BACKGROUND
methicillin resistant staphylococcus aureus  is an important pathogen in spanish hospitals.
the percentage of patients infected or colonised by mrsa among patients with nosocomial s. aureus has been estimated between  <dig> % and  <dig> % in nation-wide multicenter studies  <cit> .
in the hospital universitari de bellvitge mrsa has been endemic since  <dig>  the majority of strains isolated during the 1990- <dig> period belonged to the multiresistant iberian clone.
by multilocus sequence typing  the iberian clone showed an allelic profile or sequence type   <dig>  carrying the staphylococcal cassette chromosome mec  type i  <cit> .
isolates of the iberian clone exhibited resistance against almost all antibiotics available for mrsa therapy including clindamycin, erythromycin, gentamicin, tobramycin, tetracycline, ciprofloxacin and rifampicin.
from  <dig> to  <dig>  the iberian clone was gradually replaced by isolates of clonal complex  <dig>  related to the paediatric clone   <cit> .
unlike the iberian clone, these strains showed only consistent resistance to tobramycin and ciprofloxacin combined with variable resistance to clindamycin and/or erythromycin.
similar trends have been observed in other hospitals in spain and in other countries such as france, germany, belgium or portugal, with involvement of different clonal lineages  <cit> .
mrsa isolates resistant to clindamycin, erythromycin, gentamicin, tobramycin, and ciprofloxacin were detected in  <dig>  these isolates showed reduced susceptibility to rifampicin , according to the clinical and laboratory standards institute  criteria  <cit> .
this new phenotype of multiresistance differed from that of the iberian clone on the low level rif-r and on the tetracycline susceptibility.
the frequency of the rif-r mrsa isolates rapidly increased from  <dig> to 2006: 25%  of all mrsa clinical isolates in  <dig>  33%  in  <dig>  and 45%  in  <dig>  the percentage of rif-r mrsa decreased to 30%  in  <dig> and 25% in  <dig> .
rifampicin cannot be used as a single agent to treat mrsa infections because of the rapid selection of resistant mutants  <cit> .
however, combinations of rifampicin with other anti-staphylococcal agents such as quinolones  <cit>  or fusidic acid  <cit>  could prevent the emergence of rifampicin resistance during therapy  <cit> .
rifampicin interacts specifically with the rna polymerase beta-subunit encoded by the gene rpob  <cit> .
rifampicin resistance in s. aureus, as in other bacteria, is associated with mutations in particular regions  of the gene rpob  <cit> .
the objectives of the present study were: i) to characterise a collection of mrsa isolates expressing this new multiresistant pattern, and to determine whether they represented a novel genotype or they were the current representatives of a previously detected clone, ii) to determine the different levels of the rifampicin resistance by disk diffusion, microdilution and e-test, and iii) to analyse mutations in the rpob gene related to rifampicin resistance.
methods
hospital setting
the hospital universitari de bellvitge in barcelona, spain, is a nearly 900-bed tertiary care teaching centre.
it is the reference hospital for a geographical area with a population of approximately  <dig> million inhabitants.
the hospital universitari de bellvitge in barcelona provides medical and surgical care for adult patients with an average of  <dig>  admissions per year.
it has six intensive care units with a total of  <dig> beds and an active organ transplant program.
the control of mrsa in our institution is based on the active screening of patients at risk and contact isolation of infected or colonised patients.
in spite of this policy, the average rate of total mrsa among s. aureus clinical isolates in our hospital was 24% for the 2004- <dig> period .
the present study has been approved by the clinical research ethics committee of the hospital universitari de bellvitge.
bacterial strains
identification of s. aureus from clinical samples was performed using conventional tests: catalase, latex agglutination  and tube coagulase test .
two hundred and forty-two non-duplicate isolates resistant to clindamycin, erythromycin, gentamicin, tobramycin, ciprofloxacin and resistant to rifampicin  by the disk-diffusion or the microdilution method were recovered in the microbiology department of hospital universitari de bellvitge from january  <dig> to december  <dig>  these strains represented 34% of all mrsa isolated between  <dig> and  <dig>  and were isolated from patients admitted to the different surgical, medical and intensive care units in the hospital.
one hundred and eight isolates with rifampicin mic ≥  <dig> mg/l were selected for the present study.
the selection included the first isolates available each year  from the different hospital wards affected.
the origin of the strains was from blood cultures or catheter-related sites , wound swabs , respiratory samples , exudates , nasal swabs  and sterile fluids .
oral informed consent was given by all patients before taking the clinical specimen.
the patient acquisition of mrsa infection or colonisation was prospectively assessed.
five strains with the same resistance pattern but fully susceptible to rifampicin   were included in this study.
this rif-s pattern represented about 4% of all mrsa isolated between  <dig> and  <dig>
antimicrobial susceptibility testing
susceptibility testing of primary mrsa isolates is performed routinely by the disk-diffusion method on mueller-hinton  <dig> agar plates  to the following antibiotics: penicillin , oxacillin , cefoxitin , erythromycin , clindamycin , gentamycin , tobramycin , rifampicin , tetracycline , trimethoprim-sulfamethoxazole , chloramphenicol , ciprofloxacin , vancomycin , teicoplanin , quinupristin/dalfopristin  and linezolid .
