Geographic distribution and prevalence of potential asymptomatic Staphylococcus spp. in the nasopharyngeal cavity of elementary school boys at Al-Madinah, KSA

Nasal swaps were collected from 405 elementary school boys of eight governmental schools located within several districts in Al-Madinah city to be screened for asymptomatic carriage of potential Staphylococcus pathogens. A total of 138 Gram-positive staphylococci isolates (34%) showing blood hemolysis were selected. Based on their morphological, cultural characteristics and Restriction enzyme analysis Isolates were grouped into 12 phenotypic within four distinct clusters. The 16S rDNA sequence analysis of all isolates revealed S. aureus to be the most prevalent (80%), followed by S. epidermidis (9%) and finally S. hominis and S. simulans accounting for 6% and 5% respectively. Staphylococcus isolates were detected with higher frequency in participants of age group 7–9 years, with the lowest numbers of staphylococci (18%) isolated from the schools located in the western area. Multiplex PCR assay revealed that all S. aureus isolates (20%) which were phenotypically recognized as MRSA by the Kirby–Bauer disc diffusion method contained the mecA gene. MecA gene was also detected in three coagulase-negative staphylococci isolates.


Introduction
Gram-positive coccal bacteria are normally found as a part of the human upper respiratory tract (URT) microflora [1]. However, due to several factors like age, immunity, social and economic factors, recent drug treatments and climate changes, those opportunistic colonizers may turn into serious pathogenic microorganisms causing severe diseases that may lead to human death [2,3].
Staphylococcus species are considered to be opportunistic pathogens mainly of the skin and nasopharynx as their most favourable reservoirs [4,5]. Among Staphylococcus species, Staphylococcus aureus is believed to be significantly responsible for causing severe respiratory infections in children from various regions of the world [6][7][8]. They can also be carried asymptomatically with varying rates in the nasopharynx of some children [9][10][11].
Although there are many antibacterial agents used against S. aureus infection still, the bacterium managed to develop several mechanisms such as the methicillin resistance mechanism to counteract them [12][13][14]. There has been an alarming increasing rate of health care, community and livestock-associated infections linked to methicillin-resistant S. aureus (MRSA) worldwide [8,[13][14][15]. Such huge increase in MRSA strains isolation is mostly related to the massive administration of antibiotics in medical settings, as well as in food manufacture and animal caring facilities for several decades [16].
Methicillin resistance is associated with a specific type protein that alters penicillin binding sites (PBP2a) on bacterial cell wall hence, reducing their affinity to bind β-lactam antibiotics [17]. Such resistance to methicillin is determined by the presence of mecA gene on a genetically mobile element known as staphylococci cassette chromosome mec (SCCmec) which carries several functional genes, all together play an important role in staphylococci pathogenesis [18].
Customary culture based methods is still considered as the standard conventional microbiological procedure used for the isolation and detection of S. aureus showing resistance to methicillin, but at the expense of time. Since such technique involves the isolation of S. aureus on selective media, followed by methicillin resistance confirmation using drug testing assay, which could take all together up to 48or 72 h [19,20]. Molecular based techniques were developed to reduce time and increase the sensitivity of detecting MRSA. [21]. Multiplex PCR assay has been recently established as a suitable substitute for the rapid detection of MRSA, decreasing the examination time to approximately 18 h [22][23][24].
The 16S rRNA gene of staphylococci was found to contain highly conserved DNA sequences at the genus level hence, separating them for other bacterial genera. Since then, several primers have been designed for the detection of staphylococci using their 16S rRNA gene. Staph-756F and Staph-750R 16S rRNA gene primer pairs were previously proven to be one of the most successful in staphylococci identification [25][26][27][28].
Additionally, nuclease gene (nuc) was reported as a specific target gene for detecting S. aureus [23,29]. Also, methicillin-resistance mecA gene has been designated as a molecular marker gene extensively used for MRSA detection and typing [30][31][32]. Therefore, the combined use of all 16S rRNA, nuc and mecA gene primers in a single multiplex PCR assay could provide a reliable, rapid and accurate detection of MRSA.
This study aimed to screen boys for asymptomatic carriage of potential bacterial pathogens such as methicillin-resistant S. aureus and detect its prevalence, taking into consideration the geographical bio-map that shows the levels of microbial pathogens in several districts. Furthermore, to compare the results obtained from traditional culture method which is considered as the traditional base for the detection of MRSA with that of the multiplex PCR method used for the amplification of the genes encoding the 16S rRNA and nuclease (nuc), as well as (mecA) gene for methicillin resistance determination.

