Veterinary Hospital Dissemination of CTX-M-15 Extended-Spectrum Beta-Lactamase-Producing Escherichia coli ST410 in the United Kingdom

We characterized extended-spectrum beta-lactamases (ESBLs) and plasmid-mediated quinolone resistance (PMQR) in 32 Escherichia coli extended spectrum cephalosporin (ESC)-resistant clinical isolates from UK companion animals from several clinics. In addition, to investigate the possible dissemination of ESBL clinical isolates within a veterinary hospital, two ESBL-producing E. coli isolates from a dog with septic peritonitis and a cluster of environmental ESC-resistant E. coli isolates obtained from the same clinic and during the same time period, as these two particular ESBL-positive clinical isolates, were also included in the study. Molecular characterization identified bla(CTX-M) to be the most prevalent gene in ESC-resistant isolates, where 66% and 27% of clinical isolates carried bla(CTX-M-15) and bla(CTX-M-14,) respectively. The only PMQR gene detected was aac(6')-Ib-cr, being found in 34% of the ESC E. coli isolates and was associated with the carriage of bla(CTX-M-15). The clinical and environmental isolates investigated for hospital dissemination had a common ESBL/AmpC phenotype, carried bla(CTX-M-15), and co-harbored bla(OXA-1,) bla(TEM-1,) bla(CMY-2,) and aac(6')-Ib-cr. Multilocus sequence typing identified them all as ST410, while pulse-field gel electrophoresis demonstrated 100% homology of clinical and environmental isolates, suggesting hospital environmental dissemination of CTX-M-15–producing E. coli ST410

Biofilm forming in S. Pseudintermedius isolates from atopic dogs

S. pseudintermedius is the main colonizer of skin in dogs, with increased adherence in the ones suffering from atopic dermatitis. It is also the main pathogen involed in pyodermas and has an important zoonotic potential. Biofilm forming was tested using the microtitre plate protocol for 50 S. pseudintermedius sampled from the same number of atopic dogs. Out of these 50 isolates 40 (80%) were methicillin susceptible and 10 (20%) methicillin resistant. S. pseudintermedius isolates sampled from atopic dogs without pyoderma simptoms did not manifest an important biofilm production. Our results show that 60% of all isolates were not biofilm producers, 38% were weak biofilm producers and only one isolate was a moderate biofilm producer. Adherence capability did not show significand differences between MSSPs (methicillin susceptible S. pseudintermedius) and MRSPs (methicillin resistant S. pseudintermedius)

Increased tolerance to chlorhexidine following prolonged exposure in staphylococcus species isolated from the skin and mucosae of dogs

This study aimed to investigate if canine staphylococci isolates could develop tolerance to chlorhexidine digluconate after long-term exposure. Staphylococci (Staphylococcus pseudintermedius and coagulase-negative staphylococci, methicillin-susceptible and methicillin-resistant), with and without genotypic chlorhexidine resistance, were investigated for phenotypic chlorhexidine tolerance by determining the MBC (minimal bactericidal concentration) at various time points. The testing was performed as follows: determination of MBC for 30 minutes and 24 hours (MBC 24h-1); exposure of isolates for seven days to concentrations of chlorhexidine equal to 1/2 MBC 24h-1; determination of MBC for 24 h after the first week (MBC 24h-2); exposure of isolates for seven days to concentrations of chlorhexidine equal to 1/2 MBC 24h-2 and rest in Mueller–Hinton broth for seven days; determination of MBC for 24 h after the second week (MBC 24h-3). The MBC for 30 minutes ranged between 16–32 µg/ml compared to the MBC 24h-1 which was between 1–8 µg/ml. The MBCs for 24 h dropped from 8 µg/ml to 0.5 µg/ml for isolate 1, from 2 µg/ml to1 µg/ml for isolate 6 and from 2 µg/ml to 0.5 µg/ml for isolate 8 after being exposed for seven days to concentrations of chlorhexidine equal to 1/2 MBC 24h-1. For one CoNS (coagulase-negative staphylococci), the MBC 24h-2 increased four times from 1 µg/ml (MBC 24h-1) to 4 µg/ml and dropped again to 1 µg/ml after the second week. These results suggest that continuous exposure to chlorhexidine could lead to the selection of chlorhexidine-tolerant staphylococci that could withstand concentrations used during routine decolonisation procedures

