BACKGROUND
methicillin-resistant staphylococcus aureus  is a major cause of nosocomial and community-associated infections worldwide.
most cases of community-associated mrsa  have been associated with skin and soft-tissue infections in previously healthy individuals  <cit> .
since  <dig>  pigs  <cit>  and other animals such as horses  <cit> , poultry  <cit>  and calves  <cit>  have been identified as a new reservoir for ca-mrsa.
most of the livestock related mrsa strains share the same multi locus sequence typing  type, namely st <dig>  throughout europe  <cit> , canada  <cit>  and in the united states  <cit>  st <dig> has been found in association with animal husbandry, indicating a worldwide clonal lineage.
although the clinical importance of st <dig> is still controversial, there are reports indicating transmission and infections among humans  <cit> .
pulsed field gel electrophoresis  using smai is considered to be the gold standard for typing mrsa isolates  <cit> .
when pfge was performed on st <dig> isolates, no banding patterns could be generated, due to methylation of the smai site  <cit> .
therefore, st <dig> isolates are referred to as pfge non-typeable -mrsa.
some years ago staphylococcal protein a  typing was introduced as a highly discriminatory typing method to characterize s. aureus isolates  <cit> .
however, spa-typing of the st <dig> isolates revealed very limited variation within this group and 80% of our st <dig> isolates had either spa-type t <dig>  t <dig> or t <dig>  <cit> .
recently, a multiple-locus variable number of tandem repeat analysis  has been presented  <cit> .
although mlva is significantly more discriminatory than spa-typing, it was unable to yield a better discrimination of the isolates of the st <dig> lineage.
the lack of a typing method that can discriminate st <dig> strains has hampered studies on the origin and transmission routes of this mrsa clade.
in the netherlands all first mrsa isolates obtained from patients with staphylococcal disease and from patients that carry the pathogen are sent to the national mrsa reference centre for typing.
in  <dig>  30% of all forwarded mrsa isolates were ntsmai -mrsa  <cit> .
recently, a neoschizomer of smai, designated as cfr9i, was shown to be insensitive for the dna-methylation leading to ntsmai -mrsa isolates.
in two studies this restriction enzyme was used for generating pfge profiles of ntsmai -mrsa isolates  <cit> .
in the study presented here we optimized pfge with restriction enzyme cfr9i and evaluated its use to characterize ntsmai -mrsa isolates.
the data will yield important information about the genetic diversity of the st <dig> clonal lineage in the netherlands and demonstrates that cfr9i pfge is a powerful tool to study possible transmission and outbreaks of mrsa isolates, previously not typeable by conventional pfge approaches.
methods
bacterial isolates
the national institute for public health and the environment  serves as the dutch national mrsa reference center.
all first mrsa isolates, one per patient, are sent to the rivm for further typing.
pfge was carried out using restriction enzyme smai according to the harmony protocol  <cit> .
from this large mrsa collection a number of ntsmai -mrsa was selected to optimize and validate the cfr9i pfge.
to study the genetic diversity of the two most prevalent spa-types among ntsmai -mrsa in the netherlands,  <dig> ntsmai -mrsa isolates  and t <dig> ) in  <dig> from patients living in geographical dispersed regions in the netherlands were used.
in addition,  <dig> strains  from veterinarians and one of their family members, the latter whom did not have contact with animals and  <dig> pig and pig farmer isolates and  <dig> strains from an ntsmai -mrsa outbreak in a residential care facility  <cit>  were included in this study to assess the potential of the cfr9i pfge to identify transmissions.
to validate the cfr9i pfge method,  <dig> typeable mrsa  isolates and the reference strain nctc  <dig> were tested.
five non-typeable isolates were repeated  <dig> times with cfr9i pfge to ensure the reproducibility of the method.
molecular typing
all isolates were characterized with spa typing  <cit> .
spa-types were assigned using bionumerics software version  <dig>  .
sccmec typing of the isolates was performed using the multiplex pcr described by boye et al  <cit> .
in order to obtain clear and reproducible pfge banding patterns using cfr9i as restriction enzyme, the harmony pfge protocol had to be adjusted.
