Rapid species identification, molecular epidemiology and drug resistance gene analysis of β-hemolytic group G streptococci

• Main Authors: Liang-Chun Liu, 劉亮君
• Other Authors: 鄧麗珍
• Format: Others
• Language: zh-TW
• Published: 2006
• Online Access: http://ndltd.ncl.edu.tw/handle/47146552407705535241

碩士 === 國立臺灣大學 === 醫學檢驗暨生物技術學研究所 === 94 === Human isolate β-haemolytic group G streptococci (GGS) include two major species- Streptococcus anginosus, Streptococcus dysgalactiae subsp. equisimilis. These bacteria are usually regarded as normal flora colonized human skin, throat, intestine, and may cause serious disease such as bacteremia, endocarditis, pharyngitis, streptococcal toxic shock syndrome on immunodeficiency patients. Clinical identification of group G streptococci with conventional methods is usually to the group level, not the species level. However, species identification should not been ignored because of the different clinical significance between two species. In this report, we collected 408 clinical isolates from 1998 to 2004 in NTU hospital and used anginosus specific primers combined with conventional methods to rapidly identify two species in group G streptococci. The results showed that the number of S. dysgalactiae subsp. equisimilis was twice as many as S. anginosus, and was more invasive than S. anginosus through specimen data analysis. Due to the reports that some specific emm types are responsible for the invasion of GGS, and the recurrent infection of 14 patients, we wanted to connect the relationships between specific emm types and diseases through emm typing and PFGE genotyping. We found out that almost all the S. dysgalactiae subsp. equisimilis were emm typable (with one exception), and about 11.27% were stG6.1 or stG485 respectively, while only 0.85% (n=13) S. anginosus are emm detectable. Meanwhile, recurrent patients were mostly infected by stG485 or stG6.1 S. dysgalactiae subsp. equisimilis. Thus, we could conclude that some specific types are highly responsible for recurrence and invasive disease. In order to understand the drug susceptibility of group G streptococci, we tested with 18 antibiotics and analyzed drug resistance genes. About 20% isolates resistant to macrolide and streptogramin B, and 22% isolates resistant to lincosamide. The low MIC ranges of other antibiotics reveal that clinical drug choice is still working. After double disc testing, 2 out of 50 isolates (4%) were inducible MLSB, 96% displayed M phenotype, and 14 isolates were constitutive MLSB. To further illustrate the macrolide resistance gene, erm(B), erm(TR), mef(A) were tested. Among 64 isolates, 7.8% had erm(B), 4.7% had erm(TR), 75% had mef(A) gene, and 3.1% had both erm(B) and mef(A). At last, because erm(T) gene in S. bovis was flanked by IS1216, we try to find out whether GGS had similar structure. The results of long-term PCR, TA cloning and sequencing shows that there was a tet(S) gene, instead of erm(T), inside the IS1216. This structure was similar to Lactocococcus lactis subsp. lactis pk214. PCR screening reveals 12 isolates displayed IS1216-tet(S) fragment, and southern blotting shows that there was only a single copy of this structure. Also, the PFGE of Apa I could tell us the clonal spreading of two kinds of IS1216-tet(S) models. PFGE of I-Ceu I confirmed the chromosome position of IS1216-tet(S), and the Long-Accurate PCR sequencing shows the upstream and downstream genes of the three different truncated models. The horizontal transfer of the IS1216- tet(S) through conjugation was not detected, however, we assumed that transposon or plasmid with the IS1216-tet(S) prototype was once largely disseminated, and might have different level of deletion during the process of chromosome insertion.