Antimicrobial susceptibility of staphylococci isolated from otitis externa in dogs
W. Lilenbaum, Laboratorio de Bacteriologia, Instituto Biomédico, Universidade Federal Fluminense, Rua Prof. Hernani Mello, 101 CEP 24210-130 Niteroi, RJ, Brazil (e-mail: email@example.com).
Samples were obtained from 65 unmedicated adult dogs, processed for isolation of Staphylococcus species and tested for susceptibility to penicillin G, gentamicin, oxacillin, tetracycline, trimethoprim-sulphamethoxazole, streptomycin, ampicillin and rifampin. Forty-four isolates were obtained, which represents 67·7% of samples. Coagulase-negative species were most commonly found, and the most frequently isolated staphylococcus species were Staph. epidermidis and Staph. aureus. Other species, such as Staph. simulans, Staph. haemolyticus, Staph. saprophyticus and Staph. intermedius were also isolated. Resistance to antibiotics was frequently observed, with 90·9% of the isolates showing resistance to at least one drug. The most active antimicrobial agents against staphylococci isolated from otitis externa of dogs were rifampin and oxacillin. Multidrug resistance was a common finding, and one strain of Staph. haemolyticus species, was resistant to all tested antimicrobial agents. Resistance to three or more different drugs was a common finding, observed in 16 strains (36·4%) of both coagulase-positive and coagulase-negative staphylococci. This study highlights the emergence of cases of otitis externa determined by coagulase-negative staphylococcus strains and once more emphasizes the need for bacterial culture with species identification and susceptibility testing of swab specimens from the ear canal in order to choose appropriate antimicrobial agents.
Otitis externa is the most common disease of the ear canal in the dog with a multifactorial aetiology, which includes fungi, yeasts, parasites and bacteria, mainly from the Staphylococcus genus (Kiss et al. 1997).
Members of this genus are often found on healthy and diseased dogs, producing a variety of infections, including bacteraemia, pneumonia, furuncles, abscesses, pyoderma, conjunctivitis and otitis externa (Kloos and Bannerman 1995). The coagulase-positive species Staph. intermedius is most frequently isolated in dogs (Cox et al. 1988; Devriese 1990).
Many organisms vary in their susceptibility to antibiotics. Some strains become resistant after wide usage over a period of time. Also the overusage of a drug, frequently determined by small animal practitioners without microbiological culturing or antibiotic sensitivity testing may contribute to the emergence of resistant strains of bacteria (Blue and Wooley 1977). For these reasons, it is essential that sensitivity charts be updated frequently, based on recent reports of the literature. Many factors influence antimicrobial susceptibilities of a specific bacterium, and the continuous testing allows veterinarians to monitor the susceptibility of common bacteria to antibiotics (Hoekstra and Paulton 1996).
The purpose of this study was to evaluate the antimicrobial susceptibility in staphylococci isolated from otitis externa in dogs from Rio de Janeiro, Brazil in order to update data and evaluate best therapeutic indications for the antimicrobial drugs.
Materials and methods
Dogs Samples were obtained from 65 unmedicated adult dogs (aged > 1 year) of both sexes with clinical signs of otitis externa, e.g. local pain, pruritus, erythema, ear discharge and desquamation of the epithelium. Animals with a recent history of disease or administration of local or systemic drugs or antimicrobial agents in the last 2 months were excluded from the study.
Sample collection and processing A sterile cotton swab was used to collect samples of ear exudate from the external auditory canal and immediately inoculated in brain heart infusion (Difco, Detroit, MI, USA) broth and incubated at 37 °C. Methodology for isolation of anaerobic bacteria was not employed. If growth occurred after 24 or 48 h, smears were made, Gram-stained and examined microscopically. Samples with morphologies compatible with Staphylococcus sp. were transferred to tryptic soy agar, Streptokoken agar, 5% sheep blood agar and mannitol salt agar (Merck, Darmstadt, Germany).
