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標題: B 群鏈球菌對巨環內酯類和喹諾酮類藥物抗藥 機制之研究並探討人體細菌與 太古生物菌相變化
Mechanism of macrolide and quinolone resistance in Streptococcus agalactiae and study the bacterial and archaeal communities in human body
作者: Wen-Tsung Lee
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Streptococcus agalactiae (Group B Streptococcus, GBS), is the most common pathogen causing infections among perinatal women and neonatal babies. Nonetheless,
there are few studies on the occurrence of GBS among the women and the mechanisms of the GBS resistance to quinolones and macrolides in Taiwan.
Bacteria was identified from the vagina of 388 out of 1088 subjects, and 188 isolated were confirmed to be S. agalactiae. The isolation rate of GBS from pregnant
women was significantly higher at 21.8% compare with the non-pregnant women of 13.2%. Antibiotic susceptibility test of the 188 GBS isolates revealed a high
non-susceptible rate for erythromycin (50.0%) while the rate for levofloxacin was only 4.8%. Among 94 erythromycin non-susceptible GBS isolates, ermB gene was detected 83.1% (59/71) for those GBS that were non- susceptible to both clindamycin and tetracycline, which was significantly higher than GBS that are susceptible to clindamycin but resistant to tetracycline at 43.8% (7/16). No ermA or mef gene was detected in any isolate. Mutations were detected in the parC and gyrA genes in 14 out of 18 levofloxacin non-susceptible isolates for comparison test. The predominant mutation type was the combination of Ser79Tyr in parC and Ser81Leu mutations in gyrA. In
summary, there were various antibiotic patterns on different region, resistance to tetracycline and erythromycin is higher than the rate observed for other regions of the world, while the resistance rate for levofloxacin was relatively lower in Taiwan.
Although there has been some investigations in the Bacterial and Archaeal communities of human body and their related diseases, however there are only few directed linked the involvement of Archaea and/or Bacteria from the local resident of human body. In the present research, we studied the archaeal and bacterial 16S rDNAs using a polymerase chain reaction (PCR) amplified by specific universal primers to examine the microbial communities within the analyzing samples. The 16S rRNA gene libraries of test samples were compared with the known 16S rRNA genes for systematic analysis to determine the diversity of microorganisms within the samples. Results may also contribute to assist in comparative analyses when examining the key bacterium
related to diseases. We also used SYBR Green1 to perform a quantitative PCR to analyze the concentration and quantity of the microorganisms within the samples. Using the qPCR examination method, 22 samples of the 39 periodontal disease cases were found to contain merA gene of methanogen (56.4%) . Samples of periodontal disease were separated into different severity groups and statistical significance was found (p=0.03) with the severe group containing the highest amount of methanogen merA gene (p<0.01). In a comparison between healthy and intestinal disease cases of 29 excrement test samples, Archaea did not have any significant result (8.3% vs. 17.6%, p=0.474). Additionally, the vaginal secretion, urinal and intestinal test samples did not show Archaea findings by 16S rRNA PCR. However, Lactobacillus iners has a high
ratio that appears during the stage between the normal and abnormal transition period.
Within a total of 11 test samples of 14 colonies were Methanogen (2 were M. smithii and 11 were M. oralis and one Methanobrevibacter sp.) as examined by the 16S rRNA
gene and clone sequence. Methanogen from stool samples identification as M. smithii and only M. oralis was detected in samples with periodontal disease.

