Nasal Carriage and Methicillin Resistance of Staphylococcus aureus among Schoolchildren in Sana'a City, Yemen

Background Staphylococcus aureus (S. aureus) is a frequent cause of serious health problems with high morbidity and mortality. The risk of S. aureus infections is increased with the emergence of methicillin-resistant S. aureus (MRSA). This study aims to determine the nasal carriage rate of both S. aureus and MRSA among schoolchildren in Sana'a city. Methods This is a cross-sectional study conducted from January 2018 to May 2020. Five hundred and thirty-six students were enrolled. Their age ranged from 5 to 19 years with the mean age and standard deviation equal to 13.3 ± 3.5 years. Nasal swabs were collected from each student for culturing and methicillin susceptibility testing. Results Students with positive culture were 271 (51%) males and 265 (49%) females. S. aureus was isolated from 129 (24%) students whereas the overall prevalence of MRSA was 8 (1.5%). S. aureus was significantly recovered from students at the age group of 10–14 years (χ2 = 7.02; p=0.03), females than males (OR = 1.96; χ2 = 10.75; p=0.001), and students who were admitted into hospitals (OR = 1.6; χ2 = 4.89; p=0.03). Nevertheless, there were no significant differences between MRSA carriage and students' age (χ2 = 2.3; p=0.32), gender (OR = 1.02; χ2 = 0.001; p=0.63), and hospital admission (OR = 1.4; χ2 = 0.25; p=0.62). Conclusions The prevalence of MRSA is low among schoolchildren in Sana'a city. Age, gender, and previous hospital admission were statistically associated with nasal carriage of S. aureus but not MRSA nasal carriage.


Background
Staphylococcus aureus (S. aureus) is a Gram-positive, roundshaped, catalase-and coagulase-positive bacterium. It is a frequent cause of serious health problems with high morbidity and mortality. S. aureus colonizes the skin and nasal mucosa and thus can also be considered as normal flora. e anterior nares appear as the main reservoir site for S. aureus replication and spread to other body sites. Approximately 25-30% of healthy individuals are nasal carriers for S. aureus [1,2]. Any damage to the epithelial barriers such as trauma and medical or surgical interventions can lead to tissue invasion by S. aureus [3,4].
S. aureus expresses many virulence factors, and its resistance to antibiotics has gradually increased in recent years. Overuse and/or misuse of antibiotics led to the appearance of strains of S. aureus that are resistant to many currently used antibiotics. Penicillin-resistant S. aureus produces penicillinase, which hydrolyzes the β-lactam ring of penicillin leading to penicillin resistance. Later on, scientists developed novel semisynthetic penicillin called methicillin, which is resistant to staphylococcal β-lactamase [5,6]. Methicillin successfully controlled the infection caused by penicillin-resistant S. aureus strains. Nevertheless, only 2 years later after methicillin use in treatment, isolation of methicillin-resistant S. aureus (MRSA) strains was reported. Methicillin resistance is due to the acquisition of the methicillin resistance (mecA) gene integrated into the chromosomal element. e mecA gene encodes an altered penicillin-binding protein 2a or 2′ (PBP2a or PBP2′) which permits S. aureus to grow in the presence of methicillin and other β-lactam antibiotics [7].
MRSA strains have spread worldwide and they cause numerous nosocomial outbreaks in both hospitals and communities. MRSA carriers are predisposed to skin infections, wound infections, bone and joint infections, pneumonia, septicemia, endocarditis, and occasionally toxic shock syndrome. Infections from either hospital-acquired MRSA (HA-MRSA) or community-acquired MRSA (CA-MRSA) have increased the challenge of selecting empirical antimicrobial treatments [8][9][10]. In healthcare institutions, HA-MRSA can be transmitted between patients, or through the hands, clothes, or equipment of healthcare workers (HCWs), and the environment [11].
ere is increasing evidence that CA-MRSA is spreading among healthy persons, especially children [20]. In a study conducted by AL-Haj et al., the nasal carriage rate of S. aureus was reported to be 23.1% among public schoolchildren in Sana'a city, but they did not test the isolated bacteria for methicillin and oxacillin sensitivity [21]. erefore, this study aimed to determine the nasal carriage rate of both S. aureus and MRSA among schoolchildren in Sana'a city.

Study Design and Area.
A cross-sectional study was conducted from January 2018 to June 2020. Six primary and secondary schools at Sana'a city were randomly chosen by a cluster sampling method. ese schools included four public schools (Al Hussain Bin Ali, Roqaya, Omar Bin Al Khattab, and Khaled Bin Al Waleed) and two private schools (Al Amjad and Al Ilmiyah).

Study Population.
Five hundred and thirty-six students were enrolled in this study. Simple random sampling was used to choose students who would participate in the study.

Inclusion Criteria.
All randomly selected schoolchildren who agreed to participate were included.

Exclusion Criteria.
Students who were on antibiotic therapy for one week before sample collection or those who had ulceration or pus at the nares or skin were excluded. Diabetic students were also excluded.

