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.

Multidrug-resistant Escherichia coli causing canine pyometra and urinary tract infections are genetically related but distinct from those causing prostatic abscesses.

Despite extensive characterisation of uropathogenic Escherichia coli (UPEC) causing urinary tract infections (UTIs), the genetic background of non-urinary extraintestinal pathogenic E. coli (ExPEC) in companion animals remains inadequately understood. In this study, we characterised virulence traits of 104 E. coli isolated from canine pyometra (n = 61) and prostatic abscesses (PAs) (n = 38), and bloodstream infections (BSIs) in dogs (n = 2), and cats (n = 3). A stronger association with UPEC of pyometra strains in comparison to PA strains was revealed. Notably, 44 isolates exhibited resistance to third-generation cephalosporins and/or fluoroquinolones, 15 were extended-spectrum ß-lactamase-producers. Twelve multidrug-resistant (MDR) strains, isolated from pyometra (n = 4), PAs (n = 5), and BSIs (n = 3), along with 7 previously characterised UPEC strains from dogs and cats, were sequenced. Genomic characteristics revealed that MDR E. coli associated with UTIs, pyometra, and BSIs belonged to international high-risk E. coli clones, including sequence type (ST) 38, ST131, ST617, ST648, and ST1193. However, PA strains belonged to distinct lineages, including ST12, ST44, ST457, ST744, and ST13037. The coreSNPs, cgMLST, and pan-genome illustrated intra-clonal variations within the same ST from different sources. The high-risk ST131 and ST1193 (phylogroup B2) contained high numbers of ExPEC virulence genes on pathogenicity islands, predominating in pyometra and UTI. Hybrid MDR/virulence IncF multi-replicon plasmids, containing aerobactin genes, were commonly found in non-B2 phylogroups from all sources. These findings offer genomic insights into non-urinary ExPEC, highlighting its potential for invasive infections in pets beyond UTIs, particularly with regards to high-risk global clones.

First report of blaOXA-181-carrying IncX3 plasmids in multidrug-resistant Enterobacter hormaechei and Serratia nevei recovered from canine and feline opportunistic infections.

Whole-genome sequence analysis of six Enterobacter hormaechei and two Serratia nevei strains, using a hybrid assembly of Illumina and Oxford Nanopore Technologies sequencing, revealed the presence of the epidemic blaOXA-181-carrying IncX3 plasmids co-harboring qnrS1 and ∆ere(A) genes, as well as multiple multidrug resistance (MDR) plasmids disseminating in all strains, originated from dogs and cats in Thailand. The subspecies and sequence types (ST) of the E. hormaechei strains recovered from canine and feline opportunistic infections included E. hormaechei subsp. xiangfangensis ST171 (n = 3), ST121 (n = 1), and ST182 (n = 1), as well as E. hormaechei subsp. steigerwaltii ST65 (n = 1). Five of the six E. hormaechei strains harbored an identical 51,479-bp blaOXA-181-carrying IncX3 plasmid. However, the blaOXA-181 plasmid (pCUVET22-969.1) of the E. hormaechei strain CUVET22-969 presented a variation due to the insertion of ISKpn74 and ISSbo1 into the virB region. Additionally, the blaOXA-181 plasmids of S. nevei strains were nearly identical to the others at the nucleotide level, with ISEcl1 inserted upstream of the qnrS1 gene. The E. hormaechei and S. nevei lineages from canine and feline origins might acquire the epidemic blaOXA-181-carrying IncX3 and MDR plasmids, which are shared among Enterobacterales, contributing to the development of resistance. These findings suggest the spillover of significant OXA-181-encoding plasmids to these bacteria, causing severe opportunistic infections in dogs and cats in Thailand. Surveillance and effective hygienic practice, especially in hospitalized animals and veterinary hospitals, should be urgently implemented to prevent the spread of these plasmids in healthcare settings and communities. IMPORTANCE: blaOXA-181 is a significant carbapenemase-encoding gene, usually associated with an epidemic IncX3 plasmid found in Enterobacterales worldwide. In this article, we revealed six carbapenemase-producing (CP) Enterobacter hormaechei and two CP Serratia nevei strains harboring blaOXA-181-carrying IncX3 and multidrug resistance plasmids recovered from dogs and cats in Thailand. The carriage of these plasmids can promote extensively drug-resistant properties, limiting antimicrobial treatment options in veterinary medicine. Since E. hormaechei and S. nevei harboring blaOXA-181-carrying IncX3 plasmids have not been previously reported in dogs and cats, our findings provide the first evidence of dissemination of the epidemic plasmids in these bacterial species isolated from animal origins. Pets in communities can serve as reservoirs of significant antimicrobial resistance determinants. This situation places a burden on antimicrobial treatment in small animal practice and poses a public health threat.

