AMR in companion animals is limited.Gap statement. Scant AMR data from bacteria originating from companion animals limits an accurate assessment of the impacts of pet-animal-related AMR on public health.Purpose. This study aimed to phenotypically and genetically investigate AMR in Salmonella isolated from pet dogs and cats in Thailand.Methodology. Salmonella enterica were isolated from pet dogs (n=159) and cats (n=19) in Thailand between 2016 and 2019. All isolates were serotyped. Phenotypic and genotypic antimicrobial resistance was examined. PCR-based replicon typing, replicon sequence typing and plasmid multilocus sequence typing were conducted to characterize plasmids.Results. Seventy-seven serovars were identified, with serovars Weltevreden (9.6%) and Stockholm (9.0%) the most common. Most of the isolates (34.3%) were multidrug-resistant. The serovar Stockholm was an ESBL-producer and carried the ß-lactamase genes bla TEM-1 and bla CTX-M-55. The plasmid-mediated quinolone resistance (PMQR) gene, qnrS, was also detected (10.1%). Class 1 integrons carrying the dfrA12-aadA2 cassette array were most frequent (45.9%). Five plasmid replicon types as IncA/C (0.6%), N (1.1%), IncFIIA (28.7%), IncHI1 (2.2%), and IncI1 (3.4%) were identified. Based on the pMLST typing scheme (n=9), plasmids were assigned into five different STs including IncA/C-ST6 (n=1), IncH1-ST16 (n=4), IncI1-ST3 (n=1), IncI1-ST60 (n=1) and IncI1-ST136 (n=1). The ST 16 of IncHI1 plasmid was a novel plasmid ST. Subtyping F-type plasmids using the RST scheme (n=9) revealed four different combinations of replicons including S1:A-:B- (n=4), S1:A-:B22 (n=2), S3:A-:B- (n=1) and S-:A-:B47 (n=1).Conclusions. Our findings highlight the role of clinically healthy household dogs and cats as carriers of AMR Salmonella strains with different R plasmid. The implementation of AMR phenotypes instigation and genotypic monitoring and surveillance programmes in companion animals are imperative as integral components of the One Health framework.