Staphylococcus pseudintermedius and Staphylococcus schleiferi Subspecies coagulans from Canine Pyoderma Cases in Grenada, West Indies, and Their Susceptibility to Beta-Lactam Drugs

Over a 2-year period 66 cases of canine pyoderma in Grenada, West Indies, were examined by aerobic culture in order to ascertain the bacteria involved and their antimicrobial resistance patterns. Of the 116 total bacterial isolates obtained, the majority belonged to Gram-positive species, and the most common organism identified through biochemical and molecular methods was Staphylococcus pseudintermedius. Additionally, identification of a Staphylococcus schleiferi subspecies coagulans isolate was confirmed by molecular methods. All isolates of staphylococci were susceptible to beta-lactam drugs: amoxicillin-clavulanic acid, cefovecin, cefoxitin, cefpodoxime, and cephalothin. They were also susceptible to chloramphenicol and enrofloxacin. Resistance was highest to tetracycline. Methicillin resistance was not detected in any isolate of S. pseudintermedius or in S. schleiferi. Among the Gram-negative bacteria, the most common species was Klebsiella pneumoniae, followed by Acinetobacter baumannii/calcoaceticus. The only drug to which all Gram-negative isolates were susceptible was enrofloxacin. This report is the first to confirm the presence of S. pseudintermedius and S. schleiferi subspecies coagulans, in dogs with pyoderma in Grenada, and the susceptibility of staphylococcal isolates to the majority of beta-lactam drugs used in veterinary practice.


Introduction
Pyoderma, or bacterial infection of the skin, is the most common dermatologic problem encountered in dogs [1]. Varieties include superficial pyoderma, characterized by pustules, papules, erythema, focal crusting, and pruritus and deep pyoderma with furuncles and draining tracts. Although Gram-positive bacteria, such as staphylococci, are commonly involved in superficial pyoderma, Gram-negative bacteria can cause secondary infection, particularly in cases of deep pyoderma [2]. Empirical diagnosis of pyoderma based on history and physical examination is followed by complimentary tests, such as Gram staining and culture, with the most reliable results obtained from analysis of contents from an intact pustule [3]. Treatment usually involves antimicrobial drug therapy. Culture and antibiotic susceptibility testing are indicated in cases which do not respond to preliminary treatment and mandatory when treating deep pyoderma [4]. In addition, information on the principal organisms associated with pyoderma and their susceptibility patterns 2 Veterinary Medicine International against commonly used antimicrobial drugs is highly useful in empirical treatment. The most commonly used drugs for empirical systemic treatment of canine pyoderma are amoxicillin-clavulanic acid and cephalosporins [5].
Until recently, Staphylococcus intermedius was considered the most common organism associated with canine pyoderma [3]. In actuality, isolates originally phenotypically identified as S. intermedius could be from three different species: S. intermedius, Staphylococcus pseudintermedius, and Staphylococcus delphini [6]. For definitive identification of these species, molecular diagnostic methods, such as polymerase chain reaction (PCR) techniques, are required [7]. Given the variety of sources of dogs, ranging from local Pompeks and Pothounds originating on the island to pets brought to the island from multiple locations in the USA, it was vital to study the bacteria from cases of canine pyoderma and their drug resistance patterns in Grenada. The resulting data on pathogens and drug resistance will form a background for establishment of recommendations for prudent use of antimicrobials for companion animals [8,9].
The objectives of this study were to identify the bacteria associated with pyoderma in domestic dogs in Grenada, particularly with regard to the occurrence of S. pseudintermedius and the emerging pathogen Staphylococcus schleiferi, and to determine the extent of antimicrobial drug resistance. Although it was hypothesized that the majority of pyoderma cases would be due to staphylococci, occurrence of other Gram-positive and Gram-negative species and their antimicrobial resistance patterns were also studied. Results from these data are likely to assist towards selection of appropriate antimicrobial treatment plans.  (2), and one each of 7 breeds/types. Pustule contents or swabs applied to ulcerated lesions were obtained aseptically, using Carry Blair Transport Swabs (Becton, Dickinson and Company, Sparks, MD, USA). Transport swabs were stored at 4 ∘ C immediately after collection and were cultured within 24 h.

