Multiplex PCR for the Detection of Genes Encoding Aminoglycoside Modifying Enzymes and Methicillin Resistance among Staphylococcus Species

Abstract

We developed multiplex polymerase chain reaction (PCR) to detect aac(6 ')/aph(2 "), aph(3 ')-IIIa, and ant(4 ')-Ia, the genes encoding the most clinically relevant amino-glycoside modifying enzymes (AME), and simultaneously, the methicillin resistant gene, mecA, in Staphylococcus species. Clinical isolates of 45 S. aureus and 47 coagulase negative staphylococci (CNS) from tertiary university hospitals were tested by conventional susceptibility testing, using the agar dilution method and by multiplex PCR. Of a total of 92 isolates, 61 isolates were found to be methicillin-resistant. Of these, 54 isolates (89%) were found to be harboring mecA. Seventy-five percent of the 92 isolates demonstrated resistance to at least one of the aminoglycosides tested. Moreover, resistance to aminoglycosides was closely associated with methicillin-resistance (p<0.05). The most prevalent AME gene was aac(6 ')/aph(2 ") which was found in 65% of the isolates, and ant(4 ')-Ia and aph(3 ')-IIIa were present in 41% and 9% of the isolates, respectively. The concordance between methicillin-resistance and the presence of mecA gene was 98% in S. aureus and 81% in CNS. The concordance between gentamicin resistance and the presence of aac(6 ')/aph(2 ") gene was 100% in S. aureus and 85% in CNS. The multiplex PCR method that we developed appears to be both a more rapid and reliable than conventional method.

Aminoglycoside-resistant staphylococci in Greece: prevalence and resistance mechanisms

“…All isolates were tested for resistance to aminoglycosides by a disk diffusion method using amikacin 30 μg, gentamicin 10 μg, tobramycin 10 μg and kanamycin 30 μg, 6 according to the Clinical and Laboratory Standards Institute guidelines [3].…”

Phenotypic and Genotypic Determination of Antibiotic Resistances of Some Clinical Staphylococcus aureus Isolates

“…mecA geni heterojen olarak eksprese edilebildiği için tüm metisiline dirençli stafilokok suşları fenotipik yön-temlerle saptanmayabilmektedir. Bu yüzden mecA geninin saptanması için kullanılan PCR tekniği konvansiyonel yöntemlerle karşılaştırıldığında hem daha hızlı hem de güvenilir olan bir altın standart yöntem olarak kabul edilmektedir [14] . Çalışmamızda metisilin disk difüzyon ve Phoenix yöntemleriyle test edildiğinde metisiline direnç %19 idi ve bu izolatların hepsi de mecA genine sahipti.…”

“…Bizim çalışmamızın aksine birçok çalışma raporunda en sık genetik belirleyici olarak ermA geni tespit edilmiştir [4,10,[12][13][14][15][16][17][18][19][20][21][22] .…”

Antimicrobial susceptibility pattern and SCCmec types of methicillin-resistant coagulase-negative staphylococci from subclinical bovine mastitis in Hatay, Turkey

“…All CoNS were also screened by PCR for the presence of the mecA gene, as previously reported (1). The primers used to detect mecA gene were: 5′-CCTAG TAAAGCTCCGGAA-3′ and 5′-CTAGTCCATTC GGTCCA-3′, and the PCR reaction was as follows: initial denaturation at 95°C for 2 min, denaturation at 94°C for 2 min (30 cycles), annealing at 58°C for 30 sec and extension at 72°C for 30 sec, with a final extension step at 72°C for 7 min.…”