BACKGROUND
gram-positive cocci pose a worldwide threat to human health.
the emergence of antibiotic resistance in gram-positive cocci, including methicillin-resistant staphylococcus aureus , vancomycin-resistant staphylococci  and vancomycin-resistant enterococci , has created a clinical demand for effective novel therapeutic agents.
linezolid , the first member of the oxazolidinone class of antibiotics, was approved for clinical use in  <dig> 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  <cit> .
because of its unique antimicrobial mechanism, linezolid has been widely applied in the treatment of clinically-important gram-positive bacteria, including aerobic and anaerobic gram-positive cocci, aerobic and anaerobic gram-positive bacilli, and nocardia and mycobacteria species.
however, linezolid-resistant  staphylococcus was first reported in peritonitis patients undergoing oral linezolid treatment during peritoneal dialysis in  <dig>  <cit> .
since then, the occurrence of lr strains has been reported worldwide  <cit> .
the major mechanism of resistance to linezolid is caused by mutations in the v domain of the 23s rrna gene, with a g2576t substitution  occurring most frequently.
c2104t, g2447t, t2500a, a2503g, t2504a, g2603t and g2631t substitutions have also been found in lr strains  <cit> .
another resistance mechanism is horizontal acquisition of cfr, which encodes a methyltransferase and modifies adenosine at a <dig> in the 23s rrna.
cfr is usually plasmid-located and confers cross-resistance to phenicol, lincosamide, oxazolidinone, pleuromutilin and streptogramin a   <cit> .
alterations in the ribosomal proteins l <dig>  l <dig> and l <dig>  encoded by rplc, rpld and rplv, respectively, have also been associated with increased resistance to linezolid  <cit> .
in addition, secondary resistance mechanisms, such as biofilm formation and cell wall thickening, can enhance resistance to antibiotics as well  <cit> .
as the global emergence of lr isolates has increased  <cit> , lr gram-positive cocci have also become a problem in china  <cit> .
to comprehensively understand the current prevalence and resistance mechanisms among lr clinical isolates in china, we analysed  <dig> linezolid-resistant/intermediate strains, including 14 staphylococcus capitis, three s. aureus,  <dig> enterococcus faecalis and one enterococcus faecium isolates.
methods
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– <dig>  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  teaching hospitals in china, handling approximately  <dig> 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  teaching hospitals in china, handling about  <dig> admissions per day.
the studied isolates comprised 14 s. capitis , three s. aureus ,  <dig> e. faecalis  and one e. faecium .
among these  <dig> isolates,  <dig>   <dig>   <dig>   <dig>   <dig> and  <dig> were recovered from patients with bacteraemia, urinary tract infection, pneumonia, wound infection, biliary tract infection and prostate infection, respectively .
isolates were identified using a vitek  <dig> compact system  and a molecular method based on analysis of the 16s rrna gene sequence.
s. aureus rn <dig> was used as the recipient strain for transformation experiments.
s. aureus atcc  <dig>  e. faecalis atcc  <dig> , s. capitis hs12- <dig> and e. faecium hs13- <dig>  were included as linezolid-susceptible  strains.
all strains were stored at −70°c until use and were incubated overnight on blood agar at 37°c.table  <dig>
clinical characteristics of patients with linezolid-resistant
staphylococcus
or
enterococcus
infection
strains
organism
underlying diseases
infection type
ward
prior antibiotic use
outcome
s. capitis
s. capitis
s. capitis
s. capitis
s. capitis
s. capitis
s. capitis
s. capitis
s. capitis
s. capitis
s. capitis
s. capitis
s. capitis
s. capitis
s. aureus
s. aureus
s. aureus
e. faecalis
e. faecalis
e. faecalis
e. faecalis
e. faecalis
e. faecalis
e. faecalis
e. faecalis
e. faecalis
e. faecalis
e. faecalis
e. faecalis
e. faecalis
e. faecalis
e. faecium
hs, isolate from huashan hospital; rj, isolate from renji hospital; copd, chronic obstructive pulmonary disease; scf, cefoperazone/sulbactam; amk, amikacin; mtz, metronidazole; mem, meropenem; lzd, linezolid; dox, doxycycline; sxt, sulfamethoxazole/trimethoprim; van, vancomycin; tzp, piperacillin/tazobactam; lvx, levofloxacin; ipm, imipenem; fep, cefepime; tec, teicoplanin; caz, ceftazidime; sam, ampicillin/sulbactam; fof, fosfomycin; cxm, cefuroxime; mxf, moxifloxacin; ihy, isonicotinyl hydrazide; pyr, pyrazinamide; azm, azithromycin.
n/d, not determined.
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  <dig> clinical and laboratory standards institute guidelines  <cit> .
s. aureus atcc  <dig> was tested concurrently for quality control.
multilocus sequence typing  analysis
s. aureus, e. faecalis and e. faecium were screened using a previously described method  <cit>  to detect the seven housekeeping genes.
for s. aureus, there are carbamate kinase , shikimate dehydrogenase , glycerol kinase , guanylate kinase , phosphate acetyltransferase , triosephosphate isomerase , and acetyl coenzyme a acetyltransferase .
for e. faecalis, there are glucose-6-phosphate dehydrogenase , glyceraldehydes-3-phosphate dehydrogenase , phosphate atp binding cassette transporter , glucokinase , shikimate-5-dehydrogenase , xanthine phosphoribosyltransferase , and acetyl-coa acetyltransferase .
for e. faecium, there are adenylate kinase , atp synthase, alpha subunit , d-alanine:d-alanine ligase , glyceraldehyde-3-phosphate dehydrogenase , glucose-6-phosphate dehydrogenase , phosphoribosylaminoimidazol carboxylase atpase subunit , and phosphate atp-binding cassette transporter .
each sequence was submitted to the mlst website  to determine the allelic profile and sequence type  of each isolate  <cit> .
pfge
pfge was performed according to a previously described method  <cit>  with some modifications.
the staphylococci were treated with lysostaphin  and genomic dna was prepared in agarose blocks and then digested with the restriction enzyme smai .
the dna fragments were separated using a chef-dr ii pfge system  for 20 h at 6 v/cm and 14°c, with a pulse angle of 120° and pulse times from 5–25 s. pfge banding patterns were analysed visually.
molecular detection of resistance genes and mutations
isolates were screened for the presence of cfr and mutations in the 23s rrna and the l <dig>  l <dig> and l <dig> ribosomal proteins by pcr and dna sequencing, as previously described  <cit> .
