Intervention with impact: Reduced isolation of methicillin-resistant Staphylococcus pseudintermedius from dogs following the introduction of antimicrobial prescribing legislation in Germany.

BACKGROUND: Legislation was introduced in Germany in 2018, requiring bacterial culture and antimicrobial susceptibility testing before the prescription of fluoroquinolones and third-generation cephalosporins to dogs. We hypothesised that, following this intervention, the number of clinical samples testing positive for methicillin-resistant Staphylococcus pseudintermedius (MRSP) would reduce. METHODS: Reports of S. pseudintermedius isolated from canine clinical samples by three German veterinary diagnostic microbiology laboratories during the 38 months before the introduction of the legislation and the 46 months after were compared. Bacterial identification was performed by matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry, and antimicrobial susceptibility testing followed recognised recommendations but with changes during the study period. RESULTS: Among a total of 120,571 S. pseudintermedius isolates, MRSP accounted for 7.1% overall. Following the legislative intervention, monthly submissions yielding S. pseudintermedius increased at all three laboratories. The MRSP percentage was lower in the period after the intervention in two of the three laboratories (p < 0.001); in the third laboratory, there was no change between periods, but a year-on-year reduction in MRSP percentages occurred after the intervention (p = 0.0004). LIMITATIONS: Changing susceptibility testing methods limited the direct comparison of resistance patterns among laboratories. CONCLUSION: The reduction in MRSP in canine clinical samples following the introduction of this legislation suggests a positive impact of compulsory laboratory testing on reducing antimicrobial resistance.

Staphylococcus aureus lineages associated with a free-ranging population of the fruit bat Pteropus livingstonii retained over 25 years in captivity.

Conservation of endangered species has become increasingly complex, and costly interventions to protect wildlife require a robust scientific evidence base. This includes consideration of the role of the microbiome in preserving animal health. Captivity introduces stressors not encountered in the wild including environmental factors and exposure to exotic species, humans and antimicrobial drugs. These stressors may perturb the microbiomes of wild animals, with negative consequences for their health and welfare and hence the success of the conservation project, and ultimately the risk of release of non-native organisms into native ecosystems. We compared the genomes of Staphylococcus aureus colonising critically endangered Livingstone's fruit bats (Pteropus livingstonii) which have been in a captive breeding programme for 25 years, with those from bats in the endemic founder population free ranging in the Comoros Republic. Using whole genome sequencing, we compared 47 isolates from captive bats with 37 isolates from those free ranging in the Comoros Republic. Our findings demonstrate unexpected resilience in the bacteria carried, with the captive bats largely retaining the same two distinctive lineages carried at the time of capture. In addition, we found evidence of genomic changes which suggest specific adaptations to the bat host.

Fatal exudative dermatitis in island populations of red squirrels (Sciurus vulgaris): spillover of a virulent Staphylococcus aureus clone (ST49) from reservoir hosts.

Fatal exudative dermatitis (FED) is a significant cause of death of red squirrels (Sciurus vulgaris) on the island of Jersey in the Channel Islands where it is associated with a virulent clone of Staphylococcus aureus, ST49. S. aureus ST49 has been found in other hosts such as small mammals, pigs and humans, but the dynamics of carriage and disease of this clone, or any other lineage in red squirrels, is currently unknown. We used whole-genome sequencing to characterize 228 isolates from healthy red squirrels on Jersey, the Isle of Arran (Scotland) and Brownsea Island (England), from red squirrels showing signs of FED on Jersey and the Isle of Wight (England) and a small number of isolates from other hosts. S. aureus was frequently carried by red squirrels on the Isle of Arran with strains typically associated with small ruminants predominating. For the Brownsea carriage, S. aureus was less frequent and involved strains associated with birds, small ruminants and humans, while for the Jersey carriage S. aureus was rare but ST49 predominated in diseased squirrels. By combining our data with publicly available sequences, we show that the S. aureus carriage in red squirrels largely reflects frequent but facile acquisitions of strains carried by other hosts sharing their habitat ('spillover'), possibly including, in the case of ST188, humans. Genome-wide association analysis of the ruminant lineage ST133 revealed variants in a small number of mostly bacterial-cell-membrane-associated genes that were statistically associated with squirrel isolates from the Isle of Arran, raising the possibility of specific adaptation to red squirrels in this lineage. In contrast there is little evidence that ST49 is a common carriage isolate of red squirrels and infection from reservoir hosts such as bank voles or rats, is likely to be driving the emergence of FED in red squirrels.

