Molecular Characterization of Staphylococci Recovered from Hospital Personnel and Frequently Touched Surfaces in Tianjin, China

Staphylococci are major hospital-associated pathogens, and the dissemination of methicillin-resistant staphylococci in hospitals poses a great challenge for managing hospital-acquired infections. Little is known about the dissemination of staphylococci recovered from the hospital environment and personnel in China. In this study, antimicrobial susceptibility tests, mecA gene detection, SCCmec typing, and multilocus sequence typing (MLST) were performed to clarify the molecular epidemiology of staphylococci in a large hospital in Tianjin, China. One hundred and ninety-five staphylococci were recovered, and 94% of isolates were resistant to at least one antibiotic. Eighty-five staphylococci were mecA gene-positive, and 40% of them harbored SCCmec IV and V. The genotype of Staphylococcus aureus (S. aureus) was ST25, and the dominant genotype of methicillin-resistant Staphylococcus epidermidis (MRSE) was ST59. Three new sequence types were assigned as ST840, ST841, and ST842. One (2%) frequently touched surface was contaminated by S. aureus, which suggested that environmental contamination occurred in the hospital in China. Nineteen (39%) frequently touched surfaces were contaminated by methicillin-resistant coagulase-negative staphylococci (MRCoNS), and 46% of HP carried MRCoNS. Varied staphylococcal species and antimicrobial-resistance rates were observed between staphylococci that were recovered from hospital personnel and frequently touched surfaces. The transmission of MRSE and S. aureus between hospital personnel and frequently touched surfaces was detected. Hospital items and personnel may act as reservoirs of antimicrobial-resistant staphylococci, and cleaning strategies should be carried out to decrease the dissemination of antimicrobial-resistant staphylococci in hospitals in China.


Introduction
Staphylococcus aureus (S. aureus) and coagulase-negative staphylococci (CoNS) pose a health threat to humans worldwide. In China, staphylococci (13.4%) are on third place as a cause of clinical infection [1]. e majority (65.5%) of bloodstream infections were caused by staphylococci in a tertiary hospital in Beijing [2] and staphylococci were the most common Gram-positive cocci that were recovered from the clinic in Shanghai [3]. e clinical significance of staphylococcal species cannot be ignored. Meanwhile, studies confirm that the frequently touched surfaces (FTS) within the hospital environments might be potential reservoirs of nosocomial pathogens, and the hands of hospital personnel (HP) might be contaminated by touching the contaminated environment surfaces [4]. Afterward, HP may transfer pathogens to vulnerable patients, or pathogens may be transmitted directly from contaminated surfaces to vulnerable patients [5].
S. aureus contamination on frequently touched surfaces within the hospital environment was observed worldwide [4]. In South Africa, the S. aureus contamination rate on the frequently touched surface was 58.7%, and 9.5% were contaminated by S. aureus in the United States [4]. Moreover, 18% and 7.9% of the FTS were contaminated by S. aureus in Asia and Europe, respectively [4]. e S. aureus contamination rates on FTS in South Africa are significantly higher than in other continents. In Taiwan, the S. aureus contamination rate on the FTS was 6%, and 1.1% were contaminated by MRSA [6]. Unfortunately, no data are available to know the contamination rates of S. aureus and CoNS on FTS in mainland China, and the phylogenetic relationship between staphylococci that was recovered from HP and FTS remains unknown.
e clinical significance of S. aureus and CoNS has been widely recognized, and it can increase significant morbidity, mortality, and healthcare costs [4,7]. e dissemination of antimicrobial-resistant staphylococci in hospital personnel and the environment poses a great health threat to the vulnerable patient population. erefore, it is necessary to understand the dissemination of antimicrobial-resistant staphylococci in healthcare settings. HP and FTS may act as reservoirs of antimicrobial-resistant pathogens. erefore, this study was performed to understand the diversity, antimicrobial resistance, and genotype of S. aureus and CoNS that were recovered from hospital frequently surface and HP of a tertiary hospital in Tianjin, China.

