Main topic : Antimicrobial resistance: A worldwide concern
Detection by multiplex PCR of resistance to methicillin and production of biofilm in strains of Staphylococcus aures and Coagulase negative Staphylococcus isolated of bovine mastitis
PEREZ-GERARDO M. 1, HERNÁNDEZ ANDRADE L. 2, MENDOZA S. 3, CIPRIAN-CARRASCO A. 3, SANTILLAN-FLORES M. 2, PARRA-LACA R. 4
1 Private Professional, Mexico, Mexico; 2 INIFAP, Ciudad de México, Mexico; 3 FES Cuautitlan UNAM, Edo. de Mexico, Mexico; 4 Simon Bolivar University, Ciudad de México, Mexico
Biofilm production may be mediated by the ica operon which includes the icaA, icaB, icaC and icaD genes. The icaA and icaD genes together regulate the phenotypic expression of the biofilm forming polysaccharide (3). Treatment of bovine mastitis by Staphylococcus aureus is complicated due to resistance to ß-lactams, persistence of infection in the mammary gland due to the production of biofilm which protects microorganisms from the action of antimicrobials.
The objective of the work was to designs a multiplex PCR by means of which the biofilm production capacity, intrinsic resistance to methicillin and the expression of resistance to methicillin in Staphylococcus spp. Isolates from bovine mastitis cases could be detected from milk samples of cows with mastitis from Coahuila, Hidalgo and Queretaro, Mexico. Staphylococcus were isolated, indentified by API staph. A multiplex PCR was standardized to detect the femA, mecA and icaD genes (1). DNA extraction was performed from colonies using CTAB, standardized to 20 ng/µl. The strains of Staphylococcus aureus ATCC 976, 977, 1026, 29213 and S. aureus Cowan were used as positive control. The PCR products were evaluated by electrophoresis in a 1.5% agarose gel using ethidium bromide, the electroforesis was run for 90 min at 80 V. Finally the gel was visualized in a photodocumenter with UV light.
54 Staphylococcus were isolated, of which 22. Staphylococcus aureus were identified, of these only 9% presented the mecA gene, however 81% presented the femA gene indicating the ability to express resistance to methicillin and 27.2% were posistive for the gene icaD showing the ability to produce biofilm through the mechanism regulated by the ica operon. In the case of coagulase negative Staphylococcus 9% of the strains presented the mecA gene, 28% the femA gene, and 6% presented the icaD gene (two strains one of Staphylococcus intermedius, and one of Staphylococcus sciuri).
The femA gene was not found exlusively in Staphylococcus aureus strains, it was also found in coagulase negative Staphylococcus. The porcentage of strains positive for the mecA gene is low compared to that reported in strains of human origin, however they are similar to that reported in strains from milk. In a study carried out by Lopez (2) were isolated from bovine milk 4 strains (9%) presented the mecA gene. In this study the mecA gene is found in a smaller proportion that the femA gene. Multiplex PCR can be used for a diagnosis of biofilm production and resistance to oxacillin and methicillin for boths Staphylococcus aureus and Staphylococcus coagulase negative.