Reduced antimicrobial prescribing during autogenous staphylococcal bacterin therapy: a retrospective study in dogs with pyoderma.

Autogenous staphylococcal bacterins are commonly mentioned as treatment for canine recurrent pyoderma but little is known about their efficacy. This retrospective study describes use and assesses efficacy of an autogenous Staphylococcus (pseud)intermedius bacterin in dogs with pyoderma. Frequency and duration of systemic antimicrobial therapy were compared 12 months before and after starting bacterin (Wilcoxon signed-rank test) with data extracted from general practice medical histories.

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.

Antimicrobial Stewardship in Veterinary Medicine.

While antimicrobial resistance is already a public health crisis in human medicine, therapeutic failure in veterinary medicine due to antimicrobial resistance remains relatively uncommon. However, there are many pathways by which antimicrobial resistance determinants can travel between animals and humans: by close contact, through the food chain, or indirectly via the environment. Antimicrobial stewardship describes measures that can help mitigate the public health crisis and preserve the effectiveness of available antimicrobial agents. Antimicrobial stewardship programs have been principally developed, implemented, and studied in human hospitals but are beginning to be adapted for other applications in human medicine. Key learning from the experiences of antimicrobial stewardship programs in human medicine are summarized in this article-guiding the development of a stewardship framework suitable for adaptation and use in both companion animal and livestock practice. The antimicrobial stewardship program for veterinary use integrates infection prevention and control together with approaches emphasizing avoidance of antimicrobial agents. The 5R framework of continuous improvement that is described recognizes the importance of executive support; highly motivated organizations and teams (responsibility); the need to review the starting position, set objectives, and determine means of measuring progress and success; and a critical focus on reducing, replacing, and refining the use of antimicrobial agents. Significant issues that are currently the focus of intensive research include improved detection and diagnosis of infections, refined dosing regimens that are simultaneously effective while not selecting resistance, searches for alternatives to antimicrobial agents, and development of improved vaccines to enhance immunity and reduce disease.

Isolation and identification of Acinetobacter spp. from healthy canine skin.

BACKGROUND: Acinetobacter species can exhibit widespread resistance to antimicrobial agents. They are already recognized as important nosocomial pathogens of humans, but are becoming increasingly recognized in opportunistic infections of animals. HYPOTHESIS/OBJECTIVES: This study aimed to determine whether Acinetobacter spp. are carried on skin of healthy dogs and, if present, to identify the species. ANIMALS: Forty dogs were sampled at veterinary practices and rescue centres. They were free from skin disease and receiving no systemic or topical treatments. METHODS: Skin swab samples were collected from four sites on each dog and cultured. Acinetobacter spp. isolates were detected by biochemical tests and gas chromatography. The species was determined by sequencing the RNA polymerase ß-subunit (rpoB) gene. Isolates were screened for OXA carbapenemase genes and class 1 integrons capable of carrying resistance genes, and subjected to antimicrobial susceptibility tests. RESULTS: For 25% dogs sampled (10 of 40), Acinetobacter spp. were isolated at one or more skin sites. Thirteen Acinetobacter spp. isolates were recovered from 160 samples. The most frequently cultured was A. lwoffii (seven of 13), followed by A. baumannii (two of 13), A. junii (one of 13), A. calcoaceticus (one of 13), A. pittii (one of 13) and a novel Acinetobacter species (one of 13). Class 1 integrons and blaOXA-23-like were not detected. Isolates were susceptible to most antibiotics. CONCLUSIONS AND CLINICAL IMPORTANCE: The study confirms that Acinetobacter spp. can survive on canine skin, where they may be potential reservoirs for infection. This highlights the importance of good hygiene in veterinary practice, adhering to aseptic principles in surgery, and treatment based on culture and susceptibility testing where possible.