Clonal Dissemination of Extended-Spectrum Cephalosporin-Resistant Enterobacterales between Dogs and Humans in Households and Animal Shelters of Romania

Faecal carriage of extended-spectrum cephalosporin-resistant (ESC-R) Enterobacterales in healthy pets is a concerning issue. This study aimed to determine the prevalence, genetic background, and potential for interspecies transmission of these bacteria between dogs and humans within the same household (HH) or shelter environment in Romania. Faecal samples (n = 263) collected from healthy dogs (n = 102), their owners (n = 32), as well as dogs (n = 110) and staff (n = 19) from dog shelters, were screened for ESC-R carriage. Clonal relatedness of canine and human Escherichia coli isolates was established using Fourier Transform Infrared Spectroscopy (FTIR), followed by Illumina WGS of selected isolates. The highest prevalence of ESC-R Enterobacterales faecal carriage was identified in staff working at dog shelters (78.9%), followed by dogs from households (44.11%), dog owners (43.7%), and dogs from shelters (27%). FTIR identified 15 clusters of closely related E. coli isolates, including dog and human isolates from the same environment. Co-carriage of ESC-R isolates in both the dog and owner was identified in 12 HHs (37.5%), with two HHs (6%) having both the owner and dog carrying isolates with identical FTIR spectra, phylogroup, resistance genes, and Inc plasmids. Major ExPEC lineages such as ST127, ST10, ST155, and ST88 were detected in human and dog isolates. Our study revealed a high prevalence of faecal ESC-R E. coli carriage in both dogs and humans from Romanian households and shelters, where bidirectional clonal transmission between humans and dogs is likely. Furthermore, we identified ESC-R Enterobacterales co-carriage in people and dogs sharing the same environment using FTIR, demonstrating its value in AMR surveillance for humans and animals

Characterisation of Extended β-Lactamases and Plasmid Mediated Quinolones Resistancein Escherichia Coli from Shelter Dogs

The aim of this study was to determine the prevalence of β-lactamase (TEM, SHV, OXA), extended-spectrum β-lactamase (ESBL) and genes encoding plasmid mediated resistance to quinolones (PMQR) in extended spectrum cephalosporin (ESC)-resistant Escherichia coli isolated from dog faeces from two shelters in the North-East of Romania. Eighty-eight faecal samples from healthy dogs were analysed by cultivation on Brilliance ESBL medium (Oxoid, UK), followed by phenotipic ESBL screening using combination disc test (CDT). Identification of the E. coli strains was performed by uidA/uspA gene PCR. Susceptibility testing was performed on Mueller-Hinton Agar, with β-lactam and non-β-lactam agents. Identification of β-lactamase genes (blaCTX-M, blaTEM, blaSHV, blaOXA) and PMQR genes (qnrA, qnrB and qnrS) was performed by PCR as previously described. Twenty eight ESC-resistant E. coli (31.81%) were obtained and (n=21/28, 75%) of these were confirmed as ESBLs and showed resistance to cefpodoxime (n=21/28, 75%), amoxicillin/clavulanic acid (n=19/21; 90.48%), and enrofloxacin (n=8/21; 38.09%). Predominant ESBL types were CTX-M-1 (n=15/17, 88.24%) and CTX-M-9 (n=2/17, 11.76%) enzymes. TEM and SHV enzymes were identified in 17.86% and 14.29% of the ESC-resistant isolates, whilst some isolates (n=4) carried only blaTEM and blaSHV. The prevalence of PMQR genes was 28.57% of the 28 ESC resistant isolates, consisting of qnrS (62.5%) and qnrB (37.5%). These findings indicate a high prevalence of ESBLs and PMQR associated resistance E. coli in the normal faecal microbiota of dogs from shelters, which carries the risk for dissemination of these resistance genes to other animals, human or the environment

Resistome-based surveillance identifies ESKAPE pathogens as the predominant gram-negative organisms circulating in veterinary hospitals

