In vitro antiseptic susceptibilities for Staphylococcus pseudintermedius isolated from canine superficial pyoderma in Japan

Background –  Topical therapy, particularly with chlorhexidine, is becoming increasingly common as a treatment option for canine pyoderma; however, there are limited studies on the susceptibility of Staphylococcus pseudintermedius to chlorhexidine compounds. Objectives –  To determine the in vitro susceptibility of both meticillin‐resistant and meticillin‐susceptible S. pseudintermedius isolates to chlorhexidine and other antiseptic agents and the presence of multidrug efflux pump genes. Samples –  One hundred S. pseudintermedius isolates from 23 initial and 77 recurrent cases of canine pyoderma. Methods –  After bacterial identification and mecA testing, minimal inhibitory concentrations (MICs) of antiseptic agents were determined. Multidrug efflux pump genes, including qacA, qacB and smr, were identified. Results –  Of the 100 isolates, 57 were identified as meticillin‐resistant S. pseudintermedius. The MIC₉₀ of chlorhexidine acetate, chlorhexidine gluconate, acriflavine, ethidium bromide and benzalkonium chloride were 1, 1, 2, 0.5 and 2 μg/mL, respectively. Multidrug efflux pump genes qacA, qacB and smr were not detected in any of the isolates. Conclusions and clinical importance –  The MICs for chlorhexidine and other antiseptics remain low, and multidrug efflux pump genes were not found in the tested isolates.     

In vitro miconazole susceptibility of meticillin-resistant Staphylococcus pseudintermedius and Staphylococcus aureus

Background – The emergence and dissemination of meticillin‐resistant staphylococci has created significant treatment challenges in veterinary medicine and increased interest in topical therapy for superficial infections. Concern has been expressed regarding the use of some topical antimicrobials in animals because of the potential for emergence of resistance, and additional options are required. Miconazole has limited antibacterial properties that include antistaphylococcal activity. Hypothesis/Objectives – The objective of this study was to assess the in vitro susceptibility of Staphylococcus pseudintermedius and Staphylococcus aureus to miconazole. Methods – In vitro susceptibility of 112 meticillin‐resistant S. pseudintermedius (MRSP), 53 meticillin‐resistant S. aureus (MRSA) and 37 meticillin‐susceptible S. pseudintermedius (MSSP) to miconazole was assessed using agar dilution. Results – The minimal inhibitory concentration (MIC) range, MIC₅₀ and MIC₉₀ for MRSP were 1–8, 2 and 4 μg/mL, respectively. Corresponding results for MRSA were 1–8, 2 and 6 μg/mL, and for MSSP 1–4, 2 and 2 μg/mL. The MIC for MSSP was a significantly lower MIC than that for both MRSP (P = 0.006) and MRSA (P < 0.001), while the MIC for MRSP was significantly lower than that for MRSA (P = 0.001). Conclusions and clinical importance – These in vitro data suggest that miconazole could be a useful therapeutic option for superficial infections caused by meticillin‐susceptible and meticillin‐resistant staphylococci, but proper clinical investigation is required.     

In vitro evaluation of topical biocide and antimicrobial susceptibility of Staphylococcus pseudintermedius from dogs

Background – Staphylococcus pseudintermedius is an important canine pathogen, and the emergence and widespread dissemination of meticillin‐resistant strains (MRSP) is of significant concern. Multidrug‐resistant infections may require alternative approaches, such as the use of topical therapy. There is minimal information about the in vitro susceptibility of meticillin‐susceptible S. pseudintermedius (MSSP) and MRSP to biocides and topical antimicrobials. Hypothesis/Objectives – The hypothesis was that clinical isolates of MSSP and MRSP would not have universal susceptibility to topical biocides and antimicrobials. The goal of this study was to assess the susceptibility of a collection of S. pseudintermedius isolates to selected antimicrobials and biocides. Animals – The study was performed on clinical isolates of MSSP and MRSP from dogs with skin and soft tissue infections collected throughout North America between 2006 and 2008. Methods – The minimal inhibitory concentrations (MICs) of chlorhexidine digluconate, benzalkonium chloride, triclosan, accelerated hydrogen peroxide, geranium oil, tea tree oil and grapefruit seed extract were tested for 25 MRSP and 25 MSSP isolates from dogs using the agar dilution method. The MICs of fusidic acid, bacitracin and mupirocin were determined using Etests. Results – Triclosan demonstrated excellent activity against all bacterial isolates, with no growth at the lowest concentration evaluated (MIC ≤ 0.5 μg/mL). Conversely, grapefruit seed extract did not inhibit growth at the highest concentration tested (MIC > 3.84 μg/mL). All isolates were susceptible to mupirocin, fusidic acid and bacitracin. There were no significant differences noted in the range, MIC₅₀ or MIC₉₀ between MSSP and MRSP isolates. Conclusions and clinical importance – While isolates were susceptible to most of the tested compounds, universal susceptibility to all compounds with potential antimicrobial activity cannot be assumed, and specific testing is required.     

