
 isolates - ninety - one gas isolates recovered from oropharynx secretions 
 were included in this study .
clinical specimens were processed during routine diagnoses 
 by one clinical laboratory in rio de janeiro from january 2008-july 2012 .
the subjects 
 ages varied from two-56 years , but 46% of the isolates were recovered from children 
 two-11 years of age and 51.6% were from women . in our laboratory , these isolates were 
 cultured on blood agar plates ( difco laboratories , usa ) and submitted to conventional 
 tests ( pyr test , bacitracin susceptibility and streptococcal serogrouping ) to confirm 
 species identification . 

antimicrobial susceptibility testing - all isolates were submitted to 
 susceptibility tests to ceftriaxone ( 30 g ) , clindamycin ( 2 g ) , erythromycin ( 15 g ) , 
 levofloxacin ( 5 g ) , penicillin ( 10 u ) , tetracycline ( 30 g ) and vancomycin ( 30 g ) 
 ( cecon , brazil ) using the disk diffusion method on mueller - hinton blood agar ( difco ) 
 according to clsi guidelines ( 2013 ) .
macrolide resistance phenotypes were determined by 
 the double disk test using erythromycin ( 15 g ) and clindamycin ( 2 g ) disks placed 12 
 mm apart ( clsi 2013 ) .
the erythromycin minimum 
 inhibitory concentration ( mic ) was determined in all resistant and intermediate isolates 
 by the agar dilution method ( clsi 2009 ) . 

investigation of erythromycin resistance - associated genes - dna 
 preparation was performed as previously described ( dmitriev et al .
briefly , suspensions with turbidity 
 adjusted to mcfarland standard 3.0 were prepared in 300 l of 10 mm tris - edta buffer and 
 boiled for 5 min .
the presence of erma , ermb and 
 mefa / e genes was investigated in erythromycin - resistant isolates 
 using specific polymerase chain reaction ( pcr ) protocols ( sutcliffe et al .
cycling was carried in a geneamp 9700 
 thermocycler ( applied biosystems , usa ) .
determination of emm types - emm types were determined 
 by a sequence - based protocol ( cdc.gov/ncidod/biotech/strep/protocol_emm-type.htm ) using 
 bigdye terminator cycle sequencing kit ( applied biosystems ) .
sequences were edited using bioedit 
 software v.7.0 and compared with reference sequences using the blast algorithm 
 ( blast.ncbi.nlm.nih.gov/blast.cgi ) .
sequences that did not match with 100% similarity to 
 any sequence deposited in genbank were submitted to the centers for disease control and 
 disease ( cdc ) emm sequence database 
 ( cdc.gov/ncidod/biotech/strep/strepblast.htm ) for assignment to new emm 
 subtypes . 

analysis of dna restriction patterns by pfge - all 44 isolates 
 belonging to the five prevalent emm types 
 ( emm1 , emm4 , emm12 , 
 emm22 and emm81 ) were ana- lysed by pfge after the dna was 
 digested with smai according to a previous protocol ( teixeira et al .
a 300-l aliquot of 
 bacterial suspension in piv buffer was mixed with an equal volume of low melting point 
 agarose ( promega , usa ) and distributed into plug moulds .
plugs were incubated in 2 ml of 
 lysis solution containing 5 mg / ml lysozyme .
smai digestion was 
 performed according to the manufacturer s recommendations ( invitrogen , usa ) .
dna 
 fragments were separated by the chef - driii system ( bio - rad laboratories , usa ) .
the dice 
 coefficient was calculated by visual analysis and dendrograms based on unweighted pair 
 group method with arithmetic mean were constructed using genomes.urv.cat/upgma . 

statistical analyses - the discriminatory power of emm 
 typing , regarding the overall population as well as erythromycin and tetracycline 
 resistance , was measured using simpson s index of diversity ( sid ) by calculating the 95% 
 confidence intervals ( ci ) ( hunter & gaston 
 1988 , grundmann et al .
conventional tests identified all isolates as s. pyogenes . by the disk diffusion method , 

tetracycline - resistant and intermediate isolates comprised 18.7% and 2.2% of isolates , 
 respectively .
clindamycin resistance was observed in 15.4% of isolates , while 
 erythromycin - resistant and intermediate isolates comprised 14.3% and 1.1% of isolates , 
 respectively .
the erythromycin mic varied from 8 - 256 g / ml and therefore , the resistance 
 rate was 15.4% .
the genetic determinant ermb was 
 predominant and was detected in 78.6% of the erythromycin - resistant isolates , alone or 
 in association with erma ( 64.3% ) .
neither the m phenotype nor the 
 mefa / e gene was observed in this study .
erythromycin mic values , the 
 distribution of macrolide resistance phenotypes and genotypes and emm 
 types of erythromycin - resistant isolates are shown in table . 

