Bacteriological and Molecular Detection of Methicillin-resistant Staphylococcus Aureus Isolated From Cigarette and Hookah Smokers in Khartoum State

Objective Staphylococcus aureus is an extra-ordinarily versatile pathogen that causes a wide variety of infections in humans and animals, capable of causing supercial lesions and systemic infections. The aim of this study was to detect mecA gene in smokers of cigarette and hookah. Forty-one isolates (41%) were identied as S. aureus by traditional culture methods. non-smokers, nasal carriers of S. aureus were 16 (39%) while 25 (60%) among smokers (P = 0.103). Of the overall 25 smoker nasal carriers, 18 were hookah smokers and 7 were cigarettes smokers (P = 0.004). MRSA was 1 (2.4%) in non-smokers and 3 (7.2%) in smokers, 2 in cigarettes and 1 in hookah (P = 0.009). Only 38 isolates were conrmed as S. aureus by PCR (mecA gene). In non-smokers group mecA were 6 (15.8%) positive and 8 (21%) negative, while in smokers were 16 (42.3%) positive and 8 (20.9%) negative isolates (P = 0.187). From hookah 12 mecA positive and 4 from cigarette (P = 0.647). The correlation of MRSA and using of antibiotic during last 6 months indicates signicant (P = 0.031).


Introduction
Cigarette smoking (CS) is the leading preventable cause of death, disease, and disability worldwide. In 2010, nearly 6 million worldwide deaths were due to cigarette smoking and second-hand exposure. Forty percent of children worldwide regularly exposed to second-hand cigarette smoke [1][2][3].
Both direct and second-hand cigarette smoke exposures increase the risk and severity of developing respiratory tract and other invasive infections [4]. Invasive pneumococcal disease is 2-4 folds more common in cigarette smokers, in addition to in uenza and tuberculosis infections [2,5]. CS exposure can impair epithelial innate immune responses to microbial products, perhaps setting the stage for overgrowth and invasion [1,4]. Adverse health effects linked to cigarette smoke, including carcinogenesis [4], and chronic pulmonary disease, are commonly believed to arise from the direct action of tobacco smoke components on human cells [2]. CS contains a variety of bioactive substances, including oxidizing, genotoxic, and immunomodulatory factors [7,8]. Exposure to cigarette smoke raises the risk of many infectious diseases [9,10]. Kulkarni and his colleagues demonstrated that CS can impair epithelial innate immune reactions to microbial products [11]. Perhaps setting the stage for overcrowding and invasion [9]. Invasive pneumococcal disease is 2-4 times more common among cigarette smokers, in addition to in uenza and tuberculosis infections [12]. Moreover, direct and second-hand cigarette smoke exposures change the normal composition of the nasopharyngeal micro ora, opening the door to opportunistic pathogens such as S. aureus, Haemophilus in uenzae, and Streptococcus pneumoniae [5,12].
Tobacco in hookahs is exposed to high heat from burning carbon dioxide, and smoke is at least as toxic as cigarette smoke, this could lead to absorption of more toxic substances than those absorbed by cigarette smokers [13].
The amount of smoke inhaled during a typical hookah session is about 90,000 millilitres (ml), compared with 500-600 ml inhaled when smoking a cigarette [14].
Hookah smokers can be at risk for some of the same problems as cigarette smokers [5]. These include oral cancer, prostate cancer, stomach cancer, oesophageal cancer, diminished lung function and decreased fertility [15].
S. aureus is Gram-positive cocci that causes a broad range of infections, including pneumonia, bacteraemia, and endocarditis [5,15]. Methicillin Resistant S. aureus (MRSA) is resistant to oxacillin, methicillin and all beta lactam agent [16]. Smokers have high rates of MRSA colonization than nonsmokers, thus increasing risk of serious and di cult to treat infections [17]. The primary mechanism for this resistance is the production of an altered Penicillin Binding Protein 2a (PBP 2a). MRSA produces a PBP 2a mediated through the mecA gene, MRSA has a higher mortality rate for bacterial infections than for methicillin-sensitive S. aureus (MSSA) [17,18]. On the other hand, cigarette smoking promotes bacterial virulence, since the colonizing microbiota inhabits human mucosal spaces, microbes that share exposure to a variety of environmental stimuli, including CS [11,19]. Durmaz

Methods
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 fty non-smokers males as control were enrolled in this study. The case divided into two groups, twenty-ve 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.