Comparative in vitro efficacy of antimicrobial shampoos: a pilot study

This study compared the antimicrobial efficacy of shampoos against meticillin‐sensitive Staphylococcus pseudintermedius (MSSP), meticillin‐resistant S. pseudintermedius (MRSP), antibiotic‐sensitive Pseudomonas aeruginosa (PA), multidrug‐resistant P. aeruginosa (MDR‐PA) and Malassezia pachydermatis. Three isolates were incubated for 10, 30 and 60 min with each shampoo diluted in phosphate‐buffered saline. Aliquots were then incubated for 16–18 h on sheep blood agar (bacteria) or for 3 days on Sabouraud’s dextrose agar (Malassezia). The minimal bactericidal concentrations (MBCs) for chlorhexidine products (Malaseb^®, Pyoderm^®/Microbex^® and Hibiscrub^®) were 1:1,024–1:2,048 for MSSP and MRSP, 1:512–1:1,024 for PA and MDR‐PA, and 1:2,048–1:5,096 for Malassezia at all time points. The MBCs for benzoyl peroxide (Paxcutol^®) for MSSP and MRSP were 1:2–1:8 at 10 min, and 1:256 after 30 and 60 min. A 1:2 dilution was effective against Pseudomonas, and 1:512–1:1,024 dilutions were effective against Malassezia at all time points. The MBCs for ethyl lactate (Etiderm^®) for MSSP and MRSP were 1:2 at 10 min, and 1:2–1:16 after 30 and 60 min. A 1:2 dilution was effective against Pseudomonas, and a 1:512 dilution was effective against Malassezia at all time points. Chloroxylenol (Coatex^®) and acetic acid–boric acid (Malacetic^®) were not effective against MSSP, MRSP or Pseudomonas. Both were effective against Malassezia at 1:8–1:16 dilution at 10 min, and at 1:8–1:32 dilution after 30 and 60 min. In conclusion, chlorhexidine appeared to be the most effective topical biocide, and MRSP and MDR‐PA were no less susceptible than antibiotic‐sensitive organisms. These results should, however, be confirmed with larger numbers of isolates.     

Biofilm production by pathogens associated with canine otitis externa,and the antibiofilm activity of ionophores and antimicrobial adjuvants

Otitis externa (OE) is a frequently reported disorder in dogs associated with secondary infections by Staphylococcus, Pseudomonas and yeast pathogens. The presence of biofilms may play an important role in the resistance of otic pathogens to antimicrobial agents. Biofilm production of twenty Staphylococcus pseudintermedius and twenty Pseudomonas aeruginosa canine otic isolates was determined quantitatively using a microtiter plate assay, and each isolate was classified as a strong, moderate, weak or nonbiofilm producer. Minimum biofilm eradication concentration (MBEC) of two ionophores (narasin and monensin) and three adjuvants (N‐acetylcysteine (NAC), Tris‐EDTA and disodium EDTA) were investigated spectrophotometrically (OD_570nm) and quantitatively (CFU/ml) against selected Staphylococcus and Pseudomonas biofilm cultures. Concurrently, minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of planktonic cultures were assessed. 16/20 of the S. pseudintermedius clinical isolates were weak biofilm producers. 19/20 P. aeruginosa clinical isolates produced biofilms and were distributed almost equally as weak, moderate and strong biofilm producers. While significant antibiofilm activity was observed, no MBEC was achieved with narasin or monensin. The MBEC for NAC ranged from 5,000–10,000 µg/ml and from 20,000–80,000 µg/ml against S. pseudintermedius and P. aeruginosa, respectively. Tris‐EDTA eradicated P. aeruginosa biofilms at concentrations ranging from 6,000/1,900 to 12,000/3,800 µg/ml. The MBEC was up to 16‐fold and eightfold higher than the MIC/MBC of NAC and Tris‐EDTA, respectively. Disodium EDTA reduced biofilm growth of both strains at concentrations of 470 µg/ml and higher. It can be concluded that biofilm production is common in pathogens associated with canine OE. NAC and Tris‐EDTA are effective antibiofilm agents in vitro that could be considered for the treatment of biofilm‐associated OE in dogs.     