tablephenotypic and genotypic characteristics of macrolide resistant 
 isolatesisolate numberyear of recovery 
 emm   typemic ( g / ml)phenotypemacrolide resistance 
 genes 

 erma 

 ermb 

 mefa / e 
 186200868.1 > 256cmlsb 
 -+-274200858 >
256imlsb 
 + --302200858 > 256imlsb 
 + --40220091 > 256cmlsb 
 -+-418200958.148imlsb 
 + --425 
 20093.316cmlsb 
 -+-536201022 > 256cmlsb 
 -+-637a20111 >
256cmlsb 
 + + -638201176.7 > 256cmlsb 
 + + -710201128 > 256cmlsb 
 -+-749201173 >
256cmlsb 
 + + -75020116 > 256cmlsb 
 + + -780201211 > 256cmlsb 
 + + -798201211 > 256imlsb 
 + + -a : this isolate was intermediate by disk diffusion ; cmlsb : 
 constitutive mlsb phenotype ; imlsb : inducible 
 mlsb phenotype ; mic : minimal inhibitory concentration 
 ( susceptible < 0.25 g / ml , intermediate 0.5 g / ml , resistant > 1 
 g / ml ) ; + : presence of the gene ; - : absence of the gene . 
 
: this isolate was intermediate by disk diffusion ; cmlsb : 
 constitutive mlsb phenotype ; imlsb : inducible 
 mlsb phenotype ; mic : minimal inhibitory concentration 
 ( susceptible < 0.25 g / ml , intermediate 0.5 g / ml , resistant > 1 
 g / ml ) ; + : presence of the gene ; - : absence of the gene .
thirty - two emm types or subtypes were identified among 86 of the 91 gas 
 isolates .
the most frequent 
 types were emm1 , emm4 , emm12 , 
 emm22 and emm81 , accounting for 48% of all isolates .
three 
 new sequences were designated by the cdc streptococcus laboratory as 
 new emm subtypes ( emm1.74 , emm58.14 
 and emm76.7 ) and deposited in the emm sequence 
 database ( cdc.gov/pub/infectious_diseases/biotech/tsemm ) and in genbank ( accession 
 km364527-km364529 ) .
these new subtypes differed from their parental types in 1 - 2% of the 
 nucleotide sequence .
the emm typing revealed a high level of diversity 
 among the overall population ( sid = 0.941 ; 95% ci , 0.917 - 0.965 ) .
the frequency of each 
 emm type is shown in fig . 
 1 . 

fig . 1 : distribution of emm types and subtypes among isolates of 
 streptococcus pyogenes recovered from oropharynx secretion . 

fourteen different emm types or subtypes were found among 19 
 tetracycline non - susceptible isolates , while 11 distinct emm types or 
 subtypes were found among 14 erythromycin and clindamycin - resistant isolates .
the sid 
 values calculated for erythromycin - resistant ( 0.967 ; 95% ci , 0.929 - 1.000 ) and 
 tetracycline non - susceptible ( 0.959 ; 95% ci , 0.915 - 1.000 ) isolates were higher than 
 those calculated for erythromycin ( 0.929 ; 95% ci , 0.898 - 0.960 ) and tetracycline ( 0.898 ; 
 95% ci , 0.855 - 0.940 ) susceptible isolates .
pfge analysis of 44 isolates belonging to the five prevalent emm types 
 generated 20 restriction profiles , which are shown in fig .
three profiles , whose similarities varied from 50 - 85% , were observed among 
 emm1 isolates .
the most frequent profile was shared by eight 
 isolates , including one resistant to erythromycin .
regarding emm4 , four 
 profiles were observed among seven isolates , ranging from 85 - 96% similarity . among 
 emm22 and emm81
isolates ( 6 isolates each ) , unique 
 pfge profiles were observed for each isolate , varying from 70 - 95% similarity . 

2 : pulsed - field gel electrophoresis profiles , dendrograms , year of isolation 
 and macrolide resistance features of prevalent emm types 
 isolates . 

in this study , 91 gas isolates recovered from oropharynx secretions of residents of rio 
 de janeiro were submitted to susceptibility testing and typing methodologies . despite 
 the lack of data regarding the clinical conditions of the individuals , these results are 
 relevant because both infected and colonised individuals can transmit this species to 
 susceptible subjects .
isolates were fully susceptible to beta - lactams and glycopeptides , as observed over 
 decades of antibiotic usage .
they were also susceptible to levofloxacin , despite 
 previously detected resistance to this antimicrobial ( smeesters et al .
the tetracycline resistance rate was lower than that observed in a study conducted in 
 brazil one decade ago , in which the authors reported 43.1% resistance ( doliveira et al .
2003 ) . instead , it was very similar 
 to recent data from southern brazil and portugal ( torres 
 et al .
however , 73% resistance to this antimicrobial was reported in a recent study from india 
 ( mathur et al .
these findings may 
 reflect that , while a consistent trend of decreasing tetracycline resistance has been 
 recently observed in brazil , circulating gas isolates from other regions are highly 
 resistant to this antimicrobial .
regarding erythromycin and clindamycin resistance , the rates found here far exceeded 
 those previously reported in the same geographical area ( doliveira et al .
2011 ) , but they are very similar to those described in some recent studies 
 conducted in europe ( fries et al .
2013 ) . while a trend of 
 decreasing erythromycin resistance has been recently observed in spain and taiwan ( huang et al .
inducible and constitutive mlsb phenotypes , which are associated with 
 erma and/or ermb genes , were observed among 
 isolates belonging to a variety of emm types , reflecting the polyclonal 
 origin of such isolates .
it is worth noting that the m phenotype and the 
 mefa / e genotype , associated with emm12 , were common 
 in brazil before the year 2000 ( torres et al . 

