志賀氏宋內菌對頭孢曲松抗藥性機制之研究

Abstract

由志賀氏痢疾桿菌 (Shigella spp.) 所引起的細菌性痢疾為全球最常發生之腸胃疾病之一。台灣地區桿菌性痢疾的主要感染源為志賀氏副痢疾菌 (S. flexneri) 及志賀氏宋內菌 (S. sonnei)，臨床上以抗生素治療可縮短病程並達治癒之目的，但若未完成一完整療程，則會導致細菌在抗生素壓力下產生抗藥性。 頭孢菌素 (cephalosporin) 為全球最常使用的抗生素，而頭孢曲松 (ceftriaxone) 為頭孢菌素中血清半衰期最長的一種抗生素，其殺菌機制在於抑制細菌細胞壁之合成。細菌可能藉由排藥幫浦之表現或產生乙型單環醯胺? (beta-lactamase) 而對頭孢菌素產生抗藥性，其中又以後者所造成的抗藥性表現較為明顯。將臨床分離所得之志賀氏宋內菌培養於添加頭孢曲松的培養基進行誘導培養，以提高其抗藥性表現，觀察其最低抑制濃度，並分別以聚合?鏈反應及二維式電泳檢測其乙型單環醯胺?基因及其他蛋白質在抗藥性產生過程中的表現情形。 結果顯示 2 株臨床為敏感性之菌株在培基中之 ceftriaxone 添加濃度增加至 16 μg/mL 時，其抗生素感受性形式直接由敏感性變成抗藥性，推測在這個過程中，菌株可能啟動了某種抗藥性機制而使得對藥物的耐受性急速增加。而以聚合?鏈反應對抗藥性基因進行檢測，發現在所有臨床及誘導菌株之質體 DNA 上均帶有與 Salmonella enterica subsp. enterica serovar Choleraesuis plasmid ampC 具有 99% 相似度的基因存在。在蛋白質表現層面上，也發現高度抗藥性菌株增強表現 AmpC β-lactamase，而在敏感性菌株上則無該蛋白質點表現，證明經 ceftriaxone 誘導抗藥性產生之 S. sonnei 藉由 AmpC β- lactamase 之產生的確對該抗生素產生高度抗藥性，同時本研究中以基因及蛋白質層面探討抗藥性之表現所得之結果相符合。

Shigellosis caused by Shigella spp. is one of the most frequently happened gastrointestinal disease worldwide. The shigellosis outbreaks in Taiwan were mainly caused by S. flexneri and S. sonnei. Antibiotics are used to treat or to shorten the carrier phase, however, Shigella spp. may acquire antibiotic-resistance on account of incomplete course of treatment. Cephalosporin is the most commonly used antibiotics in the whole world. Ceftriaxone has the longest serum half-life among the cephalosporins and it impedes the synthesis of bacteria cell wall. Bacteria might acquire drug-resistance by efflux pump overexpression or beta-lactamase producing in order to fight the antibiotics pressure, and the effect of the latter one is more helpful. The clinical isolated S. sonnei was incubated in media with ceftriaxone in order to enhance the ceftriaxone-resistance. Minimal inhibition concentration (MIC) value of experimental strains is determined. Polymerase chain reaction (PCR) and two-dimensional electrophoresis are used to detect the beta-lactamase gene and protein expression. Two clinical ceftriaxone-susceptible strains became resistant ones when the ceftriaxone concentrations in the media increased to 16 μg/mL. We supposed that some mechanisms may be triggered in the process among the susceptible strains. The PCR results revealed that the clinical and ceftriaxone-resistant strains carry the plasmid-mediated ampC, having 99% homology with Salmonella enterica subsp. enterica serovar Choleraesuis plasmid ampC. The expression of AmpC beta-lactamase on ceftriaxone-resistant strains, which did not observed on susceptible strains, increased on the two-dimensional electrophoresis gel. The results of studying the drug-resistance based on both the gene and protein level are coincident in this study.