In vitro antimicrobial activity of miconazole and polymyxin B against canine meticillin-resistant Staphylococcus aureus and meticillin-resistant Staphylococcus pseudintermedius isolates

Background – Meticillin‐resistant Staphylococcus aureus (MRSA) and meticillin‐resistant Staphylococcus pseudintermedius (MRSP) infections are increasingly reported in dogs, and these bacteria may be isolated from ear infections. Hypothesis/Objectives – The main aim of the present study was to investigate the in vitro antimicrobial activity of miconazole, polymyxin B and a combination of both against 24 canine MRSA and 50 canine MRSP isolates. The minimal inhibitory concentration (MIC) values of 12 other antimicrobial agents were also determined. Methods – All MIC values were determined according to a broth microdilution assay. Results – Acquired resistance was found to all tested agents, except for linezolid, miconazole and polymyxin B. The MIC values for miconazole and polymyxin B against MRSA were in the range of 4–8 and 8–64 μg/mL, respectively, while the MIC values for miconazole and polymyxin B against MRSP were in the range of 1–2 and 0.25–4 μg/mL, respectively. Using a combination of miconazole and polymyxin B, there was no evidence for enhanced in vitro activity of the combination (i.e. synergy) of both products. Nevertheless, MIC₉₀ values of the combination of these antimicrobial agents and of a commercial product containing both agents were at least 1000 times lower than the concentration present in the commercial product. Conclusions and clinical importance – These results indicate that the topical use of a combination of miconazole and polymyxin B in a 43.5:1 ratio may have potential for the treatment of MRSA‐mediated and MRSP‐associated otitis externa in dogs.     

Staphylococcus pseudintermedius in the dog: taxonomy, diagnostics, ecology, epidemiology and pathogenicity

The dog is the natural host of Staphylococcus pseudintermedius. Many research efforts are currently being undertaken to expand our knowledge and understanding of this important canine commensal and opportunistic pathogen. The objective of this review is to summarize the current knowledge of the species, including the latest research outcomes, with emphasis on taxonomy, diagnostics, ecology, epidemiology and pathogenicity. Despite the important taxonomic changes that have occurred over the past few years, the risk of misidentification in canine specimens is low and does not have serious consequences for clinical practice. Staphylococcus pseudintermedius carriage in the dog is more frequent and genetically heterogeneous compared with that of Staphylococcus aureus in man. It appears that these staphylococcal species have evolved separately through adaptation to their respective natural hosts and differ with regard to various aspects concerning ecology, population structure and evolution of antibiotic resistance. Further understanding of the ecology and epidemiology of S. pseudintermedius is hampered by the lack of a standard method for rapid and discriminatory typing and by the limited data available on longitudinal carriage and population structure of meticillin‐susceptible strains. With regard to pathogenicity, it is only now that we are starting to explore the virulence potential of S. pseudintermedius based on genomic and proteomic approaches, and more research is needed to assess the importance of individual virulence factors and the possible existence of hypervirulent strains.     