this resulted in the following protocol: from each isolate,  <dig> μl bacterial suspension of an overnight trypton soy broth  culture, was embedded in a plug mold  with  <dig> % low-melting-point agarose .
then,  <dig> μl lysostaphine  was added and incubated for  <dig> h at 37°c.
subsequently, the plugs were incubated overnight at 55°c with  <dig> μl proteinase k .
the plugs were then washed,  <dig> to  <dig> times in a shaking incubator for  <dig> min.
in  <dig> × tris-edta buffer  at 50°c in order to remove cell debris.
finally, the plugs were equilibrated in  <dig> × cfr9i buffer  for  <dig> min.
at room temperature prior to digestion and then submerged in  <dig> μl of  <dig> × cfr9i reaction buffer containing  <dig> u of cfr9i restriction enzyme .
the reaction tubes were incubated overnight at 37°c in a shaking incubator.
further steps were carried out according to the harmony protocol  <cit> .
briefly, a 1% agarose gel was poured into a gel tray and positioned in a contour-clamped homogeneous electric field   tank and submerged in  <dig>  ml of  <dig>  × tris-borate-edta .
the total run time was  <dig> h at 14°c with an initial pulse time of  <dig> s, a final pulse time of  <dig> s and a voltage of  <dig> v/cm or  <dig> v. gels were stained in ethidium bromide  and viewed and photographed with uv transillumination.
digital images were analyzed using bionumerics software, version  <dig> .
if a difference in pfge pattern was observed, a new pulsed field type was assigned.
the definition of a pfge cluster was based on a similarity cutoff of 80%  <cit>  .
different pfge clusters were given in alphabetical order.
every band difference within a pfge cluster resulted in adding a numerical order to the pulsed field cluster.
RESULTS
optimization and validation of the cfr9i pfge method
in the initial experiments the smai restriction enzyme was replaced by cfr9i and exactly the same conditions were used as in the original pfge protocol.
this led to uninformative pfge patterns consisting mainly of smears and faint bands obtained through partial digestion of the genomic dna.
a higher lysostaphine concentration , longer incubation steps for lysis , proteinase k and digestion overnight and hot washes at 50°c - instead of washes at room temperature - produced clear and reproducible banding profiles.
after optimizing the pfge method with cfr9i, high quality banding patterns from all selected  previously non-typeable st <dig> mrsa isolates were obtained.
for validation, both pfge protocols  were performed on  <dig> typeable mrsa isolates and the reference strain nctc  <dig>  side-by-side comparison of smai and cfr9i pfge profiles yielded identical banding patterns consistent with unequivocal comparability of both restriction patterns.
reproducibility of the method was confirmed with  <dig> ntsmai -mrsa isolates which were re-analyzed  <dig> times and yielded identical banding patterns.
genetic diversity of ntsmai -mrsa
all pfge patterns of the ntsmai -mrsa were compared with a database consisting of more than  <dig> isolates containing over  <dig> different pfge types obtained with smai digestion.
surprisingly, newly-obtained banding patterns of ntsmai -mrsa isolates did not match with any known pfge cluster in the national database of mrsa isolates collected since  <dig>
thirty t <dig> isolates revealed  <dig> different pfge patterns .
the largest pfge cluster consisted of  <dig> isolates, and  <dig> patterns were found more than once .
no correlation was found between pfge cluster and geographic location.
the minimal similarity  between the different patterns was 64% .
thirty t <dig> isolates revealed  <dig> different pfge patterns .
the largest cluster contained  <dig> isolates and  <dig> patterns were found more than once .
the clusters showed no geographical correlation.
the minimal similarity of the t <dig> isolates was 50% .
one t <dig> isolate yielded a very distinct pfge pattern .
without this isolate the minimal similarity of the t <dig> isolates would be 80%.
the minimal similarity of the  <dig> ntsmai -mrsa isolates was 35%, but most isolates share 80% or more similarity .
sccmec typing of the  <dig> ntsmai -mrsa isolates showed sccmec type iv  and sccmec type v .
