Detection of PBP2a (Penicillin-binding protein 2a) and mecA gene in methicillin resistant Staphylococci originated from animals

Abstract

For the purpose of detecting methicillin (oxacillin) resistance in staphylococcal strains, in a number of microbiological laboratories only disc diffusion method with cefoxitin and/or oxacillin discs is used. Besides this method, it is desirable to determine MIC values for cefoxitin and/or oxacillin. After examination by disc diffusion and dilution methods, latex agglutination is used for the detection of PBP2a and PCR is used for the detection of mecA gene. Use of PCR is not possible in a large number of diagnostic laboratories and as method of choice, latex agglutination test for rapid detection of PBP2a is recommended. In this investigation, as confirmatory methods, latex agglutination and PCR were used for strains that were resistant to oxacillin and/or cefoxitin by disc diffusion and broth microdilution methods. In total, 14 strains of coagulase-negative staphylococci originating from clinical specimens of cats, dogs and chicken were examined. Among isolated strains, it was established that the dominating species was Staphylococcus haemolyticus with 11 isolated strains. Other isolated species were Staphylococcus epidermidis, Staphylococcus capitis and Staphylococcus vitulinus, each with one isolated strain. For all 14 strains, oxacillin MIC values ranged from 0.5 μg/mL to >64 μg/mL and cefoxitin MIC values ranged from 1 μg/mL to >256 μg/mL. Positive agglutination reaction by latex agglutination test was recorded in 13 out of 14 strains. The PCR assay for mecA gene was positive in 12 investigated strains.

Radi otkrivanja meticilin (oksacilin)-rezistentnih sojeva stafilokoka u većini mikrobioloških laboratorija koristi se najčešće samo disk difuziona metoda uz primenu antibiogram diskova oksacilina i cefoksitina. Pored navedene metode, poželjno je da se utvrde i vrednosti MIC oksacilina i/ili cefoksitina primenom dilucione metode u bujonu ili agaru. Nakon ispitivanja pomenutim metodama za definitivno utvrđivanje pripadnosti soja grupi meticilin rezistentnih stafilokoka, koriste se metoda lateks aglutinacije za utvrđivanje prisustva PBP2a i PCR metoda za detekciju mecA gena. Kako primena metode PCR nije moguća u većini laboratorija koje se bave rutinskom dijagnostikom, kao metoda izbora preporučuje se lateks aglutinacioni test za brzo otkrivanje PBP2a. U ovom ispitivanju primenjeni su lateks aglutinacioni test i PCR metod, kao potvrdne metode za ispitivanje sojeva stafilokoka koji su bili rezistentni na oksacilin i/ili cefoksitin na osnovu rezultata dobijenih disk difuzionom metodom i mikrodilucionom metodom u bujonu. Ukupno je ispitano 14 sojeva koagulaza - negativnih stafilokoka izolovanih iz kliničkih uzoraka poreklom od pasa, mačaka i kokoši. Među izolovanim sojevima, ustanovljeno je da dominira vrsta Staphylococcus haemolyticus sa 11 izolata, a po jedan soj je izolovan od vrsta Staphylococcus epidermidis, Staphylococcus capitis i Staphylococcus vitulinus . Vrednosti MIC oksacilina za 14 sojeva iznosile su od 0.5 mg/mL do >64 mg/mL, a vrednosti MIC cefoksitina iznosile su od 1 mg/mL do >256 mg/mL. Kod 13 od 14 sojeva utvrđena je pozitivna reakcija aglutinacije primenom lateks aglutinacionog testa. Kod 12 ispitivanih sojeva je utvrđeno prisustvo mecA gena PCR metodom.