Microbiological features of Staphylococcus schleiferi subsp. coagulans, isolated from dogs and possible misidentification with other canine coagulase-positive staphylococci

Staphylococcus schleiferi subsp. coagulans has only rarely been isolated and identified from the external auditory meatus of dogs suffering from external otitis. Its morphological and basic biochemical characteristics are of relatively little value for identification, as it phenotypically resembles another coagulase-positive staphylococci (CPS) and, consequently, may be easily misidentified as S. intermedius or even as S. aureus. In the present work, differentiation of S. schleiferi ssp. coagulans was therefore based on specific biochemical and genetic methods. All the strains were evaluated with the following commercial methods: Api Staph System (bioMérieux, Marcy l'Etoil, France), BBL Crystal Identification Systems (Gram-Positive ID Kit and Rapid Gram-Positive ID Kit; Becton Dickinson), and GEN-PROBE AccuProbe, Staphylococcus aureus identification test (bioMérieux). Gram-Positive ID System/GP database includes the broadest range of staphylococcal species and correctly identifies the majority of strains important in veterinary medicine. Therefore, it is an acceptable alternative to conventional methods for identification of canine staphylococcal isolates. Reliable differentiation of S. aureus from S. schleiferi ssp. coagulans and S. intermedius was feasible with AccuProbe for S. aureus, which gave positive results only for S. aureus; all other CPS tested were negative.     

Genotypic versus phenotypic identification of staphylococcal species of canine origin with special reference to Staphylococcus schleiferi subsp. coagulans

A comparative study was performed to examine the respective accuracy of 16S rDNA sequencing and of the commercial biochemical assay ID32 STAPH (bioMérieux, Marcy l'Etoile, France) in the identification of 232 staphylococcal samples representing 20 species and subspecies isolated from 367 dogs. Notable differences in species distribution were observed by comparing genotypic and phenotypic data. Partial sequencing of 16S rDNA resulted in an unambiguous identification of 226 (97.4%) of the isolates, whereas the phenotypic approach resulted in a correct diagnosis of 162 (69.8%) of the isolates. Statistical agreement between genotypic and phenotypic identification of staphylococci was substantial (Kappa coefficient of 0.6-0.8) for Staphylococcus aureus, S. hominis, S. warneri, S. cohnii subsp. urealyticus, and S. simulans, and "almost perfect" (Kappa coefficient of 0.8-1) for S. intermedius, S. epidermidis, S. equorum, S. haemolyticus, S. sciuri, and S. kloosi. No agreement above that expected by chance (Kappa coefficient=0) was observed for S. schleiferi subsp. coagulans, which was either confounded with S. intermedius and S. capitis, or categorized as unacceptable by the biochemical assay. Given the growing importance of this pathogen in veterinary medicine and its frequent misidentification with related staphylococci, a PCR-RFLP approach producing a S. schleiferi-specific restriction profile was developed. This fast and reliable assay represents a valuable tool in assisting in the monitoring of this pathogen.     

Isolation of Staphylococcus schleiferi from healthy dogs and dogs with otitis, pyoderma, or both

OBJECTIVE: To determine the frequency of isolation and susceptibility patterns of Staphylococcus schleiferi from healthy dogs and dogs with otitis, pyoderma, or both that had or had not received antimicrobial treatment. DESIGN: Prospective study. ANIMALS: 50 dogs. PROCEDURE: Dogs were allocated to 1 of 4 groups: healthy dogs (n=13), dogs without otitis but with pyoderma (10), dogs with otitis but without pyoderma (11), and dogs with otitis and pyoderma (16). Bacteriologic culture of ear swab specimens was performed in all dogs. Bacteriologic culture of skin swab specimens was also performed in dogs with concurrent pyoderma. Isolates were identified as S schleiferi subsp schleiferi or S schleiferi subsp coagulans on the basis of growth and biochemical characteristics. RESULTS: S schleiferi was not isolated from any dogs with pyoderma only. Staphylococcus schleiferi subsp schleiferi was isolated from the ears of 2 healthy dogs, and the skin and ears of 2 dogs and the skin of 1 dog with otitis and pyoderma. Staphylococcus schleiferi subsp coagulans was isolated from the ears of 3 dogs with otitis only, and the ears of 6 dogs and the skin of 2 dogs with otitis and pyoderma. One of the S schleiferi subsp schleiferi isolates from ears, 2 of the S schleiferi subsp coagulans isolates from ears, and 1 of the S schleiferi subsp coagulans isolates from the skin were resistant to methicillin. One methicillin-resistant isolate from the ears and 1 from the skin were also resistant to fluoroquinolones. CONCLUSIONS AND CLINICAL RELEVANCE: S schleiferi subsp schleiferi was detected in healthy dogs and dogs with otitis and pyoderma. Methicillin-resistant and -susceptible S schleiferi subsp schleiferi and S schleiferi subsp coagulans were detected as the predominant organisms in dogs with otitis.     

