for the protekt us study , isolates of s. pneumoniae were collected from across the united states from 2000 to 2003 . the numbers of collection centers that provided samples were 207 in year 1 ( 20002001 ) , 241 in year 2 ( 20012002 ) , and 247 in year 3 ( 20022003 ) . pathogenic respiratory tract isolates of s. pneumoniae were collected from adult and pediatric outpatients with community - acquired respiratory tract infections ( acute otitis media , pneumonia , acute exacerbations of chronic bronchitis , acute exacerbations of chronic obstructive pulmonary disease , and sinusitis ) . also included were isolates cultured from material collected from hospitalized patients within 48 hours of admission . the following sources were considered acceptable : cultures from blood , sputum , bronchoalveolar lavage , middle - ear fluid ( collected by tympanocentesis ) , nasopharyngeal swab or aspirate , and sinus aspirate . duplicate strains , or strains originating from existing collections , were also not included in the study . demographic data collected included the age and sex of the patient , infection , culture source , inpatient versus outpatient status , specimen accession number , and date of sample collection . details of the methods for isolate storage , transportation , and identification have been reported previously ( 23 ) . mics were determined at a central laboratory ( cmi , portland , or , usa ) by using the clinical and laboratory standards institute ( clsi ) broth microdilution method ( 24 ) . the following antibacterial agents were tested : amoxicillin - clavulanate ( amoxicillin alone was not tested ; however , susceptibility can be extrapolated from the amoxicillin - clavulanate results ) , azithromycin , cefuroxime , clarithromycin , clindamycin , co - trimoxazole , erythromycin , levofloxacin , linezolid , penicillin , telithromycin , and tetracycline . in all cases , susceptibility to telithromycin was determined by using the clsi breakpoints ( 25 ) : susceptible 1 g / ml ; intermediate 2 g / ml ; resistant 4 g / ml . all erythromycin - resistant ( mic 1 g / ml ) pneumococcal isolates collected from protekt us years 13 were analyzed for the presence of erm(b ) , erm(a ) subclass erm(tr ) , and mef(a ) macrolide resistance genes . isolates in year 1 were analyzed by multiplex rapid - cycle polymerase chain reaction ( pcr ) with microwell - format probe hybridization , as described previously ( 26 ) . in years 2 and 3 , isolates were analyzed by using a multiplex taqman ( applied biosystems , foster city , ca , usa ) pcr assay that was validated against the previous pcr method ( 27 ) . a proportion of dual erm(b)+mef(a ) macrolide - resistant isolates underwent serotyping and multilocus sequence type ( mlst ) determination at g.r . micro ltd ( london , uk ) . isolates were serotyped by using antisera from the statens serum institute ( ssi , copenhagen , denmark ) . serotyping and mlst determination were also conducted on 366/378 dual erm(b)+mef(a ) macrolide - resistant s. pneumoniae isolates , respectively , collected from the global protekt study ( 19992003 ) . sequence type ( st ) and alleles were analyzed by upgma ( unweighted pair group method with arithmetic mean ) and burst ( based upon related sts ) analysis by using the start program ( version 1.0.5 ) to assign lineage and clonal complexes . from 2000 to 2003 , a total of 31,001 s. pneumoniae isolates were collected as part of the protekt us study : 10,103 in year 1 , 10,012 in year 2 , and 10,886 in year 3 . the proportion of s. pneumoniae isolates resistant to erythromycin was similar across years 1 , 2 , and 3 of the protekt us study ( 29.4% overall ) . the prevalence of mef(a ) in macrolide - resistant isolates decreased from 68.8% in year 1 to 67.3% in year 2 and to 63.9% in year 3 , while the prevalence of erm(b ) alone appeared stable ( 16.9% in year 1 , 16.5% in year 2 , 16.5% in year 3 ) . by contrast , an increase was seen in the prevalence of macrolide - resistant strains carrying both erm(b ) and mef(a ) genes ; by year 3 , 16.4% of isolates were of this genotype ( table 1 ) . when considered