Geographical Variations in Virulence Factors and Antimicrobial Resistance Amongst Staphylococci Isolated From Dogs From the United Kingdom and Romania.

The objective of this study was to compare virulence and resistance factors of mucosal and cutaneous staphylococci from dogs with pyoderma in the UK and Romania, two countries with different approaches to antimicrobial use in companion animals. Staphylococcal isolates (n = 166) identified to the species level as being Staphylococcus pseudintermedius or coagulase negative (CoNS) were analyzed for their antimicrobial resistance (AMR) profile and presence of resistance and virulence genes. Of the investigated isolates, 26 were methicillin-resistant S. pseudintermedius (MRSP), 89 were methicillin-susceptible S. pseudintermedius (MSSP) and 51 were coagulase negative staphylococci (CoNS). A significantly larger number of isolates originating from Romania were resistant to clindamycin, tetracycline, and chloramphenicol compared to the UK isolates (P < 0.05). Resistance to amoxicillin-clavulanic acid, gentamicin, and trimethoprim-sulphamethoxazole was more evident in UK isolates. Fusidic acid resistance was common in Staphylococcus spp. isolates from both countries. Most isolates carried virulence factors associated with siet (exfoliative toxin) and luk (leucocidin) genes. All MRSP UK isolates exhibited fusidic acid resistance genes whilst this was very rare in the MRSP isolates from Romania. The chlorhexidine resistance gene qacA/B was frequently identified in CoNS isolates from the UK (P < 0.001). The current study documented differences in antimicrobial resistance profiles of Staphylococcus spp. isolates from dogs in two geographical locations in Europe, which could reflect differences in antimicrobial prescribing patterns. The study also highlights the need for further studies and interventions on antimicrobial use, prescribing patterns and AMR surveillance in companion animals in Romania.

Reservoirs of resistance: polymyxin resistance in veterinary-associated companion animal isolates of Pseudomonas aeruginosa.

BACKGROUND: Pseudomonas aeruginosa is an opportunistic pathogen and a major cause of infections. Widespread resistance in human infections are increasing the use of last resort antimicrobials such as polymyxins. However, these have been used for decades in veterinary medicine. Companion animals are an understudied source of antimicrobial resistant P. aeruginosa isolates. This study evaluated the susceptibility of P. aeruginosa veterinary isolates to polymyxins to determine whether the veterinary niche represents a potential reservoir of resistance genes for pathogenic bacteria in both animals and humans. METHODS AND RESULTS: Clinical P. aeruginosa isolates (n=24) from UK companion animals were compared for antimicrobial susceptibility to a panel of human-associated isolates (n=37). Minimum inhibitory concentration (MIC) values for polymyxin B and colistin in the companion animals was significantly higher than in human isolates (P=0.033 and P=0.013, respectively). Genotyping revealed that the veterinary isolates were spread throughout the P. aeruginosa population, with shared array types from human infections such as keratitis and respiratory infections, suggesting the potential for zoonotic transmission. Whole genome sequencing revealed mutations in genes associated with polymyxin resistance and other antimicrobial resistance-related genes. CONCLUSION: The high levels of resistance to polymyxin shown here, along with genetic similarities between some human and animal isolates, together suggest a need for sustained surveillance of this veterinary niche as a potential reservoir for resistant, clinically relevant bacteria in both animals and humans.

Are the Hands of Veterinary Staff a Reservoir for Antimicrobial-Resistant Bacteria? A Randomized Study to Evaluate Two Hand Hygiene Rubs in a Veterinary Hospital.

Hand hygiene (HH) is the most successful intervention for hospital infection control. HH rubs with residual action are desired. This study aimed to compare the efficacy of alcohol (A-HH) and lactic acid (LA-HH) rubs, with the latter being marketed as having residual activity. We investigated reductions in bacterial colony-forming units (CFUs), prevalence of antimicrobial-resistant (AMR) organisms, and risk factors for increased counts on the hands of veterinary staff. A randomized, crossover study (53 individuals) was performed in a referral veterinary teaching hospital. Hand plates were taken before, immediately after, and 6 hours after HH. A blinded investigator counted CFUs per plate. Methicillin-resistant Staphylococcus aureus/pseudintermedius (MRSA/MRSP), Enterobacteriaceae, and Pseudomonas species (spp.) were characterized. Gender, profession, time point, and HH product were included as variables within multivariable analyses. A significant reduction in bacterial CFU was seen immediately after A-HH rub application (p < 0.001); however, neither product showed any significant residual action. Veterinarians had higher bacterial CFUs than nurses (p = 0.005); contact with patients, rather than the environment, was also associated with higher counts (p < 0.001). MRSA, MRSP, Enterobacteriaceae spp., and Pseudomonas spp. were detected on 7%, 2%, 14%, and 2% of study participant's hands (n = 208 samples), respectively. Frequent HH administration using an A-HH rub was effective at reducing bacterial CFU on hands in vivo in this veterinary hospital setting, but its use needs further encouragement in veterinary staff. The high prevalence of antimicrobial bacteria on hands is of concern; they might act as reservoirs for patients, the environment, and in-contact people.

