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Species distribution and properties of staphylococci from canine dermatitis
Research in Veterinary Science 82 (2007) 1–6 www.elsevier.com/locate/rvsc
Species distribution and properties of staphylococci from canine dermatitis Tomasz Hauschild a
a,*
, Agnieszka Wo´jcik
b
University of Bialystok, Institute of Biology, Department of Microbiology, Swierkowa 20 B, 15-950 Bialystok, Poland b Department of Veterinary Hygiene, Zwyciestwa 26 A, 15-370 Bialystok, Poland Accepted 24 April 2006
Abstract The occurrence and phenotypic, and genotypic properties of 24 Staphylococcus isolates from canine dermatitis were investigated. The predominant staphylococcal species was Staphylococcus intermedius. The other species such as Staphylococcus chromogenes, Staphylococcus sciuri, Staphylococcus aureus, Staphylococcus saprophyticus, Staphylococcus epidermidis, and Staphylococcus capitis were only occasionally isolated. The study showed low level biochemical diversity among S. intermedius isolates. Resistance to antibiotics was frequently observed, with 87.5% of the isolates showing resistance to at least one drug. The most active antimicrobial agents against all staphylococci were amoxicillin/clavulanic acid, cephalexin and gentamicin. Resistance to carbenicillin, amoxicillin, ampicillin, cephadroxil, erythromycin, clinadmaycin and neomycin was common. No correlation was observed between antibiotic resistance and plasmid profile. PFGE analysis revealed a high degree of genetic polymorphism of S. intermedius, even among isolates collected in a restricted area over a short time. 2006 Elsevier Ltd. All rights reserved. Keywords: S. intermedius; Antibiotic resistance; Plasmid profile; PFGE
1. Introduction The normal bacterial flora of the skin has been recognised as an important factor in understanding bacterial skin diseases in animals such as pigs, horses, poultry and dogs. One of the predominant microorganisms inhabiting animal skin is staphylococci. Many studies have reported the frequent isolation of different staphylococcal species from the skin of healthy and diseased dogs (Medleau et al., 1986; Shimizu et al., 1992; Lilenbaum et al., 2000; Shimizu et al., 2001; Nagase et al., 2002). In general, the most predominant species is Staphylococcus intermedius, but in some cases from canine skin the most frequently isolated was Staphylococcus sciuri, followed by Staphylococcus xylosus, Staphylococcus capitis, Staphylococcus chromogenes, and Staphylococcus lentus (Nagase et al., 2002) or *
Corresponding author. E-mail address: [email protected] (T. Hauschild).
0034-5288/$ - see front matter 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.rvsc.2006.04.004
Staphylococcus epidermidis and Staphylococcus aureus, followed by Staphylococcus simulans, S. intermedius, Staphylococcus haemolyticus, and Staphylococcus saprophyticus (Lilenbaum et al., 2000). In veterinary dermatology, S. intermedius is one of the causative agents of canine bacterial skin infections, such as otitis externa, pyoderma and abscesses (Kloos and Bannerman, 1995). However, other staphylococcal species inhabiting skin may be involved in infections in dogs (Medleau et al., 1986; Lilenbaum et al., 2000). At present, chemotherapy is the most practical way to treat staphylococcal infections and there have been many studies on the in vitro effect of antimicrobial agents against strains of S. intermedius isolated from the dog (Wegener and Pedersen, 1992; Piriz et al., 1995; Mueller et al., 1998; Pellerin et al., 1998; Shimizu et al., 2001; Petersen et al., 2002; Tejedor Junco and Martin Barrasa, 2002). The percentage of strains resistant to various classes of antibiotics varies in different continents, even countries,
2
T. Hauschild, A. Wo´jcik / Research in Veterinary Science 82 (2007) 1–6
and changes with time (Mueller et al., 1998). Resistance to some antibiotics was sometimes, but not always, associated with previous antibiotic use (Medleau et al., 1986). Irrespective of the place of isolation, most S. intermedius strains are sensitive to cephalothin/cephalexin, amoxicillin/clavulanic acid, gentamicin, oxacillin, enrofloxacin; but resistance to penicillin, ampicillin, tetracycline, erythromycin, lincomycin and trimethoprim/sulphametoxazole is frequently observed. Genes found to be located on small plasmids carried determinants for chloramphenicol resistance (Schwarz et al., 1995), macrolide–lincosamide resistance and resistance to tetracycline (Greene and Schwarz, 1992; Schwarz et al., 1998). Detailed restriction maps of these plasmids implied marked structural homologies with those prototype plasmids initially characterised in staphylococci of human origin (Greene and Schwarz, 1992; Schwarz et al., 1995). A generally accepted typing scheme for S. intermedius does not exist, but PFGE is becoming the standard method for comparative typing of this species due to its reproducibility and discriminatory power (Hesselbarth et al., 1994; Shimizu et al., 1996; Wakita et al., 2002). Moreover, genomic DNA fingerprinting by PFGE of S. intermedius strains from various animals appears to be a useful molecular marker for epidemiological or ecological studies indicating it has a high degree of restriction fragment length polymorphism (Shimizu et al., 1996; Wakita et al., 2002). Until recently, various phenotypic and genotypic markers such: antibiograms, plasmid analysis, and PFGE have been used mainly for studies of S. intermedius strains isolated from a broad range of geographical locations or isolated over a number of years. In this study, the objective was to characterise all staphylococcal species from canine skin infections, and to state whether a high degree of polymorphism occurs among S. intermedius strains collected in a single geographical area during a limited period of time. Our studies were restricted to a city of north-east Poland. 2. Materials and methods 2.1. Samples and identification of isolates The samples were taken by vigorously swabbing diseased skin sites with a sterile cotton swab from dogs which had visited four veterinary clinics, located in one city, between January and April 2003. The swabs were sent to the microbiology laboratory at The Department of Veterinary Hygiene and were directly inoculated onto blood agar plates (blood agar base Oxoid, supplemented with 5% defibrinated sheep blood). Following incubation at 35 C, a representative colony of each morphotype (morphology and pigment) was selected. Not more than one Staphylococcus isolate per dog was chosen for this study. A sample of 24 Staphylococcus isolates were further identified by the API ID32 Staph-system (bioMe´rieux, Poland). The resulting 9-digit codes were evaluated accord-