Sample Size Determination.
Based on a study conducted by Al-Haj et al., who reported the prevalence of S. aureus among schoolchildren to be 23.1% [21], and on Central Statistical Organization at Ministry of Planning and Inter Coop (2005Coop ( -2006 which stated the number of schoolchildren in Sana'a city to be about 2240000 students, the calculated sample size was 471 students at a confidence level of 99%. However, because many students liked to participate, 536 students were enrolled in this study.

Specimen Collection and Examinations.
Samples were obtained from students by using sterile dry-cotton swabs from anterior nares. e swab was inserted 2-3 centimeters in the nasal cavity and rotated 4-5 times both clockwise and anticlockwise before swab withdrawal. Samples were labeled and transported in Amies transport media to the microbiology laboratory at the Faculty of Medicine and Health Sciences within 5-6 hours. At the microbiology laboratory, nasal swabs were inoculated on mannitol salt agar (HiMedia, India). e inoculated agar plates were incubated at 35-37°C for 24-48 h. After incubation, plates were investigated for mannitol-fermenter colonies which appear as yellow colonies on mannitol salt agar. Yellow colonies were subcultured on nutrient agar. Golden-yellow colonies on nutrient agar were further examined by Gram stain, catalase, and coagulase tests. Gram-positive cocci, arranged in grape-like clusters, catalase, and coagulase-positive were recorded as S. aureus [22,23].

Antibiotic Susceptibility Test.
All colonies confirmed to be S. aureus were tested for methicillin (5 μg) and oxacillin (1 μg) susceptibility by the modified Kirby-Bauer disc diffusion method. Using a sterile loop, colonies from nutrient agar which were confirmed to be S. aureus were picked up, suspended in sterile saline, and mixed to even turbidity. e turbidity intensity of bacterial suspension was adjusted in comparison with the 0.5 McFarland turbidity standard by adding saline or more bacteria. A sterile cotton swab was dipped into the bacterial suspension. en, the Mueller-Hinton agar plate (HiMedia, India) was inoculated by swabbing in three directions to evenly distribute the inoculum and make sure there were no gaps between streaks. 5 μg methicillin disc and 1 μg oxacillin (HiMedia, India) were applied using a sterile needle to come in contact with the agar surface. Inoculated Muller-Hinton plates were incubated at 35-37°C for 24 h [24]. Resulted inhibition zones were measured using a ruler. Inhibition zone less than or equal to 9 mm indicated S. aureus to be MRSA, while inhibition zones less than or equal to 17 mm indicated oxacillin-resistant S. aureus (Zone Size Interpretative Chart, HiMedia).

Statistical
Analysis. Data analysis was done using SPSS program version 20 (SPSS Inc., Chicago, IL, USA). Descriptive measures (mean ± standard deviation) were used for quantitative variables. Frequencies and percentages were used to present qualitative variables. Chi-square (χ 2 ) test was used for verifying the existence of associations. Probability (p) values ≤ 0.05 were considered statistically significant.

Results
Five hundred and thirty-six nasal swabs yielded bacterial growth. e age of the 536 students ranging from 5 to 19 2 International Journal of Microbiology years with the mean age and standard deviation equal to 13.3 ± 3.5 years. ey were divided into three age groups: the first age group was from 5 to 9 years, the second age group was from 10 to 14 years, and the third group was from 15 to 19 years. Out of 536, 271 (51%) students were males and 265 (49%) students were females. e number of students per class ranged from 3 to 159 students with the mean and standard deviation equal to 71 ± 34 students. Four hundred (75%) students studied at public schools while 136 (25%) studied at private schools (Table 1). S. aureus was isolated from the anterior nares of 129 (24%) students. It was recovered more frequently from students at the age group of 10-14 years (63, 30%) than from students at the age group of 5-9 years (22, 22%) and students at the age group of 15-19 years (44, 19%). is difference was statistically significant (χ 2 � 7.02; p � 0.03). S. aureus was commonly isolated from females (80, 30%) than males (49, 18%). e gender difference was statistically significant (χ 2 � 10.75; p � 0.001). S. aureus was recovered from 48 (30%) students who were admitted into hospitals for either medical treatment or surgery whereas S. aureus was found among 81 (21%) students who reported no hospital admission.
is difference was statistically significant (χ 2 � 4.89; p � 0.03). Twenty-eight (21%) students who were S. aureus carries had family members who worked at hospitals or any other health centers compared to 101 (25%) students who had no family members worked in hospitals or health centers. is difference was statistically nonsignificant (χ 2 � 1.1; p � 0.03) ( Table 2).
Out of 129 isolated S. aureus, 8 (6.2%) were resistant to both methicillin and oxacillin discs with an overall prevalence of 1.5% among school students. Table 3 shows that 3 (1.4%) students in the 10-to 14-year age group and 5 (2.2%) students in the 15-to 19-year age group but no students in the 5-to 9-year age group were carriers for MRSA. is age difference was statistically nonsignificant (χ 2 � 2.3; p � 0.32). MRSA was equally isolated from both males and females (4, 1.5%) with no significant difference (χ 2 � 0.001; p � 0.63). MRSA was isolated from 3 (1.4%) students who were admitted to hospitals while it was recovered from 5 (2.2%) who were not admitted to hospitals. is difference was statistically nonsignificant (χ 2 � 2.25; p � 0.62). Seven (1.7%) students who were MRSA carriers had no family members work at hospitals or health centers, but 1 (0.7%) student who had MRSA at his nares reported a family member works at a health center.

