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Antimicrobial resistance in staphylococci isolated from canine pyoderma/Resistencia aos antimicrobianos em estafilococos isolados de piodermites canina.

Introduction

Pyoderma is a common disease in domestic animals, especially dogs. The primary skin pathogens of dogs are the staphylococci species, of which the coagulase-positive specie Staphylococcus pseudintermedius, previously referred as S. intermedius (Devriese et al., 2009), is most frequently isolated (Kruse et al., 1996). S. pseudintermedius is present in the healthy dog's skin and besides pyoderma, can also cause urinary infection, corneal ulcer, otitis externa, otitis media and endocarditis (Penna et al., 2010; Pereira et al., 2009; Oliveira et al., 2006; Prado et al., 2006; Smith et al., 2000). Moreover, the zoonotic transmission of S. pseudintermedius to humans has been demonstrated (Pottumarthy et al., 2004).

The intensive use of antimicrobial agents has promoted an increase in the number of multi-drug resistant staphylococci strains from animal sources (Werckenthin et al., 2001). The drug resistance mechanisms present in S. pseudintermedius are similar to those found in other staphylococci species. Resistance to chloramphenicol, for example, involves the production of the enzyme chloramphenicol acetyl transferase encoded by the cat gene located in plasmid pSCS1, which has a high similarity with the S. aureus cat gene (Schwarz, 1995). Other plasmids carrying genes for resistance to chloramphenicol, macrolides, lincosamides and tetracyclines have been isolated from S. pseudintermedius and have shown structural similarities to plasmids isolated from S. aureus of human origin (Greene & Schwarz, 1992).

It is known that the erythromycin resistance in S. pseudintermedius, S. xylosus and S. hyicus is mediated by ribosomal methylase encoded by ermB gene (Eady et al., 1993). Multi-drug resistance mediated by mecA gene was also demonstrated, which encodes for an altered penicillin-binding protein (PBP2a) with low affinity to beta-lactam antibiotics in S. pseudintermedius, S. aureus and coagulase negative staphylococci isolated from dogs (Rota et al., 2011; Cohn & Middleton, 2010; Bemis et al., 2009; Coelho et al., 2007).

Antibiotic susceptibility testing allows veterinarians to monitor the susceptibility of common bacteria to antimicrobials. However, many cases of staphylococcal infections are treated empirically, thus contributing to resistant strains selection. Moreover, susceptibility studies can help to guide veterinarians in choosing the most effective antibiotics in a particular geographic region. The purpose of this study was to investigate the antimicrobial susceptibility pattern of staphylococci isolated from pyoderma canine.

Material and Methods

Samples from 34 adult dogs of both sexes were collected with sterile cotton swabs from the skin lesions at a private veterinary clinic in Natal City, Brazil. Swabs were streaked onto blood agar (Biobras, Brazil) supplemented with 5.0% sterile ovine blood and agar plates were incubated overnight at 37[degrees]C. Typical colonies were picked and bacteria were identified by standard laboratory procedures. The identification of the strains as S. pseudintermedius was based on the following characteristics: slide and tube coagulase, anaerobic utilization of mannitol and lack of acid production from D-mannitol, acid production from trehalose and sensitivity to polimixin B. Coagulase negative staphylococci were identified by API Staph identification system (BioMrieux, France) according to the manufacturer's recommendations.

The susceptibilities of the isolates to 16 antibiotics (Cecon, Brazil) commonly used in veterinary and human medicine (penicillin G 10 [micro]g, ampicillin 10 [micro]g, oxacillin 1 [micro]g, vancomycin 30 [micro]g, cephalothin 30 [micro]g, imipenem 10 [micro]g, erythromycin 15 [micro]g, clindamycin 2 [micro]g, lincomycin 2 [micro]g, streptomycin 10 [micro]g, tobramycin 10 [micro]g, amikacin 30 [micro]g, gentamicin 10 [micro]g, tetracycline 30 [micro]g, chloramphenicol 30 [micro]g and rifampicin 30 [micro]g) were performed by the disk diffusion method observing the recommendation of the Clinical and Laboratory Standards Institute (CLSI, 2007). The standard strain S. aureus ATCC 25923 was used as control in all tests.

