Molecular epidemiology and virulence characteristics of Staphylococcus aureus nasal colonization in medical laboratory staff: comparison between microbiological and non-microbiological laboratories

Abstract

Background

Medical laboratory staff are a high-risk population for colonization of Staphylococcus aureus (S. aureus) due to direct and dense contact with the pathogens; however, there is limited information about this colonization. This study sought to determine the prevalence and molecular characteristics of nasal colonization by S. aureus in medical laboratory staff in Guangzhou, southern China, and to compare the differences between microbiological laboratory (MLS) and non-microbiological laboratory (NMLS) staff.

Methods

S. aureus colonization was assessed by nasal swab cultures from 434 subjects, including 130 MLSs and 304 NMLSs from 33 hospitals in Guangzhou. All S. aureus isolates underwent the antimicrobial susceptibility test, virulence gene detection and molecular typing.

Results

The overall prevalence of S. aureus carriage was 20.1% (87/434), which was higher in MLSs than in NMLSs (26.2% vs. 17.4%, P < 0.05), while the prevalence of Methicillin-resistant S. aureus (MRSA) was similar. Living with hospital staff was associated with S. aureus carriage. The majority of the isolates harboured various virulence genes, and those in MLSs appeared less resistant to antibiotics and more virulent than their counterparts. A total of 37 different spa types were detected; among these, t338, t437, t189 and t701 were the most frequently encountered types. T338 was the main spa type contributing to nasal colonization Methicillin-sensitive S. aureus (MSSA) (13.0%), and t437-SCCmec IV was predominant in MRSA isolates (40%).

Conclusions

These findings provide insight into the risk factors, molecular epidemiology and virulence gene profiles of S. aureus nasal carriage among the medical laboratory staff in Guangzhou.

Backgrounds

Staphylococcus aureus (S. aureus) is a life-threatening pathogen and a part of the commensal flora, and its antimicrobial resistance has been a significant therapeutic challenge. Methicillin-resistant Staphylococcus aureus (MRSA) is of great concern because of its high mortality following treatment failure. S. aureus infections are usually preceded by colonization, which is most often found in the anterior nares [1]. Nasal carriage is associated with S. aureus (including MRSA) exposure and an increased risk of transmission and infection [2, 3].

S. aureus sources and transmission patterns have been extensively investigated, especially in hospital environments [4]. Our previous studies showed possible spread from HA-MRSA (hospital-acquired methicillin-resistant Staphylococcus aureus); to CA-MRSA (community-acquired methicillin-resistant Staphylococcus aureus), which may make the strains more resistant to antibiotics [5]. Therefore, nasal colonization of HA-MRSA strains in HCWs (healthcare workers) may also play a role in MRSA spread from hospital settings to the community [5]. Several studies about healthcare workers have focused on the clinical staff, such as doctors, nurses and general practitioners [4, 6], while few studies have investigated the medical laboratory staff in the hospital.

Unlike the clinical staff members who have close contact with patients, medical laboratory staff members are exposed to the infectious materials more often, including patients’ blood, fluids and tissues. The medical microbiological laboratory is an important department in the medical lab, and its main function is to isolate, culture and identify the pathogens from the patient specimens. Therefore, the microbiological lab staff members have a higher risk of exposure to S. aureus because of their direct and dense contact with pathogens [7]. Therefore, it is important to detect nasal colonization of S. aureus among the healthy medical laboratory staff, especially the medical microbiological lab staff.

The objective of this study was to assess the prevalence and factors associated with S. aureus and MRSA nasal colonization among medical laboratory staff in hospitals in Guangzhou, China. To the best of our knowledge, this is the first study performed in this population in China. Additionally, some S. aureus clones are more virulent than others, although any S. aureus genotype can become a pathogen under favourable host conditions. Molecular typing is helpful for supporting infection control measures, investigating suspected outbreaks, and preventing nosocomial transmission [8,9,10,11]. Therefore, in this study, all isolates were further genotyped by spa typing, and the MRSA isolates were evaluated with SCCmec typing. The presence of virulence genes was determined to establish their possible relationship to the epidemiological data, including the enterotoxin, haemolysin, pvl, tsst-1 and exfoliative genes as well as various adhesive protein producing genes. To date, this is the first study to indicate that medical laboratory staff may have occupational exposure to healthcare-associated S. aureus in Guangzhou, China. This knowledge will improve our understanding of the characteristics of S. aureus nasal carriage among healthy medical laboratory staff and provide a possible mode of S. aureus transmission in our region.