Population under study
The current study was conducted during autumn and winter seasons of 2016, for a period of 3 months (from beginning of October till end of December). The study population included double sets of school boys comprising 405 elementary students of age group 7-12 and 10-12 years. This total number of 405 school boys was selected from 8 governmental schools at different geographic positions, all located within Al-Madinah city, Kingdom of Saudi Arabia (Table 1 and Figure 1). Students suffering from any respiratory infection symptoms or were taking antibiotic treatment during the period of sampling were excluded from the study. Verified consent was attained from the principals of the chosen schools, parents and participating students before sample collection.

Sample collection and isolation
The specimens were collected as a single nasopharyngeal swap taken from each participant using calcium

DNA extraction
The identity of the isolates was confirmed using molecular approaches as follows: bacterial colonies showing hemolytic activity were inoculated in 10 ml Luria-Bertani (LB) media (Difco, USA) and kept overnight at 37°C for genomic DNA extraction. After incubation, genomic DNA was extracted using DNA purification kit (Promega, USA) as recommended in the protocol. An acceptable DNA concentration ( ∼ 5 μg/ml) and purity (A 260 /A 280 ratio of 1.8) was determined by UV-visible spectrophotometer (Jenway, UK).

Amplification of 16S rRNA genes
The amplification of 16S rRNA genes was carried out using universal 8F forward primer and 1492R reverse primer [33], in an automated thermal cycler (Applied Biosystems, USA). PCR amplification cycles were conducted according the following conditions: initial denature at 94°C for 5 min, then 30 cycles of denature at

Restriction enzyme analysis of the amplified 16S rDNA
To detect sequence variations among characterized isolates, restriction analysis was carried out on each of the amplified ribosomal DNA PCR product. The two double digestion reactions with both Hae III and Taqα I four base cutter restriction enzymes in the first and EcoR I and BamH I six base cutters in the second (New England Biolabs, USA), were conducted for 3 h at 37°C, then digestion was stopped for both enzymes by incubating the reactions at 80°C for 20 min. Restriction fragments were separated on 3% agarose gel and observed with the gel documentation machine (Gel Doc XR, biorad, UK). Generated restriction patterns were analysed with PyElph 1.3 software using the clustering algorithm of un-weighted pair group method of arithmetic average (UPGMA) and dice pair-wise coefficient of similarity. Bacterial isolates with similar pattern were grouped together.

DNA sequence analysis and phylogeny
Representative isolates with different ARDRA pattern were selected for DNA sequencing using Big-Dye chain terminator cycle sequencing [34] at Macrogen Inc., Seoul, Korea. FinchTV application (Geospiza, Inc.) was used for viewing and editing DNA sequencing data. The sequences of the assembled 16S rRNA were used in a blast search analysis against the nucleotide database (http://www.ncbi.nlm.nih.gov/BLASTP/) to identify the closest similarities to the DNA sequences published in the GenBank database. Sequences showing high similarity ( > 97% identity) were retrieved from Gen-Bank and used along with obtained sequences to construct a phylogenetic tree. Clustal W1.83 XP was used for sequence alignments [35] and neighbourjoining method [36] for the construction of phylogenetic trees with MEGA6 software [37]. Sequences of the 16S rRNA genes from this study were submitted in GenBank (DDBJ) under accession numbers LC529200-LC529202.