amplicons were sequenced on both strands and were compared with those from s. aureus atcc  <dig>  e. faecalis atcc  <dig>  ls s. capitis and ls e. faecium, obtained from shanghai huashan hospital during the study period, using the lasergene software package .
gene dosage
gene dosage was determined according to a previously described method  <cit>  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 .
thermal cycler conditions were 94°c for 5 min, followed by 35 cycles of 94°c , 55°c  and 72°c , with a final extension at 72°c .
the dna concentration of pcr amplicons was normalized to  <dig>  pmol prior to cloning.
then, the amplification products were ligated to plasmid pmd-18 t  and transformed into e. coli dh5α cells .
each lr s. capitis was cloned and  <dig> clones from each strain were sequenced.
plasmid analysis and cfr location
plasmid dna was extracted from all cfr-carrying isolates using a plasmid dna midi kit  and separated on 1%  agarose gel.
the migration distances of dna bands were measured directly from gel photographs in relation to the reference plasmids in e. coli v <dig>  marker.
plasmid dna bands were transferred onto a nylon membrane by southern blotting.
a cfr-specific labelled probe  was generated using a non-radioactive dig high prime dna labelling and detection kit  according to the manufacturer’s instructions, and was hybridised to the membrane.
to determine the genetic environment of cfr, the partial plasmid dna sequence of all cfr-carrying isolates was determined using the following primers, designed based on the sequence of pss- <dig> : 1 f 5′- cacttcctttattatttttc-3′; 1r 5′- cgactgaatcaagaagtacg-3′; 2 f 5′-tgaaccataacctttgtc-3′, 2r 5′-tgtttctagcctgactga-3′; 3 f 5′-actcgatggtcctcacgg-3′, 3r 5′-tcaggctcattattacttc-3′; 4 f 5′-ttaccactagagcaaatt′, 4r 5′-gaccacaagcagcgycaa-3′; 5 f 5′-aatggactactgggtgga-3′, 5r 5′-atgattcagaaagcagat-3′; 6 f 5′-ctttccgaatggactaat-3′, 6r 5′-tgagcgcagctttacgac-3′ .
amplification products from the ten cfr-carrying plasmids were sequenced on both strands and were compared with plasmid pss- <dig> using the lasergene software package .
in addition, the purified plasmids extracted from each of the ten original strains were electrotransformed  into the s. aureus recipient strain rn <dig>  transformants were incubated in fresh tryptic soy broth  at 37°c with shaking at 200 rpm for 1 h and then were selected by incubation for 24 h at 37°c on tsb agar supplemented with 6 μg/ml linezolid.
the linezolid mics of transformants were measured by the broth microdilution method.
plasmid dna was extracted from all the transformants and separated on 1% agarose gel.
southern hybridisation was then used to confirm whether the plasmids were cfr-positive as described previously  <cit> .
detection of biofilm production
all lr strains were divided into four groups by species .
s. capitis group contained  <dig> lr isolates and  <dig> ls isolates.
s. aureus group contained  <dig> lr isolates and  <dig> ls isolates.
e. faecalis group contained  <dig> lr isolates and  <dig> ls isolates.
e. faecium group contained  <dig> lr isolate and  <dig> ls isolates.
a semi-quantitative biofilm assay was conducted using flat-bottomed, tissue culture-treated, 96-well assay plates .
four wells in each assay plate contained untreated cultures, which served as negative controls.
bacteria were prepared by making a 1: <dig> dilution of an overnight culture grown in tsb containing 5 g/l glucose.
assay plates were inoculated with 200 μl of culture per well  and were incubated at 37°c for 24 h. the medium were removed from assay plates.
then assay plates were washed four times with pbs.
biofilms were fixed by bouin’s fixation for at least 1 h then assay plates were washed four times with pbs.
following crystal violet staining, the biofilms were measured using a synergy h <dig> hybrid multi-mode microplate reader  at od <dig>
transmission electron microscopy
isolates for tem were grown from a single colony in 10 ml of liquid brain-heart infusion  medium with  <dig> %  beef extract.
four groups were studied.
the s. capitis group contained strains hs12- <dig> , hs09- <dig> , hs09-17 + lzd , hs12- <dig>  and hs12-55 + lzd .
the s. aureus group contained s. aureus atcc <dig> , hs11- <dig>  and hs11-202 + lzd .
the e. faecalis group contained e. faecalis atcc <dig> , hs11- <dig> , hs12- <dig>  and hs12-309 + lzd .
finally, the e. faecium group contained strains hs13- <dig>  and hs11- <dig> .
the cells were grown at 37°c with shaking at 200 rpm for approximately 4–5 h to mid-exponential phase .
an 8-ml volume of each culture was pelleted by centrifugation at 4000 g for 15 min and suspended in 1 ml of  <dig> %  glutaraldehyde in  <dig>  m phosphate buffer, and stored at 4°c for at least 2 h. the cells were then post-fixed with 1%  osmium tetroxide in the same buffer.
following dehydration, cells were infiltrated with and embedded in eponate  <dig> resin for 24 h. ultrathin sections were cut at 70 nm, stained with uranyl acetate and lead citrate, and then examined on a jem- <dig> 80 kv tem  equipped with a gatan orius sc200w camera.
images were captured at  <dig> × magnification and measurements were made using imagej software  <cit> .
thirty cell walls were measured per sample, and means and standard deviations were calculated in excel .
RESULTS
incidence of linezolid resistance
from 2009– <dig>   <dig> staphylococcus and  <dig> enterococcus non-duplicated isolates were tested for susceptibility to linezolid.
the incidence of linezolid resistance in our study was  <dig> % of coagulase-negative staphylococci  ,  <dig> % of s. aureus ,  <dig> % of e. faecalis  and  <dig> % of e. faecium  isolates.
antimicrobial susceptibility testing
the susceptibility profiles of 14 s. capitis isolates, three s. aureus isolates and  <dig> e. faecalis isolates were similar .
the s. capitis isolates showed various levels of linezolid resistance, with mic values of 8–512 μg/ml.
the s. aureus isolates were resistant to linezolid, with mic values of 8–32 μg/ml.
other than s. aureus hs09- <dig>  all staphylococcal strains were methicillin-resistant and exhibited high-level resistance to most of the antibiotics tested, except for vancomycin, teicoplanin and tetracycline.
moreover,  <dig> staphylococcal isolates showed different levels of trimethoprim-sulfamethoxazole susceptibility data, with mic values of  <dig> / <dig> –8/152 μg/ml.
