台灣地區立即性食用點心中對頭孢菌素非敏感性細菌之特性研究

Characterization of ceftazidime non-susceptible bacteria from ready-to-eat Taiwanese snacks

詹閔喻(Min-Yu Chan)

指導教授 ： 曾嵩斌

高雄醫學大學/健康科學院/醫學檢驗生物技術學系碩士班/碩士(2015年)

摘要

食物對於微生物來說是一個很好的載體,微生物可藉由食物來找到它最佳的宿主,進而傳播出去。大多數食品安全問題以及食源性疾病包括抗藥菌株的產生,是由於人類接觸到具有抗藥能力的細菌所引發。細菌的耐藥性已經成為一個重要的公共衛生問題，使治療更加地困難。
因此為了瞭解台灣立即性食用點心中微生物的含量及分佈,總共採集了270個食物樣品,其中有90份涼麵、90個春捲及90份水果切盤,分別來自高雄、台中和台北。篩選條件為8μg/ml ceftazidime EMB。
發現到在90份涼麵中有38株對頭孢菌素非敏感性菌株 (n=38/90, 42.22%)分別在高雄, n=11株; 台中, n=10株; 台北, n=17株；在 90份春捲中有16株對頭孢菌素非敏感性菌株 (n=16/90, 17.7%) 分別為高雄, n=5株; 台中, n=8株; 台北, n=3株；而在90份的水果切盤中發現到16株對頭孢菌素非敏感性菌株 ( n=16/90, 17.7%)分別為高雄, n=3株; 台中, n=6株;台北, n=7株。在涼麵所發現到的菌株數量比另外兩種可立即食用的點心高出許多。
藉由16S rDNA定序鑑定出70株對頭孢菌素非敏感性菌株，屬於Acinetobacter spp.有22株；Pseudomonas spp.有12株 ；Stenotrophomonas maltophilia 有21株；Enterobacteriaceae 有15株。接著使用脈衝式膠體電泳來進行分子流行病學和細菌親緣性分析，可以發現到來自北中南同屬的菌株間相似性非常低，並無單一菌在台灣地區傳播擴散。藥敏試驗是根據臨床與實驗室標準協會採用凝膠稀釋分析法。結果顯示這些食物菌株大多為多重抗藥性菌株，其比例如下Acinetobacter spp.20/22,90.9%；Pseudomonas spp.12/12,100%；Stenotrophomonas maltophili,14/21,66.6%；Enterobacteriaceae,14/15,93.3%。
針對Extended-spectrum β-lactamases (ESBL) 基因 (CTX−M1, CTX−M9 , TEM , SHV , CMY , DHA , PER , VEB , OXA-1 , OXA-2 , OXA-9 , OXA-10,OXA-48 ) 使用聚合酶連鎖反應﹝PCR﹞去偵測。在春捲中的Acinetobacter soli和Acinetobacter baumannii發現到PER (1/16,6.25%) 和CTX-M9 (1/16,6.25%)基因存在；在涼麵中的Pseudomonas geniculate也偵測到CMY(1/10,10%)基因；而水果切盤中的Acinetobacter radioresistens也偵測到PER (3/16,18.7%)基因存在。除此之外，使用carbonyl cyanide m-chlorophenylhydrazone (CCCP)輸出幫浦抑制劑來檢測菌株是否有輸出幫浦過度表現 。Acinetobacter spp.中有16株(n=16/22, 72.7%)有明顯的輸出幫浦過度表現；在Pseudomonas spp.有3株(n=3/12, 25%)有明顯的輸出幫浦過度表現； 在Stenotrophomonas maltophilia 有3株(n=3/21,14.2%)有明顯表現出具有明顯的輸出幫浦過度表現；在Enterobacter cloacae發現到有1株(n=1/15, 6.66%)有明顯的輸出幫浦過度表現。

關鍵字

可立即食用點心 ； 頭孢菌素

並列摘要

Food is a good vehicle by which many bacteria can reach a suitable colonization site in a host. Most food safety problems and food-borne Illness including antibiotic resistant bacteria occur via human exposure to pathogen. Antibiotic resistance bacteria has become an important public health concern. Besides, this resistance is causing new infections and making it difficult to treat others.
In order to understand the prevalence of antibiotic-resistant bacteria from food, a total of 270 food sample (90 spring rolls,90 cold noodles and 90 fruit dishes ) were collected from 3 cities (Kaohsiung, Taichung and Taipei) in Taiwan. Samples were enriched by BHI broth 16 hrs and subsequently plated on eosin-methylene blue (EMB) agar which contains ceftazidime (8μg/ml). The 38 ceftazidime non-susceptible isolates (n=38/90, 42.22%) were isolated from 90 cold noodles (Kaohsiung, n=11; Taichung, n=10; Taipei, n=17). The 16 ceftazidime non-suscetable isolates (n=16/90, 17.7%) were isolated from 90 spring rolls (Kaohsiung, n=5; Taichung, n=8; Taipei, n=3). The 16 ceftazidime non-suscetable isolates (n=16/90, 17.7%) were isolated from 90 fruit dishes (Kaohsiung, n=3; Taichung, n=6; Taipei, n=7).
These findings suggest that cold noodles carrying ceftazidime non-susceptible bacteria more than other samples. Among 70 ceftazidime non-susceptible bacteria, Acinetobacter spp. (n=22), Pseudomonas spp. (n=12), Stenotrophomonas maltophilia (n=21) and Enterobacteriaceae (n=15) were identified by 16S rDNA sequencing, respectively. Molecular epidemiology and phylogenetic group of strains were studied by Pulsed-field gel electrophoresis (PFGE) and non-clonal spreading was found in these isolates. Antimicrobial susceptibility was determined by the Agar-dilution method according to the guidelines of the Clinical and Laboratory Standards Institute (CLSI). These findings showed that ready to eat food carrying multi-drug resistant isolates (Acinetobacter spp.20/22,90.9%；Pseudomonas spp.12/12,100%；Stenotrophomonas maltophili,14/21,66.6%；Enterobacteriaceae,14/15,93.3%).
Extended-spectrum β-lactamases (ESBL) genes (CTX−M1, CTX−M9 , TEM , SHV , CMY , DHA , PER , VEB , OXA-1 , OXA-2 , OXA-9 , OXA-10, OXA-48 ) were detected by PCR. PER (1/16,6.25%) and CTX-M9 (1/16,6.25%) were found in Acinetobacter soli and Acinetobacter baumannii in spring rolls, respectively. CMY (1/10,10%)were found in Pseudomonas geniculate in cold noodle. PER (3/16,18.7%) were found in Acinetobacter radioresistens in fruit dishes.
Contribution of efflux pumps were analyzed by carbonyl cyanide m-chlorophenylhydrazone (CCCP) which was an efflux pump inhibitor. The 16 isolates (n=16/22, 72.7%) were demonstrated efflux pumps over-expression in Acinetobacter spp.The 3 isolates (n=3/12, 25%) were demonstrated efflux pumps over-expression in Pseudomonas spp. The 3 isolates (n=3/21,14.2%) were demonstrated efflux pumps over-expression in Stenotrophomonas maltophilia. The 1 isolates (n=1/15, 6.66%) were demonstrated efflux pumps over-expression in Enterobacter cloacae.

並列關鍵字

ready-to-eat food ； ceftazidime

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