Testing for methicillin resistance
To determine S. aureus isolates resistance against methicillin, Kirby-Bauer in vitro disc diffusion assay was performed with 1 µg oxacillin discs on Mueller-Hinton plates as instructed by the Institute of Clinical and Laboratory Standards. After 24 h of incubation, the presence or absence of inhibition zones was recorded and interpreted in accordance with CLSI (2015) guidelines [38].

Conventional multiplex PCR
Multiplex PCR assay using genomic DNA of Staphylococcus isolates as template was executed to target the 16S rDNA sequences specific to genus Staphylococcus, the S. aureus species specific gene (nuc) and the methicillin resistance gene (mecA). All primers used in this study are presented in Table 2  concentration adjusted to 2.5 mM). The amplification profile was conducted as described in previous reports [23,39], accordingly the conditions were as follows: first denature step for 5 min at 94°C, followed by 10 cycles of denature at 94°C for 45 s, annealing at 55°C for 45 s and extension at 72°C for 1 min, then another 25 cycles of 94°C for 45 s, 50°C for 45 s, and 72°C for 75 s and finally an extension step of 10 min at 72°C. After PCR program was completed amplicons were loaded into 2% agarose gel stained with 0.5 μg/ml ethidium bromide and electrophoresed along with 100 bp molecular weight marker. The resulting DNA fragments were observed and photographed using gel documentation machine.

Ethical agreement
The research plane was approved by the Ethics Committees of the Taibah University and the ministry of education, Kingdom of Saudi Arabia. Consent forms were provided to each participating student and his parent or guardian explaining all the relevant details regarding the study through the school staff. Investigators also made sure that students knew they can stop or withdraw at any time. All students' information and test results will be confidentially kept.

Isolation of bacteria from nasopharyngeal cavity of elementary school boys
This study included a total of 405 participating male students of two age groups, 134 (33%) belonged to first group 7-9 years of age (average = 7.8 ± 1.19 years, mean of 8 years) and 271 (67%) belong to the second group 10-12 years (average = 11.12 ± 1.1 years, mean of = 11 years) from eight schools located within Al-Madinah city. A total of 138 catalase positive bacterial isolates identified under microscope as Gram-positive cocci obtained from all 405 participating student showed blood hemolysis on sheep blood agar plates giving an overall carriage rate of 34.1%. Isolates were grouped according to their culture characteristics, colony morphology and colour, coagulase reaction and blood hemolysis into 12 distinct groups.

Characterization of isolates
Representative isolates from each phenotypic group were subjected to restriction enzyme analysis via Amplified Ribosomal DNA Restriction Analysis (ARDRA) to reveal genotypic characteristics. The amplification of 16S rRNA gene by PCR was followed by restriction analysis in two double digestion reaction using both HaeIII and TaqαI in the first reaction and EcoRI and BamHI in the second. The digestion of the 16S rDNA region generated a complex pattern of bands in which each sample yielded from two to eight major fragments ranging between 100-700 bp. The generated fragments displayed different pattern types and revealed an obvious polymorphism among the bacterial isolates. Cluster analysis using UPGMA algorithm revealed four distinct patterns among all 138 bacteria isolates ( Figure 2). The majority of the bacterial isolates (110) belonging to five phenotypic groups displayed a unique ARDRA pattern I. All of 110 isolates showed positive growth of yellow colonies typical of staphylococci on mannitol salt agar plates. The remaining bacterial isolates showed another three different and characteristic ARDRA patterns and therefore clustered at different clades: cluster II (12 isolates), cluster III (7 isolates) and cluster IV (9 isolates).

