| Summary: | 碩士 === 國立嘉義大學 === 微生物免疫與生物藥學系研究所 === 102 === Background: Gram positive Streptococcus agalactiae (group B streptococcus; GBS) is human gastrointestinal and genitourinary normal flora and cause early-onset or late-onset sepsis in newborns. In neonates, early-onset disease (EOD) and late-onset disease (LOD) are defined as occurring within the first 7 days and occurring from days 8 to 90, respectively. GBS are categorized into ten serotypes, including Ia, Ib, II to IX based on the differences in capsular polysaccharide synthesis (cps) gene clusters. The penicillin of -lactam drug is chosen for treatment of the GBS infections, while drugs of macrolides and lincosamides are used for the patient allergy to penicillin. The major mechanisms for macrolide and lincosamide are the methylation of 23S rRNA by ermB gene encoding ribosomal methylase and the macrolide efflux coded by the mefE gene. Macrolide-resistant Streptococcus pyogenes and S. pneumonia have increased currently in clinical isolates, and the macrolide-resistant genes are located in Tn916 family transposons. These resistance genes may be transferred between Streptococcus spp. through transposition.
Purpose: GBS serotypes of human isolates were investigated for their genetic relatedness, differences in antimicrobial resistance and its related genes associated with transposon.
Materials and Methods: 562 GBS strains were collected from patients visiting Chiayi Chang-Gung Hospital (CGMH-C) during 2007~2008 and 2011~2012. GBS strains were collected from Linkou (CGMH-L) (34 strains) and Kaohsiung Chang-Gung Hospital (CGMH-G) (23 strains) and Chi-Mei Medical Center (39 strains) in 2012. The serotypes and the ermB and mefE genes were determined by multiplex PCR amplification. Antimicrobial susceptibility to β-lactam, fluoroquinolone, macrolides and lincosamide was investigated by disc diffusion method. Genotyping were determined by pulsed field gel electrophoresis (PFGE) analysis. The mutations in quinolone resistance-determining region (QRDR) of gyrA and parC in fluoroquinolone resistant isolates were determined by sequencing. The sequences flanking the ermB and mefE genes were determined by PCR, inverse PCR and sequencing.
Results and Discussion: Prevalent serotypes and their genotype changed by year and geographic regions. (1) In studying period, serotypes Ib and V were most prevalent in 2007 and 2008, while serotypes III and VI were most prevalent in 2011 and 2012. This demonstrates that the prevalent serotypes changed by years. In 2012, serotype VI become the most prevalent in four hospitals. (2) In serotype Ib, 71 (67%, 71/106) isolates belonged to pulsotype Ib-I. Pulsotype V-I was major genotype (68.3%, 112/164) among 11 pulsotypes of the serotype V of 164 GBS strains examined. In serotype VI, 79 (61.2%, 79/129) isolates belonged to pulsotype VI-I. However, there were more than 30 pulsotypes in serotype III. These results indicate major clonal dissemination of serotype Ib, V and VI and diverse genomic variations in serotype III. (3) Sequencing of QRDR fragment confirmed mutations at Ser81Leu for GyrA and Ser79Tyr, Ser79Phe and Asp83Tyr for ParC sequence in fluoroquinolone resistant isolates, suggesting point mutations in QRDR can lead to fluoroquinolone resistance. Additionally, each mutation was associated with serotype. (4) 271 GBS strains (48.2%) were resistant to erythromycin in Chiayi CGMH. The ermB gene was observed in 44.3% (249/562) isolates and mefE gene was observed in 5% (28/562) isolates. The ermB gene was major responsible for erythromycin resistance. (5) In 32 isolates, 21 isolates consisted of ermB-associated Tn6003 in which 11 isolates observed in serotype Ib.
Conclusion: (1) Pulsotype Ib-1, pulsotype V-1 and pulsotype VI-1 were main clones to disseminate and diverse genomic variations in serotype III. (2) The ermB gene is major gene for resistance to macrolides and lincosamide. (3) Change between amino acid group in QRDRs of GyrA and ParC could develop resistant to fluoroquinolones. (4) Serotype Ib isolates carried ermB-associated Tn6003 more easily.
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