ABSTRACT:

The aim of this study was to determine the prevalence and diversity of veterinary clinical isolates ofStaphylococcus and analyze their antimicrobial susceptibility. One hundredStaphylococcus spp. clinical isolates from domestic and wild animals were subjected to partial sequencing of the 16S rRNA gene to species determination. Antimicrobial susceptibility was obtained by a disk diffusion test against six antibiotics: amoxicillin (AMX), cephalexin (LEX), ciprofloxacin (CIP), erythromycin (ERY), gentamicin (GEN) and trimethoprim-sulfamethoxazole (SXT). The most common specie wasS. pseudintermedius (61%, 61/100) and resistance to ERY (57%, 57/100), SXT (50%, 50/100) and AMX (46%, 46/100) was detected most frequently. In total, 40% (40/100) of Staphylococcus spp. exhibited a multidrug-resistant (MDR) phenotype. Results of this study emphasize that animals are reservoir of MDRStaphylococcus spp.

Key words:
Staphylococcus spp.; antimicrobial resistance; multidrug resistance.

RESUMO:

O objetivo do presente trabalho foi determinar a prevalência e diversidade de isolados clínicos veterinários deStaphylococcus e analisar o perfil de suscetibilidade a antimicrobianos. Um total de 100 Staphylococcus spp. isolados de amostras clínicas de animais domésticos e silvestres foram submetidos ao sequenciamento parcial do gene 16S rRNA, para determinação da espécie. A suscetibilidade antimicrobiana foi obtida por meio da técnica de Disco Difusão contra seis antibióticos: amoxicilina, cefalexina, ciprofloxacina, eritromicina, gentamicina e sulfazotrim. A espécie mais frequente foi S. pseudintermedius (61%, 61/100) e a resistência à eritromicina (57%, 57/100), Sulfazotrim (50%, 50/100) e Amoxicilina (46%, 46/100) foi detectada mais frequentemente. No total, 40% (40/100) dos Staphylococcus spp. demonstraram um fenótipo de multirresistência a drogas (MRD). Os resultados obtidos neste trabalho reforçam o fato de que animais são reservatórios de Staphylococcus spp. MRD.

Palavras-chave:
Staphylococcus spp.; resistência a antimicrobianos; multirresistência a drogas.

Multidrug-resistant Staphylococcus spp. isolates from humans and animals have been reported in recent decades, and these pose a challenge not only in human medicine but also in veterinary medicine. GÓMEZ-SANZ et al. (2013GÓMEZ-SANZ, E. et al. Animal and human Staphylococcus aureus associated clonal lineages and high rate of Staphylococcus pseudintermedius novel lineages in Spanish kennel dogs: predominance of S. aureus ST398. Vet Microbiol, v.166, n.3-4, p.580-589, 2013. Available from: http://dx.doi.org/10.1016/j.vetmic.2013.07.014>. Accessed: Nov. 2013.
http://dx.doi.org/10.1016/j.vetmic.2013.... ) showed the presence of traditionally human strains of S. aureus in dogs, which reflect the capacity of these strains to adapt to different hosts. The increasing evidence of S. aureus and S. pseudintermedius that are resistant to many drugs, particularly oxacillin, is a serious problem in treatment and control of staphylococcal infections (DURAN et al., 2012DURAN, N. et al. Antibiotic resistance genes & susceptibility patterns in staphylococci. Indian J Med Res, v.135, p.389-396, 2012. Available from: http://www.ncbi.nlm.nih.gov/pubmed/22561627>. Accessed: Jan. 2014.
http://www.ncbi.nlm.nih.gov/pubmed/22561... ).

Studies of the antimicrobial resistance profiles of Staphylococcus spp. are very useful and can provide data that assist with strategies for avoiding the dispersion of these multidrug-resistant (MDR) microorganisms. In this context, the aims of this study were (1) to determine the prevalence and diversity of clinical isolates of Staphylococcus from domestic and wild animals and (2) to analyze their antimicrobial susceptibility.