Molecular typing and phylogenetic affiliation
Representative isolates from each ARDRA cluster were subjected to sequence-based bacterial typing via 16S rRNA gene sequence analysis. DNA sequencing of the 16S rRNA gene provided a reliable identification of the isolates after a BLAST search against the non-redundant nucleotide database hosted by the GenBank. Analysis of almost whole 16S rRNA genes revealed four specific species, namely, Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus simulans and Staphylococcus hominis. The detailed sequence similarity results are all represented in Table 3. Phylogenetic studies confirmed the association of the identified genotypes to S. aureus, S. epidermidis, S. simulans and S. hominis clusters as observed through clustering of each species to its matching group. Based on 16S rRNA gene sequences analysis, the constructed phylogenetic tree successfully illustrated the affiliation between the selected isolates and their representative closest matches (Figure 3). Clusters were defined at SD > 70% similarity level.

Prevalence of Staphylococcus spp. in elementary school boys
The prevalence of potential asymptomatic 138 Staphylococcus spp. in the nasopharyngeal cavity of elementary school boys was investigated. Of all isolates, Staphylococcus aureus accounted for 110 (carriage rate of 27.2%), followed by Staphylococcus epidermidis accounting for 12 (carriage rate of 3%), Staphylococcus hominis for 9 (carriage rate of 2.2%) and finally Staphylococcus simulans for 7 (carriage rate of 1.7%) ( Figure 4A). Staphylococcus spp. were distributed in nasopharyngeal cavity of elementary school boys with different age groups by varying degrees. S. aureus was the predominant strain, whoever, S. epidermidis, S. simulans and S. hominis were significantly present especially in students from schools located at the northern area of Al-Madinah region. It is also worthy to mention that S. simulans was absent in school boys from the western area compared with the remaining six schools ( Figure 4B). This study also revealed that the number of Staphylococcus spp. was greater in participants of age group 7-9 years compared to age group of 10-12 years ( Figure 4C). Also, the prevalence of the bacterial species identified in this study (S. aureus, S. epidermidis, S. hominis and S. simulans) was higher in students with age group 7-9 years compared with age group of 10-12 (Table 4).

Identification of isolates showing resistant to methicillin
For the detection of methicillin resistance among isolated strains along with the validation of DNA sequencing results, all 138 bacterial isolates were examined using multiplex PCR method. Results indicated that each of the primer pairs used in the reaction amplified the single-target gene amplicons that corresponds to the expected sizes ( Figure 5A). The nuc and 16S rRNA genes were only amplified from the 110 S. aureus isolates by multiplex PCR. These results corresponded precisely with those from DNA sequencing, proving the reliability of the multiplex PCR. The methicillin resistance mecA gene was detected in 22 (20%) S. aureus   South  14  31  10  7  8  1  -1  7  ---E1  East  14  36  11  8  9  1  1  -8  ---E2  East  16  29  13  11  10  1  1  1  9  2  --W1  West  18  42  10  7  8  1  1  -6  -1  -W2  West  22  38  5  3  2  1  2  -3  ---Total  134  271  80  58  59  8  8  5  51  4  strains out of the 110 isolates which coincided with their phenotypic characterization as MRSA isolates by the Kirby-Bauer method. When the DNA from Staphylococcus species other than S. aureus were examined by multiplex PCR, amplified PCR products were generated from the primers of the 16S rRNA gene only and not from those of the nuc gene. Two strains of S. epidermidis and one of S. simulans showed a positive PCR amplification of mecA gene hence detecting methicillin resistance in these two Staphylococcus species ( Figure 5B).

