This was case-control study, conducted in Khartoum state from 15th January to the 30th of July 2018.
Participant of this study were males of age from 20 to 65 years old. Fifty adult males who smoke cigarette or hookah as case, another fifty non-smokers males as control were enrolled in this study. The case divided into two groups, twenty-five were cigarette smokers and the others were hookah smokers, participants under treatment or immunocompromised were excluded from study.
Samples were collected based on probability convenience technique, then data were collected by direct non-self –constricting questionnaire from participant.
The specimens were collected from anterior nares by using sterile cotton swab emulsified by peptone water, collection was done by inserting the swab and gently rotating for several times, collected specimens were labelled by number, packaged and processed within one hour of collection to Microbiology Laboratory in Sudan University of Science and Technology.
Each nasal swab inoculated into peptone water and incubated aerobically at 37Co for 24 hours then inoculated by using sterile loop into Mannitol Salt Agar (MSA) and incubated aerobically at 37Co for overnight. After incubation, isolates show significant growth were identified using standard microbiological methods, which included colonial morphology, Gram’s stain, biochemical tests and molecular techniques (PCR).
Colonies were examined in the next day. The organisms were identified according to the morphology of the colonies, Gram stain , biochemical tests and gene detection for the 16 s RNA of S. aureus.
In-vitro Antibiotic Sensitivity Testing
Kirby-Bauer method was used in the current study; the antibiotic discs used were from HI media (HI media Laboratories Pvt. Ltd, Mumbai 400086, India). The Oxacillin antibiotic (10 mg) and Vancomycin (30 mg) were used. The discs of the antibiotics placed in the diagnostic susceptibility test agar (Muller Hinton Agar). The distance between the two adjacent discs was at least 20 mm and from the edge of the plate was 15 mm. The media were incubated aerobically for 24 hours in 37 °C. After 24 hours of incubation, the diameter of the zone inhibition was measured and compared with the published tables of the control strains according to Clinical Labratorey Stander Institute guidelines (CLSI). The results were compared with S. aureus control ATCC 25923.
Genotyping of Staphylococcus aureus resistant genes
DNA was extracted by boiling method , by tacking small inoculums of bacteria cultured on nutrient agar, dissolving it in 0.5 ml of D.W in 1.5 ml Eppendorf tube and 10 ml of proteinase K was added for overnight at 37Co, then boiled for 20 minutes, then incubated in refrigerator at -20 for 10 minutes. Repeated this process four times (heat shock), then centrifuged at 12000 rpm for 5 min. The supernatant used as template DNA for PCR.
The multiplex PCR was done by using a thermo-cycler (techne 312, England). The primer in table (1) , the following conditions: denaturation at 94Co for 10 minutes, followed by 10 cycles of denaturation at 94Co for 45 seconds, annealing at 55Co for 45 seconds, and extension at 72Co for 75 seconds. Moreover, another 25 cycles of 94Co for 45 seconds, 50Co for 45 seconds, 72Co for 45seconds, and a final extension step at 72Co for 10 minutes.
The Amplicon were separated at 120 Volt for 15 min in 1.5% (w/v) agarose gel containing ethidium bromide, bands were visualized under U.V trans-illuminator (UVitec – UK) to detect the specific amplified products by comparing with 50 base pairs standard ladders (INtRON biotechnology. Korea). All samples were confirmed as S. aureus by specific housekeeping gene primer (16 s RNA), negative sample were excluded.
Data were analysed using SPSS version-20. The Chi-squared test was performed to determine bivariate correlation and statistical significance and a P-value of less than 0.05 was considered statistically significant.