the fifteen enterococcal isolates demonstrated either linezolid-intermediate  or low-level linezolid resistance .
these strains were resistant to erythromycin, chloramphenicol, ciprofloxacin and tetracycline , but susceptible to vancomycin , teicoplanin , penicillin  and ampicillin .
eight enterococcal isolates  were not high-level resistant to gentamicin, whereas the remaining strains were resistant.table  <dig>
antimicrobial susceptibility profiles and genetic resistance markers of linezolid-resistant
staphylococcus
and
enterococcus
isolates
strain id
source
st
mic
genetic resistance markers
lzd
van
tec
oxa
fox
ery
cli
chl
tet
cip
pen
amp
gnh
sxt
23s rrna mutation
cfr
l3
l4
l22
s. capitis
s. aureus
e. faecalis
e. faecium
st, sequence typing; r, resistant; s, susceptible; lzd, linezolid; van, vancomycin; oxa, oxacillin; fox, cefoxitin; ery, erythromycin; cli, clindamycin; chl, chloramphenicol; tet, tetracycline; cip, ciprofloxacin; pen, penicillin; amp, ampicillin; gnh, high-level gentamicin; sxt, trimethoprim-sulfamethoxazole; n/d, not determined.
mlst and molecular typing
all of the isolates, except for those from s. capitis, were typed by mlst.
the sts of the three s. aureus isolates were st <dig>  st <dig> and st <dig>  the  <dig> e. faecalis isolates were classified into eight sts: two isolates each belonged to st <dig>  st <dig>  st <dig>  and st <dig>  while three were st <dig> and three others belonged to different sts.
the e. faecium isolate belonged to st <dig>  pfge analysis showed that nine s. capitis isolates  exhibited identical band pattern, and four other isolates also shared the same band pattern .
hs13- <dig> showed a unique band pattern .
in addition, e. faecalis isolates from the same st also exhibited different band patterns.figure  <dig>
pfge of
sma
i-digested chromosomal dna of linezolid-resistant isolates.
lane 17– <dig>  fourteen isolates of lr s. capitis.
<dig>  hs09-17;  <dig>  hs10-24;  <dig>  hs11-44;  <dig>  hs12-49;  <dig>  hs12-51;  <dig>  hs12-53;  <dig>  hs12-55;  <dig>  hs12-58;  <dig>  hs13-60;  <dig>  hs12-201;  <dig>  hs11-203;  <dig>  hs10-204;  <dig>  hs09-206;  <dig>  hs13- <dig>  lane 302– <dig>  fourteen isolates of lr e. faecalis.
<dig>  hs13-302;  <dig>  hs12-310;  <dig>  hs13-312;  <dig>  hs13-301;  <dig>  hs11-305;  <dig>  hs10-311;  <dig>  hs11-307;  <dig>  rj13-315;  <dig>  hs12-309;  <dig>  rj13-314;  <dig>  hs13-303;  <dig>  hs12-308;  <dig>  hs13-313;  <dig>  hs11-304;  <dig>  hs10- <dig>  lane 56– <dig>  three isolates of lr s. aureus.
<dig>  hs12-56;  <dig>  hs11-202;  <dig>  hs09- <dig>
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 .
all s. capitis isolates, except hs09- <dig>  hs09- <dig>  hs10- <dig>  hs10- <dig> and hs13- <dig>  were positive for cfr.
furthermore, s. capitis hs12- <dig> and hs12- <dig> had c316t  mutations in ribosomal protein l <dig>  but no mutations were detected in ribosomal proteins l <dig> or l <dig> in any isolates.
among s. aureus isolates, only hs12- <dig> was positive for cfr.
s. aureus hs11- <dig> had a c389g  mutation in ribosomal protein l <dig>  but proteins l <dig> and l <dig> 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.
gene dosage
gene dosage tests were performed on fourteen lr s. capitis isolates that contained mutations in domain v of the 23s rrna gene.
isolates with different linezolid resistance levels displayed diverse c2131t and g2603t mutation rates .
the relationship between linezolid resistance levels and the percentage of mutated 23s rrna genes in s. capitis is shown in figure  <dig>  clones derived from s. capitis  had mutation rates of  <dig> – <dig> %  and  <dig> –100% , and those derived from s. capitis  had mutation rates of  <dig> – <dig> %  and  <dig> –100% .figure  <dig>
mutations in the 23s rrna gene and linezolid resistance levels in lr
s. capitis
isolates.
a. the percentages of clones with c2131t and g2603t mutations.
b. the percentage of clones with c2131t mutations in isolates with various linezolid resistance levels.
c. the percentage of clones with g2603t mutations in isolates with various linezolid resistance levels.
error bars represent the mean ± sem.
genetic environment of cfr in plasmids
southern hybridisation indicated that cfr resided on plasmids of similar sizes  in all ten cfr-positive isolates .
the cfr-carrying plasmids were extracted from the isolates and transformed into s. aureus rn <dig>  plasmids were then extracted from all the transformants and were confirmed by southern hybridisation .
the linezolid mics of ten transformants were 32 μg/ml.
the genetic environment surrounding cfr in phs  was shown to be identical in all ten cfr-carrying plasmids and showed 99% identity to the corresponding region of plasmid pss- <dig> .
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 orf <dig> gene.figure  <dig>
analysis of plasmids in
cfr
-positive
s. capitis
isolates.
plasmids of ca.54 kb were detected in all ten
cfr
-positive isolates.
a. southern hybridisation of s. capitis isolates with a cfr probe.
b. m, e. coli v <dig> marker;  <dig>  hs11-203;  <dig>  hs11-44;  <dig>  hs12-49;  <dig>  hs12-201;  <dig>  hs12-51;  <dig>  hs12-53;  <dig>  hs12-55;  <dig>  hs12-58;  <dig>  hs13-60;  <dig>  hs12- <dig>
schematic representation of the genetic environment of
cfr
in phs and pss- <dig>  the arrows indicate the positions and directions of the transcription of the genes.
the regions of homology between phs and pss- <dig>  are indicated by dashed lines and grey shading.
Δ indicates a truncated 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 , whereas there were no significant differences between lr and ls isolates from the other three species .figure  <dig>
semi-quantitative biofilm analysis of
s. capitis, s. aureus, e. faecalis, e. faecium
groups.
ls strains were used as controls.
comparisons are vs. ls e. faecalis strains for lr e. faecalis strains.
