Multiple-locus variable number of tandem repeat analysis as a tool for molecular epidemiology of botulism: The Italian experience

Clostridium botulinum is the bacterial agent of botulism, a rare but severe neuro-paralytic disease. Because of its high impact, in Italy botulism is monitored by an ad hoc surveillance system. The National Reference Centre for Botulism, as part of this system, collects and analyzes all demographic, epidemiologic, microbiological, and molecular data recovered during cases and/or outbreaks occurred in Italy.A panel of 312 C. botulinum strains belonging to group I were submitted to MLVA sub-typing. Strains, isolated from clinical specimens, food and environmental samples collected during the surveillance activities, were representative of all forms of botulism from all Italian regions.Through clustering analysis isolates were grouped into 12 main clusters. No regional or temporal clustering was detected, demonstrating the high heterogeneity of strains circulating in Italy.This study confirmed that MLVA is capable of sub-typing C. botulinum strains. Moreover, MLVA is effective at tracing and tracking the source of contamination and is helpful for the surveillance system in terms of planning and upgrading of procedures, activities and data collection forms.     

2011-2012年杭州市肠道沙门菌临床分离株的分子型别分析

目的研究2011-2012年杭州市肠道沙门菌临床分离株的型别,了解本地菌株分子流行病学特征。方法对66株肠道沙门菌临床分离株进行血清分型和多位点序列分型(MLST)。对其中主要血清型:鼠伤寒、甲型副伤寒、萨雷甲尼和肠炎沙门菌菌株进行脉冲场凝胶电泳(PFGE)分型。结果分布于21个血清型的66株沙门菌分成26个ST型别。发现一株纽波特沙门菌为新型ST1690。菌株血清型与MLST型别数据库中所对应的血清型符合率为100.00%。9株甲型副伤寒沙门菌的PFGE带型完全一致(P7型),与先前杭州流行菌株有差异(P1-P6型)。6株肠炎沙门菌分成4个PFGE型,型间最小相似性为92.70%。13株鼠伤寒沙门菌分为11个PFGE型,型间最小相似性为71.70%。7株萨雷甲尼沙门菌分成4个PFGE型别,型间最小相似性为91.00%。结论近年杭州腹泻病人中流行的肠道沙门菌菌株主要血清型为鼠伤寒、甲型副伤寒、萨雷甲尼和肠炎等。甲型副伤寒沙门菌菌株在杭州出现了新PFGE型别。MLST数据可以对沙门菌血清学鉴定提供一定的帮助。     

Genetic diversity of clinical Salmonella enterica serovar Typhimurium in a university hospital of south Tunisia, 2000–2013

Typhimurium is one of the main Salmonella serovar responsible for non-typhoidal gastro-enteritis in Tunisia. Here, we aimed to assess the genetic diversity of 88 clinical Salmonella Typhimurium strains recovered during 14 years from 2000 to 2013. Phage typing, CRISPR polymorphisms (CRISPOL), pulsed-field gel electrophoresis (PFGE), multi-locus variable-number tandem repeat analysis (MLVA) and Whole genome sequencing (WGS) were used to study the relatedness and spatio-temporal evolution of Salmonella Typhimurium populations (Typhimurium (n = 81), monophasic (n = 3) and nonmotile (n = 4) variants).Seven-locus MLST from whole genome assemblies showed that all isolates, except one, belonged to ST19. The isolates were divided into 10 definitive phage (DT) types, dominated by DT104-L (39.8%), DT41 (14.8%), DT116 (11.4%) and DT120 (5.7%). Fifty-seven MLVA patterns (DI, 0.978) were obtained compared to 11 different CRISPOL types and 15 PFGE types (DI,0.845). For cgMLST analysis, 20 profiles were found. A total of 3056 SNPs were identified from the whole genome of the 88 Salmonella Typhimurium isolates. These SNPs resolved these isolates into 86 SNP haplotypes. The phylogeny result allocated most Salmonella Typhimurium isolates into four distinct clades and seven subclades. Genetic diversity between the four clades ranged in the order of 249 to 720 nucleotide changes. The prevalent phage type DT104L formed a major clade on the phylogenetic tree. Pairwise SNP differences between the strains of this clade ranged between 0 and 59.SNP-based WGS typing seems to be the most valuable molecular markers for studying the evolutionary relationships of homogeneous serovar Typhimurium isolates.     

