Mechanisms of linezolid resistance in staphylococci and enterococci isolated from two teaching hospitals in Shanghai, China

Background Linezolid is one of the most effective treatments against Gram-positive pathogens. However, linezolid-resistant/intermediate strains have recently emerged in worldwide. The purpose of this study was to analyse the prevalence and resistance mechanisms of linezolid-resistant/intermediate staphylococci and enterococci in Shanghai, China. Results Thirty-two linezolid-resistant/intermediate strains, including 14 Staphylococcus capitis, three Staphylococcus aureus, 14 Enterococcus faecalis and one Enterococcus faecium clinical isolates, were collected in this study which displayed linezolid MICs of 8 to 512 μg/ml, 8–32 μg/ml, 4–8 μg/ml and 4 μg/ml, respectively. All linezolid-resistant S. capitis isolates had a novel C2131T mutation and a G2603T mutation in the 23S rRNA region, and some had a C316T (Arg106Cys) substitution in protein L4 and/or harboured cfr. Linezolid-resistant S. aureus isolates carried a C389G (Ala130Gly) substitution in protein L3, and/or harboured cfr. The cfr gene was flanked by two copies of the IS256-like element, with a downstream orf1 gene. Linezolid-resistant/intermediate enterococci lacked major resistance mechanisms. The semi-quantitative biofilm assay showed that 14 linezolid-resistant E. faecalis isolates produced a larger biofilm than linezolid-susceptible E. faecalis strains. Transmission electron microscopy showed the cell walls of linezolid-resistant/intermediate strains were thicker than linezolid-susceptible strains. Conclusion Our data indicated that major resistance mechanisms, such as mutations in 23S rRNA and ribosomal proteins L3 and L4, along with cfr acquisition, played an important role in linezolid resistance. Secondary resistance mechanisms, such as biofilm formation and cell wall thickness, should also be taken into account.


Background
Gram-positive cocci pose a worldwide threat to human health. The emergence of antibiotic resistance in Grampositive cocci, including methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant staphylococci (VRS) and vancomycin-resistant enterococci (VRE), has created a clinical demand for effective novel therapeutic agents. Linezolid (LZD), the first member of the oxazolidinone class of antibiotics, was approved for clinical use in 2000 and has a broad spectrum of activity against a variety of Gram-positive pathogens. It acts by inhibiting protein synthesis via binding to the peptidyl transferase centre of the 50S ribosomal subunit, and preventing formation of the fMet-tRNA-30S ribosome-mRNA initiation complex [1]. Because of its unique antimicrobial mechanism, linezolid has been widely applied in the treatment of clinicallyimportant Gram-positive bacteria, including aerobic and anaerobic Gram-positive cocci, aerobic and anaerobic Gram-positive bacilli, and nocardia and mycobacteria species. However, linezolid-resistant (LR) staphylococcus was first reported in peritonitis patients undergoing oral linezolid treatment during peritoneal dialysis in 2001 [2]. Since then, the occurrence of LR strains has been reported worldwide [3][4][5].
The major mechanism of resistance to linezolid is caused by mutations in the V domain of the 23S rRNA gene, with a G2576T substitution (Escherichia coli numbering) occurring most frequently. C2104T, G2447T, T2500A, A2503G, T2504A, G2603T and G2631T substitutions have also been found in LR strains [6][7][8][9]. Another resistance mechanism is horizontal acquisition of cfr, which encodes a methyltransferase and modifies adenosine at A2503 in the 23S rRNA. cfr is usually plasmid-located and confers cross-resistance to phenicol, lincosamide, oxazolidinone, pleuromutilin and streptogramin A (known as the PhLOPSA phenotype) [10,11]. Alterations in the ribosomal proteins L3, L4 and L22, encoded by rplC, rplD and rplV, respectively, have also been associated with increased resistance to linezolid [12][13][14]. In addition, secondary resistance mechanisms, such as biofilm formation and cell wall thickening, can enhance resistance to antibiotics as well [15,16].