***p <  <dig>  .
electron microscopy
tem showed differences in cell wall thickness between ls, li and lr strains grown with and without linezolid .
the overall cell diameter had not significant difference between any of the strains of each species , but the cell walls of li and lr strains were thicker than those of ls strains ,except for li e. faecium hs11- <dig>  furthermore, the cell wall of lr strains grown with linezolid were thicker than that without linezolid , except for lr e. faecalis hs12- <dig>  .figure  <dig>
transmission electron micrographs  showing cell wall thickness in bacterial isolates.
tem images show ls s. capitis hs12- <dig> , lr s. capitis hs09- <dig>  grown without  and with  4 μg/ml linezolid, lr s. capitis hs12- <dig>  grown without  and with  32 μg/ml linezolid, wild-type atcc <dig> s. aureus
, lr s. aureus hs11- <dig>  grown without  and with  2 μg/ml linezolid, wild-type atcc <dig> e. faecalis
, li e. faecalis hs11- <dig>  grown without linezolid , lr e. faecalis hs12- <dig>  grown without  and with  2 μg/ml linezolid, ls e. faecium hs13- <dig> , li e. faecium hs11- <dig>  grown without linezolid .
cell wall thicknesses are given in table  <dig>  scale bars indicate 200 nm.
cell wall thickness measured by tem
species
strains
cell wall thickness
a
s. capitis
s. capitis
s. capitis
s. capitis
s. capitis
s. aureus
s. aureus
s. aureus
e. faecalis
e. faecalis
e. faecalis
e. faecalis
e. faecium
e. faecium
ameasurements  are means ± standard deviations from  <dig> cells per sample.
comparison between bacterial cell wall thickness in ls, lr and li isolates.
there are s. capitis
, s. aureus
, e. faecalis
and e. faecium
isolates.
<dig>  hs12-102;  <dig>  hs09-17; 17 + lzd, hs09- <dig> grown with 4 μg/ml linezolid;  <dig>  hs12-55; 55 + lzd, hs12- <dig> grown with 32 μg/ml linezolid; atcc <dig>  atcc <dig> ls s. aureus;  <dig>  hs11-202; 202 + lzd, hs11- <dig> grown with 2 μg/ml linezolid; atcc <dig>  atcc <dig> ls e. faecalis;  <dig>  hs11-304;  <dig>  hs12-309; 309 + lzd, hs12- <dig> grown with 2 μg/ml linezolid;  <dig>  hs13-194;  <dig>  hs11- <dig> .
ls reference strains hs12- <dig> , atcc <dig> , atcc <dig>  and hs13- <dig>  were used as controls for the respective groups.
<dig> versus  <dig>   <dig> versus  <dig>  17 + lzd versus  <dig>  55 + lzd versus  <dig>   <dig> versus atcc <dig>  202 + lzd versus  <dig>   <dig> versus atcc <dig>   <dig> versus atcc <dig>  309 + lzd versus hs12- <dig> showed significantly differences .
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  <cit>  and now have also emerged in china  <cit> .
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  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  <cit> .
a study in spain  <cit>  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  <dig>  only seven hospitalised patients  in our study had received linezolid therapy  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  program in europe and the linezolid experience and accurate determination of resistance  program in usa.
in  <dig>  the zaaps program analysed the linezolid activity of  <dig> g-positive clinical isolates collected over 9 years  from  <dig> medical centres in  <dig> countries on five continents  <cit> .
data showed that  <dig> % of cons ,  <dig> % of s. aureus ,  <dig> % of e. faecalis  and  <dig> % of e. faecium  isolates were resistant to linezolid.
gu et al.
<cit>  summarized the linezolid susceptibility profile of staphylococci from the leader program over 7 years .
data showed that  <dig> % of cons  and  <dig> % of s. aureus  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  <dig> times higher than that of lr e. faecium, suggesting that ongoing surveillance is necessary.
recently, multifocal outbreaks of lr staphylococci have been reported, including s. epidermidis , s. cohnii , s. haemolyticus , s. sciuri , s. capitis , s. hominis , s. lugdenesis  and s. aureus , and linezolid-resistant/intermediate enterococci, including e. faecalis , e. faecium  and e. avium   <cit> .
in our study, we detected multiple lr s. capitis, s. aureus, e. faecalis and e. faecium isolates.
notably, the clinically-derived lrsa isolates characterised in our investigation are the first to be reported in china.
previous studies  <cit>  described that most of lrcons in china exhibited high-level resistance , with very few low- and medium-level lr isolates, whereas in other countries  <cit> , low to medium-level lrcons  were more common, with a lack of high-level lrcons strains.
in contrast, the  <dig> lrcons isolates in our study exhibited a wide range of linezolid resistance levels, with mics from  <dig> to 512 μg/ml.
three lrsa isolates exhibited linezolid resistance , which were higher than lrsa isolates from italy, hong kong and brazil .
thereinto, two of the lrsa isolates were methicillin-resistant and multidrug-resistant, whereas hs09- <dig> lrsa was methicillin-susceptible.
moreover, low-level resistance in linezolid-resistant/intermediate enterococci  was similar to previous studies  <cit> .
mlst and pfge results showed that lr s. capitis, lr s. aureus, e. faecalis and e. faecium were polyclonal, suggesting diverse sts were all vulnerable to linezolid exposure.
known mechanisms of linezolid resistance in gram-positive cocci include mutations in the 23s rrna gene, acquisition of cfr and mutations of ribosomal proteins l <dig>  l <dig> and l <dig>  the novel mutation c2131t and the previously-described mutation g2603t in the v domain of the 23s rrna were identified in all  <dig> lr s. capitis clones.
although g2603t was first reported in  <dig>  <cit> , 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 gradually-increasing levels of resistance, in a mechanism known as “gene dosage”  <cit> .
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 .
moreover, the synergistic action of mutations in the 23s rrna gene and ribosomal proteins l <dig> and l <dig>  as well as acquisition of cfr gene mediated high-level linezolid resistance .