disks are supplied by bd bbl .
mics of rifampicin were determined in all strains selected for this particular study  by microdilution , following the clinical and laboratory standards institute  recommendations  <cit> , and by e-test .
isolates were interpreted as susceptible or resistant, according to the clsi criteria  <cit> .
detection of rifampicin resistance-associated mutations
an internal sequence of gene rpob of  <dig> bp  was amplified by pcr.
this region includes the rifampicin resistance-determining cluster i  and cluster ii .
the amplification was carried out in  <dig> rif-s mrsa strains , and in a selection of  <dig> rif-r strains showing different levels of rifampicin resistance: mics  <dig> mg/l,  <dig> strains; mics  <dig> mg/l, 7; mics  <dig> mg/l, 2; and mics ≥  <dig> mg/l,  <dig>  the oligonucleotide sequences used were rpobfor  and rpobrev .
amplification was carried out in a  <dig> μl volume containing  <dig> pmol of each primer,  <dig> μm deoxynucleoside triphosphates ,  <dig> μl of a template dna sample and  <dig> u of amplitaq gold dna polymerase .
thermal cycling reactions consisted of an initial denaturation  followed by  <dig> cycles of denaturation , annealing , and extension , with a final extension .
the pcr product was purified  and analysed by dna sequencing.
cycle sequencing reactions were made up in a final volume of  <dig> μl with abi bigdye terminator v <dig>  ready reaction cycle sequencing kit, following manufacturer's methodology .
the nucleotide sequences obtained were compared to the rpob wild type sequence from s. aureus subsp.
aureus  using the clustalw software http://www.ebi.ac.uk/tools/clustalw/index.html.
rifampicin-susceptible strains used as controls were: atcc <dig>  and atcc <dig> .
two representatives of the iberian clone were used as rifampicin-resistant mrsa controls: atccbaa <dig>  <cit>  and per <dig>  <cit> .
determination of spontaneous mutation frequency for rifampicin resistance
the determination of spontaneous mutation frequency for rifampicin resistance was aimed at identifying whether the presence of a first mutation conferring low level rifampicin resistance facilitated the acquisition of supplementary mutations responsible for increasing rifampicin mics.
the rifampicin mutation frequency was calculated in reference strain atcc <dig>  and in two rif-r mrsa strains carrying the low level resistance mutation his481/asn .
bacterial strains were cultured in a shaking incubator at 37°c in luria-bertani  broth  until the exponential growth phase.
each strain was plated on the selective and non-selective lb agar plates and incubated at 37°c.
rifampicin selecting concentrations were  <dig> and  <dig> mg/l for the reference strain, and  <dig> mg/l for the rif-r mrsa strains.
in these experimental conditions od <dig> =  <dig>  corresponded to  <dig> ×  <dig> cfu/ml.
the equivalent to  <dig>   <dig> and  <dig> cfu were spread on selective plates, and appropriated diluted samples were plated on non-selective plates.
after  <dig> h to  <dig> h, colonies that grew on selective and non-selective plates were counted and mutation frequencies were calculated.
three independent experiments were performed to ensure reproducibility.
molecular typing
pulsed field gel electrophoresis
was performed after smai restriction of chromosomal dna according to chung et al.
<cit> .
pulses run from  <dig> s to  <dig> s for  <dig> h for block  <dig>  and from  <dig> s to  <dig> s for  <dig> h for block  <dig>  <cit> .
isolates with pfge patterns differing in four or less restriction fragments were considered to be subtypes of a single genotype.
isolates with differences in more than four fragments were ascribed to distinct genotypes  <cit> .
sccmec typing
molecular typing based on the amplification of the mobile region mec was performed according to previously described procedures  <cit> .
control strains for sccmec typing were: atccbaa <dig>   <cit> , atccbaa- <dig>   <cit> , atccbaa- <dig>   <cit>  and hgsa <dig>   <cit> .
multilocus sequence typing
.
analysis of the seven housekeeping gene sequences was performed according to previously described procedures http://saureus.mlst.net/ <cit> .
spa typing
the polymorphic region of protein a was studied according to previously described procedures at http://spa.ridom.de/ <cit> .
the interest region was amplified with primers spa-1113f  and spa-1514r .
