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Molecular characterization of macrolide resistant Streptococcus pyogenes isolates from pharyngitis patients in Serbia
Infection, Genetics and Evolution xxx (2015) xxx–xxx
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Infection, Genetics and Evolution journal homepage: www.elsevier.com/locate/meegid
Molecular characterization of macrolide resistant Streptococcus pyogenes isolates from pharyngitis patients in Serbia Natasa Opavski a,b,⇑, Ina Gajic a,b, Anna L. Borek c,1, Katarzyna Obszan´ska c,2, Maja Stanojevic b, Ivana Lazarevic b, Lazar Ranin a,b, Izabela Sitkiewicz c, Vera Mijac a,b a b c
National Reference Laboratory for Streptococci, Medical Faculty, University of Belgrade, Dr Subotica starijeg 1, 11000 Belgrade, Serbia Institute of Microbiology and Immunology, Medical Faculty, University of Belgrade, Dr Subotica starijeg 1, 11000 Belgrade, Serbia Department of Epidemiology and Clinical Microbiology, National Medicines Institute, Chełmska 30/34, 00-750 Warszawa, Poland
a r t i c l e
i n f o
Article history: Received 14 January 2015 Received in revised form 17 April 2015 Accepted 11 May 2015 Available online xxxx Keywords: Streptococcus pyogenes Macrolide resistance emm MLST MLVA
a b s t r a c t A steady increase in macrolide resistance in Streptococcus pyogenes, group A streptococci (GAS) was reported in Serbia during 2004–2009 (9.9%). However, there are no data on the molecular epidemiology of pharyngeal macrolide resistance GAS (MRGAS) isolates. Therefore, the aims of this first nationwide study were to examine the prevalence of macrolide resistance in Serbian GAS and to determine their resistance phenotypes, genotypes and clonal relationships. Overall 3893 non-duplicate pharyngeal S. pyogenes isolates from outpatients with GAS infection were collected throughout country during 2008 and 2009. Among 486 macrolide resistant pharyngeal isolates collected, 103 were further characterized. Macrolide resistance phenotypes and genotypes were determined by double-disk diffusion test and PCR, respectively. Strain relatedness was determined by emm typing, multilocus sequence typing (MLST), multilocus variable tandem repeat analysis (MLVA), phage profiling (PP) and virulence factor profiling (VFP). Overall, macrolide resistance among GAS isolates in Serbia was 12.5%. M phenotype was the most common (71.8%), followed by iMLS (18.4%) and cMLS (9.7%). Three clonal complexes – emm75/mefA/ST49, emm12/mefA/ST36 and emm77/ermA/tetO/ST63 comprised over 90% of the tested strains. Although MLVA, PP and VFP distinguished 10, 20 and 12 different patterns, respectively, cluster analysis disclosed only small differences between strains which belonged to the same emm/ST type. Our data indicate dominance of three major internationally widely disseminated macrolide resistant clones and a high genetic homogeneity among the Serbian MRGAS population. Continued surveillance of macrolide resistance and clonal composition in MRGAS in Serbia in future is necessary to determine stability of MRGAS clones and to guide therapy strategies. Ó 2015 Elsevier B.V. All rights reserved.
1. Introduction Streptococcus pyogenes (group A Streptococcus, GAS) is an important human pathogen that causes a wide variety of diseases (Walker et al., 2014). Penicillin is still the drug of choice for GAS ⇑ Corresponding author at: Institute of Microbiology and Immunology, Faculty of Medicine, University of Belgrade, Dr Subotica 1, 11000 Belgrade, Serbia. E-mail addresses: [email protected], [email protected] (N. Opavski), [email protected] (I. Gajic), [email protected] (A.L. Borek), [email protected] (K. Obszan´ska), [email protected] (M. Stanojevic), [email protected] (I. Lazarevic), [email protected] (L. Ranin), iza.sitkiewicz@ gmail.com (I. Sitkiewicz), [email protected] (V. Mijac). 1 Current address: Department of Microbiology, National Research Institute of Tuberculosis and Lung Diseases, Płocka 26, 01-138 Warszawa, Poland. 2 Current address: Department of Microbial Biochemistry, Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Pawin´skiego 5a, 01-106 Warszawa, Poland.
infections, whereas macrolides, such as erythromycin, are recommended as a first alternative therapy for patients who are allergic to penicillin. Since the 1990s a significant increase of erythromycin resistance was observed among S. pyogenes isolates worldwide (Ardanuy et al., 2010; Cornaglia et al., 1996). However, in recent years a steady decline in macrolide resistance was recognized (Silva-Costa et al., 2008; Van Heirstraeten et al., 2012). There are two major mechanisms of macrolide resistance in S. pyogenes: (i) target modification due to methylation of the ribosomal RNA (encoded by ermA or ermB genes) which results in co-resistance to macrolides, lincosamides and streptogramins (MLS phenotype) and (ii) efflux of antibiotics (encoded by mefA gene) that affects 14- and 15-membered macrolides only and gives rise to the so-called M phenotype of resistance (Leclercq, 2002). The MLS phenotype could be further subdivided into inducible (iMLS) and constitutive (cMLS).
http://dx.doi.org/10.1016/j.meegid.2015.05.011 1567-1348/Ó 2015 Elsevier B.V. All rights reserved.
Please cite this article in press as: Opavski, N., et al. Molecular characterization of macrolide resistant Streptococcus pyogenes isolates from pharyngitis patients in Serbia. Infect. Genet. Evol. (2015), http://dx.doi.org/10.1016/j.meegid.2015.05.011
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N. Opavski et al. / Infection, Genetics and Evolution xxx (2015) xxx–xxx
M protein gene (emm) sequence typing is the most widely used method for epidemiological classification of GAS strains (Beall et al., 1996). The correlation between macrolide resistance and emm types has been noted worldwide (Michos et al., 2009; Silva-Costa et al., 2008). However, for the precise detection of the clonal relationships between the macrolide-resistant strains, additional methods, such as pulse field gel electrophoresis (PFGE) or multilocus sequence typing (MLST) should be used (Carrico et al., 2006). Beside these techniques there are several other approaches for GAS typing – multiple-locus variable number tandem repeat analysis (MLVA) (Obszanska et al., 2011) and recently designed phage profiling (PP) and virulence factor profiling (VFP) (Borek et al., 2011). The incidence of macrolide resistance and the distribution of resistance phenotypes and genotypes are significantly variable in different parts of the world (Green et al., 2006; Silva-Costa et al., 2008; Van Heirstraeten et al., 2012). In Serbia macrolide consumption has substantially increased during 2000s and a significant rise in macrolide resistance was observed in S. pyogenes pediatric isolates (9.9%) (Mijac et al., 2014). However, there are no data on the molecular epidemiology of pharyngeal macrolide resistant GAS (MRGAS). The aims of this study were to examine the prevalence of macrolide resistance in Serbian pharyngeal isolates of S. pyogenes and to investigate phenotypic and genotypic characteristics and genetic similarity of MRGAS isolates.
