2.4 Staphylococcus aureus methicillin-resistant strains identification

With the identified resistant oxacillin and methicillin antibiotypes (ORSA/ MRSA), the in vitro susceptibility test on antimicrobials with 90n S. aureus isolates was carried out by the agar diffusion method of National Committee for Clinical Laboratory Standards [30], S. aureus isolates were incubated in Mueller Hilton broth for 4 hours at 37°C, compared to McFarland standard 0.5; Mueller Hinton agar plates were inoculated by applying on the plates the antibiotic unidisks (BBL, Lawrence, KS, USA): 10 U penicillin, 30 μg ampicillin, 1 μg oxacillin, and 10 μg cephalothin. The S. aureus strains of ATCC25923 and ATCC 29213 and the S. epidermidis strain ATCC 12228 were used as controls. The strain of S. aureus ATCC 43300 was used as a control of methicillin resistance. To confirm the MRSA strains, Mueller Hinton agar plates containing 4% NaCl were inoculated and incubated at 35 and 42°C for 24 hours. The bacterial inhibition halos of the bacterial growth on the agar plates were expressed in mm and compared to the established values for the test with a difference >4 mm in diameter between the halos of inhibition containing unidisks with 1, 2, 4, and 6 μg of oxacicline [31]. The in vitro production of β-lactamase was determined by the modified iodometric method [32].

## 2.4.1 Identification of the mec A gene in Staphylococcus aureus by polymerase chain reaction (PCR) test

Isolation of chromosomal DNA was obtained from S. aureus 90n isolates, from the study groups of work strains 1, 56, 305, and 123, with the control strains of S. epidermidis ATCC 12228 and S. aureus ATCC 25923 as negative controls, and S. aureus ATCC 43300 as a positive control. The washed bacterial pellet was suspended in a buffered phosphate solution of pH 7.2 to proceed to bacterial lysis. The bacterial DNA solution was standardized at 0.5 ng per 1 μL and stored at �20° C. The PCR reactions were carried out with a commercial PCR kit (BioTecnologías Universitarias, México), using a thermal cycler (Genius Techne, Duckford, UK). The PCR for the identity of the S. aureus isolates was carried out by the amplification of nuc gene, using the oligonucleotides (Gibco BRL, Rockville, MD), Sa1 (5<sup>0</sup> GCGATTGATGGTGATACGGTT-3<sup>0</sup> ), and Sa2 (5<sup>0</sup> AGCCAAGCCTTGACGAA CTAAAGC-3<sup>0</sup> ) [33]; the amplification product of PCR obtained was 270 bp related to the nuc gene. The amplification of the mec A gene was performed to identify the MRSA strains of S. aureus, using the primers (Gibco BRL, Rockville, MD), mec1 (5<sup>0</sup> -TGGCTATCGTGTCACAATCG3<sup>0</sup> ), and mec2 (5<sup>0</sup> -CTGGAACTTGTTGAGCA-GAG-3<sup>0</sup> ) to obtain an amplification product of 310 bp; initial denaturation was performed at 92°C for 3 minutes and 30 cycles under the amplification conditions: 92°C for 1 minute, 56°C for 1 minute, 72°C for 1 minute, and a final extension of 2 minutes at 72°C. The amplified product was kept at 4°C, before being visualized on an agarose gel. The amplification conditions of the gene were similar with an alignment temperature of 56°C. The nuclear DNA amplification products were separated by electrophoresis in a horizontal chamber (Horizon 5B, Gibco BRL, Goithersburg, MD, USA), with a power supply of 70 Volts for 90 minutes applied to 2% agarose gel (Gibco BRL, Rockville, MD. USA), in TBE in a run with a 100 bp molecular weight marker (Gene Ruler, Fermentas, Burlington, Ontario, CA). The strains of S. aureus ATCC 29213, ATCC 43300, and S. epidermidis ATCC 12228 were used as a control. The gels were stained with ethidium bromide. The gels were photographed in a UV transluminator (UVP, Upland, CA. USA), visualized with a DC 120 digital camera (Kodak Eastman, Rochester, NY, USA) and image analysis program (ID image analysis software Windows; and 3.0, Kodak digital science, Rochester, NY, USA). The analysis of results was made from the frequency of

isolation and the expression of virulence factors of S. aureus related to the biotype, by estimating the absolute and relative frequency; The statistical evaluation was performed with the Pearson's Chi-square test (χ2), with an α level of 0.05 and a 95% confidence interval using Epi-Info 6.0 software (CDS, Atlanta, GA, USA). The in vitro sensitivity was estimated from the mean ˜ standard deviation of the inhibition halos (in mm) of the antibiotics.
