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Antimicrobial resistance within Staphylococcus pseudintermedius poses a significant risk for the treatment of canine pyoderma and as a reservoir for resistance and potential zoonoses, but few studies examine long-term temporal trends of resistance. This study assesses the antimicrobial resistance prevalence and minimum inhibitory concentration (MIC) trends in S. pseudintermedius (n=1804) isolated from canine skin samples at the Cornell University Animal Health Diagnostic Center (AHDC) between 2007 and 2020. Not susceptible (NS) prevalence, Cochran-Armitage tests, logrank tests, MIC50 and MIC90 quantiles, and survival analysis models were used to evaluate resistance prevalence and temporal trends to 23 antimicrobials. We use splines as predictors in accelerated failure time (AFT) models to model non-linear temporal trends in MICs. Multidrug resistance was common among isolates (47%), and isolates had moderate to high NS prevalence to the beta-lactams, chloramphenicol, the fluoroquinolones, gentamicin, the macrolides/lincosamides, the tetracyclines, and trimethoprim-sulfamethoxazole. However, low levels of NS to amikacin, rifampin, and vancomycin were observed. Around one third of isolates (38%) were found to be methicillin resistant S. pseudintermedius (MRSP), and these isolates had a higher prevalence of NS to all tested antimicrobials than methicillin susceptible isolates. Amongst the MRSP isolates, one phenotypically vancomycin resistant isolate (MIC >16 µg/mL) was identified, but genomic sequence data was unavailable. AFT models showed increasing MICs across time to the beta-lactams, chloramphenicol, the fluoroquinolones, gentamicin, and the macrolides/lincosamides, and decreasing temporal resistance (decreasing MICs) to doxycycline was observed amongst isolates. Notably, ATF modeling showed changes in MIC distributions that were not identified using Cochran-Armitage tests on prevalence, MIC quantiles, and logrank tests. Increasing resistance amongst these S. pseudintermedius isolates highlights the need for rational, empirical prescribing practices and increased antimicrobial resistance (AMR) surveillance to maintain the efficacy of current therapeutic agents. AFT models with non-linear predictors may be a useful, breakpoint-independent, surveillance tool alongside other modeling methods and antibiograms.

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ETHNOPHARMACOLOGICAL RELEVANCE: Atractylodis Rhizoma is extensively employed in Traditional Chinese Medicine for the treatment of skin and gastrointestinal ailments. Its active components have been proven to demonstrate numerous beneficial properties, including antibacterial, antiviral, anti-inflammatory, anti-tumor, and anti-ulcer activities. Furthermore, the volatile oil from Atractylodis Rhizoma (VOAR) has been reported to effectively inhibit and eradicate pathogens such as Staphylococcus aureus, Escherichia coli and Candida albicans. Of particular concern is Staphylococcus pseudintermedius, the predominant pathogen responsible for canine pyoderma, whose increasing antimicrobial resistance poses a serious public health threat. VOAR merits further investigation regarding its antibacterial potential against Staphylococcus pseudintermedius. AIM OF THE STUDY: The study aims to verify the in vitro antibacterial activity of VOAR against Staphylococcus pseudintermedius. And a superficial skin infection model in mice was established to assess the in vivo therapeutic effect of VOAR. MATERIALS AND METHODS: Thirty strains of S. pseudintermedius were isolated from dogs with pyoderma, and the drug resistance was analyzed by disc diffusion method. The Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) of VOAR were determined through the broth dilution method. The growth curve of bacteria in a culture medium containing VOAR was monitored using a UV spectrophotometer. Scanning electron microscopy was employed to observe the effects of VOAR on the microstructure of S. pseudintermedius. The impact of VOAR on the antibiotic resistance of S. pseudintermedius was assessed using the disc diffusion method. Twenty mice were randomly divided into four groups: the control group, the physiological saline group, the VOAR group, and the amikacin group. With the exception of the control group, the skin barrier of mice was disrupted by tap stripping, and the mice were subsequently inoculated with S. pseudintermedius to establish a superficial skin infection model. The modeled mice were treated with normal saline, VOAR, and amikacin for 5 days. Following the treatment period, the therapeutic effect of each group was evaluated based on the measures of body weight, skin symptoms, tissue bacterial load, tissue IL-6 content, and histopathological changes. RESULTS: The MIC and MBC of VOAR against 30 clinical isolates of S. pseudintermedius were found to be 0.005425% and 0.016875%, respectively. VOAR could exhibit the ability to delay the entry of bacteria into the logarithmic growth phase, disrupt the bacterial structure, and enhance the antibacterial zone in conjunction with antibiotic drugs. In the superficial skin infection model mice, VOAR significantly reduced the scores for skin redness (P < 0.0001), scab formation (P < 0.0001), and wrinkles (P < 0.0001). Moreover, VOAR markedly reduced the bacterial load (P < 0.001) and IL-6 content (P < 0.0001) in the skin tissues of mice. Histopathological observations revealed that the full-layer skin structure in the VOAR group was more complete, with clearer skin layers, and showed significant improvement in inflammatory cell infiltration and fibroblast proliferation compared to other groups. CONCLUSION: The results demonstrate that VOAR effectively inhibits and eradicates Staphylococcus pseudintermedius in vitro while also enhancing the pathogen's sensitivity to antibiotics. Moreover, VOAR exhibits a pronounced therapeutic effect in the superficial skin infection model mice.

