BACKGROUND
cystic fibrosis  is caused by a mutation in the cftr-gene leading to dysfunction of the exocrine glands.
the disease is responsible for chronic airway obstruction in the lung, a favourable condition for pulmonary infections during childhood.
in different studies investigating pathogens in cf, s. aureus was observed in  <dig> to 60% of patients frequently in association with other bacteria, such as pseudomonas aeruginosa  <cit> .
since the introduction of methicillin in  <dig>  methicillin-resistant s. aureus  clones have rapidly emerged and spread worldwide and account for  <dig> to 30% of s. aureus infections  <cit> .
molecular epidemiology studies using multi locus sequence typing  on clinical strains of s. aureus have shown that they are distributed into  <dig> major clonal complexes   <cit> .
mrsa strains represent the most threatening challenge as they are frequently resistant to many antibiotics and there is evidence that antibiotic treatments not only facilitate the spreading of these clones but also enhance their pathogenicity  <cit> .
patients with cf are at particular risk for pulmonary colonization of mrsa, both because of their difficulty in clearing mucus and because of their frequent hospital visits, which can increase exposure to mrsa.
several studies reported that  <dig> to 35% of cf patients harbored a mrsa strain and described the emergence of community-acquired mrsa   <cit> .
methicillin-susceptible strains  also constitute a risk in cf patients, particularly because of the existence of biofilms in the infected lung in which they can escape from antibiotic treatment  <cit> .
the epidemiology of s. aureus in cf patients has been investigated in different studies, but mostly mrsa were analysed and the role of mssa was not assessed.
in order to extend the knowledge of the population of s. aureus chronically infecting cf patients, all the isolates should be systematically genotyped with a high degree of discrimination which is difficult using the currently available techniques.
the polymorphism of the staphylococcus protein a gene , first used by frenay et al.
<cit>  to genotype s. aureus and further evaluated by shopsin et al.
<cit>  has proven to be very useful to investigate s. aureus genetic diversity.
subsequently mlst became the most widely used technique to analyse the epidemiology of s. aureus and to perform phylogenetic studies  <cit> .
although the combined discriminatory power of spa typing and mlst is high, these techniques do not sufficiently discriminate within the major ccs and their cost is elevated.
new approaches have been developed which use variable number of tandem repeats  either to produce a multiple band pattern in a technique called mlvf  <cit>  or to perform multiple loci vntr analysis .
mlva consists in the analysis of individual vntrs allowing the description of a strain in the form of a code easily exchangeable between laboratories  <cit> .
mlva with  <dig> vntrs could correctly assigned isolates to outbreaks or identified isolates as unlinked  <cit> .
schouls et al.
using  <dig> vntrs showed that mlva was at least as discriminatory as pulse field gel electrophoresis  and twice as discriminatory as spa-sequence typing  <cit> .
finally we recently described a very informative mlva scheme which makes use of  <dig> vntrs  and demonstrated that its discriminatory power was much higher than those of mlst and spa typing  <cit> .
in the present work we used the proposed mlva- <dig> assay to genotype s. aureus isolates recovered from firstly as well as chronically colonized cf patients, over a period of  <dig> months.
the aim of our study was to investigate the genetic diversity of mrsa and mssa present in the sputum of cf children whether sporadically or chronically.
the longitudinal survey of genotypes provided information on the variations in those strains recovered from some patients over a maximum period of  <dig> months.
RESULTS
clinical characteristics of s. aureus colonization
from a total number of  <dig> patients attending the armand trousseau cf centre during the  <dig> months study period,  <dig> provided sputum of which  <dig> showed one or several cultures positive for s. aureus.
it is likely that most were community-acquired s. aureus contaminations as the majority of patients were outpatients.
in addition there was no outbreak episode during the study period.
although this study was not designed to correlate the bacteria recovered from the sputum with the respiratory evolution of the patients, the following features may be underlined: among the  <dig> patients,  <dig> were co-infected with p. aeruginosa, as observed in a previous investigation  <cit> , making it difficult to determine the role of s. aureus in broncho-pulmonary exacerbations.
twenty-four of these patients harboured mssa,  <dig> patients harboured mrsa and  <dig> patients harboured both.
mrsa were mainly isolated from older patients who were treated by regular intravenous antibiotic courses, as recommended by the international guidelines.
in the  <dig> other patients, s. aureus was the single species recovered from sputum cultures ; mssa isolates were found in  <dig> patients, mrsa in  <dig> patients and both mssa and mrsa in  <dig> other patients.
these  <dig> patients were younger than those co-infected with p. aeruginosa.