Identification of isolates After growth, staphylococci isolates were repeatedly subcultured in mannitol salt agar plates, in order to ensure homogeneity of the tested colonies and to avoid the possibility of mixed cultures. Purified isolates were then identified on the basis of colony characteristics, Gram stain, pigment production, haemolysis on 5% bovine blood agar and biochemical reactions; namely catalase activity, tube coagulase test (rabbit plasma) mannitol fermentation – Chapman (Merck), urease (Difco), novobiocin resistance (Pimenta Abreu) and deoxyribonuclease test (BBL, Cockeysville, MD, USA), following MacFaddin (1977). Staphylococcus species were classified as described in previous studies (Lilenbaum et al. 1998; Lilenbaum et al. 1999) and according to the Bergey's Manual of Determinative Bacteriology (Holt et al. 1994). Isolates of other bacterial genera (e.g. Gram-negative rods) were not considered in this study. One colony from each isolate was transferred to stock agar.
Susceptibility tests All the staphylococci strains were tested for susceptibility to antimicrobial agents by the agar disc diffusion method on Mueller Hinton agar (Difco) incubated at 37 °C, as described (Anonymous 1993). Discs of penicillin G (10 units), gentamicin (10 µg), oxacillin (5 µg), tetracycline (30 µg), trimethoprim-sulphamethoxazole (25 µg), streptomycin (10 µg), ampicillin (10 µg) and rifampin (30 µg) were tested. After measuring the antimicrobial zone diameters and following the standards (Anonymous 1999), the strains were categorized as susceptible or resistant to the drug.
Forty-four isolates with the typical characteristics of the genus Staphylococcus were obtained from 65 dogs (67·7% growth positivity). No isolates were obtained from 12 samples, and nine dogs presented isolates of Gram-negative rods or yeasts, not considered in this study.
The coagulase-negative species were the most common, representing 61·3% of the isolates. The most frequently isolated (11 samples) coagulase-negative staphylococcus (CoNS) species was Staph. epidermidis. Other CoNS, such as Staph. simulans, Staph. haemolyticus and Staph. saprophyticus were also isolated in seven, five and four samples, respectively. Coagulase-positive species of staphylococci (CoPS) were isolated from 17 samples (38·6%), distributed among Staph. aureus (11 samples) and Staph. intermedius (six samples). The frequency of isolation of the different staphylococci species is shown in Table 1.
Table 1. Frequency of different staphylococcal species isolated from otitis externa in dogs
| Staph. aureus||Positive||11||25|
| Staph. intermedius ||Positive||6||13·6|
| Staph. epidermidis ||Negative||11||25|
| Staph. simulans ||Negative||7||15·9|
| Staph. haemolyticus ||Negative||5||11·4|
| Staph. saprophyticus ||Negative||4||9·1|
The results of the antimicrobial susceptibility tests are given in Tables 2 and 3. Resistance to antibiotics was frequently observed, and 90·9% of the isolates (40 samples) showed resistance to at least one drug. Resistance to penicillin G was a common finding, and was observed on 17 (38·6%) of the 44 isolates. The most active antimicrobial agents against staphylococci isolated from otitis externa of dogs were rifampin and oxacillin, with only one and two samples, respectively, showing resistance to those drugs.
Table 2. Susceptibility rate of isolated staphylococci against eight antibiotics
Table 3. Resistance pattern of strains of staphylococci species isolated from otitis externa in dogs
| Staph. aureus ||11/11 (100%)||PN TS GN ST AP (2); PN TS AP (1); TS GN ST (1); TS ST (1); GN ST (1); TS (2); PN (1); ST (2)|
| Staph. intermedius ||5/6 (83·3%)||PN TS ST AP (1); PN TS AP (1); PN AP (1); TS ST (1); TS (1)|
| Staph. haemolyticus ||4/5 (80%)||PN TS GN OX ST AP RP TT (1) PN TS AP (2) ST (1)|
| Staph. epidermidis ||10/11 (90·9%)||PN TS AP TT (1); PN TS ST (1); PN TS AP (2); PN TS (1); TS (5)|
| Staph. simulans ||7/7 (100%)||PN TS ST (1); TS GN ST (1); GN (1); TS (4)|
| Staph. saprophyticus ||3/4 (75%)||PN OX ST (1); TS (2)|
|Total ||40/44 (90·9%) ||PN TS GN OX ST AP RP TT (1); PN TS GN ST AP (2); PN TS ST AP (1); PN TS AP TT (1); |
PN TS AP (6); TS GN ST (2); PN TS ST (2); PN OX ST (1); TS ST (2); GN ST (1); PN AP (1); PN TS (1);
TS (14); PN (1); ST (3); GN (1)
Multidrug resistance was a common finding, and one strain, of Staph. haemolyticus species, was resistant to all tested antimicrobial agents. Two strains showed resistance to five drugs, and other two were resistant to four different drugs. Resistance to three different drugs was a common finding, observed in 11 strains of both CoNS and CoPS.