Multiple-locus variable-number tandem-repeat analysis of meticillin-resistant Staphylococcus aureus discriminates within USA pulsed-field gel electrophoresis types

Many isolates of meticillin-resistant Staphylococcus aureus (MRSA) are indistinguishable when compared using the standard pulsed-field gel electrophoresis (PFGE) typing method. This may present a problem when investigating local outbreaks of MRSA transmission in a healthcare setting. It also impedes investigation of the widely disseminated community-acquired MRSA (USA 300-0114) in the inpatient setting, which is displacing other traditional hospital-acquired PFGE types. Combination of methods, including multiple-locus sequence typing (MLST), spa typing and staphylococcal cassette chromosome mec (SCCmec) typing, have been used with, or in place of, PFGE to characterise MRSA for epidemiological purposes. These methods are technically challenging, time-consuming and expensive and are rarely feasible except in large laboratories in tertiary care medical centres. Another method, which is simpler and with faster turnaround time, is multiple-locus variable-number tandem-repeat analysis (MLVA). We investigated the utility of MLVA to distinguish common PFGE types. The results suggest that MLVA can be used to identify unrelated strains with identical PFGE patterns or confirm close genetic composition of linked isolates. MLVA could potentially be used in conjunction with PFGE to validate relationships, but further prospective evaluation of these relationships will be required in order to define the proper role, if any, for use of this method in hospital epidemiology.     

Phylogenetic assignment of Mycobacterium tuberculosis Beijing clinical isolates in Japan by maximum a posteriori estimation

Intra-species phylogeny of Mycobacterium tuberculosis has been regarded as a clue to estimate its potential risk to develop drug-resistance and various epidemiological tendencies. Genotypic characterization of variable number of tandem repeats (VNTR), a standard tool to ascertain transmission routes, has been improving as a public health effort, but determining phylogenetic information from those efforts alone is difficult. We present a platform based on maximum a posteriori (MAP) estimation to estimate phylogenetic information for M. tuberculosis clinical isolates from individual profiles of VNTR types. This study used 1245 M. tuberculosis clinical isolates obtained throughout Japan for construction of an MAP estimation formula. Two MAP estimation formulae, classification of Beijing family and other lineages, and classification of five Beijing sublineages (ST11/26, STK, ST3, and ST25/19 belonging to the ancient Beijing subfamily and modern Beijing subfamily), were created based on 24 loci VNTR (24_Beijing-VNTR) profiles and phylogenetic information of the isolates. Recursive estimation based on the formulae showed high concordance with their authentic phylogeny by multi-locus sequence typing (MLST) of the isolates. The formulae might further support phylogenetic estimation of the Beijing lineage M. tuberculosis from the VNTR genotype with various geographic backgrounds. These results suggest that MAP estimation can function as a reliable probabilistic process to append phylogenetic information to VNTR genotypes of M. tuberculosis independently, which might improve the usage of genotyping data for control, understanding, prevention, and treatment of TB.     

Evidence of circulation of an epidemic strain of Pasteurella multocida in Jiangsu,China by multi-locus sequence typing (MLST)

Pasteurella multocida, the causative agent of fowl cholera, is a serious threat to poultry farming. In this study, we isolated and identified 40 P. multocida strains in fowl cholera outbreaks in Jiangsu province, China. The identified P. multocida was further characterized using multi-locus sequence typing (MLST). All of the 40 P. multocida strains studied are genetically identical and belong to the ST129 sequence type based on seven MLST loci. Our study provides evidence of a circulating epidemic strain of P. multocida in Jiangsu, China.     