Results and Discussion

In all pyoderma canine cases studied, it was possible to isolate staphylococci strains. The distribution percentage of isolated species is presented in Table 1. S. pseudintermedius was the predominant species, what agrees with other researchers (Scott et al., 2006; Pedersen & Wegener, 1995). Coagulase-negative staphylococci (S. chromogenes, S. epidermidis, S. haemolyticus, S. warneri and S. sciuri) were isolated with lower frequency, which is in agreement with other studies that have isolated these species from canine pyoderma (Penna et al., 2009; Hauschild & Wojcik, 2007; Medleau et al., 1986). Coagulase-negative staphylococci species are common inhabitants of the skyn endogenous microflora of healthy dogs and can act as opportunistic pathogens (Malik et al., 2005).

The S. pseudintermedius strains showed a significant percentage of resistance to lincomycin, penicillin G, ampicillin and tetracycline (Table 2). Less significant levels of resistance were observed for streptomycin, erythromycin, rifampin, clindamycin and oxacillin. The resistance to lincomycin, penicillin G and ampicillin was also prevalent among coagulase-negative strains. The S. warneri strain has shown susceptibility to all antimicrobial tested.

Resistance to lincomycin, penicillins and tetracycline is often reported in staphylococci isolated from dogs, what is probably related to the frequent use of these antibiotics in veterinary practice, including the treatment of pyoderma in dogs (Hoekstra & Paulton, 1996; Harvey et al., 1993). Penna et al. (2009) have found that a percentage of 71.8% of strains isolated from canine pyoderma were resistant to penicillin.

At the present study, the aminoglycosides gentamicin, tobramycin and amikacin were effective in inhibiting all isolated strains. The streptomycin was the only antibiotic in this class to which the occurrence of resistant strains has been verified. Gentamicin and other aminoglycoside antibiotics have been reported as highly effective against staphylococcal infections in dogs (Oliveira et al., 2006; Oliveira et al., 2005). Since aminoglycosides can induce nephrotoxicity, the low percentage of resistance observed for the antibiotics of this class, probably, is related to the limited use of these drugs in the treatment of canine pyoderma (Noli & Morris, 2011). However, high levels of resistance were observed in aminoglycosides staphylococci isolated from canine pyoderma (Penna et al., 2009). These discrepant results may be related to the local habits of antibiotic therapy.

Regarding the resistance patterns (Table 3), it was found that 50.0% (17/34) of strains have shown resistance to one or two classes of antibiotics and 38.23% (13/34) were multi-drug resistant (have shown resistance to three or more classes of antibiotics). A similar percentage of multi-drug resistant strains from canine pyoderma was obtained by Ganiere et al. (2005). However, a more recent study has shown a higher rate (94.9%) of multi-drug resistant strains isolated from canine pyoderma (Penna et al., 2009).

The multi-drug resistant strains emergence is a phenomenon that is occurring worldwide and that has hindered the treatment of human and animal staphylococcal infections. Different mechanisms, such as efflux pumps capable of causing the extrusion of several types of antibiotics out of the bacterial cell, encoded by genes located on plasmids or the acquisition of mobile genetic elements, such as the SCCmec, which contains additional insertion sequences that allow the incorporation of several resistance genes, contribute to the acquisition of multi-drug resistance phenotype (Cohn & Middleton, 2010). The antibiotic therapy not careful and empirical contributes to rising the prevalence of multi-drug resistant strains, since the intensive use of antibiotics acts by exerting a selective pressure on the resistant strains, which can progressively acquire new resistance genes (Morar & Wright, 2010). Since 2010, the Brazilian government has started controlling the antibiotics sale, requiring the prescriptions retention by pharmacies, an important measure which has inhibited the self-medication practice (Brazil, 2010). The results obtained in this study indicate the necessity of implementing more effective control measures to reduce the use of antibiotics in veterinary practice.

Successful treatment of canine pyoderma depends on the proper choice of antimicrobial agent for which the etiologic agent presents susceptibility. This study has found that all S. pseudintermedius isolated were susceptible to amikacin, tobramycin, gentamicin, cephalothin, vancomycin, imipenem and chloramphenicol. oxacillin, clindamycin and rifampin have also shown high efficacy against these species. Coagulase negative strains, as well as susceptibility to antibiotics previously mentioned, were also susceptible to tetracycline and streptomycin.