The emm types of MRGAS isolates were determined according to the recommendations of Centers for Disease Control and Prevention http://www.cdc.gov/streplab/protocol-emm-type.html. MLST was carried out according to Enright et al. (2001). Allele numbers and the sequence type were assigned using the MLST website http://spyogenes.mlst.net. Multilocus variable tandem repeat analysis (MLVA) was performed using the procedure described by Obszanska et al. (2011). Virulence factor profiling (VFP) and phage profiling (PP) was done using the set of multiplex PCR reactions which detect simultaneously 20 GAS virulence factors (spd3, sdc, sdaB, sdaD, speB, spyCEP, scpA, mac, sic, speL, speK, speM, speC, speI, speA, speH, speG, speJ, smeZ and ssa) and 21 phage end integrative and conjugative element (ICE) integration sites as previously described (Borek et al., 2011). The results of MLVA, VFP and PP were analyzed using BioNumerics software (Applied Maths). Similarity coefficient between strains was calculated using ranked Pearson correlation. MLVA based clusters were assessed using UPGMA method. 2.4. Cluster typing Cluster designation was done by a typing system recently developed by Sanderson-Smith et al. (2014). It is based on emm typing results and distinguishes 48 GAS clusters containing closely related M proteins.
2. Materials and methods 2.5. Statistics 2.1. Strain collection and identification Serbia is Southeastern European country with roughly 7 million inhabitants. It could be arbitrary divided into three regions: Belgrade (the capital, 2 million), Central Serbia (3 million) and Vojvodina (2 million). During 2008 and 2009, 5 laboratories from Belgrade and 6 laboratories located throughout Vojvodina and Central Serbia collected 3893 non-duplicate pharyngeal S. pyogenes isolates from outpatients with GAS infection. Among those strains, MRGAS isolates were identified and sent into the National Reference Laboratory. One hundred and three (21.2% of all MRGAS) were randomly chosen for further characterization: 25 out of 123 (20.3%) from Belgrade; 29 out of 139 (20.9%) from Vojvodina and 49 out of 224 (21.9%) from the Central Serbia. The majority of these isolates (89%) originated from school age children. Isolates were identified by slide agglutination with group A antisera (Slidex Streptokit; bioMerieux, France), susceptibility to bacitracin and positive PYR test (Rosco, Denmark). 2.2. Antimicrobial susceptibility testing Antimicrobial susceptibility was tested using a disk diffusion test for penicillin (10 IU), erythromycin (15 lg), clindamycin (2 lg), tetracycline (30 lg), ofloxacin (5 lg) and chloramphenicol (30 lg) (BioRad, USA), in accordance with the Clinical and Laboratory Standard Institute guidelines (CLSI) guidelines (2012). Macrolide resistance phenotypes were determined by the double-disk diffusion test (CLSI, 2012). MICs of erythromycin and clindamycin were determined by E test (bioMerieux, France). Streptococcus pneumoniae ATCC 49619 was used as a control strain. 2.3. Antibiotic resistance gene detection, emm typing, MLST, MLVA, VFP and PP Detection of the macrolide resistance genes mefA, ermA, ermB, and tetracycline resistance genes tetM, tetO was performed by PCR as described previously (Perez-Trallero et al., 2007).
The v2 test was used for statistical analysis when appropriate. A p < 0.05 was considered significant.
3. Results 3.1. Antibiotic resistance and detection of resistance determinants In this study 486 (12.5%) macrolide resistant strains were detected among 3893 GAS pharyngeal isolates identified over a two-year (2008 and 2009) period. The erythromycin resistance rates were 15% in Belgrade, 14% in Central Serbia and 10% in Vojvodina. Among 103 analyzed MRGAS strains, the M phenotype, moderately resistant to erythromycin, was detected in 74 strains, iMLS pattern in 19 strains, while the cMLS phenotype, characterized by high level of erythromycin and clindamycin resistance was represented by only 10 isolates (Table 1). The mefA gene was found in three-quarters of isolates among which 97.4% expressed M phenotype. The ermA and ermB genes were present in a minority of strains (Table 1). Resistance genes were not found in one MLS-phenotype strain. All ermA positive isolates expressed iMLS, while all ermB positive strains showed cMLS phenotype. The mefA/ermA and mefA/ermB genotypes were observed in two isolates, displaying an iMLS and cMLS phenotype, respectively. Strains with M phenotype were predominant in Belgrade (68%) and in the Central Serbia (89.8%), whereas MLS was the most prevalent phenotype in Vojvodina (55.2%). Strains with iMLS were more frequent than cMLS isolates. The overall frequency of tetracycline resistance (TR) among the 103 studied MRGAS strains was 19.4%. TR was detected in 20 isolates with MLS phenotype. All tetO genes were found in 15 ermA positive iMLS strains, while a total of 5 tetM genes were identified among ermB positive cMLS isolates (Table 1). All 103 analyzed MRGAS strains were uniformly susceptible to penicillin, vancomycin, ofloxacin and chloramphenicol.
Please cite this article in press as: Opavski, N., et al. Molecular characterization of macrolide resistant Streptococcus pyogenes isolates from pharyngitis patients in Serbia. Infect. Genet. Evol. (2015), http://dx.doi.org/10.1016/j.meegid.2015.05.011
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N. Opavski et al. / Infection, Genetics and Evolution xxx (2015) xxx–xxx Table 1 emm types distribution and correlation between emm types and resistance genes among 103 erythromycin-resistant S. pyogenes isolates, Serbia, 2008–2009. Macrolide resistance genotypes
mefA
ermA
ermB
Macrolide resistance phenotypes
Tetracycline resistance genotypes No resistant genes
tetO
tetM
MIC 50 (lg/ml)
Er Cl
M 8 0.047
iMLS >256 0.047
cMLS >256 >256
emm types
No (%)
75 12 77 28 1 118 94 11
47 (45.6) 34 (33) 15 (14.6) 2 (1.9) 2 (1.9) 1 (1) 1 (1) 1 (1)
45 29 1 0 1 0 0 0
1 1 15 0 1 0 1 0
1 4 0 2 0 1 0 1
0 1 0 0 0 0 0 0
0 0 15 0 0 0 0 0
0 4 0 0 0 1 0 0
45 28 0 0 1 0 0 0
1 1 15 0 1 0 1 0
1 5 0 2 0 1 0 1
Total
103
76
19
9
1
15
5
74
19
10
Er – erythromycin. Cl – clindamycin.
