Prevalence and molecular epidemiology of methicillin-resistant Staphylococcus aureus colonization with and without methicillin-resistant coagulase-negative Staphylococci in HIV-infected patients

Background The global prevalence of methicillin-resistant Staphylococcus aureus (MRSA) colonization in human immunodeciency virus (HIV)-infected patients is increasing, but data about it was limited in mainland China. This study aims to investigate the epidemiology of MRSA nasal colonization among HIV-infected patients in mainland China and also evaluate the impact of methicillin-resistant coagulase-negative Staphylococci (MRCoNS). Methods This cross-sectional study was designed to collect nasal samples and individual information for HIV-infected participants. Risk factors and phenotypic and molecular characteristics among those with MRSA colonization comparing those with and without MRCoNS co-colonization were analyzed. Results We found 119 (11.89%) out of 1001 HIV-infected patients were colonized with MRSA, including 41 (4.10%) with MRCoNS and 78 (7.79%) without MRCoNS. Having a history of respiratory tract infection in the previous 6 months(adjusted OR=1.64, 95% CI: 1.04-2.57) and male gender (aOR=3.92, 95% CI: 1.20-12.85) were risk factors for the overall MRSA and MRSA without MRCoNS colonization, respectively. No risk factor was associated with co-colonization of MRSA and MRCoNS. The proportions of antibiotic resistance and toxin genes as well as the distribution of molecular types for MRSA isolates were equivalent between subjects with and without MRCoNS isolates (P-value ≥ 0.05).

agents for nosocomial bloodstream infection in humans, especially immunocompromised patients.
Studies suggested methicillin-resistant CoNS (MRCoNS) as a source of mecA, the methicillin resistance gene, and has the potential contribution to the emergence of MRSA [7]. Studies also reported the horizontal cross-propagation of resistance genes could lead to the co-colonization of MRSA and MRCoNS in humans [8]. In other words, MRCoNS might affect the risk of MRSA colonization.
Study have shown that the global and regional prevalence of MRSA colonization in HIV-infected patients increasing [9]. Moreover, the epidemiology of MRSA in HIV-infected patients have been explored in Taiwan, China [10][11]. But data on the prevalence and molecular characteristics of MRSA isolated among HIVinfected patients was limited in mainland China. we thus aimed to investigate the prevalence and molecular epidemiology of MRSA colonization among this vulnerable population in mainland China and also examine the impact of MRCoNS by risk factors as well as phenotypic, genotypic and molecular characteristics on MRSA colonization.

Study design and population
HIV-infected participants were enrolled from a large public HIV clinic with a population of more than 9000 of HIV-infected patients, about 90% of all HIV-infected patients in Guangzhou city, that is a liated with the Guangzhou Eighth People's Hospital. Patients in the clinic were eligible for enrollment if they were ≥ 18 years of age and agreed with participation. The study was approved by the Ethics Committee of Guangdong Pharmaceutical University. All participants signed an informed consent.

Data collection
Nasal cultures were obtained from enrolled participants, and a face-to-face questionnaire was administered. The questionnaire included demographics (age, gender, martial status and education level), behavior-related information (smoking status, number of people living in the household, use of shared items and living with mammalian pets), details of community-based risk factors (location and type of residence, sexual behavior, street drug abuse and incarceration), medical information (hospitalization, surgeries, skin infection and respiratory tract infection) and HIV-related information (CD4 count, viral load, trimethoprim-sulfamethoxazole prophylaxis, antiretroviral therapy, antifungal medication and tuberculosis medication). Nasal swabs were obtained by swabbing both nares of participants for further experiments.

Laboratory processing
Swabs were inoculated into 7.5% NaCl broth and incubated overnight at 37℃ for enrichment. Isolates were identi ed as Staphylococci if they were positive for chromogenic reaction in sodium chloride mannitol medium, Gram staining, catalase test and hemolysis test. All Staphylococci isolates were then classi ed into Staphylococcus aureus (S. aureus) and CoNS by the coagulase test. Both S. aureus and CoNS isolates were tested for antibiotic resistance by using Kirby-Bauer disk diffusion method based on the 2017 Clinical Laboratory Standards Institute guidelines. Twelve antibiotics were tested, including cefoxitin, penicillin, erythromycin, clindamycin, tetracycline, rifampicin, macrodantin, moxi oxacin, trimethoprim-sulfamethoxazole, teicoplanin, linezolid and gentamicin. Isolates resistant to cefoxitin were con rmed as methicillin resistant. Genotypic analysis with pulsed-eld gel electrophoresis was performed on all con rmed MRSA isolates, including the Panton-Valentine leukocidin gene (pvl), two exfoliating genes (eta and etb) and the toxic shock syndrome toxin gene (tst) [12]. Staphylococcal cassette chromosome mec (SCCmec) typing [13] and multilocus sequence typing (MLST) [14] for all MRSA isolates were also analyzed.