Sampling and Isolation.
e research protocol and informed consent were approved by the Ethics Committee of Tianjin Xiqing Hospital (approval No TMUaMEC2017017 and xqyyll-2021-17). Verbal permission was acquired from all participants before sampling. e hospital is a large (1400 bed and 1258 hospital staff) teaching hospital that handles about 2500 patients per day and is located in the north of Tianjin, China. e collection date was from September to December in 2018. Hospital personnel was from the Chinese medicine department, dermatology department, endocrinology department, emergency room, ultrasound medicine, medical exam center, orthopedics department, proctology department, rehabilitation department, and thoracic surgery department (Table S1). Frequently touched surfaces included water tap, door handles, medical equipment, checking bed, supporter, and registration machine of 19 departments (Table S2). e personnel were trained by practical demonstration before sample collection. Sterilized cotton dry swabs (n � 128) were moistured with sterilized saline and then used to collect hand or nasal samples of HP and frequently touched surface samples randomly, and the samples were transferred back to the lab within 2 hours with sterilized saline. e swabs were plated on mannitol salt agar and incubated at 37°C for 24-48 hours.
e single colony with different morphology was inoculated and purified on nutrient agar (OXOID, UK) and incubated at 37°C for 24 hours.

Identification.
Staphylococci isolated from FTS were identified by matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS) in a positive linear mode (2000-20,000 m/z range) as described previously [8]. e resulting spectra were compared with reference spectra using the Biotyper 3.0 software (Bruker Daltonics, Germany). Escherichia coli DH5α (Bruker Daltonics, Germany) was used as a standard for calibration and quality control. 16S rRNA genes of isolates recovered from HP were amplified and sequenced by Sangon Biotech (Shanghai Ltd.). e sequences were then compared with the NCBI database [9].

Antimicrobial Susceptibility Test.
e disc diffusion method was used to test the antimicrobial susceptibility of staphylococci that were recovered from the hospital environment and personnel, and a panel of 11 antibiotics, in-

mecA Gene Detection and SCCmec
Typing. mA1 and mA2 primers were used to amplify the mecA gene, and eight pairs of primers were used for amplification of class A, B, and C mec complex and the ccr 1-5 complex.
e PCR products were visualized by gel electrophoresis. e combination of the mec gene complex and the ccr gene complex was used to determine the SCCmec types [11]. Sequences of primers and amplification protocols were referred to Kondo et al. [12].

Multilocus Sequence
Typing of S. aureus and S. epidermidis. S. aureus and S. epidermidis were the most common staphylococcal species that were identified in clinics in China (https://www.carss.cn), and thus, S. aureus and S. epidermidis were selected for MLST typing. Housekeeping genes of S. aureus and S. epidermidis were amplified according to the standard guidelines [13,14] and sequenced by Sangon Biotech (Shanghai, China). e sequence type (ST) was assigned by the MLST database (https://pubmlst. org/).

Statistical Analysis.
e χ 2 test was used to analyze the quantitative variables. A P value <0.05 was considered statistically significant.

Staphylococcal Isolates.
Seventy-nine samples were collected from the hands or noses of 79 hospital personnel, and 49 samples were collected randomly from frequently touched surfaces. One hundred and ninety-five staphylococci were recovered, including 109 isolates recovered from HP, and 86 were isolated from FTS of the hospital. S. epidermidis (n � 72) was the dominant species that was recovered from HP, followed by S. hominis (n � 27). Only 1 S. capitis and 1 S. pasteuri were identified in HP samples. e main species that were isolated from FTS were S. epidermidis (n � 27), followed by S. hominis (n � 13) and S. capitis (n � 13). In contrast, only 1 S. aureus, 1 S. pettenkoferi, and 1 S. succinus were recovered from FTS samples.

Antimicrobial Susceptibility Profile.
Antimicrobial susceptibility profiles of 195 staphylococcal isolates are listed in Table 1. Staphylococcal isolates showed the highest resistance rate (67%) towards penicillin and the lowest resistance rate (3%) towards linezolid. e rest 9 antibiotics were ordered by their resistant rate as follows: erythromycin Tables 1, S1, and S2).

mecA Gene Detection and SCCmec
Typing. Eighty-five staphylococcal isolates harbored the mecA gene, including 55 (50.5%) isolates that were recovered from HP and 30 (35.3%) isolates that were isolated from FTS. e SCCmec type V (n � 16, 29%), I (n � 9, 16%), and II (n � 9, 16%) were mainly identified in MRCoNS that were recovered from HP, and SCCmec IV (n � 7, 23%) and V (n � 6, 20%) were the dominant types that were found in MRCoNS isolated from FTS. e SCCmec types were not determined in 10 isolates due to the lack of either the mec complex or the ccr complex (Tables 2, S1, and S2).