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Time-kill curves (TKCs) are more informative compared with the use of minimum inhibitory concentration (MIC) as they allow the capture of bacterial growth and the development of drug killing rates over time, which allows to compute key pharmacodynamic (PD) parameters. Our study aimed, using a semi-mechanistic mathematical model, to estimate the best pharmacokinetic/pharmacodynamic (PK/PD) indices (ƒAUC/MIC or %ƒT > MIC) for the prediction of clinical efficacy of veterinary FQs in Staphylococcus pseudintermedius, Staphylococcus aureus, and Escherichia coli collected from canine pyoderma cases with a focus on the comparison between marbofloxacin and pradofloxacin. Eight TCKs for each bacterial species (4 susceptible and 4 resistant) were analysed in duplicate. The best PK/PD index was ƒAUC24h/MIC in both staphylococci and E. coli. For staphylococci, values of 25-40 h were necessary to achieve a bactericidal effect, whereas the calculated values (25-35 h) for E. coli were lower than those predicting a positive clinical outcome (100-120 h) in murine models. Pradofloxacin showed a higher potency (lower EC50) in comparison with marbofloxacin. However, no difference in terms of a maximal possible pharmacological killing rate (Emax) was observed. Taking into account in vivo exposure at the recommended dosage regimen (3 and 2 mg/kg for pradofloxacin and marbofloxacin, respectively), the overall killing rates (Kdrug) computed were also similar in most instances.

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Staphylococcus pseudintermedius is the most common opportunistic pathogen in dogs and methicillin resistance (MRSP) has been identified as an emerging problem in canine pyoderma. Here, we evaluated the antimicrobial resistance (AMR) features and phylogeny of S. pseudintermedius isolated from canine pyoderma cases in Argentina (n = 29) and the United States (n = 29). 62% of isolates showed multi-drug resistance. The AMR genes found: mecA, blaZ, ermB, dfrG, catA, tetM, aac(6')-aph(2″), in addition to tetK and lnuA (only found in U.S. isolates). Two point mutations were detected: grlA(S80I)-gyrA(S84L), and grlA(D84N)-gyrA(S84L) in one U.S. isolate. A mutation in rpoB (H481N) was found in two isolates from Argentina. SCCmec type III, SCCmec type V, ΨSCCmec57395 were identified in the Argentinian isolates; and SCCmec type III, SCCmec type IVg, SCCmec type V, and SCCmec type VII variant in the U.S. cohort. Sequence type (ST) ST71 belonging to a dominant clone was found in isolates from both countries, and ST45 only in Argentinian isolates. This is the first study to comparatively analyze the population structure of canine pyoderma-associated S. pseudintermedius isolates in Argentina and in the U.S. It is important to maintain surveillance on S. pseudintermedius populations to monitor AMR and gain further understanding of its evolution and dissemination.

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Staphylococcus pseudintermedius is an opportunistic pathogen commonly associated with skin infections in dogs. Twenty-three methicillin-resistant S. pseudintermedius (MRSP) isolated in Argentina from dogs with pyoderma were analyzed using whole genome sequencing (WGS) and classified into sequence types (ST) by multilocus sequence typing (MLST) and staphylococcal chromosome cassette mec (SCCmec) types.Based on the WGS analysis, MLST, and SCCmec type results, we report for the first time in Argentina two MRSP strains, one each, belonging to ST71-SCCmec III and ST45-ΨSCCmec57395 from dogs with pyoderma. We also identified seven isolates with ST339, which had been previously reported in only two isolates in Argentina. Additionally, we identified ten MRSP isolates harboring variants of the SCCmec V found in S. aureus, seven SCCmec V (5C2&5) with two ccrC1 recombinases, and three SCCmec V (5C2) with one ccrC1 recombinase.Our findings provide important insights into the evolution and geographic spread of these hypervirulent dominant clones that threaten the health of our companion animals and represent a significant risk for zoonotic infections.