Discussion
Nasal carriage of S. aureus is approximately 20-30% of healthy individuals with high permanent colonization among children [25,26]. e emergence of MRSA infections has become a worrying problem in the clinical field because MRSA strains are resistant to many antibiotics particularly β-lactam classes [27]. e purpose of this study was to estimate the nasal carriage rate and methicillin resistance of S. aureus among schoolchildren in Sana'a city.
In the present study, the prevalence of S. aureus among primary and secondary schoolchildren was 24% (129/536).
is finding is consistent with a study conducted by Al-Haj et al., who found the colonization rate of S. aureus among public schoolchildren in Sana'a city to be 23.1% [21]. Our finding was also similar to those reported from other countries [28][29][30]. However, the nasal carriage rate of S. aureus in this study tends to be lower than that reported from Nigeria (56.3%), India (46.67%), the United States (39.6%), the Netherlands (36%), and Nepal (31%) [31][32][33][34][35]. On the other hand, the prevalence of S. aureus among schoolchildren in our study was higher than that reported from China (5.1%), Serbia (2.59%), and Iraq (17.75%) [36][37][38]. Variation in the S. aureus nasal carriage from one country to another might be attributed to differences in geographical distribution, sampling, culturing, and diagnostic techniques used by the researchers. e prevalence of MRSA among schoolchildren in our study was 1.5% (8/536) which was half lower (3%) than that found by Shetty et al., in Deralakatte, India, who used oxacillin to detect MRAS among schoolchildren [39].  Moreover, the prevalence of MRSA in this study was much lower than that reported by a study performed in southwest Ethiopia (18.8%) in which the researchers used cefoxitin (30 μg) discs to detect MRSA nasal carriage [40].
Comparing the prevalence of S. aureus in our study to the prevalence of S. aureus among hospitalized patients at ICU reported by Abdelmonem et al., the prevalence of S. aureus among schoolchildren was much less than that among hospitalized patients [12]. In addition, the prevalence of CA-MRSA among schoolchildren in our study was less than the prevalence of HA-MRSA among ICU patients (86.2% and 100%) found by Goudarzi et al. and Eftekhar et al.,respectively [41,42]. is reflects the higher prevalence of HA-MRSA than CA-MRSA. e current study showed the nasal carriage rate of S. aureus in the 5-to 9-year age group to be 22% which increased significantly to 30% in the 10-to 14-year age group and then decreased to 19% in the 15-to 19-year age group. Our finding is in agreement with a study conducted by Esposito et al., who reported nasal carriage of S. aureus decreases while oropharyngeal carriage increases with age [43]. Decreased nasal carriage in older students may be attributed to their well-developed and stronger immune system.
Concerning gender, our study revealed a significantly higher prevalence of S. aureus among females than males but no difference between nasal carriage of MRSA among females and males. Al-Haj et al. reported a higher nasal carriage among females than males, but the difference was nonsignificant [21]. Moreover, Okwu et al. found nasal carriage of S. aureus and MRSA to be nonsignificantly higher among females than males [44]. Nevertheless, many studies conducted on schoolchildren found the frequency of S. aureus and MRSA nasal carriage to be nonsignificantly higher in males than in females [35,42,45]. Tigabu et al. described a higher prevalence of S. aureus among males than females but a slightly higher MRSA prevalence among females than males [30]. Statistically significant nasal colonization of S. aureus among females than males in our study might be due to wearing a veil among females as an obligatory custom. Wearing a veil might make the anterior nares environment warmer and more humid which in turn may favor S. aureus colonization.
Regarding hospitalization, this study showed a highly significant association between nasal colonization of S. aureus with hospitalization; however, nasal carriage of MRSA among schoolchildren showed a nonsignificant association with hospitalization. Nevertheless, no association was found between the presence of first-degree relatives who worked in hospitals or health centers and nasal colonization with S. aureus and MRSA. Our study is in disagreement with the study conducted in Argentina by Gardella et al., who reported no statistical difference between nasal carriage of S. aureus and hospitalization [46]. However, our study is consistent with a study performed in Ethiopia by Reta et al., who reported no association between MRSA prevalence and hospitalization [45].
is finding might imply S. aureus acquisition during hospital admission. On the other hand, the low frequency of isolated MRSA makes it difficult to conclude whether MRSA is HA-MRSA or CA-MRSA.

Conclusions
In conclusion, the prevalence of MRSA among schoolchildren in Sana'a city is low. Age, gender, and previous hospital admission were statistically associated with nasal carriage of S. aureus but not with MRSA nasal carriage.
Data Availability e data that support the findings of this study are available. Anyone interested can get upon reasonable request from the corresponding author.

Conflicts of Interest
e authors declare that they have no competing interests.