ing to the database provided by the manufacturer. Haemolytic activity was determined according to the recommendation given by Freney et al. (1999). 2.2. Antibiotic susceptibility testing All isolates were tested by the agar disk diffusion method for their susceptibility to a panel of 10 antimicrobial agents with different mechanism of action. The following disks obtained from Oxoid (Poznan, Poland) were used: amoxycillin (AML-25 lg), amoxycillin/clavulanic acid (AMC—30 lg), ampicillin (AMP—10 lg), carbenicillin (CAR—100 lg), cephalexin (CL—30 lg), neomycin (N—30 lg), erythromycin (E—15 lg), gentamicin (CN— 10 lg), cefadroxil (CFR—30 lg) and clindamycin (DA— 2 lg). Performance of the susceptibility testing as well as evaluation of the results followed the recommendations given in the National Committee for Clinical Laboratory Standards (2002). 2.3. Plasmid profiling All Staphylococcus spp. isolates were investigated for their plasmid content by a staphylococcal-specific modification of the alkaline lysis procedure as described by Schwarz et al. (1990). Plasmids were electrophoretically separated in 1.0% (w/v) agarose gels (Sigma, Poland) at 6 V cm 1 using 1 · TAE as running buffer. The plasmids of Escherichia coli V517 (Macrina et al., 1978) served as size standards for the determination of approximate plasmid sizes. 2.4. Pulsed-field gel electrophoresis Whole cell DNA was prepared as described by Shimizu et al. (1996). Slices of the DNA-containing agarose plugs were subjected to restriction endonuclease digestion in a total volume of 150 ll which contained 40 U of SmaI (MBI Fermentas, Lithuania). Digestion of the DNA was performed at 30 C for 4 h. The SmaI fragments were electrophoretically separated in a 1% (w/v) agarose (Sigma, Poland) gel by using a CHEF-DR II system (BioRad, Poland) and 0.5 · TBE as running buffer at 14 C. The gel was exposed to a pulse time of 5–40 s at 6 V cm 1 for 20 h. The pulsed-field DNA marker 50–1000 kbp (Sigma, Poland) served as a size standard. 3. Results The characteristics of the 24 isolates obtained from dogs with canine dermatitis are summarized in Table 1. Seventeen isolates were identified by ID32 Staph-system as S. intermedius with probabilities of 95.3–65.1%. Two isolates were identified as S. chromogenes with probabilities of 76.2–57.2%, and the remaining six strains as S. sciuri, S. aureus, S. saprophyticus, S. epidermidis, and S. capitis with probabilities of 99.7%, 99.8%, 99.9%, 97.3% and 91.5%,
T. Hauschild, A. Wo´jcik / Research in Veterinary Science 82 (2007) 1–6
3
Table 1 Characteristic of staphylococci from canine dermatitis included in this study Isolate no.
Staphylococcus spp.
4 5 15 34 38 42 43 70 87 89 101 104 113 115 118 135 136 49 93 6 17 28 65 99
Staphylococcus Staphylococcus Staphylococcus Staphylococcus Staphylococcus Staphylococcus Staphylococcus Staphylococcus Staphylococcus Staphylococcus Staphylococcus Staphylococcus Staphylococcus Staphylococcus Staphylococcus Staphylococcus Staphylococcus Staphylococcus Staphylococcus Staphylococcus Staphylococcus Staphylococcus Staphylococcus Staphylococcus
intermedius intermedius intermedius intermedius intermedius intermedius intermedius intermedius intermedius intermedius intermedius intermedius intermedius intermedius intermedius intermedius intermedius chromogenes chromogenes sciuri aureus saprophyticus epidermidis capitis
Antimicrobial resistance pattern
Plasmid profile
PFGE pattern
CAR
3.1 2.1
A B C D1 E K L F G H B I D2 J M N O P Q R S T U V
AML, AMP, DA, E, AML, CAR, AMP DA, CL DA, E AML, CAR, AMP, DA, E, N, CFR CAR, DA, E, N, AML, E, N, CL, CFR AML, CAR, AMP AML, CAR, DA, E, N, CN N AML, CAR, AMP, N, CFR CAR, E, N, CFR, CN DA AML, CAR, AMP, N AML, CAR, AMP AML, CAR, AMP AML, CAR, AMP, DA, E, CL, CFR, AMC AML, CAR, AMP N, CL, CN
respectively. By using the ID32 Staph-system it was possible to distinguish five biochemical profiles among the 17 S. intermedius isolates included in this study. Variations were observed in the fermentation of mannitol and turanose 71% and 59% of strains gave a positive reaction, respectively. One strain gave a negative reaction in the fermentation of ribose. All isolates were positive for maltose acidification. All isolates excluding one isolate of S. intermedius and S. sciuri produced b-haemolysin. Analysis of the antimicrobial susceptibility patterns (Table 1) showed a high level of antimicrobial resistance in both S. intermedius and coagulase-negative staphylococci (CoNS), with 88% of the isolates showing resistance to at least one drug. Most of the S. intermedius isolates under study were resistant to b-lactam antibiotics, 47%, 41%, 29% and 24% of the isolates were resistant to carbenicillin, amoxicillin, ampicillin, and cephadroxil, respectively. Resistance to macrolides and lincosamides was also frequently observed, and 41% and 35% of S. intermedius strains were resistant to erythromycin and clindamycin, respectively. The most active antimicrobial agent against staphylococci isolated from dermatitis of dogs was amoxicillin/clavulanic acid. All S. intermedius and CoNS isolates were sensitive to this drug. Moreover, cephalexin and gentamicin were very active against S. intermedius strains, with only two isolates, showing resistance to these agents. Antimicrobial susceptibilities of the CoNS was also a common finding (Table 1); S. saprophyticus was resistant to the greatest number of antibiotics tested. The plasmid profiles of isolates are reported in Table 1. The 11 isolates were plasmid-free, but the remaining iso-
2.3
3.2
2.1 3.1 2.6
3.0 3.0 4.6 2.1, 3.2, 5.0, 20.0
