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

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.