广州市两例B群流脑病例分离株的MLST分型研究

目的对从广州市报告的两例B群流行性脑脊髓膜炎（流脑）病例中分离的4株脑膜炎奈瑟菌（Neisseria meningitidis,Nm）进行多位点序列分型（multi—locus sequence typing,MLST）研究。方法分纯并提取菌株DNA、运用MI。ST分析对7个管家基因进行扩增并测序,确定菌株的序列型,对其相关性进行分析。结果两例病例分离株7个管家基因位点序列均不相同,第一例病例分离株的序列型为ST-7型,属高致病性ST-5complex／subgroupIII克隆系;第二例病例分离株的序列型为新发现的ST一9804型。结论广州两例B群流脑病例虽然发生时间相近,但分属不同的序列型,无同源性。MLST分型技术对研究不同流脑病例间的流行病学关系有重要意义。     

Discrimination between epidemic and non-epidemic glycopeptide-resistant E. faecium in a post-outbreak situation

Vancomycin-resistant enterococci (VRE) have been isolated in increasing numbers. Hospital-adapted VRE exhibit relatively high pathogenicity by expressing factors like enterococcal surface protein (Esp), which facilitates epidemic spread. By contrast, 'community-acquired' VRE show low pathogenicity and non-epidemic features. In 2004 and 2005 an extended outbreak of VRE occurred at a university hospital in Southwestern Germany and an infection control programme was implemented to confine the outbreak. Pulsed-field gel electrophoresis (PFGE), esp PCR, multiple-locus variable number of tandem repeat analysis (MLVA), purK1 typing and multiple-locus sequence typing (MLST) were performed on representative VRE isolates. Twenty-six non-epidemic and two epidemic VRE types (MLST203, MLST280) were identified by PFGE. Seven of the non-outbreak VRE types were esp gene negative, whereas 19 non-outbreak and both epidemic VRE types were esp positive. Eight MLVA types were identified. MLVA type 1 included five PFGE types and MLVA type 159 included 16 PFGE types. Currently there is no efficient method available to identify non-epidemic VRE and avoid unnecessary isolation of patients. More than 50% non-epidemic clones were esp positive; nevertheless, esp PCR appears to be the most promising approach to identify non-epidemic VRE.     

Vaccination of cattle with a recombinant bivalent toxoid against botulism serotypes C and D

Cattle botulism is a fatal intoxication caused by botulinum neurotoxins (BoNTs) produced by Clostridium botulinum serotypes C and D resulting in economic losses. Vaccination is the most effective way to control botulism. However, the commercially available vaccines are difficult and hazardous to produce. Neutralizing antibodies against the C-terminal fragment of the BoNT heavy chain (H_C) are known to protect against lethal doses of BoNTs. We report the vaccination of cattle with a previously tested recombinant chimera consisting of Escherichia coli heat-labile enterotoxin B subunit and the H_C of BoNTs C and D. Vaccinated animals produced neutralizing antibodies against serotypes C and D averaging 5 ± 0 and 6.14 ± 1.06 IU/mL, respectively. For BoNT D, the titers were greater than those measured for the commercial vaccine, which induced titers of 5 ± 0 and 2.85 ± 1.35 against the respective serotypes, suggesting that this chimera is effective against cattle botulism.     

Molecular Typing of Mycobacterium intracellulare Using Pulsed-Field Gel Electrophoresis,Variable-Number Tandem-Repeat Analysis,Mycobacteria Interspersed Repetitive-Unit-Variable-Number Tandem Repeat Typing,and Multilocus Sequence Typing: Molecular Characterization and Comparison of Each Typing Methods

Objectives

Mycobacterium intracellulare is the major causative agent of nontuberculous mycobacteria-related pulmonary infections. The strain typing of M. intracellulare is important for the treatment and control of its infections. We compared the discrimination capacity and effective value of four different molecular typing methods.

Methods

Antibiotic susceptibility testing, hsp65 and rpoB sequencing, pulsed-field gel electrophoresis (PFGE), multilocus sequence typing (MLST), mycobacteria interspersed repetitive-unit-variable-number tandem-repeat analysis (MIRU-VNTR), and VNTR assay targeting 44 M. intracellulare isolates obtained from patients with pulmonary infections were performed.

Results

All the antibiotic susceptibility patterns had no association with the molecular and sequence types tested in this study; however, the molecular and sequence types were related with each other. PFGE gave best results for discriminatory capacity, followed by VNTR, MLST, and MIRU-VNTR.

Conclusion

The high discriminatory power of PFGE, VNTR, and MLST is enough for differentiating between reinfection and relapse, as well as for other molecular epidemiological usages. The MLST could be regarded as a representative classification method, because it showed the clearest relation with the sequence types.     