RESULTS
rifampicin resistance levels and associated rpob mutations
the majority  of the  <dig> rif-r mrsa isolates, showed rifampicin mics between  <dig> and  <dig> mg/l.
two isolates had rifampicin mics of  <dig> mg/l and the remaining two had mics ≥  <dig> mg/l.
corresponding e-test and disk diffusion results are shown in table  <dig>  on the basis of these results and following other authors' categorisation  <cit>  the strains were classified into categories of rifampicin susceptible , low-level rifampicin resistance , and high-level rifampicin resistance .
interestingly,  <dig> strains with rifampicin mics of  <dig> mg/l showed inhibition zones between  <dig> and  <dig> mm, borderline to the susceptible clsi breakpoint .
the five rif-s mrsa isolates, with the same multi-resistance pattern, had rifampicin mics of  <dig>  mg/l and inhibition zones >  <dig> mm.
the mutations in the rifampicin resistance-determining region of rpob gene were studied in  <dig> rif-r and in  <dig> rif-s mrsa strains.
results are shown in table  <dig>  all  <dig> strains presented the mutational change 481his/asn, determined by a mutation in cluster i of rpob gene, conferring a low-level rifampicin resistance.
the four isolates with mic≥  <dig> mg/l had an additional amino acid substitution: 468gln/lys , 477ala/thr  or 527ile/leu , associated with a high level rifampicin resistance.
mutational changes  <dig> and  <dig> were determined by mutations located in cluster i and substitution  <dig> was determined by a mutation located in cluster ii.
rif-s mrsa isolates, had no mutations related to rifampicin resistance.
all isolates, including rif-s isolates, and  <dig>  out of  <dig> control strains, presented a silent mutation in amino acid  <dig> with the substitution ala per ala.
genotype
st247/i
per88
st247/i
atccbaa44
frequency of spontaneous mutation for rifampicin resistance
the rifampicin mutation frequency was calculated in reference strain atcc <dig>  and in two rif-r mrsa strains carrying the low-level resistant amino acid substitution 481his/asn .
rifampicin high level resistant mutants occurred with frequencies of around 10- <dig> to 10- <dig> in the rif-r mrsa strains after selection by rifampicin concentration of  <dig> mg/l.
an identical mutational ratio was found in the control strain atcc <dig> at both selective concentrations .
rif-r mrsa genotypes by pfge and epidemiology
all  <dig> rif-r mrsa isolates belonged to the same genotype by pfge.
this specific restriction pattern  was unique, distinct from both the pfge patterns obtained for the multi-resistant rif-s mrsa isolates  and from representatives of the iberian clone .
the rif-r mrsa isolates were classified into eight subtypes  with pattern b- <dig> being the most frequent , followed by subtype b- <dig> .
subtype b- <dig> was dominant during  <dig> representing 76%  of studied isolates, then decreased during  <dig>  and rose in  <dig> to 48% .
subtype b- <dig> represented 52%  in  <dig>  and 48%  in  <dig>  no correlation could be established between rifampicin resistance levels and pfge subtypes.
this rif-r clone was not restricted to a specific hospital ward.
isolates were obtained from patients admitted to intensive care, medical and surgical units.
almost all patients included in this study  acquired the mrsa in our hospital.
seven patients acquired the rif-r mrsa infection or colonisation in a prior admission to another hospital.
sccmec typing, mlst and spa typing
sccmec typing was carried out in the  <dig> strains where rpob mutations were characterised.
this selection included representatives of the eight pfge b subtypes.
also rif-s mrsa strains were analysed for sccmec type.
all  <dig> rif-r mrsa strains carried a sccmec type i. the  <dig> rif-s of pfge pattern a carried a sccmec type iv-a.
interestingly, all strains belonged to a common mlst type: st <dig>  defined by alleles arcc  <dig>  aroe  <dig>  glpf  <dig>  gmk  <dig>  pta  <dig>  tpi  <dig>  and yqi  <dig> .
mlst
in parallel, a selection of  <dig> rif-r mrsa strains and the  <dig> rif-s mrsa were further genotyped by spa typing.
all rif-r strains belonged to spa-type t <dig>  among the rif-s mrsa strains, three belonged to spa-type t <dig> and two showed novel spa-types .
discussion
the multi-resistant nature of most mrsa clones found in hospitals represents a therapeutical challenge for treating serious mrsa infections.
the burden that the iberian clone posed in spanish hospitals in the early  <dig> s  <cit> , shifted to other clones susceptible to more antibiotics, which have been dominant in recent years  <cit> .
in this paper, we described the emergence and spread of a mrsa clone resistant to clindamycin, erythromycin, gentamicin, tobramycin, ciprofloxacin and rifampicin which has reduced substantially the number of effective antibiotics for treatment of serious mrsa infections.
rifampicin is an antibiotic of substantial interest in the rise of mrsa infections, but cannot be used as a single agent to treat such infections because rapid emergence of resistance can occur, even during therapy  <cit> .
although the role of rifampicin as adjunctive therapy is controversial  <cit> , the combined therapy seems beneficial as long as the bacteria exhibit susceptibility to the antibiotics combined  <cit> .
the distinctive phenotypic feature in the particular clone of st- <dig> described here was the borderline resistance to rifampicin that could be missed by some methods of antimicrobial susceptibility testing .
hence our interest in studying whether this low-level rif-r was an adaptive phenomenon or to the contrary, known rpob mutations underlay such phenotype.
almost all isolates belonging to this multi-resistant mrsa clone  showed a low-level rifampicin resistance  and carried the amino acid substitution 481his/asn in the rna polymerase.
