Nasal Carriage Rate, Antimicrobial Susceptibility Pattern, and Associated Factors of Staphylococcus aureus with Special Emphasis on MRSA among Urban and Rural Elementary School Children in Gondar, Northwest Ethiopia: A Comparative Cross-Sectional Study

Introduction Staphylococcus aureus is a Gram-positive, catalase-positive, and coagulase-positive bacterial species commonly found on the skin and in the nose of most healthy individuals. The anterior nares of nose are the most frequent carriage sites for S. aureus in both adults and children. Methicillin resistance among S. aureus isolates has steadily increased worldwide. Objective The main objective of this study was to determine nasal carriage rate, antimicrobial susceptibility pattern, and associated risk factors of Staphylococcus aureus with special emphasis on MRSA among urban and rural elementary school children in Gondar, Northwest Ethiopia. Method A community based comparative cross-sectional study was conducted on 622 urban and rural elementary school children in Gondar from January 1st to March 30th, 2018. Data was collected using a questionnaire and nasal swab samples were collected by sterile cotton tip swab moistened with sterile normal saline. Collected samples were inoculated on mannitol salt agar and incubated aerobically at 37°C for 24 hrs. S. aureus was confirmed by observing colony characteristics and biochemical tests. MRSA was detected using cefoxitin disc by Modified Kirby-Bauer disk diffusion technique. Finally data was entered, cleared, and checked using Epi-info version 7 and exported to SPSS version 20 for analysis. Odds ratio and logistic regression were used for statistical association. P-value ≤ 0.05 at 95% CI was considered for statistical association. Result Of the 622 school children, the overall prevalence of S. aureus was 143/622 (23%). Of them, 14/143 (9.79%) were MRSA. The carriage rate in urban schools was 83/622 (13.3%) whereas it was 60/622 (9.6%) in rural schools. The prevalence of MRSA among urban schools, 9.1%, was higher than their urban counterparts, 0.7%. Gentamycin, clindamycin, and ciprofloxacin were the most effective whereas penicillin and tetracycline were resistant. Children's fathers' educational status and number of children in class room were significantly associated with S. aureus but only living in urban of children significantly associated with MRSA. Conclusion This study showed high prevalence of S. aureus and MRSA, 143/622 (23%) and 14/143 (9.79%), respectively. So, decolonization of nasal carriers of MRSA and reducing the number of students per classroom should be addressed. Moreover, regular large scale survey should be conducted to assess the burden and intervene accordingly.


Introduction
Staphylococcus aureus (S. aureus) is a Gram-positive, catalasepositive, and coagulase-positive bacteria commonly found mainly on the skin and nasal mucosa of most healthy individuals [1]. It is a frequent cause of clinically important infections ranging in severity from superficial infections to severe invasive diseases [2]. Approximately 20% of individuals are persistently nasal carriers of S. aureus and the rest 30% are intermittently colonized [3]. The anterior nares of nose 2 Advances in Preventive Medicine are the primary reservoir for replication and spread to other body sites [4,5].
Staphylococcus aureus exhibits increasing virulence and resistance to various antibiotics, complicating prevention, and treatment of infections [6]. Virulence factors are tightly regulated and act to degrade host cells, tissues, change the immune response, and enable for the dissemination inside and outside the host cell [7]. Overuse and misuse of antibiotics led to the emergence of bacterial antibiotic resistance [8]. Methicillin-resistant S. aureus (MRSA) carriage in healthy children is a major asymptomatic reservoir with an ability to quickly spread of MRSA within the community [9,10].
MRSA is resistant to a large group of antibiotics called the beta lactams. Methicillin is a -lactam antibiotic produced to treat penicillin resistant S. aureus. Methicillin-resistant S. aureus is a pathogenic strain responsible for difficult to treat infections in human [11]. MRSA resistance to antibiotics encoded by the mobile genetic element Staphylococcal chromosomal cassette (SCC) which carry the mecA gene and these elements vary in size and genetic content. The mecA gene encodes an altered penicillin binding protein (PBP2a) which permits the bacteria S. aureus to grow in the presence of methicillin and other -lactam antibiotics [12,13]. PBP2a is located in the bacterial cell wall and has a low binding affinity for -lactam antibiotics [14].
In Ethiopia, no population based study had been carried out on nasal carriage rate, antibiotic susceptibility pattern, and associated risk factors of S. aureus and MRSA among school children particularly in Gondar, Northwest Ethiopia. Thus, this study is intended to assess and fill the information gap of the current nasal carriage rate, antibiotic susceptibility pattern, and associated risk factors of S. aureus and MRSA among elementary school children in Gondar town. Also to indicate the prevention and control measures in the general community, it will also be used as preliminary information for future studies.

