Chapter 4.4
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.
Introduction
Canine superficial pyoderma is a common skin disease of dogs, and the primary pathogen is Staphylococcus pseudintermedius.1 In addition to addressing the underlying cause, treatment of superficial pyoderma includes the use of systemic antimicrobials and topical therapy. Following the emergence of multidrug-resistant mecA-positive S. pseudintermedius, there has been increased interest in the use of topical antiseptics as sole or adjuvant therapy. In previous studies, we demonstrated the efficacy of 2% chlorhexidine acetate (Nolvasan® Surgical Scrub; Fort Dodge Animal Health, Fort Dodge, IA, USA) as monotherapy for canine superficial pyoderma associated with meticillin-resistant (MR) S. pseudintermedius group (SIG), but not in all cases.2–4 In those studies, 2-4% chlorhexidine acetate resulted in a positive clinical response in 60-70% of the dogs.2–4 In humans, there are reports of topical treatment with chlorhexidine not being successful in eliminating meticillin-resistant Staphylococcus aureus (MRSA),5 and a low level of resistance to chlorhexidine is a concern.6 A possible explanation is the existence of multidrug efflux pump genes, which have been detected in S. aureus isolates, that confer resistance to antiseptic agents, including chlorhexidine, quaternary ammonium compounds (e.g. benzalkonium chloride) and dyes (e.g. acriflavine and ethidium bromide).7–10 The aims of this study were to assess the in vitro susceptibilities of both meticillin-resistant and meticillin-susceptible (MS) S. pseudintermedius to chlorhexidine acetate, as well as other antiseptics, and to determine whether multidrug efflux pump genes (qacA, qacB and smr) were present.
Materials and methods
Bacterial isolates
One hundred clinical isolates were obtained from dogs with either a first occurrence of pyoderma (n = 23) or recurrent pyoderma (n = 77) between May 2009 and September 2010. Samples were collected from dogs presented to the ASC Dermatology Service, Tokyo, Japan. These samples have been used in previous studies.2–4 Pyoderma was confirmed based on clinical signs, cytological findings and bacterial culture. Samples for bacterial culture and susceptibility testing were collected from skin lesions compatible with pyoderma. Swabs (Sterile BBL CultureSwab; Becton, Dickinson and Co., Franklin Lakes, NJ, USA) were rubbed vigorously against the sampling site and stored at 4°C, and were processed within 7 days. Each swab was inoculated onto blood agar (Nissui Pharmaceutical Co., Ltd, Tokyo, Japan) and mannitol salt agar (Nissui Pharmaceutical Co., Ltd), and incubated aerobically at 37°C for 18-24 h. Staphylococcal isolates were putatively identified using colony morphology, the ability to grow on mannitol salt agar, Gram-stain characteristics and coagulase reaction. The strains were stored in skimmed milk at -80°C until further use.
Identification of S. pseudintermedius
Bacterial speciation was performed as previously described using a multiplex PCR method.11 (See Table S1 in Supplementary material for primers).
Determination of meticillin resistance by detection of mecA
Primers for mecA PCRs, mA1 (5′-TGCTATCCACCCTCAAACAGG-3′) and mA2 (5′-AACGTTGTAACCACCCCAAGA-3′), were used in the present study with a previously published method.12 All results were confirmed by at least two independent experiments.
Antimicrobial and antiseptic susceptibility testing
Antimicrobial susceptibility testing was used to identify MR and MS staphylococci. The minimal inhibitory concentrations (MICs) of antimicrobial and antiseptic agents were determined by the standard agar plate dilution method according to the Clinical and Laboratory Standards Institute (CLSI) document M31-A3.13 Isolates with a MIC for oxacillin ≥0.5 μg/mL or positive for mecA by PCR were regarded as MR strains.14 The antiseptic agents tested included chlorhexidine acetate and chlorhexidine gluconate, acriflavine, ethidium bromide and benzalkonium chloride.
Determination of multidrug efflux pump genes, including qacA, qacB and smr
A search for qacA/B and smr genes was performed by multiple PCRs with the following sets of primers: 5′-GCAGAAAGTGCAGAGTTCG-3′ and 5′-CCAGTCCAATCAGCCTG-3′ for qacA/B (product size 361 bp);15 and 5′-GCCATAAGTACTGAAGTTATTGGA-3′ and 5′-GACTACGGTTGTTAAGACTAAACCT-3′ for smr (product size 195 bp).16
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