| 摘要: | 沙門氏菌是一種常見的人畜共通病原菌，可在人類與動物之間傳播。然而，跟其他細菌病原體一樣，沙門氏菌的抗藥性也成為治療上日漸嚴重的問題。為釐清台灣沙門氏菌的抗藥情形與抗藥性的傳播方式，我們蒐集了1997-2009年間在台灣各種動物和人類的499株沙門氏菌分離株，包括S. Choleraesuis（205株）、S. Typhimurium（191株）、S. Enteritidis（20株）、S. Albany（20株）、S. Newport（19株）、S. Heron（17株）、S. Bredeney（11株）、S. Treforest（8株）及S. 4,[5],12:i:-（8株）。在這些分離株中發現共有417株（83.6%）有class I integron，其中有360株（86.3%）分離株帶有gene cassette。由gene cassette的種類來看，各血清型主要流行型別在S. Choleraesuis及S. Enteritidis為A1 type，S. Typhimurium、S. Albany、S. Heron、S. Bredeney、S. Treforest及S. 4,[5],12:i:-為D type，而S. Newport則為C type及D type。在台灣的沙門氏菌株主要流行的是D type（42.2%），其次為A1 type（40.3%）。D type上帶有ampicillin及streptomycin的抗藥基因，而A1 type上帶有streptomycin及sulfamethoxazole的抗藥基因。在ACSSuT的敏感性試驗結果，在499株菌株中共有157株（31.5%） 對ACSSuT等抗生素（ampicillin、chloramphenicol、strptomycin、sulfamethoxazole及tetracycline）均有抗藥性，僅有168株（33.7%）均為敏感性，而抗ACSSuT其中4種以上的抗生素的菌株則高達325株（65.1%）。其中帶有D type的菌株中，對ampicillin及streptomycin具抗藥性的菌株佔72.4%，但有16.4%是完全不抗ampicillin及streptomycin的，所以我們推測，在class I integron中的gene cassette不一定會將其抗藥基因表現出來。同時E1及E2 type的為首見之gene cassette型別，分別帶有ampicillin－chloramphenicol－streptomycin抗藥基因與chloramphenicol－streptomycin－sulfamethoxazole抗藥基因。而在fluoroquinolone類藥物的敏感性試驗結果中，所有分離株有20.4% 對4種fluoroquinolone類藥物（nalidixic acid、norfloxacin、ciprofloxacin和enrofloxacin）均產生抗藥性，而有37.7%則均具感受性。其中在S. Choleraesuis發現有101株（49.3%）分離株對4種fluoroquinolone類藥物皆有抗藥性，而有87株（42.4%）只對nalidixic acid具有抗藥性，只有2株（3.7%）是完全不具有4種fluoroquinolone類藥物的抗藥性，這跟S. Typhimurium的108株（50.5%）不具fluoroquinolone類藥物的抗藥性比較起來，S. Choleraesuis對fluoroquinolone類藥物的有顯著較強之抗藥性。為此我們進行沙門氏菌分離株的gyrA、gyrB、parC及parE的quinolone-resistance determining region (QRDR)序列定序，以找出其序列與抗藥性相對應的關係。結果僅在S. Choleraesuis分離株中gyrA及parC的QRDR發現其的DNA序列與fluoroquinolone類藥物的抗藥性有關，而在gyrB及parE則沒有發現類似的規律存在，因此，我們利用single-nucleotide polymorphism (SNP)的原理檢測gyrA及parC的DNA序列，用以快速篩選S. Choleraesuis的fluoroquinolone類藥物抗藥性。在本研究中，我們進行台灣沙門氏菌各血清型的抗藥性之與class I integron的流行率之分析，並且成功建立一種快速的篩選具fluoroquinolone類抗藥性之S. Choleraesuis菌株之分子診斷方式。以上結果可得知台灣沙門氏菌各血清型對常用藥物之抗藥性，以供臨床治療上使用抗生素之參考。另外篩檢具有fluoroquinolone類藥物抗藥性菌株的分子診斷方法，提供快速得知沙門氏菌的抗藥性的方，可縮短菌株抗藥性檢測的時間，在擴大QRDR序列的篩選與比對後，也可應用在S. Choleraesuis外的血清型。
Salmonella is a common zoontic pathogen which can be transmitted by humans to animals and vice versa. Antimicrobial treatment is used in clinical management. Recently, multidrug - resistance Salmonella have been found frequently in clinical settings. In order to investigate the prevalence of antibiotic resistance strains and study the mechanism involved the spread of antibiotic resistance, we analyzed 499 Salmonella isolates in Taiwan from 1997 to 2009, including 205 isolates of S. Choleraesuis, 191 isolates of S. Typhimurium, 20 isolates of S. Enteritidis, 20 isolates of S. Albany, 19 isolates of S. Newport, 17 isolates of S. Heron, 11 isolates of S. Bredeney, 8 isolates of S. Treforest and 8 isolates of S. 4,[5],12:i:-. The intI1 gene as detected in 417 of all 499 Salmonella isolates (83.6%). Among these intI1-positive strains, 360 isolates (86.3%) harbored class I integron containing various lengths