Genotyping of community-associated methicillin resistant Staphylococcus aureus (CA-MRSA) in a tertiary care centre in Mysore,South India: ST2371-SCCmec IV emerges as the major clone

The burden of community-associated methicillin resistant Staphylococcus aureus (CA-MRSA) is on the rise in population and clinical settings on account of the adaptability and virulence traits of this pathogen. We characterized 45 non-duplicate CA-MRSA strains implicated mainly in skin and soft tissue infections (SSTIs) in a tertiary care hospital in Mysore, South India. All the isolates were genotyped by staphylococcal cassette chromosome mec (SCCmec) typing, staphylococcal protein A (spa) typing, accessory gene regulator (agr) typing, and multi-locus sequence typing (MLST). Four sequence types (STs) belonging to three major clonal complexes (CCs) were identified among the isolates: CC22 (ST2371 and ST22), CC1 (ST772) and CC8 (ST8). The majority (53.3%) of the isolates was of the genotype ST2371-t852-SCCmec IV [sequence type-spa type-SCCmec type], followed by ST22-t852-SCCmec IV (22.2%), ST772-t657-SCCmec V (13.3%) and ST8-t008-SCCmec IV (11.1%). ST237I, a single locus variant of ST22 (EMRSA-15 clone), has not been reported previously from any of the Asian countries. Our study also documents for the first time, the appearance of ST8-SCCmec IV (USA300) strains in India. Representative strains of the STs were further analyzed by pulsed field gel electrophoresis (PFGE). agr typing detected type I or II alleles in the majority of the isolates. All the isolates were positive for the leukotoxin gene, pvl (Panton–Valentine leukocidin) and the staphylococcal enterotoxin gene cluster, egc. Interestingly, multidrug resistance (resistance to ⩾3 classes of non-beta-lactam antibiotics) was observed in 77.8% (n = 35) of the isolates. The highest (75.5%) resistance was recorded for ciprofloxacin, followed by erythromycin (53.3%), and quinupristin–dalfopristin (51.1%). Inducible clindamycin-resistance was identified in 37.7% of the isolates and it was attributed to the presence of erm(A), erm(C) and a combination of erm(A) and erm(C) genes. Isolates which showed a phenotypic pattern of M^R/L^S (macrolide-resistance/lincosamide-sensitivity) harbored the msr(A) gene. In conclusion, we report a high rate of multidrug resistance among Indian strains of CA-MRSA and the emergence of the lineages ST2371 and ST8 in India.     

Molecular Typing and Epidemiology of Human Listeriosis Cases,Denmark, 2002–2012

Denmark has a high incidence of invasive listeriosis (0.9 cases/100,000 population in 2012). We analyzed patient data, clinical outcome, and trends in pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST) of Listeria monocytogenes strains isolated in Denmark during 2002–2012. We performed 2-enzyme PFGE and serotyping on 559 isolates and MLST on 92 isolates and identified some correlation between molecular type and clinical outcome and patient characteristics. We found 178 different PFGE types, but isolates from 122 cases belonged to just 2 closely related PFGE types, clonal complex 8 and sequence type 8. These 2 types were the main cause of a peak in incidence of invasive listeriosis during 2005–2009, possibly representing an outbreak or the presence of a highly prevalent clone. However, current typing methods could not fully confirm these possibilities, highlighting the need for more refined discriminatory typing methods to identify outbreaks within frequently occurring L. monocytogenes PFGE types.     

Genomic characterization of Italian Clostridium botulinum group I strains

Clostridium botulinum is a gram-positive bacterium capable of producing the botulinum neurotoxin, a powerful poison that causes botulism, a severe neuroparalytic disease. Its genome has been sequenced entirely and its gene content has been analyzed. To date, 19 full genomes and 64 draft genomes are available. The geographical origin of these genomes is predominantly from the US. In the present study, 10 Italian genomes of C. botulinum group I were analyzed and compared with previously sequenced group I genomes, in order to genetically characterize the Italian population of C. botulinum group I and to investigate the phylogenetic relationships among different lineages. Using the suites of software ClonalFrame and ClonalOrigin to perform genomic analysis, we demonstrated that Italian C. botulinum group I population is phylogenetically heterogeneous encompassing different and distant lineages including overseas strains, too. Moreover, a high recombination rate was demonstrated in the evolution of C. botulinum group I species. Finally, genome sequencing of the strain 357 led us to identify a novel botulinum neurotoxin subtype, F8.     