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,  <dig> lr staphylococcal isolates  harboured cfr gene and all the s. aureus rn <dig> transformants showed that cfr-carrying phs mediate medium-level linezolid resistance .
wang et al.
had indicated the two copies of is256-like elements played an important role in the dissemination of cfr in animal isolates  <cit> .
subsequently, similar structures were found in lr s. capitis from zhejiang  <cit> , lr s. cohnii from beijing  <cit> , 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  in this study.
moreover, compared with plasmid s. capitis mhz  from zhejiang  <cit> , s. capitis phs possessed additional upstream aminoglycoside resistance gene aaca-aphd.
considering the first cfr-positive lr s. capitis strain was detected in  <dig> and hs12- <dig> lrsa which carried the same plasmid emerged in  <dig>  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 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 l <dig>  l <dig> and l <dig> of the peptidyltransferase centre also contribute to decreased susceptibility to linezolid  <cit> .
in our study, mutation c316t  in l <dig> was first detected in two s. capitis clones  and mutation c389g  in l <dig> was first detected in s. aureus .
none of the lr isolates contained mutations in l <dig>  because no other mutation was detected in s. aureus hs11- <dig>  which has a linezolid mic of 16 μg/ml, suggesting that the ala130gly mutation in l <dig> 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  <cit> .
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 .
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- <dig> and li hs11- <dig> 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.
CONCLUSIONS
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.
competing interests
the authors declare that they have no competing interests.
authors’ contributions
yt performed all experiments.
tl assisted in mlst and gene dosage experiments.
yz assisted in pfge experiment.
bw and xz assisted in antimicrobial susceptibility testing.
ml and yt conceived the study and analyzed the results.
ml supervised the study and wrote the manuscript.
all authors read and approved the final manuscript.

 gram-positive cocci pose a worldwide threat to human health. the emergence of antibiotic resistance in gram-positive cocci, including methicillin-resistant staphylococcus aureus, vancomycin-resistant staphylococci and vancomycin-resistant enterococci, has created a clinical demand for effective novel therapeutic agents. linezolid, the first member of the oxazolidinone class of antibiotics, was approved for clinical use in 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. because of its unique antimicrobial mechanism, linezolid has been widely applied in the treatment of clinically-important gram-positive bacteria, including aerobic and anaerobic gram-positive cocci, aerobic and anaerobic gram-positive bacilli, and nocardia and mycobacteria species. however, linezolid-resistant staphylococcus was first reported in peritonitis patients undergoing oral linezolid treatment during peritoneal dialysis in. since then, the occurrence of lr strains has been reported worldwide. the major mechanism of resistance to linezolid is caused by mutations in the v domain of the 23s rrna gene, with a g2576t substitution occurring most frequently. c2104t, g2447t, t2500a, a2503g, t2504a, g2603t and g2631t substitutions have also been found in lr strains. another resistance mechanism is horizontal acquisition of cfr, which encodes a methyltransferase and modifies adenosine at a in the 23s rrna. cfr is usually plasmid-located and confers cross-resistance to phenicol, lincosamide, oxazolidinone, pleuromutilin and streptogramin a. alterations in the ribosomal proteins l l and l encoded by rplc, rpld and rplv, respectively, have also been associated with increased resistance to linezolid. in addition, secondary resistance mechanisms, such as biofilm formation and cell wall thickening, can enhance resistance to antibiotics as well. as the global emergence of lr isolates has increased, lr gram-positive cocci have also become a problem in china. to comprehensively understand the current prevalence and resistance mechanisms among lr clinical isolates in china, we analysed linezolid-resistant/intermediate strains, including 14 staphylococcus capitis, three s. aureus, enterococcus faecalis and one enterococcus faecium isolates. methods 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– 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 teaching hospitals in china, handling approximately 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 teaching hospitals in china, handling about admissions per day. the studied isolates comprised 14 s. capitis, three s. aureus, e. faecalis and one e. faecium. among these isolates, and were recovered from patients with bacteraemia, urinary tract infection, pneumonia, wound infection, biliary tract infection and prostate infection, respectively. isolates were identified using a vitek compact system and a molecular method based on analysis of the 16s rrna gene sequence. s. aureus rn was used as the recipient strain for transformation experiments. s. aureus atcc e. faecalis atcc, s. capitis hs12- and e. faecium hs13- were included as linezolid-susceptible strains. all strains were stored at −70°c until use and were incubated overnight on blood agar at 37°c.table clinical characteristics of patients with linezolid-resistant staphylococcus or enterococcus infection strains organism underlying diseases infection type ward prior antibiotic use outcome s. capitis s. capitis s. capitis s. capitis s. capitis s. capitis s. capitis s. capitis s. capitis s. capitis s. capitis s. capitis s. capitis s. capitis s. aureus s. aureus s. aureus e. faecalis e. faecalis e. faecalis e. faecalis e. faecalis e. faecalis e. faecalis e. faecalis e. faecalis e. faecalis e. faecalis e. faecalis e. faecalis e. faecalis e. faecium hs, isolate from huashan hospital; rj, isolate from renji hospital; copd, chronic obstructive pulmonary disease; scf, cefoperazone/sulbactam; amk, amikacin; mtz, metronidazole; mem, meropenem; lzd, linezolid; dox, doxycycline; sxt, sulfamethoxazole/trimethoprim; van, vancomycin; tzp, piperacillin/tazobactam; lvx, levofloxacin; ipm, imipenem; fep, cefepime; tec, teicoplanin; caz, ceftazidime; sam, ampicillin/sulbactam; fof, fosfomycin; cxm, cefuroxime; mxf, moxifloxacin; ihy, isonicotinyl hydrazide; pyr, pyrazinamide; azm, azithromycin. n/d, not determined. 