as a proportion of all s. pneumoniae isolates collected in year 3 , a total of 520 ( 4.8% ) of 10,886 were positive for both erm(b ) and mef(a ) . * north - central = illinois , iowa , kansas , minnesota , missouri , nebraska , north dakota , south dakota , wisconsin ; northeast = connecticut , delaware , indiana , maryland , massachusetts , michigan , new jersey , new york , ohio , pennsylvania , rhode island , vermont , washington dc ; northwest = alaska , idaho , montana , oregon , washington , wyoming ; south - central = alabama , arkansas , louisiana , oklahoma , tennessee , texas ; southeast = florida , georgia , kentucky , north carolina , puerto rico , south carolina , virginia , west virginia ; southwest = arizona , california , colorado , nevada , new mexico , utah . geographic differences were observed in the prevalence of erm(b)+mef(a)encoded resistance across the united states , from 10.3% in the southeast to 23.9% in the north - central region ( year 3 ) . the prevalence of this genotype increased in all regions between years 1 and 3 ( table 1 ) . the largest increases in erm(b)+mef(a)-encoded resistance during the 3-year study period occurred in isolates collected from pediatric patients ( table 2 ) . by year 3 , isolates exhibiting this genotype made up 254 ( 22.7% ) of 1,119 isolates obtained from pediatric patients ( 14 years of age ) compared with 98 ( 12.3% ) of 794 isolates collected from patients > 64 years of age . patients in the 0- to 2-year age group had the highest prevalence ( 23.9% ) of dual erm(b)+mef(a ) resistance ( table 2 ) . across the 3-year study period , the dual erm(b)+mef(a ) genotype was found most frequently in isolates collected from the ear or middle - ear fluid ( table 3 ) . in year 3 , the prevalence of this form of macrolide resistance was > 30% in isolates collected from either of these sources . by contrast , isolates cultured from blood samples had the lowest proportion of dual erm(b)+mef(a)-encoded resistance ( 92 [ 4.6% ] of 2,014 isolates in the 3 years ) . * bal , bronchoalveolar lavage ; csf , cerebrospinal fluid ; mef , middle - ear fluid ; nap , nasopharyngeal swab or aspirate ;  , no isolates collected . in addition to exhibiting almost universal resistance to the macrolides tested ( azithromycin , clarithromycin , erythromycin ) , isolates carrying both erm(b ) and mef(a ) were highly resistant ( > 90% ) to penicillin , cefuroxime , tetracycline , and co - trimoxazole ( table 4 ) . resistance to amoxicillin - clavulanate ( and hence amoxicillin ) was also common in these isolates ( table 4 ) , and the longitudinal data showed that the rate of resistance to this antibacterial drug increased from 29.9% to 43.9% from year 1 to year 3 . almost all ( 1,150 [ 99.2% ] of 1,159 ) of the erm(b)+mef(a ) isolates were multidrug - resistant ( i.e. , resistant to 2 classes of antibacterial drugs ) . * susceptibility was defined according to clinical and laboratory standards institute interpretive criteria ( 25 ) . amoxicillin alone was not tested ; however , susceptibility can be extrapolated from the amoxicillin - clavulanate results . a total of 16 ( 1.4% ) of the 1,159 dual erm(b)+mef(a ) isolates collected were resistant to levofloxacin ; mic values for these were as follows : 8 g / ml ( 2 isolates ) , 16 g / ml ( 9 isolates ) , 32 g / ml ( 4 isolates ) , and 128 g / ml ( 1 isolate ) . one dual erm(b)+mef(a ) isolate ( < 0.1% of the total ) was resistant to telithromycin ( mic 4 g / ml ) . the results of mlst determination on 518 s. pneumoniae isolates ( 366 from protekt global [ including 35 from the united states ] and 152 from protekt us ) with dual erm(b)+mef(a)encoded resistance showed 82 st variants ( figure ) . of these , 21 were in the mlst database , and 61 were submitted to the database and assigned a new st ( sts 14071467 ) . all of the unique sts were serotyped and , together with the 20 s. pneumoniae clones listed by the pneumococcal molecular epidemiology network ( 29 ) , were analyzed for clonal relatedness by using upgma and burst ( figure ) . both the serotype distribution and range of mlsts in these isolates