Skin disease in captive bats: results of an online survey of zoos and rehabilitators in Europe, North America and Australasia.

BACKGROUND: Bats may be held captive in zoos and breeding programmes, and for rehabilitation due to illness, abandonment or injury. OBJECTIVES: To describe the frequency and characteristics of skin disease in captive bats. METHODS: Zoos (n = 164) in Europe, North America, Australia and New Zealand, Wildlife Disease Association members and rehabilitators were invited to complete online questionnaires on skin lesions and housing. Associations between lesion type and site, frequency, species, age, suspected cause and season, and their association with housing in zoos were tested using chi-squared and two-sample z-tests. RESULTS: Skin lesions were seen by 38.5% (15 of 39) of responding zoos and more frequently by rehabilitators (66.7%, 18 of 27; P = 0.024). Of the total of 153 lesions of any type reported by zoos and rehabilitators, almost two thirds occurred on the pinnae (49 of 153, 32%) or wing membranes (45 of 153, 29%). Amongst pinnal lesions, crusting (27%), swelling and redness (25%) and necrosis (20%) were most frequent. In zoos, pinnal (P = 0.001) and wing lesions (P = 0.045) were associated with "season", being more common in winter. Pruritus was rare but more often reported from rehabilitation centres (12 of 77 observed lesions) than from zoos (1 of 76) (P = 0.0015). Lesions most often affected adult and geriatric bats in zoos, and juveniles and adults in rehabilitation. Eight respondents reported that skin disease necessitated euthanasia in individual bats. There was no significant association between type of housing and lesions. CONCLUSION: Pinnal and wing lesions were common in captive bats, often with necrosis. Further research into the causes is needed to improve health and welfare of captive bats.

Oxacillin sensitization of methicillin-resistant Staphylococcus aureus and methicillin-resistant Staphylococcus pseudintermedius by antisense peptide nucleic acids in vitro.

BACKGROUND: Antibiotic resistance genes can be targeted by antisense agents, which can reduce their expression and thus restore cellular susceptibility to existing antibiotics. Antisense inhibitors can be gene and pathogen specific, or designed to inhibit a group of bacteria having conserved sequences within resistance genes. Here, we aimed to develop antisense peptide nucleic acids (PNAs) that could be used to effectively restore susceptibility to ß-lactams in methicillin-resistant Staphylococcus aureus (MRSA) and methicillin-resistant Staphylococcus pseudintermedius (MRSP). RESULTS: Antisense PNAs specific for conserved regions of the mobilisable gene mecA, and the growth essential gene, ftsZ, were designed. Clinical MRSA and MRSP strains of high oxacillin resistance were treated with PNAs and assayed for reduction in colony forming units on oxacillin plates, reduction in target gene mRNA levels, and cell size. Anti-mecA PNA at 7.5 and 2.5 µM reduced mecA mRNA in MRSA and MRSP (p < 0.05). At these PNA concentrations, 66 % of MRSA and 92 % of MRSP cells were killed by oxacillin (p < 0.01). Anti-ftsZ PNA at 7.5 and 2.5 µM reduced ftsZ mRNA in MRSA and MRSP, respectively (p ≤ 0.05). At these PNA concentrations, 86 % of MRSA cells and 95 % of MRSP cells were killed by oxacillin (p < 0.05). Anti-ftsZ PNAs resulted in swelling of bacterial cells. Scrambled PNA controls did not affect MRSA but sensitized MRSP moderately to oxacillin without affecting mRNA levels. CONCLUSIONS: The antisense PNAs effects observed provide in vitro proof of concept that this approach can be used to reverse ß-lactam resistance in staphylococci. Further studies are warranted as clinical treatment alternatives are needed.