lates, except S. saprophyticus, harboured only one plasmid ranging in size between 2.1 kb and 4.6 kb. Digestion of genomic DNA with SmaI and separation of the fragments by PFGE revealed the presence of 16 different patterns among the 17 S. intermedius isolates (Table 1, Fig. 1). As expected, the SmaI fragment patterns of the six CoNS species differed distinctly from one another, including the two S. chromogenes isolates, and from those of 17 S. intermedius isolates (Fig. 1). Isolates no. 5 and 101 were indistinguishable by their PFGE patterns, and no. 34 and 113 showed the PFGE patterns D1 and D2, respectively, differed from one another by only two bands. 4. Discussion The biochemical properties of the all isolates were examined by using the ID 32 Staph-system. The seventeen S. intermedius strains differed in fermentation of mannitol, turanose and ribose. The diversity of S. intermedius in fermentation of turanose was also shown by Bes et al. (2002) using the ID 32 Staph-system. They also found variable results in the production of acid from maltose. In our study all isolates were positive for maltose acidification. The diversity in fermentation of mannitol matched the description of S. intermedius species reported by Hajek (1976). Our study showed that the biochemical activity of the canine S. intermedius strains was independent of the country of isolation and confirmed low level biochemical diversity among this staphylococcal species. All isolates excluding one isolate of S. intermedius and S. sciuri were b-haemolytic. This property is commonly found
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T. Hauschild, A. Wo´jcik / Research in Veterinary Science 82 (2007) 1–6
Fig. 1. SmaI macrorestriction patterns of the 24 Staphylococcus spp. isolates included in this study. Lanes A–V represent each PFGE-type (Table 1). Lane m contain the size standard 50–1000 kbp.
in the strains isolated from the skin of diseased dogs (Kruse et al., 1996). With regards to the susceptibility to antimicrobial agents of the strains isolated in this study, a high frequency of resistant strains was observed amongst S. intermedius as well as CoNS, since 88% of the isolates were resistant to at least one drug. Similar results were obtained by Lilenbaum et al. (2000), who detected drug resistance in 90.9% of the isolates from otitis externa in dogs. The majority of S. intermedius isolates under study were resistant to b-lactams such as carbenicillin, amoxicillin, ampicillin, and cephadroxil. These results are in agreement with several investigators which revealed a high percentage of resistance to the same class of antibiotics (Piriz et al., 1995; Pellerin et al., 1998; Petersen et al., 2002). However, a total of 90 strains of S. intermedius isolated from dogs in Japan were sensitive to ampicillin and cephalexin (Shimizu et al., 2001). Resistance to macrolides is also common finding among canine S. intermedius strains. Many studies have reported a high percentage of resistance to erythromycin among strains tested, ranging from 7.8% in Japan, 10% in Australia to 21% in the USA. However, in the present study, as much as 41.2% and 35.3% of S. intermedius strains were resistant to erythromycin and clindamycin, respectively. This observation is consistent with the observed continuous increase in macrolide resistance amongst S. intermedius isolates as described by Cox et al. (1984) and Kruse et al. (1996). On the other hand, this fact may be explained because the use of these drugs in canine dermatology has increased steadily over the last years. The frequency of neomycin resistance (41%), was higher than in other studies (Kruse et al., 1996). This finding may be explained by the possibility of correlation between macrolide resistance and resis-
tance to neomycin described by Boerlin et al. (2001). The most active antimicrobial agents against staphylococci isolated from dogs was amoxicillin/clavulanic acid, cephalexin and gentamicin. The resistance patterns of the CoNS were similar to S. intermedius strains but the small numbers of CoNS species precluded making meaningful comparison with S. intermedius. However, it is noteworthy to mention, that one strain of S. saprophyticus was resistant to the greatest number of antibiotics tested. No correlation has been observed between antibiotic resistance and occurrence of plasmids. In investigations where plasmid profiling has been performed, a striking variation in plasmid patterns has also been detected (Greene and Schwarz, 1992; Wegener and Pedersen, 1992). Resistant strains may be plasmid-less, indicating that resistance may also be chromosomally-mediated (Greene and Schwarz, 1992; Wegener and Pedersen, 1992; Schwarz et al., 1995). This can be explained by the observation of either self-movable or movable genetic elements occurring in staphylococci (Pellerin et al., 1998). Resistance in S. intermedius from dogs may be plasmidmediated, but is most frequently chromosomally-located (Greene and Schwarz, 1992). A mechanism probably exists in this staphylococcal species which encourages chromosomal acquisition of genes, or which discourages plasmids. Dog strains commonly carry single copies of the IS257 insertion element. The genes coding for penicillinases are located in plasmids or transposable elements that can be located in bacteriophages or in the chromosome (Pellerin et al., 1998). On the other hand, resistance to penicillin in staphylococci of animal origin could not be related to plasmids in any of strains tested (Wegener
T. Hauschild, A. Wo´jcik / Research in Veterinary Science 82 (2007) 1–6