3.2. emm type distribution A total of eight different emm types were identified among the 103 tested isolates: emm75, emm12, emm77, emm28, emm1, emm11, emm94 and emm118 (Table 1). However, 93.2% of macrolide resistant GAS isolates belonged to the three predominant emm types: emm75, emm12 and emm77. The remaining 6.8% isolates represented five emm types. The greatest diversity of emm types was observed in the capital, Belgrade, where 7 out of 8 emm types were found. The emm75 was the most prevalent (14 out of 25 strains), while the other 6 emm types (emm12, emm1, emm11, emm18, emm77 and emm94) were presented in 65 isolates. In the rest of the country the distribution of emm types was more uniform and up to four different emm types were recovered. In the erythromycin- and tetracycline-resistant population, 3 emm types were detected: emm77, emm12, and emm118; the majority (75%) being emm77. The statistically significant association between TR and emm77 was found (p < 0.05). A correlation between emm types and the antibiotic resistance genes was observed. Majority of emm75 (95.7%) and emm12 (85.3%) were mefA positive, while all 15 emm77 isolates were ermA and tetO positive. The ermB gene was randomly distributed among 5 emm types, although most belonged to emm12. Also, the tetM gene was significantly linked to emm12 (p < 0.05) (Table 1). 3.3. Clonal characterization and clustering of MRGAS In order to investigate whether the erythromycin-resistant isolates represent only a few clonal types, since they belonged mainly to 3 emm types, we used multiple methods of typing: MLST, MLVA, PP and VFP. Seventy-two MRGAS isolates assigned to the three predominant emm types: 30 emm75, 27 emm12 and 15 emm77 strains, were chosen for further analysis. MLST divided isolates into 3 previously described sequence types (STs): ST36, ST49, ST63, and one new ST (N1), that was a single allele variant of ST49 in yqiL (allele 96 instead of 7) (Fig. 1). Three STs were associated exclusively with the predominant emm types. ST49 accounted for 29 strains, all belonged to emm75; ST36 for 27 emm12 isolates and ST63 for 15 emm77 isolates. One emm75 strain represented variant of ST49. Relatedness analysis of the 72 MRGAS isolates revealed the presence of 3 major clones: emm75/mefA/ST49 (40.3%),
emm12/mefA/ST36 (37.5%) and emm77/ermA/tetO/ST63 (20.8%) (Fig. 1). The last one expressed co-resistance to macrolides and tetracycline. Although MLVA identified a total of 10 patterns, isolates were clustered into 3 major complexes correlating well with emm/MLST patterns (Fig. 1). The emm77/ST63 was the most homogenous, since all strains belonging to this type share one MLVA pattern (cluster 1). Isolates representing emm12/ST36 and emm75/ST49 types were more diverse – 4 and 5 different MLVA patterns were found among them, respectively. However, MLVA patterns within each of these two types differ by only one band within the cluster. Therefore, emm75/ST49 is placed in cluster 2 and emm12/ST36 in cluster 3. The new STN1 was placed in cluster 2, together with strains emm75/ST. PP revealed the presence of 20 profiles, denoted by a lower-case letters (Fig. 1). Ten different PP were identified within emm75 strains, 7 in emm12 and 3 in emm77 isolates. However, cluster analysis disclosed only small differences in PP between strains which belonged to the same emm type, while considerable differences were observed between strains associated with different emm types (Fig. 2). On the basis on differences in virulence factors, 12 different patterns, marked with the capital letters (Fig. 1) were identified. Three VFP were found among emm12, four among emm75 and five among emm77 strains. However, the majority of isolates within same emm type shared a similar toxin gene composition. Furthermore, major differences in superantigen (SAg) toxin gene content were linked to emm type (Table 2). The following associations were noticed: speL, speM and speC with emm75 type; speI, and speH with emm12; and speC with emm77. All the isolates were positive for smeZ gene, but negative for speA. The chromosomal genes (sdaB, speB, mac and spyCEP) were detected in all isolates but one emm75, which lacked speB and sdaB. Cluster analysis revealed that emm75 and emm77 belong to E6 and E4 clusters (clade X), respectively, while emm12 belong to A-C4 cluster (clade Y). Other less frequently encountered emm types were distributed as follows: emm28-E4, emm1-A-C3, emm11-E6, emm94-E6 and emm118-E3. 4. Discussion In the present nationwide study we found that the prevalence of macrolide resistance among pharyngeal GAS isolates in Serbia in 2008 and 2009 was 12.5%. This result confirmed our previous
Please cite this article in press as: Opavski, N., et al. Molecular characterization of macrolide resistant Streptococcus pyogenes isolates from pharyngitis patients in Serbia. Infect. Genet. Evol. (2015), http://dx.doi.org/10.1016/j.meegid.2015.05.011
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Fig. 1. Analysis of clonal relationship based on MLVA, PP and VF profiling among 72 erythromycin-resistant S. pyogenes isolates, Serbia, 2008–2009. Dendrogram was created based on MLVA clusters. Black boxes denote presence of antibiotic resistance determinant/virulence factor detected/phage integration. A cutoff value of 80% similarity was applied to define MLVA clusters (named MLVA cluster 1 to MLVA cluster 3).
Please cite this article in press as: Opavski, N., et al. Molecular characterization of macrolide resistant Streptococcus pyogenes isolates from pharyngitis patients in Serbia. Infect. Genet. Evol. (2015), http://dx.doi.org/10.1016/j.meegid.2015.05.011
N. Opavski et al. / Infection, Genetics and Evolution xxx (2015) xxx–xxx
Fig. 2. Minimum spanning trees based on the results obtained by phage typing of 72 erythromycin-resistant S. pyogenes strains, Serbia, 2008–2009. Each circle represents single strain; numbers within circles denote number of identical strains.
