Nasal carriage of methicillin resistant Staphylococcus aureus among health care workers at Al Shifa hospital in Gaza Strip

A cross sectional study involving 200 health care personnel who are working at different departments in Al Shifa hospital during the period from February to April 2015.

HCWs with a history of upper respiratory tract infection, fever, recent nasal surgery, diabetes, use of nasal medications or antimicrobial therapy and immunocompromised patients were excluded.

The study was approved by the Ministry of Health, and an informed consent was obtained from each participant.

Nasal swabs were collected from the anterior nares of the HCWs using a cotton swab in AMIES transport medium.

Antibiotic susceptibility testing of all isolates was performed by modified Kirby Bauer disc diffusion method as recommended by CLSI guidelines [14] using Mueller-Hinton agar. The antibiotics used in this study were penicillin (10 Units), amoxicillin-calvulanic (30 μg) acid, erythromycin (15 μg), vancomycin (30 μg), tetracycline (30 μg), cefuroxime (30 μg), gentamycin (10 μg), rifampicin (5 μg), clindamycin (2 μg), and ciprofloxacin (5 μg) (Himedia - India). Methicillin resistance was detected using oxacillin (1 μg) disc diffusion test. Briefly, single isolated colonies were selected and inoculated in Mueller-Hinton broth until its turbidity is comparable to 0.5 McFarland turbidity standard. Then the plates were inoculated with each broth culture and left to dry at room temperature before the application of antibiotic discs. The plates were incubated at 35 ± 2 °C in ambient air for 16–18 h. Plates with oxacillin were incubated at 33–35 °C because testing at temperatures above 35 °C may not detect MRSA as recommended by CLSI guidelines [14]. Zone of inhibition for each antimicrobial agent was interpreted, reporting the organism as resistant, intermediate or susceptible. S. aureus ATCC 33,592 (mecA positive, resistant) and S. aureus ATCC 29,213 (mecA negative, susceptible) were used as quality control strains.

DNA was extracted from cultured isolates by alkaline lysis as previously described [15]. Briefly, one bacterial colony was suspended in 20 μl of lysis buffer (0.25% sodium dodecyl sulfate, 0.05 N NaOH) and heated at 95 °C for 15 min. The cell lysate was diluted by 180 μl of distilled water. The cell debris was pelted by centrifugation at 16,000 xg for 5 min. and the supernatants were used for PCR or frozen at −20 °C until further use.

The mecA gene and femA endogenous control gene were simultaneously amplified in the same reaction. The primers used to amplify the mecA gene were (forward: 5’- AAGCGACTTCATCTATTAGGTTAT-3’ and reverse: 5’-TATATTCTTCGTTACTCATGCCATAC-3’; [16]. The primers used to amplify the femA gene were (forward: 5’-AACTGTTGGCCACTATGAGT-3’ and reverse: 5’-CCAGCATTACCTGTAATCTCG-3’; [17]. The reactions were performed in 25 μl final volumes in the presence of 1 μM of each primer, 2 μl DNA and 1X of the GoTaq Green MMX (Promega, USA).

The thermal cycling program for detection of both genes was as follows: one cycle of initial denaturation at 95 °C for 5 min; 34 cycles of denaturation at 95 °C for 30s, annealing at 60 °C for 30s, and extension at 72 °C for 1 min followed by a final extension at 72 °C for 5 min. The amplified products (femA: 306 bp and mecA: 402 bp) were resolved on a 2% agarose gel. The fragments were stained with ethidium bromide and visualized and photographed using gel documentation system. A 100 bp ladder was run as a molecular weight marker.

Another set of primers targeting the mecA gene was used (forward: 5’- AAAATCGATGGTAAAGGTTGGC-3’ and reverse: 5’-GTTCTGCAGTACCGGATTTGC-3’) for confirmation of mecA gene expression using the reaction components described above [18]. After initial denaturation at 95 °C for 1 min, the samples were subjected to 35 cycles of denaturation at 95 °C for 1 min, annealing at 54 °C for 1 min and extension at 72 °C for 1 min. A final extension was performed at 72 °C for 5 min. The expected amplicon size is 533 bp.

Positive and negative controls were added in each run, we used reference strains that are mecA positive and negative. S. aureus ATCC 33,592 (positive control for mec gene, MRSA); S. aureus ATCC 29,213 negative control.