Selection and evaluation of lactic acid bacteria from chicken feces in Thailand as potential probiotics.

Background: Lactic acid bacteria (LAB) are widely used as probiotics in poultry production due to their resilience to low pH and high bile salt concentrations, as well as their beneficial effects on growth performance and antagonistic activity against enteric pathogens. However, the efficacy of probiotics depends on strain selection and their ability to colonize the host's intestine. This study aimed to select, identify, and evaluate LAB strains isolated from chicken feces in Thailand for potential use as probiotics in the chicken industry. Methods: LAB strains were isolated from 58 pooled fresh fecal samples collected from chicken farms in various regions of Thailand, including commercial and backyard farms. Gram-positive rods or cocci with catalase-negative characteristics from colonies showing a clear zone on MRS agar supplemented with 0.5% CaCO3 were identified using MALDI-TOF mass spectrometry. The LAB isolates were evaluated for acid (pH 2.5 and pH 4.5) and bile salt (0.3% and 0.7%) tolerance. Additionally, their cell surface properties, resistance to phenol, antimicrobial activity, hemolytic activity, and presence of antimicrobial resistance genes were determined. Results: A total of 91 LAB isolates belonging to the Pediococcus, Ligilactobacillus, Limosilactobacillus, and Lactobacillus genera were obtained from chicken feces samples. Backyard farm feces exhibited a greater LAB diversity compared to commercial chickens. Five strains, including Ligilactobacillus salivarius BF12 and Pediococcus acidilactici BF9, BF14, BYF20, and BYF26, were selected based on their high tolerance to acid, bile salts, and phenol. L. salivarius BF12 and P. acidilactici BF14 demonstrated strong adhesion ability. The five LAB isolates exhibited significant cell-cell interactions (auto-aggregation) and co-aggregation with Salmonella. All five LAB isolates showed varying degrees of antimicrobial activity against Salmonella strains, with P. acidilactici BYF20 displaying the highest activity. None of the LAB isolates exhibited beta-hemolytic activity. Whole genome analysis showed that L. salivarius BF12 contained ermC, tetL, and tetM, whereas P. acidilactici strains BF9 and BF14 carried ermB, lnuA, and tetM. Conclusion: The selected LAB isolates exhibited basic probiotic characteristics, although some limitations were observed in terms of adhesion ability and the presence of antibiotic resistance genes, requiring further investigation into their genetic location. Future studies will focus on developing a probiotic prototype encapsulation for application in the chicken industry, followed by in vivo evaluations of probiotic efficacy.

Rapid identification of canine uropathogens by matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry and the clinical factors that correlated bacterial species and antimicrobial resistance.

Using the matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF MS) method for bacterial diagnosis, rapid urine sample preparation can reduce time relapsing of diagnosis and improve discriminatory power in coinfection cases. We aimed to evaluate rapid urine preparation procedures before MALDI-TOF MS application using dog clinical urine samples in comparison with standard microbiological diagnostic methods by agreement analysis. We determined the frequency and distribution of bacteria and bacterial resistance and their correlations to clinical history. Three experimental procedures comprising direct centrifugation, 10% sodium dodecyl sulfate digestion, and ultrasonic preparation were performed for method validation and sensitivity. Sterile urine containing Escherichia coli and/or Staphylococcus aureus were used as simulated samples. By ultrasonic preparation, the microorganisms could be detected 1.46-1.51 × 105 CFU, which was considered the most suitable technique. This preparation was significantly consistent with the routine method based on data from Hospital Information Systems for 50 urine samples from canine cystitis. By standard protocol, Enterobacteriaceae and Staphylococcus pseudintermedius were found in most of the 155 urine samples with cystitis. Extended-spectrum beta-lactamase-producing Enterobacteriaceae was found in 25-30% of the samples. Imipenem resistance was found in 70% of Acinetobacter baumannii cases; almost all were resistant to second-generation fluoroquinolones and tetracyclines. The most efficient antibiotic for treating bacterial urinary tract infection was amoxicillin plus clavulanic acid. A. baumannii and Pseudomonas aeruginosa were susceptible to pradofloxacin. Prolonged urine catheterization was linked to lower urinary tract infections by Enterobacter spp., which also correlated with chronic kidney disease.