Discussion
The human nasopharynx is occupied by a large number of bacteria some of which are commensal while others are considered to be potential pathogens [40].
Genus Staphylococcus comprises many unique species of which, S. aureus and other certain coagulase-negative staphylococci species have been implicated as main causal agents of community acquired as well as nosocomial infections. The coagulase-negative S. epidermidis, S. hominis and S. simulans species isolated in this study have been frequently reported to be involved in human infections, with S. epidermidis accounting for most infections [41][42][43][44][45][46].
So far only limited number of studies was conducted to highlight the nasal carriage of pathogenic Staphylococcus spp. among children in KSA. Instead, most of the research regarding staphyloccocal species in KSA mainly focused only on infections caused by methicillin resistant S. aureus in the outpatient clinics of tertiary care hospitals for children [47][48][49]. Hence, we the authors believe that none of the previous studies discussed the asymptomatic distribution of Staphylococcus spp. nasal carriage among school children in KSA.
Colonization of the upper respiratory tract with potential Staphylococcus pathogens is very common in healthy children [50][51][52]. Our nasopharyngeal carriage study among the 405 elementary schools participants indicated that four blood hemolytic Staphylococcus spp. with carriage rate of 34.1% was detected among the participating school boys, while nasal cavities of the remaining participants (65.9%) did not bear any blood hemolytic bacterial species. When comparing this rate of carriage among the two age groups, we find the prevalence of nasopharyngeal colonization by each species to be affected by the student's age. All four Staphylococcus spp. were slightly more prevalent (mean of 53%) in younger students attending schools and less prevalent (mean of 47%) in students of grades 4-6. Of the four species, Staphylococcus aureus was the most highly detected (79.7%) in both age groups of the elementary school boys. These carriage patterns coincide with most of the preceding studies showing the younger age group to have a higher carriage rate than the older ones in elementary schools [50][51][52][53]. Such findings propose that school children in this age group (7-9 years) have higher exposure risk most probably because of their less evolved adaptive immune response. This adaptive immunity is usually stimulated as a result of immunological memory over time, hence, providing an increased protection for young adult students of age groups 10-12 years [54]. It is fairly known that the results of epidemiological studies vary significantly from one study to another. The difficulty to compare the colonization rates of potential respiratory pathogens between different studies is related to the influence of numerous factors apart from the age, such as geographical area, climate, sampling technique, level of immunity, social and economic conditions [55,56]. Still, in contrast to many studies that focused on young children under 2 years of age, who frequently have high prevalence of Staphylococcus spp. carriage [47][48][49]56], this study paid more attention to two age groups that have been predisposed to the crowded urban life environment represented by elementary school boys in grade 1-3 (7-9 years) and older boys in grades 4-6 of school (10-12 years).
This study demonstrated that the colonization rate of Staphylococcus spp. varies according to the geographical location of the school. Furthermore, it was noticed that the lowest carriage number of Staphylococcus spp. was isolated from participants attending the two schools located in the west part of Al-Madinah city (18%) while higher carriage numbers were isolated from the remaining six schools (23-31%). The reason behind these differences may be related to the poor infrastructure of the schools as well as their overcrowding with students. Therefore, students in these schools are expected to have close contacts during most of the school activities which will subsequently lead to a higher risk of respiratory pathogens transmission among them. Another explanation may be related to the standards of living and socioeconomic status since school boys of the west part of Al-Madinah are more likely to be from higher social as well as economic status which may reflect on their proper hygiene practices. One drawback of this study was the low number of participating students (only 25 participants) from one of the schools located in the northern area of Al-Madinah city. This occurred due to the fact that some of the participating students' parent or legal guardians hesitated after signing the consent forms, and since the authors made sure to inform the students as well as parent or legal guardians that they can stop or withdraw at any time, the results regarding the previous group of participants had to be excluded from the study. Still, the samples that were successfully collected from 60 participants in a second school that shares the same geographical location (reaching a total of 85 participants) has reassured the authors that an accurate investigation of the carriage was properly undertaken.
Collectively, the epidemiological data from this study supports that there is a significant increase in S. aureus carriage among the participating students. Such result has also been acknowledged in previous studied since S. aureus is well known to primarily reside in the anterior nares. Therefore, S. aureus carriage is more frequently detected (36-65%) in nasal, oral or nasopharyngeal swabs [57]. On the other hand, the different carriage rate among coagulase-negative staphylococci in this study may involve a multifarious combination of factors that influence the participants exposure to a specific bacterial species, most important are participants immune responses and receptor-binding sites as well as direct competitive interactions between coagulasenegative staphylococci isolated in this study and other bacterial species in the same local niche [58].
Since S. aureus was the most frequently isolated staphylococcal bacteria in this study, a simple and rapid technique for identifying as well as discriminating of S. aureus from other coagulase-negative staphylococci was accomplished through conventional multiplex PCR. Amplifying the 16S rRNA and nuclease (nuc) encoding genes allowed the accurate identification of S. aureus in a single step. The results of the conventional multiplex PCR performed in this study, confirmed the specificity of the 16S rRNA gene primer pair in amplifying all Staphylococcus species and the nuc primers specificity for S. aureus isolates as reported previously [21,23,34,39].
The detection of S. aureus isolates showing resistance towards methicillin is essential especially when it involves the frequently targeted subjects (school age children) by these bacteria as reported in this study as well as several other studies [50][51][52]. The importance of such screening for MRSA will be reflected upon both, proper patient care through a sufficient supply of effective antibacterial drugs and infection control by restraining the use ineffective classes of antibiotics. The examination of MRSA can be done either phenotypically by Kirby-Bauer disc diffusion method or genotypically through detecting the presence of mecA gene which normally indicates a possible resistance to beta-lactam antibiotics.
In this study, it was found that all 20% (22/110) of the S. aureus isolates that were identified as methicillin resistant by the Kirby-Bauer method showed the presence of mecA gene. Such data indicates a close association between resistance towards methicillin and the presence of mecA gene in the tested isolates. Other studies on S. aureus isolates showed similar results in which most of the investigated isolates of MRSA certainly had mecA gene [59][60][61]. Such resistance towards methicillin along with all beta-lactam groups of antibiotics is related to a mutation that resulted in changing the normal antibiotic binding protein which resulted in lowering the binding affinity of beta-lactams to their site [62]. Additionally, during the same study mecA gene amplification was identified in two S. epidermidis isolates and one S. simulans isolate. The detection of mecA gene in Staphylococcus species apart from S. aureus was previously confirmed [63,64].