of note, those harbouring mrsa had more respiratory exacerbations and worse lung function than those harbouring mssa.
both methicillin-susceptible and methicillin resistant isolates were repeatedly recovered over several months.
forty percent of patients were suffering from their first colonization with s. aureus while in 60% the recurrent isolation of the bacteria was indicative of colonization with exacerbations.
in the later cases genotyping could show in several instances that the strain was different and therefore that several independent infections took place .
patients were treated by antimicrobial drugs, however in most cases s. aureus was still recovered from sputum samples despite clinical improvement.
investigation of mrsa
in total a mrsa isolate was found at least once in  <dig> patients  with a positive culture.
both screening techniques used here failed to detect the presence of the penicillin binding protein pbp 2a  in the resistant strains from five patients .
methicillin-resistance in these strains  was caused by a high production of β-lactamase, leading to a characteristic bor-sa  phenotype.
isolates from patient cfu_ <dig> did not show bor-sa characteristics, suggesting that methicillin resistance resulted from a new modified penicillin-binding capacity   <cit> , though this modified capacity was not investigated.
in patients colonized by bor-sa or mod-sa, isolates showing the same genotype but different susceptibility patterns were occasionally recovered from the same sputum sample, or over time in successive samples.
the genetic diversity of strains as assessed by mlva
a total of  <dig> isolates were genotyped by mlva using fourteen vntrs .
overall the pcr efficiency was very satisfying and there was no difficulty in evaluating the amplicon size on 2% agarose gels.
in one case, the presence of several bands with sa <dig>  suggested the existence of two different variants of a strain in the sample.
indeed this could be confirmed by testing several colonies from a culture .
a the chromosomal position on the mu <dig> genome, in kilobase-pairs is indicated in the vntr name, for example sa <dig> is at position  <dig> .
b primers different from  <cit>
c primers different from  <cit>
d star stands for s. aureus repeat
e primers different from siru <dig>  <cit>
the s. aureus population diversity is shown in the minimum spanning tree representation on figure  <dig>  the presence of the  <dig> available sequenced genomes in the analysis allowed the identification of several mlst ccs .
mlst was performed on two strains  of the second largest cluster, showing that it belonged to cc <dig> .
a comparison made with a collection of  <dig> strains previously analysed by mlst and by mlva- <dig>  <cit>  confirmed the concordance of the ccs defined by the two techniques .
therefore, in the present study it was decided to use the mlst nomenclature to designate the largest ccs.
to facilitate the comparison of isolates, one strain of a given genotype per patient  and  <dig> reference strains were used to perform a clustering analysis.
with a cut-off value of 45%   <dig> clusters, or clonal complexes , were observed.
figure  <dig> shows the first part of a dendrogram in which all the strains belonging to cc <dig>  cc <dig>  cc <dig>  cc <dig>  cc <dig> and cc <dig> fall.
the second part of the dendrogram shown on figure  <dig> displays all the cc <dig>  cc <dig> and cc <dig> isolates.
four ccs comprised 71%  of the strains .
five clusters contained only one genotype each.
mrsa were distributed into  <dig> genotypes, mssa into  <dig> genotypes whereas  <dig> genotypes were assigned to both mrsa and mssa strains.
spa typing
the spa repeats were sequenced in  <dig> selected isolates on the basis of their distribution into the different clusters and of their polymorphism within these clusters.
the sequence was submitted to the ridom spaserver in order to identify the spa type.
seven new spa types were given a number by the ridom spaserver.
the result confirmed the correct clustering of strains by mlva, as shown by the almost perfect correlation between the two genotyping techniques .
strain trsa <dig> was positioned near cc <dig> strains and its spa type was characteristic of st <dig> strains  a bacterial population resulting from a large chromosomal rearrangement between cc <dig> and cc <dig>  <cit> .
three identical isolates from patient cfu_ <dig> which spa type corresponded to cc <dig> were branched in an ancestral position to the cc <dig> cluster.
interestingly, in cc <dig> a larger diversity was observed for spa , as compared to the other large clusters, cc <dig>  and cc <dig> .
longitudinal survey
in  <dig> patients , isolates that were repeatedly recovered over the  <dig> months study period belonged to the same lineage, i.e.
they were either identical or differed at only one vntr.
in the other patients the isolates belonged to  <dig>   <dig> or even  <dig> different ccs.
for example isolates belonging to  <dig> different ccs were found in patient cfu_ <dig> and only one of them was observed more than once.
table  <dig> shows the number of ccs and genotypes from patients for which at least  <dig> isolates were recovered.
