School environmental contamination of methicillin-sensitive Staphylococcus aureus as an independent risk factor for nasal colonization in schoolchildren: An observational, cross-sectional study

Objective We aim to assess the similarities of proportional, phenotypic, and molecular characteristics between the school environment and schoolchildren on methicillin-sensitive S. aureus (MSSA) isolates. Methods A cross-sectional study was conducted between March 2016 and August 2016 in eight elementary schools in Guangzhou, China. Nasal swabs from students and environmental swabs from school environments were collected. Univariate and multivariate logistic regression analyses under a multistage stratified cluster cross-sectional survey design were performed to access the prevalence relationship and influencing factors, respectively. Phenotypic and molecular characterizations of MSSA isolates were conducted using the Kirby-Bauer disk diffusion method and polymerase chain reaction assays, respectively. Results In total, 1705 schoolchildren and 1240 environmental samples from 40 classes in eight elementary schools obtained between March and August 2016 were include in this study. The rates of MSSA prevalence among schoolchildren and the environment were 11.44% (195/1705) and 4.60% (57/1240), respectively. The odds ratios and 95% confidence intervals (CIs) on the prevalence of MSSA isolates were 1.11 (95% CI, 1.05–1.29; P = 0.010) and 1.04 (95% CI, 1.01–1.07; P = 0.003) for the school or class environment and students, respectively. Similar phenotypic and molecular characteristics were identified between schoolchildren and the environment. A cause and effect relationship could not be established because the study design was cross-sectional. Conclusions Because of the cross-sectional design, we can reveal the association between school environment and schoolchildren on MSSA, but it is not a cause and effect relationship.


Results
In total, 1705 schoolchildren and 1240 environmental samples from 40 classes in eight elementary schools obtained between March and August 2016 were include in this study. The rates of MSSA prevalence among schoolchildren and the environment were 11.44% (195/ 1705) and 4.60% (57/1240), respectively. The odds ratios and 95% confidence intervals (CIs) on the prevalence of MSSA isolates were 1.11 (95% CI, 1.05-1.29; P = 0.010) and 1.04 (95% CI, 1.01-1.07; P = 0.003) for the school or class environment and students, respectively. Similar phenotypic and molecular characteristics were identified between schoolchildren and the environment. A cause and effect relationship could not be established because the study design was cross-sectional. PLOS  Introduction selected using a multistage stratified cluster sampling design. First, we randomly selected three administrative districts from the urban region (Haizhu, Huangpu, and Huadu District) and one administrative district (Conghua District) from the rural region. Second, two elementary schools from each administrative district and eight schools were included. The buildings of these schools were constructed from reinforced concrete and brick and surrounded by trees. Third, the number of classes in grades four and five were proportional and randomly selected. All schoolchildren who obtained consent from their legal representatives were included in the study. Schoolchildren from grades four and five were selected given their better compliance and lighter academic burden in China. Schoolchildren who were absent were excluded. The study was approved by the Ethics Committee of Guangdong Pharmaceutical University, and performed in accordance with the approved guidelines of the Declaration of Helsinki. All legal representatives of participants signed an informed written consent form. We selected classrooms, toilets, and public places of each school for environmental sampling. We sampled five locations (floor, light switch, door handle, desk, chair, and air) of the classroom, four locations (floor, light switch, door handle, faucet, and toilet flush handle) of the two toilets (men's and women's), and three locations (handle of stairs, floor of stairs, and floor of corridor) of the public place from each class. Overall, we obtained 31 samples from each class. These locations are frequently touched by people, easily contacted by skin, and amenable to cleaning and disinfection. To avoid bias of sampling time, we performed sampling between two and four in the afternoon on weekdays.

Questionnaires and sampling
We collected information through questionnaires answered by parents of schoolchildren. Information regarding demographic characteristics, personal characteristics, characteristics of family members, and household characteristics was collected. The questionnaire was first prepared in English and then translated into the local language (Chinese), and back to English to ensure consistency. Sterile swabs (BBL Culture Swab; Becton Dickinson) moistened with sterile saline water were used to sample both nasal vestibules of the schoolchildren and surfaces of environmental locations by trained personnel. Floor, desks, and chairs were swiped in an approximately 10×10 cm 2 sample area given their regular surfaces. The entire surfaces of light switches, handles, and toilet faucets were swiped given their irregular surfaces. Each swab was placed into a sterile tube with 7.5% sodium chloride broth, and the tubes were transported to the laboratory immediately.
We used the natural sedimentation method to collect air samplings. We placed five mannitol salt agar plates on five desktops in each classroom (one plate in the center and four at the four corners of the classroom) ten minutes after the classroom became empty. After ten minutes of exposure, we closed the cover of plates and transferred them to the laboratory for further analyses. To ensure the acquisition of quality data and the quality of sampling, all investigators were trained by the principal investigator for three days before the survey, and random assessments were performed by the principal investigator. We conducted sampling during the afternoon (2-4 p.m.) on weekdays to avoid the bias of the time.