A Comparison of Subtyping Methods for Differentiating Salmonella enterica Serovar Enteritidis Isolates Obtained from Food and Human Sources

PurposeTo evaluate the abilities of these subtyping methods, we distinguished Salmonella Enteritidis (S. Enteritidis) isolated from food products and human clinical samples between 2009 and 2010 in Seoul using five subtyping methods.MethodsWe determined the subtypes of 20 S. Enteritidis isolates from food and human sources using phage typing, antimicrobial susceptibility, pulsed-field gel electrophoresis (PFGE), repetitive sequence-based PCR (rep-PCR), and multilocus sequence typing (MLST).ResultsA total of 20 tested isolates were differentiated into six antimicrobial susceptibility patterns, three different phage types, four different PFGE profiles, seven rep-PCR patterns, and one MLST type. Food isolates were considerably more susceptible to antibiotics than human isolates. We were best able to discriminate among S. Enteritidis isolates using rep-PCR, and obtained the highest Simpson's diversity index of 0.82, whereas other methods produced indices that were less than 0.71. PFGE pattern appeared to be more related to antimicrobial resistance and phage types of S. Enteritidis isolates than rep-PCR. MLST revealed identical alleles in all isolates at all seven loci examined, indicating no resolution.ConclusionThe results of this study suggest that rep-PCR provided the best discriminatory power for phenotypically similar S. Enteritidis isolates of food and human origins, whereas the discriminatory ability of MLST may be problematic because of the high sequence conservation of the targeted genes.     

Evaluation and Selection of Multilocus Variable-Number Tandem-Repeat Analysis Primers for Genotyping Brucella abortus Biovar 1 Isolated from Human Patients

ObjectivesBrucellosis is the most common bacterial zoonosis in the world. Multilocus variable-number tandem-repeat (VNTR) analysis (MLVA) is a molecular method for genotyping bacterial species. Brucella abortus biovar I was isolated from most of the brucellosis-suspected patients in Korea. This study was conducted to investigate the ability of various MLVA primers that are used for molecular typing B. abortus isolates and for analyzing their epidemiological data.MethodsA total of 80 human isolates of B. abortus biovar I isolated from human patients and the reference strain were used for MLVA. Genetic diversity was determined by calculating the Simpson's diversity index (DI) of each VNTR locus. The Brucella strains were subcultured 30 times to determine the stability of each locus. The DNA of the strains cultivated in each passage was extracted and subjected to MLVA for further investigation.ResultsThe 15 VNTR loci were selected based on high DI values. The DIs of the 15 VNTR loci showed considerable discrimination power ranging from 59% for Bruce 43 to 87% for Bruce 22. Bruce 09, Bruce 11, Bruce 16, Bruce 42, and Bruce 43 were confirmed to remain stable in vitro among the 15 VNTR loci selected.ConclusionThe results of this study suggest that the five loci subsets may be a useful epidemiological tool for investigating B. abortus biovar 1 outbreak.     

Allelic diversity of variable number of tandem repeats provides phylogenetic clues regarding the Mycobacterium tuberculosis Beijing family

The Beijing family is the putative hypervirulent lineage of Mycobacterium tuberculosis that has been endemic in East Asia and has disseminated worldwide. The genetic population structure of Beijing family strains with regard to Japan is notable in its high diversity and dominance of the ancestral sublineage, in contrast to the modern sublineage found worldwide. Therefore, it is expected to be a suitable population model for investigating the microevolutionary process of the lineage. Variable number of tandem repeats (VNTR) has become a reliable genotyping method for M. tuberculosis, but its dynamics in the phylogenetic process remains unclear. Using 355 clinical Beijing family isolates in Japan, genetic traits, including VNTR, were analyzed and subjected to minimum spanning tree (MST) reconstruction. In the results, the topology of the tree was tightly related to other genotypic characters. We also found that some VNTR alleles were specific to each sublineage and provided clues for reconstructing a valid MST topology for the population. It is suggested that VNTR typing can elucidate genetic markers representing the phylogenetic classification in the lineage.     