Genomic insights into the rapid emergence and evolution of MDR in Staphylococcus pseudintermedius.

OBJECTIVES: MDR methicillin-resistant Staphylococcus pseudintermedius (MRSP) strains have emerged rapidly as major canine pathogens and present serious treatment issues and concerns to public health due to their, albeit low, zoonotic potential. A further understanding of the genetics of resistance arising from a broadly susceptible background of S. pseudintermedius is needed. METHODS: We sequenced the genomes of 12 S. pseudintermedius isolates of varied STs and resistance phenotypes. RESULTS: Nine distinct clonal lineages had acquired either staphylococcal cassette chromosome (SCC) mec elements and/or Tn5405-like elements carrying up to five resistance genes [aphA3, sat, aadE, erm(B), dfrG] to generate MRSP, MDR methicillin-susceptible S. pseudintermedius and MDR MRSP populations. The most successful and clinically problematic MDR MRSP clones, ST68 SCCmecV(T) and ST71 SCCmecII-III, have further accumulated mutations in gyrA and grlA conferring resistance to fluoroquinolones. The carriage of additional mobile genetic elements (MGEs) was highly variable, suggesting that horizontal gene transfer is frequent in S. pseudintermedius populations. CONCLUSIONS: Importantly, the data suggest that MDR MRSP evolved rapidly by the acquisition of a very limited number of MGEs and mutations, and that the use of many classes of antimicrobials may co-select for the spread and emergence of MDR and XDR strains. Antimicrobial stewardship will need to be comprehensive, encompassing human medicine and veterinary disciplines to successfully preserve antimicrobial efficacy.

Extensive horizontal gene transfer during Staphylococcus aureus co-colonization in vivo.

Staphylococcus aureus is a commensal and major pathogen of humans and animals. Comparative genomics of S. aureus populations suggests that colonization of different host species is associated with carriage of mobile genetic elements (MGE), particularly bacteriophages and plasmids capable of encoding virulence, resistance, and immune evasion pathways. Antimicrobial-resistant S. aureus of livestock are a potential zoonotic threat to human health if they adapt to colonize humans efficiently. We utilized the technique of experimental evolution and co-colonized gnotobiotic piglets with both human- and pig-associated variants of the lineage clonal complex 398, and investigated growth and genetic changes over 16 days using whole genome sequencing. The human isolate survived co-colonization on piglets more efficiently than in vitro. During co-colonization, transfer of MGE from the pig to the human isolate was detected within 4 h. Extensive and repeated transfer of two bacteriophages and three plasmids resulted in colonization with isolates carrying a wide variety of mobilomes. Whole genome sequencing of progeny bacteria revealed no acquisition of core genome polymorphisms, highlighting the importance of MGE. Staphylococcus aureus bacteriophage recombination and integration into novel sites was detected experimentally for the first time. During colonization, clones coexisted and diversified rather than a single variant dominating. Unexpectedly, each piglet carried unique populations of bacterial variants, suggesting limited transmission of bacteria between piglets once colonized. Our data show that horizontal gene transfer occurs at very high frequency in vivo and significantly higher than that detectable in vitro.

Guidelines for the diagnosis and antimicrobial therapy of canine superficial bacterial folliculitis (Antimicrobial Guidelines Working Group of the International Society for Companion Animal Infectious Diseases).

