Dissemination of blaNDM-5-carrying IncX3-type plasmid among non-clonal Escherichia coli strains colonising a dog with a skin infection caused by a carbapenem-resistant Klebsiella pneumoniae, United Kingdom.

A successful outcome of a post-surgical wound infection management by a carbapenem-resistant Klebsiella pneumoniae is described in a dog. Four multidrug-resistant and carbapenem-resistant Escherichia coli strains belonging to ST410 (n = 1) and ST648 (n = 3) were isolated from faecal samples and nasal swabs of this dog at admission to a veterinary hospital in the United Kingdom, and one month after discharge. Whole-genome sequencing analysis suggests dissemination of a 46,161-bp IncX3 blaNDM-5-carrying plasmid among E. coli strains from the different lineages. In this study, the E. coli ST648 strains were virtually identical to each other (5 SNPs difference) indicating dissemination and persistence of this clone over time and across different anatomical sites in the same dog maybe due to the prolonged antimicrobial therapy. The carbapenemase carrying plasmid also showed homology with other publicly available plasmid sequences from Asian countries. These results suggests that plasmids may be a major vehicle in mediating the dissemination of carbapenem-resistance. Further studies investigating the selection and flow of plasmids carrying important resistance genes amongst companion animals are needed as it may further contaminate other environments posing a threat to public health.

Longitudinal study of ESBL/AmpC-producing Enterobacterales strains sharing between cohabiting healthy companion animals and humans in Portugal and in the United Kingdom.

Extended-spectrum beta-lactamase (ESBL)- and plasmid-mediated cephalosporinase (AmpC)-producing Enterobacterales (ESBL/AmpC-E) are an increasing healthcare problem in both human and veterinary medicine. The aim of this study was to investigate the possible sharing of ESBL/AmpC-E strains between healthy companion animals and humans of the same household in Portugal (PT) and the United Kingdom (UK). In a prospective longitudinal study, between 2018 and 2020, faecal samples were collected from healthy dogs (n=90), cats (n=20) and their cohabiting humans (n=119) belonging to 41 PT and 44 UK households. Samples were screened for the presence of ESBL/AmpC-E and carbapenemase-producing bacteria. Clonal relatedness between animal and human strains was established by using REP-PCR fingerprinting method, followed by whole-genome sequencing (WGS) of selected strains. ESBL/AmpC-E strains were detected in companion animals (PT=12.7%, n=8/63; UK=8.5%, n=4/47) and humans (PT=20.7%, n=12/58; UK=6.6%, n=4/61) in at least one timepoint. REP-PCR identified paired multidrug-resistant ESBL/AmpC-producing Escherichia coli strains from companion animals and owners in two Portuguese households (4.8%) and one UK household (2.3%). WGS analysis of nine E. coli strains from these three households confirmed that interhost sharing occurred only between the two animal-human pairs from Portugal. Three shared strains were identified: one CTX-M-15-producing E. coli strain in a cat-human pair (O15-H33-ST93) and two CTX-M-15- and CTX-M-55/CMY-2-producing E. coli strains, in a dog-human pair (O8:H9-ST410 and O11:H25-ST457, respectively) at different timepoints. These E. coli clonal lineages are human pandemic, highlighting the role of companion animals living in close contact with humans in the dissemination and persistence of antimicrobial resistance in the household environment.

mcr-1 colistin resistance gene sharing between Escherichia coli from cohabiting dogs and humans, Lisbon, Portugal, 2018 to 2020.