Ethics statement
The collection of the linezolid-resistant/intermediate bacterial isolates from patients and the related information of patients were approved by the ethics committee of Huashan Hospital, Shanghai Medical College, Fudan University and the ethics committee of Renji Hospital, Shanghai Jiaotong University School of Medicine, Shanghai, People's Republic of China. All subjects provided written informed consent before their inclusion in the study.

Bacterial isolates
Thirty-two non-duplicated linezolid-resistant/intermediate isolates were collected from patients of two Shanghai comprehensive teaching hospitals in China from 2009-2013. One of the hospitals was Huashan Hospital, which is a tertiary care hospital affiliated with Fudan University, located in the centre of Shanghai. It is one of the largest (1300 beds) teaching hospitals in china, handling approximately 8000 admissions per day. The other was Renji Hospital, a tertiary care hospital affiliated with Shanghai Jiaotong University, which is located in the east of Shanghai, and is also one of the largest (1800 beds) teaching hospitals in china, handling about 9000 admissions per day. The studied isolates comprised 14 S. capitis (from Huashan Hospital), three S. aureus (from Huashan hospital), 14 E. faecalis (12 isolates from Huashan Hospital and two from Renji Hospital) and one E. faecium (from Huashan Hospital). Among these 32 isolates, 15, 10, 3, 2, 1 and 1 were recovered from patients with bacteraemia, urinary tract infection, pneumonia, wound infection, biliary tract infection and prostate infection, respectively (Table 1).
Isolates were identified using a VITEK 2 compact system (bioMérieux, Marcy l'Etoile, France) and a molecular method based on analysis of the 16S rRNA gene sequence. S. aureus RN4220 was used as the recipient strain for transformation experiments. S. aureus ATCC 29213, E. faecalis ATCC 29212 (both with linezolid MIC of 2 μg/ml, American type culture collection, USA), S. capitis HS12-102 and E. faecium HS13-194 (both with linezolid MIC of <2 μg/ml, from Huashan Hospital) were included as linezolid-susceptible (LS) strains. All strains were stored at −70°C until use and were incubated overnight on blood agar at 37°C.

Antibiotic susceptibility testing
The antimicrobial agents tested were linezolid, vancomycin, teicoplanin, oxacillin, cefoxitin, trimethoprim-sulfamethoxazole, erythromycin, clindamycin, chloramphenicol, tetracycline, ciprofloxacin, penicillin, ampicillin and high-level gentamicin. The MIC of each antimicrobial agent was determined by the broth microdilution MIC method and interpretation of MIC results was based on 2013 Clinical and Laboratory Standards Institute guidelines [20]. S. aureus ATCC 29213 was tested concurrently for quality control.

PFGE
PFGE was performed according to a previously described method [25] with some modifications. The staphylococci were treated with lysostaphin (Sigma-Aldrich, Saint Louis, MO, USA) and genomic DNA was prepared in agarose blocks and then digested with the restriction enzyme SmaI (NEB, Hitchin, UK). The DNA fragments were separated

Molecular detection of resistance genes and mutations
Isolates were screened for the presence of cfr and mutations in the 23S rRNA and the L3, L4 and L22 ribosomal proteins by PCR and DNA sequencing, as previously described [26,27]. Amplicons were sequenced on both strands and were compared with those from S. aureus ATCC 29213, E. faecalis ATCC 29212, LS S. capitis and LS E. faecium, obtained from Shanghai Huashan Hospital during the study period, using the Lasergene software package (DNAStar; Madison, WI, USA).

Gene dosage
Gene dosage was determined according to a previously described method [28,29] with some modifications. Isolates of LR S. capitis containing mutations in the central loop of the 23S rRNA gene were amplified using primers based on the S. capitis 23S rRNA gene (primer F, 5′-AAGGCGTAACGATTTGGG-3′; primer R, 5′-CAGCA CTTATCCCGTCCA-3′; expected PCR product size: 720 bp). Thermal cycler conditions were 94°C for 5 min, followed by 35 cycles of 94°C (30 s), 55°C (30 s) and 72°C (30 s), with a final extension at 72°C (7 min). The DNA concentration of PCR amplicons was normalized to 0.3 pmol prior to cloning. Then, the amplification products were ligated to plasmid pMD-18 T (Takara Biotechnology, Dalian, China) and transformed into E. coli DH5α cells (Sangon Biotech, Shanghai, China). Each LR S. capitis was cloned and 30 clones from each strain were sequenced.