Emergence and multi-lineages of carbapenemase-producing Acinetobacter baumannii-calcoaceticus complex from canine and feline origins.

The carbapenemase-producing Acinetobacter baumannii is an important opportunistic bacterium and frequently causes hospital-acquired infections in humans. It also has increasingly been reported in veterinary medicine. This study illustrates multiple clones of carbapenemase-producing A. baumannii disseminating and causing diseases in dogs and cats in Thailand. Between 2016 and 2020, 44 A. baumannii and two A. pittii isolates exhibiting imipenem resistance (MIC≥16 µg/mL) from diagnostic samples were characterized by Pasteur multilocus sequence typing (MLST), sequence grouping (SG), repetitive extragenic palindromic element (rep)-PCR fingerprint analysis and antimicrobial resistance (AMR) profiling. All isolates contained blaOXA-23 in the Tn2006 family, and A. baumannii showed the sequence type (ST) 16 (14/44), ST149 (12/44), ST25 (6/44), ST2 (4/44), ST1581 (3/44), ST23 (2/44), ST1575 (1/44) and ST1576 (1/44). DNA fingerprint analysis and SG illustrated clonal relationships in the STs and its single locus variants, and AMR gene profiles, including tetracycline and aminoglycoside resistance genes, showed minor variations in the clones. The findings suggest that blaOXA-23 has been spread in multiple clones of A. baumannii and A. pittii from canine and feline hosts. With the collection of multiple AMR genes and intrinsic resistance, antimicrobial options are limited for treatment, and pets can be a potential reservoir of extensively drug-resistant, carbapenemase-producing A. baumannii in the community. Epidemiological tracking by passive and active surveillance in animals, veterinary personnel and hospital environment and preventive measurements should be promoted to decrease the risk of infection and transmission to humans.

Diversity and antimicrobial susceptibility profiles of Aeromonas spp. isolated from diseased freshwater fishes in Thailand.

Motile Aeromonas septicemia (MAS), a disease caused by Aeromonas spp., is recognized as a major disease in freshwater aquaculture. This study aimed to investigate the distribution and diversity of Aeromonas spp. and their antimicrobial susceptibility patterns. A total of 86 isolates of Aeromonas spp. were recovered from diseased freshwater fishes from 13 farms in Thailand. All isolates were identified using biochemical characteristics, matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS), polymerase chain reaction assays, and the gyrB gene sequence analysis. The result of MALDI-TOF MS showed 100% (86 isolates) accuracy at genus-level identification, and 88.4% (76 isolates) accuracy at species-level identification. Six species of Aeromonas were confirmed through nucleotide sequencing and phylogenetic analysis of the gyrB gene Aeromonas veronii (72.1%), Aeromonas jandaei (11.6%), Aeromonas schubertii (9.3%), Aeromonas diversa (3.5%), Aeromonas hydrophila (2.3%), and Aeromonas punctata (1.2%). Antimicrobial susceptibility tests for all isolates revealed resistance against amoxicillin (99%), ampicillin (98%), oxolinic acid (81.4%), oxytetracycline (77%), trimethoprim-sulfamethoxazole (24%), and enrofloxacin (21%). The multiple antibiotic resistance (MAR) index varied between 0.14 and 0.86, with MAR values more than 0.2 in 99% of isolates. Furthermore, four diverse multidrug-resistant (MDR) patterns were found among Aeromonas isolates. Our finding show that A. veronii is the most abundant species in Thai cultured freshwater fish with the highest MDR patterns.

Frequency, Distribution, and Antimicrobial Resistance of Coagulase-Negative Staphylococci Isolated from Clinical Samples in Dogs and Cats.