were limited ( table 5 ) , with 3 clonal complexes predominating . a phylogenetic analysis of these variations showed that 45 of the 82 sts were closely related , either serotype 19f or 19a ( table 5 ) . these strains were of ancestral st 271 and hence were designated clonal complex ( cc ) 271 , which is equivalent to cc i ( 15 ) and cc 236 ( 22 ) . overall , 305 ( 83.3% ) of 366 global isolates had mlst profiles consistent with this clone ( table 6 ) . phylogenetic relationships of the 82 different sequence type variations found in 518 streptococcus pneumoniae isolates with combined erm(b)- and mef(a)-mediated macrolide resistance collected during the protekt global study ( 19992003 , n = 366 ) and the protekt us study ( 20002003 , n = 152 ) compared with the 20 pmen ( pneumococcal molecular epidemiology network ) clones . sequence type 236 was previously described as cc 236 by ko and song ( 22 ) but is described as cc 271 in this study based on burst analysis . * no clonal lineage found except for 2 isolates ( st 1467 ) and 1 isolate ( st 1452 ) , which were clonally related ( cc 1467 ) . 8 isolates were clonally related ( st 1426 ) . of the 35 isolates collected in the united states from the protekt global study , all exhibited mlst profiles and serotypes characteristic of cc 271 ( table 6 ) . moreover , analysis of a geographically and chronologically varied sample of 152 s. pneumoniae isolates with dual erm(b)+mef(a)encoded resistance collected from the protekt us study suggested that > 90% of dual - resistant isolates in the united states belong to cc 271 . from 2000 to 2003 , a total of 31,001 s. pneumoniae isolates were collected as part of the protekt us study : 10,103 in year 1 , 10,012 in year 2 , and 10,886 in year 3 . the proportion of s. pneumoniae isolates resistant to erythromycin was similar across years 1 , 2 , and 3 of the protekt us study ( 29.4% overall ) . the prevalence of mef(a ) in macrolide - resistant isolates decreased from 68.8% in year 1 to 67.3% in year 2 and to 63.9% in year 3 , while the prevalence of erm(b ) alone appeared stable ( 16.9% in year 1 , 16.5% in year 2 , 16.5% in year 3 ) . by contrast , an increase was seen in the prevalence of macrolide - resistant strains carrying both erm(b ) and mef(a ) genes ; by year 3 , 16.4% of isolates were of this genotype ( table 1 ) . when considered as a proportion of all s. pneumoniae isolates collected in year 3 , a total of 520 ( 4.8% ) of 10,886 were positive for both erm(b ) and mef(a ) . * north - central = illinois , iowa , kansas , minnesota , missouri , nebraska , north dakota , south dakota , wisconsin ; northeast = connecticut , delaware , indiana , maryland , massachusetts , michigan , new jersey , new york , ohio , pennsylvania , rhode island , vermont , washington dc ; northwest = alaska , idaho , montana , oregon , washington , wyoming ; south - central = alabama , arkansas , louisiana , oklahoma , tennessee , texas ; southeast = florida , georgia , kentucky , north carolina , puerto rico , south carolina , virginia , west virginia ; southwest = arizona , california , colorado , nevada , new mexico , utah . geographic differences were observed in the prevalence of erm(b)+mef(a)encoded resistance across the united states , from 10.3% in the southeast to 23.9% in the north - central region ( year 3 ) . the prevalence of this genotype increased in all regions between years 1 and 3 ( table 1 ) . the largest increases in erm(b)+mef(a)-encoded resistance during the 3-year study period occurred in isolates collected from pediatric patients ( table 2 ) . by year 3 , isolates exhibiting this genotype made up 254 ( 22.7% ) of 1,119 isolates obtained from pediatric patients ( 14 years of age ) compared with 98 ( 12.3% ) of 794 isolates collected from patients > 64 years of age . patients in the 0- to 2-year age group had the highest prevalence ( 23.9% ) of dual erm(b)+mef(a ) resistance ( table 2 ) . across the 3-year study period , the dual erm(b)+mef(a ) genotype was found most frequently in isolates collected from the ear or middle - ear fluid ( table 3 ) . in year 3 , the prevalence of this