1 Detection of the mecA gene in Staphylococcus sp . isolated from dog skin with atopic dermatitis and recurrent pyoderma

PALAVRAS-CHAVE DAC Gene mecA Staphylococcus PCR ABSTRACT: Canine atopic dermatitis (CAD) is a multifactorial, allergic and inflammatory skin disorder with a genetic predisposition manifested by clinical signs, and it is generally associated with the production of specific IgE antibodies, mainly against environmental allergens. The objective of this study was to report the occurrence of mecA gene in an isolate of Staphylococcus sp. from a dog with canine atopic dermatitis and recurrent pyoderma. Clinical specimen was collected for culture and antimicrobial susceptibility analysis. The isolate of Staphylococcus was then submitted to PCR for the detection of the mecA gene. The isolation of Staphylococcus sp. from this patient’s skin is worrisome, since the presence of such multi-resistant bacterium in such circumstance is a potentially grave risk for public health.

ABSTRACT: Canine atopic dermatitis (CAD) is a multifactorial, allergic and inflammatory skin disorder with a genetic predisposition manifested by clinical signs, and it is generally associated with the production of specific IgE antibodies, mainly against environmental allergens.The objective of this study was to report the occurrence of mecA gene in an isolate of Staphylococcus sp. from a dog with canine atopic dermatitis and recurrent pyoderma.Clinical specimen was collected for culture and antimicrobial susceptibility analysis.The isolate of Staphylococcus was then submitted to PCR for the detection of the mecA gene.The isolation of Staphylococcus sp. from this patient's skin is worrisome, since the presence of such multi-resistant bacterium in such circumstance is a potentially grave risk for public health.Skin is the largest and most visible organ of the body.It works as an anatomical and physiological barrier between the animal and the environment.Its interactions with the internal organs will often be indicative of pathological events that are occurring in such organs and their associated tissues (Miller et al., 2013).

RESUMO:
Canine atopic dematitis (CAD) is a multifactorial, allergic and inflammatory skin disease that is associated with genetic predisposition and manifests itself with characteristic clinical signs, associated with the production of specific IgE antibodies, mainly against environmental allergens (Dethioux, 2006).
Methicillin-resistant Staphylococcus pseudointermedius (MRSP) has been described since the 1980s, and it is common in recurrent pyoderma, making it difficult to treat.The prevalence of methicillin-resistant strains of Staphylococcus has recently increased, and, in some areas of the United States of America, more than 50% of dog's skin cultures are composed of methicillin-resistant Staphylococcus (Guardabassi et al., 2010).
Staphylococcus sp. is a very frequent bacterium in nosocomial infections, and since dogs are increasingly prevalent in Brazilian homes, and CAD is being more frequently diagnosed, the recovery of this bacterial pathogen from a dog skin it is a case of extreme importance.
The objective of this study was to report the presence of the mecA gene in Staphylococcus sp.isolated from the skin of a dog with CAD and recurrent pyoderma.

Material and Methods
The paper is registered in Ethics Committee of Federal Rural University of Amazonia under protocol nº.004/2014 (CEUA) -23084.001959/ 2014-99.
A female Poodle, two years old, presented a chronic / recurrent condition, for almost a year, of intense pruritus associated with pyoderma that subsided with the use of systemic antibiotic therapy.
Erythematous, dry, scaly skin with pustular lesions and numerous epidermal collars and pruritus were observed during clinical examination.
Skin scrapings were negative for mites and fungi.Cytology revealed frequent Gram-positive cocci and Malassezia sp.Following exam results, a clinical diagnosis of CAD was determined, using the criteria of Favrot (2010).
The microbial species were identified based on colony morphology, Gram staining, pigment production and biochemical tests (Ikram, 2005).
The culture confirmed the presence of coagulase positive Staphylococcus sp., and the antimicrobial susceptibility indicated resistance to oxacillin.
The bacterium was submitted to polymerase chain reaction (PCR) to detect the mecA gene.
DNA was extracted with kit Purelink Genomic DNA Mini Kit (Invitrogen), commercial kit.
The generated amplicon was analyzed by electrophoresis on a 2% agarose gel, at a voltage of 100 v / 40min and viewed under ultraviolet (UV) light, with a 533 base pairs fragment.
Purification of amplified PCR-DNA in PCR was performed with the Quick Gel extraction kit (Promega, USA).
Purified DNA was sequenced in cycles with the ABI PRISM Dye Terminator Version 3.0 Kit, which employs the dideoxyribonucleotide chain termination method labeled with different fluorophores for each nucleotide at the 3 'end.The mixture for each sequencing reaction contained 4 μL of Terminator ready reaction mix, 50 ng PCR product, 3.5 pmol oligonucleotides used for PCR (mecA-F1 and mecA-R primers), and sufficient amount of water for one final volume of 20 μL.
Amplification was performed using a 25 cycle program, each cycle consisting of denaturation steps at 96°C for 10 seconds, primer hybridization at 52°C for 5 seconds and extension at 60°C for 4 min.Following this procedure, the sequencing product was purified using a commercial X-terminator kit (Applied Biosystems, USA).Assembly and curation of the obtained readings were made using the Geneious v.9.1.6program.The nucleotide sequences obtained from sequencing were aligned and compared against each other and against other sequences from various Staphylococcus spp.available at Genbank (http:// www.ncbi.nlm.nih.gov) using the Mafft v.7 program.

Results and Discussion
Because it is a cutaneous, multifactorial condition with allergic and inflammatory characteristics, CAD causes a lot of discomfort for the animal according to Dethioux (2006).The patient showed pruritus throughout the consultation.One of the characteristics of this disease is the loss of the integrity of the epidermal barrier, which, according to Olivry et al. (2010) is one of the key factors triggering the clinical signs.In the current study this was manifested by the patient in the form of erythema, desquamation and secondary bacterial infection.As noted by Maluta (2008), bacterial pyoderma has been widely reported as a baseline cause of such dermal reactions as allergies and endocrinopathies.Furthermore, as shown experimentally by Pianta et al. (2006), inflamed skin with pyoderma is associated with the decrease of antimicrobial peptides, which favors colonization by coagulase-positive staphylococci more rapidly than on healthy skin.
A cytology test found Gram positive cocci.This is significant as this method is regarded by Hillier (2002), as highly conclusive for the confirmation of bacterial colonization, who emphasize the use of cultures with antibiogram.
The presence of Staphylococcus sp. in the patient sample of the present report is according Pereira et al. (2009), who detected resistance in 48.6% of the isolates of Staphylococcus sp. of different infectious sites of companion animals.Tunon et al. (2008) found strains of Staphylococcus sp. in canine otitis and found 83% of strains resistant to at least one of the antibiotics used in research.In this study, the detection of the mecA gene in Staphylococcus sp. with resistance to oxacillin showed that the disc diffusion test may be an indication for the