Multilocus Sequence
Typing of S. aureus and S. epidermidis. One S. aureus recovered from FTS was determined to be ST25, and 44 out of 57 S. epidermidis recovered from HP and FTS belonged to clonal complex 2 (CC2). Four S. epidermidis belonged to minor CCs, and 6 S. epidermidis were categorized into other CCs. In addition, three new STs were determined, including ST840 (n � 1), ST841 (n � 1), and ST842 (n � 1) (Tables 3, S1 and S2).

Discussion
Few data are available to understand the contamination rate of S. aureus and MRCoNS on FTS in hospitals in mainland China, and the phylogenetic relationship of staphylococci that was recovered from HP and FTS remains unknown. In this study, we had an insight into the genotypes of staphylococci that were recovered from FTS and HP.
S. capitis, S. epidermidis, and S. hominis are species that are frequently recovered from the body surface of humans [7]. In this study, S. epidermidis and S. hominis were the main species that were recovered from HP. In contrast, S. capitis, S. epidermidis, and S. hominis were the major species that were recovered from FTS. It is suggested that the FTS was significantly affected by human microbiota, and surface cleaning should be carried out from time to time to decrease the transmission of antimicrobial-resistant bacteria. e colonization rate of S. epidermidis among HP was significantly higher than that of FTS (P < 0.000003124), and no significant difference was observed in other staphylococcal species. S. equorum, S. haemolyticus, S. pettenkoferi, S. saprophyticus, and S. succinus were exclusively found in FTS, which suggested that FTS might be contaminated by diverse sources. In this study, 1 out of 49 (2%) FTS samples was contaminated by S. aureus, which was lower than the S. aureus contamination rate on the frequently touched surface in Taiwan and other countries [4,6]. Fifteen out of 79 (19%) HP carried S. aureus, which was cited from published work [9]. Diverse staphylococcal species were found in samples collected from FTS, and attention should be paid to hospital environment monitoring and cleaning.
In addition, CoNS was detected in 75.5% (37/49) samples of FTS, and 39% (19/49) of FTS were contaminated by MRCoNS. In this study, 46% of HP carried MRCoNS, which was higher than the rate of the international report [15].
us, hand hygiene practices should be emphasized by hospitals in China. Four (4%) staphylococci that were recovered from HP were fully susceptible to all tested antibiotics, while 11 (13%) isolates that were recovered from FTS were fully susceptible to all tested antibiotics. For isolates recovered from HP, the resistance rate of clindamycin, tetracycline, gentamicin, and linezolid was lower than in previous international surveillance reports [15]. In contrast, the resistance rate of penicillin, erythromycin, cefoxitin, and levofloxacin was higher than in the previous report [15]. e resistance rates of isolates that were recovered from FTS towards gentamicin were much higher than that of isolates recovered from HP (χ 2 � 16.852, P < 0.05). In contrast, the resistance rates of isolates that were recovered from HP towards erythromycin (χ 2 � 5.1809, P < 0.05), cefoxitin (χ 2 � 4.1302, P < 0.05), and penicillin (χ 2 � 33.203, P < 0.05) were significantly higher than that of isolates recovered from FTS (Table 3). Multiresistant CoNS may limit the medicinal options and aggravate treatment strategies [16], and the wide dissemination of multiple-resistant staphylococci in HP and FTS was a worrisome finding.
mecA gene encodes penicillin-binding protein 2a, which confers methicillin resistance [17]. e carriage rate (50.5%) of the mecA gene in staphylococci that were recovered from HP was significantly higher than the rate (35.3%) of staphylococci that were isolated from FTS (χ 2 � 4.1302, P < 0.05). MRCoNS may act as concealed reservoirs for antibiotic resistance and virulence genes [18], and the high prevalence of MRCoNS in this study is an alerting finding. mecA gene is located on a mobile genetic element, which is named staphylococcal cassette chromosome mec. e SCCmec types I, II, and III were hospital-associated and SCCmec IV and V were community-associated [19]. e SCCmec types I, II, IV, and V were identified in S. epidermidis that were recovered from HP and FTS. In contrast, SCCmec VI was exclusively identified in S. epidermidis that were recovered from FTS, and SCCmec I and IX were exclusively detected in S. epidermidis that were isolated from HP. e SCCmec types VI and IX were first identified in staphylococci that were recovered from Portugal and ailand, respectively [11]. In this study, the majority of staphylococci that were recovered from HP and FTS harbored community-associated SCCmec IV and V; however, it is worth noting that hospital-associated SCCmec I and II were mainly identified in staphylococci that were recovered from HP ( Table 2). It is reported that SCCmec IV and V were higher than SCCmec I, II, and III due to their smaller size [20], and the difference in dominant SCCmec identified in staphylococci that were recovered from HP and FTS may be due to their varied transmissible abilities. ST25 was identified in S. aureus that was recovered from HP and FTS (Table 3) [9], which suggested the cross-transmission of S. aureus between HP and FTS.
In this study, 32 sequence types were determined in S. epidermidis, and ST59, ST200, and ST210 were found in S. epidermidis that were recovered from samples of HP and FTS. In contrast, 12 sequence types were exclusively identified in FTS, and 17 sequence types were only identified in HP. However, most STs belonged to CC2, which is the dominant clonal complex in HP in China [21]. us, it is suggested that CC2 was also the dominant clone that disseminated in the hospital environment. It is worth noting that S. epidermidis ST210 was identified in samples that were collected from HP and FTS, respectively, and the samples were both gathered from the emergency room. us, the cross-transmission of S. epidermidis between HP and FTS was highly plausible. Microbiota of patients might affect the microbiota of FTS [21] and thus might explain the inconsistency of ST between staphylococci that were recovered from HP and FTS. No data were collected from patients which was a limitation of this study. ST5, ST17, ST20, ST89, ST152, ST173, ST192, ST218, ST235, ST251, and ST291 were previously identified in staphylococci that were recovered from HP in Shanghai [21]. Consistent with the previous study, ST5, ST20, ST89, ST152, ST173, ST192, ST235, ST251, and ST291 were detected in HP of our study, while ST17 and ST218 were identified in FTS only. Moreover, ST35, ST247, ST249, ST454, ST840, ST841, and ST842 that were identified in HP of this study were not reported by the previous study [21]. ST6 was reported to be identified in patients [21], which was detected in the FTS of this study. In addition, some STs of S. epidermidis that were isolated from FTS, such as ST4,