Routine antibiotic therapy in dogs increases the detection of antimicrobial-resistant faecal Escherichia coli.

Background: Antimicrobial resistance (AMR) is a critical health problem, with systemic antimicrobial therapy driving development of AMR across the host spectrum. Objectives: This study compares longitudinal carriage, at multiple timepoints, of AMR faecal Escherichia coli in dogs undergoing routine antimicrobial treatment. Methods: Faecal samples (n = 457) from dogs (n = 127) were examined pretreatment, immediately after treatment and 1 month and 3 months post-treatment with one of five antimicrobials. Isolates were tested for susceptibility to a range of antimicrobials using disc diffusion for each treatment group at different timepoints; the presence/absence of corresponding resistance genes was investigated using PCR assays. The impact of treatment group/timepoint and other risk factors on the presence of resistance [MDR, fluoroquinolone resistance, third-generation cephalosporin resistance (3GCR) and ESBL and AmpC production] was investigated using multilevel modelling. Samples with at least one AMR E. coli from selective/non-selective agar were classed as positive. Resistance was also assessed at the isolate level, determining the abundance of AMR from non-selective culture. Results: Treatment with ß-lactams or fluoroquinolones was significantly associated with the detection of 3GCR, AmpC-producing, MDR and/or fluoroquinolone-resistant E. coli, but not ESBL-producing E. coli, immediately after treatment. However, 1 month post-treatment, only amoxicillin/clavulanate was significantly associated with the detection of 3GCR; there was no significant difference at 3 months post-treatment for any antimicrobial compared with pretreatment samples. Conclusions: Our findings demonstrated that ß-lactam and fluoroquinolone antibiotic usage is associated with increased detection of important phenotypic and genotypic AMR faecal E. coli following routine therapy in vet-visiting dogs. This has important implications for veterinary and public health in terms of antimicrobial prescribing and biosecurity protocols, and dog waste disposal.

Impact of systemic antimicrobial therapy on mucosal staphylococci in a population of dogs in Northwest England.

BACKGROUND: Antimicrobial-resistant bacteria are increasingly isolated from veterinary patients. OBJECTIVES: To determine risk factors for antimicrobial resistance (AMR) among canine mucosal staphylococci following routine antimicrobial treatment with cefalexin (CFX), clavulanate-amoxicillin (AC), cefovecin (CVN), clindamycin (CD) or a fluoroquinolone (FQ). ANIMALS: Mucosal swab samples (n = 463) were collected from 127 dogs pre-treatment, immediately, and at one- and three-months post-treatment. METHODS: Staphylococci were identified phenotypically and biochemically as coagulase negative (CoNS) or coagulase positive (CoPS); CoPS were speciated by nuc gene PCR. Antimicrobial susceptibility was determined using disc diffusion and mecA gene carriage by PCR. Multilevel, multivariable models examined associations between risk factors and presence/absence of CoPS, meticillin resistance (MR), multidrug-resistance (MDR) and fluoroquinolone resistance (FQR). RESULTS: The percentage of samples with CoNS increased and with CoPS (including S. pseudintermedius) decreased immediately post-treatment with CFX, CVN and CD (P ≤ 0.001) and one month post-treatment with CD (P = 0.003). By three months post-treatment, there was no significant difference compared to pre-treatment samples. Immediately post-treatment with FQs there was significantly increased risk of isolating MRS (P = 0.002), MDR (P = 0.002) or FQR (P = 0.013) staphylococci and of MDR following CFX treatment (P = 0.019). The percentage of samples with AMR staphylococci declined from immediately to three months post-treatment and there was no significant difference between resistance prevalence at one or three months post-treatment for most AMR traits and treatment groups. Exceptions include increased MDR following FQ (P = 0.048) or CFX (P = 0.021), at one and three months post-treatment, respectively. CONCLUSIONS AND CLINICAL IMPORTANCE: Systemic antimicrobials impact on mucosal staphylococci. Immediately after therapy, the mucosa may be a reservoir for AMR staphylococci that are a source of mobile genetic elements carrying AMR genes.