Specimens Collection
The specimens were collected from anterior nares by using sterile cotton swab emulsi ed 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 signi cant growth were identi ed using standard microbiological methods, which included colonial morphology, Gram's stain, biochemical tests and molecular techniques (PCR).

Bacterial Identi cation
Colonies were examined in the next day. The organisms were identi ed according to the morphology of the colonies, Gram stain [27], 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) [28]. The results were compared with S. aureus control ATCC 25923.
Genotyping of Staphylococcus aureus resistant genes DNA was extracted by boiling method [29], 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 37C o , 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) [30], 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 nal 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 speci c ampli ed products by comparing with 50 base pairs standard ladders (INtRON biotechnology. Korea). All samples were con rmed as S. aureus by speci c housekeeping gene primer (16 s RNA), negative sample were excluded.

Statistical analysis
Data were analysed using SPSS version-20. The Chi-squared test was performed to determine bivariate correlation and statistical signi cance and a P-value of less than 0.05 was considered statistically signi cant.

Phenotypic analysis
S. aureus were found in the nasal of smokers 7 (17.1) and 18 (43.9) in Ciggrate and Hookahs respectively and non-smokers 16 (39) with insigni cant association (P.value = 0.103). The distribution of S. aureus in cigarette and hookah smokers and it was very high in hookah smokers, 7 (28%) and 18 (72%) respectively. There was association between hookah and nasal carriers (P = 0.000).
S. aureus was susceptible to Vancomycin (41 were sensitive). In this study four participants from total number of S. aureus carriers were MRSA, while 35 carriers were MSSA and (2) isolates were showed intermediate result.
MRSA presence was higher in cigarette smokers than in hookah (2 folds), while only one case of MRSA was found in non-smokers as showed in Table 2. The relationship between nasal carriers of S. aureus and smokers group indicated signi cant relationship (P = 0.001), Moreover, the relationship between nasal carriers and number of cigarette or hookah per day indicated signi cant (P = 0.008), while, the relationship between nasal carriers and smoking years indicated insigni cant (P = 0.479).

Genotypic Analysis
Forty one isolate of S. aureus were examined by the multiplex PCR to detect the presence of 16 s rRNA and mecA gene (Fig. 1), three samples were negative to 16 s rRNA, so were excluded from the study, and mecA was detected in the remainder 38 isolates.
From (38) S. aureus isolates (22) were MRSA positive, which was observed to be higher in smokers than non-smokers (16 and 6) respectively as showed in Table 2.
These ndings indicate a substantial rise in S. aureus in the smoker group rather than the non-smoker group. The distribution of S. aureus among smokers' group (n = 25) was: hookah group 18 (72%) while in cigarette 7 (28%), this result has shown an increase in the colonization of S. aureus in the hookah group more than a cigarette; this may be due to an increase in inhalation of more than a cigarette in the hookah, it may also be due to the sharing of hookah devices between different personnel, which may increase the colonization of S. aureus in this group.
There was signi cant association between nasal carriage and smoking (P. value = 0.001), and number of smoking per day (P. value = 0.008), this means that, smoking increases the colonization of S. aureus by smoking per day (P. value = 0.479).
This study revealed 6 (15.8%) isolates were mecA positive from non smoker, while there were 16 (42.3%) S. aureus in smokers. This genotypic analysis removes 3 phenotypically reported samples as S. aureus, which indicates that it is more accurate than phenotypic analysis. In addition, genotypic study reveals a sharp rise in Oxacillin resistant to more than phenotypic study, because of its ability to identify an unexpressed gene, it is an innovative diagnostic method and more e cient than a phenotypic study. In addition, the genotypic study revealed a higher concentration of mecA in the hookah than the cigarette according to the phenotypic study; This may increase the amount of nicotine and inhaled smoke in the hookah by more than a cigarette due to the presence of toxic substance in the hookah. Furthermore, the way in which genes are transmitted (through plasmid) and the way in which hookah is used (different people share one device) helps to spread the mecA gene between different S. aureus carriers.