form of macrolide resistance was > 30% in isolates collected from either of these sources . by contrast , isolates cultured from blood samples had the lowest proportion of dual erm(b)+mef(a)-encoded resistance ( 92 [ 4.6% ] of 2,014 isolates in the 3 years ) . * bal , bronchoalveolar lavage ; csf , cerebrospinal fluid ; mef , middle - ear fluid ; nap , nasopharyngeal swab or aspirate ;  , no isolates collected . in addition to exhibiting almost universal resistance to the macrolides tested ( azithromycin , clarithromycin , erythromycin ) , isolates carrying both erm(b ) and mef(a ) were highly resistant ( > 90% ) to penicillin , cefuroxime , tetracycline , and co - trimoxazole ( table 4 ) . resistance to amoxicillin - clavulanate ( and hence amoxicillin ) was also common in these isolates ( table 4 ) , and the longitudinal data showed that the rate of resistance to this antibacterial drug increased from 29.9% to 43.9% from year 1 to year 3 . almost all ( 1,150 [ 99.2% ] of 1,159 ) of the erm(b)+mef(a ) isolates were multidrug - resistant ( i.e. , resistant to 2 classes of antibacterial drugs ) . * susceptibility was defined according to clinical and laboratory standards institute interpretive criteria ( 25 ) . amoxicillin alone was not tested ; however , susceptibility can be extrapolated from the amoxicillin - clavulanate results . a total of 16 ( 1.4% ) of the 1,159 dual erm(b)+mef(a ) isolates collected were resistant to levofloxacin ; mic values for these were as follows : 8 g / ml ( 2 isolates ) , 16 g / ml ( 9 isolates ) , 32 g / ml ( 4 isolates ) , and 128 g / ml ( 1 isolate ) . one dual erm(b)+mef(a ) isolate ( < 0.1% of the total ) was resistant to telithromycin ( mic 4 g / ml ) . the results of mlst determination on 518 s. pneumoniae isolates ( 366 from protekt global [ including 35 from the united states ] and 152 from protekt us ) with dual erm(b)+mef(a)encoded resistance showed 82 st variants ( figure ) . of these , 21 were in the mlst database , and 61 were submitted to the database and assigned a new st ( sts 14071467 ) . all of the unique sts were serotyped and , together with the 20 s. pneumoniae clones listed by the pneumococcal molecular epidemiology network ( 29 ) , were analyzed for clonal relatedness by using upgma and burst ( figure ) . both the serotype distribution and range of mlsts in these isolates were limited ( table 5 ) , with 3 clonal complexes predominating . a phylogenetic analysis of these variations showed that 45 of the 82 sts were closely related , either serotype 19f or 19a ( table 5 ) . these strains were of ancestral st 271 and hence were designated clonal complex ( cc ) 271 , which is equivalent to cc i ( 15 ) and cc 236 ( 22 ) . overall , 305 ( 83.3% ) of 366 global isolates had mlst profiles consistent with this clone ( table 6 ) . phylogenetic relationships of the 82 different sequence type variations found in 518 streptococcus pneumoniae isolates with combined erm(b)- and mef(a)-mediated macrolide resistance collected during the protekt global study ( 19992003 , n = 366 ) and the protekt us study ( 20002003 , n = 152 ) compared with the 20 pmen ( pneumococcal molecular epidemiology network ) clones . * pmen , pneumococcal molecular epidemiology network ; nt , nontypeable . sequence type 236 was previously described as cc 236 by ko and song ( 22 ) but is described as cc 271 in this study based on burst analysis . * no clonal lineage found except for 2 isolates ( st 1467 ) and 1 isolate ( st 1452 ) , which were clonally related ( cc 1467 ) . of the 35 isolates collected in the united states from the protekt global study , all exhibited mlst profiles and serotypes characteristic of cc 271 ( table 6 ) . moreover , analysis of a geographically and chronologically varied sample of 152 s. pneumoniae isolates with dual erm(b)+mef(a)encoded resistance collected from the protekt us study suggested that > 90% of dual - resistant isolates in the united states belong to cc 271 . pneumococcal macrolide resistance in the united states is predominantly mediated by the mef(a ) gene , which encodes for lower - level , efflux - mediated resistance ( 