Antimicrobial susceptibility and antibiotic resistance genes
A total of 227 S.aureus isolates were tested for antimicrobial susceptibility. The antimicrobial resistance profiles of the S. aureus, MRSA, MSSA and MDR isolates are shown in Figure 1. No S. aureus isolate was resistant to VAN or LZD, but most were resistant to GEN (14.1%), LEV (10.6%), and RIF (19.8%). Less than 50% of isolates were resistant to the remaining antibiotics, except for PEN, to which 92.5% had resistance. All of 76 FOX-resistant isolates, including an OXA susceptible-MRSA (OS-MRSA), were found to be mecA-positive, and were thus classified as MRSA isolates. Statistical analysis showed that the MRSA isolates had significantly higher resistance rates to PEN than the MSSA isolates (100.0% vs. 88.7%, p = 0.002), ERY (75.0% vs. 35.8%, p<0.001), CLI (64.5% vs. 29.8%, p<0.001), GEN (18.4% vs. 8.6%, p = 0.031), RIF (17.1% vs. 4.6%, p = 0.002), and LEV (15.8% vs. 6.6%, p = 0.028).
A total of 111 ERY-resistant S. aureus isolates were found and used to examine the presence of erm gene. The most prevalent erm gene was ermC (90.1%, 100/111), followed by ermB (38.7%, 43/111) and ermA (21.6%, 24/111). In the ERY-resistant MSSA isolates, the frequencies of the three ERY-resistant genes showed the same trend as in the ERY-resistant MRSAs. ermC (87.0%, 47/54) was the most prevalent gene, followed by ermB (38.9%, 21/54) and ermA (11.1%, 6/54). The ERY-resistant MRSA isolates had higher frequencies of ermA than the ERY-resistant MSSA isolates (c2=6.855, p<0.05). All of 85 TET-resistant isolates carried the TET-resistant genes tet. The prevalences of tetK, tetM, tetL and tetO were 91.8% (78/85), 67.1% (57/85), 23.5% (20/85) and 0.0% (0/85), respectively. In the TET-resistant MSSA isolates, the most prevalent TET-resistant gene was still tetK (93.2%, 55/59), followed by tetM (59.3%, 35/59), tetL (23.7%, 14/59), and tetO (0.0%, 0/59). TET-resistant MRSA isolates had higher frequencies tetM than the TET-resistant MSSA isolates(c2=5.227, p<0.05).
One hundred thirteen (49.8%) S. aureus isolates were found to be MDR, defined as having resistance to more than three classes of antibiotics (Table 2). The chi-square test showed that the prevalence of MDR was significantly higher in the MRSA isolates than in the MSSA isolates (Table 2)(c2=26.115, p<0.05). In addition, when comparing the S. aureus isolates collected in 2013-2014 with those from 2018-2019, the resistance rates to all antibiotics except SXT were broadly similar. Compared with those collected in 2018-2019, the S. aureus isolates from 2013-2014 had a higher resistance rate to SXT (64.8% vs. 5.9%, p<0.05) and a greater prevalence of MDR (61.5% vs. 41.9%, p<0.05).
MLST, spa, and SCCmec typing
Forty STs belonging to 19 CCs and 2 singletons were identified by eBURST. As shown in Table 3 and Figure 2, ST398 (14.1%, 32/227) was the most prevalent, followed by ST188 (13.2%, 30/227) and ST45 (10.1%, 23/227). It was found that 78.1% (25/32) of the ST398 isolates, 80.0% (24/30) of the ST188 isolates, and 91.3% (21/23) of the ST45 isolates were derived from Hainan General Hospital. In addition, three isolates could not be assigned to any known ST, so these novel alleles were submitted to the MLST database, and three new STs, ST5489, ST5492 and ST5493, were assigned. By spa typing, 79 spa types were found. The most prevalent was t189 (12.3%, 28/227), followed by t437 (7.9%, 18/227), t116 (7.5%, 17/227), and t011 (6.6%, 15/227). When the STs and spa typing were combined, the predominant combinations were ST188-t189 (12.3%, 28/227), ST45-t116 (7.5%, 17/227), ST59-t437 (7.0%, 16/227), ST398-t011 (6.6%, 15/227), ST398-t034 (4.8%, 11/227), and ST7-t091 (4.8%, 11/227). A strong association was observed between certain STs and spa types: ST188 was primarily associated with t189 (93.3%, 28/30); ST45 was associated mainly with t116 (73.9%, 17/23); and ST59 was associated mainly with t437 (72.7%, 16/22).