and Schwarz, 1993). The genes which confer erythromycin-resistance in dog staphylococci are almost entirely ermB, and for the first described by Boerlin et al. (2001), that can be physically linked to Tn5405-like elements known as resistance determinants for streptomycin, neomycin and kanamycin. The absolute correlation between macrolide resistance and resistance to neomycin observed by these authors was partially confirmed in our study; five of seven S. intermedius strains were resistant to both drugs (Table 1). Digestion of genomic DNA with SmaI revealed the presence of 16 different patterns among the 17 S. intermedius isolates. According to criteria proposed by Ternover et al. (1995), two isolates were indistinguishable by their PFGE patterns, and two showed the PFGE patterns D1 and D2, differed from one another by only two bands. These two last isolates are genetically related and could be exchanged between dogs. At present, genomic DNA fingerprinting by PFGE appears to be an effective technique for discriminating canine S. intermedius strains and may provide detailed information for epidemiological investigations of this species (Hesselbarth et al., 1994; Shimizu et al., 1996; Wakita et al., 2002). Moreover, the PFGE is also useful for discriminating S. intermedius implicated in food-related disease from strains of veterinary origin (Khambaty et al., 1994). The high genetic polymorphism of S. intermedius detected in PFGE studies were also confirmed in our study, even the isolates were collected in a restricted area over a short time. We expected that the diversity of our strains would be much lower than that of the population of S. intermedius collected over a few years in diverse areas. These results might indicate that genotypically unrelated S. intermedius isolates spread within and beyond populations of dogs and obviously could not also be exchanged between dogs, with the exception of two cases of canine dermatitis (strains no. 5 and 101). In conclusion, our work highlights the emergence of CoNS-determined canine dermatitis and shows that the population of canine S. intermedius from Poland is biochemically homogenous, but indicate considerable genetic diversity within this species. The many different resistance patterns observed confirm other studies, reporting a great variety of resistance patterns in staphylococci depending on the country of its isolation. References Bes, M., Slim, L.S., Becharnia, F., Meugnier, H., Vandenesch, F., Etienne, J., Freney, J., 2002. Population diversity of Staphylococcus intermedius isolates from various host species: typing by 16S-23S intergenic ribosomal DNA spacer polymorphism analysis. Journal of Clinical Microbiology 40, 2275–2277. Boerlin, P., Burnens, A.P., Frey, J., Kuhnert, P., Nicolet, J., 2001. Molecular epidemiology and genetic linkage of macrolide and aminoglycoside resistance in Staphylococcus intermedius of canine origin. Veterinary Microbiology 79, 155–169.
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Cox, H.U., Hoskins, J.D., Roy, A.F., Newman, S.S., Luther, D.G., 1984. Antimicrobial susceptibility of coagulase-positive staphylococci isolated from Louisiana dogs. American Journal of Veterinary Research 45, 2039–2042. Freney, J., Kloos, W.E., Hajek, V., Webster, J.A., 1999. Recommended minimal standards for description of new staphylococcal species. International Journal of Systematic Bacteriology 49, 489–502. Greene, R.T., Schwarz, S., 1992. Small antibiotic resistance plasmids in Staphylococcus intermedius. Zentralblatt fu¨r Bacteriologie 276, 380– 389. Hajek, V., 1976. Staphylococcus intermedius, a new species isolated from animals. International Journal of Systematic Bacteriology 26, 401–408. Hesselbarth, J., Witte, W., Cuny, C., Rohde, R., Amtsberg, G., 1994. Characterization of Staphylococcus intermedius from healthy dogs and cases of superficial pyoderma by DNA restriction endonuclease patterns. Veterinary Microbiology 41, 259–266. Khambaty, F.M., Bennett, R.W., Shah, D.B., 1994. Application of pulsed-field gel electrophoresis to the epidemiological characterization of Staphylococcus intermedius implicated in a food-related outbreak. Epidemiology and Infections 113, 75–81. Kloos, W.E., Bannerman, T.L., 1995. Staphylococcus and Micrococcus. In: Manual of Clinical Microbiology, sixth ed. ASM Press, Washington, DC, pp. 282–298. Kruse, H., Hofshagen, M., Thorensen, S.I., Bredal, W.P., Vollset, I., Soli, N.E., 1996. The antimicrobial susceptibility of Staphylococcus species isolated from canine dermatitis. Veterinary Research Communications 20, 205–214. Lilenbaum, W., Veras, M., Blum, E., Souza, G.N., 2000. Antimicrobial susceptibility of staphylococci isolated from otitis externa in dogs. Letters in Applied Microbiology 31, 42–45. Macrina, F.L., Kopecko, D.J., Jones, K.R., Ayers, D.J., McCowen, S.M., 1978. A multiple plasmid-containing Escherichia coli strain: convenient source of size reference plasmid molecules. Plasmid 1, 417–420. Medleau, L., Long, R.E., Brown, J., Miller, W.H., 1986. Frequency and antimicrobial susceptibility of Staphylococcus species isolated from canine pyoderma. American Journal of Veterinary Research 47, 229– 231. Mueller, R.S., Bettenay, S.V., Lording, P., Dell’Osa, D., 1998. Antibiotic sensitivity of Staphylococcus intermedius isolated from canine pyoderma. Australian Veterinary Practitioner 28, 10–13. Nagase, N., Sasaki, A., Yamashita, K., Shimizu, A., Wakita, Y., Kitai, S., Kawano, J., 2002. Isolation and species distribution of staphylococci from animal and human skin. Journal of Veterinary Medical Science 64, 245–250. National Committee for Clinical Laboratory Standards, 2002. Performance Standards for Antimicrobial Disk and Dilution Susceptibility Tests for Bacteria Isolated from Animals; Approved Standards, second ed., NCCLS document M31-A2. National Committee for Laboratory Standards, Wayne, PA, USA. Pellerin, J.L., Bourdeau, P., Sebbag, H., Person, J.M., 1998. Epidemiosurveillance of antimicrobial compound resistance of Staphylococcus intermedius clinical isolates from canine pyodermas. Comparative Immunology, Microbiology and Infectious Diseases 21, 115–133. Petersen, A.D., Walker, R.D., Bowman, M.M., Schott, H.C., Rosser Jr., E.J., 2002. Frequency of isolation and antimicrobial susceptibility patterns of Staphylococcus intermedius and Pseudomonas aeruginosa isolates from canine skin and ear samples over a 6-year period (1992– 1997). Journal of the American Animal Hospital Association 38, 407– 413. Piriz, S., De La Fuente, R., Valle, J., Mateos, E., Hurtado, M.A., Cid, D., Ruiz-Santaquiteria, J.A., Vadillo, S., 1995. Comparative in vitro activity of 11 b-lactam antibiotics against 91 Staphylococcus intermedius strains isolated from staphylococcal dermatitis in dogs. Journal of Veterinary Medicine B 42, 293–300. Schwarz, S., Cardoso, M., Blobel, H., 1990. Detection of a novel chloramphenicol resistance plasmid from ‘‘equine’’ Staphylococcus sciuri. Journal of Veterinary Medicine B 37, 674–679.