Interaction of chlorhexidine with trisEDTA or miconazole in vitro against canine meticillin-resistant and -susceptible Staphylococcus pseudintermedius isolates from two UK regions.

BACKGROUND: Topical therapy is an important alternative to systemic antibacterial therapy for treatment of canine superficial pyoderma in light of the emergence of multidrug-resistant staphylococci. Chlorhexidine is widely used in shampoo products alone or in combination with miconazole or tromethamine-ethylenediaminetetraacetic acid (trisEDTA). Comparisons of these combinations have not been made. HYPOTHESIS/OBJECTIVES: To determine minimum inhibitory concentrations (MICs) of combinations of chlorhexidine/miconazole and chlorhexidine/trisEDTA in vitro in a collection of Staphylococcus pseudintermedius (SP) from northern (NUK) and southeastern (SEUK) United Kingdom (UK) sources. METHODS: MICs of chlorhexidine, miconazole, trisEDTA and combinations of chlorhexidine/miconazole (1:1) or chlorhexidine/trisEDTA (80:16:1 and 80:5:1) were determined for 196 canine SP isolates from NUK [49 meticillin-resistant (MRSP), 50 meticillin-susceptible (MSSP)] and fom SEUK (48 MRSP, 49 MSSP) using agar dilution. RESULTS: TrisEDTA alone did not inhibit growth. Chlorhexidine/miconazole MICs (median = 0.5 mg/L) were lower than those of either drug alone (P < 0.05) and lower than chlorhexidine/trisEDTA MICs (median = 1 mg/L; P < 0.0005) in each bacterial type and from both regions, except for miconazole in NUK MSSP. An additive interaction was noted between chlorhexidine and miconazole or trisEDTA (80:16:1 ratio) in 79 and 43 isolates, respectively, whereas antagonism between chlorhexidine and trisEDTA was noted for three isolates. NUK isolates were more susceptible than SEUK isolates (P < 0.05), except MRSP exposed to chlorhexidine and the chlorhexidine/trisEDTA (80:16:1) combination. CONCLUSIONS AND CLINICAL IMPORTANCE: These low MICs are likely to be exceeded by topical therapy. Evaluation of the mechanisms by which chlorhexidine combinations interact to reduce MICs is warranted, in view of increasing concerns of biocide tolerance in staphylococci.

A pilot study comparing in vitro efficacy of topical preparations against veterinary pathogens.

BACKGROUND: Topical antimicrobial agents are important for the management of cutaneous infections. For topical antimicrobial agents, in vitro efficacy data are limited. OBJECTIVES: To determine and compare the minimum bactericidal/fungicidal concentrations (MBCs/MFCs) of several topical antimicrobial agents against veterinary pathogens. MATERIALS AND METHODS: Two chlorhexidine, two oxychlorine based products (NaOCl & HOCl) lime sulfur (LS), manuka honey (MH) and hydrocortisone aceponate (HCA) were tested against American Type Culture Collection (ATCC) and clinical isolates: meticillin susceptible and resistant Staphylococcus pseudintermedius (MSSP), qac A/B carrying MSSP, antimicrobial susceptible and extended spectrum beta-lactamase producing Escherichia coli, multidrug-resistant Pseudomonas aeruginosa and Malassezia pachydermatis. The MBCs/MFCs were measured, where available, using a broth microdilution method; isolates were incubated for 3 and 10 min. RESULTS: Chlorhexidine and isopropyl alcohol (Chl(1) ) showed significantly lower MBCs (0.46 mg/L -937.50 mg/L, P = 0.027) compared to chlorhexidine and climbazole (Chl², 58.59 mg/L-1875 mg/L). NaOCl and HOCl showed excellent antimicrobial activity with HOCl having significantly lower MBCs compared to NaOCl (0.03 mg/L-1.72 mg/L and 0.03 mg/L-1.95 mg/L, respectively, P = 0.042). The detectable MBCs for LS and HCA were high, being close to the starting concentration (5,000 mg/L and 146 mg/L, respectively). The MBC/MFC for MH was not detectable. Amongst all test products there was no significant effect of contact time or isolate resistance status. CONCLUSIONS AND CLINICAL IMPORTANCE: Chlorhexidine, NaOCl and HOCl demonstrated low MBCs against tested organisms, suggesting potential in vivo efficacy. The selection of an appropriate antimicrobial agent, however, cannot be based exclusively upon MBC/MFC data; other factors should be considered.