Comparative activity of pradofloxacin and marbofloxacin against coagulase‐positive staphylococci in a pharmacokinetic–pharmacodynamic model based on canine pharmacokinetics

Körber‐Irrgang, B., Wetzstein, H.‐G., Bagel‐Trah, S., Hafner, D., Kresken, M. Comparative activity of pradofloxacin and marbofloxacin against coagulase‐positive staphylococci in a pharmacokinetic–pharmacodynamic model based on canine pharmacokinetics. J. vet. Pharmacol. Therap.  35 , 571–579. Pradofloxacin (PRA), a novel veterinary 8‐cyano‐fluoroquinolone (FQ), is active against Staphylococcus pseudintermedius, the primary cause of canine pyoderma. An in vitro pharmacokinetic–pharmacodynamic model was used to compare the activities of PRA and marbofloxacin (MAR) against three clinical isolates of S. pseudintermedius and reference strain Staphylococcus aureus ATCC 6538. Experiments were performed involving populations of 10¹⁰ CFU corresponding to an inoculum density of approximately 5 × 10⁷ CFU/mL. The time course of free drug concentrations in canine serum was modelled, resulting from once daily standard oral dosing of 3 mg of PRA/kg and 2 mg of MAR/kg. In addition, experimentally high doses of 6 mg of PRA/kg and 16 mg of MAR/kg were tested against the least susceptible strain. Viable counts were monitored over 24 h. At concentrations associated with standard doses, PRA caused a faster and more sustained killing than MAR of all strains. The ratios of free drug under the concentration–time curve for 24 h over MIC and the maximum concentration of free drug over MIC were at least 90 and 26, and 8.5 and 2.1 for PRA and MAR, respectively. At experimentally high doses, PRA was superior to MAR in terms of immediate killing. Subpopulations with reduced susceptibility to either FQ did not emerge. We conclude that PRA is likely to be an efficacious therapy of canine staphylococcal infections.     

Determination of enrofloxacin stability and in vitro efficacy against Staphylococcus pseudintermedius and Pseudomonas aeruginosa in four ear cleaner solutions over a 28 day period

Chemical stability and in vitro bactericidal efficacy of 0.9% enrofloxacin‐compounded solutions were evaluated following storage at room temperature for 28 days. Chemical stability of enrofloxacin was determined by high‐performance liquid chromatography (HPLC) in five compounded solutions, including sterile water. Bactericidal efficacy was determined by spiral plating serial 10‐fold dilutions of bacteria and solutions followed by colony counts. Tris–EDTA [TrizEDTA^® (TE)], Tris–EDTA and 0.15% chlorhexidine [TrizChlor^® (TC)], 2.5% lactic acid, 0.1% salicylic acid and 0.1% parachlorometaxylenol [Epi‐Otic (EO)], and 0.1% free salicylic acid, 0.1% parachlorometaxylenol and 0.5% EDTA [Epi‐Otic Advanced (EA)] were used. High‐performance liquid chromatography was carried out with one‐step liquid/liquid extraction to detect and quantify enrofloxacin stability. Mean recoveries for compounded samples run in triplicate at 28 days were 97.7% (TE), 99.9% (TC), 98.1% (EO) and 97.8% (EA). Kruskal–Wallis analysis showed no significant difference in the percentage recovery (H = 0.0539, df = 3, P = 0.9967). American Type Culture Collection strains of Staphylococcus pseudintermedius and Pseudomonas aeruginosa were used to evaluate in vitro efficacy following 30 min incubation on days 0, 14 and 28. Consistent in vitro bactericidal efficacy of all compounded solutions, indicated by killing >2.3 × 10⁷ colony‐forming units/mL, was seen; however, bactericidal efficacy decreased for compounded TC on day 14. Pseudomonas aeruginosa was more sensitive to the ear cleaners and enrofloxacin than S. pseudintermedius. The HPLC and in vitro data suggest that 0.9% enrofloxacin compounded with sterile water, TE, EO and EA maintains chemical stability and bactericidal efficacy for 28 days.     