Conclusions
In conclusion, species of staphylococci that were isolated from FTS were much more diverse than that from HP. e antimicrobial-resistance rate, mecA-genes carriage rate, and the sequence types differed between staphylococci recovered from HP and FTS. S. epidermidis STs identified in HP samples were associated with both community and hospital clones, while STs of S. epidermidis that were isolated from FTS were partially associated with hospital clones. e sources of some S. epidermidis isolated from FTS remain unknown, and attention should be paid to tracing the sources of clones that were disseminated in the hospital environment in China. S. aureus ST25 was identified in samples of HP and FTS, which suggested the transmission of S. aureus between HP and FTS. ree new MLST types were identified in this study. e limitation of this study was that no patients were recruited in this study; whereas, the crosstransmission of S. aureus and S. epidermidis between HP and FTS in the hospital was observed, and cleaning strategies should be carried out to decrease the dissemination of antimicrobial-resistant staphylococci in hospitals in China.

Data Availability
e data generated or analyzed during this study are included within the article and supplemented materials.
Ethical Approval e research protocol and informed consent were approved by the Ethics Committee of Tianjin Medical University and the Ethics Committee of Tianjin Xiqing Hospital (approval no. TMUaMEC2017017 and xqyyll-2021-17).

Disclosure
A preprint has previously been published [23].

Conflicts of Interest
e authors declare that they have no conflicts of interest.