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Schwarz, S., Werckenthin, C., Pinter, L., Kent, L.E., Noble, W.C., 1995. Chloramphenicol resistance in Staphylococcus intermedius from a single veterinary centre: evidence for plasmid and chromosomal location of the resistance genes. Veterinary Microbiology 43, 151–159. Schwarz, S., Roberts, M.C., Werckenthin, C., Pand, Y., Lange, C., 1998. Tetracycline resistance in Staphylococcus spp. from domestic animals. Veterinary Microbiology 63, 217–227. Shimizu, A., Ozaki, J., Kawano, J., Saitoh, Y., Kimura, S., 1992. Distribution of Staphylococcus species on animal skin. Journal of Veterinary Medical Science 54, 355–357. Shimizu, A., Berkhoff, H.A., Kloos, W.E., George, C.G., Ballard, D.N., 1996. Genomic DNA fingerprinting, using pulsed-field gel electrophoresis, of Staphylococcus intermedius isolated from dogs. American Journal of Veterinary Research 57, 1458–1462. Shimizu, A., Wakita, Y., Nagase, S., Okabe, M., Koji, T., Hayashi, T., Nagase, N., Sasaki, A., Kawano, J., Yamashita, K., Takagi, M., 2001. Antimicrobial susceptibility of Staphylococcus intermedius isolated from healthy and diseased dogs. Journal of Veterinary Medical Science 63, 357–360.
Tejedor Junco, M.T., Martin Barrasa, J.L., 2002. Identification and antimicrobial susceptibility of coagulase positive staphylococci isolated from healthy dogs and dogs suffering from otitis externa. Journal of Veterinary Medicine B 49, 419–423. Ternover, F.C., Arbeit, R.D., Goering, R.V., Mickelsen, P.A., Murray, B.E., Persing, D.H., Swaminathan, B., 1995. Interpreting chromosomal DNA restriction patterns produced by pulsed-field gel electrophoresis: criteria for bacterial strain typing. Journal of Clinical Microbiology 33, 2233–2239. Wakita, Y., Shimizu, A., Hajek, V., Kawano, J., Yamashita, K., 2002. Characterization of Staphylococcus intermedius from pigeons, dogs, foxes, mink, and horses by pulsed-field gel electrophoresis. Journal of Veterinary Medicine Sciences 64, 237–243. Wegener, H.C., Pedersen, K., 1992. Variations in antibiograms and plasmid profiles among multiple isolates of Staphylococcus intermedius from pyoderma in dogs. Acta Veterinaria Scandinavica 33, 391–394. Wegener, H.C., Schwarz, S., 1993. Antibiotic-resistance and plasmids in Staphylococcus hyicus isolated from pigs with exudative epidermidis and from healthy pigs. Veterinary Microbiology 34, 363–372.
Species distribution and properties of staphylococci from canine dermatitis Tomasz Hauschild a
a,*
, Agnieszka Wo´jcik
b
University of Bialystok, Institute of Biology, Department of Microbiology, Swierkowa 20 B, 15-950 Bialystok, Poland b Department of Veterinary Hygiene, Zwyciestwa 26 A, 15-370 Bialystok, Poland Accepted 24 April 2006
Abstract The occurrence and phenotypic, and genotypic properties of 24 Staphylococcus isolates from canine dermatitis were investigated. The predominant staphylococcal species was Staphylococcus intermedius. The other species such as Staphylococcus chromogenes, Staphylococcus sciuri, Staphylococcus aureus, Staphylococcus saprophyticus, Staphylococcus epidermidis, and Staphylococcus capitis were only occasionally isolated. The study showed low level biochemical diversity among S. intermedius isolates. Resistance to antibiotics was frequently observed, with 87.5% of the isolates showing resistance to at least one drug. The most active antimicrobial agents against all staphylococci were amoxicillin/clavulanic acid, cephalexin and gentamicin. Resistance to carbenicillin, amoxicillin, ampicillin, cephadroxil, erythromycin, clinadmaycin and neomycin was common. No correlation was observed between antibiotic resistance and plasmid profile. PFGE analysis revealed a high degree of genetic polymorphism of S. intermedius, even among isolates collected in a restricted area over a short time. 2006 Elsevier Ltd. All rights reserved. Keywords: S. intermedius; Antibiotic resistance; Plasmid profile; PFGE
1. Introduction The normal bacterial flora of the skin has been recognised as an important factor in understanding bacterial skin diseases in animals such as pigs, horses, poultry and dogs. One of the predominant microorganisms inhabiting animal skin is staphylococci. Many studies have reported the frequent isolation of different staphylococcal species from the skin of healthy and diseased dogs (Medleau et al., 1986; Shimizu et al., 1992; Lilenbaum et al., 2000; Shimizu et al., 2001; Nagase et al., 2002). In general, the most predominant species is Staphylococcus intermedius, but in some cases from canine skin the most frequently isolated was Staphylococcus sciuri, followed by Staphylococcus xylosus, Staphylococcus capitis, Staphylococcus chromogenes, and Staphylococcus lentus (Nagase et al., 2002) or *
Corresponding author. E-mail address: [email protected] (T. Hauschild).