one example of stability is observed in patient cfu_ <dig> for which  <dig> mod-sa cc <dig> isolates with the same genotype were recovered from january  <dig> to july  <dig>  patient cfu_ <dig> had  <dig> isolates with two different spa alleles.
in  <dig> and early  <dig>  a single genotype with seven repeats at the spa locus was observed, whereas after march  <dig>  isolates with two repeats at the spa locus were also found.
on several occasions, both were present in equal amounts giving rise to two products upon pcr amplification .
from march  <dig> to january  <dig>   <dig> cc <dig> isolates were recovered from patient cfu_ <dig>  with three variants differing at vntrs sa <dig> and sa1132: one genotype was found in  <dig> isolates in  <dig> and early  <dig>  another one in  <dig> isolates from  <dig> to  <dig> and the third corresponded to a single isolate in  <dig>  patient cfu_ <dig> had  <dig> isolates with three variants differing at vntr sa1132: one was found only in  <dig> in two isolates, a second one was found once in  <dig>   <dig> times in  <dig> and once in  <dig>  and the third one was found twice in  <dig>  patient cfu_ <dig> had  <dig> isolates with two variants differing at vntr sa2039: one genotype was observed in  <dig> in two isolates, and the other was found in five isolates once in  <dig> and in  <dig>
a indicates the loci where there are vntr variants within identical cc.
in parentheses are shown the number of repeats at the variants.
b un <dig> corresponds to st109
c un <dig> corresponds to st398
genotypes and mrsa
on figures  <dig> and  <dig> are shown the sensitivity to methicillin and the presence/absence of the meca gene carried by staphylococcal cassette chromosome mec , as tested by pcr.
the large majority of mrsa isolates fall inside cc <dig>  cc <dig> and cc <dig>  in cc <dig>  all strains were mssa except for trsa <dig> which is placed outside of the cluster and is meca negative.
interestingly, in patient cfu_ <dig>   <dig> isolates were of the same genotype, of which  <dig> were meca positive and  <dig> were meca negative, suggesting a recent transfer of the meca gene or sccmec instability in this particular strain.
in five patients, isolates with identical genotypes were apparently either resistant or sensitive to methicillin but meca was not detected while the phenotypic resistance aspects were bor-sa or mod-sa.
in four patients only mssa strains were isolated over more than  <dig> months .
the genetic diversity among mssa isolates was larger than among mrsa, but both could be found in large ccs.
discussion
mlva
the mlva procedure used in the present study allowed the systematic investigation of all s. aureus isolates recovered from cf patients attending a french centre during a period of  <dig> months.
in the present study a total of  <dig> isolates from  <dig> children were genotyped, with a great variation within the number of s. aureus isolates per patient: one or two isolates for the firstly colonized patients , to more than  <dig> isolates for some chronically colonized patients repeatedly hospitalized for acute exacerbation.
this type of study had never been conducted before because the available techniques were either time consuming or too expensive.
generally only mrsa isolates were studied and consequently, the mssa diversity was insufficiently known although they account for a large proportion of strains responsible for chronic colonization in cf patients.
mlva using  <dig> vntrs is a very informative technique which compares favourably with mlst and spa typing.
more genotypes are observed and it is possible to see the emergence of variants.
the size of the vntrs repeats ranges from  <dig> bp  to  <dig> bp, which makes the technique very easy to implement using agarose gel electrophoresis as well as high throughput approaches.
the allelic size differences for such markers can be estimated directly by eye and compared to a chart where all the known alleles have been indicated.
this information is accessible on a dedicated web page in "the bacterial mlva-genotyping-on-the-web service" .
for epidemiology purposes, a simpler scheme could be performed with a selection of  <dig> informative markers .
however, it is important to keep a large collection of markers with different degrees of variability for the investigation of outbreaks or for phylogenetic studies.
in the present work each vntr was amplified in a separate pcr reaction but our preliminary experiments showed that  <dig> vntrs could be amplified simultaneously and the size automatically determined using a capillary electrophoresis apparatus  <cit> .
this opens the way to automatized genotyping similarly to the protocol described by schouls et al.