14 ) . however , the latest surveillance data from protekt us presented in this article show that the prevalence of this form of resistance is decreasing . this trend coincides with the emergence of multidrug - resistant clones of s. pneumoniae that express both erm(b ) and mef(a ) . these strains increased in prevalence from 9.7% of macrolide - resistant isolates in 20002001 to 16.4% in 20022003 . moreover , geographic data indicate that dual erm(b)+mef(a ) isolates are currently even more prevalent in some regions of the united states ( accounting for > 20% of macrolide - resistant strains ) . by 20022003 , s. pneumoniae strains with this dual mechanism of resistance made up almost 5% of all isolates collected . the major clinical implication of the present report is the increased potential for treatment failure with most antibacterial drugs currently recommended to empirically treat community - acquired respiratory tract infections ( 30,31 ) . ear isolates are more prone to represent treatment failure , and blood isolates represent primary infection ; thus , the dramatic increase in cc 271 in ear isolates compared to blood isolates ( table 2 ) is noteworthy . almost all dual erm(b)+mef(a ) isolates were highly resistant to multiple antibacterial drugs , including penicillin , macrolides , tetracycline , and co - trimoxazole . . furthermore , resistance to amoxicillin - clavulanate ( and hence amoxicillin ) increased in these isolates from 29.9% to 43.9% during the 3-year surveillance period , which raises concerns about the potential selection of resistant isolates through widespread use of this agent for community - acquired infections , particularly acute otitis media . the prevalence of resistance to fluoroquinolones , such as levofloxacin , was low ( 1.4% overall ) in the dual erm(b)+mef(a ) isolates ; however , when present , this resistance was often high ( mic 8128 g / ml ) . telithromycin resistance was rare ( < 0.1% ) in s. pneumoniae isolates with dual erm(b)+mef(a)encoded macrolide resistance . previous studies have indicated that a small number of clonal groups account for most penicillin- , macrolide- , and multidrug - resistant s. pneumoniae in the united states ( 18,32 ) . the mlst analysis conducted in the present study shows that the dual erm(b)+mef(a ) macrolide - resistant s. pneumoniae isolates collected in the united states from 1999 to 2003 are associated with 3 major global clones , in addition to a wide variety of other mlst variations . most of these isolates belong to 1 major clonal group ; the genotypic profile and serotype distribution of this predominant group show that it is highly related to an international erm(b)+mef(a ) clonal strain , taiwan-14 , first found in the far east ( 22 ) . the designation of clonal groups is determined by burst analysis , which assigns ancestral lineage by the most common st . for this reason , the pneumococcal clone designated cc 271 in the present study was named cc 236 in the study by ko and song ( 22 ) . to avoid confusion , a common cc nomenclature ( such as the original designation of the clone , cc the pneumococcal clone discussed in this paper was previously identified in the first year ( 19992000 ) of the protekt global study ( 15 ) . the most recent data from this survey , which covered the period 19992003 , confirm that this clone now has a worldwide distribution , with particularly high incidences in south africa and south korea , as reported in previous studies ( 15,19,21 ) . strains carrying both genes have also been recorded recently in new zealand ( 33 ) , canada ( 34 ) , italy ( 35 ) , and scotland ( 36 ) . together with the regional genotyping data , the epidemiologic analyses we describe show that this multidrug - resistant cc 271 is now widespread and increasing in prevalence across the united states . the widespread emergence of the erm(b)+mef(a ) genotype into varying lineages at the apparent expense of strains expressing only 1 resistance determinant suggests that s. pneumoniae carrying this form of resistance has an evolutionary advantage . since dual resistant isolates have drug mics