three isolates yielded a variant of sccmec type v  and no sccmec types i, ii or iii were found .
transmission of st <dig> isolates
the results of cfr9i pfge of  <dig> pairs of veterinarians and one of their close family members showed that  <dig> pairs gave indistinguishable banding patterns suggesting possible transmission of st <dig> .
two pairs that did not match also had different spa-types .
one pair which had the same spa-type differed in a single pfge band .
six isolates belonging to an outbreak in a residential care facility with spa-types t <dig> and t <dig> all shared the same banding pattern .
furthermore, the transmission between pigs, pig farmers and their family on  <dig> different pig farms  was studied.
farms  <dig> to  <dig> shared the same spa-type whereas on farms  <dig> to  <dig>  two or more different spa-types were present.
the number of different pfge patterns  differed between farms, ranging from indistinguishable patterns  to  <dig> different pfge patterns .
pfge patterns b <dig>  d <dig>  d <dig>  d <dig> and e <dig> were found on several farms .
the minimal similarity within the farms varied from 52%  to 100%  and the minimal similarity between the farms was 61% .
figure  <dig> shows the pfge results of farm  <dig> with  <dig> different pfge patterns and from farm  <dig> which all had indistinguishable pfge patterns.
* dice similarity coefficient, using upgma.
optimization  <dig> %, position tolerance
discussion
mrsa isolates belonging to the st <dig> clonal lineage are hard to discriminate based on spa-typing and/or mlst, hampering the assessment of transmission and outbreaks.
therefore, other techniques such as a modified pfge could provide a new opportunity to differentiate st <dig> isolates.
the restriction enzyme smai does not cut the dna of ntsmai -mrsa isolates, due to methylation of the smai site.
however, cfr9i, a neoschizomer of smai, can be used for generating pfge profiles of the ntsmai -mrsa isolates.
when the standard smai protocol was used for cfr9i, banding patterns with smears and partial digests appeared.
other recently published articles seemed to have encountered similar problems with their cfr9i pfge  <cit> .
the results indicated that lysis of st <dig> isolates and digestion with restriction enzyme cfr9i is more cumbersome than lysis of typeable mrsa and digestion with smai  <cit> .
after modifying the protocol, banding patterns of similar quality as those of typeable mrsa isolates digested with smai were obtained.
all previously non-typeable mrsa isolates can be typed with the optimized pfge method providing a new opportunity to differentiate the st <dig> clonal lineage.
from april  <dig> until january  <dig>  all mrsa isolates sent to the rivm have been typed with pfge using smai as restriction enzyme creating a database with more than  <dig> isolates with over  <dig> different pfge types.
since cfr9i recognizes the same restriction site as smai, cfr9i enables analysis and comparison of the patterns with other profiles in our database.
no comparison was found when comparing banding patterns of ntsmai -mrsa with known pfge patterns, suggesting that smai restriction modification is confined to a defined clonal lineage.
recently, st <dig> isolates were typed using amplified fragment length polymorphism .
these data also suggested that st <dig> is a distinct cluster recently introduced into the dutch patient population  <cit> .
the pfge patterns of the two most prevalent spa-types  within the ntsmai -mrsa isolates showed more variation than spa-typing or mlst.
the genetic diversity within the st <dig> clonal lineage of mrsa sharing the same spa-type creates an opportunity for improved investigation of outbreak and potential transmission events.
spa-typing, which is currently used as a mrsa typing standard, cannot differentiate these isolates further.
using cfr9i pfge, spa-type t <dig> seemed to be more diverse than t <dig>  although the minimal similarity of the t <dig> isolates was 50%, this was mainly caused by a single isolate with a very distinct pfge pattern .
without this isolate the minimal similarity of the t <dig> isolates was 80%.
the t <dig> isolates showed a minimal similarity of 64% .
sccmec typing showed an almost equal distribution between sccmec type iv  and v  for t <dig> isolates, whereas all t <dig> isolates carried sccmec type v or a sccmec type v variant.