Treatment outcome of dogs with meticillin-resistant and meticillin-susceptible Staphylococcus pseudintermedius pyoderma

Background – The prevalence of meticillin‐ and multidrug‐resistant Staphylococcus pseudintermedius (MRSP) in canine pyoderma has been increasing in recent years; thus, treatment of these cases has become challenging. Hypothesis/Objectives – To compare treatment outcome (clinical resolution and treatment duration), adverse effects of medication, and concurrent diseases and medications in dogs with meticillin‐susceptible S. pseudintermedius (MSSP) and MRSP pyoderma. Animals/Methods – Medical records were reviewed retrospectively, and 123 MSSP and 93 MRSP clinical cases between January 2008 and April 2010 were included. Results – In MSSP infections, cefalexin and cefpodoxime were the most commonly prescribed antimicrobials, accounting for 43.2 and 34.4% of cases, respectively. In MRSP infections, chloramphenicol and doxycycline were most commonly prescribed, accounting for 52.6 and 14.4% of cases, respectively. Adverse effects were reported in seven MSSP and 31 MRSP cases. The most commonly reported adverse effects were gastrointestinal, prompting antibiotic discontinuation in three MSSP and 20 MRSP cases. Chloramphenicol was associated with the highest incidence of adverse reactions (27 of 51 cases). Of 164 cases with follow up, 43 of 88 MSSP infections and 29 of 76 MRSP infections achieved complete clinical resolution at the first recheck examination. Three MSSP and seven MRSP cases failed to improve or resolve at subsequent visits assessed at 3–4 week intervals. Conclusions and clinical importance – Results from this study showed that the majority of pyodermas resolved regardless of meticillin susceptibility. Although some cases of MRSP pyoderma took longer to treat, this is likely to be because of chronicity and not the organism. In addition, adverse effects were frequently associated with chloramphenicol administration.     

Antimicrobial resistance of Staphylococcus pseudintermedius

Staphylococcus pseudintermedius, Staphylococcus intermedius and Staphylococcus delphini together comprise the S. intermedius group (SIG). Within the SIG, S. pseudintermedius represents the major pathogenic species and is involved in a wide variety of infections, mainly in dogs, but to a lesser degree also in other animal species and humans. Antimicrobial agents are commonly applied to control S. pseudintermedius infections; however, during recent years S. pseudintermedius isolates have been identified that are meticillin‐resistant and have also proved to be resistant to most of the antimicrobial agents approved for veterinary applications. This review deals with the genetic basis of antimicrobial resistance properties in S. pseudintermedius and other SIG members. A summary of the known resistance genes and their association with mobile genetic elements is given, as well as an update of the known resistance‐mediating mutations. These data show that, in contrast to other staphylococcal species, S. pseudintermedius seems to prefer transposon‐borne resistance genes, which are then incorporated into the chromosomal DNA, over plasmid‐located resistance genes.     

P‐74 Evaluation of the in vivo effects of Tris‐EDTA and chlorhexidine digluconate 0.15% solution in chronic bacterial otitis externa: 11 cases

The objectives of this study were to evaluate in vivo tolerance, and antimicrobial and clinical activities of a topical otic preparation containing EDTA tromethamine (Tris) and chlorhexidine digluconate 0.15% solution (Otodine^®) in dogs with chronic bacterial otitis externa. Eleven dogs were included. The affected ears were filled with the solution once daily during a 2‐week period. Dogs were evaluated on days 0, 14 and 28. Three clinical parameters (exudate, erythema, pain) and three cytologic parameters (Malassezia, cocci, rods) were scored (0–4 scale) by otoscopic and cytological examinations of otic exudate. Bacterial cultures were performed at each time point. If there were bacteria on cytological examination on day 14, the dogs were treated with the original product, with the addition of enrofloxacin (5%) applied 10 min after the original product, for a further 2 weeks. All 11 cases yielded isolates of resistant gram‐negative bacteria; gram‐positive bacteria were also isolated from six of 11 dogs. On day 14, six of 11 dogs were negative on culture examination; on day 28, 10 of 11 were negative and only one case had a positive culture. On day 14, clinical and microbial scores (cytology) were reduced by 54.6 and 71.1%, respectively, and by 85.7 and 94% on day 28. All cases reported good tolerance of the treatment. The results show that this ear solution was helpful in the management of chronic bacterial otitis externa in dogs and was well tolerated. There seems to be a synergistic effect of the combination of Tris‐EDTA/chlorhexidine digluconate 0.15% solution, and an antimicrobial agent (enrofloxacin) against resistant gram‐positive and gram‐negative bacteria. Funding: Self‐funded.     