Table 2 Distribution of virulence genes among 72 erythromycin-resistant S. pyogenes isolates, Serbia, 2008–2009. Number of MRGAS isolates carrying virulence genes emm12 (n = 27) Virulence genes speA 0 speB 27 speC 4 speG 27 speH 27 speI 26 speJ 0 speK 0 speL 0 speM 0 ssa 0 sic 0 smeZ 27 mac 27 spD3 0 sdc 0 spyCEP 27 scpA 27 sdaB 27 sdaD 27
emm77 (n = 15)
emm75 (n = 30)
Total
0 15 15 6 5 0 0 2 0 0 0 0 15 15 0 15 15 15 15 15
0 29 30 30 17 17 0 0 30 29 1 0 30 30 30 13 30 30 29 0
0 71 49 63 49 43 0 2 30 29 1 0 72 72 30 28 72 72 71 42
finding that macrolide resistance among S. pyogenes in our country had increased significantly since the beginning 2000s, when it was
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almost absent (<2%) to 9.9% during the 6 years period (2004–2009) (Mijac et al., 2014). Extensive use of long-acting macrolides, such as azithromycin, which was the first on the list of all antibiotics used in 2008 in Serbia (Mijac et al., 2014) and the availability of antibiotics over the counter during previous period might have contributed to the rapidly rising rates of macrolide resistance. Nonetheless, the overall rate of macrolide resistance in Serbia was moderate in comparison with other European countries (Richter et al., 2008) and similar to the one found in Portugal in the mid 2000s (Silva-Costa et al., 2008). Lower rates of resistance were continuously present in Northern countries in Europe in contrast to Mediterranean region with the highest rates of resistance (Grivea et al., 2006; Richter et al., 2008; Rubio-Lopez et al., 2012). However, in recent years a decrease of macrolide resistance among GAS has been recorded in several countries (Silva-Costa et al., 2008; Van Heirstraeten et al., 2012). In the present study M phenotype harboring mefA gene was the most prevalent, accounting for roughly 70% of all MRGAS isolates. iMLS phenotype associated with the presence of ermA gene was dominant among MLS strains, while cMLS phenotype was less frequent. In one strain none of the tested genes was found indicating other mechanism of macrolide resistance. The dominance of the M phenotype of resistance was found in many regions of the world especially during the 1990s. This was the most common phenotype in Spain (76.9%) and Greece (50.9%) (Grivea et al., 2006; Rubio-Lopez et al., 2012) and in regions with the low level of macrolide resistance (Green et al., 2006). However, MLS phenotype is increasingly reported in Europe (Richter et al., 2008) and recent studies showed its dominance in Belgium (Van Heirstraeten et al., 2012) and Germany (Bley et al., 2011). All 103 examined MRGAS isolates were fully susceptible to penicillin, which confirmed that penicillin is still considered first-line therapy in the treatment of most GAS infections. Although, fluoroquinolone resistance could be found in GAS isolates at a low frequency (Ardanuy et al., 2010; Montes et al., 2006), it was not present among our MRGAS isolates. Resistance was observed to tetracyclines only, and its incidence was 19.4%. It was related to MLS phenotype exclusively: tetO and tetM were present among isolates with ermA and ermB genes, respectively. Macrolide and tetracycline co-resistance is commonly present worldwide (Bessen, 2009; Silva-Costa et al., 2008). Different combinations of genes conferring co-resistance are possible due to the simultaneous presence of resistance genes on the same transposons (Ardanuy et al., 2010; Giovanetti et al., 2003). High homogeneity of emm types was found in the present study: 3 emm types were responsible for more than 90% of all MRGAS isolates: emm75, emm12 and emm77. Among macrolide susceptible GAS isolates collected in the same period, emm1 and emm6 dominated (data not shown) demonstrating the substantial differences among these GAS populations. Macrolide resistant clones could have significant survival advantage in conditions of increased macrolide usage (Bessen, 2009), which was probably the case in this study. Data from other countries also indicate that a few emm types account for much of macrolide resistance (Green et al., 2006; Michos et al., 2009; Perez-Trallero et al., 2007; Rubio-Lopez et al., 2012). The relation of 3 predominant emm types from Serbia to macrolide resistance was found in numerous reports from different regions in the world: emm75 was the most frequent among MRGAS isolates from USA (Green et al., 2006), while emm12 and emm77 were prevailing resistant genotypes found in Greece (Grivea et al., 2006; Michos et al., 2009) and Germany (Reinert et al., 2004). MLST typing results revealed that three main emm types from our study, emm12 emm75 and emm77 corresponded only to three STs, ST36, ST49 and ST63, respectively. The most prevalent clone was emm75/mefA/ST49. Interestingly, in reports from other
Please cite this article in press as: Opavski, N., et al. Molecular characterization of macrolide resistant Streptococcus pyogenes isolates from pharyngitis patients in Serbia. Infect. Genet. Evol. (2015), http://dx.doi.org/10.1016/j.meegid.2015.05.011
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European countries, this clone was sporadic (Reinert et al., 2004.). However, the emm75/T25/mefA/ST150 clone was found at a considerable frequency in Portugal (Silva-Costa et al., 2008) and probably in Spain (Rubio-Lopez et al., 2012), but not in our study. On the other hand, emm12/mefA/ST36 and emm77/ermA/tetO/ST63 clones appear to be widely disseminated across Europe (Ardanuy et al., 2010; Reinert et al., 2004, Van Heirstraeten et al., 2012). Both clones were predominant MRGAS clones found in German study (Reinert et al., 2004) and clonal spread of emm77/ermA/tetO was described in Italy and Norway (Palmieri et al., 2006). Recent increase in the proportion of this clone among MRGAS population was also observed in Belgium (Van Heirstraeten et al., 2012) indicating its potential for propagation. MLVA, PP and VPF of our strains yielded a grouping similar to those obtained by emm/MLST, and only minor differences between strains belonging to the same emm type were observed. All methods of typing used in this study revealed that there was a high genetic homogeneity among the isolates sharing the same emm type. Even among the isolates belonging to emm12, regardless of the resistance genotype, toxin gene profile was similar: all isolates were speH and speI positive, the observation already made by other authors (Commons et al., 2008). Our results point to the relation of superantigens to emm type, rather than to a particular resistance mechanism. This association has already been suggested by others (Commons et al., 2008). Cluster analysis showed that all emm types encountered among MRGAS isolates belonged to different clusters. As the new typing system is based on grouping of closely related M proteins that share similar structural and binding properties (Sanderson-Smith et al., 2014), this result indicates a functional difference between emm types expressing macrolide resistance from Serbia. In conclusion, our results show that there is a moderate level of macrolide resistance among GAS isolates in Serbia and that the efflux-mediated mechanism is the most widespread. The dominance of three major resistant clones and a high genetic homogeneity characterize Serbian MRGAS population. These results demonstrate that macrolide resistance in Serbia is due to spread of a few internationally widely disseminated clones. Further surveillance of macrolide resistance in S. pyogenes in Serbia is necessary in order to monitor the trends of macrolide resistance and to facilitate preventive measures. Acknowledgments The research was partially financed by Serbian Ministry of Education and Science (Project Nos. 175039, and 175024) and from N N401 536140 Grant from National Science Center (Poland). We gratefully acknowledge all colleagues for providing group A streptococcal isolates. References Ardanuy, C., Domenech, A., Rolo, D., Calatayud, L., Tubau, F., Ayats, J., Martiin, R., Linnares, J., 2010. Molecular characterization of macrolide- and multidrugresistant Streptococcus pyogenes isolated from adult patients in Barcelona, Spain (1993–2008). J. Antimicrob. Chemother. 65, 634–643. http://dx.doi.org/ 10.1093/jac/dkq006. Beall, B., Facklam, R., Thompson, T., 1996. Sequencing emm-specific PCR product for routine and accurate typing of group A streptococci. J. Clin. Microbiol. 34, 953– 958. Bessen, D.E., 2009. Population biology of the human restricted pathogen, Streptococcus pyogenes. Infect. Genet. Evol. 9, 581–593. http://dx.doi.org/ 10.1016/j.meegid.2009.03.002. Bley, C., van der Linden, M., Reinert, R.R., 2011. Mef(A) is the predominant macrolide resistance determinant in Streptococcus pneumoniae and Streptococcus pyogenes in Germany. Int. J. Antimicrob. Agents 37, 425–431. http://dx.doi.org/10.1016/ j.ijantimicag.2011.01.019.