Isolation of Staphylococcus schleiferi from dogs with pyoderma

OBJECTIVE: To determine frequency with which Staphylococcus schleiferi could be isolated from dogs with pyoderma and antimicrobial susceptibility patterns of isolates that were obtained. DESIGN: Prospective study. ANIMALS: 54 dogs with a first (n = 14) or recurrent (40) episode of pyoderma. PROCEDURE: Specimens were obtained and submitted for bacterial culture. Isolates were identified as S schleiferi on the basis of growth and biochemical characteristics. Two isolates were submitted for DNA sequencing to confirm identification. Methicillin susceptibility was determined by means of disk diffusion with oxacillin-impregnated disks. RESULTS: 3 of 14 dogs examined because of a first episode of pyoderma and 12 of 40 dogs examined because of a recurrent episode of pyoderma were receiving antimicrobials at the time of specimen collection. Staphylococcus schleiferi was not isolated from any dog with first-time pyoderma but was isolated from 5 dogs with recurrent pyoderma that were not receiving antimicrobials at the time of specimen collection and 10 dogs with recurrent pyoderma that were receiving antimicrobials. Nine isolates were identified as S schleiferi subsp schleiferi, and 6 were identified as S schleiferi subsp coagulans. All S schleiferi subsp schleiferi isolates were resistant to methicillin, but only 2 S schleiferi subsp coagulans isolates were. Two methicillin-resistant isolates were also resistant to fluoroquinolones, and 1 isolate had intermediate susceptibility to fluoroquinolones. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggest that S schleiferi subsp schleiferi and S schleiferi subsp coagulans may be isolated from dogs with recurrent pyoderma. Although isolates from dogs with pyoderma were frequently resistant to methicillin, multiple drug resistance was uncommon.     

Comparison of microbial isolates and susceptibility patterns from the external ear canal of dogs with otitis externa

Otic exudate was obtained from 33 dogs with otitis externa for cytopathology and culture. Two samples were taken from the same location in the external ear canal, for a total of 100 samples. Thirty-six (36%) samples isolated only a single organism, of which 21 (21%) were Malassezia spp. Two organisms were present in 23 (23%) of the samples. Cultures of the two samples agreed in 40 (80%) of the 50 pairs. Cytopathology agreed with culture results only 68% of the time. Cytopathology and culture may not be as definitive as previously assumed because of apparent variability of the microbial population within the external canal.     

Antimicrobial susceptibility of Pseudomonas aeruginosa isolates from dogs with otitis externa

Pseudomonas aeruginosa is a common cause of otitis externa in dogs, and treatment of these infections is becoming problematic because of the increasing number of multiresistant strains. The aim of the present study was to compare the in vitro activities of cefepime, ceftazidime, enrofloxacin, ciprofloxacin, gentamicin and ticarcillin/clavulanic acid against 104 strains of P aeruginosa isolated from dogs with otitis externa. Antimicrobial susceptibility and minimum inhibitory concentrations, in μg/ml, were evaluated by the E test (bioMérieux). The most active compound was ceftazidime, with 100 per cent efficiency. The majority of tested strains were susceptible to ticarcillin/clavulanic acid (89.4 per cent), followed by ciprofloxacin (88.5 per cent) and cefepime (60.6 per cent). The highest resistance was observed to enrofloxacin (51.9 per cent) and gentamicin (43.3 per cent). Large numbers of strains were intermediately susceptible to antibiotics registered for use in veterinary medicine in Croatia--enrofloxacin (47.1 per cent) and gentamicin (41.3 per cent).     