huijsdens and colleagues performed sccmec typing on  <dig> ntsmai -mrsa isolates and they showed similar results  <cit> .
this variation in sccmec types may also indicates a higher diversity among t <dig> mrsa isolates compared to t <dig> isolates.
the minimal similarity of the cfr9i pfge patterns among st <dig> isolates was 35% and showed variation within spa-types, but the diversity within this lineage is still limited.
furthermore, one isolate with spa-type t <dig> yielded a very distinct pfge pattern which causes the similarity to be 35% .
when excluding this isolate from the dendrogram the minimal similarity was 62%.
comparing the pfge results using the criteria by tenover et al.
and when a similarity cut-off of 80% was applied, most ntsmai -mrsa isolates should be classified as one pfge cluster  <cit> .
however, the cfr9i pfge is still better in discriminating possible differences between ntsmai -mrsa isolates.
no geographical relation could be found in either spa-type.
however, most ntsmai -mrsa isolates are found in areas with the highest pig density.
this could be explained by the frequent movement of pigs between farms in the netherlands.
this facilitates the dissemination of st <dig> mrsa on a national scale.
a similar situation took place during the foot- and -mouth epidemic in england of  <dig>  <cit> .
to provide additional resolution on the molecular evolution and dissemination of mrsa lineages, several typing techniques such as pfge, sccmec- and spa-typing have been developed.
since pfge with smai does not digest the dna of st <dig> isolates, spa-typing has been the method of choice for characterizing ntsmai -mrsa isolates.
however, given the low diversity in spa-types it is hard to ascertain health care-associated transmission if two or more different spa-types are present in the same institution.
fanoy et al.
described an outbreak in a residential care facility where two spa-types  were prevalent  <cit> .
after re-examination of the same isolates the pfge profiles using cfr9i were indistinguishable, indicating isogenicity.
moreover, the discriminatory ability of spa-typing of ntsmai -mrsa is compromised by the fact that more than 80% of the ntsmai -mrsa in the netherlands belong either to spa-type t <dig> or t <dig>  <cit> .
with the modified cfr9i pfge a better tool for epidemiological investigation has become available.
the results obtained by cfr9i pfge of isolates from veterinarians and their close family members showed possible transmission of st <dig>  five out of eight pairs had identical profiles.
the family members had themselves no contact with animals and were presumably infected by the occupationally exposed veterinarian.
two pairs of pfge patterns among family members were not identical.
their isolates also had different spa-types.
family members may have been colonized by one mrsa through the veterinarian and subsequently the veterinarian may have been re-colonized by another mrsa after occupational exposure.
one pair differed only in a single pfge band probably as a consequence of micro-evolution.
a study on nine different farms revealed that the pfge patterns of isolates from seven farms were related, but pfge patterns varied within and between the farms.
for example, farm  <dig>  yielded only  <dig> very closely related pfge patterns , while other farms, like farm  <dig>  showed  <dig> different pfge patterns  and had a similarity of only 66%.
different batches of animals entering the farm, carrying different ntsmai -mrsa, could have caused variation within farms.
further study is needed to confirm that farms with a fast turnover of pigs indeed show a higher diversity of pfge patterns of ntsmai -mrsa.
CONCLUSIONS
in conclusion, the modified pfge protocol for cfr9i provided highly informative banding patterns and showed good reproducibility.
the pfge results showed diversity within and between the two most prevalent spa-types among ntsmai -mrsa.
pfge confirmed transmission of the st <dig> clonal lineage within families and in a residential care facility.
the modified pfge approach can be used as a method for selecting important and distinct st <dig> isolates for further research.
the adjustments in the pfge protocol using cfr9i are easy to implement in laboratories which already have a pfge facility, creating a powerful tool to study the st <dig> clonal lineage.
authors' contributions
tb carried out all molecular typing and drafted the manuscript.
ajn participated in the design of the study and revised the manuscript critically for important intellectual content.
lms has made substantial contributions to conception and design of the study.
kwz was responsible for analysis and interpretation of the data and revised the manuscript critically.
jajwk has been involved in drafting the manuscript and revising it critically for important intellectual content.
hg participated in the design of the study and has given final approval of the version to be published.
xwh participated in the design of the study, has been involved in drafting the manuscript and revising it critically for important intellectual content.
all authors read and approved the final manuscript.

