Metro system in Guangzhou as a hazardous reservoir of methicillin-resistant Staphylococci: findings from a point-prevalence molecular epidemiologic study

Staphylococci are common causes of healthcare-associated and community-associated infections. However, limited data are available on the prevalence, phenotypes and molecular characteristics of Staphylococci in metro system around the world. 320 surface samples were collected from the Guangzhou metro system to isolate and characterize Staphylococci strains. Of the samples, 75.6% (242/320) were contaminated with Staphylococci. The Staphylococci isolates, especially the methicillin resistant isolates, were resistance to most of the antibiotics, with 79.8% (193/242) classified as multidrug resistant (MDR) strains. 8 strains of methicillin-resistant Staphylococcus aureus (MRSA) carried a range of staphylococcal cassette chromosome mec (SCCmec) types [I (1), II (3), III (2) and NT (2)]. Staphylococcus aureus isolates were classified into several ST types and showed possible cross transmissions of strains from various sources. All MRSA strains were positive for the qac gene, and only one methicillin-susceptible Staphylococci aureus (MSSA) strain was positive for the Panton-Valentine leukocidin (PVL) genes. This study demonstrated that environmental surfaces in the Guangzhou metro system may be a hazardous reservoir for transmission of Staphylococci to passengers. The resistance to antibiotics and disinfectants observed among isolates was also noteworthy.

The Guangzhou metro system has an average of 5 million passengers daily and the environment is highly enclosed, which creates an ideal setting for the accumulation and transmission of Staphylococci among passengers. In this cross-sectional study, the aims were to elucidate the prevalence, antimicrobial susceptibilities, and molecular characteristics of Staphylococci strains contaminating metro surfaces in Guangzhou, China.

Methods
Environmental sample collection. Surface sampling in the metro system was conducted in November of 2013. Environmental samples were collected from five predetermined locations: hand rails, seats, stanchions, Ticket Vending Machines (TVMs), and escalators. These locations were chosen because they are frequently touched and are more likely to be contaminated with bacteria. The sampling points covered 32 metro stations in 7 lines (line 1, 2, 3, 4, 5, 8 and APM).
Swabs moistened with saline were used to sample surfaces, and the sample area of each swab was approximately 10 cm × 10 cm. To streamline the collection and processing, we used the composite surface sampling method. Each swab was placed into a sterile tube with 7.5% sodium chloride broth and the tubes were transported to the laboratory immediately after sampling.
Isolation and identification. After 24 hours of incubation at 37 °C, the swabs were transferred to mannitol salt agar plates for another 24 hours of incubation. Colonies were identified as Staphylococci through morphology, color and catalase reaction. All Staphylococci isolates were further screened for β -haemolysm and were verified as S. aureus by tube coagulase testing. The strains were regarded as CoNS if they were negative for coagulase testing. Those Staphylococci strains that were positive for the mecA gene and/or resistance to cefoxitin were labelled as methicillin-resistant.
Detection of mecA, qac, Panton-Valentine leukocidin, and SCCmec typing. All Staphylococci isolates were further tested to confirm the presence of the mecA gene 12 . S. aureus isolates were tested for the presence of the qac gene, an anti-disinfectant gene, and the Panton-Valentine leukocidin (PVL) genes using polymerase chain reaction (PCR) assays 15 .
A multiplex PCR technique was used to confirm and type the staphylococcal cassette chromosome mec (SCCmec) gene 16 . The results were reported as types I-V, and those isolates that were not type I-V were deemed nontypeable (NT).
Multilocus sequence typing. The multilocus sequence typing (MLST) PCR assays were carried using previously published primers and conditions 17 . Allelic profiles and sequence types (ST) were assigned using MLST database (http://www. mlst.net). Dendrogram analysis was performed based on ST types to determine the clonal relatedness and potential epidemiologic origin.
Statistical analysis. Statistical analysis was conducted using Stata 13.0 (College Station, Texas, USA).
Data were analyzed using descriptive statistics and χ 2 tests. A P value < 0.05 was considered statistically significant, and all statistical analyses were 2-sided.

Discussion
To the best of our knowledge, this is the first systematic study to report the occurrence and characteristics of Staphylococci strains from environmental surfaces in a metro system of the world. In the current study, 11.25% of the samples were MRSA-positive (2.5%) and/or MSSA-positive (8.75%), whereas the majority (> 60%) of the environmental samples were positive for MRCoNS or MSCoNS. The MRSA isolation rate in our work is in similar to that from a Japanese study conducted on trains (2.5% vs 2.3%) 18 . However, considerably higher MRSA detection levels were noted in several previous studies. In two Portuguese studies, 26% (22/85) and 36% (72/199) of sampled buses were tested positive for MRSA contamination 19,20 . Additionally, an American study found that 14.8% (35/237) of the surfaces sampled from buses were contaminated with MRSA isolates 8 . The considerable differences in the reported value for MRSA prevalence could be affected by factors such as limited sampling locations, varying sampling techniques, and different regional hygiene measures.
The findings of drug resistance in this study are also noteworthy. The levels of resistance to some common antibiotics, such as penicillin, erythromycin and rifampicin, are alarming, especially among methicillin resistant strains. In addition, some isolates were even resistant to teicoplanin and linezolid, both of which are final effective agents against Staphylococci infections 21 . Even more intriguing is that most of the Staphylococci isolates displayed the characteristics of multidrug resistance, suggesting that they are more likely to have originated from healthcare-associated settings.
The results of the analyses of molecular features further broadened our insights into Staphylococci in a non-hospital environment. ST 188, the most predominant ST type of MSSA in our study, is widely disseminated in China and originated from community and hospital settings 22,23 . Some of the isolates, such as MSSA-16, MSSA-87, MSSA-127 and MSSA-170, have same molecular characteristics, suggesting they may represent a single clone. It is possible that the strains were transferred to the contaminated surfaces by discharged patients and/or healthcare workers who carried the recent epidemic strains in Guangzhou, although the sources of these strains are unclear. In contrast, ST398 and ST97 were also observed in the present study, and they have been reported as typical animal clones in many countries [24][25][26][27] . The appearance of these clones in an urban environment is rarely reported and, thus, we cannot rule out the possibility that those five ST398 and ST97 isolates were transmitted by passengers who have close contacts with animals or meats. Most of the MRSA isolates were typed as SCCmec I-III, and all of them were negative for the PVL genes; both of these features are indicators of hospital-acquired isolates. This phenomenon is in agreement with the findings of the drug resistance testing. A high frequency of the anti-disinfectant qac gene was discovered among all MRSA isolates in our study, and this gene was previously detected in airborne Staphylococci isolates from Shanghai metro stations 15 . Hence, the efficiency of sterilization processes in metro systems should be improved by reselection of suitable disinfectants.
In conclusion, our study demonstrated that the frequently touched surfaces in metro system may be a reservoir for Staphylococci transmissions and the resistance to antimicrobials and disinfectants is prevalent, especially among methicillin resistant strains. Cross transmissions of Staphylococci isolates from various sources, including hospitals, communities and livestock, are possible. More stringent infection control and surveillance measures are urgently needed.