The major types of S. aureus collected in 2013-2014 were ST188 (14.3%), ST45 (14.3%), ST59 (8.8%), and ST88 (8.8%), whereas in 2018-2019, ST398 (19.9%), ST188 (12.5%), ST59 (10.3%), ST45 (7.4%), and ST7 (7.4%) were the top five types. Among the STs that exhibited OXA sensitivity, the two predominant types in 2013-2014 were ST188-MSSA (14.3%) and ST45-MRSA (12.1%), whereas in 2018-2019 they were ST398-MSSA (18.4%) and ST59-MRSA (8.1%). The prevalence of ST398-MSSA markedly increased from 2013-2014 (5.5%) to 2018-2019 (18.4%), and this increase was significant (p<0.05).
Among the 76 MRSA isolates, 6 SCCmec types or subtypes, namely types I, II, III, IVa, IVc, and V, were found. The most common SCCmec type was IVa, which was found in 43 isolates (56.6%, 43/76), whereas types I, II, III, IVc, and V were found in 1, 3, 6, 5, and 9 isolates, respectively. Nine isolates, including OS-MRSA, were classified as NT for SCCmec typing. When the STs and SCCmec typing were combined, the predominant combination was ST45-SCCmec IVa (8.8%, 20/227), and no significant difference was found in the positive rate of ST45-SCCmec IVa between the S. aureus isolates collected in 2013-2014 and 2018-2019 (12.1% vs. 6.6%, p>0.05) (Table 3).
Virulence gene profiles
The frequencies of the virulence genes identified in the 227 S. aureus isolates are listed in Table 4. ClfA was present in all S. aureus isolates, hla, hlb, and eta were detected in 98.7%, 70.9%, and 57.3% of these isolates, respectively, whereas the remaining ones were found in less than 50%. One hundred and twenty (52.9%) S. aureus isolates harbored six or more virulence genes. Of those 120 isolates, 11 contained 9 virulence genes, 31 had 8 such genes, 38 carried 7, and 40 carried 6. The frequencies of fnbA, sea, and sec were significantly higher in the MRSA isolates than in the MSSA isolates, but no significant difference was found in the likelihood of harboring six or more virulence genes between the MRSA and MSSA isolates (56.6% vs. 51.0%, p>0.05). Compared with those collected in 2013-2014, the S. aureus isolates from 2018-2019 had higher frequency of pvl, fnbB, hlb, seb, eta, and etb and higher rates of harboring six or more virulence genes.
Characteristics of the major clones ST398, ST188, and ST45
The most abundant sequence type found in this study was ST398 (14.1%, 32/227) followed by ST188 (13.2%, 30/227) and ST45 (10.1%, 23/227). Most ST398 (93.8%, 30/32) and ST188 (96.7%, 29/30) isolates were MSSA, whereas most ST45 (87.0%, 20/23) isolates were MRSA and all ST45-MRSA isolates belonged to the SCCmec IVa type (Table 2, Table 3). The ST398 (c2=17.685, p<0.01) and ST188 isolates (p<0.01) had higher resistance rates to TET than the ST45 isolates. In addition, no significant difference was seen in the resistance rate to any antibiotics between ST398, ST188 and ST45 isolates.
Of the 11 tested virulence genes, pvl and fnbB were more frequent in ST398 isolates than in ST45 (c2=22.010 and c2=30.457, respectively, p<0.01) and ST188 isolates (c2=12.790 and c2=38.027, respectively, p<0.01). The prevalence of sec in ST45 isolates was higher than that in ST398 (c2=43.487, p<0.01) and ST188 isolates (c2=32.500, p<0.01), whereas the prevalence of eta in ST45 isolates was higher than in ST188 isolates (c2=14.339, p<0.01). On the contrary, the positive rate of hlb in ST45 isolates was lower than in ST398 (c2=7.118, p<0.01) and ST188 isolates (c2=7.248, p<0.01). No significant difference was found in the positive rate of any other virulence genes between any two of the three STs (Table 4).