<cit> .
however in this latest study only  <dig> vntrs  were analysed which, in our opinion may not be sufficiently discriminant for epidemiological studies.
indeed the simpson's diversity index  in the mlva- <dig> assay was  <dig> % whereas we obtained a  <dig> % di using the mlva- <dig> assay.
other published vntr-based genotyping methods either did not use enough markers or analyzed fingerprints which makes the comparison of profiles between laboratories very difficult  <cit> .
in addition failure to amplify some vntrs in a relatively important number of samples led to partial profiles in up to 27% of isolates in one study  <cit> .
genetic diversity of strains and population structure
in the present collection of isolates,  <dig> genotypes were observed , 68% belonging to  <dig> main clusters.
the genotypes in the mlva cluster corresponding to cc <dig> were very stable over a period of more than  <dig> years.
in contrast, more variability was observed in isolates of cc <dig> and cc <dig>  in cc <dig>  several vntrs showed very small alleles as compared to the other clonal complexes which could be the result of frequent loss of repeats due to recombination.
the majority of cc <dig> isolates were mrsa, whereas these represented about 50% of cc <dig> and cc <dig> isolates.
recent studies have shown that cc <dig> contained both community and hospital-acquired mrsa strains whereas the other ccs mainly contained hospital-acquired strains  <cit> .
in a recent survey of cf patients in spain, 67% of mrsa isolates were st <dig> which belong to cc <dig>  and the second largest group corresponded to st <dig> belonging to cc <dig>  <cit> .
colonization
the precise identification of s. aureus genotypes colonizing the lungs of cf patients is of importance to trace the source of contamination and eventually modify the management of patients in the hospital.
it is also important to know whether a single strain is present over time despite antibiotic treatment, or if different strains are involved in patients exacerbations.
in two patients, isolates belonging to four different ccs were observed, but the most frequent situation was colonization by a single strain which could vary over time.
in some patients, the same strain was recovered over a two years period, with a few variants differing at a single vntr.
in several cases the variants seemed to appear sequentially suggesting that they acquired an advantage over the first isolate.
on the contrary, in patient cfu_ <dig>  two variants were alternatively isolated during the two years period, and in patient cfu_ <dig>  the presence of two spa amplicons in a single pcr reaction pointed to the coexistence in equal amounts of two variants in the sputum sample.
interestingly variants were more frequently observed in cc <dig> strains than in other ccs, again indicating the existence of a higher degree of instability in this cc.
it was shown that the adaptation to chronic colonization requires the expression of virulence factors and a higher mutation capacity resulting in an increase of the genetic diversity  <cit> .
in  <dig> cases, a given genotype was shared by different patients, but it is difficult to define the origin of the contamination as most of these strains belonged to common ccs.
indeed, in a recent study by sakwinska et al., it was shown that cc <dig> and cc <dig> colonized each 24% of the carrier population  <cit> .
in the armand trousseau center, the risk of p. aeruginosa cross-colonization has led to the increased use of isolation protocols among the patients since many years.
the source of s. aureus lung colonization could be either the nose, or the oro-pharynx, as suggested by recent studies  <cit> .
the simplicity of mlva genotyping should allow a systematic analysis of the first oro-pharyngal or nasal isolates of young cf patients and those chronically found in purulent sputum, as this may contribute to an early diagnosis of s. aureus infection.
however searching for potential sources of s. aureus from the patients and their family members, the medical staff, the environmental home and hospital setting requires a laborious sampling work and needs another study.
thirty eight patients were also colonized by p. aeruginosa as observed in a previous investigation  <cit> .
there was apparently no link between the s. aureus genotype and the presence of p. aeruginosa.
however, the patients from whom we analyzed a large number of s. aureus isolates, reflecting a long-term colonization, were usually coinfected with p. aeruginosa, with the exception of patient cfu_ <dig> .
in a few patients, chronic colonization by a single strain was not observed although strains from up to  <dig> different ccs could be isolated during the study period.
antibiotic resistance
mrsa were found in more than 30% of patients, while some of them also carried mssa.
the presence of mrsa can limit the inscription of a patient on a lung transplant list  <cit> , therefore, it is important to investigate the status and mechanisms of methicillin resistance.
in some mrsa strains methicillin resistance was not associated with presence of meca  <cit>  and the resistance phenotype for most of these strains was bor-sa, with overproduction of β-lactamase.
vancomycin was frequently used to treat mrsa infection, though pulmonary diffusion of this drug was not excellent.
eradication of s. aureus was rarely observed and chronic colonization was confirmed from repetitive sputum samples over time.
CONCLUSIONS
in the present study, using the mlva- <dig> procedure, we genotyped rapidly and with a simple equipment a large number of s. aureus isolates, allowing the longitudinal survey of  <dig> cf patients.
a large proportion of isolates belonged to a limited number of ccs, and in most cases a single strain, either a mrsa or a mssa, chronically colonized the patient.
over time variants appeared and it will be interesting to test whether they show selective advantages.
the performances of mlva open the way to additional studies to investigate the contamination sources and to identify s. aureus isolates responsible for colonization and clinical exacerbations.