Materials and Methods
. . Study Area. The study was conducted in five governmental urban elementary schools in Gondar town and five governmental rural elementary schools surrounding Gondar town; Gondar town is the capital of North Gondar administrative zone, in Amhara region, Northwest Ethiopia.

. . Study Design and Periods.
A community-based comparative cross-sectional study was conducted to determine nasal carriage rate, antimicrobial susceptibility pattern, and associated factors of Staphylococcus aureus with special emphasis on MRSA among elementary school children from January 1 st to May 30 th , 2018.
. . Study Population. School children from recruited schools who gave sociodemographic information and nasal swab samples were included whereas children who were unable to give sociodemographic information, nasal swab specimen, and those who were on antibiotic therapy at the time of data collection were excluded . . Inclusion and Exclusion Criteria . . . Inclusion Criteria. All randomly selected school children from recruited schools who have agreed to give sociodemographic information and nasal swab samples were included.
. . . Exclusion Criteria. Children who were unable to give sociodemographic information and nasal swab specimen and those who were involuntary to participate in the study due to different reasons and children who were on antibiotic therapy at the time of data collection were also excluded.
. . Sample Size Determination and Sampling Technique. Single population proportion formula was used to determine the sample size and the calculated sample size was 258. However, adding 20% nonresponse rate and a design effect of two, the final calculated sample size was 622. A multistage sampling technique was used to select schools by using simple random sampling technique and stratifying the schools to grades and sections. The number of study participants were allocated proportionally to each schools and grades based on the school sampling frame and the study subjects were selected by simple random sampling technique (lottery method).

. . Sociodemographic Data and Nasal Swab Specimen Collection.
A pretested questionnaire based on postulated or known risk factors were developed and modified to explore the objectives of the study. Then sociodemographic characteristics and other relevant information were collected. Nasal swab specimen were collected by using sterile cotton tip swabs prewetted with sterile saline for each anterior nares by inserting the swab and gently rotating four to five times both in clockwise and in anticlockwise direction from each study participants. The nasal swabs were collected and inoculated immediately in a properly labeled sterile Tryptone Soya Broth (TSY) (Oxoid Ltd. England) and transported by using vaccine carrier which has ice box to maintain the temperature at 2-8 ∘ C until it reaches the laboratory.
. . Laboratory Inoculation and Identification. Each nasal sample was inoculated onto mannitol salt agar (Oxoid Ltd. England) and the plate was incubated aerobically at 37 ∘ C for 24 hrs. The sample that was positive for mannitol fermentation and golden yellow colonies on mannitol salt agar was further inoculated on blood agar (Oxoid Ltd. England) plate and incubated at 37 ∘ C for 24 hrs. The isolates obtained were identified using standard microbiological methods including colony morphology, Gram's stain reaction, and biochemical tests such as catalase and coagulase. Finally isolates that were golden yellow colony on mannitol salt agar and blood agar pate, Gram-positive cocci in clusters, catalase, and coagulase positive were confirmed as S. aureus.
. . Antimicrobial Susceptibility Testing. A suspension of pure colony from each confirmed culture isolate was done in sterile normal saline and incubated at 37 ∘ C for at least 15 minutes. The suspension was adjusted at 0.5% MacFarland standard. Then Modified Kirby-Bauer disk diffusion technique was implemented for antibiotic susceptibility pattern using different antibiotics such as penicillin (10 g), erythromycin (15 g), clindamycin (2 g), gentamicin (10 g), cotrimoxazole (1.25/23.75 g), cefoxitin (30 g), ciprofloxacin (30 g), and tetracycline (30 g). Moreover, inducible clindamycin resistance was tested using D-test by putting erythromycin and clindamycin discs at a distance of 15 mm from each other on Muller Hinton agar plate (Oxoid Ltd. England). Then after incubation at 37 ∘ C for 24 hrs, flattening of zone (D-shaped) around clindamycin in the area between erythromycin and clindamycin discs was observed and interpreted as inducible clindamycin resistant.
All Staphylococcus aureus isolates were tested for methicillin susceptibility patterns by using Modified Kirby-Bauer disc diffusion technique. Cefoxitin (30 g) (Oxoid Ltd. England) discs were placed in the plates and then incubated aerobically at 35 ∘ C for 24 hrs. Zone inhibition in millimeters was measured with a ruler. Isolates were classified as resistant, intermediate, and sensitive based on CLSI interpretation [15]. Finally those which were resistant to cefoxitin (≤ 21 mm) were confirmed as MRSA. Known strains of S. aureus (ATCC 25923) was inoculated to cheek the performance of culture media as a positive control for catalase, coagulase, and antibiotic susceptibility testing. And Escherichia coli (ATCC 25922) strain was used as a negative control. Gram stain reagents were also cheeked by preparing smears from known S. aureus (ATCC 25923) strain and observing them under the microscope.
. . Data Entry and Analysis. Data was entered using EPI-Info version 7 and its completeness and clearance was checked and then transferred to SPSS version 20 for analysis. The characteristics of the study population were summarized using frequencies, mean, and standard deviation. Binary logistic regression was done to determine the association of variables with S. aureus and MRSA. Crude odds ratio was calculated. Moreover, adjusted odds ratio was computed using multivariate logistic regression for variables with p value ≤ 0.2. P-value ≤0.05 at 95% CI was considered as statistically significant.