Bacterial
Culture. All samples were plated on blood and MacConkey agar and incubated aerobically at 37 ∘ C for up to 72 h. Culture plates were examined daily for the number and types of colonies. Samples with 2 colonies or fewer after 72 h were considered negative. Bacterial growth was classified as pure or mixed. Bacteria were initially identified based on colony morphology; Gram stain; and other preliminary testing methods, including catalase, coagulase, and oxidase tests [10]. Further classification of bacteria involved the use of API bacterial identification strips (bioMérieux Inc., Durham, NC, USA), multiplex PCR, and sequencing. API strips were not able to distinguish among S. intermedius, S. pseudintermedius, and S. delphini.

DNA Isolation.
Bacterial DNA from all isolates phenotypically identified as S. intermedius was extracted using the DNeasy Blood & Tissue kit (Qiagen, Valencia, CA, USA) and the manufacturer's Gram-positive protocol. As a modification, preliminary incubation was performed in enzymatic lysis buffer, consisting of 20 mg/mL lysozyme (Sigma-Aldrich, St. Louis, MO, USA) and 1.2% Triton X-100 (Sigma) in phosphate-buffered saline, pH 7.4. DNA content was checked by a NanoDrop2000C (Thermo Scientific, Wilmington, DE, USA). DNA was forwarded to the Veterinary Teaching Hospital Microbiology Laboratory at The Ohio State University College of Veterinary Medicine, Columbus, OH, USA.

Multiplex PCR.
Multiplex PCR was performed as previously described [11]. Multiplex PCR utilized Invitrogen Platinum Mastermix (Life Technologies, Grand Island, NY, USA) in 25 L total volume reactions. Controls for multiplex PCR included Staphylococcus aureus (ATCC 29213) (American Type Culture Collection, Manassas, VA, USA), S. pseudintermedius (LMG 22221) (LMG Bacteria Collection, Laboratory of Microbiology, Gent, Belgium), and S. intermedius (ATCC 29663). The result for S. schleiferi subspecies coagulans was based on comparison of the band size relative to the DNA ladder and other control staphylococci.

Sequencing of the rpoB Gene Fragment.
For confirmation of the identification of S. schleiferi subsp. coagulans, the single isolate was tested by sequencing a fragment of the rpoB gene as previously described [12]. This method was additionally applied to just one isolate originally identified as S. intermedius by API strip, but identified as S. aureus by multiplex PCR. Sequencing results were identified using the National Center for Biotechnology Information Basic Local Alignment Search Tool (BLAST) against highly similar sequences (megablast) and protein-protein BLAST (blastp) using the corresponding databases.
2.6. Antimicrobial Susceptibility Testing. Antimicrobial susceptibility tests were performed on isolates using the disk diffusion method on Mueller-Hinton agar, as recommended by the Clinical and Laboratory Standards Institute (CLSI), and the zone sizes were interpreted per CLSI guidelines [13]. Antimicrobial drugs used against Gram-positive isolates, and the disk potencies were as follows: amoxicillin-clavulanic acid (30 g), ampicillin (10 g), cefovecin (30 g), cefpodoxime (10 g), cefoxitin (30 g), cephalothin (30 g), chloramphenicol (30 g), clindamycin (2 g), enrofloxacin (5 g), erythromycin (30 g), gentamicin (10 g), neomycin (30 g), penicillin (10 units), sulfamethoxazole-trimethoprim (25 g),  and tetracycline (30 g). For enrofloxacin, zone sizes were interpreted per CLSI guidelines for bacteria from animals [14]. All antimicrobial disks, except cefovecin were obtained from Becton, Dickinson, and Company. Cefovecin disks were obtained from Pfizer Animal Health (New York City, NY, USA). As per the manufacturer's instructions, isolates giving growth inhibition zone diameters less than or equal to 19 mm with cefovecin were considered resistant. Gram-negative isolates were tested only against amoxicillin-clavulanic acid, ampicillin, cephalothin, enrofloxacin, gentamicin, neomycin, sulfamethoxazole-trimethoprim, and tetracycline, and the disk potencies were the same as indicated above. All S. pseudintermedius and the single S. schleiferi subsp. coagulans isolates were tested for methicillin resistance using 2 methods. A zone of growth inhibition of ≤17 mm against an oxacillin 1 g disk was considered indicative of resistance as recommended by Bemis et al. [15] and Gold et al. [16]. Growth on MRSA ID chromagar (bioMérieux) was the second test used as per the recommended by Horstmann et al. [17]. The test was conducted as per the manufacturer's directions and as outlined by Diederen et al. [18].