 methicillin-resistant staphylococcus aureus is a major cause of nosocomial and community-associated infections worldwide. most cases of community-associated mrsa have been associated with skin and soft-tissue infections in previously healthy individuals. since pigs and other animals such as horses, poultry and calves have been identified as a new reservoir for ca-mrsa. most of the livestock related mrsa strains share the same multi locus sequence typing type, namely st throughout europe, canada and in the united states st has been found in association with animal husbandry, indicating a worldwide clonal lineage. although the clinical importance of st is still controversial, there are reports indicating transmission and infections among humans. pulsed field gel electrophoresis using smai is considered to be the gold standard for typing mrsa isolates. when pfge was performed on st isolates, no banding patterns could be generated, due to methylation of the smai site. therefore, st isolates are referred to as pfge non-typeable -mrsa. some years ago staphylococcal protein a typing was introduced as a highly discriminatory typing method to characterize s. aureus isolates. however, spa-typing of the st isolates revealed very limited variation within this group and 80% of our st isolates had either spa-type t t or t. recently, a multiple-locus variable number of tandem repeat analysis has been presented. although mlva is significantly more discriminatory than spa-typing, it was unable to yield a better discrimination of the isolates of the st lineage. the lack of a typing method that can discriminate st strains has hampered studies on the origin and transmission routes of this mrsa clade. in the netherlands all first mrsa isolates obtained from patients with staphylococcal disease and from patients that carry the pathogen are sent to the national mrsa reference centre for typing. in 30% of all forwarded mrsa isolates were ntsmai -mrsa. recently, a neoschizomer of smai, designated as cfr9i, was shown to be insensitive for the dna-methylation leading to ntsmai -mrsa isolates. in two studies this restriction enzyme was used for generating pfge profiles of ntsmai -mrsa isolates. in the study presented here we optimized pfge with restriction enzyme cfr9i and evaluated its use to characterize ntsmai -mrsa isolates. the data will yield important information about the genetic diversity of the st clonal lineage in the netherlands and demonstrates that cfr9i pfge is a powerful tool to study possible transmission and outbreaks of mrsa isolates, previously not typeable by conventional pfge approaches. methods bacterial isolates the national institute for public health and the environment serves as the dutch national mrsa reference center. all first mrsa isolates, one per patient, are sent to the rivm for further typing. pfge was carried out using restriction enzyme smai according to the harmony protocol. from this large mrsa collection a number of ntsmai -mrsa was selected to optimize and validate the cfr9i pfge. to study the genetic diversity of the two most prevalent spa-types among ntsmai -mrsa in the netherlands, ntsmai -mrsa isolates and t ) in from patients living in geographical dispersed regions in the netherlands were used. in addition, strains from veterinarians and one of their family members, the latter whom did not have contact with animals and pig and pig farmer isolates and strains from an ntsmai -mrsa outbreak in a residential care facility were included in this study to assess the potential of the cfr9i pfge to identify transmissions. to validate the cfr9i pfge method, typeable mrsa isolates and the reference strain nctc were tested. five non-typeable isolates were repeated times with cfr9i pfge to ensure the reproducibility of the method. molecular typing all isolates were characterized with spa typing. spa-types were assigned using bionumerics software version. sccmec typing of the isolates was performed using the multiplex pcr described by boye et al. in order to obtain clear and reproducible pfge banding patterns using cfr9i as restriction enzyme, the harmony pfge protocol had to be adjusted. this resulted in the following protocol: from each isolate, μl bacterial suspension of an overnight trypton soy broth culture, was embedded in a plug mold with % low-melting-point agarose. then, μl lysostaphine was added and incubated for h at 37°c. subsequently, the plugs were incubated overnight at 55°c with μl proteinase k. the plugs were then washed, to times in a shaking incubator for min. in × tris-edta buffer at 50°c in order to remove cell debris. finally, the plugs were equilibrated in × cfr9i buffer for min. at room temperature prior to digestion and then submerged in μl of × cfr9i reaction buffer containing u of cfr9i restriction enzyme. the reaction tubes were incubated