BACKGROUND: Superficial bacterial folliculitis (SBF) is usually caused by Staphylococcus pseudintermedius and routinely treated with systemic antimicrobial agents. Infection is a consequence of reduced immunity associated with alterations of the skin barrier and underlying diseases that may be difficult to diagnose and resolve; thus, SBF is frequently recurrent and repeated treatment is necessary. The emergence of multiresistant bacteria, particularly meticillin-resistant S. pseudintermedius (MRSP), has focused attention on the need for optimal management of SBF. OBJECTIVES: Provision of an internationally available resource guiding practitioners in the diagnosis, treatment and prevention of SBF. DEVELOPMENT OF THE GUIDELINES: The guidelines were developed by the Antimicrobial Guidelines Working Group of the International Society for Companion Animal Infectious Diseases, with consultation and advice from diplomates of the American and European Colleges of Veterinary Dermatology. They describe optimal methods for the diagnosis and management of SBF, including isolation of the causative organism, antimicrobial susceptibility testing, selection of antimicrobial drugs, therapeutic protocols and advice on infection control. Guidance is given for topical and systemic modalities, including approaches suitable for MRSP. Systemic drugs are classified in three tiers. Tier one drugs are used when diagnosis is clear cut and risk factors for antimicrobial drug resistance are not present. Otherwise, tier two drugs are used and antimicrobial susceptibility tests are mandatory. Tier three includes drugs reserved for highly resistant infections; their use is strongly discouraged and, when necessary, they should be used in consultation with specialists. CONCLUSIONS AND CLINICAL IMPORTANCE: Optimal management of SBF will improve antimicrobial use and reduce selection of MRSP and other multidrug-resistant bacteria affecting animal and human health.

Case-control risk factor study of methicillin-resistant Staphylococcus pseudintermedius (MRSP) infection in dogs and cats in Germany.

Methicillin-resistant Staphylococcus pseudintermedius (MRSP) has emerged as a highly drug-resistant small animal veterinary pathogen. Although often isolated from outpatients in veterinary clinics, there is concern that MRSP follows a veterinary-hospital-associated epidemiology. This study's objective was to identify risk factors for MRSP infections in dogs and cats in Germany. Clinical isolates of MRSP cases (n=150) and methicillin-susceptible S. pseudintermedius (MSSP) controls (n=133) and their corresponding host signalment and medical data covering the six months prior to staphylococcal isolation were analysed by multivariable logistic regression. The identity of all MRSP isolates was confirmed through demonstration of S. intermedius-group specific nuc and mecA. In the final model, cats (compared to dogs, OR 18.5, 95% CI 1.8-188.0, P=0.01), animals that had been hospitalised (OR 104.4, 95% CI 21.3-511.6, P<0.001), or visited veterinary clinics more frequently (>10 visits OR 7.3, 95% CI 1.0-52.6, P=0.049) and those that had received topical ear medication (OR 5.1, 95% CI 1.8-14.9, P=0.003) or glucocorticoids (OR 22.5, 95% CI 7.0-72.6, P<0.001) were at higher risk of MRSP infection, whereas S. pseudintermedius isolates from ears were more likely to belong to the MSSP-group (OR 0.09, 95% CI 0.03-0.34, P<0.001). These results indicate an association of MRSP infection with veterinary clinic/hospital settings and possibly with chronic skin disease. There was an unexpected lack of association between MRSP and antimicrobial therapy; this requires further investigation but may indicate that MRSP is well adapted to canine skin with little need for selective pressure.

Whole-genome comparison of meticillin-resistant Staphylococcus aureus CC22 SCCmecIV from people and their in-contact pets.

BACKGROUND: Meticillin-resistant Staphylococcus aureus (MRSA) infections remain important medical and veterinary challenges. The MRSA isolated from dogs and cats typically belong to dominant hospital-associated clones, in the UK mostly EMRSA-15 (CC22 SCCmecIV), suggesting original human-to-animal transmission. Nevertheless, little is known about host-specific genetic variation within the same S. aureus lineage. HYPOTHESIS/OBJECTIVES: To identify host-specific variation amongst MRSA CC22 SCCmecIV by comparing isolates from pets with those from in-contact humans using whole-genome microarray. METHODS: Six pairs of MRSA CC22 SCCmecIV from human carriers (owners and veterinary staff) and their respective infected in-contact pets were compared using a 62-strain whole-genome S. aureus microarray (SAM-62). The presence of putative host-specific genes was subsequently determined in a larger number of human (n = 47) and pet isolates (n = 93) by PCR screening. RESULTS: Variation in mobile genetic elements (MGEs) occurred frequently and appeared largely independent of host and in-contact pair. A plasmid (SAP078A) encoding heavy-metal resistance genes (arsR, arsA, cadA, cadC, mco and copB) was found in three of six human and none of six animal isolates. However, only two of four resistance genes were associated with human hosts (P = 0.015 for arsA and cadA). CONCLUSIONS AND CLINICAL IMPORTANCE: The variation found amongst MGEs highlights that genetic adaptation in MRSA continues. However, host-specific MGEs were not detected, which supports the hypothesis that pets may not be natural hosts of MRSA CC22 and emphasizes that rigorous hygiene measures are critical to prevent contamination and infection of dogs and cats. The host specificity of individual heavy-metal resistance genes warrants further investigation into different selection pressures in humans and animals.