2011 ) , but they have not been detected since that time , either by those 
 authors or in this study .
in contrast , mlsb emerged after the 2000s and is 
 linked to a variety of emm types , such as emm11 , 
 emm22 , emm28 and emm73 ( torres et al .
in contrast to this local 
 replacement of the m phenotype with the cmlsb phenotype , the m phenotype has 
 been prevalent over 12 years of survey ( 1998 - 2010 ) in taiwan ( huang et al .
2014 ) . however , the authors observed the replacement of 
 emm12 with emm22 as the prevalent type associated 
 with macrolide resistance , as well as a decreasing rate in macrolide resistance from 
 53.1% before 2000 to 10.7% from 2006 - 2010 .
these variations demonstrate how dynamic the 
 bacterial population is worldwide and also highlight the need for changing therapeutic 
 recommendations in geographical areas where mlsb phenotypes predominate . a wide variety of emm types , including new alleles ,
the diversity of emm 
 types and the detection of new alleles from gas isolates recovered from oropharynx 
 secretions have been described in previous studies from brazil ( teixeira et al .
these findings reflect the genetic heterogeneity of such isolates and 
 contribute to the expansion of the emm type database .
the most frequent 
 emm types found here are among the prevalent types in high - income 
 countries , where the majority of studies have been performed ( steer et al .
. however , they differ significantly from those types observed in recent 
 studies from india and the pacific region ( baroux et al . 
 2014 , mathur et al . 2014 ) .
due to the 
 limited amount of data from latin america , our results contribute to a better 
 understanding of emm type distribution in a poorly studied area .
this 
 issue is particularly important considering that m protein - based vaccines are under 
 development ( dale et al .
2013 ) . when comparing the 
 two most promising 26 and 30-valent m vaccines , 49% and 64% of our isolates , 
 respectively ,
the 
 potentially low impact of these vaccines can be explained by the fact that the m protein 
 fragments for the 26-valent m vaccine were selected based on emm type 
 distribution in north america ( steer et al . 

there was strong agreement between emm type and pfge clustering , as 
 previously observed ( torres et al .
no 
 identical pfge profile was observed among isolates belonging to distinct 
 emm types , which could be related to the horizontal transfer of 
 emm genes ( whatmore et al . 

regarding erythromycin - resistant isolates , a great diversity of pfge 
 profiles and emm types were observed , illustrating that this 
 characteristic is not due to the expansion of a specific clone
. moreover , both 
 erythromycin - susceptible and erythromycin - resistant emm1 isolates 
 shared a single pfge profile .
this finding suggests that the emm type , 
 not erythromycin resistance , is more likely to be a determinant of clonality . in conclusion
, susceptibility testing and epidemiological typing techniques revealed an 
 incidence of macrolide resistance not yet observed in this area and a great diversity of 
 emm types , including new alleles , among gas isolates circulating in 
 brazil .
these data contribute to the improvement of prevention and treatment strategies 
 of gas infections .
 
 streptococcus pyogenes is responsible for a variety of infectious 
 diseases and immunological complications . in this study , 
 91 isolates of s. 
 pyogenes recovered from oropharynx secretions were submitted to 
 antimicrobial susceptibility testing , emm typing and pulsed - field 
 gel electrophoresis ( pfge ) analysis . 
 all isolates were susceptible to ceftriaxone , 
 levofloxacin , penicillin g and vancomycin . 
 resistance to erythromycin and clindamycin 
 was 15.4% , which is higher than previous reports from this area , while 20.9% of the 
 isolates were not susceptible to tetracycline . 
 the macrolide resistance phenotypes 
 were cmlsb ( 10 ) and imlsb ( 4 ) . 
 the ermb gene was predominant , 
 followed by the erma gene . 
 thirty - two emm types and 
 subtypes were found , but five ( emm1 , emm4 , 
 emm12 , emm22 , emm81 ) were detected in 
 48% of the isolates . 
 three new emm subtypes were identified 
 ( emm1.74 , emm58.14 , emm76.7 ) . 
 there was a strong association between emm type and pfge clustering . 
 
 a variety of pfge profiles as well as emm types were found among 
 tetracycline and erythromycin - resistant isolates , demonstrating that antimicrobial 
 resistant strains do not result from the expansion of one or a few clones . 
 this study 
 provides epidemiological data that contribute to the development of suitable 
 strategies for the prevention and treatment of such infections in a poorly studied 
 area . 
SUBJECTS, MATERIALS AND METHODS
RESULTS
DISCUSSION