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Please cite this article in press as: Opavski, N., et al. Molecular characterization of macrolide resistant Streptococcus pyogenes isolates from pharyngitis patients in Serbia. Infect. Genet. Evol. (2015), http://dx.doi.org/10.1016/j.meegid.2015.05.011
Contents lists available at ScienceDirect
Infection, Genetics and Evolution journal homepage: www.elsevier.com/locate/meegid
Molecular characterization of macrolide resistant Streptococcus pyogenes isolates from pharyngitis patients in Serbia Natasa Opavski a,b,⇑, Ina Gajic a,b, Anna L. Borek c,1, Katarzyna Obszan´ska c,2, Maja Stanojevic b, Ivana Lazarevic b, Lazar Ranin a,b, Izabela Sitkiewicz c, Vera Mijac a,b a b c
National Reference Laboratory for Streptococci, Medical Faculty, University of Belgrade, Dr Subotica starijeg 1, 11000 Belgrade, Serbia Institute of Microbiology and Immunology, Medical Faculty, University of Belgrade, Dr Subotica starijeg 1, 11000 Belgrade, Serbia Department of Epidemiology and Clinical Microbiology, National Medicines Institute, Chełmska 30/34, 00-750 Warszawa, Poland
a r t i c l e
i n f o
Article history: Received 14 January 2015 Received in revised form 17 April 2015 Accepted 11 May 2015 Available online xxxx Keywords: Streptococcus pyogenes Macrolide resistance emm MLST MLVA
a b s t r a c t A steady increase in macrolide resistance in Streptococcus pyogenes, group A streptococci (GAS) was reported in Serbia during 2004–2009 (9.9%). However, there are no data on the molecular epidemiology of pharyngeal macrolide resistance GAS (MRGAS) isolates. Therefore, the aims of this first nationwide study were to examine the prevalence of macrolide resistance in Serbian GAS and to determine their resistance phenotypes, genotypes and clonal relationships. Overall 3893 non-duplicate pharyngeal S. pyogenes isolates from outpatients with GAS infection were collected throughout country during 2008 and 2009. Among 486 macrolide resistant pharyngeal isolates collected, 103 were further characterized. Macrolide resistance phenotypes and genotypes were determined by double-disk diffusion test and PCR, respectively. Strain relatedness was determined by emm typing, multilocus sequence typing (MLST), multilocus variable tandem repeat analysis (MLVA), phage profiling (PP) and virulence factor profiling (VFP). Overall, macrolide resistance among GAS isolates in Serbia was 12.5%. M phenotype was the most common (71.8%), followed by iMLS (18.4%) and cMLS (9.7%). Three clonal complexes – emm75/mefA/ST49, emm12/mefA/ST36 and emm77/ermA/tetO/ST63 comprised over 90% of the tested strains. Although MLVA, PP and VFP distinguished 10, 20 and 12 different patterns, respectively, cluster analysis disclosed only small differences between strains which belonged to the same emm/ST type. Our data indicate dominance of three major internationally widely disseminated macrolide resistant clones and a high genetic homogeneity among the Serbian MRGAS population. Continued surveillance of macrolide resistance and clonal composition in MRGAS in Serbia in future is necessary to determine stability of MRGAS clones and to guide therapy strategies. Ó 2015 Elsevier B.V. All rights reserved.
1. Introduction Streptococcus pyogenes (group A Streptococcus, GAS) is an important human pathogen that causes a wide variety of diseases (Walker et al., 2014). Penicillin is still the drug of choice for GAS ⇑ Corresponding author at: Institute of Microbiology and Immunology, Faculty of Medicine, University of Belgrade, Dr Subotica 1, 11000 Belgrade, Serbia. E-mail addresses: [email protected], [email protected] (N. Opavski), [email protected] (I. Gajic), [email protected] (A.L. Borek), [email protected] (K. Obszan´ska), [email protected] (M. Stanojevic), [email protected] (I. Lazarevic), [email protected] (L. Ranin), iza.sitkiewicz@ gmail.com (I. Sitkiewicz), [email protected] (V. Mijac). 1 Current address: Department of Microbiology, National Research Institute of Tuberculosis and Lung Diseases, Płocka 26, 01-138 Warszawa, Poland. 2 Current address: Department of Microbial Biochemistry, Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Pawin´skiego 5a, 01-106 Warszawa, Poland.
infections, whereas macrolides, such as erythromycin, are recommended as a first alternative therapy for patients who are allergic to penicillin. Since the 1990s a significant increase of erythromycin resistance was observed among S. pyogenes isolates worldwide (Ardanuy et al., 2010; Cornaglia et al., 1996). However, in recent years a steady decline in macrolide resistance was recognized (Silva-Costa et al., 2008; Van Heirstraeten et al., 2012). There are two major mechanisms of macrolide resistance in S. pyogenes: (i) target modification due to methylation of the ribosomal RNA (encoded by ermA or ermB genes) which results in co-resistance to macrolides, lincosamides and streptogramins (MLS phenotype) and (ii) efflux of antibiotics (encoded by mefA gene) that affects 14- and 15-membered macrolides only and gives rise to the so-called M phenotype of resistance (Leclercq, 2002). The MLS phenotype could be further subdivided into inducible (iMLS) and constitutive (cMLS).
http://dx.doi.org/10.1016/j.meegid.2015.05.011 1567-1348/Ó 2015 Elsevier B.V. All rights reserved.
Please cite this article in press as: Opavski, N., et al. Molecular characterization of macrolide resistant Streptococcus pyogenes isolates from pharyngitis patients in Serbia. Infect. Genet. Evol. (2015), http://dx.doi.org/10.1016/j.meegid.2015.05.011
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N. Opavski et al. / Infection, Genetics and Evolution xxx (2015) xxx–xxx
M protein gene (emm) sequence typing is the most widely used method for epidemiological classification of GAS strains (Beall et al., 1996). The correlation between macrolide resistance and emm types has been noted worldwide (Michos et al., 2009; Silva-Costa et al., 2008). However, for the precise detection of the clonal relationships between the macrolide-resistant strains, additional methods, such as pulse field gel electrophoresis (PFGE) or multilocus sequence typing (MLST) should be used (Carrico et al., 2006). Beside these techniques there are several other approaches for GAS typing – multiple-locus variable number tandem repeat analysis (MLVA) (Obszanska et al., 2011) and recently designed phage profiling (PP) and virulence factor profiling (VFP) (Borek et al., 2011). The incidence of macrolide resistance and the distribution of resistance phenotypes and genotypes are significantly variable in different parts of the world (Green et al., 2006; Silva-Costa et al., 2008; Van Heirstraeten et al., 2012). In Serbia macrolide consumption has substantially increased during 2000s and a significant rise in macrolide resistance was observed in S. pyogenes pediatric isolates (9.9%) (Mijac et al., 2014). However, there are no data on the molecular epidemiology of pharyngeal macrolide resistant GAS (MRGAS). The aims of this study were to examine the prevalence of macrolide resistance in Serbian pharyngeal isolates of S. pyogenes and to investigate phenotypic and genotypic characteristics and genetic similarity of MRGAS isolates.