Alternatives to conventional antimicrobial drugs: a review of future prospects.

BACKGROUND: Growing antimicrobial resistance poses the threat that before long no suitable drugs will be available for treatment of common infections. This review examines promising new strategies for treatment and control of microbial diseases, with an emphasis on staphylococcal infection. NEW DRUGS AND TARGETS: Advances in microbial genomics have provided tools identifying many new targets for antimicrobial drugs. Of particular interest amongst these are inhibition of microbial efflux pump activity, interruption or diversion of riboswitches controlling bacterial metabolism, and metagenomics, which allows analysis of genes from unculturable organisms. BIOLOGICAL APPROACHES: Advances are also being made in biological systems for disease control, with the exploitation of antimicrobial peptides to attack micro-organisms and modulate immune responses, and the use of bacteriophages or their lysins to eliminate bacteria. There are new approaches in the development and targeting of vaccines and immunoglobulin preparations based on advanced knowledge of microbial physiology and immunoregulation. WORKING WITH THE BIOME: With increasing recognition of the value of the normal microbiota in modulating immunity and the establishment of pathogens, there is growing interest in understanding the mammalian microbiome. Strategies are being developed to promote or maintain the normal microbiota, including the use of probiotics, and there is re-evaluation of the potential of bacterial interference. LOOKING AHEAD: Whilst these approaches are likely to generate new methods of disease control, few will yield usable products within the near future. There will be a continuing need for careful use of existing drugs based on firm diagnosis, rigorous hygiene and prudent antimicrobial stewardship.

Foxes as a potential wildlife reservoir for mecA-positive Staphylococci.

Methicillin-resistant staphylococci (MRS), and methicillin-resistant Staphylococcus aureus (MRSA) in particular, have become a public and veterinary health concern. The search for MRS reservoirs outside human hospitals is needed in order to understand the reasons for their persistence and to control their spread. MRS have been isolated from rats, but little is known about their occurrence in foxes. In view of the perceived increasing proximity between people and foxes in the U.K. and the well-documented potential of foxes as hosts for zoonotic pathogens, this study examined whether foxes can be a reservoir for MRS. This study examined the carriage of staphylococci and their antimicrobial resistance patterns in 38 foxes (Vulpes vulpes) from rural and semirural areas in the U.K. Staphylococci were isolated by enrichment culture from nasal, oral, axillary, and perineal swabs and speciated by standard bacteriological tests and API ID32 STAPH (bioMérieux, Marcy l'Etoile, France). Antimicrobial resistance was investigated by disc diffusion tests and identification of mecA. Thirty-seven staphylococcal isolates were identified from 35 of the 38 foxes. All isolates were coagulase-negative and most frequently included species from the S. sciuri group (35%), S. equorum (27%), and S. capitis (22%). All were phenotypically resistant to methicillin, and mecA was detected in 33 (89%) of isolates, but only 10 (27%) showed broad ß-lactam antibiotic resistance. Methicillin-resistant S. aureus was not identified. These results indicate that foxes are a potential wildlife reservoir for mecA-positive staphylococci. Selection pressure from environmental contamination with antimicrobials should be considered.