Campylobacter spp.的抗藥性、血清型、基因分型、致病及毒素基因檢測之研究

Abstract

本研究利用Etest方法針對75株Campylobacter jejuni和29株Campylobacter coli進行11種抗菌物質之抗藥性圖譜分析和最小抑制濃度（MIC）檢測。結果顯示所有菌株可區分為28種抗藥性圖譜型別，其中有1株C. jejuni臨床菌株（AkApCpEGKNaSSuTTm）和1株C. coli雞肉相關檢體分離菌株（AkApCpEKNaSSuTTm）具有最多重抗藥性（multidrug resistant）。有81.3%的C. jejuni和96.6% C. coli分離菌株對於4種以上（含4種）抗生素具有抗藥性。自雞肉相關檢體和臨床分離的C. jejuni對amikacin與gentamicin抗藥性最低（皆為3.0%；皆為9.1%），其MIC90 (μg/mL)分別為16和1.5及32和1.5。而所有的C. jejuni對sulphadiazine與trimethoprim皆具有100.0%的抗藥性。至於雞肉相關檢體的C. coli，對amikacin之抗藥性最低（4.0%）（MIC90 = 12），而對tetracycline及trimethoprim則有100%抗藥性。1998年以後分離之C. jejuni和C. coli雞肉相關檢體菌株，奎喏隆類（quinolone）抗藥性皆遠高於1998年以前者。1998年以後分離的臨床菌株亦有相同的增加趨勢。在抗藥性機制方面，奎喏隆類有高抗藥性者 (ciprofloxacin MIC＞32且nalidixic acid＞256) 皆可檢測出具有gyrA的Thr-86→Ile（ACT→ATT）點突變。另外各選取2株奎喏隆類高抗藥性的C. jejuni和C. coli菌株進行QRDR（quinolone resistance determining region）PCR及定序，結果顯示奎喏隆類高抗藥性菌株確實具有Thr-86→Ile點突變。 在AFLP分型分析方面，發現AFLP的分析結果並不會因為使用不同DNA抽取套組而受到影響，而且其重複性良好。以40%相似度進行65株C. jejuni群組分析時，可分成2個AFLP分群，42種AFLP分型，而大多數的菌株屬於A分群。所有的C. jejuni分離菌株中，有7.9%菌株具有AFLP分型1，AFLP分型5和12則皆佔有6.3%。至於35株C. coli分離菌株可區分成3個AFLP分群，33種AFLP分型。AFLP分型方法可應用於區分遺傳關係相近的C. jejuni和C. coli分離菌株。由AFLP的親緣樹狀圖中，可發現臨床檢體的C. jejuni菌株有可能來自於雞肉加工廠和雞肉相關產品。某些雞肉相關檢體分離出多株Campylobacter spp.菌株（multiple isolates），有檢體同時含有奎喏隆類抗藥性和奎喏隆類敏感性的C. jejuni分離菌株，也有檢體同時存在C. jejuni和C. coli菌株，且都具有奎喏隆類抗藥性。經Penner血清型分群分析發現有33株C. jejuni（52.4%）具有血清型，其中B:2血清型佔最多（14株），其次為Y:37血清型（7株）。有3株O:19血清型菌株皆歸屬B分群的AFLP分型39。 C. jejuni菌株具有cdtB基因的比例（100%）高於cdtA（90.5%）和cdtC（82.5%）。在致病基因部分，flaA（100%）高於ceuE（98.4%）和cadF（96.8%）。在C. coli菌株的毒素基因方面，cdtB基因（97.1%）遠高於cdtA（42.9%）和cdtC（22.9%）。在致病基因部分，依次是flaA基因（100%）＞ceuE（97.1%）和cadF（97.1%）。C. jejuni的雞肉相關檢體分離菌株含有cdtC基因的比例（24/32, 75.0%）明顯少於臨床來源者（26/27, 96.3%）。將不含C. jejuni的培養液上清液（cell-free bacterial culture supernatant, CFBCS）經1:32稀釋後進行Vero細胞培養，可觀察到有部分Vero細胞呈現擴張膨大的現象。由於CDT（cytolethal distending toxin）毒性之細胞株試驗是針對CFBCS進行檢測，所以探討不同培養液於Vero細胞株檢測五株C. jejuni分離菌株產生CDT含量之影響，結果可發現brucella broth明顯優於其他三種培養基（p < 0.05）。另外，有80%（24/30）C. jejuni分離菌株以Vero細胞株檢測法檢測出具有CDT活性，而18株C. coli分離菌株則皆沒有產生CDT活性。依菌株來源區分，C. jejuni雞肉相關檢體和臨床分離菌株分別有78.6%（11/14）和86.7%（13/15）檢測出具有CDT活性。