only  <dig> isolates showed additional substitutions known to be involved in a high-level rifampicin resistance: two isolates  carried mutational change 477ala/thr, and one isolate  468gln/lys  <cit> .
the fourth isolate  showed substitution 527ile/leu, the only one which mutation was found in the rifampicin resistance-determining cluster ii, described recently among japanese mrsa isolates  <cit> .
it is noteworthy that  <dig> isolates  of the rif-r isolates, carrying rpob mutation resulting in amino acid substitution in position  <dig>  were detected as rifampicin susceptible by the disk-diffusion test.
however, the inhibition zones of these strains were between  <dig> and  <dig> mm, closer to the susceptibility breakpoint established by clsi  than inhibition zones among rif-s mrsa isolates that were usually ≥  <dig> mm.
therefore, if screening for rifampicin resistance is made only by disk diffusion, special attention needs to be paid to strains borderline to the clsi susceptibility breakpoint to avoid reporting false susceptibility results.
mics by e-test failed to detect rifampicin resistance following clsi guidelines  <cit>  in a group of  <dig> strains .
these isolates showed mics by microdilution of  <dig> mg/l and carried the rpob mutation responsible for amino acid substitution in position  <dig>  thus, and according to other authors, it would be advisable to apply ≤  <dig>  and ≥  <dig> mg/l as new breakpoints to classify rifampicin susceptibility or resistance in s. aureus clinical isolates  <cit> .
high-level rifampicin resistance could be attributable to double mutations within rpob, as previously described  <cit> .
we did not find in this particular clone that the presence of a prior mutational change  increased the frequency of acquisition of additional mutations responsible for a higher level of rifampicin resistance, when compared to a reference strain.
the multi-resistance pattern exhibited by the iberian clone, dominant lineage in our hospital during the 90's, also included resistance to tetracycline.
the new rif-r mrsa isolates were resistant to clindamycin, erythromycin, gentamicin, tobramycin, ciprofloxacin and susceptible to tetracycline.
however, molecular typing showed that the iberian clone and the new rif-r mrsa clone had different genetic backgrounds represented by st- <dig> and st- <dig>  respectively, with only a single locus in common.
although both clones carried a sccmec element type i, pfge patterns and spa-types were clearly different.
all strains with the multi-resistant phenotype described in this work, showing resistance or decreased susceptibility to rifampicin, belonged to st- <dig>  carried a sccmec element type i and were spa-type t <dig>  this clone seems to be related to the southern germany clone  reported in germany in 1997- <dig>  <cit> .
in the same period, strains of st- <dig> and sccmec type i were reported at several hospitals located in seven italian cities  <cit> , although these isolates also showed resistance to multiple antibiotics, rifampicin resistance was not stated.
recently, strains of st- <dig> have spread epidemically in finland in 2002- <dig> and in hungary in 2003- <dig>  <cit> .
also, st- <dig> has been reported in other european countries: belgium, slovenia or switzerland  <cit> .
the first isolate st- <dig>  sccmec type i was isolated in our hospital in september  <dig>  from a patient admitted to the icu.
however, it was not until march  <dig> that this clone spread epidemically in our hospital and currently represents one third of all clinical mrsa isolates in our institution.
strains belonging to st- <dig> have been reported in other hospitals in spain since  <dig>  <cit> .
however, none of these reports  analysed the decreased susceptibility to rifampicin among representative strains of st- <dig>  during the 2004- <dig> period, we did not find significant changes in the rifampin consumption in our institution, which was on average  <dig>  ddd/ <dig> patients-days for intravenous and  <dig>  ddd/ <dig> patients-days for oral administration.
a set of  <dig> strains resistant to clindamycin, erythromycin, gentamicin, tobramycin, ciprofloxacin, but fully susceptible to rifampicin with mics of  <dig>  mg/l were included in this study.
on average, this rif-s pattern represented 4% of all mrsa isolated between  <dig> and  <dig>  however this resistance phenotype can be traced back to  <dig> in our hospital.
the rif-s isolates were classified as st- <dig>  the same as the rif-r mrsa.
isolates of st- <dig>  belong to the clonal complex  <dig>  as well as isolates of st- <dig>  which was the dominant mrsa clone in hospital universitari de bellvitge from  <dig> to  <dig>  consequently, an alternative hypothesis to explain the emergence of multiresistant clone st- <dig>  sccmec type i would be the sccmec type i transfer from st- <dig> to the st- <dig> background, with an intermediate stage where rif-s isolates could be found belonging to st- <dig>
CONCLUSIONS
our study describes the hospitalary spread of an mrsa clone , related to the southern-germany clone   <cit> .
in this particular case, the studied strains were resistant to many more antibiotics than any previous mrsa clone spread in our institution, with the exception of the iberian clone.
in addition, the study of the rpob mutations demonstrated that rifampin was not a suitable option for treatment of infections caused by this clone.
authors' contributions
md and jl conceived the study and participated in its design.
md, ft, rm, mp and jl participated in field and clinical aspects of the study.
vm and md carried out the molecular genetic studies and sequence alignment.
md and vm wrote the manuscript which was co-ordinated by jl and critically reviewed by ft, rm and mp.
all authors read and approved the final version of the manuscript.