IntroductionHealthcare-associated infections (HCAIs) associated with extended-spectrum cephalosporin-resistant gram-negative (ESC-R GN) bacteria are an emerging concern in veterinary hospitals, especially in companion animal intensive care units (ICUs).MethodsTo understand the molecular epidemiology of ESC-R GN isolates in two veterinary hospitals (equine and small animal), a 6-month pilot study was performed during which fecal and environmental samples were obtained twice from selected patients, upon ICU admission and after 48 h of hospitalization. In total, 295 ESC-R GNs were analyzed using the Acuitas Resistome® Test (OpGen, Maryland, US), a PCR-based assay screening for 50 antimicrobial resistance gene families encoding for production of extended-spectrum beta-lactamase (ESBLs), TEM/SHV/OXA or AmpC beta-lactamases and carbapenemases. Combining organism identification and antimicrobial susceptibility data to genotyping results, unique “Acuitas profiles” were generated that can be used for fast typing the isolates and tracking transmission events.ResultsESKAPE GN pathogens were the most prevalent ESC-R GN isolates circulating in both the small animal and equine hospitals, consisting of Enterobacter cloacae complex (21.7%), Pseudomonas aeruginosa (20%), Klebsiella pneumoniae (15.9%), and Acinetobacter baumannii complex (13.6%) followed by Escherichia coli (12.2%), most harboring a combination of genes encoding for beta-lactamases and ESBLs. Some ESKAPE genotypes showed likely intra-hospital transmission, including E. cloacae (two genotypes, one carrying SHV4, SHV5, and TEM7 and the other TEM1, TEM3, and TEM7 enzymes) in the equine and K. pneumoniae (SHV1, SHV5, and DHA1-positive) in the small animal ICUs, respectively. Furthermore, P. aeruginosa (carrying OXA-50), A. baumannii complex (OXA-51), and E. coli (CTX-M-1) genotypes were isolated across both hospitals, suggesting possible transfer mediated via movement of staff and students. Importantly, isolates carrying transmissible resistance to last-resort antimicrobials (i.e. carbapenems) were identified within the hospital environments, consisting of three environmental Acinetobacter spp. harboring blaOXA − 23 and one clinical E. coli with blaOXA − 48.ConclusionWe describe the widespread occurrence of ESKAPE gram-negative organisms in veterinary ICU patients and hospital environments. Findings from this project provide baseline data on the epidemiology of ESKAPE pathogens in veterinary settings, which can inform infection control policies to aid in patient management and prevent transmission of nosocomial infections associated with these pathogens

Investigation of In Vitro Susceptibility and Resistance Mechanisms in Skin Pathogens: Perspectives for Fluoroquinolone Therapy in Canine Pyoderma

Fluoroquinolones (FQ) are commonly used in dogs with bacterial skin infections. Their use as first choice, along with the increased incidence of FQ-resistance, represents a risk to animal and public health. Our study determined minimum inhibitory (MIC) and bactericidal (MBC) concentrations of five FQs in Staphylococcus aureus, Staphylococcus pseudintermedius, and Escherichia coli, together with FQ-resistance mechanisms. MICs, efflux pump (EP) overexpression and MBCs were measured in 249 skin infection isolates following CLSI guidelines (CLSI VET01-A4, CLSI M26-A). Chromosomal and plasmid-mediated resistance genes were investigated after DNA extraction and sequencing. FQ-resistance was detected in 10% of methicillin-susceptible (MS), 90% of methicillin-resistant (MR) staphylococci and in 36% of E. coli. Bactericidal effect was observed except in 50% of MRSA/P for ciprofloxacin and in 20% of MRSPs for enrofloxacin. Highest MICs were associated with double mutation in gyrA (Ser83Leu + Asp87Asn), efflux pumps and three PMQR genes in E. coli, and grlA (Ser80Phe + Glu84Lys) in S. aureus. EP overexpression was high among E. coli (96%), low in S. aureus (1%) and absent in S. pseudintermedius. Pradofloxacin and moxifloxacin showed low MICs with bactericidal effect. Since in vitro FQ resistance was associated with MR, FQ use should be prudently guided by susceptibility testing