MOLECULAR TYPING OF HUNGARIAN STREPTOCOCCUS PYOGENES ISOLATES BARBARA
MOLECULAR TYPING OF HUNGARIAN STREPTOCOCCUS PYOGENES ISOLATES BARBARA 1 KRUCSÓ , 1 Abstract The aim of the authors was the examination of the genetic relatedness of the Hungarian Streptococcus pyogenes clinical isolates by emm amplicon characterisation and PFGE analysis, as well as the determination of the prevalent emm types among the strains originated from different infections. Methods From 2003 to 2004 40 S. pyogenes strains were isolated from two Hungarian counties. Out of the isolates, 15 originated from respiratory, 17 from superficial pyogen, and 8 from invasive infections, like Streptococcal shock syndrome (STSS), meningitis and sepsis. Emm gene detection and restriction analysis of the emm amplicon (with Hinc. II and Hae. III; Dde. I enzymes) was performed in all strains. The sequence of the emm amplicon from all invasive isolates and from one strain of all RFLP groups was determined, and macrorestriction chromosomal analysis by PFGE (Pulsed Field Gel Electrophoresis) method for these isolates was also performed. Results As a result of the restriction analysis of the emm amplicons 17 restriction patterns were differentiated. Most emm types were standard types: emm 1. 0, emm 12. 0, emm 28. 0, emm 59. 0, emm 66. 0, emm 75. 0, emm 76. 0, emm 77. 0, emm 80. 0, emm 81. 1, emm 84. 0, emm 110. 1, emm 118. 0, plus one new allelic variant (emm 110. 2) was identified. Moreover, two GAS strains had a type (stg 6 and stg 485) previously found in group G streptococci. The emm types varied among the isolates originated from different sources. The next emm types were identified among the invasive isolates: emm 1. 0, emm 28. 0, emm 66. 0, emm 80. 0, emm 81. 1, emm 84. 0, stg 6. Some emm type could be further differentiated into several PFGE genotypes, and within a PFGE genotype different emm types could be observed. Conclusions This report provides epidemiological information from a geographic area has not been examined by emm characterisation. PFGE and emm type analysis showed high genetic diversity among the 40 GAS strains. The most frequent emm types were emm 1. 0, emm 28. 0, emm 81. 1 and emm 84. 0 in the two counties. Introduction and purpose In the 1960’s streptococcal epidemiological work has mainly focused on the scarlet fever, the incidence of which has been recorded in Hungary ever since 1881. In 1962 the number of the reported scarlet fever cases was 21, 000, beside the unrecorded but presumably high number of cases of streptococcal sore throat, erysipelas, nephritis etc. Nationwide surveys were performed by applying M and T protein typing in 1964 -65 and in 1968 -69. M types M 1, M 3 and M 12 has been observed as predominant types. From 1970 to 2003 epidemiological typing has not been performed in Hungary at all. The number of the reported cases in the last years in the Hungarian public health and hospital laboratories were as follows: 9334 in 2000, 7394 in 2001 and 5913 in 2002. About 0, 9 percent of the strains were originated from invasive sources. MÁRIA 3 GACS , JUDIT 1 PÁSZTI Department of Phage and Molecular Typing, ”Johan Béla” National Center for Epidemiology, Hungary 2 Nógrád 3 KÁROLY 2 CSISZÁR , County Institute of the National Public Health Service, Salgótarján, Hungary Department of Bacteriology, ”Johan Béla” National Center for Epidemiology, Hungary Though in our days the number of the infections caused by S. pyogenes are not as high as in the 1960’s, the occurent serious clinical pictures makes it desirable to perform more detailed investigations applying modern epidemiological methods. Our purpose was: Fig. II Dendrogram generated by Quantity One software showing the results of cluster analysis on the basis of PFGE fingerprinting Results Similarity % Table I RFLP-types and their distribution according to the different secretion sources 0. 40 0. 46 0. 5 0. 26 0. 89 0. 54 0. 32 0. 61 0. 7 0. 41 0. 57 • to establish a database from the DNA fingerprints of the isolates as a basis for an improved laboratory study of streptococcal diseases and their clinical and epidemiological problems in Hungary, • to examine the genetic relatedness of the Hungarian Streptococcus pyogenes clinical isolates by emm amplicon characterisation and PFGE analysis among the strains originated from different infections. 0. 48 0. 37 0. 54 0. 56 0. 58 0. 44 0. 48 0. 56 9, emm 80 3, stg 485 26, emm 77 7, emm 28 28, emm 1 14, emm 1 3 11, emm 1 2, emm 28 4, emm 76 6, emm 28 5, emm 28 17, emm 110. 2 10, emm 81 27, emm 84 13, emm 66 12, emm 84 22, emm 85 18, emm 75 21, emm 12 15, stg 6 24, emm 76 19, emm 118 20, emm 110. 1 25, emm 59 23, emm 81. 1 The method of UPGMA was used to generate the family tree. Lane serial numbers correspond to lane numbers in Figure I. The corresponding emm types are indicated on the right side of the dendrogram. Conclusions Seventeen emm types and PFGE profiles were observed. The prevalent emm types were: emm 1, emm 81. 1, emm 84 and emm 28, out of which emm 1 and emm 28 are reported as worldwide 40 S. pyogenes isolates from: disseminated. - respiratory (2 chest punctates, 13 throats), The following emm types were observed in the - superficial pyogen (13 wounds, 1 postoperative invasive samples: emm 1, emm 66, emm 80, emm 81 Fig. I infection, 2 infected burned skin, 1 fasciitis necrotisans), and stg 6. - and invasive infections (8 blood ) Pulsed-field Gel Electrophoresis patterns We identified a new allelic variant: emm 110. 2. originated from two Hungarian counties were examined. of the invasive strains and that of the representatives of In the case of two GAS strains the emm the different RFLP-types stg 6 and stg 485 were identified which • PCR and RFLP analysis of the emm gene was types were previously reported as found in M 2 3 4 5 6 7 M 9 10 11 12 13 14 15 M 17 18 19 20 21 22 23 24 25 26 27 28 M performed for all of the strains according to 436, 5(kb) group G streptococci only. Further 388 examination of these isolates is going on. www. cdc. gov/ncidod/biotech/strep/protocols. html. 339, 5 291 Our examinations proved that using PFGE 242, 5 and RFLP methods the genetic relatedness of • RFLP-groups were differentiated on the basis of the Hungarian strains can be well 194 RFLP patterns obtained for the emm amplicons. characterised and by sequencing reactions the 145, 5 emm types can be determined and thereby • Pulsed-field Gel Electrophoresis and emm sequence 97 become comparable with international data. The determination were performed for all invasive samples results obtained in this pilot study provide basis for (n=8) and for 1 representative sample of all (n=17) RFLP 48, 5 further epidemiological studies on the Hungarian S. groups. M: Lambda Ladder, Chromosomal DNA was digested with Sma. I pyogenes strains. PFGE- was used according to Goering and Winters 1992, JCM, 30: 577 -580 Methods
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