overnight at 37°c in a shaking incubator. further steps were carried out according to the harmony protocol. briefly, a 1% agarose gel was poured into a gel tray and positioned in a contour-clamped homogeneous electric field tank and submerged in ml of × tris-borate-edta. the total run time was h at 14°c with an initial pulse time of s, a final pulse time of s and a voltage of v/cm or v. gels were stained in ethidium bromide and viewed and photographed with uv transillumination. digital images were analyzed using bionumerics software, version. if a difference in pfge pattern was observed, a new pulsed field type was assigned. the definition of a pfge cluster was based on a similarity cutoff of 80%. different pfge clusters were given in alphabetical order. every band difference within a pfge cluster resulted in adding a numerical order to the pulsed field cluster.  optimization and validation of the cfr9i pfge method in the initial experiments the smai restriction enzyme was replaced by cfr9i and exactly the same conditions were used as in the original pfge protocol. this led to uninformative pfge patterns consisting mainly of smears and faint bands obtained through partial digestion of the genomic dna. a higher lysostaphine concentration, longer incubation steps for lysis, proteinase k and digestion overnight and hot washes at 50°c - instead of washes at room temperature - produced clear and reproducible banding profiles. after optimizing the pfge method with cfr9i, high quality banding patterns from all selected previously non-typeable st mrsa isolates were obtained. for validation, both pfge protocols were performed on typeable mrsa isolates and the reference strain nctc side-by-side comparison of smai and cfr9i pfge profiles yielded identical banding patterns consistent with unequivocal comparability of both restriction patterns. reproducibility of the method was confirmed with ntsmai -mrsa isolates which were re-analyzed times and yielded identical banding patterns. genetic diversity of ntsmai -mrsa all pfge patterns of the ntsmai -mrsa were compared with a database consisting of more than isolates containing over different pfge types obtained with smai digestion. surprisingly, newly-obtained banding patterns of ntsmai -mrsa isolates did not match with any known pfge cluster in the national database of mrsa isolates collected since thirty t isolates revealed different pfge patterns. the largest pfge cluster consisted of isolates, and patterns were found more than once. no correlation was found between pfge cluster and geographic location. the minimal similarity between the different patterns was 64%. thirty t isolates revealed different pfge patterns. the largest cluster contained isolates and patterns were found more than once. the clusters showed no geographical correlation. the minimal similarity of the t isolates was 50%. one t isolate yielded a very distinct pfge pattern. without this isolate the minimal similarity of the t isolates would be 80%. the minimal similarity of the ntsmai -mrsa isolates was 35%, but most isolates share 80% or more similarity. sccmec typing of the ntsmai -mrsa isolates showed sccmec type iv and sccmec type v. three isolates yielded a variant of sccmec type v and no sccmec types i, ii or iii were found. transmission of st isolates the results of cfr9i pfge of pairs of veterinarians and one of their close family members showed that pairs gave indistinguishable banding patterns suggesting possible transmission of st. two pairs that did not match also had different spa-types. one pair which had the same spa-type differed in a single pfge band. six isolates belonging to an outbreak in a residential care facility with spa-types t and t all shared the same banding pattern. furthermore, the transmission between pigs, pig farmers and their family on different pig farms was studied. farms to shared the same spa-type whereas on farms to two or more different spa-types were present. the number of different pfge patterns differed between farms, ranging from indistinguishable patterns to different pfge patterns. pfge patterns b d d d and e were found on several farms. the minimal similarity within the farms varied from 52% to 100% and the minimal similarity between the farms was 61%. figure shows the pfge results of farm with different pfge patterns and from farm which all had indistinguishable pfge patterns. * dice similarity coefficient, using upgma. optimization %, position tolerance discussion mrsa isolates belonging to the st clonal lineage are hard to discriminate based on spa-typing and/or mlst, hampering the assessment of transmission and outbreaks. therefore, other techniques such as a modified pfge could provide a new opportunity to differentiate st isolates. the restriction enzyme smai does not cut the dna of ntsmai -mrsa isolates, due to