Isolation of Malassezia pachydermatis and M. sympodialis from the external ear canal of cats with and without otitis externa

The objective of this study was to determine the presence of Malassezia spp. in the external ear canal of cats with and without otitis. Forty-five animals were studied, 20 with and 25 without otitis externa (OE). Cerumen or secretion from external ear canal samples was cultured on modified Mycosel agar and sterile olive oil was added to the surface of the medium before specimen seeding. The isolates were analysed for macro- and micromorphology and identified by catalase tests and on the basis of growth on Tween 20, 40, 60 and 80. Malassezia spp. were isolated from 15 out of 20 (75%) animals with otitis and from 7 out of 25 (28%) cats without OE; the difference between the two groups was statistically significant (P < or = 0.05). Malassezia pachydermatis and M. sympodialis were isolated from 60% (12/20) and 40% (8/20) of cats with otitis, respectively, with no significant difference in the frequency of isolation between the two species. In the microflora of the healthy ear canal M. pachydermatis was significantly more common (6/25, 24%) than M sympodialis (1/25, 4%). The present investigation confirms that M. sympodialis can also act as an aetiological agent of feline OE, and if commercial veterinary laboratories do not use media with added lipids for the isolation of Malassezia spp., this might lead to false-negative results.     

Identification and antimicrobial susceptibility of coagulase positive staphylococci isolated from healthy dogs and dogs suffering from otitis externa

A total of 67 strains of coagulase positive staphylococci isolated from healthy dogs and dogs suffering from otitis externa were studied. Twenty-two isolates were from healthy dogs (five from hound dogs and 17 from companion dogs) and 45 from dogs suffering otitis externa (14 from hound dogs and 31 from companion dogs). Presumptive identification was attempted using the following tests: production of acetoin, anaerobic utilization of mannitol, acid production from mannitol, presence of beta-galactosidase, and growth on P agar supplemented with different concentrations of acriflavine. Susceptibility of staphylococci to 16 antibiotics was determined. Most effective antibiotics were imipenem, amoxycillin/clavulanic acid, ciprofloxacin, tobramycin, gentamicin and marbofloxacin. Penicillin, ampicillin and polymyxin B showed the lowest activity. There were no significant differences in antimicrobial susceptibility among isolates from healthy dogs and dogs suffering from otitis externa.     

Microbiological Features of Staphylococcus schleiferi subsp. coagulans,Isolated from Dogs and Possible Misidentification with Other Canine Coagulase‐positive Staphylococci

Staphylococcus schleiferi subsp. coagulans has only rarely been isolated and identified from the external auditory meatus of dogs suffering from external otitis. Its morphological and basic biochemical characteristics are of relatively little value for identification, as it phenotypically resembles another coagulase‐positive staphylococci (CPS) and, consequently, may be easily misidentified as S. intermedius or even as S. aureus. In the present work, differentiation of S. schleiferi ssp. coagulans was therefore based on specific biochemical and genetic methods. All the strains were evaluated with the following commercial methods: Api Staph System (bioMérieux, Marcy l'Etoil, France), BBL Crystal Identification Systems (Gram‐Positive ID Kit and Rapid Gram‐Positive ID Kit; Becton Dickinson), and GEN‐PROBE^® AccuProbe, Staphylococcus aureus identification test (bioMérieux). Gram‐Positive ID System/GP database includes the broadest range of staphylococcal species and correctly identifies the majority of strains important in veterinary medicine. Therefore, it is an acceptable alternative to conventional methods for identification of canine staphylococcal isolates. Reliable differentiation of S. aureus from S. schleiferi ssp. coagulans and S. intermedius was feasible with AccuProbe for S. aureus, which gave positive results only for S. aureus; all other CPS tested were negative.     

Variability of laboratory identification and antibiotic susceptibility reporting of Pseudomonas spp. isolates from dogs with chronic otitis externa

The purpose of this study was to evaluate interlaboratory variation in isolation and antibiotic susceptibility pattern of Pseudomonas spp. as reported to veterinarians for cases of canine chronic bacterial otitis externa. Twenty-six dogs with unilateral or bilateral bacterial otitis externa from multiple referral practices were included in this prospective study. Triplicate samples collected simultaneously from the same location in the external ear canal were randomly submitted to three laboratories for culture and susceptibility testing. Pseudomonas spp. were isolated from 18 of 34 (53%) ears. All three laboratories agreed on the presence of Pseudomonas spp. in 15 (83.3%) ears sampled. However, two laboratories agreed on two (11.1%) occasions, and on one occasion (5.5%) Pseudomonas spp. were identified in only one laboratory. Minimum inhibitory concentration (MIC) susceptibilities to 11 antibiotics were compared between laboratories B and C. Using laboratory-defined susceptibility of sensitive (S), intermediate (I) and resistant (R), none of the 16 Pseudomonas spp. with MIC data reported had identical patterns of antibiotic susceptibility. Agreement in susceptibility to individual antibiotics was observed in 13 of 16 (81%) occasions for amikacin and gentamicin, 10 of 16 (63%) occasions for ticarcillin, and nine of 16 (56%) for enrofloxacin. These results indicate that Pseudomonas spp. were identified by all three laboratories chosen for this study in 83% of the time. Moreover, antibiotic susceptibility patterns and MIC values reported to veterinarians may not agree between laboratories. Veterinarians should interpret bacterial culture and susceptibility results with multiple caveats including variability between laboratories.     