Transmission electron microscopy (TEM)
Isolates for TEM were grown from a single colony in 10 mL of liquid brain-heart infusion (BHI) medium with 0.5% (w/v) beef extract. Four groups were studied.    exhibited identical band pattern, and four other isolates also shared the same band pattern (HS09-206, HS09-17, HS10-204, HS10-24). HS13-207 showed a unique band pattern ( Figure 1). In addition, E. faecalis isolates from the same ST also exhibited different band patterns.

Resistance genes and mutations
A novel C2131T mutation and a previously reported G2603T mutation, both in domain V of the 23S rRNA gene, were identified in all S. capitis isolates ( Table 2). All S. capitis isolates, except HS09-206, HS09-17, HS10-204, HS10-24 and HS13-207, were positive for cfr. Furthermore, S. capitis HS12-201 and HS12-55 had C316T (Arg106Cys) mutations in ribosomal protein L4, but no mutations were detected in ribosomal proteins L3 or L22 in any isolates. Among S. aureus isolates, only HS12-56 was positive for cfr. S. aureus HS11-202 had a C389G (Ala130Gly) mutation in ribosomal protein L3, but proteins L4 and L22 were wild-type. No mutation was identified in any of the studied genes in the E. faecalis and E. faecium isolates, nor was cfr detected.

Genetic environment of cfr in plasmids
Southern hybridisation indicated that cfr resided on plasmids of similar sizes (ca. 54 kb) in all ten cfr-positive isolates ( Figure 3). The cfr-carrying plasmids were extracted from the isolates and transformed into S. aureus RN4220. Plasmids were then extracted from all the transformants and were confirmed by Southern hybridisation (data not shown). The linezolid MICs of ten transformants were 32 μg/ml. The genetic environment surrounding cfr in pHS (plasmid from Huashan Hospital) was shown to be identical in all ten cfr-carrying plasmids and showed 99% identity to the corresponding region of plasmid pSS-01 (GenBank accession no. JQ041372, Figure 4). The cfr gene was located downstream of aminoglycoside resistance gene aacA-aphD and was flanked by two copies of the IS256-like element, with a downstream orf1 gene.

Biofilm production
The semi-quantitative biofilm assay showed that the mean biofilm production in LR E. faecalis strains was significantly higher than that in LS E. faecalis strains (P < 0.0001, one-way ANOVA), whereas there were no significant differences between LR and LS isolates from the other three species ( Figure 5).

Electron microscopy
TEM showed differences in cell wall thickness between LS, LI and LR strains grown with and without linezolid ( Figure 6, Table 3). The overall cell diameter had not significant difference between any of the strains of each species (data not shown), but the cell walls of LI and LR strains were thicker than those of LS strains (P < 0.0001, two-tailed t-test),except for LI E. faecium HS11-306. Furthermore, the cell wall of LR strains grown with linezolid were thicker than that without linezolid (P < 0.0001, twotailed t-test), except for LR E. faecalis HS12-309 (P ≤ 0.001, two-tailed t-test) (Figure 7).