methylation of the smai site. however, cfr9i, a neoschizomer of smai, can be used for generating pfge profiles of the ntsmai -mrsa isolates. when the standard smai protocol was used for cfr9i, banding patterns with smears and partial digests appeared. other recently published articles seemed to have encountered similar problems with their cfr9i pfge. the results indicated that lysis of st isolates and digestion with restriction enzyme cfr9i is more cumbersome than lysis of typeable mrsa and digestion with smai. after modifying the protocol, banding patterns of similar quality as those of typeable mrsa isolates digested with smai were obtained. all previously non-typeable mrsa isolates can be typed with the optimized pfge method providing a new opportunity to differentiate the st clonal lineage. from april until january all mrsa isolates sent to the rivm have been typed with pfge using smai as restriction enzyme creating a database with more than isolates with over different pfge types. since cfr9i recognizes the same restriction site as smai, cfr9i enables analysis and comparison of the patterns with other profiles in our database. no comparison was found when comparing banding patterns of ntsmai -mrsa with known pfge patterns, suggesting that smai restriction modification is confined to a defined clonal lineage. recently, st isolates were typed using amplified fragment length polymorphism. these data also suggested that st is a distinct cluster recently introduced into the dutch patient population. the pfge patterns of the two most prevalent spa-types within the ntsmai -mrsa isolates showed more variation than spa-typing or mlst. the genetic diversity within the st clonal lineage of mrsa sharing the same spa-type creates an opportunity for improved investigation of outbreak and potential transmission events. spa-typing, which is currently used as a mrsa typing standard, cannot differentiate these isolates further. using cfr9i pfge, spa-type t seemed to be more diverse than t although the minimal similarity of the t isolates was 50%, this was mainly caused by a single isolate with a very distinct pfge pattern. without this isolate the minimal similarity of the t isolates was 80%. the t isolates showed a minimal similarity of 64%. sccmec typing showed an almost equal distribution between sccmec type iv and v for t isolates, whereas all t isolates carried sccmec type v or a sccmec type v variant. huijsdens and colleagues performed sccmec typing on ntsmai -mrsa isolates and they showed similar results. this variation in sccmec types may also indicates a higher diversity among t mrsa isolates compared to t isolates. the minimal similarity of the cfr9i pfge patterns among st isolates was 35% and showed variation within spa-types, but the diversity within this lineage is still limited. furthermore, one isolate with spa-type t yielded a very distinct pfge pattern which causes the similarity to be 35%. when excluding this isolate from the dendrogram the minimal similarity was 62%. comparing the pfge results using the criteria by tenover et al. and when a similarity cut-off of 80% was applied, most ntsmai -mrsa isolates should be classified as one pfge cluster. however, the cfr9i pfge is still better in discriminating possible differences between ntsmai -mrsa isolates. no geographical relation could be found in either spa-type. however, most ntsmai -mrsa isolates are found in areas with the highest pig density. this could be explained by the frequent movement of pigs between farms in the netherlands. this facilitates the dissemination of st mrsa on a national scale. a similar situation took place during the foot- and -mouth epidemic in england of. to provide additional resolution on the molecular evolution and dissemination of mrsa lineages, several typing techniques such as pfge, sccmec- and spa-typing have been developed. since pfge with smai does not digest the dna of st isolates, spa-typing has been the method of choice for characterizing ntsmai -mrsa isolates. however, given the low diversity in spa-types it is hard to ascertain health care-associated transmission if two or more different spa-types are present in the same institution. fanoy et al. described an outbreak in a residential care facility where two spa-types were prevalent. after re-examination of the same isolates the pfge profiles using cfr9i were indistinguishable, indicating isogenicity. moreover, the discriminatory ability of spa-typing of ntsmai -mrsa is compromised by the fact that more than 80% of the ntsmai -mrsa in the netherlands belong either to spa-type t or t. with the modified cfr9i pfge a better tool for epidemiological investigation has become available. the results obtained by cfr9i pfge of isolates from veterinarians and their close family members showed possible transmission of st five out of eight