The study was performed in the Veterinary Microbiology Laboratory at Universidade Federal de Mato Grosso (UFMT), between 2012 and 2013. One hundred isolates of Staphylococcus were obtained from swabs and biopsies of lesions at different sites (abscess, cornea, ear, fluids, fracture, lung, lymph node, oral mucosa, nail, rectum, skin, and urine) of domestic and wild animals (Cockatiel, Crab-eating fox, Crab-eating raccoon, Hoary fox, Maned wolf and Rabbit). Samples were inoculated into eight per cent sheep blood agar plates (Sigma-Aldrich) according QUINN et al. (1994QUINN, P.J. et al. Clinical veterinary microbiology. Spain:Wolfe, 1994. Chapt.8, p.118-126. ), and incubated at 37°C for 48h. Identification of staphylococci was on the basis of colony morphology, Gram staining and biochemical tests such catalase test (QUINN et al., 1994QUINN, P.J. et al. Clinical veterinary microbiology. Spain:Wolfe, 1994. Chapt.8, p.118-126. ). DNA extraction was performed using the phenol-chloroform extraction method (SAMBROOK & RUSSELL, 2001SAMBROOK, J.; RUSSELL, D. Molecular cloning: a laboratory manual. 3.ed. New York: Cold spring Habor Laboratory, 2001. V.3.).

Species were identified using 16S rRNA partial sequence analysis, according to LANE (1991LANE, D.J. 16S/23S rRNA sequencing. In: STACKEBRANDT, E.; GOODFELLOW, M. (Ed.). Nucleic acid techniques in bacterial systematics. New York: John Wiley & Sons, 1991. p.115-175.). Sequences obtained by ABI 3500 Genetic Analyzer (Applied Biosystems) were compared to the sequences available in GenBank using BLAST <http://www.ncbi.nlm.nih.gov/blast/Blast.cgi>. Positive control was an American Type Culture Collection (ATCC) S. aureus strain 25923 and ultrapure water was negative control.

Antimicrobial susceptibility tests were performed using disk diffusion on Mueller-Hinton agar. All procedures and interpretation of antibiotic susceptibility were followed as previously described by the Clinical and Laboratory Standards Institute (CLSI) documents M100-S22 (CLSI, 2012CLSI. Performance standards for antimicrobial disk and dilution susceptibility tests for bacteria isolated from animals; approved standard. 4.ed. Wayne, PA: Clinical and Laboratory Standards Institute, 2013. (CLSI document VET01-A4).) and VET01-A4 (CLSI, 2013CLSI. Performance standards for antimicrobial susceptibility testing; twenty-second informational supplement. Wayne, PA: Clinical and Laboratory Standards Institute , 2012. (CLSI document M100-S22).). The following antibiotic disks (Cefar Diagnóstica Ltda) were tested: gentamicin (10μg, GEN), cephalexin (30μg, LEX), erythromycin (15μg, ERY), ciprofloxacin (5μg, CIP), amoxicillin (10μg, AMX) and trimethoprim-sulfamethoxazole (25μg, SXT). Isolates resistant to three or more antimicrobial classes were classified as MDR (MAGIORAKOS et al., 2012MAGIORAKOS, A.P. et al. Multidrug-resistant, extensively drug-resistant and pandrug-resistant bacteria: an international expert proposal for interim standard definitions for acquired resistance. Clin Microbiol Infect, v.18, n.3, p.268-2681, 2012. Available from: http://www.ncbi.nlm.nih.gov/pubmed/21793988>. Accessed: Sept. 2014. doi: 10.1111/j.1469-0691.2011.03570.x.
http://www.ncbi.nlm.nih.gov/pubmed/21793... ).

Association between phenotypic resistance, host characteristics (species and site of infection) and Staphylococcus species were evaluated by the ϰ² test and Fisher's exact test, using the software R (R-3.0.2). Differences were considered significant when P<0.05.

Coagulase positive Staphylococcus species such as S. pseudintermedius (61%, 61/100), S. schleiferi (15%, 15/100), S. aureus (5%, 5/100), and S. delphini (3%, 3/100) were more commonly detected than coagulase negative species (Table 1). Similar results were reported by DETWILER et al. (2013DETWILER, A. et al. Multi-drug and methicillin resistance of staphylococci from canine patients at a veterinary teaching hospital (2006-2011). Vet Q, v.33, n.2, p.60-67, 2013. Available from: http://www.ncbi.nlm.nih.gov/pubmed/23721478>. Accessed: Apr. 2014. doi: 10.1080/01652176.2013.799792.
http://www.ncbi.nlm.nih.gov/pubmed/23721... ).

Isolates were more frequently associated to canine samples (78%, 78/100) followed by wild animals (9%, 9/100) and cats (8%, 8/100). S. pseudintermedius (68%, 53/78) and S. schleiferi (18%, 14/78) were associated mainly with canine samples. Other species like S. cohnii , S. delphini ,S. arlettae andS. pasteuri that occasionally or rarely were associated to disease in animals were detected from lesions.