Antimicrobial resistance risk factors and characterisation of faecal E. coli isolated from healthy Labrador retrievers in the United Kingdom.

Antimicrobial resistant bacteria are increasingly detected from canine samples but few studies have examined commensal isolates in healthy community dogs. We aimed to characterise faecal Escherichia coli from 73 healthy non-veterinarian-visiting and non-antimicrobial treated Labrador retrievers, recruited from dog shows in the North West United Kingdom between November 2010 and June 2011. Each enrolled dog provided one faecal sample for our study. E. coli were isolated from 72/73 (99%) faecal samples. Disc diffusion susceptibility tests were determined for a range of antimicrobials, including phenotypic extended-spectrum beta-lactamase (ESBL) and AmpC-production. PCR assay detected phylogenetic groups and resistance genes (blaCTX-M, blaSHV, blaTEM, blaOXA, blaCIT, qnr), and conjugation experiments were performed to investigate potential transfer of mobile genetic elements. Multivariable logistic regression examined potential risk factors from owner-questionnaires for the presence of antimicrobial resistant faecal E. coli. Antimicrobial resistant, multi-drug resistant (≥3 antimicrobial classes; MDR) and AmpC-producing E. coli were detected in 63%, 30% and 16% of samples, respectively. ESBL-producing E. coli was detected from only one sample and conjugation experiments found that blaCTX-M and blaCIT were transferred from commensal E. coli to a recipient strain. Most isolates were phylogenetic groups B1 and A. Group B2 isolates were associated with lower prevalence of resistance to at least one antimicrobial (P<0.001) and MDR (P<0.001). Significant at P<0.003, was the consumption of raw meat for clavulanate-amoxicillin (OR: 9.57; 95% CI: 2.0-45.7) and third generation cephalosporin resistance (3GCR) (OR: 10.9; 95% CI: 2.2-54.0). AMR E. coli were surprisingly prevalent in this group of non-antimicrobial treated and non-veterinarian-visiting dogs and consumption of raw meat was a significant risk factor for antimicrobial resistance. These findings are of concern due to the increasing popularity of raw-meat canine diets, and the potential for opportunistic infection, zoonotic transmission and transmission of antimicrobial resistant determinants from commensal isolates to potential pathogenic bacteria.

Antimicrobial resistance and characterisation of staphylococci isolated from healthy Labrador retrievers in the United Kingdom.

BACKGROUND: Coagulase-positive (CoPS) and coagulase-negative (CoNS) staphylococci are normal commensals of the skin and mucosa, but are also opportunist pathogens. Meticillin-resistant (MR) and multidrug-resistant (MDR) isolates are increasing in human and veterinary healthcare. Healthy humans and other animals harbour a variety of staphylococci, including MR-CoPS and MR-CoNS. The main aims of the study were to characterise the population and antimicrobial resistance profiles of staphylococci from healthy non-vet visiting and non-antimicrobial treated Labrador retrievers in the UK. RESULTS: Nasal and perineal samples were collected from 73 Labrador retrievers; staphylococci isolated and identified using phenotypic and biochemical methods. They were also confirmed by matrix-assisted laser desorption ionisation time-of-flight mass spectrometry (MALDI-TOF-MS), PCR of the nuc gene and PCR and sequencing of the tuf gene. Disc diffusion and minimum inhibitory concentration (MIC) susceptibility tests were determined for a range of antimicrobials. In total, 102 CoPS (S. pseudintermedius n = 91, S. aureus n = 11) and 334 CoNS isolates were detected from 99% of dogs in this study. In 52% of dogs CoNS only were detected, with both CoNS and CoPS detected in 43% dogs and CoPS only detected in 4% of dogs. Antimicrobial resistance was not common among CoPS, but at least one MDR-CoNS isolate was detected in 34% of dogs. MR-CoNS were detected from 42% of dogs but no MR-CoPS were isolated. S. epidermidis (52% of dogs) was the most common CoNS found followed by S. warneri (30%) and S. equorum (27%), with another 15 CoNS species isolated from ≤ 15% of dogs. S. pseudintermedius and S. aureus were detected in 44% and 8% of dogs respectively. CONCLUSIONS: MR- and MDR-CoPS were rare. However a high prevalence of MR- and MDR-CoNS were found in these dogs, even though they had no prior antimicrobial treatment or admission to veterinary premises. These findings are of concern due to the potential for opportunistic infections, zoonotic transmission and transmission of antimicrobial resistant determinants from these bacteria to coagulase positive staphylococci.