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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.

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Staphylococcal cassette chromosome mec (SCCmec) confers methicillin resistance and shows ability for horizontal transfer. However, little is known about the potential transfer of SCCmec between different species of staphylococci in a clinical setting. In this study, we investigated the genetic relationship of SCCmec between staphylococci isolated from dogs affected with pyoderma and their owners. Clinical isolates were collected from pyoderma lesions of dogs and from the nasal cavity and finger of owners. Clonal lineages were characterized using multi-locus sequence typing. Genetic relatedness of SCCmec in the isolates from dogs and owners was first evaluated with dru and SCCmec typing, and whole-genome sequencing (WGS) was used to confirm the similarity of DNA sequences and the structural composition of SCCmec. A total of 100 Staphylococcus strains were isolated from 31 dog-owner pairs. One pair with isolates carrying the same SCCmec type V and dru type 11a was detected: 18D20-1 (S. pseudintermedius, dog), 18D20-2 (S. schleiferi, dog), and 18H20-F2 (S. epidermidis, dog owner). WGS revealed that these three isolates showed remarkable genetic similarity in SCCmec with respect to DNA sequences, dru type, structure composition of ccrC and the mec complex, and DR-1 in orfX, which is considered to be the insertion site of SCCmec. Entire identical nucleotide sequences of the whole SCCmec region in different Staphylococcus strains were absent between dogs and owners. However, the remarkable genetic similarity of SCCmec from staphylococci isolated from a dog and owner pair emphasizes that antimicrobial resistance surveillance adopted One Health concept should be continuously performed.

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Although Staphylococcus pseudintermedius is considered the major pathogen associated with superficial canine pyoderma, no study has investigated the entire bacterial community in these lesions with molecular techniques. The objectives of this study were to characterize the bacterial microbiota in two forms of superficial canine pyoderma lesions, superficial bacterial folliculitis (SBF) and epidermal collarette (EC), especially in terms of the staphylococcal community. Swabs from 12 SBF and 9 EC lesions were obtained from eight and six atopic dogs, respectively. Eight samples from the axilla and groin of four healthy dogs served as controls. DNA was extracted for 16S rRNA gene sequencing and quantitative polymerase chain reaction of Staphylococcus spp. and S. pseudintermedius. Healthy skin samples harbored significantly more diverse bacterial communities than pyoderma samples. Healthy samples had communities that were more similar to each other, and were distinct from pyoderma samples. Staphylococcus spp. abundance was increased in pyoderma samples, especially those from EC samples. Although determining species-level identities of staphylococcal sequences revealed many species, S. pseudintermedius was the primary staphylococcal species found in all sample types. As expected, there are many differences in the microbiota when comparing healthy and canine pyoderma lesions samples. These lesions do not seem to be associated with a change in the relative abundance of specific Staphylococcus species, but simply an overall increase in Staphylococcus spp. abundance. The results of this study provide a starting point for future studies investigating how antimicrobial treatments may further change the microbiota associated with these lesions.

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The emergence of staphylococcal canine pathogens resistant to multiple antimicrobial agents is a growing and urgent problem in veterinary practice. Antimicrobial peptides (AMPs) seem to be a promising alternative to conventional antibiotics. The aim of this in vitro study was to evaluate the antimicrobial activity of selected AMPs against pathogenic staphylococcal strains, including multidrug- and methicillin-resistant strains isolated from canine pyoderma cases. Seven antimicrobial peptides (aurein 1.2, CAMEL, citropin 1.1, protegrin-1, pexiganan, temporin A and uperin 3.6) synthesized by the 9-fluorenylmethoxycarbonyl (Fmoc) solid-phase method were tested. The minimal inhibitory and minimal bactericidal concentrations (MIC and MBC) were determined by the broth microdilution method. The study showed that analyzed AMPs exerted an extensive effect against canine pathogens, with the most active peptide being uperin 3.6. The tested AMPs were equally efficient against both resistant- and susceptible staphylococcal strains and were more efficient against Staphylococcus pseudintermedius than against Staphylococcus aureus strains. Our findings are particularly interesting from a clinical perspective, as they point to AMPs as potential therapeutic topical agents in canine pyoderma cases associated with antimicrobial resistance of staphylococci.