0034-5288/$ - see front matter 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.rvsc.2006.04.004
Staphylococcus epidermidis and Staphylococcus aureus, followed by Staphylococcus simulans, S. intermedius, Staphylococcus haemolyticus, and Staphylococcus saprophyticus (Lilenbaum et al., 2000). In veterinary dermatology, S. intermedius is one of the causative agents of canine bacterial skin infections, such as otitis externa, pyoderma and abscesses (Kloos and Bannerman, 1995). However, other staphylococcal species inhabiting skin may be involved in infections in dogs (Medleau et al., 1986; Lilenbaum et al., 2000). At present, chemotherapy is the most practical way to treat staphylococcal infections and there have been many studies on the in vitro effect of antimicrobial agents against strains of S. intermedius isolated from the dog (Wegener and Pedersen, 1992; Piriz et al., 1995; Mueller et al., 1998; Pellerin et al., 1998; Shimizu et al., 2001; Petersen et al., 2002; Tejedor Junco and Martin Barrasa, 2002). The percentage of strains resistant to various classes of antibiotics varies in different continents, even countries,
2
T. Hauschild, A. Wo´jcik / Research in Veterinary Science 82 (2007) 1–6
and changes with time (Mueller et al., 1998). Resistance to some antibiotics was sometimes, but not always, associated with previous antibiotic use (Medleau et al., 1986). Irrespective of the place of isolation, most S. intermedius strains are sensitive to cephalothin/cephalexin, amoxicillin/clavulanic acid, gentamicin, oxacillin, enrofloxacin; but resistance to penicillin, ampicillin, tetracycline, erythromycin, lincomycin and trimethoprim/sulphametoxazole is frequently observed. Genes found to be located on small plasmids carried determinants for chloramphenicol resistance (Schwarz et al., 1995), macrolide–lincosamide resistance and resistance to tetracycline (Greene and Schwarz, 1992; Schwarz et al., 1998). Detailed restriction maps of these plasmids implied marked structural homologies with those prototype plasmids initially characterised in staphylococci of human origin (Greene and Schwarz, 1992; Schwarz et al., 1995). A generally accepted typing scheme for S. intermedius does not exist, but PFGE is becoming the standard method for comparative typing of this species due to its reproducibility and discriminatory power (Hesselbarth et al., 1994; Shimizu et al., 1996; Wakita et al., 2002). Moreover, genomic DNA fingerprinting by PFGE of S. intermedius strains from various animals appears to be a useful molecular marker for epidemiological or ecological studies indicating it has a high degree of restriction fragment length polymorphism (Shimizu et al., 1996; Wakita et al., 2002). Until recently, various phenotypic and genotypic markers such: antibiograms, plasmid analysis, and PFGE have been used mainly for studies of S. intermedius strains isolated from a broad range of geographical locations or isolated over a number of years. In this study, the objective was to characterise all staphylococcal species from canine skin infections, and to state whether a high degree of polymorphism occurs among S. intermedius strains collected in a single geographical area during a limited period of time. Our studies were restricted to a city of north-east Poland. 2. Materials and methods 2.1. Samples and identification of isolates The samples were taken by vigorously swabbing diseased skin sites with a sterile cotton swab from dogs which had visited four veterinary clinics, located in one city, between January and April 2003. The swabs were sent to the microbiology laboratory at The Department of Veterinary Hygiene and were directly inoculated onto blood agar plates (blood agar base Oxoid, supplemented with 5% defibrinated sheep blood). Following incubation at 35 C, a representative colony of each morphotype (morphology and pigment) was selected. Not more than one Staphylococcus isolate per dog was chosen for this study. A sample of 24 Staphylococcus isolates were further identified by the API ID32 Staph-system (bioMe´rieux, Poland). The resulting 9-digit codes were evaluated accord-
ing to the database provided by the manufacturer. Haemolytic activity was determined according to the recommendation given by Freney et al. (1999). 2.2. Antibiotic susceptibility testing All isolates were tested by the agar disk diffusion method for their susceptibility to a panel of 10 antimicrobial agents with different mechanism of action. The following disks obtained from Oxoid (Poznan, Poland) were used: amoxycillin (AML-25 lg), amoxycillin/clavulanic acid (AMC—30 lg), ampicillin (AMP—10 lg), carbenicillin (CAR—100 lg), cephalexin (CL—30 lg), neomycin (N—30 lg), erythromycin (E—15 lg), gentamicin (CN— 10 lg), cefadroxil (CFR—30 lg) and clindamycin (DA— 2 lg). Performance of the susceptibility testing as well as evaluation of the results followed the recommendations given in the National Committee for Clinical Laboratory Standards (2002). 2.3. Plasmid profiling All Staphylococcus spp. isolates were investigated for their plasmid content by a staphylococcal-specific modification of the alkaline lysis procedure as described by Schwarz et al. (1990). Plasmids were electrophoretically separated in 1.0% (w/v) agarose gels (Sigma, Poland) at 6 V cm 1 using 1 · TAE as running buffer. The plasmids of Escherichia coli V517 (Macrina et al., 1978) served as size standards for the determination of approximate plasmid sizes. 2.4. Pulsed-field gel electrophoresis Whole cell DNA was prepared as described by Shimizu et al. (1996). Slices of the DNA-containing agarose plugs were subjected to restriction endonuclease digestion in a total volume of 150 ll which contained 40 U of SmaI (MBI Fermentas, Lithuania). Digestion of the DNA was performed at 30 C for 4 h. The SmaI fragments were electrophoretically separated in a 1% (w/v) agarose (Sigma, Poland) gel by using a CHEF-DR II system (BioRad, Poland) and 0.5 · TBE as running buffer at 14 C. The gel was exposed to a pulse time of 5–40 s at 6 V cm 1 for 20 h. The pulsed-field DNA marker 50–1000 kbp (Sigma, Poland) served as a size standard. 3. Results The characteristics of the 24 isolates obtained from dogs with canine dermatitis are summarized in Table 1. Seventeen isolates were identified by ID32 Staph-system as S. intermedius with probabilities of 95.3–65.1%. Two isolates were identified as S. chromogenes with probabilities of 76.2–57.2%, and the remaining six strains as S. sciuri, S. aureus, S. saprophyticus, S. epidermidis, and S. capitis with probabilities of 99.7%, 99.8%, 99.9%, 97.3% and 91.5%,
T. Hauschild, A. Wo´jcik / Research in Veterinary Science 82 (2007) 1–6
3
Table 1 Characteristic of staphylococci from canine dermatitis included in this study Isolate no.