 methicillin resistant staphylococcus aureus is an important pathogen in spanish hospitals. the percentage of patients infected or colonised by mrsa among patients with nosocomial s. aureus has been estimated between % and % in nation-wide multicenter studies. in the hospital universitari de bellvitge mrsa has been endemic since the majority of strains isolated during the 1990- period belonged to the multiresistant iberian clone. by multilocus sequence typing the iberian clone showed an allelic profile or sequence type carrying the staphylococcal cassette chromosome mec type i. isolates of the iberian clone exhibited resistance against almost all antibiotics available for mrsa therapy including clindamycin, erythromycin, gentamicin, tobramycin, tetracycline, ciprofloxacin and rifampicin. from to the iberian clone was gradually replaced by isolates of clonal complex related to the paediatric clone. unlike the iberian clone, these strains showed only consistent resistance to tobramycin and ciprofloxacin combined with variable resistance to clindamycin and/or erythromycin. similar trends have been observed in other hospitals in spain and in other countries such as france, germany, belgium or portugal, with involvement of different clonal lineages. mrsa isolates resistant to clindamycin, erythromycin, gentamicin, tobramycin, and ciprofloxacin were detected in these isolates showed reduced susceptibility to rifampicin, according to the clinical and laboratory standards institute criteria. this new phenotype of multiresistance differed from that of the iberian clone on the low level rif-r and on the tetracycline susceptibility. the frequency of the rif-r mrsa isolates rapidly increased from to 2006: 25% of all mrsa clinical isolates in 33% in and 45% in the percentage of rif-r mrsa decreased to 30% in and 25% in. rifampicin cannot be used as a single agent to treat mrsa infections because of the rapid selection of resistant mutants. however, combinations of rifampicin with other anti-staphylococcal agents such as quinolones or fusidic acid could prevent the emergence of rifampicin resistance during therapy. rifampicin interacts specifically with the rna polymerase beta-subunit encoded by the gene rpob. rifampicin resistance in s. aureus, as in other bacteria, is associated with mutations in particular regions of the gene rpob. the objectives of the present study were: i) to characterise a collection of mrsa isolates expressing this new multiresistant pattern, and to determine whether they represented a novel genotype or they were the current representatives of a previously detected clone, ii) to determine the different levels of the rifampicin resistance by disk diffusion, microdilution and e-test, and iii) to analyse mutations in the rpob gene related to rifampicin resistance. methods hospital setting the hospital universitari de bellvitge in barcelona, spain, is a nearly 900-bed tertiary care teaching centre. it is the reference hospital for a geographical area with a population of approximately million inhabitants. the hospital universitari de bellvitge in barcelona provides medical and surgical care for adult patients with an average of admissions per year. it has six intensive care units with a total of beds and an active organ transplant program. the control of mrsa in our institution is based on the active screening of patients at risk and contact isolation of infected or colonised patients. in spite of this policy, the average rate of total mrsa among s. aureus clinical isolates in our hospital was 24% for the 2004- period. the present study has been approved by the clinical research ethics committee of the hospital universitari de bellvitge. bacterial strains identification of s. aureus from clinical samples was performed using conventional tests: catalase, latex agglutination and tube coagulase test. two hundred and forty-two non-duplicate isolates resistant to clindamycin, erythromycin, gentamicin, tobramycin, ciprofloxacin and resistant to rifampicin by the disk-diffusion or the microdilution method were recovered in the microbiology department of hospital universitari de bellvitge from january to december these strains represented 34% of all mrsa isolated between and and were isolated from patients admitted to the different surgical, medical and intensive care units in the hospital. one hundred and eight isolates with rifampicin mic ≥ mg/l were selected for the present study. the selection included the first isolates available each year from the different hospital wards affected. the origin of the strains was from blood cultures or catheter-related sites, wound swabs, respiratory samples, exudates, nasal swabs and sterile fluids. oral informed consent was given by all patients before taking the clinical specimen. the patient acquisition of mrsa infection or colonisation was prospectively assessed. five strains with the same resistance pattern but fully susceptible to rifampicin were included in this study. this rif-s pattern represented about 4% of all mrsa isolated between and antimicrobial susceptibility testing susceptibility testing of primary mrsa isolates is performed routinely by the disk-diffusion method on mueller-hinton agar plates to the following antibiotics: penicillin, oxacillin, cefoxitin, erythromycin, clindamycin, gentamycin, tobramycin, rifampicin, tetracycline, trimethoprim-sulfamethoxazole, chloramphenicol, ciprofloxacin, vancomycin, teicoplanin, quinupristin/dalfopristin and linezolid. disks are supplied by bd bbl. mics of rifampicin were determined in all strains selected for this particular study by microdilution, following the clinical and laboratory standards institute recommendations, and by e-test. isolates were interpreted as susceptible or resistant, according to the clsi criteria. detection of