The present study confirms the occurrence of staphylococcal strains in otitis externa of dogs, due to the isolation of 44 strains of Staphylococcus from ear discharge of 65 dogs, which represents a rate of 67·7% growth positivity. The coagulase-negative species were the most prevalent, representing 61·3% of the isolates. This was an unexpected result, since previous studies usually reports coagulase-positive species, such as Staph. intermedius and Staph. aureus as the most frequent Staphylococcus isolates in otitis externa of dogs (Blue and Wooley 1977; Lilenbaum et al. 1987; Kiss et al. 1997; Cole et al. 1998).
Coagulase-negative species as a group constitute a major component of the normal microflora of human beings, dogs and cats (Devriese 1990; Kloos and Bannerman 1995; Lilenbaum et al. 1998) and are considered as important opportunistic pathogens in those species. Nevertheless, in contrast with humans, the presence of these staphylococcal species in animals have received little attention to date (Devriese 1990). We believe that further studies are necessary in order to evaluate the real role of those species in the aetiology of the major infections of the dog. The distribution of the coagulase-positive species among Staph. aureus (25%) and Staph. intermedius (13·6%) is in agreement with the majority of previous studies.
With regard to the susceptibility to antimicrobial agents of the strains isolated in this study, a high frequency of resistant strains was observed, since 90·9% of the isolates were resistant to at least one drug. Multidrug resistance was a common finding and 16 strains of both CoPS and CoNS (36·4%) showed resistance to three or more different drugs. This is a very alarming finding and confirms the tendency observed by Love et al. (1981), who reported that 85% of the staphylococcus isolated from canine and feline infections were resistant to at least one drug.
Resistance to penicillin G was observed in 38·7% of the isolates. This rate is in agreement with previous studies (Blue and Wooley 1977; Lilenbaum et al. 1987) and demonstrates the large frequency of β-lactamase producers strains among both CoPS (41·2%) and CoNS (37·0%) isolated from animals. Some strains of methicillin-resistant staphylococci has been recently reported in the normal microbiota of the skin and saliva of cats (Lilenbaum et al. 1998; Lilenbaum et al. 1999). In the present study, we identified two strains resistant to oxacillin, from the coagulase-negative species Staph. saprophyticus and Staph. haemolyticus.
Gentamicin susceptibility rate was 84·1%. Gentamicin has been frequently indicated for the therapeutics of staphylococcal infections. Unfortunately, in the present study we observed a slight reduction of the susceptibility rate to gentamicin when compared with previous studies that had observed 94·4% (Blue and Wooley 1977) and 96·3% of susceptibility (Cole et al. 1998), possibly indicating an overusage of gentamicin. The same phenomena was observed in the susceptibility rate to trimethoprim-sulphamethoxazole. A 27·3% susceptibility rate was reported in the present study compared with reported as 51·9% (Cole et al. 1998).
One strain of Staph. haemolyticus was resistant to all tested antimicrobials, which represents serious evidence of emergence of multidrug resistant strains in dogs infections. Clinicians and other practitioners should be advised of the risk of selection of such multidrug-resistant strains after overusage of antimicrobial drugs without previous testing.
In conclusion, we believe that, although laboratory assistance in determining antibiotic sensitivity patterns of bacteria from cases of otitis externa has been suggested for at least 20 years (Blue and Wooley 1977), a greater proportion of bacterial isolates are resistant to antibacterial agents commonly used to treat otitis externa in the dog. This study highlights the emergence of CoNS-determined otitis externa and once more emphasizes the need for bacterial culture with species identification and susceptibility testing of swab specimens from the ear canal in order to choose appropriate antimicrobial agents.