Conclusion
In view of the findings presented in this study, asymptomatic nasopharyngeal colonization by blood haemolytic staphylococcal especially S. aureus among elementary school boys was found to be high. Unfortunately, those asymptomatic carriers are considered to be a potential threat to other healthy students, emphasizing the need to develop crucial approaches in order to reduce the carriage of such potential respiratory pathogen. Hence, constant screening for carriers is necessary to minimize the transmission of respiratory infections as well as the occurrence of bacterial pathogens among school students.
The examination of the antimicrobial susceptibility pattern among the isolated blood haemolytic staphylococcal in this study revealed an epidemiological shift in S. aureus isolates with 20% of them showing mecA gene occurrence accompanied by resistance towards methicillin which prompts the need for sufficient control measures to avoid the spread of these MRSA isolates. The multiplex-PCR based identification technique used in our study has proven to be both quick and precise for the differentiation between coagulase-positive and negative staphylococci as well as the detection of MRSA isolates that might be circulating in the schools.
Based on the previous data, surveillance programs must be carried out in different parts of KSA for a better understanding of the URT colonization rate by the targeted bacteria and the frequency of successive respiratory infection. This may be important for future prediction of any changes in the epidemiology of URT infectious staphylococci and will help implementing accurate guidelines for control strategies of potential pathogenic bacteria colonizing the URT. their collaboration in providing us with the required permissions necessary for sample collection. The authors also extend their sincere thanks to all staff members of Biology Department College of Science, Taibah University who allowed us to use all the equipments and materials needed to complete our research. Finally, the authors are grateful to all those who participated in this study during data collection: the directors, administration staff, teachers, students and their legal guardians of the eight respective elementary schools in Al-Madinah city. It was our pleasure to be associated with them through this study.

Disclosure statement
No potential conflict of interest was reported by the author(s).

Author contributions
Each one of the authors has made an equal contribution in conceptualizing and implementing the methodology, analysing the data as well as writing and revising the manuscript.