 cystic fibrosis is caused by a mutation in the cftr-gene leading to dysfunction of the exocrine glands. the disease is responsible for chronic airway obstruction in the lung, a favourable condition for pulmonary infections during childhood. in different studies investigating pathogens in cf, s. aureus was observed in to 60% of patients frequently in association with other bacteria, such as pseudomonas aeruginosa. since the introduction of methicillin in methicillin-resistant s. aureus clones have rapidly emerged and spread worldwide and account for to 30% of s. aureus infections. molecular epidemiology studies using multi locus sequence typing on clinical strains of s. aureus have shown that they are distributed into major clonal complexes. mrsa strains represent the most threatening challenge as they are frequently resistant to many antibiotics and there is evidence that antibiotic treatments not only facilitate the spreading of these clones but also enhance their pathogenicity. patients with cf are at particular risk for pulmonary colonization of mrsa, both because of their difficulty in clearing mucus and because of their frequent hospital visits, which can increase exposure to mrsa. several studies reported that to 35% of cf patients harbored a mrsa strain and described the emergence of community-acquired mrsa. methicillin-susceptible strains also constitute a risk in cf patients, particularly because of the existence of biofilms in the infected lung in which they can escape from antibiotic treatment. the epidemiology of s. aureus in cf patients has been investigated in different studies, but mostly mrsa were analysed and the role of mssa was not assessed. in order to extend the knowledge of the population of s. aureus chronically infecting cf patients, all the isolates should be systematically genotyped with a high degree of discrimination which is difficult using the currently available techniques. the polymorphism of the staphylococcus protein a gene, first used by frenay et al. to genotype s. aureus and further evaluated by shopsin et al. has proven to be very useful to investigate s. aureus genetic diversity. subsequently mlst became the most widely used technique to analyse the epidemiology of s. aureus and to perform phylogenetic studies. although the combined discriminatory power of spa typing and mlst is high, these techniques do not sufficiently discriminate within the major ccs and their cost is elevated. new approaches have been developed which use variable number of tandem repeats either to produce a multiple band pattern in a technique called mlvf or to perform multiple loci vntr analysis. mlva consists in the analysis of individual vntrs allowing the description of a strain in the form of a code easily exchangeable between laboratories. mlva with vntrs could correctly assigned isolates to outbreaks or identified isolates as unlinked. schouls et al. using vntrs showed that mlva was at least as discriminatory as pulse field gel electrophoresis and twice as discriminatory as spa-sequence typing. finally we recently described a very informative mlva scheme which makes use of vntrs and demonstrated that its discriminatory power was much higher than those of mlst and spa typing. in the present work we used the proposed mlva- assay to genotype s. aureus isolates recovered from firstly as well as chronically colonized cf patients, over a period of months. the aim of our study was to investigate the genetic diversity of mrsa and mssa present in the sputum of cf children whether sporadically or chronically. the longitudinal survey of genotypes provided information on the variations in those strains recovered from some patients over a maximum period of months.  clinical characteristics of s. aureus colonization from a total number of patients attending the armand trousseau cf centre during the months study period, provided sputum of which showed one or several cultures positive for s. aureus. it is likely that most were community-acquired s. aureus contaminations as the majority of patients were outpatients. in addition there was no outbreak episode during the study period. although this study was not designed to correlate the bacteria recovered from the sputum with the respiratory evolution of the patients, the following features may be underlined: among the patients, were co-infected with p. aeruginosa, as observed in a previous investigation , making it difficult to determine the role of s. aureus in broncho-pulmonary exacerbations. twenty-four of these patients harboured mssa, patients harboured mrsa and patients harboured both. mrsa were mainly isolated from older patients who were treated by regular intravenous antibiotic courses, as recommended by the international guidelines. in the other patients, s. aureus was the single species recovered from sputum cultures; mssa isolates were found in patients, mrsa in patients and both mssa and mrsa in other patients. these patients were younger than those co-infected with p. aeruginosa. of note, those harbouring mrsa had more respiratory exacerbations and worse lung function than those harbouring mssa. both methicillin-susceptible and methicillin resistant isolates were repeatedly recovered over several months. forty percent of patients were suffering from their first colonization with s. aureus while in 60% the recurrent isolation of the bacteria was indicative of colonization with exacerbations. in the later cases genotyping could show in several instances that the strain was different and therefore that several independent infections took place. patients were treated by antimicrobial drugs, however in most cases s. aureus was still recovered from sputum samples despite clinical improvement. investigation of mrsa in total