Methods

Population and study design

A cross-sectional prevalence study was conducted in 33 hospitals’ medical laboratories in Guangzhou, China between February 22, 2016, and March 17, 2016. All the subjects were the staff working in medical laboratory; the ones with acute and chronic respiratory tract infection were excluded. As the main metropolises with a large population of more than 10,000,000 in southern China, Guangzhou holds a prominent position in the healthcare of local and surrounding areas. As the main tertiary hospitals, the 33 hospitals are distributed in urban and suburban in Guangzhou, making them representative (Fig. 1).

Fig. 1

Map of the 33 hospitals in Guangzhou, China, and the subjects, S. aureus and MRSA isolates distribution

A total of 434 medical laboratory staff, aged 20–56 years, in the 33 hospitals participated in this study; 130 microbiological laboratory staffs (MLS) were randomly recruited from the microbiological labs, and 304 non-microbiological laboratory staffs (NMLS) were randomly recruited from the non-microbiological labs. All volunteers signed informed consent documents approving the use of their samples for research purposes, and the Ethics Committee of Sun Yat-Sen Memorial Hospital approved the study.

Using a standardized questionnaire, the pertinent demographic, medical information and potential factors that are related to S. aureus nasal colonization and transmission were collected from each participant. The “Standard working protection” meant the basic lab rule of hygiene, including hand washing, use of gloves, long sleeves and masks. Before sampling, the subjects were independently required to finish the questionnaires. No surveys were eliminated during data collation, and 434 nasal swabs were sampled.

Bacterial strains

Both anterior nares were swabbed by rotating a sterile dry cotton swab 5 times inside the nostril. The samples were immediately stored in Copan eSwab Liquid Amies preservation medium (eSwab Collection and Preservation System, Copan Italia, Brescia, Italy) and transported at room temperature to the department of bacteriology in 4 h. The swabs were streaked on blood agar plates at 35 °C for 24 h. Gram-positive, β-haemolytic, coagulase-positive isolates were confirmed as S. aureus using a Vitek®2 microbial identification system (bioMérieux, Marcy l’Etoile, France) according to the manufacturer’s instructions.

Antimicrobial susceptibility testing

Antimicrobials used for susceptibility testing included penicillin, erythromycin, clindamycin, tetracycline, cefoxitin, chloramphenicol, rifampicin, ciprofloxacin, gentamicin, trimethoprim/sulfamethoxazole, teicoplanin, linezolid, levofloxacin and vancomycin. Susceptibilities were determined using the disk diffusion method in accordance with the performance standards for antimicrobial susceptibility testing, 26th informational supplement (M100-S26), recommended by the Clinical and Laboratory Standards Institute. The susceptibility for vancomycin was determined using the E-test method. Inducible clindamycin resistance was determined by the D-test. All disks and E-tests were obtained from Oxoid Ltd. (Oxoid, Basingstoke, England), and S. aureus ATCC 25923 and ATCC29213 were used as the quality control strains.

Molecular characterization

Bacterial DNA was extracted using a DNA extraction kit (Tiangen Biotech, Beijing) with lysostaphin according to the manufacturer’s instructions. PCR of the spa locus with subsequent sequencing was performed on the 87 S. aureus isolates, including clustering of the spa types into spa clonal complexes (using the Ridom Staph Type version 1.5 software package, www.spaserver.ridom.de) as previously reported [12]. SCCmec typing of MRSA strains was conducted by a multiplex PCR method as previously described [13]. The presence of the genes that code for Panton-Valentine leukocidin (lukF-PV/lukS-PV or pvl), twelve staphylococcal enterotoxins (sea~see, seg~sej and sem-seo), two exfoliative toxins (eta and etb), four haemolysins (α-hemolysin, β-hemolysin, δ-hemolysin, and γ-hemolysin), three biofilm information-related genes (ica A, ica D and bap), and the toxic shock syndrome toxin (tsst-1) and mecA gene were determined using PCR as previously reported, with some modification [14, 15].