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.     

Screening for skin carriage of methicillin-resistant coagulase-positive staphylococci and Staphylococcus schleiferi in dogs with healthy and inflamed skin

Methicillin resistance rates of 41% for Staphylococcus aureus, 16% for S. intermedius, and 40% for S. schleiferi have recently been reported in canine patients. These were deemed to be reflective of referral and clinician-selection biases, which would imply significantly lower methicillin-resistant staphylococcal carriage rates in less-biased canine populations. In this study, swabs for bacterial culture were collected from five cutaneous sites on each of 50 healthy dogs and 59 dogs with inflammatory skin disease to determine prevalence of carriage and relative frequency of methicillin resistance in coagulase-positive staphylococci and S. schleiferi ssp. schleiferi. These were identified morphologically and by Gram's staining, catalase and coagulase testing, and biochemical speciation. Coagulase-positive staphylococci and S. schleiferi ssp. schleiferi were isolated from 88% (52 of 59) of affected dogs. Species identified in the culture-positive dogs were: S. aureus in 12%, S. intermedius (92%), S. schleiferi ssp. schleiferi (10%), and S. schleiferi ssp. coagulans (10%) with methicillin resistance rates of 17%, 8%, 20% and 20%, respectively. Coagulase-positive staphylococci were isolated from 74% (37 of 50) of healthy dogs: S. aureus (16%), S. intermedius (92%) and S. schleiferi ssp. coagulans (5%). Methicillin resistance rates were 0%, 3% and 50%, respectively. Of total methicillin-resistant isolates, 11 of 13 were positive for PBP2a via latex agglutination. Methicillin-resistant S. intermedius and S. schleiferi ssp. schleiferi isolates were all positive for the mecA gene via PCR. There was no significant difference in staphylococcal isolation or methicillin resistance between study groups. While present, methicillin-resistant coagulase-positive staphylococci are significantly less common in these less-biased populations than in the clinical isolates previously reported from this institution which provided the impetus for this study.     

FC-46 Methicillin-resistant Staphylococcus intermedius organisms from the vertical ear canal of dogs with end-stage otitis externa

Different mite species generally found on animals may temporarily also infest humans. Consequently, these arthropods may be responsible for pruritic skin reactions that are often misdiagnosed. Mite dermatitis caused by the tropical rat mite Ornithonyssus bacoti occurs in several small mammals and rodents under tropical and temperate climatic conditions. According to various observations in Germany, O. bacoti appears in wild rodents more frequently than previously thought. In most cases, symptoms of mites are recognized only when they attack humans, but the diagnosis of rat mite dermatitis requires identification of the parasite, which is more likely to be found in the environment than on the hosts' skin itself. Here, five different outbreaks from Bavaria are reported. A clinical example is the case of a 23-year-old medical student and several other residents inhabiting a rat- and mouse-infested house in Munich. The arthropods originally came from an Italian restaurant and surrounding facilities. Mites were found in large numbers in the students' flat. The patient was suffering from severe itching and papular urticaria. He consulted a dermatology clinic complaining of a pruritic dermatitis of 2-weeks duration. Dermatitis was misdiagnosed as allergy and treatment with an anti-inflammatory agent was unsuccessful. Eradication of rodents and treatment of the house with a pyrethroid were performed to prevent reinfestation. Ornithonyssus bacoti is a periodic haematophageous parasite and spends a relatively short time on the host. Causal therapy with antiparasitic agents on human patients is not necessary. If indicated, treatment should be symptomatic.
Funding: Self-funded.     

Identification and antimicrobial susceptibility of bacteria and yeasts isolated from healthy dogs and dogs with otitis externa

The bacterial and fungal flora of the external ear canal of dogs with otitis externa and of healthy dogs were studied. The most frequently isolated microorganism from otitic ears was Staphylococcus intermedius (58.8%), followed by Malassezia pachydermatis (30.9%), Streptococcus canis (29.9%), Proteus spp. (14.4%) and Escherichia coli (10.3%). A statistical analysis of our results showed that the prevalence of these microorganisms is significant in dogs with otitis externa. Furthermore, the antimicrobial susceptibility patterns of isolated strains were determined. Majority of all bacterial isolates were most susceptible to gentamicin. Malassezia pachydermatis, the most prevalent yeast in this study, showed an excellent level of susceptibility to all antifungal agents tested.     