History of Antimicrobial Use.
Histories of previous antimicrobial use for pyoderma cases were collected when these were available.

Bacterial Identification.
A total of 116 bacterial isolates were obtained from the 66 canine pyoderma cases. The number of isolates per case ranged from 1 colony type (pure culture) to 4 colony types (mixed infection). Twenty-one of the 66 cases (31.8%) yielded pure culture. Of the total 116 isolates, 62 (53.5%) were Gram-positive, and 54 (46.5%) were Gram-negative. Distribution of bacterial isolates is provided in Table 1.
Staphylococci were the most commonly isolated Grampositive bacteria. Of the 43 isolates of staphylococci, 18 (41.9%) were from cases yielding a pure culture and 25 (58.1%) were from mixed infections. Using multiplex PCR, 27 of the 29 isolates identified by API strips as S. pseudintermedius were identified as S. pseudintermedius, 1 as S. aureus, and 1 as S. schleiferi subsp. coagulans. The isolate identified as Table 2: Antimicrobial resistance patterns of staphylococci from canine pyoderma cases in Grenada, West Indies. S. aureus, which had phenotypic properties of S. intermedius, was subjected to rpoB gene sequencing, and it was identified as S. pseudintermedius. The identity of the S. schleiferi subsp. coagulans isolate was also confirmed by sequencing. Of the 28 S. pseudintermedius isolates, 11 (39.3%) were obtained in pure culture, and 17 (60.7%) were from mixed infections.
Only 9 dogs with pyoderma (13.6%) had histories of previous antimicrobial use. Four of five dogs with histories of previous treatment with cephalexin yielded S. pseudintermedius, and the fifth had Streptococcus dysgalactiae, and all were susceptible to cephalothin and other cephalosporins. Two dogs had prior treatment with lincomycin, and the staphylococcal isolates from these showed no resistance to cephalosporins, but one isolate was resistant to clindamycin. Of the remaining 2 dogs, one was treated earlier with tetracycline, and it was positive for tetracycline-resistant S. pseudintermedius, and the other treated with amoxicillinclavulanic acid yielded only S. pseudintermedius susceptible to all drugs.