Statistical analysis

In descriptive statistics, the frequency and proportions were calculated for categorical variables. The frequency of the SCCmec type, specimen type and virulence genes were treated as categorical variables. The chi-square or two-sided Fisher’s exact test was used to discriminate whether the distributions of the studied genes or types were significantly different between the MLS and NMLS groups. The only continuous variable, the duration of employment, was transformed into a categorical variable using the quartiles of the frequency distribution (< 1, 1~ 5, 6~ 10, 11~ 20, and > 20). The odds ratios (OR), 95% confidence intervals (CI), and P-values were calculated. A finding was considered statistically significant for a two-sided P-value< 0.05. Univariable logistic regression models were applied to determine the independent risk factors. Multiple logistic regression analysis was performed by stepwise backward selection of variables with biological plausibility and a significance level < 0.10 for entry into the model. All statistical analyses were performed using SPSS 19.0 for Windows (IBM).

Results

S. aureus Nasal colonization

A total of 434 medical laboratory staff members in the 33 hospitals were enrolled in this study. The demographic characteristics of the study population are shown in Table 1. The average age of the participating volunteers was 34.2 ± 12.2 years, and 187 (43.1%) of the volunteers were male. S. aureus was detected in the nasal swabs of 87 participants (20.0%, 87/434). The overall prevalence of S. aureus nasal colonization was 26.2% (34/130) in MLS, which was higher than that in NMLS (17.4%, 53/304) (P = 0.03) (OR = 1.677, 95% CI: 1.027–2.740). Additionally, there were no differences for other demographic characteristics and potential risk factors between the two comparison groups. A proper nasal cavity cleaning habit could help to decrease the S. aureus nasal carriage [16], but it is interesting to note that the S. aureus nasal carriage rate in people who cleaned their nasal cavity daily or weekly was higher than for those who rarely or never cleaned their nasal cavity (P = 0.032) (OR = 1.753, 95% CI: 1.050–2.927) in this study, and those living with hospital staff were more likely to be colonized by S. aureus in the nasal cavity (P = 0.008) (OR = 1.909, 95% CI: 1.182–3.083). In multiple logistic regression analysis, nasal colonization of S. aureus was also significantly associated with working in a microbiological laboratory and living with hospital staff.

Table 1 Prevalence of S. aureus and MRSA carriage by population group and factors associated with nasal carriage

Ten MRSA strains were isolated in this study, two in the MLS group (1.5%, 2/130) and eight in its counterparts (2.6%, 8/304), while no significant differences were found between the two groups (P = 0.487). MRSA accounted for 11.5% of all S. aureus isolates, and its distribution was centred in three hospitals, Hospital A (three isolates, typed as t008, t437 and t701 respectively), Hospital B (two isolates, typed as t571 and t441 respectively) and Hospital ZE (two isolates, typed as t437 and t037 respectively), with relative hospital clustering. The distributions of S. aureus and MRSA carriers stratified by population characteristics are shown in Table 1 and Fig. 1.

Spa typing and SCCmec typing

The 87 S. aureus strains were spa typed, resulting in 37 different types, including two new types (Fig. 2 and Additional file 1: Table S1). The largest observed spa clusters were types t338 (n = 10, 11.5%) and t437 (n = 10, 11.5%), which were followed by t189 (n = 8, 9.2%) and t701 (n = 8, 9.2%). All types t338 and t189 were MSSA isolates, and type 701 was also mainly found in the MSSA group. MRSA isolates clustered in spa type t437 (4/10, 40%). All other S. aureus isolates had a distinct spa type. Additionally, two new spa types, t16614 (26–23–12-34-12-23-02-02-12-23) (Kreiswirth IDs: TJGBGJAAGJ) and t16615 (11–10–21-17-17-34-24-34-22-25-25) (Kreiswirth IDs: YC2FMMBQBLOO), were found in the MSSA isolates, and they were obtained from nasal swabs of a 26-year-old female non-microbiological lab staff member and a 33-year-old male microbiological lab staff member, respectively. There was no clustering of the isolate spa types within different departments.