Staphylococcus pseudintermedius expresses surface proteins that closely resemble those from Staphylococcus aureus

Staphylococcus pseudintermedius is a commensal of dogs that is implicated in the pathogenesis of canine pyoderma. This study aimed to determine if S. pseudintermedius expresses surface proteins resembling those from Staphylococcus aureus and to characterise them. S. pseudintermedius strain 326 was shown to adhere strongly to purified fibrinogen, fibronectin and cytokeratin 10. It adhered to the α-chain of fibrinogen which, along with binding to cytokeratin 10, is the hallmark of clumping factor B of S. aureus, a surface protein that is in part responsible for colonisation of the human nares. Ligand-affinity blotting with cell-wall extracts demonstrated that S. pseudintermedius 326 expressed a cell-wall anchored fibronectin binding protein which recognised the N-terminal 29 kDa fragment. The ability to bind fibronectin is an important attribute of pathogenic S. aureus and is associated with the ability of S. aureus to colonise skin of human atopic dermatitis patients. S. pseudintermedius genomic DNA was probed with labelled DNA amplified from the serine-aspartate repeat encoding region of clfA of S. aureus. This probe hybridised to a single SpeI fragment of S. pseudintermedius DNA. In the cell-wall extract of S. pseudintermedius 326, a 180 kDa protein was discovered which bound to fibrinogen by ligand-affinity blotting and reacted in a Western blot with antibodies raised against the serine-aspartate repeat region of ClfA and the B-repeats of SdrD of S. aureus. It is proposed that this is an Sdr protein with B-repeats that has an A domain that binds to fibrinogen. Whether it is the same protein that binds cytokeratin 10 is not clear.     

Prevalence of genes for enterotoxins,toxic shock syndrome toxin 1 and exfoliative toxin among clinical isolates of Staphylococcus pseudintermedius from canine origin

A total of 74 Staphylococcus pseudintermedius strains were isolated from the 99 clinical cases of canine pyoderma or chronic otitis in our veterinary teaching hospital during May 2006–February 2008. In this study, we examined the genetic distribution of staphylococcal pyogenic toxins such as staphylococcal enterotoxins A (sea), B (seb), C (sec), D (sed), E (see), and toxic shock syndrome toxin 1 (tst) as well as the previously characterized S. intermedius exfoliative toxin (siet) among those isolates. The polymerase chain reaction analyses with the toxin gene‐specific primers revealed that 18 (24.3%) of 74 S. pseudintermedius isolates carried the sec genes, but none of the sea, seb, sed, see and tst genes. Further DNA sequencing analysis of the amplified sec genes revealed that they all belonged to the canine type C staphylococcal enterotoxin (SEC_canine) whose superantigenic activity has been demonstrated. In addition to the sec_canine genes, our polymerase chain reaction results showed that all the 74 isolates carried the siet gene. Since both SEC_canine and SIET toxins are known to be biologically active, it would be interesting to investigate how those toxins are involved in the pathogenesis of the canine diseases by S. pseudintermedius such as pyoderma or chronic otitis.     

Comparative assessment of genotypic and phenotypic correlates of Staphylococcus pseudintermedius strains isolated from dogs with otitis externa and healthy dogs

Staphylococcus pseudintermedius is considered a primary pathogen of canine skin and soft tissue infections, and the rapid emergence of methicillin-resistant S. pseudintermedius worldwide is a major issue. In the current study, genotypic and phenotypic correlates associated with S. pseudintermedius causing canine otitis externa were evaluated using 41 S. pseudintermedius strains isolated from dogs with otitis externa (n = 26) and healthy dogs (n = 15). The S. pseudintermedius strains were subjected to a comparative analysis of (i) genotypes (multilocus sequence typing, agr, and spa types), (ii) methicillin resistance and SCCmec types, (iii) multidrug resistance (MDR), (iv) biofilm formation, and (v) susceptibility to canine cathelicidin (K9CATH). A high degree of genetic diversity was observed in both groups of S. pseudintermedius strains, regardless of methicillin resistance. Almost all methicillin-resistant strains (>95%) harbored SCCmec V and displayed MDR. Although there was no difference in biofilm formation, S. pseudintermedius strains derived from otitis externa exhibited enhanced resistance to cationic antimicrobial peptide (K9CATH) compared with strains from healthy dogs. The high degree of heterogeneity in MLST, agr, and spa types prevented the identification of correlations between any specific genotype and virulence phenotype in otitis externa caused by S. pseudintermedius, These findings provide an important basis for monitoring and treating canine skin and soft tissue infections in Korea.     