pairs had identical profiles. the family members had themselves no contact with animals and were presumably infected by the occupationally exposed veterinarian. two pairs of pfge patterns among family members were not identical. their isolates also had different spa-types. family members may have been colonized by one mrsa through the veterinarian and subsequently the veterinarian may have been re-colonized by another mrsa after occupational exposure. one pair differed only in a single pfge band probably as a consequence of micro-evolution. a study on nine different farms revealed that the pfge patterns of isolates from seven farms were related, but pfge patterns varied within and between the farms. for example, farm yielded only very closely related pfge patterns, while other farms, like farm showed different pfge patterns and had a similarity of only 66%. different batches of animals entering the farm, carrying different ntsmai -mrsa, could have caused variation within farms. further study is needed to confirm that farms with a fast turnover of pigs indeed show a higher diversity of pfge patterns of ntsmai -mrsa.  in conclusion, the modified pfge protocol for cfr9i provided highly informative banding patterns and showed good reproducibility. the pfge results showed diversity within and between the two most prevalent spa-types among ntsmai -mrsa. pfge confirmed transmission of the st clonal lineage within families and in a residential care facility. the modified pfge approach can be used as a method for selecting important and distinct st isolates for further research. the adjustments in the pfge protocol using cfr9i are easy to implement in laboratories which already have a pfge facility, creating a powerful tool to study the st clonal lineage. authors' contributions tb carried out all molecular typing and drafted the manuscript. ajn participated in the design of the study and revised the manuscript critically for important intellectual content. lms has made substantial contributions to conception and design of the study. kwz was responsible for analysis and interpretation of the data and revised the manuscript critically. jajwk has been involved in drafting the manuscript and revising it critically for important intellectual content. hg participated in the design of the study and has given final approval of the version to be published. xwh participated in the design of the study, has been involved in drafting the manuscript and revising it critically for important intellectual content. all authors read and approved the final manuscript.
the pfge results showed diversity within and between the two most prevalent spa-types of ntsmai -mrsa. the pfge patterns of st isolates showed more diversity than with spa-typing and/or mlst. after optimizing and evaluating the cfr9i pfge, clear and reproducible banding patterns were obtained from all previously non-typeable mrsa isolates. furthermore, possible transmission among veterinarians and their family members was investigated and an outbreak of st mrsa in a residential care facility was confirmed with the cfr9i pfge. livestock has recently been identified as a new reservoir of methicillin-resistant staphylococcus aureus. the adjustments in the pfge protocol using cfr9i are easy to implement to study the st clonal lineage in laboratories which already have a pfge facility. no match was found, when comparing banding patterns of the ntsmai -mrsa with different pfge types, obtained with smai digestion, in our database of more than strains.
 livestock has recently been identified as a new reservoir of methicillin-resistant staphylococcus aureus. most isolates belong to st and are non-typeable with pfge using smai, making it difficult to study transmission and outbreaks. therefore, a new pfge using cfr9i, a neoschizomer of smai was optimized and evaluated to investigate st isolates.  after optimizing and evaluating the cfr9i pfge, clear and reproducible banding patterns were obtained from all previously non-typeable mrsa isolates. the pfge patterns of st isolates showed more diversity than with spa-typing and/or mlst. the pfge results showed diversity within and between the two most prevalent spa-types of ntsmai -mrsa. no match was found, when comparing banding patterns of the ntsmai -mrsa with different pfge types, obtained with smai digestion, in our database of more than strains. furthermore, possible transmission among veterinarians and their family members was investigated and an outbreak of st mrsa in a residential care facility was confirmed with the cfr9i pfge.  the adjusted pfge can be used as a method for selecting important and distinct st isolates for further research. the adjustments in the pfge protocol using cfr9i are easy to implement to study the st clonal lineage in laboratories which already have a pfge facility. 