S. sciuri (3%, 3/100) were isolated only in wild animals (Cerdocyon thous ,Lycalopex vetulus ,Chrysocyon brachyurus ). This species was associated to mastitis in dairy cattle (RAHMAN et al., 2005RAHMAN, M.T. et al. Genetic analysis of mec A homologues in Staphylococcus sciuri strains derived from mastitis in dairy cattle. Microb Drug Resist, v.11, n.3, p.205-214, 2005. Available from: http://www.ncbi.nlm.nih.gov/pubmed/16201922>. Accessed: Sept. 2015. doi: 10.1089/mdr.2005.11.205.
http://www.ncbi.nlm.nih.gov/pubmed/16201... ) and have been reported in wild animals, such as rodents and insectivores (HAUSCHILD & SCHWARZ, 2003HAUSCHILD, T.; SCHWARZ, S. Differentiation of Staphylococcus sciuri strains isolated from free-living rodents and insectivores. J Vet Med B Infect Dis Vet Public Health, v.50, n.5, p.241-246, 2003. Available from: <http://www.ncbi.nlm.nih.gov/pubmed/12864900>. Accessed: Sept. 2015. doi: 10.1046/j.1439-0450.2003.00662.x.
https://doi.org/10.1046/j.1439-0450.2003... ), but not in wild canidae.

Drug resistance frequencies were described in figure 1. ERY (57%, 57/100), SXT (50%, 50/100) and AMX (46%, 46/100) were more frequent resistant and LEX (12%, 12/100) was less frequent. In a study performed in Tunisia using S. pseudintermedius isolates from healthy dogs, only 1.8% of isolates were erythromycin-resistant (GHARSA et al., 2013GHARSA, H. et al. Antimicrobial resistance, virulence genes, and genetic lineages of Staphylococcus pseudintermedius in healthy dogs in tunisia. Microb Ecol, v.66, n.2, p.363-368, 2013. Available from: http://www.ncbi.nlm.nih.gov/pubmed/23686400>. Accessed: Apr. 2014. doi: 10.1007/s00248-013-0243-y.
http://www.ncbi.nlm.nih.gov/pubmed/23686... ); however, DÉGI et al. (2013DÉGI, J. et al. Frequency of isolation and antibiotic resistance of staphylococcal flora from external otitis of dogs. Vet Rec, v.173, n.2, p.42, 2013. Available from: http://www.ncbi.nl-m.nih.gov/pubmed/23740316>. Accessed: Apr. 2014. doi: 10.1136/vr.101426.
http://www.ncbi.nl-m.nih.gov/pubmed/2374... ) studied otitis isolates from dogs in Romania and reported that 61.3% of isolates were resistant to this drug and YOON et al. (2010YOON, J.W. et al. Antibiotic resistance profiles of Staphylococcus pseudintermedius isolates from canine patients in Korea. J Microbiol Biotechnol, v.20, n.12, p.1764-1768, 2010. Available from: http://www.ncbi.nlm.nih.gov/pubmed/21193835>. Accessed: Apr. 2014.
http://www.ncbi.nlm.nih.gov/pubmed/21193... ) in Korea observed 60.8% of resistance.

Figure 1
Antimicrobial susceptibility profiles of Staphylococcus isolates from animals. Breakpoints used for interpretation of each antimicrobial resistance were a zone of inhibition of: ≤19mm (AMX); ≤15mm (CIP); ≤13mm (ERY); ≤12mm (GEN); ≤14mm (LEX); ≤10mm (SXT), according to CLSI documents M100-S22 (CLSI, 2012CLSI. Performance standards for antimicrobial susceptibility testing; twenty-second informational supplement. Wayne, PA: Clinical and Laboratory Standards Institute , 2012. (CLSI document M100-S22).) and VET01-A4 (CLSI, 2013CLSI. Performance standards for antimicrobial disk and dilution susceptibility tests for bacteria isolated from animals; approved standard. 4.ed. Wayne, PA: Clinical and Laboratory Standards Institute, 2013. (CLSI document VET01-A4).).