similar to those observed in strains harboring erm(b ) alone , such an advantage can not be explained on the basis of increased macrolide resistance alone . this clone has previously been shown to contain 2 mobile genetic elements , tn1545 and " mega " ( 15 ) . while the erm(b ) gene is most often present on tn1545 , " mega " is known to contain the mef(e ) variant of mef(a ) , and this variant has been shown to be present in cc 271 ( 15 ) . hence , acquisition of 2 mobile genetic elements and associated resistance genes is a possible explanation for the successful emergence of this clone over isolates with only erm(b ) or mef(a ) ; it is not solely the acquisition of the efflux or methylase gene but the associated resistance genes on the genetic elements that lead to a multidrug - resistant clone in which prevalence is driven by greater environmental pressures . of particular concern is the finding that dual erm(b)+mef(a)encoded resistance was most prevalent in isolates collected from pediatric patients . by year 3 of the study , 8.7% of all s. pneumoniae isolates collected from children 14 years of age and 10.7% of those collected from children 2 years of age exhibited this form of macrolide resistance . the introduction of the 7-valent pneumococcal vaccine ( pcv7 ) in 2000 was aimed primarily at reducing the incidence of disease in this vulnerable group . while recent evidence suggests that this reduction has occurred ( 37,38 ) , the vaccine does not provide coverage against all s. pneumoniae serotypes . as discussed above , most dual erm(b)+mef(a ) isolates characterized in this study are of serotype 19a ( the prevalence of which increased from years 1 to 3 ) or 19f . although serotype 19f is represented in the pcv7 vaccine , it affords low levels of protection against upper respiratory infections such as otitis media ( 39 ) and has been shown recently to be the least immunogenic of the vaccine serotypes ( 40 ) . the trends reported in this article indicate that the introduction of routine immunization has not prevented the spread of this nonvaccine serotype multidrug - resistant clone in the pediatric population and may have contributed to the selection of serotype 19a strains . in summary , although pneumococcal macrolide resistance rates appear to have stabilized in the united states , prevalence of clonal isolates with the combined erm(b)+mef(a ) genotype is increasing . these strains show high - level macrolide and multidrug resistance , and their spread across the united states represents a serious public health concern . these findings also highlight the critical need for continued monitoring of pneumococcal resistance patterns over time , in particular , the spread of these multidrug - resistant clones , and for physicians to be aware of local or regional resistance patterns when selecting empiric antibacterial treatment for community - acquired respiratory tract infections .
streptococcus pneumoniae isolates ( n = 31,001 ) were collected from patients with community - acquired respiratory tract infections during the protekt us surveillance study ( 20002003 ) . while the macrolide ( erythromycin ) resistance rate remained stable at 29% , the prevalence of resistant isolates containing both erm(b ) and mef(a ) 
 increased from 9.7% in year 1 to 16.4% in year 3 , with substantial regional variability . 
 almost all ( 99.2% ) dual erm(b)+mef(a ) macrolide - resistant isolates exhibited multidrug resistance , whereas 98.6% and 99.0% were levofloxacin- and telithromycin - susceptible , respectively . 
 these strains were most commonly isolated from the ear or middle - ear fluid of children . 
 of 152 representative erm(b)+mef(a ) isolates , > 90% were clonally related to the multidrug - resistant international taiwan19f-14 clonal complex 271 ( cc271 ) . of 366 erm(b)+mef(a ) isolates from the protekt global study ( 19992003 ) , 83.3% were cc271 , with the highest prevalence seen in south africa , south korea , and the united states . 
 this study confirms the increasing global emergence and rapidly increasing us prevalence of this multidrug - resistant pneumococcal clone .