Discussion
The prevalence of MRSA, VRS and VRE strains has presented a new challenge in antimicrobial medication.
Linezolid is one of the few treatment options that is highly active against Gram-positive pathogens. However, LR strains have been increasingly reported in worldwide [2][3][4][5][6][7][9][10][11][12][13][14] and now have also emerged in China [8,[17][18][19]. Although the incidence of linezolid resistance among Gram-positive organisms remains low, the emergence of LR strains is still of great concern. Surveillance data in our study suggested that all the LR S. capitis were recovered from bacteraemia patients, whereas LR S. aureus (LRSA) came from respiratory tract specimens. Linezolid-resistant/intermediate enterococci were collected from blood, urine, wound, bile and prostatic fluid. All of the patients infected with LR staphylococci were critically ill, which is consistent with findings from previous studies [8,[17][18][19]. A study in Spain [31] described that LR strains emerged after patients received three  courses of linezolid for 600 mg every 12 h for 14 days. Interestingly, as shown in Table 1, only seven hospitalised patients (HS09-17, HS10-24, HS11-44, HS12-51, HS13-60, HS13-207 and HS11-307) in our study had received linezolid therapy (range 13-40 days) prior to the appearance of LR strains. The remaining patients, other than outpatients whose antibiotic drug histories were not determined, had not been exposed to linezolid. So, except for linezolid exposure, if there were other factors involved worth considering.
Nowadays, linezolid susceptibility of Gram-positive clinical isolates is mainly monitored through two surveillance programmes, the Zyvox Annual Appraisal of Potency and Spectrum (ZAAPS) Program in Europe and the Linezolid Experience and Accurate Determination of Resistance (LEADER) Program in USA. In 2014, the ZAAPS Program analysed the linezolid activity of 7972 g-positive clinical isolates collected over 9 years (2004-2012) from 73 medical centres in 33 countries on five continents [32]. Data showed that 0.11% of CoNS (8/6909 isolates), 0.02% of S. aureus (4/25148 isolates), 0.92% of E. faecalis (4/434 isolates) and 0.03% of E. faecium (1/333 isolates) isolates were resistant to linezolid. Gu et al. [33] summarized the linezolid susceptibility profile of staphylococci from the LEADER Program over 7 years (2004)(2005)(2006)(2007)(2008)(2009)(2010). Data showed that 1.4% of CoNS (73/5202 isolates) and 0.05% of S. aureus (13/23077 isolates) isolates were resistant to linezolid. Our data showed that the incidence of LR strains in China was higher than that of the two surveillance programs, except for LRCoNS which was similar to that of the LEADER program. In particular, the incidence of LRCoNS in China was nine times higher than that of LRSA, and the incidence of LR E. faecalis was 15 times higher than that of LR E. faecium, suggesting that ongoing surveillance is necessary.
Known mechanisms of linezolid resistance in Grampositive cocci include mutations in the 23S rRNA gene, acquisition of cfr and mutations of ribosomal proteins L3, L4 and L22. The novel mutation C2131T and the previously-described mutation G2603T in the V domain of the 23S rRNA were identified in all 14 LR S. capitis clones. Although G2603T was first reported in 2009 [9], this is the first report of this mutation in China. The novel mutation C2131T, whether it plays a role in linezolid resistance is uncertain and needs a further confirmation. But this revelation may provide new information for investigating the mechanism of linezolid resistance caused by mutations in the V domain of 23S rRNA gene. Because staphylococci possess five or six copies of the 23S rRNA gene, linezolid resistance caused by 23S rRNA mutations develop slowly. Besier et al. indicated that accumulation of single point mutations might be associated with graduallyincreasing levels of resistance, in a mechanism known as "gene dosage" [28]. To gain insight into the relationship between gene dosage and 23S rRNA mutations, we examined the proportion of cloned PCR products with mutations. Our data showed that the percentage of clones with G2603T mutation was significantly higher than that with the C2131T mutation. Independent of other resistance mechanisms, various levels of the 23S rRNA mutations mediated low-to medium-level linezolid resistance (HS09-206, HS09-17, HS10-204, HS10-24, HS13-207; MICs: 8-64 μg/ml). Moreover, the synergistic action of mutations in the 23S rRNA gene and ribosomal proteins L3 and L4, as well as acquisition of cfr gene mediated high-level linezolid resistance (MICs: 128-512 μg/ml). However, the complicated background of resistance mechanisms involved and the biological characteristics of the clinical bacteria overlaid the relationship between gene dosage and linezolid resistance levels.