BackgroundThe emergence of colistin resistance is a One Health antimicrobial resistance challenge worldwide. The close contact between companion animals and humans creates opportunities for transmission and dissemination of colistin-resistant bacteria.AimTo detect potential animal reservoirs of colistin-resistant Escherichia coli and investigate the possible sharing of these bacteria between dogs, cats and their cohabiting humans in the community in Lisbon, Portugal.MethodsA prospective longitudinal study was performed from 2018 to 2020. Faecal samples from dogs and cats either healthy or diagnosed with a skin and soft tissue or urinary tract infection, and their cohabiting humans were screened for the presence of colistin-resistant E. coli. All isolates were tested by broth microdilution against colistin and 12 other antimicrobials. Colistin-resistant isolates were screened for 30 resistance genes, including plasmid-mediated colistin resistance genes (mcr-1 to mcr-9), and typed by multilocus sequence typing. Genetic relatedness between animal and human isolates was analysed by whole genome sequencing.ResultsColistin-resistant E. coli strains harbouring the mcr-1 gene were recovered from faecal samples of companion animals (8/102; 7.8%) and humans (4/125; 3.2%). No difference between control and infection group was detected. Indistinguishable multidrug-resistant E. coli ST744 strains harbouring the mcr-1 gene were found in humans and their dogs in two households.ConclusionsThe identification of identical E. coli strains containing the plasmid-mediated mcr-1 gene in companion animals and humans in daily close contact is of concern. These results demonstrate the importance of the animal-human unit as possible disseminators of clinically important resistance genes in the community setting.

The nose is not enough: Multi-site sampling is best for MRSP detection in dogs and households.

BACKGROUND: Following recovery from meticillin-resistant Staphylococcus pseudintermedius (MRSP) infection of any type, dogs may continue to carry MRSP asymptomatically on skin and mucosae, contributing to the spread of this multidrug-resistant, veterinary hospital-associated pathogen with zoonotic potential to others and into the environment. OBJECTIVES: This study determined which canine anatomic and household environmental sites are most sensitive for sampling to identify carriage and contamination. METHODS AND MATERIALS: Fifty-one dogs and 22 households, MRSP-positive on at least one tested site, were sampled on 132 and 40 occasions over time, respectively. Dogs were swabbed at six sites (mouth, nose, conjunctiva, skin, prepuce/vulva, perianal area); household environments were sampled using contact plates (mannitol salt agar [MSA] and MSA + 6 mg/L oxacillin [MS+]) on five sites. MRSP was isolated after enrichment, grown on MSA/MS+ and was confirmed by PCR. Generalized estimating equations were used for calculation of sensitivity (95% confidence interval) for each site/combination. RESULTS: Each anatomical and environmental site yielded MRSP at least once. MRSP was isolated from only a single site in 27.3% of dogs, with the buccal mucosa showing the highest sensitivity (63.8%). Multi-site sampling of a minimum of four canine anatomical or four environmental sites, respectively, was needed to achieve >95% sensitivity. CONCLUSIONS AND CLINICAL RELEVANCE: The canine buccal mucosa should be included in MRSP sampling protocols, ideally in addition to at least three other anatomical sites. Likewise, environment sampling should be of multiple household sites in cases where it is used as a part of clinical case management.

Effect of topical antimicrobial therapy and household cleaning on meticillin-resistant Staphylococcus pseudintermedius carriage in dogs.

BACKGROUND: Meticillin-resistant Staphylococcus pseudintermedius (MRSP) is a multidrug-resistant canine pathogen with a low zoonotic potential. This study investigated MRSP carriage and clearance through topical antimicrobial therapy and household cleaning in dogs recovered from MRSP infection. METHODS: Dogs were swabbed for MRSP carriage; household contamination was assessed using contact plates. Carrier dogs were allocated randomly to receive topical fusidic acid and chlorhexidine/miconazole treatment combined with owners implementing a household hygiene protocol (H&T) or implementation of hygiene alone (H) over three weeks. Carriage-negative dogs were monitored monthly. The relatedness of isolates over time was investigated by pulsed-field gel electrophoresis (PFGE). RESULTS: At inclusion, MRSP carriage was confirmed in 31/46 (67.4%) index dogs and 16/24 (66.7%) contact dogs, and contamination was found in 18/40 (45%) environments. In dogs completing all cycles, interventions cleared carriage in 5/9 (55.6%) dogs in group H&T and 2/6 (33.3%) in group H. Environmental contamination was infrequent but associated with carrier dogs (p = 0.047). Monthly monitoring of initially negative dogs showed intermittent carriage in 9/14 dogs. PFGE-concordance was found among all 34 MRSP isolated from eight index dogs over time. CONCLUSION: MRSP carriage was common in dogs after recovery from infection. Topical antimicrobial therapy temporarily eliminated carriage but recurrence was frequent. Management efforts must include the prevention of recurrent infections and hygiene.