Laboratory methods
Details on our laboratory methods can be found in our previous study [19]. Air samples were incubated for 48 hours at 37±1˚C. The swabs were transferred to mannitol salt agar plates for a 24-hourincubation after a first 24-hour incubation at 37±1˚C. Samples, including air samples, were identified as S. aureus isolates through specific colony morphology and positivity for Gram-staining, catalase reaction, hemolysis test, DNase test, and coagulase test.
All S. aureus isolates underwent cefoxitin susceptibility testing using the Kirby-Bauer disk diffusion method, following the Clinical and Laboratory Standards Institute guidelines from 2015. S. aureus isolates that were sensitive to cefoxitin were identified as MSSA isolates. All MSSA isolates were then subject to specific antibiotic susceptibility testing using the Kirby-Bauer disk diffusion method and 11 antimicrobial agents: cefoxitin, penicillin, linezolid, gentamicin, teicoplanin, erythromycin, trimethoprim-sulfamethoxazole, moxifloxacin, rifampin, chloramphenicol, tetracycline, and clindamycin.
DNA was obtained from all MSSA isolates using the sodium dodecyl sulfate method [20] and were further tested to confirm the presence toxin genes [Tst, Eta, Etb, and Panton-valentine leukocidin (Pvl)] [21,22] using polymerase chain reaction (PCR) assays.
Multilocus sequence typing (MLST) was performed using previously published primers and conditions [23]. Allelic profiles, clonal complex (CC) types, and sequence types (STs) were assigned using the MLST database (http://www.mlst.net). Singletons or members of a clonal complex were determined using a related sequence types algorithm (http://eburst.mlst.net). Dendrogram analysis was performed based on STs to determine the clonal relatedness and potential epidemiologic origin.

Definitions of obesity
Anthropometric measurements were obtained using standardized techniques and calibrated equipment. Schoolchildren were weighted to the nearest 0.1 kg in light indoor clothing and bare feet or with stockings. Height was measured using a stadiometer. Schoolchildren stood in erect posture without shoes, and the results were recorded to the nearest 0.1 cm. Measures were obtained twice, and the average was considered in the analysis. Body mass index (BMI) was calculated as the ratio of weight in kilograms to the square of height in meters (kg/m 2 ). Age-and gender-specific BMI cutoff points recommended by the Working Group on Obesity in China were used to define obesity [24].

Statistical analysis
The significance of the difference between proportions was assessed using Pearson's Chisquare test for categorical variables. There were two clusters in the study; a class cluster and a school cluster. We drew linear prediction plots to intuitively elucidate the dose-response relationship of MSSA isolates between nasal colonization among schoolchildren and environment in class or school clusters. The association between influencing factors and MSSA nasal colonization was examined using multivariable logistic regression models. We performed multivariable logistic regression analysis of all variables with a P-value of <0.2 indication significance. We removed variables that were not significant at this level. All statistical measures were estimated using survey data analysis methods (SVY commands) from STATA package. A twosided P-value for statistical significance was defined as P-value of < 0.05. All analyses were performed using STATA 14.2 (College Station, Texas, USA).

Prevalence of MSSA in both schoolchildren and the environment
In total, 1705 schoolchildren and 1240 environmental samples from 40 classes in eight elementary schools were included in the study. The prevalence of MSSA nasal colonization among schoolchildren was 11.44% (195/1705). A significant difference in MSSA (school: χ 2 = 53.01, P<0.001; class: χ 2 = 95.23, P<0.001) nasal colonization was noted among schoolchildren in different schools and classes. More details of the characteristics of participants are presented in Table 1.