Result
. . Sociodemographic Characteristics. A total of 622 urban and rural elementary school children were included in the study; of these, 317/622 (51%) were males. The ages of the study participants ranged from 6 to 25 years with a mean age of 11.9 years (SD+2.9). Most of the study participants, 345/622 (55.5%), belonged to the age group of 11-15 years of age and 345/622 (55.5%) were from urban elementary schools while the rest, 277/622 (44.5%), were from rural schools. Among the study participants, 321/622 (51.6%) had a family size of greater than five members and 271/622 (43.6%) had mothers who were unable to read and write ( In urban elementary schools, the S. aureus carriage rate was 83/622 (13.3%) whereas in the rural schools, it was 60/622 (9.6%). The frequency of MRSA among S. aureus positive children in urban and rural school children was 13/143 (9.1%) and 1/143 (0.7%), respectively. The overall prevalence of inducible clindamycin resistant S. aureus in this study was 12/622 (1.93%).

Discussion
According to studies which were conducted on nasal colonization of Staphylococcus aureus, approximately 20-30% of healthy persons are persistent nasal carriers with high colonization rates among children [3,9]. There was an important association between nasal carriage rates of S. aureus and development of subsequent infections [6]. MRSA had emerged in various geographically distinct communities outside of health care settings without known health care associated risk factors [12].
In this study, among 143 children who had S. aureus, 79/143(55.2%) were male which indicates higher proportion of nasal carriage rate of S. aureus among male students than females, 64/143 (44.8%). This result is in agreement with previous studies which were conducted in Nigeria and Iraq [1,8]. This higher prevalence in males might be due to male students participated in sport activities more often than females which makes them more vulnerable to colonization. Nasal carriage of S. aureus and MRSA, 83/143 (58%) and 13/83(15.66%), respectively, was higher in urban than rural residents, 60/143(42%) and 1/60 (1.67%). This might be due to overcrowding and the presence of large number of governmental and nongovernmental health care facilities  Advances in Preventive Medicine such as hospitals and health centers/clinics in the urban than rural community. Regarding the antimicrobial susceptibility pattern result there were more than 99% and 70% resistance strains to penicillin and tetracycline, respectively. In this study antimicrobial susceptibility test showed that penicillin and tetracycline were the least effective agent with more than 70% bacterial resistance. This result of penicillin resistant was in line with various studies which was conducted on healthy children in China (87.5%), Nigeria (100%), Ghana (95%), Jimma (100%), and Bahir Dar (100%) [2,16,19,24,25]. Majority of S. aureus isolates showed 99.3% resistance to penicillin. Similarly, MRSA isolates showed 100% resistance to penicillin and cefoxitin. This result was similar to a study conducted in Nigeria (100%), Jimma (100%), and Bahir Dar (100%) [1,24,25].
The majority of the isolated S. aureus strains were sensitive for tested antimicrobial agents. However, 44/143 (30.8%) of isolates of S. aureus showed multidrug resistance patterns. The highest MDR pattern, 27/143 (18.9%), was observed for penicillin, tetracycline, and erythromycin. 5/143 (3.5%) of isolates were resistant for penicillin, cotrimoxazole, tetracycline, and erythromycin. One isolate was resistant for six antibiotics: penicillin, cotrimoxazole, tetracycline, erythromycin, clindamycin, and gentamycin. The differences in susceptibility pattern of the isolates might be due to frequent and irrational use of antibiotics, illegal drug healing in our setup, and differences in geographical area.