B 群鏈球菌 (group B streptococcus, GBS) 是懷孕婦女及新生兒最常見的侵襲性病原菌,新生兒如感染 B 群鏈球菌,容易造成新生兒敗血症而導致死亡。然而,台灣對於婦女感染 B 群鏈球菌之盛行率及 B 群鏈球菌對巨環內酯類 (macrolide)和喹諾酮類 (quinolone) 藥物抗藥機制卻很少有相關之研究。因此,本研究目的除了針對台灣婦女感染 B 群鏈球菌盛行率及藥物感受性之研究,並探討 B 群鏈球菌對巨環內酯類、喹諾酮類這兩類抗生素產生抗藥性之可能機制。
1088 位受試者的陰道檢體有 388 個檢體分離出微生物病原菌,並鑑定分離出188 株 B 群鏈球菌。分析結果發現懷孕婦女 B 群鏈球菌之帶菌率為 21.8% 顯著高於非懷孕婦女的 13.2%,兩者統計上具有顯著差異。B 群鏈球菌對紅黴素非感受性之比率高達 50%。不過對屬於喹諾酮類抗生素的左氧氟沙星 (levofloxacin) 非感受性比率只有 4.8%。進一步分析 94 株對紅黴素非感受性之 B 群鏈球菌,有 71 株同時對克林達黴素 (clindamycin) 及四環黴素 (tetracycline) 非感受性之 B 群鏈球菌中,且有 83.1% (59/71) 帶有對紅黴素產生抗藥作用的 ermB 基因比率,顯著高於16 株對克林達黴素感受性但對四環黴素非感受性之 B 群鏈球菌只有 43.8% (7/16)帶有 ermB 基因。但所有分離之 B 群鏈球菌株並未測得其他可對紅黴素產生抗藥作用之 ermA 和 mef 基因。另外,有關 B 群鏈球菌對左氧氟沙星之藥物感受性試驗,台灣台南與中國浙江比對試驗之 18 株對左氧氟沙星非感受性 B 群鏈球菌中,測得14 株在 parC 和 gyrA 基因發生序列突變以致結構改變而產生抗藥性,主要胺基酸變異型態為 parC 基因第 79 胺基酸位置 (Ser79Tyr),同時在 gyrA 基因第 81 胺基酸位置 (Ser81Leu) 發生變異。總結來說,我們觀察到 B 群鏈球菌在不同地區呈現不同藥物感受性型態,國內 B 群鏈球菌對四環黴素及紅黴素兩種抗生素出現非感受
有關人體中細菌與太古生物菌相變化的探討上,目前已有少數研究分析人體中的太古生物甲烷菌及其與人類疾病的相關性,但是並沒有對人體與人類疾病相關的整體太古生物與細菌分析。本研究分別以太古生物與細菌 16S rRNA 基因通用引子(universal primer),進行聚合酶連鎖反應(Polymerase Chain Reaction, PCR),分析各檢體中太古生物與細菌的微生物種類與組成,以建立各檢體之 16S rRNA 基因的基因庫,並與已知 16S rRNA 基因資料庫比對與系統演化分析,確定各檢體中微生物種類與多樣性,以研究與疾病相關之重要關鍵菌種。另外也採用 SYBR GreenI 嵌合螢光法進行即時定量 PCR (quantitative PCR, qPCR),分別分析檢體中太古生物與細菌定量濃度與微生物量的組成差異。
牙周病患 39 個檢體,利用 qPCR 方法 (mcrA gene) 檢測出 22 個檢體含有甲烷菌 (56.4%)。甲烷菌 mcrA 基因在牙周病嚴重度不同部位檢體之檢出率,具有統計上差異 (p=0.03),牙周病嚴重部位檢體甲烷菌之含量,相較上亦比嚴重度低的部位為高 (p<0.01)。在健康人與腸胃道疾病之 29 個糞便檢體,太古生物 16S rRNA檢出率則未出現顯著差異 (8.3% vs.17.6%, p=0.474)。有關陰道分泌物、尿液檢體
及腸胃道疾病病理檢體均未測得太古生物,值得注意的,惰性乳酸菌 (Lactobacillus iners) 這種介於正常與不正常過渡期才會出現之乳酸菌,於陰道分泌物檢體之微生物出現的比率很高。綜合以上 11 個陽性檢體共 14 個轉殖株,經 16S rRNA 基因選殖定序確認後均為甲烷菌 (2 個 16S rRNA 序列比對結果為 Methanobrevibacter smithii、11 個 16S rRNA 序列比對結果為 M. orlais、另一個為 Methanobrevibacter sp.)。糞便檢體 DNA 進行 16S rRNA PCR 反應,選殖定序所得為 M. smithii 16S rRNA序列,牙周病檢體 DNA 選殖定序幾乎均為 M. oralis,推測菌種與檢體部位有高度相關性。
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