Development of resistant bacteria isolated from dogs with otitis externa or urinary tract infections after exposure to enrofloxacin in vitro

Minimum inhibitory concentrations for enrofloxacin were determined for 63 bacterial isolates from dogs with otitis externa or urinary tract infections. Development of resistant mutants was determined after exposing the isolates to enrofloxacin in vitro for up to five serial passages. Results indicated that Pseudomonas aeruginosa and Enterococcus spp isolates exposed to enrofloxacin developed resistance rapidly, whereas Klebsiella, Proteus, and Streptococcus spp were less likely to develop resistance. Despite the presence of enrofloxacin pressure, no resistant bacteria developed in the Escherichia coli and staphylococcal isolates. In many isolates, susceptibility patterns changed from susceptible to intermediate.     

Antibiotic resistance and molecular characterization of ophthalmic Staphylococcus pseudintermedius isolates from dogs

The prevalence, virulence potential, and antibiotic resistance of ophthalmic Staphylococcus pseudintermedius (SP) isolated from dogs were examined. Sixty-seven Staphylococcus species were isolated from ophthalmic samples and surveyed for species-specific sequences in the Staphylococcus intermedius group (SIG) nuclease gene (SInuc), exfoliative toxin gene for SIG (siet), and antibiotic resistance genes (blaZ and mecA). PCR-restriction fragment length polymorphism analysis of the pta gene was also performed. Fifty isolates were identified as SIG strains, all of which were found to be SP. The blaZ gene was detected in 42 of the 50 SP strains and mecA gene was observed in 18 of the 50 SP strains. The 50 SP strains were most susceptible to amoxicillin/clavulanic acid (94%) and chlorampenicol (70%), and highly resistant to tetracycline (94%) and penicillin (92%). It was also found that 16 (88.9%) mecA-positive SP strains were resistant to oxacillin, tetracycline and penicillin. All mecA-positive SP were resistant to more than four of the eight tested antibiotics and therefore considered SP with multi-drug resistance (MDR). Our results indicate a high prevalence of antibiotic resistance genes in ophthalmic SP along with a close relationship between MDR SP strains and the mecA gene. Based on our findings, judicious administration of antibiotics to companion dogs is necessary.     

Surveillance of healthy cats and cats with inflammatory skin disease for colonization of the skin by methicillin-resistant coagulase-positive staphylococci and Staphylococcus schleiferi ssp. schleiferi

In this study, bacterial cultures were collected from five sites on each of 50 healthy cats and 48 cats with inflammatory skin disease (ISD), to determine prevalence of carriage and relative frequency of methicillin resistance in coagulase-positive staphylococci and Staphylococcus schleiferi ssp. schleiferi. Latex agglutination testing for penicillin-binding protein 2a (PBP2a) and pulsed field gel electrophoresis (PFGE) were performed on all methicillin-resistant (MR) isolates. Polymerase chain reaction (PCR) for the mecA gene was performed on MR S. intermedius and S. schleiferi isolates. Staphylococcal chromosomal cassette (SCCmec) typing was performed on all MR S. aureus isolates. Coagulase-positive staphylococci and S. schleiferi ssp. schleiferi were isolated from 24 of 48 cats with ISD: Staphylococcus aureus (14 of 24, 58%), Staphylococcus intermedius (11 of 24, 46%), Staphylococcus schleiferi ssp. schleiferi (1 of 24, 4%), and Staphylococcus hyicus (1 of 24, 4%). Prevalence of MR was 7% for S. aureus, 0% for S. intermedius, 100% for S. schleiferi ssp. schleiferi, and 0% for S. hyicus. Coagulase-positive staphylococci were isolated from 17 of 50 healthy cats: S. aureus (10 of 17, 59%), S. intermedius (11 of 17, 65%), and S. schleiferi ssp. coagulans (1 of 17, 6%). Prevalence of MR was 20% for S. aureus, 18% for S. intermedius, and 0% for S. schleiferi ssp. coagulans. All MR isolates were positive for PBP2a via latex agglutination. Methicillin-resistant S. intermedius and S. schleiferi ssp. schleiferi isolates were also positive for the mecA gene via PCR. Methicillin-resistant S. aureus isolates were identified as SCCmec type II. Results of PFGE indicated heterogeneity among isolates. There was no significant difference in staphylococcal isolation or methicillin resistance between study groups. While present, MR coagulase-positive staphylococci are significantly less common in these study populations.