The emm types of MRGAS isolates were determined according to the recommendations of Centers for Disease Control and Prevention http://www.cdc.gov/streplab/protocol-emm-type.html. MLST was carried out according to Enright et al. (2001). Allele numbers and the sequence type were assigned using the MLST website http://spyogenes.mlst.net. Multilocus variable tandem repeat analysis (MLVA) was performed using the procedure described by Obszanska et al. (2011). Virulence factor profiling (VFP) and phage profiling (PP) was done using the set of multiplex PCR reactions which detect simultaneously 20 GAS virulence factors (spd3, sdc, sdaB, sdaD, speB, spyCEP, scpA, mac, sic, speL, speK, speM, speC, speI, speA, speH, speG, speJ, smeZ and ssa) and 21 phage end integrative and conjugative element (ICE) integration sites as previously described (Borek et al., 2011). The results of MLVA, VFP and PP were analyzed using BioNumerics software (Applied Maths). Similarity coefficient between strains was calculated using ranked Pearson correlation. MLVA based clusters were assessed using UPGMA method. 2.4. Cluster typing Cluster designation was done by a typing system recently developed by Sanderson-Smith et al. (2014). It is based on emm typing results and distinguishes 48 GAS clusters containing closely related M proteins.
2. Materials and methods 2.5. Statistics 2.1. Strain collection and identification Serbia is Southeastern European country with roughly 7 million inhabitants. It could be arbitrary divided into three regions: Belgrade (the capital, 2 million), Central Serbia (3 million) and Vojvodina (2 million). During 2008 and 2009, 5 laboratories from Belgrade and 6 laboratories located throughout Vojvodina and Central Serbia collected 3893 non-duplicate pharyngeal S. pyogenes isolates from outpatients with GAS infection. Among those strains, MRGAS isolates were identified and sent into the National Reference Laboratory. One hundred and three (21.2% of all MRGAS) were randomly chosen for further characterization: 25 out of 123 (20.3%) from Belgrade; 29 out of 139 (20.9%) from Vojvodina and 49 out of 224 (21.9%) from the Central Serbia. The majority of these isolates (89%) originated from school age children. Isolates were identified by slide agglutination with group A antisera (Slidex Streptokit; bioMerieux, France), susceptibility to bacitracin and positive PYR test (Rosco, Denmark). 2.2. Antimicrobial susceptibility testing Antimicrobial susceptibility was tested using a disk diffusion test for penicillin (10 IU), erythromycin (15 lg), clindamycin (2 lg), tetracycline (30 lg), ofloxacin (5 lg) and chloramphenicol (30 lg) (BioRad, USA), in accordance with the Clinical and Laboratory Standard Institute guidelines (CLSI) guidelines (2012). Macrolide resistance phenotypes were determined by the double-disk diffusion test (CLSI, 2012). MICs of erythromycin and clindamycin were determined by E test (bioMerieux, France). Streptococcus pneumoniae ATCC 49619 was used as a control strain. 2.3. Antibiotic resistance gene detection, emm typing, MLST, MLVA, VFP and PP Detection of the macrolide resistance genes mefA, ermA, ermB, and tetracycline resistance genes tetM, tetO was performed by PCR as described previously (Perez-Trallero et al., 2007).
The v2 test was used for statistical analysis when appropriate. A p < 0.05 was considered significant.
3. Results 3.1. Antibiotic resistance and detection of resistance determinants In this study 486 (12.5%) macrolide resistant strains were detected among 3893 GAS pharyngeal isolates identified over a two-year (2008 and 2009) period. The erythromycin resistance rates were 15% in Belgrade, 14% in Central Serbia and 10% in Vojvodina. Among 103 analyzed MRGAS strains, the M phenotype, moderately resistant to erythromycin, was detected in 74 strains, iMLS pattern in 19 strains, while the cMLS phenotype, characterized by high level of erythromycin and clindamycin resistance was represented by only 10 isolates (Table 1). The mefA gene was found in three-quarters of isolates among which 97.4% expressed M phenotype. The ermA and ermB genes were present in a minority of strains (Table 1). Resistance genes were not found in one MLS-phenotype strain. All ermA positive isolates expressed iMLS, while all ermB positive strains showed cMLS phenotype. The mefA/ermA and mefA/ermB genotypes were observed in two isolates, displaying an iMLS and cMLS phenotype, respectively. Strains with M phenotype were predominant in Belgrade (68%) and in the Central Serbia (89.8%), whereas MLS was the most prevalent phenotype in Vojvodina (55.2%). Strains with iMLS were more frequent than cMLS isolates. The overall frequency of tetracycline resistance (TR) among the 103 studied MRGAS strains was 19.4%. TR was detected in 20 isolates with MLS phenotype. All tetO genes were found in 15 ermA positive iMLS strains, while a total of 5 tetM genes were identified among ermB positive cMLS isolates (Table 1). All 103 analyzed MRGAS strains were uniformly susceptible to penicillin, vancomycin, ofloxacin and chloramphenicol.
Please cite this article in press as: Opavski, N., et al. Molecular characterization of macrolide resistant Streptococcus pyogenes isolates from pharyngitis patients in Serbia. Infect. Genet. Evol. (2015), http://dx.doi.org/10.1016/j.meegid.2015.05.011
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N. Opavski et al. / Infection, Genetics and Evolution xxx (2015) xxx–xxx Table 1 emm types distribution and correlation between emm types and resistance genes among 103 erythromycin-resistant S. pyogenes isolates, Serbia, 2008–2009. Macrolide resistance genotypes
mefA
ermA
ermB
Macrolide resistance phenotypes
Tetracycline resistance genotypes No resistant genes
tetO
tetM
MIC 50 (lg/ml)
Er Cl
M 8 0.047
iMLS >256 0.047
cMLS >256 >256
emm types
No (%)
75 12 77 28 1 118 94 11
47 (45.6) 34 (33) 15 (14.6) 2 (1.9) 2 (1.9) 1 (1) 1 (1) 1 (1)
45 29 1 0 1 0 0 0
1 1 15 0 1 0 1 0
1 4 0 2 0 1 0 1
0 1 0 0 0 0 0 0
0 0 15 0 0 0 0 0
0 4 0 0 0 1 0 0
45 28 0 0 1 0 0 0
1 1 15 0 1 0 1 0
1 5 0 2 0 1 0 1
Total
103
76
19
9
1
15
5
74
19
10
Er – erythromycin. Cl – clindamycin.