rifampicin resistance-associated mutations an internal sequence of gene rpob of bp was amplified by pcr. this region includes the rifampicin resistance-determining cluster i and cluster ii. the amplification was carried out in rif-s mrsa strains, and in a selection of rif-r strains showing different levels of rifampicin resistance: mics mg/l, strains; mics mg/l, 7; mics mg/l, 2; and mics ≥ mg/l, the oligonucleotide sequences used were rpobfor and rpobrev. amplification was carried out in a μl volume containing pmol of each primer, μm deoxynucleoside triphosphates, μl of a template dna sample and u of amplitaq gold dna polymerase. thermal cycling reactions consisted of an initial denaturation followed by cycles of denaturation, annealing, and extension, with a final extension. the pcr product was purified and analysed by dna sequencing. cycle sequencing reactions were made up in a final volume of μl with abi bigdye terminator v ready reaction cycle sequencing kit, following manufacturer's methodology. the nucleotide sequences obtained were compared to the rpob wild type sequence from s. aureus subsp. aureus using the clustalw software http://www.ebi.ac.uk/tools/clustalw/index.html. rifampicin-susceptible strains used as controls were: atcc and atcc. two representatives of the iberian clone were used as rifampicin-resistant mrsa controls: atccbaa and per. determination of spontaneous mutation frequency for rifampicin resistance the determination of spontaneous mutation frequency for rifampicin resistance was aimed at identifying whether the presence of a first mutation conferring low level rifampicin resistance facilitated the acquisition of supplementary mutations responsible for increasing rifampicin mics. the rifampicin mutation frequency was calculated in reference strain atcc and in two rif-r mrsa strains carrying the low level resistance mutation his481/asn. bacterial strains were cultured in a shaking incubator at 37°c in luria-bertani broth until the exponential growth phase. each strain was plated on the selective and non-selective lb agar plates and incubated at 37°c. rifampicin selecting concentrations were and mg/l for the reference strain, and mg/l for the rif-r mrsa strains. in these experimental conditions od = corresponded to × cfu/ml. the equivalent to and cfu were spread on selective plates, and appropriated diluted samples were plated on non-selective plates. after h to h, colonies that grew on selective and non-selective plates were counted and mutation frequencies were calculated. three independent experiments were performed to ensure reproducibility. molecular typing pulsed field gel electrophoresis was performed after smai restriction of chromosomal dna according to chung et al.. pulses run from s to s for h for block and from s to s for h for block. isolates with pfge patterns differing in four or less restriction fragments were considered to be subtypes of a single genotype. isolates with differences in more than four fragments were ascribed to distinct genotypes. sccmec typing molecular typing based on the amplification of the mobile region mec was performed according to previously described procedures. control strains for sccmec typing were: atccbaa, atccbaa-, atccbaa- and hgsa. multilocus sequence typing. analysis of the seven housekeeping gene sequences was performed according to previously described procedures http://saureus.mlst.net/. spa typing the polymorphic region of protein a was studied according to previously described procedures at http://spa.ridom.de/. the interest region was amplified with primers spa-1113f and spa-1514r.  rifampicin resistance levels and associated rpob mutations the majority of the rif-r mrsa isolates, showed rifampicin mics between and mg/l. two isolates had rifampicin mics of mg/l and the remaining two had mics ≥ mg/l. corresponding e-test and disk diffusion results are shown in table on the basis of these results and following other authors' categorisation the strains were classified into categories of rifampicin susceptible, low-level rifampicin resistance, and high-level rifampicin resistance. interestingly, strains with rifampicin mics of mg/l showed inhibition zones between and mm, borderline to the susceptible clsi breakpoint. the five rif-s mrsa isolates, with the same multi-resistance pattern, had rifampicin mics of mg/l and inhibition zones > mm. the mutations in the rifampicin resistance-determining region of rpob gene were studied in rif-r and in rif-s mrsa strains. results are shown in table all strains presented the mutational change 481his/asn, determined by a mutation in cluster i of rpob gene, conferring a low-level rifampicin resistance. the four isolates with mic≥ mg/l had an additional amino acid substitution: 468gln/lys, 477ala/thr or 527ile/leu, associated with a high level rifampicin resistance. mutational changes and were determined by mutations located in cluster i and substitution was determined by a mutation located in cluster ii. rif-s mrsa isolates, had no mutations related to rifampicin resistance. all isolates, including rif-s isolates, and out of control strains, presented a silent mutation in amino acid with the substitution ala per ala. genotype st247/i per88 st247/i atccbaa44 frequency of spontaneous mutation for rifampicin resistance the rifampicin mutation frequency was calculated in reference strain atcc and in two rif-r mrsa strains carrying the low-level resistant amino acid substitution 481his/asn. rifampicin high level resistant mutants occurred with frequencies of around 10- to 10- in the rif-r mrsa strains after selection by rifampicin concentration of mg/l. an identical mutational ratio was found in the control strain atcc at both selective concentrations. rif-r mrsa genotypes by pfge and epidemiology all rif-r mrsa isolates belonged to