of regions (gene cassettes). Serotypes under the Type D category was the most commonly found gene cassette (42.2%) among the integron-positive S. Typhimurium, S. Newport, S. Heron, S. Bredeney, S. Treforest, and S. 4,[5],12:i:- isolates. Type A was a high prevalent gene cassette as well (40.3%), most commonly found in S. Choleraesuis and S. Enteritidis. Type D gene cassette carried ampicillin and streptomycin resistance genes and Type A harbored streptomycin and sulfamethoxazole resistance genes. In ACSSuT (ampicillin, chloramphenicol, streptomycin, sulfamethoxazole and tetracycline) susceptibility analysis, with 31.5% (157/499) of isolates were ACSSuT resistance, 33.7% (168/499) were susceptibility to all ACSSuT, and 65.1% (325/499) were resistant to 4 kinds of ACSSuT antibiotics. Among the isolates carrying Type D gene cassettes, only 72.4% of them were resistance to ampilcllin and streptomycin, with 16.4% of isolates were susceptibility to both. These data indicated that the genes in the cassettes might not be involved in the resistant phenotype of isolates. Type E1 and E2 cassettes were the first ones reported in class I integron and they carry ampicillin - chloramphenicol - streptomycin and chloramphenicol - streptomycin - sulfamethoxazole resistance genes, respectively. In fluoroquinolone susceptibility analysis, with 20.4% of isolates were fluoroquinolone (nalidixic acid, norfloxacin, ciprofloxacin and enrofloxacin) resistance, with 37.7% were susceptibility to all. Among S. Choleraesuis, 101 isolates (49.3%) were resistant to four fluoroquinolone drugs and 87 isolates (42.4%) were resistant to only nalidixic acid only. Compared with S. Typhimurium (50.5% of resistance), S. Choleraesuis was more resistant to fluoroquinolone drugs. In order to study the mechanism of fluoroquinolone resistance, quinolone-resistance determining region (QRDR) of gyrA, gyrB, parC, and parE genes in Salmonella isolates were sequenced. However, only in S. Choleraesuis, unique single nucleotide differences were identified within gyrA and parE genes between RRRR type (the isolates resistant to all 4 fluoroquinolone) and RSSS type (the isolates resistant to only nalidixic acid). There was no consistent sequence pattern in rest of serotypes in order to distinguish fluoroquinolone resistance and susceptibility strains. A single-nucleotide polymorphism based genotyping was established to diagnose fluoroquinolone resistance strains in S. Choleraesuis. In this study, we explore the relationship between class I integron positivity and antimicrobial resistance in different Salmonella serotypes from humans and the various animal hosts and successfully developed an effective molecular diagnostic method to detect antibiotic resistance strains. Based on the investigation of the prevelance of commonly used antibiotic resistance patterns among different serotypes in Taiwan Salmonella isolates, we should be more cautious with the antibiotic treatment in Salmonella infection. |