Statistical analysis
Frequencies and proportions were calculated to describe the characteristics of HIV-infected patients by MRSA colonization. Risk factors for MRSA colonization was examined by using logistics regression analysis. Signi cant factors in univariate analysis were selected into multivariate models. Differences of phenotypic and genotypic characteristics for MRSA isolates between with and without MRCoNS were examined by Pearson Chi-square test or Fisher's exact test. Two-sided P-values < 0.05 were considered as statistically signi cant. All statistical analyses were performed using Stata v15.1 (Collage Station, TX). The sequence type relationship of MRSA isolates between with and without MRCoNS isolates was visualized using Phyloviz 2.0 (http://www.phyloviz.net/).

Participant characteristics
A total of 1001 HIV-infected patients were eligible and enrolled. We found 119 (11.89%) patients were with MRSA colonization, including 41 (4.10%) with MRCoNS and 78 (7.79%) without MRCoNS (Fig. 1). The participant characteristics for MRSA colonization are shown in Table 1. The mean age was 37.24 (SD, 11.35) years, and 84.42% of the study population was male. Most patients (91.41%) had antiretroviral therapy. The median and interquartile range of CD4 count was 330 (500-1022) cells/mL, and the median and interquartile range of viral load was 20 (20-1630000) copies/mL. Only 6.6% and 5.9% of our patients had antifungal and tuberculosis medication by self-report. The proportions of diagnosis with skin and respiratory tract infections in the previous 6 months were 44.58% and 67.47%.

Discussion
As the rst study to determine epidemiology of MRSA nasal colonization for HIV-infected patients in mainland China, we found the prevalence (11.89%) was higher than the result of a global meta-analysis which showed the pooled worldwide prevalence was 7% (95% CI 5%-7%) in adult HIV-infected patients [9].
Moreover, the prevalence in our study was also higher than other high-risk populations such as dialysis patients (6.2%; 95% CI 4.2%-8.5%) [15], men who have sex with men (1.6% ; 95% CI: 0.5-2.6%) [16] and injection drug users (7.4%) [17]. This high prevalence of MRSA colonization could be explained by high rates of antibiotic use and not effective infection control programs. Further studies need to con rm the reasons.
We found HIV-infected patients with diagnosis of respiratory tract infection in the previous 6 months were a more likely to have MRSA nasal colonization, which could be associated with the more frequently respiratory episode occur in HIV-infected population and bacteria colonize in the nasal cavity as the respiratory barrier weakens [18]. Male gender was a risk factor for MRSA colonization without MRCoNS, which was consistent with the previous results [19,20]. It may be related to the fact that 428 MSM out of 843 men recruited for this study (data are not shown), study reported that MSM was found to have a high rate of MRSA infection in other contexts regardless of HIV status [21]. According to some studies, young people have higher risks of bacterial colonization [20,22], however, we did not nd the same result in this study.
High proportions of resistance to penicillin, erythromycin, clindamycin and tetracycline were found in MRSA isolates, which is similar to observed studies [23][24][25]. This nding suggests medical professionals should pay more attention to the rational use of these antibiotics.
The detection rates of toxin genes for MRSA isolates in HIV-infected patients were lower than hospital patients [26][27][28][29], but similar to general populations [30][31][32]. Base on the results of SCCmec typing we found most MRSA isolates in this study were community-associated, which was similar to other studies [33,34]. However, hospital-associated SCCmec types were also found in this study, indicating a cross transmission between the community and hospital facilities. Moreover, the dominant STs (ST5, ST45, ST59 and ST188) in this study were consistent with other community-based populations [26, [35][36], but some other STs were also previously reported in hospital patients (ST338 and ST1) [37][38][39] and animals ( ST398 and ST1) [40][41][42]. The MLST ndings also indicated the cross transmission between communities and hospital settings.
Comparisons of characteristics, including antibiotic resistance and toxin genes as well as results of molecular typing, for MRSA isolates between with and without MRCoNS isolates were of no signi cant difference, suggesting the phenotypic and molecular characteristics of MRSA colonization were not affected by co-colonizing with MRCoNS.  Declarations YZJ conceptualized the study. LY and LJL were responsible for data curation, formal analysis and wrote the original draft. LLH and CWP were responsible for resources and supervision. WYY and YJP were responsible for validation and visualization. HSP and ZWC were responsible for investigation and Laboratory operation. YZJ and YXH reviewed and edited the original draft.All authors read and approved the nal manuscript.

Funding
This research was funded by Guangdong Science and Technology Plan Project (grant number 2014A020213013) and National Natural Science Foundation of China (grant number 81973069, 81602901).

Availability of data and materials
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Ethics approval and consent to participate
This study was approved by the Ethics Committee of Guangdong Pharmaceutical University. ll participants signed an informed consent.