the same genotype by pfge. this specific restriction pattern was unique, distinct from both the pfge patterns obtained for the multi-resistant rif-s mrsa isolates and from representatives of the iberian clone. the rif-r mrsa isolates were classified into eight subtypes with pattern b- being the most frequent, followed by subtype b-. subtype b- was dominant during representing 76% of studied isolates, then decreased during and rose in to 48%. subtype b- represented 52% in and 48% in no correlation could be established between rifampicin resistance levels and pfge subtypes. this rif-r clone was not restricted to a specific hospital ward. isolates were obtained from patients admitted to intensive care, medical and surgical units. almost all patients included in this study acquired the mrsa in our hospital. seven patients acquired the rif-r mrsa infection or colonisation in a prior admission to another hospital. sccmec typing, mlst and spa typing sccmec typing was carried out in the strains where rpob mutations were characterised. this selection included representatives of the eight pfge b subtypes. also rif-s mrsa strains were analysed for sccmec type. all rif-r mrsa strains carried a sccmec type i. the rif-s of pfge pattern a carried a sccmec type iv-a. interestingly, all strains belonged to a common mlst type: st defined by alleles arcc aroe glpf gmk pta tpi and yqi. mlst in parallel, a selection of rif-r mrsa strains and the rif-s mrsa were further genotyped by spa typing. all rif-r strains belonged to spa-type t among the rif-s mrsa strains, three belonged to spa-type t and two showed novel spa-types. discussion the multi-resistant nature of most mrsa clones found in hospitals represents a therapeutical challenge for treating serious mrsa infections. the burden that the iberian clone posed in spanish hospitals in the early s, shifted to other clones susceptible to more antibiotics, which have been dominant in recent years. in this paper, we described the emergence and spread of a mrsa clone resistant to clindamycin, erythromycin, gentamicin, tobramycin, ciprofloxacin and rifampicin which has reduced substantially the number of effective antibiotics for treatment of serious mrsa infections. rifampicin is an antibiotic of substantial interest in the rise of mrsa infections, but cannot be used as a single agent to treat such infections because rapid emergence of resistance can occur, even during therapy. although the role of rifampicin as adjunctive therapy is controversial, the combined therapy seems beneficial as long as the bacteria exhibit susceptibility to the antibiotics combined. the distinctive phenotypic feature in the particular clone of st- described here was the borderline resistance to rifampicin that could be missed by some methods of antimicrobial susceptibility testing. hence our interest in studying whether this low-level rif-r was an adaptive phenomenon or to the contrary, known rpob mutations underlay such phenotype. almost all isolates belonging to this multi-resistant mrsa clone showed a low-level rifampicin resistance and carried the amino acid substitution 481his/asn in the rna polymerase. only isolates showed additional substitutions known to be involved in a high-level rifampicin resistance: two isolates carried mutational change 477ala/thr, and one isolate 468gln/lys. the fourth isolate showed substitution 527ile/leu, the only one which mutation was found in the rifampicin resistance-determining cluster ii, described recently among japanese mrsa isolates. it is noteworthy that isolates of the rif-r isolates, carrying rpob mutation resulting in amino acid substitution in position were detected as rifampicin susceptible by the disk-diffusion test. however, the inhibition zones of these strains were between and mm, closer to the susceptibility breakpoint established by clsi than inhibition zones among rif-s mrsa isolates that were usually ≥ mm. therefore, if screening for rifampicin resistance is made only by disk diffusion, special attention needs to be paid to strains borderline to the clsi susceptibility breakpoint to avoid reporting false susceptibility results. mics by e-test failed to detect rifampicin resistance following clsi guidelines in a group of strains. these isolates showed mics by microdilution of mg/l and carried the rpob mutation responsible for amino acid substitution in position thus, and according to other authors, it would be advisable to apply ≤ and ≥ mg/l as new breakpoints to classify rifampicin susceptibility or resistance in s. aureus clinical isolates. high-level rifampicin resistance could be attributable to double mutations within rpob, as previously described. we did not find in this particular clone that the presence of a prior mutational change increased the frequency of acquisition of additional mutations responsible for a higher level of rifampicin resistance, when compared to a reference strain. the multi-resistance pattern exhibited by the iberian clone, dominant lineage in our hospital during the 90's, also included resistance to tetracycline. the new rif-r mrsa isolates were resistant to clindamycin, erythromycin, gentamicin, tobramycin, ciprofloxacin and susceptible to tetracycline. however, molecular typing showed that the iberian clone and the new rif-r mrsa clone had different genetic backgrounds represented by st- and st- respectively, with only a single locus in common. although both clones carried a sccmec element type i, pfge patterns and spa-types were clearly different. all strains with the multi-resistant phenotype described in this work, showing resistance or decreased susceptibility to rifampicin, belonged to st- carried a sccmec element type i and were spa-type t this clone seems to be related to the southern germany clone reported in germany in 1997-. in the