Conclusions

The results indicate the occurrence of resistant staphylococci on canine pyoderma and that canine host may contribute to the maintenance and dissemination of drug resistant staphylococci in our midst. They also warn about the need of a careful selection of antibiotics based on results of susceptibility testing, to reduce the selection of multi-drug resistant staphylococci strains.

Received: 31 May 2011

Accepted: 23 January 2012

Acknowledgements

The authors are grateful to Dr. Euclides Leandro de Castro, owner of Prontocan Veterinary Clinic for providing clinical material and to M.Sc. Leonardo Souza Carvalho for his helpful suggestions and his revision of the English version of this manuscript.

References

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Luciana Fernandes de Albuquerque Lima (1), Andrea Cysneiros Lira (1), Henrique Douglas Melo Coutinho (2), Jos Pinto de Siqueira Junior (3), Humberto Medeiros Barreto (4) *

(1) Department of Health Sciences and Biology, University Potiguar, Natal, RN, Brazil

(2) Departament of Biological Sciences, Biology Sciences and Health Center, Regional University of Cariri, Crato, CE, Brazil

(3) Departament of Molecular Biology, Exact Sciences and Nature Center, Federal University of Paraiba, Joao Pessoa, PB, Brazil

(4) Campus Amilcar Ferreira Sobral, Federal University of Piaui, Floriano, PI, Brazil

* Corresponding author, email: hmbarreto@ufpi.edu.br
Table 1. Distribution percentage of staphylococci
isolates obtained from canine pyoderma.

Isolation                         N      %

Staphylococcus pseudintermedius   28   82.4
Staphylococcus chromogenes        2     6.0
Staphylococcus epidermidis        1     2.9
Staphylococcus warneri            1     2.9
Staphylococcus haemolyticus       1     2.9
Staphylococcus sciuri             1     2.9
Total                             34   100.0

Table 2. Antimicrobial susceptibility rate of staphylococci
isolated from canine pyoderma *.

Antimicrobial   S. pseudintermedius   S. chromogenes  S. epidermidis
                    (n = 28) %          (n = 2) %       (n = 1) %

Oxacillin              96.4               100.0           100.0
Rifampicin             92.9               100.0           100.0
Clindamycin            85.7               100.0           100.0
Erythromycin           78.6               100.0           100.0
Streptomycin           71.4               100.0           100.0
Tetracicline           53.6               100.0           100.0
Penicillin G           50.0               50.0            100.0
Ampicillin             46.4               50.0            100.0
Lincomycin             42.9               50.0             --

Antimicrobial   S. sciuri   S. haemolylicus
                (n = 1) %      (n = 1) %

Oxacillin         100.0          100.0
Rifampicin        100.0          100.0
Clindamycin       100.0          100.0
Erythromycin      100.0           --
Streptomycin      100.0          100.0
Tetracicline      100.0          100.0
Penicillin G       --             --
Ampicillin         --             --
Lincomycin         --             --

* The S. warneri strain was susceptible to all antimicrobial tested.
(--) No susceptible strain recovery.

Table 3. Resistance pattern of staphylococci resistant strains
isolated from canine pyoderma.

Staphylococci isolates   Sensitive   Non multi-drug resistant
                           n (%)              n (%)

S. pseudintermedius      3 (10.7)           13 (46.4)
S.chromogenes             0 (0.0)           2 (100.0)
S. epidermidis            0 (0.0)           1 (100.0)
S. warneri               1 (100.0)           0 (0.0)
S. haemolyticus           0 (0.0)            0 (0.0)
S. sciuri                 0 (0.0)           1 (100.0)
Total                    4 (11.8)           17 (50.0)

Staphylococci isolates   Multi-drug resistant *     Total
                                 n (%)              n (%)

S. pseudintermedius            12 (42.9)          28 (82.4)
S.chromogenes                   0 (0.0)            2 (6.0)
S. epidermidis                  0 (0.0)            1 (2.9)
S. warneri                      0 (0.0)            1 (2.9)
S. haemolyticus                1 (100.0)           1 (2.9)
S. sciuri                       0 (0.0)            1 (2.9)
Total                          13 (38.2)          34 (100.0)

* Resistant to three or more class of antibiotics.
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Title Annotation:Original Article
Author:Lima, Luciana Fernandes de Albuquerque; Lira, Andrea Cysneiros; Coutinho, Henrique Douglas Melo; de
Publication:Comunicata Scientiae
Date:Sep 1, 2012
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