3.2. emm type distribution A total of eight different emm types were identified among the 103 tested isolates: emm75, emm12, emm77, emm28, emm1, emm11, emm94 and emm118 (Table 1). However, 93.2% of macrolide resistant GAS isolates belonged to the three predominant emm types: emm75, emm12 and emm77. The remaining 6.8% isolates represented five emm types. The greatest diversity of emm types was observed in the capital, Belgrade, where 7 out of 8 emm types were found. The emm75 was the most prevalent (14 out of 25 strains), while the other 6 emm types (emm12, emm1, emm11, emm18, emm77 and emm94) were presented in 65 isolates. In the rest of the country the distribution of emm types was more uniform and up to four different emm types were recovered. In the erythromycin- and tetracycline-resistant population, 3 emm types were detected: emm77, emm12, and emm118; the majority (75%) being emm77. The statistically significant association between TR and emm77 was found (p < 0.05). A correlation between emm types and the antibiotic resistance genes was observed. Majority of emm75 (95.7%) and emm12 (85.3%) were mefA positive, while all 15 emm77 isolates were ermA and tetO positive. The ermB gene was randomly distributed among 5 emm types, although most belonged to emm12. Also, the tetM gene was significantly linked to emm12 (p < 0.05) (Table 1). 3.3. Clonal characterization and clustering of MRGAS In order to investigate whether the erythromycin-resistant isolates represent only a few clonal types, since they belonged mainly to 3 emm types, we used multiple methods of typing: MLST, MLVA, PP and VFP. Seventy-two MRGAS isolates assigned to the three predominant emm types: 30 emm75, 27 emm12 and 15 emm77 strains, were chosen for further analysis. MLST divided isolates into 3 previously described sequence types (STs): ST36, ST49, ST63, and one new ST (N1), that was a single allele variant of ST49 in yqiL (allele 96 instead of 7) (Fig. 1). Three STs were associated exclusively with the predominant emm types. ST49 accounted for 29 strains, all belonged to emm75; ST36 for 27 emm12 isolates and ST63 for 15 emm77 isolates. One emm75 strain represented variant of ST49. Relatedness analysis of the 72 MRGAS isolates revealed the presence of 3 major clones: emm75/mefA/ST49 (40.3%),
emm12/mefA/ST36 (37.5%) and emm77/ermA/tetO/ST63 (20.8%) (Fig. 1). The last one expressed co-resistance to macrolides and tetracycline. Although MLVA identified a total of 10 patterns, isolates were clustered into 3 major complexes correlating well with emm/MLST patterns (Fig. 1). The emm77/ST63 was the most homogenous, since all strains belonging to this type share one MLVA pattern (cluster 1). Isolates representing emm12/ST36 and emm75/ST49 types were more diverse – 4 and 5 different MLVA patterns were found among them, respectively. However, MLVA patterns within each of these two types differ by only one band within the cluster. Therefore, emm75/ST49 is placed in cluster 2 and emm12/ST36 in cluster 3. The new STN1 was placed in cluster 2, together with strains emm75/ST. PP revealed the presence of 20 profiles, denoted by a lower-case letters (Fig. 1). Ten different PP were identified within emm75 strains, 7 in emm12 and 3 in emm77 isolates. However, cluster analysis disclosed only small differences in PP between strains which belonged to the same emm type, while considerable differences were observed between strains associated with different emm types (Fig. 2). On the basis on differences in virulence factors, 12 different patterns, marked with the capital letters (Fig. 1) were identified. Three VFP were found among emm12, four among emm75 and five among emm77 strains. However, the majority of isolates within same emm type shared a similar toxin gene composition. Furthermore, major differences in superantigen (SAg) toxin gene content were linked to emm type (Table 2). The following associations were noticed: speL, speM and speC with emm75 type; speI, and speH with emm12; and speC with emm77. All the isolates were positive for smeZ gene, but negative for speA. The chromosomal genes (sdaB, speB, mac and spyCEP) were detected in all isolates but one emm75, which lacked speB and sdaB. Cluster analysis revealed that emm75 and emm77 belong to E6 and E4 clusters (clade X), respectively, while emm12 belong to A-C4 cluster (clade Y). Other less frequently encountered emm types were distributed as follows: emm28-E4, emm1-A-C3, emm11-E6, emm94-E6 and emm118-E3. 4. Discussion In the present nationwide study we found that the prevalence of macrolide resistance among pharyngeal GAS isolates in Serbia in 2008 and 2009 was 12.5%. This result confirmed our previous
Please cite this article in press as: Opavski, N., et al. Molecular characterization of macrolide resistant Streptococcus pyogenes isolates from pharyngitis patients in Serbia. Infect. Genet. Evol. (2015), http://dx.doi.org/10.1016/j.meegid.2015.05.011
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Fig. 1. Analysis of clonal relationship based on MLVA, PP and VF profiling among 72 erythromycin-resistant S. pyogenes isolates, Serbia, 2008–2009. Dendrogram was created based on MLVA clusters. Black boxes denote presence of antibiotic resistance determinant/virulence factor detected/phage integration. A cutoff value of 80% similarity was applied to define MLVA clusters (named MLVA cluster 1 to MLVA cluster 3).
Please cite this article in press as: Opavski, N., et al. Molecular characterization of macrolide resistant Streptococcus pyogenes isolates from pharyngitis patients in Serbia. Infect. Genet. Evol. (2015), http://dx.doi.org/10.1016/j.meegid.2015.05.011
N. Opavski et al. / Infection, Genetics and Evolution xxx (2015) xxx–xxx
Fig. 2. Minimum spanning trees based on the results obtained by phage typing of 72 erythromycin-resistant S. pyogenes strains, Serbia, 2008–2009. Each circle represents single strain; numbers within circles denote number of identical strains.