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 clinical and laboratory standards institute guidelines. s. aureus atcc was tested concurrently for quality control. multilocus sequence typing analysis s. aureus, e. faecalis and e. faecium were screened using a previously described method to detect the seven housekeeping genes. for s. aureus, there are carbamate kinase, shikimate dehydrogenase, glycerol kinase, guanylate kinase, phosphate acetyltransferase, triosephosphate isomerase, and acetyl coenzyme a acetyltransferase. for e. faecalis, there are glucose-6-phosphate dehydrogenase, glyceraldehydes-3-phosphate dehydrogenase, phosphate atp binding cassette transporter, glucokinase, shikimate-5-dehydrogenase, xanthine phosphoribosyltransferase, and acetyl-coa acetyltransferase. for e. faecium, there are adenylate kinase, atp synthase, alpha subunit, d-alanine:d-alanine ligase, glyceraldehyde-3-phosphate dehydrogenase, glucose-6-phosphate dehydrogenase, phosphoribosylaminoimidazol carboxylase atpase subunit, and phosphate atp-binding cassette transporter. each sequence was submitted to the mlst website to determine the allelic profile and sequence type of each isolate. pfge pfge was performed according to a previously described method with some modifications. the staphylococci were treated with lysostaphin and genomic dna was prepared in agarose blocks and then digested with the restriction enzyme smai. the dna fragments were separated using a chef-dr ii pfge system for 20 h at 6 v/cm and 14°c, with a pulse angle of 120° and pulse times from 5–25 s. pfge banding patterns were analysed visually. molecular detection of resistance genes and mutations isolates were screened for the presence of cfr and mutations in the 23s rrna and the l l and l ribosomal proteins by pcr and dna sequencing, as previously described. amplicons were sequenced on both strands and were compared with those from s. aureus atcc e. faecalis atcc ls s. capitis and ls e. faecium, obtained from shanghai huashan hospital during the study period, using the lasergene software package. gene dosage gene dosage was determined according to a previously described method 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. thermal cycler conditions were 94°c for 5 min, followed by 35 cycles of 94°c, 55°c and 72°c, with a final extension at 72°c. the dna concentration of pcr amplicons was normalized to pmol prior to cloning. then, the amplification products were ligated to plasmid pmd-18 t and transformed into e. coli dh5α cells. each lr s. capitis was cloned and clones from each strain were sequenced. plasmid analysis and cfr location plasmid dna was extracted from all cfr-carrying isolates using a plasmid dna midi kit and separated on 1% agarose gel. the migration distances of dna bands were measured directly from gel photographs in relation to the reference plasmids in e. coli v marker. plasmid dna bands were transferred onto a nylon membrane by southern blotting. a cfr-specific labelled probe was generated using a non-radioactive dig high prime dna labelling and detection kit according to the manufacturer’s instructions, and was hybridised to the membrane. to determine the genetic environment of cfr, the partial plasmid dna sequence of all cfr-carrying isolates was determined using the following primers, designed based on the sequence of pss-: 1 f 5′- cacttcctttattatttttc-3′; 1r 5′- cgactgaatcaagaagtacg-3′; 2 f 5′-tgaaccataacctttgtc-3′, 2r 5′-tgtttctagcctgactga-3′; 3 f 5′-actcgatggtcctcacgg-3′, 3r 5′-tcaggctcattattacttc-3′; 4 f 5′-ttaccactagagcaaatt′, 4r 5′-gaccacaagcagcgycaa-3′; 5 f 5′-aatggactactgggtgga-3′, 5r 5′-atgattcagaaagcagat-3′; 6 f 5′-ctttccgaatggactaat-3′, 6r 5′-tgagcgcagctttacgac-3′. amplification products from the ten cfr-carrying plasmids were sequenced on both strands and were compared with plasmid pss- using the lasergene software package. in addition, the purified plasmids extracted from each of the ten original strains were electrotransformed into the s. aureus recipient strain rn transformants were incubated in fresh tryptic soy broth at 37°c with shaking at 200 rpm for 1 h and then were selected by incubation for 24 h at 37°c on tsb agar supplemented with 6 μg/ml linezolid. the linezolid mics of transformants were measured by the broth microdilution method. plasmid dna was extracted from all the transformants and separated on 1% agarose gel. southern hybridisation was then used to confirm whether the plasmids were cfr-positive as described previously. detection of biofilm production all lr strains were divided into four groups by species. s. capitis group contained lr isolates and ls isolates. s. aureus group contained lr isolates and ls isolates. e. faecalis group contained lr isolates and ls isolates. e. faecium group contained lr isolate and ls isolates. a semi-quantitative biofilm assay was conducted using flat-bottomed, tissue culture-treated, 96-well assay plates. four wells in each assay plate contained untreated cultures, which served as negative controls. bacteria were prepared by making a 1: dilution of an overnight culture grown in tsb containing 5 g/l glucose. assay plates were inoculated with 200 μl of culture per well and were incubated at 37°c for 24 h. the medium were removed from assay plates. then assay plates were washed four times with pbs. biofilms were fixed by bouin’s fixation for at least 1 h then assay plates were washed four times with pbs. following crystal violet staining, the biofilms were measured using a synergy h hybrid multi-mode microplate reader at od transmission electron microscopy isolates for tem were grown from a single colony in 10 ml of liquid brain-heart infusion medium with % beef extract. four groups were studied. the s. capitis group contained strains hs12-, hs09-, hs09-17 + lzd, hs12- and hs12-55 + lzd. the s. aureus group contained s. aureus atcc, hs11- and hs11-202 + lzd. the e. faecalis group contained e. faecalis atcc, hs11-, hs12- and hs12-309 + lzd. finally, the e. faecium group contained strains hs13- and hs11-. the cells were grown at 37°c with shaking at 200 rpm for approximately 4–5 h to mid-exponential phase. an 8-ml volume of each culture was pelleted by centrifugation at 4000 g for 15 min and suspended in 1 ml of % glutaraldehyde in  m phosphate buffer, and stored at 4°c for at least 2 h. the cells were then post-fixed with 1% osmium tetroxide in the same buffer. following dehydration, cells were infiltrated with and embedded in eponate resin for 24 h. ultrathin sections were cut at 70 nm, stained with uranyl acetate and lead citrate, and then examined on a jem- 80 kv tem equipped with a gatan orius sc200w camera. images were captured at × magnification and measurements were made using imagej software. thirty cell walls were measured per sample, and means and standard deviations were calculated in excel.  incidence of linezolid resistance from 2009– staphylococcus and enterococcus non-duplicated isolates were tested for susceptibility to linezolid. the incidence of linezolid resistance in our study was % of coagulase-negative staphylococci, % of s. aureus, % of e. faecalis and % of e. faecium isolates. antimicrobial susceptibility testing the susceptibility profiles of 14 s. capitis isolates, three s. aureus isolates and e. faecalis isolates were similar. the s. capitis isolates showed various levels of linezolid resistance, with mic values of 8–512 μg/ml. the s. aureus isolates were resistant to linezolid, with mic values of 8–32 μg/ml. other than s. aureus hs09- all staphylococcal strains were methicillin-resistant and exhibited high-level resistance to most of the antibiotics tested, except for vancomycin, teicoplanin and tetracycline. moreover, staphylococcal isolates showed different levels of trimethoprim-sulfamethoxazole susceptibility data, with mic values of / –8/152 μg/ml. the fifteen enterococcal isolates demonstrated either linezolid-intermediate or low-level linezolid resistance. these strains were resistant to erythromycin, chloramphenicol, ciprofloxacin and tetracycline, but susceptible to vancomycin, teicoplanin, penicillin and ampicillin. eight enterococcal isolates were not high-level resistant to gentamicin, whereas the remaining strains