Discussion
It has been demonstrated that phenotypically identified S. intermedius strains could include not only true S. intermedius strains, but also 2 other species in this group, namely, S. pseudintermedius and S. delphini. In fact, most of the canine strains of this group have been identified as S. pseudintermedius, not S. intermedius [6,7]. S. pseudintermedius was the predominant isolate from 66 cases of canine pyoderma in the present study. This is in accordance with the recent findings from a study in Japan [19], in which none of the 190 isolates from canine pyoderma cases belonged to S. intermedius or S. aureus species.
In the present study, a single isolate phenotypically identified by API strip as S. intermedius was identified by both multiplex PCR and DNA sequencing as S. schleiferi subsp. coagulans. Historically, the early isolations of S. schleiferi were from a canine otitis case in Japan in 1990 [20] and 2 canine pyoderma cases in Europe in 2002 [21]. Since then, isolation of this staphylococcus from dogs has been reported in other countries including USA, and S. schleiferi has been noted to occur in recurring pyoderma cases, dogs with both otitis and pyoderma, and apparently healthy dogs [22]. Recent findings suggest that the 2 subspecies: S. schleiferi subsp. coagulans (coagulase positive) and subsp. schleiferi (coagulase negative) are not genetically distinct and are likely variations of coagulase-producing strains within one species [23].
In this study, 11.6% of staphylococci showed resistance to penicillin, whereas resistance to ampicillin was only 2.3%. In a recent study [24], of 67 S. pseudintermedius isolates form dogs, penicillin resistance was 61%, whereas ampicillin resistance was 40%. Methicillin resistance was not tested in their study, but all clinical isolates were included. It is possible that resistance mechanisms other than beta-lactamase production may play a role. Modification of penicillin-binding proteins is increasingly important as another mechanism of resistance to penicillins [25]. Further studies are needed to elucidate the mechanisms involved. The antibiotics of choice for extended therapy in canine pyoderma are cephalexin, amoxicillin-clavulanic acid, and enrofloxacin [1]. All 28 S. pseudintermedius isolates in the present study were susceptible to amoxicillin-clavulanic acid, cephalosporins, including cephalothin (which indicates susceptibility to cephalexin), and enrofloxacin. Cephalexin is bactericidal, has a low potential for development of resistance, and has minimal side effects [2]. Cephalexin and cefpodoxime are the most commonly prescribed drugs for S. pseudintermedius pyoderma in dogs [26]. The usage of cephalexin is justifiable as long as pyoderma is not complicated by Gram-negative bacteria. Moreover, the limited data in the present study on the history of antimicrobial use also showed susceptibility of all isolates from the treated animals to cephalosporins. It is known that canine pyoderma may be complicated by Gram-negative organisms such as E. coli, Proteus spp., and Pseudomonas spp. [3]. The role breeds of dogs and high humidity and heat on the role of Gram-negative bacteria in canine pyoderma may be worth investigating. Breed may be relevant in the pathogenesis of canine pyoderma [27]. Nearly 58% of the 6 most commonly-occurring Gram-negative species in the present study were resistant to cephalexin. Whether or not cephalothin-resistant Gram-negative bacteria are susceptible to other cephalosporins, such as cefovecin is worth investigating in future studies. In a recent study [28], cefovecin was found to have excellent in-vitro activity against a variety of both Gram-positive and Gram-negative pathogens from dogs.
Enrofloxacin was effective against all tested bacterial isolates in the present study. Both enrofloxacin and orbifloxacin, 2 fluoroquinolone drugs with similar antimicrobial activity, are safe and effective in treating superficial and deep pyoderma in dogs [29]. Fluoroquinolones have modest immunomodulating properties as well [30]. In a study published in 2006 from Canada, only 1% of 651 S. intermedius isolates from canine otitis were resistant to enrofloxacin [31].
Isolates resistant to clindamycin, another drug used for treatment of staphylococcal infections in dogs [32], were found in the present study. Same was the case with erythromycin. The macrolide antibiotics erythromycin and tylosin have been used to treat pyoderma cases [33].

Conclusions
This report is the first to confirm the presence of S. pseudintermedius and S. schleiferi subsp. coagulans in dogs with pyoderma in Grenada, and the susceptibility of staphylococcal isolates to the majority of beta-lactam drugs used in veterinary practice. Nearly a quarter of the S. pseudintermedius isolates in the present study showed resistance to more than one drug. Monitoring drug resistance trends is essential as new drug-resistant strains from other parts of the world could be introduced in dogs located in the island nation of Grenada. It is also important to monitor emergence of S. schleiferi and other new species, as well as beta-lactam resistance in canine strains of staphylococci.