Heterogeneity of Staphylococcus pseudintermedius isolates from atopic and healthy dogs

Staphylococcus pseudintermedius is part of the normal canine flora but frequently causes pyoderma in canine atopic dermatitis (AD). This study aimed to determine whether particular S. pseudintermedius strains were associated with AD and/or pyoderma. Ninety‐six S. pseudintermedius isolates from the ear, nares, perineum and lesions of 21 atopic and 16 healthy dogs were lysed with proteinase K and digested with 40 U SmaI. Restriction products were separated using pulsed‐field gel electrophoresis (PFGE) with an Oxford S. aureus control and lambda‐ladder DNA concatomer markers. A dendrogram was constructed by the unweighted pair group method. All isolates showed a ≥56% similarity coefficient. Nine distinct PFGE clusters were identified, as follows: five from both atopic and healthy dogs; three from atopic dogs only; and one from healthy dogs only. Nine clusters were isolated from the nares, eight from the perineum, five from the ears and six from pyoderma lesions. There were no significant differences in the frequency of isolation from atopic or healthy skin, body sites or infected lesions for any of the clusters. Two of six healthy dogs and 18 of 20 atopic dogs with multiple isolates had closely related isolates (less than three band differences) at more than one sampling site. Isolates from pyoderma lesions were closely related to at least one mucosal isolate in 11 of 16 dogs. Staphylococcus pseudintermedius isolates appear to be heterogeneous, and colonization or infection of atopic skin was not associated with any particular strain or cluster of strains.     

Antimicrobial effect of combinations of EDTA-Tris and amikacin or neomycin on the microorganisms associated with otitis externa in dogs

Combinations of EDTA-Tris and two aminoglycoside antibiotics (amikacin and neomycin) were tested for synergistic activities against the microorganisms associated with otitis externa in dogs and for the solutions' stability over time. Synergistic activity was observed when EDTA-Tris plus amikacin and EDTA-Tris plus neomycin were tested againstStaphylococcus intermedius, Proteus mirabilis, Pseudomonas aeruginosa, andEscherichia coli, but not againstCandida albicans. Stability studies over a 3-month period indicated that the test solutions were stable at room temperature and that their antimicrobial activity was maintained.Abbreviations CFU colony-forming units - EDTA (ethylenedinitrilo)tetraacetic acid, disodium salt - FIC fractional inhibitory concentration - MBC minimal bactericidal concentration - MIC minimal inhibitory concentration - Tris tris(hydroxymethyl)aminomethane     

In vitro antimicrobial efficacy of β‐defensin 3 against Staphylococcus pseudintermedius isolates from healthy and atopic canine skin

β‐Defensins (BDs) are highly conserved antimicrobial peptides important in innate defence against bacteria. β‐Defensin 3 has a specific role in protecting the skin. This study quantified the minimal inhibitory concentration (MIC) of human (h)BD3 against Staphylococcus pseudintermedius isolates from atopic and healthy dogs. Single colony isolates (1 × 10⁵ colony‐forming units/mL log phase) were cultured with doubling dilutions of hBD3 in sodium phosphate buffer from 0.8 to 50 μg/mL at 37 °C for 2 h, before adding 100 μL of tryptone soy broth and incubating for a further 20 h. Bacterial growth was assessed as the mean optical density at 540 nm corrected for background. The median MIC was 12.5 μg hBD3/mL (range 3.125–25 μg/mL; n = 22). Forty‐five percent of the isolates were inhibited at ≤6.25 μg hBD3/mL, and 90% were inhibited at ≤12.5 μg hBD3/mL. Bacterial growth was not inhibited at ≤1.6 μg hBD3/mL. There were no significant differences in the inhibition by hBD3 of isolates from atopic (median MIC 12.5 μg/mL, range 6.25–25 μg/mL, n = 14) and healthy dogs (median MIC 9.4 μg/mL, range 3.125–12.5 μg/mL, n = 8); from noninfected colonized sites (median MIC 12.5 μg/mL, range 3.125–25 μg/mL, n = 16) and infected lesions (median MIC 9.4 μg/mL, range 6.25–12.5 μg/mL, n = 6); or between sample sites (nose median MIC 12.5 μg/mL, range 6.25–25 μg/mL, n = 5; perineum median MIC 12.5 μg/mL, range 3.125–25 μg/mL, n = 7; ear median MIC 6.25 μg/mL, range 6.25–12.5 μg/mL, n = 4; lesions median MIC 9.4 μg/mL, range 6.25–12.5 μg/mL, n = 6). In conclusion, hBD3 inhibited the growth of canine S. pseudintermedius isolates in vitro irrespective of origin.     