The resistance profiles (R-type) and MICs of 75 Campylobacter jejuni and 29 C. coli isolates to 11 antimicrobial agents were determined by the Etest. All Campylobacter spp. isolates were differentiated into 28 different R-types, and one C. jejuni, isolated from clinical sample, was most multidrug resistant (AkApCpEGKNaSSuTTm). Among C. coli isolates, the R-type of the most multidrug resistant strain, isolated from chicken-related sample, was AkApCpEKNaSSuTTm. Nearly 81.3% C. jejuni isolates and 96.6% C. coli isolates were resistant to four or more antimicrobial agents. For chicken-related and clinical isolates, C. jejuni were most susceptible to amikacin and gentamicin (both 3.0%; both 9.1%) with MIC90 (μg/mL) = 16 and 1.5 as well as 32 and 1.5, respectively. However, all C. jejuni isolates were completely resistant to sulphadiazine and trimethoprim. C. coli chicken-related isolates were most susceptible to amikacin (4.0%; MIC90 = 12) and most resistant to tetracycline and trimethoprim (both 100%). The resistance to quinolone (ciprofloxacin and nalidixic acid) of C. jejuni and C. coli chicken-related isolates collected after 1998 was much higher than those of isolates collected before 1998. The resistance to quinolone also has been on an increasing trend in C. coli clinical isolates collected after 1998. The Thr-86→Ile (ACT→ATT) mutation of gyrA was found in high quinolone-resistant isolates (ciprofloxacin MIC＞32 and nalidixic acid＞256). When each two isolates of C. jejuni and C. coli were examined the QRDR by the QRDR PCR and then sequenced, only the high quinolone-resistant isolates possessed the Thr-86→Ile mutation. Three different kinds of DNA extraction kits had no effect on the repeatability of the AFLP method. All C. jejuni isolates were separated into two distinct genetic clusters at 40% genetic similarity and 42 different AFLP types at 90% similarity by AFLP technique. However, three clusters at 40% genetic similarity and 33 different AFLP types at 90% similarity were observed in C. coli isolates. These results showed that AFLP analysis could be used to identify individual isolates of two Campylobacter species. Among C. jejuni isolates, the predominant AFLP type 1 was observed in 5 (7.9%) isolates, and type 5 and 12 in 4 (both 6.3%) isolates. AFLP fingerprints of chicken-related isolates were closely related genetically to those of isolates from humans with gastroenteritis. The predominant serotypes in C. jejuni isolates were B:2 and Y:37. All isolates belonging to serotype O:19 grouped into one single AFLP type. Some chicken samples yielded multiple isolates of Campylobacter, harboring simultaneously a quinolone-resistant and a quinolone-sensitive isolates attributed to the same species, or harboring C. jejuni and C. coli that have the characteristics of quinolone resistance. By the PCR methods, the cdtB, cdt genes, cdtA and cdtC were detected in 100.0%, 98.4%, 90.5% and 82.5% of C. jejuni isolates, respectively. The flaA, ceuE and cadF genes were found in 100%, 98.4% and 96.8% of C. jejuni isolates, respectively. About C. coli isolates, 97.1%, 42.9% and 22.9% of isolates contained cdtB, cdtA and cdtC genes, respectively. Besides, 100%, 97.1% and 97.1% of them contained flaA, ceuE and cadF genes, respectively. The rate (24/32, 75.0%) of cdtC PCR-positive C. jejuni isolates from chicken-related samples was apparently lower than that (26/27, 96.3%) of isolates from humans. After Vero cells interacted with 1:32 dilution of cell-free bacterial culture supernatant (CFBCS) of C. jejuni for 5 days, distended or rounded cells were observed by the inverted microscope. To compare the effect of four different broths (brucella broth, heart infusion broth, brain heart infusion broth and veal infusion broth) on the CDT titres of CFBCSes obtained from 5 C. jejuni isolates, the result showed that the efficiency of brucella broth was significantly superior to those of the others (p<0.05). There were 24 (80%) C. jejuni isolates (including from 11 chicken-related samples and 13 clinical samples) to produce CDT. However, all C. coli isolates produced no toxin.