Influencing factors of MSSA nasal colonization among schoolchildren
We found that obese [odds ratio (OR), 2.01; 95% confidence interval (CI), 1.72-5.59; P = 0.029] and family members who were healthcare workers (OR, 0.45; 95% CI, 0.26-0.81; P = 0.028) were associated with MSSA nasal colonization among schoolchildren. To account for the potential confounding factors among influencing factors, we further analyzed the relationship between the potential predictors using a multivariate logistic regression model. This model demonstrated that when controlling for the effects of the other influencing factors (gender, region, allergic diseases, outpatient service, and had physical exercise every week), the relationships found in the univariable analyses changed. Obese and family members who were healthcare workers were no longer associated with MSSA nasal colonization. More specific details are presented in Table 1.

Relationship of MSSA isolates between schoolchildren and the environment
A positive relationship between MSSA isolates from the school or class environment and schoolchildren was observed. ORs and 95% CIs for the school or class environment and schoolchildren on the prevalence of MSSA isolates were 1.11 (95% CI, 1.05-1.29; P = 0.010) and 1.04 (95% CI, 1.01-1.07; P = 0.003), respectively.
Overall, the prevalence of MSSA contamination in the school environment of this study was not high. Nevertheless, there were positive prevalence relationships of MSSA isolates between the environment and schoolchildren. Of note, relevant departments should be aware of the hygiene in the entire school environment, including classrooms, toilets, and public places.
Our study contributes to the existing literature because we assessed a population with a high burden of obesity and MSSA colonization in which the association has not been examined in previous publications. Obesity was not a risk factor for MSSA nasal colonization among schoolchildren in the model of multivariate logistic regression analysis, which differed from other observed studies [10,32]. Our study revealed that higher body fat percentage was related to increased frequency of S. aureus colonization [33]. Obesity may perturb the microbiome of the anterior nares, rendering them more conducive to colonization by S. aureus [10]. Accumulating evidence suggests that the human microbiome is influenced by and influences different pathological states [34,35]. Other studies suggested that the interaction between site- specific microbiota and tissue-specific immune responses could hinder or propagate the proliferation of pathological species such as S. aureus [36,37]. Family members who were healthcare workers was not an influencing factor for MSSA nasal colonization in this study. This finding needs to be further explored.
Given that the proportions of antibiotic resistance in MSSA isolates from schoolchildren and the environment were not different, the antibiotic resistance patterns of MSSA isolates from schoolchildren and the environment were similar, which might indicate the homology of MSSA isolates between schoolchildren and the environment.
The proportions of toxin genes in MSSA isolates were increased compared with other observed studies [27,38], revealing increased toxicity of MSSA isolates in this study. Furthermore, the proportions of all toxin genes in MSSA isolates between schoolchildren and the environment were not significantly different, which might also indicate the homology of MSSA isolates between schoolchildren and the environment.
The dominant CC types and STs in this study were similar to other studies [27,39]. The findings regarding 22 CC types and 71 STs of MSSA isolates were not different between schoolchildren and the environment. In addition, 17 STs were found in MSSA isolates from both schoolchildren and the environment. Dendrogram analysis also revealed the homologous relatedness of MSSA isolates between schoolchildren and the environment.
Therefore, according to the positive relationship of MSSA isolates, prevalence between schoolchildren and the environment and the homology of MSSA isolates between schoolchildren and environment based on phenotypic, genetic, and molecular characteristics, we suggest that both the decontamination of MSSA in the environment and the increasing hygiene of schoolchildren could be beneficial to reduce the prevalence of MSSA nasal colonization among schoolchildren.
One of the strengths of this study is that the sample size is relatively large and representative. Given considerable variability in BMI, it enabled us to make robust estimates of the relationships. The relatively large number of schoolchildren with a high response rate yields high statistical power to infer the characteristics of the study population. Moreover, this study is the first to elucidate the quantitative association of isolates between schoolchildren and the environment. We also performed phenotypic and molecular characterization of MSSA isolates to further assess the relationship.
This study also has several limitations. First, we cannot establish a cause and effect relationship because the study design is cross-sectional. Second, one of the strengths of the study is the use of multistage stratified cluster regression analysis, which overcomes the limitations of the standard regression analysis previously performed. Finally, given limited financial support, we could not offer decolonization measurements to schoolchildren, and whether such intervention would improve health of schoolchildren could not be scientifically proven.

Conclusions
Despite these limitations, our findings suggest that both schoolchildren and the school environment were colonized with MSSA. Therefore, environmental decontamination, including enhancement of disinfection and cleaning, may be considered as strategies to decrease and prevent MSSA nasal colonization in schoolchildren.
Supporting information S1 Table.