Forty isolates exhibited an MDR phenotype (Table 1) and the most common associations were AMX-ERY-SXT (17,5%, 7/40), AMX-CIP-ERY-GEN-SXT (17,5%, 7/40) and AMX-CIP-ERY-SXT (15%, 6/40). This result was higher than the result reported by GHARSA et al. (2013GHARSA, H. et al. Antimicrobial resistance, virulence genes, and genetic lineages of Staphylococcus pseudintermedius in healthy dogs in tunisia. Microb Ecol, v.66, n.2, p.363-368, 2013. Available from: http://www.ncbi.nlm.nih.gov/pubmed/23686400>. Accessed: Apr. 2014. doi: 10.1007/s00248-013-0243-y.
http://www.ncbi.nlm.nih.gov/pubmed/23686... ) (18%) but lower than the result reported by BARDIAU et al. (2013BARDIAU, M. et al. Characterization of methicillin-resistant Staphylococcus pseudintermedius isolated from dogs and cats. Microbiol Immunol, v.57, n.7, p.496-501, 2013. Available from: http://www.ncbi.nlm.nih.gov/pubmed/23607810>. Accessed: Apr. 2014. doi: 10.1111/1348-0421.12059.
http://www.ncbi.nlm.nih.gov/pubmed/23607... ) in isolates from animals in Japan (100%) and by YOUN et al. (2011YOUN, J.H. et al. Prevalence and antimicrogram of Staphylococcus intermedius group isolates from veterinary staff, companion animals, and the environment in veterinary hospitals in Korea. J Vet Diagn Invest, v.23, n.2, p.268-274, 2011. Available from: http://www.ncbi.nl-m.nih.gov/pubmed/21398446>. Accessed: Apr. 2014. doi: 10.4142/jvs.2011.12.3.221.
http://www.ncbi.nl-m.nih.gov/pubmed/2139... ) in Korea (71.9%). In a study of 103 MRSP (methicillin-resistant Staphylococcus pseudintermedius ) isolates from dogs from several countries in Europe, USA and Canada, resistance to multiple antimicrobials routinely used in pets was observed (PERRETEN et al., 2010PERRETEN, V. et al. Clonal spread of methicillin-resistant Staphylococcus pseudintermedius in Europe and North America: an international multicentre study. J Antimicrob Chemother, v.65, p.1145-1154, 2010. doi: 10.1093/jac/dkq078.
https://doi.org/10.1093/jac/dkq078... ).

Emergence of Staphylococcus spp. multidrug resistance, especially oxacillin-resistant, can complicate treatment of infections in animals such as otitis, dermatitis, urinary tract infections, leading to recurrent disease. Furthermore, the transmissions of these strains from animals to humans and from humans to animals are potential risks that have to be considered. Transmission of S. pseudintermedius between dogs and their owners was recently reported (GÓMEZ-SANZ et al., 2011GÓMEZ-SANZ, E. et al. Detection and characterization of methicillin-resistant Staphylococcus pseudintermedius in healthy dogs in La Rioja, Spain. Comp Immunol Microbiol Infect Dis, v.34, n.5, p.447-453, 2011. Available from: http://www.sciencedirect.com/science/article/pii/S0147957111000592>. Accessed: May 2014. doi: 10.1016/j.cimid.2011.08.002.
http://www.sciencedirect.com/science/art... ). No significant association was observed (P>0.05) between Staphylococcus species, host type and resistance profile.

Identification of staphylococci species by conventional methods requires a minimum of two days period or more, can be influenced by different variables and different species with similar phenotypic features. For these reasons, molecular methods, like partial sequence analysis of 16S rRNA gene, have been used to confirm the results obtained in the phenotypic tests (FORSMAN et al., 1997FORSMAN, P. et al. Identification of staphylococcal and streptococcal causes of bovine mastitis using 16S-23S rRNA spacer regions. Microbiology, v.143, p.3491-500, 1997. Available from: http://www.ncbi.nlm.nih.gov/pubmed/9387227>. Accessed: May 2016. doi: 10.1099/00221287-143-11-3491.
http://www.ncbi.nlm.nih.gov/pubmed/93872... ). Regarding the antimicrobial susceptibility tests, the disk diffusion is a simple and practical method used in many veterinary diagnostic laboratories; however, the results obtained are only qualitative. Broth microdilution is a standard reference method (Minimal Inhibitory Concentration-MIC) that can be used to confirm and measure quantitatively the in vitro activity of an antimicrobial agent against bacterium isolates (CLSI, 2013CLSI. Performance standards for antimicrobial disk and dilution susceptibility tests for bacteria isolated from animals; approved standard. 4.ed. Wayne, PA: Clinical and Laboratory Standards Institute, 2013. (CLSI document VET01-A4)., VET01-A4).

In conclusion, this study emphasize that animals are reservoir of MDR Staphylococcus spp. and may become an important source of contamination of these microorganisms to both humans and other animals. Thus, monitoring and surveillance programs related to dispersion of these microorganisms are needed.

ACKNOWLEDGEMENTS

We are grateful to Programa de Apoio a Núcleos de Excelência (Pronex)/ Fundação de Amparo à Pesquisa do Estado de Mato Grosso (FAPEMAT), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) for support.

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