Molecular and microbiological characterization of Clostridium difficile isolates from single, relapse, and reinfection cases

Abstract

In this study, we investigated the correlation between the microbiological characteristics of Clostridium difficile clinical isolates and the recurrence of C. difficile-associated disease (CDAD). Twenty C. difficile isolates recovered from 20 single infection cases and 53 isolates from 20 recurrent cases were analyzed by pulsed-field gel electrophoresis (PFGE) and PCR ribotyping, and the cytotoxicity, antimicrobial susceptibility, and sporulation/germination rates of the isolates were examined. Recurrent cases were divided into relapse or reinfection cases by the results of C. difficile DNA typing. Among the 20 recurrent cases, 16 cases (80%) were identified to be relapse cases caused by the initial strain and the remaining 4 cases (20%) were identified to be reinfection cases caused by different strains. All 73 isolates were susceptible to both vancomycin and metronidazole, but resistance against clindamycin, ceftriaxone, erythromycin, and ciprofloxacin was found in 87.7%, 93.2%, 87.7%, and 100% of the isolates, respectively. No correlations between DNA typing group, cytotoxicity, and sporulation rate of isolates and infection status, i.e., single, relapse, or reinfection, were observed. However, the isolates recovered from relapse cases showed a significantly higher germination rate when incubated in medium lacking the germination stimulant sodium taurocholate. These results indicate that the germination ability of C. difficile may be a potential risk factor for the recurrence of CDAD.

Fig 1

PCR ribotype patterns of C. difficile isolates. Lanes R1 to R11, representative pattern of each ribotype; lanes M and n.c., 50-bp size markers and negative control, respectively.

Fig 2

Dendrogram and PFGE patterns of C. difficile isolates. Lanes P1a to P12, representative pattern of each PFGE type; lanes M, bacteriophage lambda ladder size marker. The dendrogram was constructed with Fingerprinting II software (Bio-Rad Laboratories, Inc.).

Fig 3

Spore formation rate (A), germination rate on GAM agar without sodium taurocholate (B), and germination rate on GAM agar supplemented with 0.1% sodium taurocholate (C) of C. difficile isolates compared to PFGE subtype.

Fig 4

Spore formation rate (A), germination rate on GAM agar without sodium taurocholate (B), and germination rate on GAM agar supplemented with 0.1% sodium taurocholate (C) of C. difficile isolates compared to infection status. S (n = 20), Rl (n = 43), and Ri (n = 10) indicate isolates from single, relapse, and reinfection cases, respectively. Bars represent mean ± SDs. Asterisks denote statistical significance (P < 0.05).