same period, strains of st- and sccmec type i were reported at several hospitals located in seven italian cities, although these isolates also showed resistance to multiple antibiotics, rifampicin resistance was not stated. recently, strains of st- have spread epidemically in finland in 2002- and in hungary in 2003-. also, st- has been reported in other european countries: belgium, slovenia or switzerland. the first isolate st- sccmec type i was isolated in our hospital in september from a patient admitted to the icu. however, it was not until march that this clone spread epidemically in our hospital and currently represents one third of all clinical mrsa isolates in our institution. strains belonging to st- have been reported in other hospitals in spain since. however, none of these reports analysed the decreased susceptibility to rifampicin among representative strains of st- during the 2004- period, we did not find significant changes in the rifampin consumption in our institution, which was on average ddd/ patients-days for intravenous and ddd/ patients-days for oral administration. a set of strains resistant to clindamycin, erythromycin, gentamicin, tobramycin, ciprofloxacin, but fully susceptible to rifampicin with mics of mg/l were included in this study. on average, this rif-s pattern represented 4% of all mrsa isolated between and however this resistance phenotype can be traced back to in our hospital. the rif-s isolates were classified as st- the same as the rif-r mrsa. isolates of st- belong to the clonal complex as well as isolates of st- which was the dominant mrsa clone in hospital universitari de bellvitge from to consequently, an alternative hypothesis to explain the emergence of multiresistant clone st- sccmec type i would be the sccmec type i transfer from st- to the st- background, with an intermediate stage where rif-s isolates could be found belonging to st-  our study describes the hospitalary spread of an mrsa clone, related to the southern-germany clone. in this particular case, the studied strains were resistant to many more antibiotics than any previous mrsa clone spread in our institution, with the exception of the iberian clone. in addition, the study of the rpob mutations demonstrated that rifampin was not a suitable option for treatment of infections caused by this clone. authors' contributions md and jl conceived the study and participated in its design. md, ft, rm, mp and jl participated in field and clinical aspects of the study. vm and md carried out the molecular genetic studies and sequence alignment. md and vm wrote the manuscript which was co-ordinated by jl and critically reviewed by ft, rm and mp. all authors read and approved the final version of the manuscript. 
isolates of clonal complex gradually replaced the iberian clone from to a new multiresistant mrsa phenotype showing rifampicin resistance emerged in and rapidly increased from 25% in to 45% in the aims of this study were i) the molecular characterisation of rifampicin resistant mrsa isolates, ii) the study of the rifampicin resistance expression by disk diffusion, microdilution and e-test, and iii) the analysis of the rpob gene mutations involved in rifampicin resistance. isolates with a high-level rifampicin resistance carried additional mutations in the rpob gene. decreased susceptibility to rifampicin in mrsa strains of st228-sccmeci was associated with one or two specific mutations in the rpob gene. the emergence of mrsa clone st228-sccmeci, related to the southern germany clone, involved a therapeutical challenge for treating serious mrsa infections. disk diffusion and e-test methods failed to identify a low-level rifampicin resistance in and isolates, respectively. methicillin-resistant s. aureus has been endemic in hospital universitari de bellvitge, barcelona, since during the 1990- period the iberian clone was dominant. a sample of representative rifampicin-resistant mrsa isolates belonged to a single pfge genotype, st- sccmec type i and spa type t of isolates, had a low-level rifampicin resistance and a high-level rifampicin resistance. a low-level rifampicin resistance was associated with amino acid substitution 481his/asn in the beta-subunit of rna polymerase.
 methicillin-resistant s. aureus has been endemic in hospital universitari de bellvitge, barcelona, since during the 1990- period the iberian clone was dominant. isolates of clonal complex gradually replaced the iberian clone from to a new multiresistant mrsa phenotype showing rifampicin resistance emerged in and rapidly increased from 25% in to 45% in the aims of this study were i) the molecular characterisation of rifampicin resistant mrsa isolates, ii) the study of the rifampicin resistance expression by disk diffusion, microdilution and e-test, and iii) the analysis of the rpob gene mutations involved in rifampicin resistance.  a sample of representative rifampicin-resistant mrsa isolates belonged to a single pfge genotype, st- sccmec type i and spa type t of isolates, had a low-level rifampicin resistance and a high-level rifampicin resistance. disk diffusion and e-test methods failed to identify a low-level rifampicin resistance in and isolates, respectively. a low-level rifampicin resistance was associated with amino acid substitution 481his/asn in the beta-subunit of rna polymerase. isolates with a high-level rifampicin resistance carried additional mutations in the rpob gene.  the emergence of mrsa clone st228-sccmeci, related to the southern germany clone, involved a therapeutical challenge for treating serious mrsa infections. decreased susceptibility to rifampicin in mrsa strains of st228-sccmeci was associated with one or two specific mutations in the rpob gene. one fifth of isolates with low-level rifampicin-resistance were missed by the diffusion methods. 