中国2000-2008年间甲型副伤寒沙门菌脉冲场凝胶电泳和多位点序列分析

目的 分析我国2000-2008年间甲型副伤寒沙门菌流行株的分子分型及病原进化上的特征.方法 应用以Spe Ⅰ为限制性内切酶的脉冲场凝胶电泳(pulsed-field gel electrophoresis,PFGE)方法 和基于9个管家基因位点(aroC、thrA、hisD、purE、sucA、dnaN、hemD、adk和purA)的多位点序列分析(multilocus sequence typing,MLST)分型方法 ,对我国2000-2008年间分离自10个地区的118株甲型副伤寒沙门菌流行株进行分析.结果 应用PFGE方法 将118株甲型副伤寒沙门菌分为32个型,其中包含5株以上的优势PFGE带型有5种.而应用MLST方法 ,所有菌株只分出了2个序列分型(sequence type,ST),各菌株的管家基因序列高度保守,呈现高度克隆化.结论 中国2000-2008年间甲型副伤寒沙门菌菌株应用MLST这种分型方法 很难区分,MLST不适于甲型副伤寒沙门菌的暴发调查和流行病学监测.目前中国的甲型副伤寒是由高度克隆化的菌株引起全国范围的扩散 流行.随着年份的变迁,也积累了散在的变异.     

The molecular epidemiology of serial Candida tropicalis isolates from ICU patients as revealed by multilocus sequence typing and pulsed-field gel electrophoresis

The genetic profiles of 50 Candida tropicalis isolates serially collected from 14 patients during a prospective surveillance study in adult intensive care units (ICUs) were characterized by multilocus sequence typing (MLST) and pulsed-field gel electrophoresis (PFGE) of NaeI restriction fragments. A total of 21 diploid sequence types (DSTs) and 43 genotypes were differentiated by MLST and PFGE, respectively. Significant correlations were found between PFGE genotypes and DST types (P < 0.05). Dendrograms generated by either MLST or PFGE-NaeI showed that most isolates from the same patient co-clustered with high similarity regardless of the anatomical source of isolation. Maintenance, microvariation or replacement of C. tropicalis isolates could be observed within the individual patients by further analysis of variations in MLST sequence data. Antifungal susceptibility testing revealed that 17 (34%) of 50 isolates presented high MICs to flucytosine (MIC ≥ 8 μg/mL). Sixteen (94%) of these isolates belonged to DST 164, and these were collected from four patients with different PFGE genotypes. Isolates sharing the same DST may represent a common clone that underwent extensive mutation over time to cope with drug selection pressure, different hosts or different geographic environments.     

A reliable combination method to identification and typing of epidemic and endemic clones among clinical isolates of Acinetobacter baumannii

The multi-drug resistant (MDR) Acinetobacter baumannii as an important nosocomial pathogen has emerged a global health concern in recent years. In this study, we applied three easier, faster, and cost-effective methods including PCR-based open reading frames (ORFs) typing, sequence typing of bla_OXA-51-like and RAPD-PCR method to rapid typing of A. baumannii strains. Taken together in the present study the results of ORFs typing, PCR-sequencing of bla_OXA-51-like genes and MLST sequence typing revealed there was a high prevalence (62%, 35/57) of ST2 as international and successful clone which detected among clinical isolates of multi-drug resistant A. baumannii with ORF pattern B and bla_OXA-66 gene. Only 7% (4/57) of MDR isolates belonged to ST1 with ORF pattern A and bla_OXA-69 gene. Interestingly, we detected singleton ST513 (32%, 18/57) that encoded bla_OXA-90 and showed the ORF pattern H as previously isolated in Middle East. Moreover, our data showed RAPD-PCR method can detect divergent strains of the STs. The Cl-1, Cl-2, Cl-3, Cl-4, Cl-10, Cl-11, Cl-12, Cl-13 and Cl-14 belonged to ST2. While the Cl-6, Cl-7, Cl-8 and Cl-9 belonged to ST513. Only Cl-5 belonged to ST1. It seems that the combination of these methods have more discriminatory than any method separately and could be effectively applied to rapid detection of the clonal complex (CC) of A. baumannii strains without performing of MLST or PFGE.     

Molecular characterization of macrolide resistant Streptococcus pyogenes isolates from pharyngitis patients in Serbia

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.