a mrsa isolate was found at least once in patients with a positive culture. both screening techniques used here failed to detect the presence of the penicillin binding protein pbp 2a in the resistant strains from five patients. methicillin-resistance in these strains was caused by a high production of β-lactamase, leading to a characteristic bor-sa phenotype. isolates from patient cfu_ did not show bor-sa characteristics, suggesting that methicillin resistance resulted from a new modified penicillin-binding capacity, though this modified capacity was not investigated. in patients colonized by bor-sa or mod-sa, isolates showing the same genotype but different susceptibility patterns were occasionally recovered from the same sputum sample, or over time in successive samples. the genetic diversity of strains as assessed by mlva a total of isolates were genotyped by mlva using fourteen vntrs. overall the pcr efficiency was very satisfying and there was no difficulty in evaluating the amplicon size on 2% agarose gels. in one case, the presence of several bands with sa suggested the existence of two different variants of a strain in the sample. indeed this could be confirmed by testing several colonies from a culture. a the chromosomal position on the mu genome, in kilobase-pairs is indicated in the vntr name, for example sa is at position. b primers different from c primers different from d star stands for s. aureus repeat e primers different from siru the s. aureus population diversity is shown in the minimum spanning tree representation on figure the presence of the available sequenced genomes in the analysis allowed the identification of several mlst ccs. mlst was performed on two strains of the second largest cluster, showing that it belonged to cc. a comparison made with a collection of strains previously analysed by mlst and by mlva- confirmed the concordance of the ccs defined by the two techniques. therefore, in the present study it was decided to use the mlst nomenclature to designate the largest ccs. to facilitate the comparison of isolates, one strain of a given genotype per patient and reference strains were used to perform a clustering analysis. with a cut-off value of 45% clusters, or clonal complexes, were observed. figure shows the first part of a dendrogram in which all the strains belonging to cc cc cc cc cc and cc fall. the second part of the dendrogram shown on figure displays all the cc cc and cc isolates. four ccs comprised 71% of the strains. five clusters contained only one genotype each. mrsa were distributed into genotypes, mssa into genotypes whereas genotypes were assigned to both mrsa and mssa strains. spa typing the spa repeats were sequenced in selected isolates on the basis of their distribution into the different clusters and of their polymorphism within these clusters. the sequence was submitted to the ridom spaserver in order to identify the spa type. seven new spa types were given a number by the ridom spaserver. the result confirmed the correct clustering of strains by mlva, as shown by the almost perfect correlation between the two genotyping techniques. strain trsa was positioned near cc strains and its spa type was characteristic of st strains a bacterial population resulting from a large chromosomal rearrangement between cc and cc. three identical isolates from patient cfu_ which spa type corresponded to cc were branched in an ancestral position to the cc cluster. interestingly, in cc a larger diversity was observed for spa, as compared to the other large clusters, cc and cc. longitudinal survey in patients, isolates that were repeatedly recovered over the months study period belonged to the same lineage, i.e. they were either identical or differed at only one vntr. in the other patients the isolates belonged to or even different ccs. for example isolates belonging to different ccs were found in patient cfu_ and only one of them was observed more than once. table shows the number of ccs and genotypes from patients for which at least isolates were recovered. one example of stability is observed in patient cfu_ for which mod-sa cc isolates with the same genotype were recovered from january to july patient cfu_ had isolates with two different spa alleles. in and early a single genotype with seven repeats at the spa locus was observed, whereas after march isolates with two repeats at the spa locus were also found. on several occasions, both were present in equal amounts giving rise to two products upon pcr amplification. from march to january cc isolates were recovered from patient cfu_ with three variants differing at vntrs sa and sa1132: one genotype was found in isolates in and early another one in isolates from to and the third corresponded to a single isolate in patient cfu_ had isolates with three variants differing at vntr sa1132: one was found only in in two isolates, a second one was found once in times in and once in and the third one was found twice in patient cfu_ had isolates with two variants differing at vntr sa2039: one genotype was observed in in two isolates, and the other was found in five isolates once in and in a indicates the loci where there are vntr variants within identical cc. in parentheses are shown the number of repeats at the variants. b un corresponds to st109 c un corresponds to st398 genotypes and mrsa on figures and are shown the sensitivity to methicillin and the presence/absence of the meca gene carried by staphylococcal cassette chromosome mec, as tested by pcr. the large majority of mrsa isolates fall inside cc cc and cc in cc all strains were mssa except for trsa which is placed outside of the cluster and is meca negative. interestingly, in patient cfu_ isolates were of the same genotype, of which were meca