Staphylococcus spp.
4 5 15 34 38 42 43 70 87 89 101 104 113 115 118 135 136 49 93 6 17 28 65 99
Staphylococcus Staphylococcus Staphylococcus Staphylococcus Staphylococcus Staphylococcus Staphylococcus Staphylococcus Staphylococcus Staphylococcus Staphylococcus Staphylococcus Staphylococcus Staphylococcus Staphylococcus Staphylococcus Staphylococcus Staphylococcus Staphylococcus Staphylococcus Staphylococcus Staphylococcus Staphylococcus Staphylococcus
intermedius intermedius intermedius intermedius intermedius intermedius intermedius intermedius intermedius intermedius intermedius intermedius intermedius intermedius intermedius intermedius intermedius chromogenes chromogenes sciuri aureus saprophyticus epidermidis capitis
Antimicrobial resistance pattern
Plasmid profile
PFGE pattern
CAR
3.1 2.1
A B C D1 E K L F G H B I D2 J M N O P Q R S T U V
AML, AMP, DA, E, AML, CAR, AMP DA, CL DA, E AML, CAR, AMP, DA, E, N, CFR CAR, DA, E, N, AML, E, N, CL, CFR AML, CAR, AMP AML, CAR, DA, E, N, CN N AML, CAR, AMP, N, CFR CAR, E, N, CFR, CN DA AML, CAR, AMP, N AML, CAR, AMP AML, CAR, AMP AML, CAR, AMP, DA, E, CL, CFR, AMC AML, CAR, AMP N, CL, CN
respectively. By using the ID32 Staph-system it was possible to distinguish five biochemical profiles among the 17 S. intermedius isolates included in this study. Variations were observed in the fermentation of mannitol and turanose 71% and 59% of strains gave a positive reaction, respectively. One strain gave a negative reaction in the fermentation of ribose. All isolates were positive for maltose acidification. All isolates excluding one isolate of S. intermedius and S. sciuri produced b-haemolysin. Analysis of the antimicrobial susceptibility patterns (Table 1) showed a high level of antimicrobial resistance in both S. intermedius and coagulase-negative staphylococci (CoNS), with 88% of the isolates showing resistance to at least one drug. Most of the S. intermedius isolates under study were resistant to b-lactam antibiotics, 47%, 41%, 29% and 24% of the isolates were resistant to carbenicillin, amoxicillin, ampicillin, and cephadroxil, respectively. Resistance to macrolides and lincosamides was also frequently observed, and 41% and 35% of S. intermedius strains were resistant to erythromycin and clindamycin, respectively. The most active antimicrobial agent against staphylococci isolated from dermatitis of dogs was amoxicillin/clavulanic acid. All S. intermedius and CoNS isolates were sensitive to this drug. Moreover, cephalexin and gentamicin were very active against S. intermedius strains, with only two isolates, showing resistance to these agents. Antimicrobial susceptibilities of the CoNS was also a common finding (Table 1); S. saprophyticus was resistant to the greatest number of antibiotics tested. The plasmid profiles of isolates are reported in Table 1. The 11 isolates were plasmid-free, but the remaining iso-
2.3
3.2
2.1 3.1 2.6
3.0 3.0 4.6 2.1, 3.2, 5.0, 20.0
lates, except S. saprophyticus, harboured only one plasmid ranging in size between 2.1 kb and 4.6 kb. Digestion of genomic DNA with SmaI and separation of the fragments by PFGE revealed the presence of 16 different patterns among the 17 S. intermedius isolates (Table 1, Fig. 1). As expected, the SmaI fragment patterns of the six CoNS species differed distinctly from one another, including the two S. chromogenes isolates, and from those of 17 S. intermedius isolates (Fig. 1). Isolates no. 5 and 101 were indistinguishable by their PFGE patterns, and no. 34 and 113 showed the PFGE patterns D1 and D2, respectively, differed from one another by only two bands. 4. Discussion The biochemical properties of the all isolates were examined by using the ID 32 Staph-system. The seventeen S. intermedius strains differed in fermentation of mannitol, turanose and ribose. The diversity of S. intermedius in fermentation of turanose was also shown by Bes et al. (2002) using the ID 32 Staph-system. They also found variable results in the production of acid from maltose. In our study all isolates were positive for maltose acidification. The diversity in fermentation of mannitol matched the description of S. intermedius species reported by Hajek (1976). Our study showed that the biochemical activity of the canine S. intermedius strains was independent of the country of isolation and confirmed low level biochemical diversity among this staphylococcal species. All isolates excluding one isolate of S. intermedius and S. sciuri were b-haemolytic. This property is commonly found
4
T. Hauschild, A. Wo´jcik / Research in Veterinary Science 82 (2007) 1–6
Fig. 1. SmaI macrorestriction patterns of the 24 Staphylococcus spp. isolates included in this study. Lanes A–V represent each PFGE-type (Table 1). Lane m contain the size standard 50–1000 kbp.
