Effects of ciprofloxacin on the expression and production of exotoxins by Clostridium difficile

Abstract

Hypervirulent BI/NAP1/027 strains of Clostridium difficile have been associated with increased mortality of C. difficile infection (CDI). The emergence of highly fluoroquinolone (FLQ)-resistant BI/NAP1/027 strains suggests that FLQ exposure may be a risk factor for CDI development. However, the mechanism for this is not clear. We compared the effects of subinhibitory concentrations of ciprofloxacin on Toxin A and B gene expression and protein production in recent (strain 039) and historical (strain 5325) BI/NAP1/027 clinical isolates with high- and low-level ciprofloxacin resistance, respectively. In the highly ciprofloxacin-resistant isolate (strain 039), ciprofloxacin significantly and dose-dependently increased Toxin A gene expression and shifted its expression to earlier in its growth cycle; TcdB gene expression also increased but was less sensitive to low-dose ciprofloxacin. Maximal Toxin A/B production (4 ng ml(-1)) was increased twofold and occurred significantly earlier than in the untreated control. In strain 5325, ciprofloxacin at 0.25×MIC markedly increased both tcdA and tcdB expression but their temporal dynamics were unchanged. Maximal toxin production (250 ng ml(-1)) was reduced approximately threefold compared with that of the untreated control. These results demonstrate significant differences in ciprofloxacin-induced toxin gene expression and protein production among BI/NAP1/027 isolates, and offer a new paradigm for FLQ-associated CDI caused by recent, highly antibiotic-resistant strains.

Fig. 1.

Effects of ciprofloxacin on the growth, toxin gene expression and soluble toxin production by the C. difficile NAP1 039 strain. (a) Antibiotic-free cultures, (b) 0.0625×MIC (16 µg ml⁻¹), (c) 0.125×MIC (32 µg ml⁻¹) and (d) 0.25×MIC (64 µg ml⁻¹). Ciprofloxacin was added, at the final concentrations indicated, during early exponential phase growth (designated time 0). Samples were collected in duplicate over 48 h for quantification of viable C. difficile, and for measurement of toxin gene expression by real-time RT-PCR, and TcdA/B production by ELISA. Fold change in mRNA expression for each strain is made relative to that in its respective ‘No treatment’ control at 6 h (which was virtually zero in both strains). Filled line, tcdA mRNA; dotted line, tcdB mRNA.

Fig. 2.

Effects of ciprofloxacin on the growth, toxin gene expression and soluble toxin production by the C. difficile NAP1 5325 strain. (a) Antibiotic-free cultures, (b) 0.0625×MIC (0.5 µg ml⁻¹), (c) 0.125×MIC (1 µg ml⁻¹) and (d) 0.25×MIC (2 µg ml⁻¹). Ciprofloxacin was added, at the final concentrations indicated, during early exponential phase growth (designated time 0). Samples were collected in duplicate over 48 h for quantification of viable C. difficile, and for measurement of toxin gene expression by real-time RT-PCR, and TcdA/B production by ELISA. Fold change in mRNA expression for each strain is made relative to that in its respective ‘No treatment’ control at 6 h (which was virtually zero in both strains). Filled line, tcdA mRNA; dotted line, tcdB mRNA.

Fig. 3.

Effects of ciprofloxacin on Toxin A and B production by the C. difficile NAP1 039 strain. Soluble Toxin A and B measured by ELISA in the 64 µg ml⁻¹-treated (0.25×MIC) culture. Ciprofloxacin was added at the final concentration during early exponential phase growth, designated time 0. Culture supernatant samples were collected over 48 h for detection of TcdA/TcdB by ELISA. Data shown are the means±sd of three replicates. •, No treatment; ○, 64 mg ml⁻¹.

Fig. 4.

Dose-dependent decreases in Toxin A and B production in C. difficile NAP1 5325 strain following ciprofloxacin exposure. Culture supernatant samples were collected at 24 and 48 h following the addition of ciprofloxacin at final concentrations of 0, 0.5, 1.0 and 2.0 µg ml⁻¹ (antibiotic free, 0.0625, 0.125 and 0.25×MIC, respectively) for detection of TcdA/TcdB by ELISA. Data shown are the means±sd of three replicates. ○, 24 h; •, 48 h.