Fig. 2

Thirty-seven spa types grouped into four groups based on DNA microarray analysis. Group A: t002, t571,t304, t008, t116, t5132, t437, t954, t085, t796, t091, t1839, t4938; Group B: t078, t148, t1346, t548, t563, t127, t8457, t10247, t701, t16615(new type); Group C: t189, t441, t3349, t287, t803, t769; t364, t338, t037, t1184, t13960, t034, t1451; Group D: t16614 (new type)

Based on homology analysis, 37 spa types were grouped into four groups (Fig. 2). Group A contained 12 spa types, including t437, t571 and t002, which evolved based on t002. Group B consisted of ten types, including t701 and t127. Group C contained 13 types, including t189 and t338. The new spa type t16614 formed group D alone. By sequence alignment, the homology of the spa types in the same group was more than 90%. Another new type, t16615, has high homology with t701. The isolates in groups A, B and C were obtained from both the NMLS and MLS groups, while the isolates from the microbiological laboratory were mainly from groups A and C, accounting for 85.3% (29/34). Ten MRSA isolates were dispersed in different groups.

The distribution of SCCmec types in the ten MRSA strains is shown in Table 2. Among these, SCCmec type IV was found in eight isolates (80.0%), MRSA-t437-IV was the most prevalent of all MRSA isolates (40.0%, 4/10) (Fig. 2 and Table 2). Two MRSA isolates in the NMLS group were SCCmec I MRSA and SCCmec III MRSA, respectively, which were related to hospital-acquired MRSA (HA-MRSA). As a common virulence factor of community-acquired MRSA (CA-MRSA), the pvl gene was positive in 50% of isolates in the ten MRSA isolates.

Table 2 Distribution of the ten MRSA strains in details

Antimicrobial susceptibility

All isolates were β-lactamases positive. A high resistance rate to penicillin was detected (92.0%, 80/87) in the 87 S. aureus isolates, which was followed by the erythromycin (69.0%, 60/87) and clindamycin (69.0%, 60/87). All isolates were susceptible to levofloxacin, vancomycin, teicoplanin and linezolid. A total of 44 (50.6%, 44/87) isolates were MDRSA [17]; 22 in MLS (64.7%, 22/34) and 22 in NMLS (41.5%, 22/53). Penicillin-erythromycin-clindamycin (49.4%, 43/87) was the predominant resistance profile. Only ten isolates (11.5%, 10/87) were resistant to cefoxitin, and they were confirmed to be MRSA by mecA PCR screening. Nine MRSA isolates were resistant to erythromycin and clindamycin. Only one MRSA isolate was resistant to erythromycin, clindamycin, tetracycline, and gentamycin. Notably, no MRSA was resistant to quinolone in this study. The resistant percentage to 14 antibiotics of the S. aureus isolates is shown in Fig. 3 and Additional file 2: Table S2.

Fig. 3

Comparison of the resistance rate to 14 antibiotics of the S. aureus isolates between MLS and NMLS group. MLS vs. NMLS, *P < 0.05. MLS, microbiological lab staff; NMLS, non-microbiological lab staff

Virulence factors

Almost all the isolates contained haemolysin gene hla (97.7%, 85/87), hld (98.9%, 86/87) and hlg (98.9%, 86/87) and all isolates in the MLS group were hla-hld-hlg positive. Twelve staphylococcal enterotoxins genes (sea, seg, seh, sem, sen, seo, sec, sei, sej, sem, sen and seo) make up the enterotoxin gene cluster (egc). Sea, seg, seh, sem, sen, and seo genes were found in more than 20% isolates in the 87 S. aureus isolates. Additionally, sec, sei and sej genes were mainly found in the 34 S. aureus isolates in MLS (17.7%, 23.5% and 11.8% respectively), while they were rarely in NMLS. The see gene was not found. Toxic shock syndrome toxin gene (tsst-1) was found in 20 isolates (23.0%), and the exfoliative toxins genes (eta or etb) were rarely found (2.3% and 0, respectively). The pvl gene was found in 13 isolates (14.9%), and five isolates were MRSA. The icaA and bap genes were not found, while another biofilm formation related gene icaD widely existed in the S. aureus isolates in this study (Table 3).