were resistant.table antimicrobial susceptibility profiles and genetic resistance markers of linezolid-resistant staphylococcus and enterococcus isolates strain id source st mic genetic resistance markers lzd van tec oxa fox ery cli chl tet cip pen amp gnh sxt 23s rrna mutation cfr l3 l4 l22 s. capitis s. aureus e. faecalis e. faecium st, sequence typing; r, resistant; s, susceptible; lzd, linezolid; van, vancomycin; oxa, oxacillin; fox, cefoxitin; ery, erythromycin; cli, clindamycin; chl, chloramphenicol; tet, tetracycline; cip, ciprofloxacin; pen, penicillin; amp, ampicillin; gnh, high-level gentamicin; sxt, trimethoprim-sulfamethoxazole; n/d, not determined. mlst and molecular typing all of the isolates, except for those from s. capitis, were typed by mlst. the sts of the three s. aureus isolates were st st and st the e. faecalis isolates were classified into eight sts: two isolates each belonged to st st st and st while three were st and three others belonged to different sts. the e. faecium isolate belonged to st pfge analysis showed that nine s. capitis isolates exhibited identical band pattern, and four other isolates also shared the same band pattern. hs13- showed a unique band pattern. in addition, e. faecalis isolates from the same st also exhibited different band patterns.figure pfge of sma i-digested chromosomal dna of linezolid-resistant isolates. lane 17– fourteen isolates of lr s. capitis. hs09-17; hs10-24; hs11-44; hs12-49; hs12-51; hs12-53; hs12-55; hs12-58; hs13-60; hs12-201; hs11-203; hs10-204; hs09-206; hs13- lane 302– fourteen isolates of lr e. faecalis. hs13-302; hs12-310; hs13-312; hs13-301; hs11-305; hs10-311; hs11-307; rj13-315; hs12-309; rj13-314; hs13-303; hs12-308; hs13-313; hs11-304; hs10- lane 56– three isolates of lr s. aureus. hs12-56; hs11-202; hs09- 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. all s. capitis isolates, except hs09- hs09- hs10- hs10- and hs13- were positive for cfr. furthermore, s. capitis hs12- and hs12- had c316t mutations in ribosomal protein l but no mutations were detected in ribosomal proteins l or l in any isolates. among s. aureus isolates, only hs12- was positive for cfr. s. aureus hs11- had a c389g mutation in ribosomal protein l but proteins l and l 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. gene dosage gene dosage tests were performed on fourteen lr s. capitis isolates that contained mutations in domain v of the 23s rrna gene. isolates with different linezolid resistance levels displayed diverse c2131t and g2603t mutation rates. the relationship between linezolid resistance levels and the percentage of mutated 23s rrna genes in s. capitis is shown in figure  clones derived from s. capitis had mutation rates of – % and –100%, and those derived from s. capitis had mutation rates of – % and –100%.figure mutations in the 23s rrna gene and linezolid resistance levels in lr s. capitis isolates. a. the percentages of clones with c2131t and g2603t mutations. b. the percentage of clones with c2131t mutations in isolates with various linezolid resistance levels. c. the percentage of clones with g2603t mutations in isolates with various linezolid resistance levels. error bars represent the mean ± sem. genetic environment of cfr in plasmids southern hybridisation indicated that cfr resided on plasmids of similar sizes in all ten cfr-positive isolates. the cfr-carrying plasmids were extracted from the isolates and transformed into s. aureus rn plasmids were then extracted from all the transformants and were confirmed by southern hybridisation. the linezolid mics of ten transformants were 32 μg/ml. the genetic environment surrounding cfr in phs was shown to be identical in all ten cfr-carrying plasmids and showed 99% identity to the corresponding region of plasmid pss-. 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 orf gene.figure analysis of plasmids in cfr -positive s. capitis isolates. plasmids of ca.54 kb were detected in all ten cfr -positive isolates. a. southern hybridisation of s. capitis isolates with a cfr probe. b. m, e. coli v marker; hs11-203; hs11-44; hs12-49; hs12-201; hs12-51; hs12-53; hs12-55; hs12-58; hs13-60; hs12- schematic representation of the genetic environment of cfr in phs and pss- the arrows indicate the positions and directions of the transcription of the genes. the regions of homology between phs and pss- are indicated by dashed lines and grey shading. Δ indicates a truncated 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, whereas there were no significant differences between lr and ls isolates from the other three species.figure semi-quantitative biofilm analysis of s. capitis, s. aureus, e. faecalis, e. faecium groups. ls strains were used as controls. comparisons are vs. ls e. faecalis strains for lr e. faecalis strains. ***p < . electron microscopy tem showed differences in cell wall thickness between ls, li and lr strains grown with and without linezolid. the overall cell diameter had not significant difference between any of the strains of each species, but the cell walls of li and lr strains were thicker than those of ls strains,except for li e. faecium hs11- furthermore, the cell wall of lr strains grown with linezolid were thicker than that without linezolid, except for lr e. faecalis hs12-.figure transmission electron micrographs showing cell wall thickness in bacterial isolates. tem images show ls s. capitis hs12-, lr s. capitis hs09- grown without and with 4 μg/ml linezolid, lr s. capitis hs12- grown without and with 32 μg/ml linezolid, wild-type atcc s. aureus, lr s. aureus hs11- grown without and with 2 μg/ml linezolid, wild-type atcc e. faecalis, li e. faecalis hs11- grown without linezolid, lr e. faecalis hs12- grown without and with 2 μg/ml linezolid, ls e. faecium hs13-, li e. faecium hs11- grown without linezolid. cell wall thicknesses are given in table  scale bars indicate 200 nm. cell wall thickness measured by tem species strains cell wall thickness a s. capitis s. capitis s. capitis s. capitis s. capitis s. aureus s. aureus s. aureus e. faecalis e. faecalis e. faecalis e. faecalis e. faecium e. faecium ameasurements are means ± standard deviations from cells per sample. comparison between bacterial cell wall thickness in ls, lr and li isolates. there are s. capitis, s. aureus, e. faecalis and e. faecium isolates. hs12-102; hs09-17; 17 + lzd, hs09- grown with 4 μg/ml linezolid; hs12-55; 55 + lzd, hs12- grown with 32 μg/ml linezolid; atcc atcc ls s. aureus; hs11-202; 202 + lzd, hs11- grown with 2 μg/ml linezolid; atcc atcc ls e. faecalis; hs11-304; hs12-309; 309 + lzd, hs12- grown with 2 μg/ml linezolid; hs13-194; hs11-. ls reference strains hs12-, atcc, atcc and hs13- were used as controls for the respective groups. versus versus 17 + lzd versus 55 + lzd versus versus atcc 202 + lzd versus versus atcc versus atcc 309 + lzd versus hs12- showed significantly differences. 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 and now have also emerged in china. 