Canine pyoderma: mecA persists autogenous bacterin formulation from meticillin-resistant Staphylococcus pseudintermedius (MRSP) and S. aureus (MRSA).

OBJECTIVE: Autogenous Staphylococcus pseudintermedius bacterins can reduce prescribing of antimicrobials in the management of canine recurrent pyoderma. However, increasing prevalence of meticillin-resistant, mecA-positive S. pseudintermedius (MRSP) raises concern over dispersal of mecA through bacterin therapy. We investigated the presence and integrity of mecA in bacterin formulations after manufacturing. MATERIAL AND METHODS: Twenty clinical isolates (12 MRSP, 7 MR-S. aureus, 1 meticillin-susceptible SP) were investigated. Pellets from overnight growth were washed 3 times with 0.5 % phenol saline, followed by addition of 0.1 ml 10 % formal-saline to 10 ml phenol-saline. Sterility was confirmed, and DNA extracted using both a standard genomic extraction kit and one recommended for formalin-fixed tissue samples (FFPE). The presence of mecA was determined after PCR and its integrity examined in 5 randomly selected samples after sequencing. RESULTS: In all bacterins from meticillin-resistant isolates, mecA was detected following FFPE extraction; products aligned fully to a reported mecA sequence. After standard DNA extraction, mecA was seen in 16/19 samples. CONCLUSION: Persistence of mecA in MRSP bacterins suggests that dispersal of this important resistance mediator through therapy may be possible. While the ability of skin bacteria to uptake naked DNA remains unclear, it seems prudent to only formulate autogenous bacterins from mecA-negative S. pseudintermedius to avoid unnecessary spread of mecA.

Genes on the Move: In Vitro Transduction of Antimicrobial Resistance Genes between Human and Canine Staphylococcal Pathogens.

Transmission of methicillin-resistant Staphylococcus aureus (MRSA) and methicillin-resistant Staphylococcus pseudintermedius (MRSP) between people and pets, and their co-carriage, are well-described. Potential exchange of antimicrobial resistance (AMR) genes amongst these staphylococci was investigated in vitro through endogenous bacteriophage-mediated transduction. Bacteriophages were UV-induced from seven donor isolates of canine (MRSP) and human (MRSA) origin, containing tet(M), tet(K), fusB or fusC, and lysates filtered. Twenty-seven tetracycline- and fusidic acid- (FA-) susceptible recipients were used in 122 donor-recipient combinations (22 tetracycline, 100 FA) across 415 assays (115 tetracycline, 300 FA). Bacteriophage lysates were incubated with recipients and presumed transductants quantified on antimicrobial-supplemented agar plates. Tetracycline resistance transduction from MRSP and MRSA to methicillin-susceptible S. pseudintermedius (MSSP) was confirmed by PCR in 15/115 assays. No FA-resistance transfer occurred, confirmed by negative fusB/fusC PCR, but colonies resulting from FA assays had high MICs (≥32 mg/L) and showed mutations in fusA, two at a novel position (F88L), nine at H457[Y/N/L]. Horizontal gene transfer of tetracycline-resistance confirms that resistance genes can be shared between coagulase-positive staphylococci from different hosts. Cross-species AMR transmission highlights the importance of good antimicrobial stewardship across humans and veterinary species to support One Health.

DIVERSITY OF STAPHYLOCOCCAL SPECIES CULTURED FROM CAPTIVE LIVINGSTONE'S FRUIT BATS (PTEROPUS LIVINGSTONII) AND THEIR ENVIRONMENT.