Table 3 Detection of the 23 virulence genes of 87 S. aureus isolates in this study

Among isolates in MLS, the detection rates of sec and sei were higher than in the NMLS group (17.6% vs. 1.9% and 23.5% vs. 1.9%, respectively) (P < 0.05), and all sej positive isolates were found in the MLS group (11.8%, 4/34). On the other hand, the eta gene was only found in isolates of NMLS (3.8%, 2/53). Furthermore, more than half (51.7%) of the 87 S. aureus isolates carried more than five virulence genes, and 26.5% (9/34) of the 34 isolates in MLS group carried more than seven virulence genes, which were obviously higher than that in the NMLS group (18.9%, 10/53) (P < 0.05). Additionally, 80% of MRSA (8/10) carried more than five virulence genes, which was obviously higher than in MSSA (48.05%, 37/77) (P < 0.05) (Table 3).

Discussion

To the best of our knowledge, this is the first study that focused on the prevalence of S. aureus and MRSA colonization in medical laboratory staff in China, especially a comparison between microbiological laboratory and non-microbiological laboratory staff. This population had a high risk of S. aureus colonization. The overall prevalence of S. aureus colonization was 20.0% in this study. Compared with the general population, this is higher than the recorded prevalence among healthy adults in northern China (16.5%) and in adults in community settings in southern China (13.7%) [18, 19], while it agrees with the rates in America and Europe (20–30%) [20]. Compared with the other HCWs (working mainly in the hospital), the prevalence of S. aureus colonization in this study coincides with a review that calculated 23.7% of 10,589 HCWs worldwide carried S. aureus [4]. We expected a higher prevalence of S. aureus colonization in the MLS, as there is a higher risk of exposure to S. aureus because of the direct, dense contact with pathogens. The MLSs in this study had a high prevalence of S. aureus colonization (26.2%), which was significantly higher than the NMLSs (non-microbiological laboratory staff) (17.4%) and higher than that in our other study that focused on the prevalence of S. aureus colonization in the nurses and clinical doctors in Guangzhou (21.6%) [16]. The MRSA colonization rate in this study (2.3%) was higher than that in northern Europe (0.2%) and southern China (0.3%) for healthy adults [21], and it was higher than the 1.0% in our other study on clinical nurses and doctors [16]. But both the colonization rate of S. aureus and MRSA were lower than the research about the carriage among health care professionals (HCPs) attending an international symposium (32.4% and 5.3%, respectively), 66.88% of which subjects worked in MLS, and major of which came from Western Europe [7].

Youth, male gender and chronic disease were the most significant risk factors for nasal S. aureus colonization in the healthy population according to past studies [2, 18]; however, in our study, there were no significant differences between different ages and underlying disease conditions. For the medical lab staff, our study found that higher S. aureus colonization rates were associated with living with hospital staff. Hospital staff had high risks of pathogen carriage and could act as a “vector” and transmit S. aureus to their family, roommates and colleagues [4]. In this study, the prevalence of S. aureus colonization appeared to have relative area and hospital clustering, especially MRSA, 70% of which come from three hospitals, and two isolates were detected in two student technicians in training at Hospital A who were roommates.

The spa types revealed a broad range of types with a few more prevalent ones, such as t338, t437, t189 and t701, in this study. There were some unique spa clusters in MSSA (t338, t189 and t701) and MRSA (t437) isolates. As the main spa clusters of MSSA in this study, t189 and t338 had high homology, which coincides with the past reports about the molecular epidemiology in seven main cities in China during this time [22]. As a variant of t002, t437 was the predominant spa type of MRSA. According to past reports, MLST CC59-MRSA, which is primarily linked with the spa type t437, was the predominant community-associated MRSA clone in Asia, and it represented a genetically tight cluster in Europe [23]. The main SCCmec type of MRSA in this study was SCCmec IV, including IVa and IVd, which showed the isolates were mainly CA-MRSA. Combined with spa typing, MRSA-t437-IV was the most prevalent of all MRSA isolates, which was reported to be a prevalent clone in the community setting in northern China and Taiwan [24, 25]. Two new spa types, t16614 and t16615, were detected in this study; both isolates were detected in MSSA isolates and had characteristics of low virulence and low resistance to antibiotics. Although the spa types are evolving over time in the same person [1, 26, 27], the clustering of the spa type in spa complexes could also be used to describe the transmission direction in our study because all sampling was performed within a short time period (three weeks). There was no obvious difference in the molecular type between MLS and NMLS.