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 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. a study in spain 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  only seven hospitalised patients in our study had received linezolid therapy 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 program in europe and the linezolid experience and accurate determination of resistance program in usa. in the zaaps program analysed the linezolid activity of g-positive clinical isolates collected over 9 years from medical centres in countries on five continents. data showed that % of cons, % of s. aureus, % of e. faecalis and % of e. faecium isolates were resistant to linezolid. gu et al. summarized the linezolid susceptibility profile of staphylococci from the leader program over 7 years. data showed that % of cons and % of s. aureus 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 times higher than that of lr e. faecium, suggesting that ongoing surveillance is necessary. recently, multifocal outbreaks of lr staphylococci have been reported, including s. epidermidis, s. cohnii, s. haemolyticus, s. sciuri, s. capitis, s. hominis, s. lugdenesis and s. aureus, and linezolid-resistant/intermediate enterococci, including e. faecalis, e. faecium and e. avium. in our study, we detected multiple lr s. capitis, s. aureus, e. faecalis and e. faecium isolates. notably, the clinically-derived lrsa isolates characterised in our investigation are the first to be reported in china. previous studies described that most of lrcons in china exhibited high-level resistance, with very few low- and medium-level lr isolates, whereas in other countries, low to medium-level lrcons were more common, with a lack of high-level lrcons strains. in contrast, the lrcons isolates in our study exhibited a wide range of linezolid resistance levels, with mics from to 512 μg/ml. three lrsa isolates exhibited linezolid resistance, which were higher than lrsa isolates from italy, hong kong and brazil. thereinto, two of the lrsa isolates were methicillin-resistant and multidrug-resistant, whereas hs09- lrsa was methicillin-susceptible. moreover, low-level resistance in linezolid-resistant/intermediate enterococci was similar to previous studies. mlst and pfge results showed that lr s. capitis, lr s. aureus, e. faecalis and e. faecium were polyclonal, suggesting diverse sts were all vulnerable to linezolid exposure. known mechanisms of linezolid resistance in gram-positive cocci include mutations in the 23s rrna gene, acquisition of cfr and mutations of ribosomal proteins l l and l the novel mutation c2131t and the previously-described mutation g2603t in the v domain of the 23s rrna were identified in all lr s. capitis clones. although g2603t was first reported in, 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 gradually-increasing levels of resistance, in a mechanism known as “gene dosage”. 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. moreover, the synergistic action of mutations in the 23s rrna gene and ribosomal proteins l and l as well as acquisition of cfr gene mediated high-level linezolid resistance. 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, lr staphylococcal isolates harboured cfr gene and all the s. aureus rn transformants showed that cfr-carrying phs mediate medium-level linezolid resistance. wang et al. had indicated the two copies of is256-like elements played an important role in the dissemination of cfr in animal isolates. subsequently, similar structures were found in lr s. capitis from zhejiang, lr s. cohnii from beijing, 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 in this study. moreover, compared with plasmid s. capitis mhz from zhejiang, s. capitis phs possessed additional upstream aminoglycoside resistance gene aaca-aphd. considering the first cfr-positive lr s. capitis strain was detected in and hs12- lrsa which carried the same plasmid emerged in 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 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 l l and l of the peptidyltransferase centre also contribute to decreased susceptibility to linezolid. in our study, mutation c316t in l was first detected in two s. capitis clones and mutation c389g in l was first detected in s. aureus. none of the lr isolates contained mutations in l because no other mutation was detected in s. aureus hs11- which has a linezolid mic of 16 μg/ml, suggesting that the ala130gly mutation in l 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. 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. 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- and li hs11- 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.  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. competing interests the authors declare that they have no competing interests. authors’ contributions yt performed all experiments. tl assisted in mlst and gene dosage experiments. yz assisted in pfge experiment. bw and xz assisted in antimicrobial susceptibility testing. ml and yt conceived the study and analyzed the results. ml supervised the study and wrote the manuscript. all authors read and approved the final manuscript. 
our data indicated that major resistance mechanisms, such as mutations in 23s rrna and ribosomal proteins l and l 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. 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 substitution in protein l and/or harboured cfr. thirty-two linezolid-resistant/intermediate strains, including 14 staphylococcus capitis, three staphylococcus aureus, enterococcus faecalis and one enterococcus faecium clinical isolates, were collected in this study which displayed linezolid mics of to 512 μg/ml, 8–32 μg/ml, 4–8 μg/ml and 4 μg/ml, respectively. the purpose of this study was to analyse the prevalence and resistance mechanisms of linezolid-resistant/intermediate staphylococci and enterococci in shanghai, china. linezolid-resistant/intermediate enterococci lacked major resistance mechanisms. linezolid resistancemutations of 23s rrna and ribosomal proteinscfrbiofilm productioncell wall thicknessissue-copyright-statement© the author 2014 the cfr gene was flanked by two copies of the is256-like element, with a downstream orf gene. transmission electron microscopy showed the cell walls of linezolid-resistant/intermediate strains were thicker than linezolid-susceptible strains. the semi-quantitative biofilm assay showed that linezolid-resistant e. faecalis isolates produced a larger biofilm than linezolid-susceptible e. faecalis strains. linezolid is one of the most effective treatments against gram-positive pathogens.
 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.  thirty-two linezolid-resistant/intermediate strains, including 14 staphylococcus capitis, three staphylococcus aureus, enterococcus faecalis and one enterococcus faecium clinical isolates, were collected in this study which displayed linezolid mics of 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 substitution in protein l and/or harboured cfr. linezolid-resistant s. aureus isolates carried a c389g substitution in protein l and/or harboured cfr. the cfr gene was flanked by two copies of the is256-like element, with a downstream orf gene. linezolid-resistant/intermediate enterococci lacked major resistance mechanisms. the semi-quantitative biofilm assay showed that 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.  our data indicated that major resistance mechanisms, such as mutations in 23s rrna and ribosomal proteins l and l 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. keywords linezolid resistancemutations of 23s rrna and ribosomal proteinscfrbiofilm productioncell wall thicknessissue-copyright-statement© the author 2014 