positive and were meca negative, suggesting a recent transfer of the meca gene or sccmec instability in this particular strain. in five patients, isolates with identical genotypes were apparently either resistant or sensitive to methicillin but meca was not detected while the phenotypic resistance aspects were bor-sa or mod-sa. in four patients only mssa strains were isolated over more than months. the genetic diversity among mssa isolates was larger than among mrsa, but both could be found in large ccs. discussion mlva the mlva procedure used in the present study allowed the systematic investigation of all s. aureus isolates recovered from cf patients attending a french centre during a period of months. in the present study a total of isolates from children were genotyped, with a great variation within the number of s. aureus isolates per patient: one or two isolates for the firstly colonized patients, to more than isolates for some chronically colonized patients repeatedly hospitalized for acute exacerbation. this type of study had never been conducted before because the available techniques were either time consuming or too expensive. generally only mrsa isolates were studied and consequently, the mssa diversity was insufficiently known although they account for a large proportion of strains responsible for chronic colonization in cf patients. mlva using vntrs is a very informative technique which compares favourably with mlst and spa typing. more genotypes are observed and it is possible to see the emergence of variants. the size of the vntrs repeats ranges from bp to bp, which makes the technique very easy to implement using agarose gel electrophoresis as well as high throughput approaches. the allelic size differences for such markers can be estimated directly by eye and compared to a chart where all the known alleles have been indicated. this information is accessible on a dedicated web page in "the bacterial mlva-genotyping-on-the-web service". for epidemiology purposes, a simpler scheme could be performed with a selection of informative markers. however, it is important to keep a large collection of markers with different degrees of variability for the investigation of outbreaks or for phylogenetic studies. in the present work each vntr was amplified in a separate pcr reaction but our preliminary experiments showed that vntrs could be amplified simultaneously and the size automatically determined using a capillary electrophoresis apparatus. this opens the way to automatized genotyping similarly to the protocol described by schouls et al.. however in this latest study only vntrs were analysed which, in our opinion may not be sufficiently discriminant for epidemiological studies. indeed the simpson's diversity index in the mlva- assay was % whereas we obtained a % di using the mlva- assay. other published vntr-based genotyping methods either did not use enough markers or analyzed fingerprints which makes the comparison of profiles between laboratories very difficult. in addition failure to amplify some vntrs in a relatively important number of samples led to partial profiles in up to 27% of isolates in one study. genetic diversity of strains and population structure in the present collection of isolates, genotypes were observed, 68% belonging to main clusters. the genotypes in the mlva cluster corresponding to cc were very stable over a period of more than years. in contrast, more variability was observed in isolates of cc and cc in cc several vntrs showed very small alleles as compared to the other clonal complexes which could be the result of frequent loss of repeats due to recombination. the majority of cc isolates were mrsa, whereas these represented about 50% of cc and cc isolates. recent studies have shown that cc contained both community and hospital-acquired mrsa strains whereas the other ccs mainly contained hospital-acquired strains. in a recent survey of cf patients in spain, 67% of mrsa isolates were st which belong to cc and the second largest group corresponded to st belonging to cc. colonization the precise identification of s. aureus genotypes colonizing the lungs of cf patients is of importance to trace the source of contamination and eventually modify the management of patients in the hospital. it is also important to know whether a single strain is present over time despite antibiotic treatment, or if different strains are involved in patients exacerbations. in two patients, isolates belonging to four different ccs were observed, but the most frequent situation was colonization by a single strain which could vary over time. in some patients, the same strain was recovered over a two years period, with a few variants differing at a single vntr. in several cases the variants seemed to appear sequentially suggesting that they acquired an advantage over the first isolate. on the contrary, in patient cfu_ two variants were alternatively isolated during the two years period, and in patient cfu_ the presence of two spa amplicons in a single pcr reaction pointed to the coexistence in equal amounts of two variants in the sputum sample. interestingly variants were more frequently observed in cc strains than in other ccs, again indicating the existence of a higher degree of instability in this cc. it was shown that the adaptation to chronic colonization requires the expression of virulence factors and a higher mutation capacity resulting in an increase of the genetic diversity. in cases, a given genotype was shared by different patients, but it is difficult to define the origin of the contamination as most of these strains belonged to common ccs. indeed, in a recent study by sakwinska et al., it was shown that cc and cc colonized each 24% of the carrier population. in the