According to the antimicrobial susceptibility test, we found there was a high rate of resistance to penicillin, erythromycin and clindamycin and more than half of the isolates had MDR (multi drug resistance). This resistance pattern is associated with the excessive use of penicillin and macrolides in China and requires substantial attention [28, 29]. Spa types t189 and t701 appeared relatively sensitive to antibiotics, and their main resistance pattern was to penicillin. While t338 and t437 isolates had higher resistance, and their main resistance pattern was to penicillin-clindamycin-erythromycin.

In contrast to the low prevalence of the exfoliative toxins genes (eta or etb) and pvl gene, the enterotoxin genes, tsst-1 gene and haemolysin genes were widely distributed among the isolates; more than a half of the isolates carried over five virulence genes. Previous studies have confirmed that the sea and seb genes are the most abundant toxin genes in clinical S. aureus isolates from patients in China [5, 30, 31]; in this study, seg and sem~n were the predominant enterotoxin genes, which may be related to the different studied populations. Haemolysin of S. aureus contributes to bacterio-lyticenzyme release that can damage the nearby organizations through affecting the red blood cells, platelets and neutrophils [32]. Consistent with a report in China [32], almost all isolates contained hla, hld and hlg genes. The tsst-1 gene was found in 23.0% of isolates in this study, suggesting that once infection or cross-infection occurred, these patients may have a greater potential for developing fatal toxic shock syndrome (TSS) [33]. Of note, the isolates in MLS appeared to carry more virulence determinants than the ones in NMLS, which may relate to their more heavy and direct exposure to pathogens. MRSA isolates harboured more virulence genes than MSSA isolates in this study. Spa type t189 and t701 isolates harboured fewer virulence genes in this study, and the main toxin pattern was hemolysis α/γ/δ/icaD (data not shown). As the predominant spa type, t437 appeared more virulent, and it mainly harboured seb/seh/tsst-1/pvl/hemolysis α/β/γ/δ/icaD genes. Among the main spa types in this study, the most virulent type was t338, which harboured more toxin genes (sea/seg/ sem~o/tsst-1/hemolysis α/γ/δ/icaD). Notably, one MRSA isolate typed as t548-SCCmec IVd harboured up to 13 virulence genes and appeared to have high resistance to antibiotics; it was isolated from a 50-year-old male microbiology lab staff member who came from hospital ZD, had worked in this department for 27 years and smoked, poor work protection (sometimes didn’t wear mask), rarely nasal cleaning and no underlying diseases; additionally, there were HCWs in his family. This colonization of a healthy medical staff member by a high toxin producing and highly resistant strain is worrisome. In the absence of effective function control practices, these strains may become widely disseminated in the hospital and community.

The current study had several limitations. Most importantly, the small size limited the broad representative significance of the research. Second, sampling of only the nostrils without including other body parts may underestimate the frequency of MRSA colonization. Third, MLST typing was not performed in the current study, and it should be conducted in future analyses. Despite the above limitations, our results confirmed the nasal S. aureus colonization in a special population, medical lab staff in local region.

Conclusions

These findings demonstrate an average prevalence of S. aureus nasal colonization in medical lab staff as for other HCWs according to the previous reports. Living with hospital staff members and improper nasal hygiene could increase the colonization risk. Some unique spa clusters (t338, t437, t189 and t701), high antibiotic resistance and high virulence were found in the S. aureus isolates. Furthermore, MLS had a higher prevalence of S. aureus colonization, and the isolates appeared more virulent than the ones in NMLS, which may relate to their more heavy, direct exposure to the pathogens. Some of the many infection prevention partitions, which influence the S. aureus/MRSA epidemiology, should be handled by the medical lab staff, especially in the microbiology department, because they have appropriate work protection, nasal clean and hand washing habits.