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Methicillin-resistant Staphylococcus pseudintermedius (MRSP) is of worldwide concern in veterinary medicine. The identification of resistant strains is necessary for proper treatment and the prevention of its propagation among animals. This study aimed to identify S. pseudintermedius isolated from canine pyoderma and evaluate their resistance profiles. Lesions from 25 dogs with pyoderma were sampled. Bacterial isolates were subjected to phenotypic and genotypic analysis for identification of the causative agent. S. pseudintermedius isolates were subjected to SmaI macrorestriction analysis and PFGE for genetic grouping, and PCR to identify the presence of the mecA gene. Their resistance profiles against 12 antimicrobials were also assessed. According to the microbiological analysis, 70 of the 75 isolates obtained were S. pseudintermedius. The isolates presented PFGE patterns, with similarity varying between 84.6 and 100%, and were grouped into 19 clusters. Despite a high frequency of mecA-positive isolates (66 out 70), only 12 presented resistances to oxacillin. Multi-resistance was identified in 29 isolates. The high frequency of MRSP isolated in this study highlights the relevance of identifying resistant strains to lead proper clinical treatment.

Staphylococcus pseudintermedius meticilina-resistente (MRSP) é de preocupação mundial na medicina veterinária. A identificação de cepas resistentes é necessária a um tratamento adequado e à prevenção da sua propagação entre os animais. O objetivo do estudo foi identificar S. pseudintermedius isolados de piodermite canina e avaliar o perfil de resistência. Foram coletadas amostras de lesões de 25 cães com piodermite. Os isolados bacterianos foram submetidos a análises fenotípicas e genotípicas para identificação do agente causador. Isolados de S. pseudintermedius foram submetidos à análise de macrorrestrição por SmaI e PFGE para agrupamento genético e à PCR para identificar a presença do gene mecA. Seu perfil de resistência contra 12 antimicrobianos também foi avaliado. De acordo com a análise microbiológica, 70 dos 75 isolados obtidos foram identificados como S. pseudintermedius. Os isolados apresentaram padrões de PFGE com similaridade variando entre 84.6 e 100% e foram agrupados em 19 grupos genéticos. Apesar da frequência alta de isolados mecA positivos (66 em 70), apenas 12 apresentaram resistência à oxacilina. Multirresistência foi identificada em 29 isolados. A alta frequência de MRSP isolados neste estudo destaca a relevância de se identificarem cepas resistentes para se conduzir um tratamento clínico adequado.

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CASE HISTORY: A 14-year-old neutered male Sealyham terrier was referred for assessment of a persistent pyoderma. It had experienced numerous episodes of dermatitis involving pododermatitis, pyoderma and otitis over the previous 6 years. CLINICAL FINDINGS: Superficial, focally deep and mucocutaneous pyoderma were present, with yellow mucoid exudate on both nares and the lower lips crusted with haemopurulent exudate. Epidermal collarettes were present on the dorsal and lateral trunk. There were peri-anal crusts and mild erythema was present on the concave aspect of both pinnae. MICROBIOLOGICAL FINDINGS: Culture and microbiological testing identified Staphylococcus pseudintermedius as the infecting organism. Kirby-Bauer disc susceptibility testing revealed the isolate was resistant to numerous antimicrobials including oxacillin. PCR testing of the isolate identified the presence of the mecA gene which confers resistance to ß-lactam antimicrobials. Pulsed field gel electrophoresis typing suggested the isolate was not related to the methicillin-resistant S. pseudintermedius that had been reported to be associated with canine infections in Western Australia. DIAGNOSIS: Superficial, deep and mucus membrane pyoderma associated with a multi-drug resistant S. pseudintermedius. CLINICAL RELEVANCE: This is the first recorded case of canine pyoderma involving methicillin-resistant multidrug-resistant S. pseudintermedius in New Zealand. Treatment of such cases is difficult because the number of effective and available antimicrobials is limited. This finding should raise the awareness of the veterinary and medical professions to the presence of such organisms in New Zealand and stimulate a discussion about possible biosecurity barriers, treatment strategies and prevention of zoonotic and nosocomial infections.

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