This study aimed to investigate the frequency, distribution, and antimicrobial resistance of coagulase-negative staphylococci (CoNS) obtained from clinical samples from dogs and cats and to classify any methicillin-resistant CoNS (MRCoNS). The samples were collected in 2017-2018, and species identification and antimicrobial susceptibility testing were routinely performed using the Vitek2 system. Among 1,056 staphylococci, 185 CoNS (17.5%) were obtained and included 18 species from dogs (n = 116) and 14 species from cats (n = 69). The predominant species were Staphylococcus chromogenes (31.4%), Staphylococcus hominis ssp. hominis (16.2%), Staphylococcus warneri (10.8%), and Staphylococcus epidermidis (8.1%). The primary isolation sites were the skin and urinary tract. High levels of resistance to ß-lactams (65.4%), tetracycline (44.3%), clindamycin (36.8%), and erythromycin (30.8%) were observed. Twenty-five MRCoNS (13.4%), mainly Staphylococcus haemolyticus (n = 8), S. epidermidis (n = 6), and S. hominis ssp. hominis (n = 5), were identified. SCCmec type V (n = 8) was the most common type, followed by SCCmec type IV (n = 6) and SCCmec type III (n = 2), whereas nontypable SCCmec were classified into nine MRCoNS. Some CoNS have been recorded in humans, and these might be transferred to and cause subsequent infections in humans. Moreover, the diversity of SCCmec types and resistant strains suggested that they may serve as a reservoir of resistance genes among staphylococci.

Multiple and High-Risk Clones of Extended-Spectrum Cephalosporin-Resistant and blaNDM-5-Harbouring Uropathogenic Escherichia coli from Cats and Dogs in Thailand.

Resistance to extended-spectrum cephalosporins (ESC) and carbapenems in Escherichia coli (E. coli), increasingly identified in small animals, indicates a crisis of an antimicrobial resistance situation in veterinary medicine and public health. This study aimed to characterise the genetic features of ESC-resistant E. coli isolated from cats and dogs with urinary tract infections in Thailand. Of 72 ESC-resistant E. coli isolated from diagnostic samples (2016-2018), blaCTX-M including group 1 (CTX-M-55, -15 and -173) and group 9 (CTX-M-14, -27, -65 and -90) variants were detected in 47 isolates (65.28%) using PCR and DNA sequencing. Additional antimicrobial resistance genes, including plasmid-mediated AmpC (CIT and DHA), blaNDM-5, mcr-3, mph(A) and aac(6')-Ib-cr, were detected in these isolates. Using a broth microdilution assay, all the strains exhibited multidrug-resistant phenotypes. The phylogroups were F (36.11%), A (20.83%), B1 (19.44%), B2 (19.44%) and D (4.17%), with several virulence genes, plasmid replicons and an integrase gene. The DNA fingerprinting using a repetitive extragenic palindromic sequence-PCR presented clonal relationships within phylogroups. Multiple human-associated, high-risk ExPEC clones associated with multidrug resistance, including sequence type (ST) 38, ST131, ST224, ST167, ST354, ST410, ST617 and ST648, were identified, suggesting clonal dissemination. Dogs and cats are a potential reservoir of ESC-resistant E. coli and significant antimicrobial resistance genes.

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.

Methicillin-Resistant Staphylococcus aureus Clonal Complex 398 as a Major MRSA Lineage in Dogs and Cats in Thailand.

The aim of this study was to present molecular and antimicrobial resistance characteristics of methicillin-resistant Staphylococcus aureus (MRSA) clonal complex (CC) 398 isolated from diseased dogs and cats in Thailand. A total of 20 MRSA isolates of 134 Staphylococcus aureus isolated from canine and feline clinical samples during 2017-2020 were CC398, consisting of sequence type (ST) 398 (18 isolates), ST5926 (1 isolate), and ST6563 (1 isolate) by multilocus sequence typing. spa t034 and staphylococcal cassette chromosome mec (SCCmec) V were predominantly associated with ST398. Intraclonal differentiation was present by additional spa (t1255, t4653), non-detectable spa, composite SCCmec with a hybrid of ccrA1B1+ccrC and class A mec complex, and DNA fingerprints by pulsed-field gel electrophoresis. The isolates essentially carried antimicrobial resistance genes, mediating multiple resistance to ß-lactams (mecA, blaZ), tetracyclines [tet(M)], aminoglycosides [aac(6')-Ie-aph(2')-Ia], and trimethoprim (dfr). Livestock-associated MRSA ST398 resistance genes including lnu(B), lsa(E), spw, fexA, and tet(L) were heterogeneously found and lost in subpopulation, with the absence or presence of additional erm(A), erm(B), and ileS2 genes that corresponded to resistance phenotypes. As only a single CC398 was detected with the presence of intraclonal variation, CC398 seems to be the successful MRSA clone colonizing in small animals as a pet-associated MRSA in Thailand.

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.