Prevalence, risk factors, phenotypic and molecular characteristics for methicillin-resistant Staphylococcus aureus carriage in community-based drug users in Guangzhou, China CURRENT STATUS: POSTED

Background: Staphylococcus aureus ( S. aureus ) and methicillin-resistant Staphylococcus aureus (MRSA) remained the predominant cause of infections in drug users. The cross-sectional study aimed to elucidate the prevalence, risk factors, phenotypic and molecular characteristics of S. aureus and MRSA carriage among community-based drug users. Methods: Eligible drug users were asked to complete questionnaires and collect nasal swabs during May and December 2017 in Guangzhou, China. Swabs were processed for identification of S. aureus and MRSA. Antimicrobial susceptibility test and polymerase chain reaction assays were used to detect phenotypic and molecular characteristics for identified isolates. Univariate and multivariate logistic regression analyses were used to assess risk factors for S. aureus and MRSA carriage. Results: Overall, the prevalence of S. aureus and MRSA carriage in 353 drug users were 15.01% and 6.79%, respectively. Cohabitation was a risk factor for S. aureus (adjusted OR=8.80, 95% CI: 1.89-40.99) and MRSA (adjusted OR=14.30, 95% CI: 2.67-76.46) carriage. The proportions of multidrug resistance were respectively 72.41% and 89.47% for S. aureus and MRSA isolates and were simultaneously resistant to penicillin, erythromycin and clindamycin. The results of clonal complexes and sequence types for S. aureus and MRSA isolates were diverse. The proportions of virulence genes were high for MRSA isolates. Conclusion: The prevalence of S. aureus nasal carriage was lower while the prevalence of MRSA nasal carriage was moderate. Phenotypic and molecular characteristics of MRSA isolates revealed serious antibiotic resistance, indicating the cross-circulation of MRSA isolates, and imply high opportunity of virulence-related diseases. Decolonization might be considered for drug users with MRSA carriage, especially for those with risk factors.

Based on the latest World Drug Report, an estimated 271 million people aged 15-64 had used drugs in the previous year, while 35 million people are estimated to be suffering from drug use disorders in 2017 [4]. Obviously, illicit Drug use is a global public health problem. In recent studies, the prevalence of S. aureus and MRSA carriage among drug users is higher compared to the general population [5,6]. The phenotypic-molecular characteristics of S. aureus and MRSAcarriage in drug users were little reported. Most of these studies were conducted in developed countries, including the United States of America, Canada, and European countries.
According to what we mentioned above, it is necessary to investigate the epidemiology of S. aureus and MRSA carriage among drug users, in China. Therefore, in this study, we aimed to elucidate the prevalence, risk factors, phenotypic and molecular characteristics for S. aureus and MRSA nasal carriage in community-based drug users in Guangzhou, China.

Ethics statement
The study was approved by the Ethics Committee of Guangdong Pharmaceutical University, and it was performed in accordance with the approved guidelines. Written informed consent were obtained from all participants.

Study design and participants
A cross-sectional study of S. aureus and MRSA nasal carriage among drug users was conducted between May and December 2017 in three communities, Guangzhou, China. Participants had drug use in the previous 12 months were voluntarily recruited in the study. Those participants were with psychiatric illness or acute diseases were excluded. A face-to-face questionnaire was used to collect relevant information, including demographics (age, sex), socio-related characteristics (employed status, living conditions, income level, history of homelessness, and history of incarceration), behavior (history of sex and the number of sexual partner), health-related characteristics (human immunodeficiency virus (HIV) status, hepatitis, antibiotic use, skin infection, hospitalization, and history of needle exchange), and period and route of drug use.

Isolation and identification of S. aureus and MRSA
After completing the questionnaire, trained personnel collected swabs from both anterior nares of participants. The swabs were soaked in enrichment broth at 4 °C during transportation, and incubated for 24 hours for further experiments. The swabs were then transferred to mannitol salt agar for 24-48 hours incubation. Samples were identified as S. aureus isolates when they had specific colony morphology and were positive for gram staining, catalase reaction, hemolysis test, DNase test, coagulase tests, and 16S rRNA and nuc genes. Those S. aureus isolates that were resistant to cefoxitin and positive for mecA gene were identified as MRSA isolates. More details were described previously [7].

Phenotypic characterization
The antimicrobial susceptibility of all S. aureus isolates was determined by the disk diffusion method.
The following antibiotics were tested: clindamycin, erythromycin, penicillin, linezolid, gentamicin, teicoplanin, moxifloxacin, trimethoprim-sulfamethoxazole, rifampin, chloramphenicol, and tetracycline. The reference S. aureus strain ATCC 25923 and ATCC 29213 were respectively used for quality and positive control. We classified the isolates as susceptible or resistant to each antibiotic.
Those isolates resistant to ≥3 classes of antibiotics were identified as multidrug resistant (MDR) [8].