Prevalence of meticillin-resistant Staphylococcus pseudintermedius (MRSP) from skin and carriage sites of dogs after treatment of their meticillin-resistant or meticillin-sensitive staphylococcal pyoderma

Background – Meticillin‐resistant staphylococci are significant pathogens in veterinary dermatology, yet longitudinal studies of the impact of routine antimicrobial therapy on emergence or resolution of resistance are lacking. Objectives – To determine the prevalence of meticillin‐resistant staphylococci on skin and carriage sites in dogs with bacterial pyoderma and evaluate the prevalence of meticillin‐resistant Staphylococcus pseudintermedius (MRSP) colonization after successful treatment of pyoderma. Animals – One hundred and seventy‐three dogs that presented to a dermatology referral service with pyoderma and 41 healthy control dogs. Methods – Skin, nasal and rectal swabs for bacterial culture were collected at the time of referral and after clinical resolution of the pyoderma. Meticillin resistance was confirmed by demonstration of penicillin binding protein 2a antigen. Results – Initially, skin cultures yielded MRSP in 70 (40.5%) dogs, meticillin‐resistant Staphylococcus aureus (MRSA) in three (1.7%) and meticillin‐resistant Staphylococcus schleiferi ssp. coagulans (MRSScoag) in five (2.9%). Samples collected from the nose and rectum (carriage sites) yielded MRSP in 59 (34.1%) dogs, MRSA in 11 (6.4%) and MRSScoag in seven (4.0%). One hundred and two dogs were available for follow‐up cultures after clinical cure. Of 42 dogs initially diagnosed with MRSP pyoderma, MRSP was isolated at follow‐up from skin in 19 (45.2%) and carriage sites in 20 (47.6%). Of 60 dogs that did not have MRSP pyoderma initially, MRSP was isolated post‐treatment from the skin in 17 (28.3%), and MRSP from carriage sites increased from 7.8% (initially) to 26.7% (P = 0.0022). Conclusions and clinical importance – Colonization by MRSP often persists after resolution of MRSP pyoderma. Acquisition of MRSP during treatment appears to be common.     

Determination of staphylococcal exotoxins, SCCmec types, and genetic relatedness of Staphylococcus intermedius group isolates from veterinary staff, companion animals, and hospital environments in Korea

The Staphylococcus (S.) intermedius group (SIG) has been a main research subject in recent years. S. pseudintermedius causes pyoderma and otitis in companion animals as well as foodborne diseases. To prevent SIG-associated infection and disease outbreaks, identification of both staphylococcal exotoxins and staphylococcal cassette chromosome mec (SCCmec) types among SIG isolates may be helpful. In this study, it was found that a single isolate (one out of 178 SIG isolates examined) harbored the canine enterotoxin SEC gene. However, the S. intermedius exfoliative toxin gene was found in 166 SIG isolates although the S. aureus-derived exfoliative toxin genes, such as eta, etb and etd, were not detected. SCCmec typing resulted in classifying one isolate as SCCmec type IV, 41 isolates as type V (including three S. intermedius isolates), and 10 isolates as non-classifiable. Genetic relatedness of all S. pseudintermedius isolates recovered from veterinary staff, companion animals, and hospital environments was determined by pulsed-field gel electrophoresis. Strains having the same band patterns were detected in S. pseudintermedius isolates collected at 13 and 18 months, suggesting possible colonization and/or expansion of a specific S. pseudintermedius strain in a veterinary hospital.