in the strains isolated from the skin of diseased dogs (Kruse et al., 1996). With regards to the susceptibility to antimicrobial agents of the strains isolated in this study, a high frequency of resistant strains was observed amongst S. intermedius as well as CoNS, since 88% of the isolates were resistant to at least one drug. Similar results were obtained by Lilenbaum et al. (2000), who detected drug resistance in 90.9% of the isolates from otitis externa in dogs. The majority of S. intermedius isolates under study were resistant to b-lactams such as carbenicillin, amoxicillin, ampicillin, and cephadroxil. These results are in agreement with several investigators which revealed a high percentage of resistance to the same class of antibiotics (Piriz et al., 1995; Pellerin et al., 1998; Petersen et al., 2002). However, a total of 90 strains of S. intermedius isolated from dogs in Japan were sensitive to ampicillin and cephalexin (Shimizu et al., 2001). Resistance to macrolides is also common finding among canine S. intermedius strains. Many studies have reported a high percentage of resistance to erythromycin among strains tested, ranging from 7.8% in Japan, 10% in Australia to 21% in the USA. However, in the present study, as much as 41.2% and 35.3% of S. intermedius strains were resistant to erythromycin and clindamycin, respectively. This observation is consistent with the observed continuous increase in macrolide resistance amongst S. intermedius isolates as described by Cox et al. (1984) and Kruse et al. (1996). On the other hand, this fact may be explained because the use of these drugs in canine dermatology has increased steadily over the last years. The frequency of neomycin resistance (41%), was higher than in other studies (Kruse et al., 1996). This finding may be explained by the possibility of correlation between macrolide resistance and resis-
tance to neomycin described by Boerlin et al. (2001). The most active antimicrobial agents against staphylococci isolated from dogs was amoxicillin/clavulanic acid, cephalexin and gentamicin. The resistance patterns of the CoNS were similar to S. intermedius strains but the small numbers of CoNS species precluded making meaningful comparison with S. intermedius. However, it is noteworthy to mention, that one strain of S. saprophyticus was resistant to the greatest number of antibiotics tested. No correlation has been observed between antibiotic resistance and occurrence of plasmids. In investigations where plasmid profiling has been performed, a striking variation in plasmid patterns has also been detected (Greene and Schwarz, 1992; Wegener and Pedersen, 1992). Resistant strains may be plasmid-less, indicating that resistance may also be chromosomally-mediated (Greene and Schwarz, 1992; Wegener and Pedersen, 1992; Schwarz et al., 1995). This can be explained by the observation of either self-movable or movable genetic elements occurring in staphylococci (Pellerin et al., 1998). Resistance in S. intermedius from dogs may be plasmidmediated, but is most frequently chromosomally-located (Greene and Schwarz, 1992). A mechanism probably exists in this staphylococcal species which encourages chromosomal acquisition of genes, or which discourages plasmids. Dog strains commonly carry single copies of the IS257 insertion element. The genes coding for penicillinases are located in plasmids or transposable elements that can be located in bacteriophages or in the chromosome (Pellerin et al., 1998). On the other hand, resistance to penicillin in staphylococci of animal origin could not be related to plasmids in any of strains tested (Wegener
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and Schwarz, 1993). The genes which confer erythromycin-resistance in dog staphylococci are almost entirely ermB, and for the first described by Boerlin et al. (2001), that can be physically linked to Tn5405-like elements known as resistance determinants for streptomycin, neomycin and kanamycin. The absolute correlation between macrolide resistance and resistance to neomycin observed by these authors was partially confirmed in our study; five of seven S. intermedius strains were resistant to both drugs (Table 1). Digestion of genomic DNA with SmaI revealed the presence of 16 different patterns among the 17 S. intermedius isolates. According to criteria proposed by Ternover et al. (1995), two isolates were indistinguishable by their PFGE patterns, and two showed the PFGE patterns D1 and D2, differed from one another by only two bands. These two last isolates are genetically related and could be exchanged between dogs. At present, genomic DNA fingerprinting by PFGE appears to be an effective technique for discriminating canine S. intermedius strains and may provide detailed information for epidemiological investigations of this species (Hesselbarth et al., 1994; Shimizu et al., 1996; Wakita et al., 2002). Moreover, the PFGE is also useful for discriminating S. intermedius implicated in food-related disease from strains of veterinary origin (Khambaty et al., 1994). The high genetic polymorphism of S. intermedius detected in PFGE studies were also confirmed in our study, even the isolates were collected in a restricted area over a short time. We expected that the diversity of our strains would be much lower than that of the population of S. intermedius collected over a few years in diverse areas. These results might indicate that genotypically unrelated S. intermedius isolates spread within and beyond populations of dogs and obviously could not also be exchanged between dogs, with the exception of two cases of canine dermatitis (strains no. 5 and 101). In conclusion, our work highlights the emergence of CoNS-determined canine dermatitis and shows that the population of canine S. intermedius from Poland is biochemically homogenous, but indicate considerable genetic diversity within this species. The many different resistance patterns observed confirm other studies, reporting a great variety of resistance patterns in staphylococci depending on the country of its isolation. References Bes, M., Slim, L.S., Becharnia, F., Meugnier, H., Vandenesch, F., Etienne, J., Freney, J., 2002. Population diversity of Staphylococcus intermedius isolates from various host species: typing by 16S-23S intergenic ribosomal DNA spacer polymorphism analysis. Journal of Clinical Microbiology 40, 2275–2277. Boerlin, P., Burnens, A.P., Frey, J., Kuhnert, P., Nicolet, J., 2001. Molecular epidemiology and genetic linkage of macrolide and aminoglycoside resistance in Staphylococcus intermedius of canine origin. Veterinary Microbiology 79, 155–169.
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