Statistical analysis
The data were entered using Epidata 3.1 (EpiData Associa-tion, Odense Denmark) and exported to Stata 14.2 (College Station, Texas, USA) software for further analysis. We assessed the associations between non-S. aureus and S. aureus /MRSA carriers by the following methods. Univariate analyses were used by Pearson's chi-squared test or Fisher's exact test when appropriate. Multivariate logistic regression models were then used to assess the adjusted odds ratios (aORs) and 95% confidence intervals (CIs). Potential confounding covariates were adjusted in the models. A two-sided P-value of ≤0.05 was defined as statistical significance.

Prevalence of S. aureus and MRSA carriage
A total of 353 drug users were eligible for inclusion in the study. The prevalence of S. aureus nasal carriage in drug users was 15.01% (53/353). Twenty-nine drug users carried with MDR S. aureus and the prevalence was 8.22%. The prevalence of MRSA carriage was 6.79% (24/353). Seventeen drug users were with MDR MRSA carriage and the prevalence was 4.82%.
A total of four SCCmec types were detected from the 24 MRSA isolates, in which 12 isolates were type IVd, five were type IVa, one was type V, one was type II, and five were non-typeable ( Figure 2).

Discussion
To our knowledge, this is a relatively comprehensive study which contributes to the prevalence, risk factors, and phenotypic-molecular characteristics for S. aureus and MRSA nasal carriage among community-based drug users in China. The prevalence of S. aureus carriage in the study (15.01%) is lower than previously reported estimates ranged from 19.79% to 45.05% [5,[9][10][11][12]. Participants of those previous studies were injection drug users. In the study, however, only 64.02% of participants had history of using infection drugs in previous 3 months. Additionally, we found that a majority of long-term drug users who took drugs by snorting had few vibrissae. This might also be a potential factor leading to a low prevalence of S. aureus carriage. Further studies need to explore in the future.
The prevalence of MRSA nasal carriage (6.79%) in the study is similar to observed studies in other countries [5,[13][14][15]. Notably, the proportion of MRSA in S. aureus isolates were higher than observed studies [5,15].
In the present study, we found that cohabitation was a risk factor for S. aureus carriage and MRSA carriage in drug users, which is different from other study [5]. One of the possible reasons might be that most drug users cohabitated with other drug users. This could provide more opportunities for sharing drugs [12]. HIV infection has been reported to be a risk factor for S. aureus carriage [16], however, we did not find any significance. This could be caused by the limited number of drug users with HIV infection. Therefore, further studies need to identify the risk factors for S. aureus carriage and MRSA carriage in drug users.
The proportions of antibiotic resistance on S. aureus and MRSA isolates are consistent to limited available studies [9,17,18]. However, the proportion of MDR MRSA (70.83%) is high. The most 8 predominant MDR pattern could partially demonstrate the high use of antibiotics in community-based drug users and provide evidence provides evidence that healthcare workers need to be more careful with selection of antibiotics for drug users. The high proportions of erythromycin and tetracycline resistance and low proportions of erythromycin-and tetracycline-resistance genes could demonstrate that resistant genes are always attributed to the presence of antibiotic resistance.
The proportions of virulence genes were higher in MRSA than in S. aureus isolates, suggesting the higher risks of MRSA isolates in casing virulence-related diseases, including Staphylococcal scalded skin syndrome, toxic shock syndrome, Staphylococcal food poisoning, etc. [19][20][21]. The proportions of virulence genes for MRSA isolates were higher than observed studies [7,[22][23][24]. The results implied that drug users with MRSA carriage harbouring virulence associated genes, might have higher risks for relevant disease and should be paid greater attention to.
The results of CCs and STs for S. aureus and MRSA isolates could demonstrate the multiple transmissions among human beings, livestock and environment, which are similar to previous studies [7,22]. Although we included community-based drug users in the study, according to the results of SCCmec types we could know the source of MRSA isolates were from both communities and healthcare settings, which is similar to observed studies [5,10]. MRSA isolates could circulate between communities and healthcare settings and this might be a potential risk for other populations. Therefore, relevant decolonization methods could be used for drug users with MRSA carriage, which would help prevent further MRSA circulation.
Our study contributes to the prevalence, risk factors, phenotypic and molecular characteristics for S. aureus and MRSA carriage among drug users in China. Despite of the strengths, there are several limitations. Firstly, is was a cross-sectional study, thus, we could not determine the persistence of S. aureus and MRSA carriage. Secondly, we only obtained cultures from the anterior nares, which may lead to an underestimation of the prevalence of S. aureus and MRSA carriage. Finally, the generality of this study is limited owing to the small number of drug users.

Conclusion
In summary, the prevalence of S. aureus nasal carriage was lower while the prevalence of MRSA nasal carriage was moderate among community-based drug users, China. Cohabitation is a risk factor for S.