Table 2 Distribution of virulence genes among 72 erythromycin-resistant S. pyogenes isolates, Serbia, 2008–2009. Number of MRGAS isolates carrying virulence genes emm12 (n = 27) Virulence genes speA 0 speB 27 speC 4 speG 27 speH 27 speI 26 speJ 0 speK 0 speL 0 speM 0 ssa 0 sic 0 smeZ 27 mac 27 spD3 0 sdc 0 spyCEP 27 scpA 27 sdaB 27 sdaD 27
emm77 (n = 15)
emm75 (n = 30)
Total
0 15 15 6 5 0 0 2 0 0 0 0 15 15 0 15 15 15 15 15
0 29 30 30 17 17 0 0 30 29 1 0 30 30 30 13 30 30 29 0
0 71 49 63 49 43 0 2 30 29 1 0 72 72 30 28 72 72 71 42
finding that macrolide resistance among S. pyogenes in our country had increased significantly since the beginning 2000s, when it was
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almost absent (<2%) to 9.9% during the 6 years period (2004–2009) (Mijac et al., 2014). Extensive use of long-acting macrolides, such as azithromycin, which was the first on the list of all antibiotics used in 2008 in Serbia (Mijac et al., 2014) and the availability of antibiotics over the counter during previous period might have contributed to the rapidly rising rates of macrolide resistance. Nonetheless, the overall rate of macrolide resistance in Serbia was moderate in comparison with other European countries (Richter et al., 2008) and similar to the one found in Portugal in the mid 2000s (Silva-Costa et al., 2008). Lower rates of resistance were continuously present in Northern countries in Europe in contrast to Mediterranean region with the highest rates of resistance (Grivea et al., 2006; Richter et al., 2008; Rubio-Lopez et al., 2012). However, in recent years a decrease of macrolide resistance among GAS has been recorded in several countries (Silva-Costa et al., 2008; Van Heirstraeten et al., 2012). In the present study M phenotype harboring mefA gene was the most prevalent, accounting for roughly 70% of all MRGAS isolates. iMLS phenotype associated with the presence of ermA gene was dominant among MLS strains, while cMLS phenotype was less frequent. In one strain none of the tested genes was found indicating other mechanism of macrolide resistance. The dominance of the M phenotype of resistance was found in many regions of the world especially during the 1990s. This was the most common phenotype in Spain (76.9%) and Greece (50.9%) (Grivea et al., 2006; Rubio-Lopez et al., 2012) and in regions with the low level of macrolide resistance (Green et al., 2006). However, MLS phenotype is increasingly reported in Europe (Richter et al., 2008) and recent studies showed its dominance in Belgium (Van Heirstraeten et al., 2012) and Germany (Bley et al., 2011). All 103 examined MRGAS isolates were fully susceptible to penicillin, which confirmed that penicillin is still considered first-line therapy in the treatment of most GAS infections. Although, fluoroquinolone resistance could be found in GAS isolates at a low frequency (Ardanuy et al., 2010; Montes et al., 2006), it was not present among our MRGAS isolates. Resistance was observed to tetracyclines only, and its incidence was 19.4%. It was related to MLS phenotype exclusively: tetO and tetM were present among isolates with ermA and ermB genes, respectively. Macrolide and tetracycline co-resistance is commonly present worldwide (Bessen, 2009; Silva-Costa et al., 2008). Different combinations of genes conferring co-resistance are possible due to the simultaneous presence of resistance genes on the same transposons (Ardanuy et al., 2010; Giovanetti et al., 2003). High homogeneity of emm types was found in the present study: 3 emm types were responsible for more than 90% of all MRGAS isolates: emm75, emm12 and emm77. Among macrolide susceptible GAS isolates collected in the same period, emm1 and emm6 dominated (data not shown) demonstrating the substantial differences among these GAS populations. Macrolide resistant clones could have significant survival advantage in conditions of increased macrolide usage (Bessen, 2009), which was probably the case in this study. Data from other countries also indicate that a few emm types account for much of macrolide resistance (Green et al., 2006; Michos et al., 2009; Perez-Trallero et al., 2007; Rubio-Lopez et al., 2012). The relation of 3 predominant emm types from Serbia to macrolide resistance was found in numerous reports from different regions in the world: emm75 was the most frequent among MRGAS isolates from USA (Green et al., 2006), while emm12 and emm77 were prevailing resistant genotypes found in Greece (Grivea et al., 2006; Michos et al., 2009) and Germany (Reinert et al., 2004). MLST typing results revealed that three main emm types from our study, emm12 emm75 and emm77 corresponded only to three STs, ST36, ST49 and ST63, respectively. The most prevalent clone was emm75/mefA/ST49. Interestingly, in reports from other
Please cite this article in press as: Opavski, N., et al. Molecular characterization of macrolide resistant Streptococcus pyogenes isolates from pharyngitis patients in Serbia. Infect. Genet. Evol. (2015), http://dx.doi.org/10.1016/j.meegid.2015.05.011
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European countries, this clone was sporadic (Reinert et al., 2004.). However, the emm75/T25/mefA/ST150 clone was found at a considerable frequency in Portugal (Silva-Costa et al., 2008) and probably in Spain (Rubio-Lopez et al., 2012), but not in our study. On the other hand, emm12/mefA/ST36 and emm77/ermA/tetO/ST63 clones appear to be widely disseminated across Europe (Ardanuy et al., 2010; Reinert et al., 2004, Van Heirstraeten et al., 2012). Both clones were predominant MRGAS clones found in German study (Reinert et al., 2004) and clonal spread of emm77/ermA/tetO was described in Italy and Norway (Palmieri et al., 2006). Recent increase in the proportion of this clone among MRGAS population was also observed in Belgium (Van Heirstraeten et al., 2012) indicating its potential for propagation. MLVA, PP and VPF of our strains yielded a grouping similar to those obtained by emm/MLST, and only minor differences between strains belonging to the same emm type were observed. All methods of typing used in this study revealed that there was a high genetic homogeneity among the isolates sharing the same emm type. Even among the isolates belonging to emm12, regardless of the resistance genotype, toxin gene profile was similar: all isolates were speH and speI positive, the observation already made by other authors (Commons et al., 2008). Our results point to the relation of superantigens to emm type, rather than to a particular resistance mechanism. This association has already been suggested by others (Commons et al., 2008). Cluster analysis showed that all emm types encountered among MRGAS isolates belonged to different clusters. As the new typing system is based on grouping of closely related M proteins that share similar structural and binding properties (Sanderson-Smith et al., 2014), this result indicates a functional difference between emm types expressing macrolide resistance from Serbia. In conclusion, our results show that there is a moderate level of macrolide resistance among GAS isolates in Serbia and that the efflux-mediated mechanism is the most widespread. The dominance of three major resistant clones and a high genetic homogeneity characterize Serbian MRGAS population. These results demonstrate that macrolide resistance in Serbia is due to spread of a few internationally widely disseminated clones. Further surveillance of macrolide resistance in S. pyogenes in Serbia is necessary in order to monitor the trends of macrolide resistance and to facilitate preventive measures. Acknowledgments The research was partially financed by Serbian Ministry of Education and Science (Project Nos. 175039, and 175024) and from N N401 536140 Grant from National Science Center (Poland). We gratefully acknowledge all colleagues for providing group A streptococcal isolates. References Ardanuy, C., Domenech, A., Rolo, D., Calatayud, L., Tubau, F., Ayats, J., Martiin, R., Linnares, J., 2010. Molecular characterization of macrolide- and multidrugresistant Streptococcus pyogenes isolated from adult patients in Barcelona, Spain (1993–2008). J. Antimicrob. Chemother. 65, 634–643. http://dx.doi.org/ 10.1093/jac/dkq006. Beall, B., Facklam, R., Thompson, T., 1996. Sequencing emm-specific PCR product for routine and accurate typing of group A streptococci. J. Clin. Microbiol. 34, 953– 958. Bessen, D.E., 2009. Population biology of the human restricted pathogen, Streptococcus pyogenes. Infect. Genet. Evol. 9, 581–593. http://dx.doi.org/ 10.1016/j.meegid.2009.03.002. Bley, C., van der Linden, M., Reinert, R.R., 2011. Mef(A) is the predominant macrolide resistance determinant in Streptococcus pneumoniae and Streptococcus pyogenes in Germany. Int. J. Antimicrob. Agents 37, 425–431. http://dx.doi.org/10.1016/ j.ijantimicag.2011.01.019.
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Please cite this article in press as: Opavski, N., et al. Molecular characterization of macrolide resistant Streptococcus pyogenes isolates from pharyngitis patients in Serbia. Infect. Genet. Evol. (2015), http://dx.doi.org/10.1016/j.meegid.2015.05.011