armand trousseau center, the risk of p. aeruginosa cross-colonization has led to the increased use of isolation protocols among the patients since many years. the source of s. aureus lung colonization could be either the nose, or the oro-pharynx, as suggested by recent studies. the simplicity of mlva genotyping should allow a systematic analysis of the first oro-pharyngal or nasal isolates of young cf patients and those chronically found in purulent sputum, as this may contribute to an early diagnosis of s. aureus infection. however searching for potential sources of s. aureus from the patients and their family members, the medical staff, the environmental home and hospital setting requires a laborious sampling work and needs another study. thirty eight patients were also colonized by p. aeruginosa as observed in a previous investigation. there was apparently no link between the s. aureus genotype and the presence of p. aeruginosa. however, the patients from whom we analyzed a large number of s. aureus isolates, reflecting a long-term colonization, were usually coinfected with p. aeruginosa, with the exception of patient cfu_. in a few patients, chronic colonization by a single strain was not observed although strains from up to different ccs could be isolated during the study period. antibiotic resistance mrsa were found in more than 30% of patients, while some of them also carried mssa. the presence of mrsa can limit the inscription of a patient on a lung transplant list, therefore, it is important to investigate the status and mechanisms of methicillin resistance. in some mrsa strains methicillin resistance was not associated with presence of meca and the resistance phenotype for most of these strains was bor-sa, with overproduction of β-lactamase. vancomycin was frequently used to treat mrsa infection, though pulmonary diffusion of this drug was not excellent. eradication of s. aureus was rarely observed and chronic colonization was confirmed from repetitive sputum samples over time.  in the present study, using the mlva- procedure, we genotyped rapidly and with a simple equipment a large number of s. aureus isolates, allowing the longitudinal survey of cf patients. a large proportion of isolates belonged to a limited number of ccs, and in most cases a single strain, either a mrsa or a mssa, chronically colonized the patient. over time variants appeared and it will be interesting to test whether they show selective advantages. the performances of mlva open the way to additional studies to investigate the contamination sources and to identify s. aureus isolates responsible for colonization and clinical exacerbations. 
methicillin-resistance was observed in 60% of the isolates, 90% of which belonged to the main clonal complexes cc cc and cc in patients, methicillin-resistance of s. aureus isolates was not associated with the meca gene: for four patients, it was due to overproduction of β-lactamase, leading to bor-sa isolates, while a strain showing probably a new modified penicillin-binding capacity was observed from one patient. systematic genotyping of s. aureus isolates recovered from sputum of cf children allows a thorough analysis of the strains responsible for sporadic as well as chronic colonization and the follow up of their evolution over time. on six occasions, s. aureus isolates with the same genotype were shared by different patients and they belonged to one of the four main clusters. mssa as well as mrsa, bor-sa and mod-sa isolates can persist over several years, despite antibiotic treatments. in patients, strains from two to four different ccs were recovered over time. staphylococcus aureus infection in patients with cystic fibrosis is frequent and may be due to colonization by a few pathogenic lineages.
 staphylococcus aureus infection in patients with cystic fibrosis is frequent and may be due to colonization by a few pathogenic lineages. systematic genotyping of all isolates, methicillin-susceptible s. aureus as well as methicillin-resistant s. aureus is necessary to identify such lineages and follow their evolution in patients. multiple-locus variable-number tandem repeat analysis was used to survey s. aureus clinical isolates in a french paediatric cf centre.  during a months period, patients, aged to years, regularly followed up at the centre, provided sputum for culture. from patients, a total of isolates were genotyped by mlva, resolving into genotypes and clonal complexes composed of similar or closely related isolates. 71% of the strains were distributed into four main ccs, in term of number of isolates and number of genotypes. spa typing was performed on representative samples, showing an excellent concordance with mlva. in patients, strains from two to four different ccs were recovered over time. on six occasions, s. aureus isolates with the same genotype were shared by different patients and they belonged to one of the four main clusters. methicillin-resistance was observed in 60% of the isolates, 90% of which belonged to the main clonal complexes cc cc and cc in patients, methicillin-resistance of s. aureus isolates was not associated with the meca gene: for four patients, it was due to overproduction of β-lactamase, leading to bor-sa isolates, while a strain showing probably a new modified penicillin-binding capacity was observed from one patient.  systematic genotyping of s. aureus isolates recovered from sputum of cf children allows a thorough analysis of the strains responsible for sporadic as well as chronic colonization and the follow up of their evolution over time. we show here that more than 70% of these strains belong to major ccs. mssa as well as mrsa, bor-sa and mod-sa isolates can persist over several years, despite antibiotic treatments. 