The cfr gene is often located on transferable plasmid and is thus considered to promote horizontal spread of linezolid resistance. In our study, 10 LR staphylococcal isolates (nine S. capitis and one S. aureus) harboured cfr gene and all the S. aureus RN4220 transformants showed that cfr-carrying pHS mediate medium-level linezolid resistance (MIC 32 μg/ml). Wang et al. had indicated the two copies of IS256-like elements played an important role in the dissemination of cfr in animal isolates [17]. Subsequently, similar structures were found in LR S. capitis from Zhejiang [18], LR S. cohnii from Beijing [19], and now LR S. capitis and LR S. aureus from Shanghai in the current study. Notably, the pHS with cfr flanked by IS256-like elements is the first to be reported in a LRSA strain (HS12-56) in this study. Moreover, compared with plasmid S. capitis MHZ (GenBank accession no. JX232067) from Zhejiang [18], S. capitis pHS possessed additional upstream aminoglycoside resistance gene aacA-aphD. Considering the first cfr-positive LR S. capitis strain was detected in 2011 and HS12-56 LRSA which carried the same plasmid emerged in 2012, it suggested that the pHS was most probably transferred from LRCoNS to LRSA. This finding presented significant concerns about the possibility of cfr-positive LRCoNS acting as reservoirs for linezolid resistance. Based on the information  Table 3. Scale bars indicate 200 nm. above, cfr flanked by two IS256-like elements may be transmitted from animal-derived isolates to human-derived isolates and spread horizontally within, or even between, bacterial species. Therefore, ongoing surveillance is essential to avoid the dissemination of linezolid resistance. Mutations in ribosomal proteins L3, L4 and L22 of the peptidyltransferase centre also contribute to decreased susceptibility to linezolid [7,[12][13][14]. In our study, mutation C316T (Arg106Cys) in L4 was first detected in two S. capitis clones (HS12-201, HS12-55) and mutation C389G (Ala130Gly) in L3 was first detected in S. aureus (HS11-202). None of the LR isolates contained mutations in L22. Because no other mutation was detected in S. aureus HS11-202, which has a linezolid MIC of 16 μg/ml, suggesting that the Ala130Gly mutation in L3 might be associated with low-level linezolid resistance.
Biofilm could provide a microenvironment for bacteria to survive. Bacteria in a biofilm aggregated into micelles which could withstand attacks by innate host defence mechanisms and evaded the threats of antibiotics [15].
We performed a semi-quantitative biofilm assay, which only showed that biofilm formation was significantly higher in LR E. faecalis isolates than in LS E. faecalis isolates. This suggested that formation of biofilm might increase low-level resistance to linezolid in LR E. faecalis (MICs 4-8 μg/ml).
We performed TEM to determine whether the decreased susceptibility of LI and/or LR strains was related to the thickness of the cell wall. We found that the cell walls of LI and LR strains were significantly thicker than LS strains. Also, the cell walls of LR strains grown with linezolid were thicker than those of LR strains grown without linezolid. These thicked cell wall bacteria were probably selected by the linezolid pressure and might be able to avoid clearance in patients undergoing antibiotic therapy. There was no significant differences in cell wall thickness between LI and LR strains. Data suggested that the thickness of the cell wall might decrease susceptibility to linezolid to a certain degree, but not play a major role in linezolid resistance. Furthermore, no significant differences in cell wall thickness were observed between LS HS13-194 and LI HS11-306 E. faecium isolates, however, we observed a thick transparent substance around the bacteria. Whether this substance might hinder linezolid absorption and result in resistance is unknown.

Conclusion
In summary, various levels of LR strains have emerged in Shanghai, China in our study. Mutiple resistance mechanism were involved in these LR strains. Although the prevalence of resistance to linezolid remains low, the emergence of LR staphylococcal and enterococcal clinical isolates should prompt increased attention, especially for the horizontal dissemination of cfr, and surveillance of linezolid resistance in Gram-positive bacteria is of increasing importance.