Livingstone's fruit bats (Pteropus livingstonii) are critically endangered and a captive population has been established as part of the International Union for Conservation of Nature Species Action Plan. The largest colony, in Jersey Zoo, was sampled for staphylococcal carriage and at infection sites, as disease associated with staphylococci had previously been found. Staphylococci were cultured from swabs from 44 bats (skin, oropharynx, mouth ejecta, skin lesions) and from their enclosure. The isolates were identified by matrix-assisted laser desorption-ionization time of flight mass spectrometry; antimicrobial susceptibility testing was performed by disc diffusion and screening for mecA and mecC. Seventeen species of coagulase-negative staphylococci including Staphylococcus xylosus, S. kloosii, S. nepalensis, and S. simiae were isolated. Staphylococcus aureus was identified from both carriage and lesional sites. These findings suggest S. nepalensis may be part of the normal carriage flora of bats. Antimicrobial resistance rates were low and methicillin-resistant Staphylococcus aureus (MRSA) was not identified. Sampling of mouth ejecta for staphylococci may provide results representative for carriage sites.

Activity In Vitro of Clotrimazole against Canine Methicillin-Resistant and Susceptible Staphylococcus pseudintermedius.

Emergence of multidrug-resistance in Staphylococcus pseudintermedius (SP) has increased interest in topical therapy as an alternative to systemic antibiotics in canine pyoderma. The antifungal imidazole, clotrimazole, is contained in numerous licensed canine ear preparations. Its in vitro activity against SP has not been evaluated, although previous studies have shown that the related imidazole, miconazole, has significant anti-staphylococcal efficacy. We therefore determined minimum inhibitory concentrations (MICs) of clotrimazole amongst 50 SP isolates (25 methicillin-resistant [MR]SP and susceptible [MS]SP) collected from dogs in Germany during 2010-2011 using an agar dilution method (CLSI VET01-A4). MICs amongst MRSP and MSSP were comparable (MIC50 and MIC90 = 1mg/L for both groups, p = 0.317); overall, 49 isolates had MIC = 1 mg/L and one had MIC = 0.5 mg/L. The relatively low MICs obtained in this study are likely to be exceeded by topical therapy and thus further clinical evaluation of clotrimazole use in canine superficial pyoderma and otitis externa caused by MRSP and MSSP is now warranted.

Opportunities for topical antimicrobial therapy: permeation of canine skin by fusidic acid.

BACKGROUND: Staphylococcal infection of the canine epidermis and hair follicle is amongst the commonest reasons for antimicrobial prescribing in small animal veterinary practice. Topical therapy with fusidic acid (FA) is an attractive alternative to systemic therapy based on low minimum inhibitory concentrations (MICs, commonly <0.03 mg/l) documented in canine pathogenic staphylococci, including strains of MRSA and MRSP (methicillin-resistant Staphylococcus aureus and S. pseudintermedius). However, permeation of canine skin by FA has not been evaluated in detail. This study aimed to define the degree and extent of FA permeation in canine skin in vitro from two sites with different hair follicle density following application of a licensed ophthalmic formulation that shares the same vehicle as an FA-betamethasone combination product approved for dermal application in dogs. Topical FA application was modelled using skin held in Franz-type diffusion cells. Concentrations of FA in surface swabs, receptor fluid, and transverse skin sections of defined anatomical depth were determined using high-performance liquid chromatography and ultraviolet (HPLC-UV) analysis. RESULTS: The majority of FA was recovered by surface swabs after 24 h, as expected (mean ± SEM: 76.0 ± 17.0%). FA was detected within 424/470 (90%) groups of serial sections of transversely cryotomed skin containing follicular infundibula, but never in 48/48 (100%) groups of sections containing only deeper follicular structures, nor in receptor fluid, suggesting that FA does not permeate beyond the infundibulum. The FA concentration (mean ± SEM) in the most superficial 240 µm of skin was 2000 ± 815 µg/g. CONCLUSIONS: Topically applied FA can greatly exceed MICs for canine pathogenic staphylococci at the most common sites of infection. Topical FA therapy should now be evaluated using available formulations in vivo as an alternative to systemic therapy for canine superficial bacterial folliculitis.