Among elementary school children, high rate of isolation of MRSA was reported in urban elementary schools. The high prevalence of MRSA in urban schools than rural might be due to reasons; for example, urban schools were more closer to health care facility; high antibiotic selective pressure in urban settings and overcrowding was more prominent in urban than rural schools.
Several studies showed that sociodemographic characteristics and risk factors contribute a lot to the nasal carriage of S. aureus and MRSA [5,21,22,24,25,27,28]. The result of this study showed children's father's educational status significantly associated with nasal carriage of S. aureus which was in contrast with a study in Nigeria [12]. The less the level of education among fathers of school children, the more likely for nasal carriage of S. aureus was observed. The possible reason might be that the less level of the fathers education, the less they care their children socioeconomically and hygienically.
Number of students in classroom was significant risk factors for nasal colonization of S. aureus. Majority of the students who had high nasal carriage of S. aureus belonged among children with 41-60 classmates in the class room which showed that the increase in the number of students per class room, the more likely the nasal carriage of S. aureus was observed in school children. This result was consistent with study conducted in Ethiopia, Jimma [24]. The possible reason might be due to the fact that high number of students in one class room makes them more frequent contact among each other, overcrowding, and greater sharing of nasal flora which causes the more spread of the bacteria.
Living in urban of children involved in this study was statistically significant risk factor for nasal colonization of MRSA. This study showed that being urban elementary school children had more likely hood for nasal carriage of MRSA than rural school children. The possible reason might be due to children living in the urban community which makes them contact with health care workers more frequently and more exposed to health care facilities and overcrowding in the urban area which increases nasal carriage of MRSA in urban schools.

Limitation of the Study
We did not perform vancomycin minimum inhibitory concentration due to resource constraints and advanced molecular techniques like PCR which may underestimate the true prevalence in the study population.

Conclusion
The prevalence of S. aureus and MRSA among primary school children was 23% and 9.79%, respectively. Father's educational status and number of students in class room were independent risk factors for nasal colonization of S. aureus. Moreover, being urban school children's were at risk of MRSA nasal colonization. Therefore, health education, screening of target population, and decolonization of carriers are effective prevention strategies to control the spread and burden of drug resistant S. aureus and MRSA within elementary schools and community. Large community-based studies, molecular distinction between hospital acquired-MRSA and community acquired-MRSA, continuous surveillance, screening of target population, and decolonization of carriers should be conducted to decrease the spread and burden of drug resistant S. aureus and MRSA in the elementary schools and the community at large.

Data Availability
All data generated or analyzed during this study are included in this article. Data that support the findings of this study are also available from the corresponding author upon reasonable request.

Ethical Approval
Ethical clearance was obtained from University of Gondar Ethical Review Committee. Written permission was obtained from Gondar Town Administration Health Office.

Consent
A written informed consent from parents and assent from children were obtained. Additionally confidentiality of information was assured.