Antimicrobial susceptibility of Brazilian Clostridium difficile strains determined by agar dilution and disk diffusion

Abstract

Clostridium difficile is a leading cause of diarrhea in hospitalized patients worldwide. While metronidazole and vancomycin are the most prescribed antibiotics for the treatment of this infection, teicoplanin, tigecycline and nitazoxanide are alternatives drugs. Knowledge on the antibiotic susceptibility profiles is a basic step to differentiate recurrence from treatment failure due to antimicrobial resistance. Because C. difficile antimicrobial susceptibility is largely unknown in Brazil, we aimed to determine the profile of C. difficile strains cultivated from stool samples of inpatients with diarrhea and a positive toxin A/B test using both agar dilution and disk diffusion methods. All 50 strains tested were sensitive to metronidazole according to CLSI and EUCAST breakpoints with an MIC90 value of 2μg/mL. Nitazoxanide and tigecycline were highly active in vitro against these strains with an MIC90 value of 0.125μg/mL for both antimicrobials. The MIC90 were 4μg/mL and 2μg/mL for vancomycin and teicoplanin, respectively. A resistance rate of 8% was observed for moxifloxacin. Disk diffusion can be used as an alternative to screen for moxifloxacin resistance, nitazoxanide, tigecycline and metronidazole susceptibility, but it cannot be used for testing glycopeptides. Our results suggest that C. difficile strains from São Paulo city, Brazil, are susceptible to metronidazole and have low MIC90 values for most of the current therapeutic options available in Brazil.

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“…The statistically validated observations of this study revealed that BBRHCl's concentrations 5 and 10 (mg/ml) corresponds to EUCAST's epidemiological cut-off diameter for vancomycin and the concentrations 2.5, 5, and 10 (mg/ml) to the susceptibility breakpoint diameter of teicoplanin (the standard antimicrobial agents used for treating CDI) [14]. This finding is noteworthy especially in present day scenario with emerging C. difficile isolates showing reduced susceptibility to metronidazole [23].…”

“…It also highlights the use of agar diffusion through punch well method as a simple susceptibility testing method for this nosocomial pathogen for routine microbiology laboratories as mentioned in previous literature [13,14].…”

“…1). The inhibitory zone diameters of BBRHCl used, in our study, were compared with that of teicoplanin (mean inhibitory zone diameter 15 mm) [14] and the breakpoint correlates for the EUCAST epidemiological cutoff values of metronidazole and vancomycin (mean inhibitory zone diameters being 23,19 [mm], respectively) [13]. The diameters of zone of inhibition for BBRHCl (all the test concentrations) were significantly lesser (P≤0.0001) than the EUCAST epidemiological cutoff value for metronidazole (data not shown).…”

“…The test concentrations of BBRHCl, lower than 1.25 mg/ml were not considered since they were significantly lesser than (i) EUCAST's epidemiological cutoff value for metronidazole and vancomycin [17], (ii) susceptibility breakpoint zone diameter for teicoplanin [14], and (iii) susceptibility breakpoint zone diameter for 2% black seed oil against toxigenic C. difficile [12]. Hence, a trend line (linear regression) with R 2 value 0.9506 was generated for the diameter of zones of inhibition (mm) plotted against log concentrations (1.25, 2.5, 5, and 10 [mg/ml]) of BBRHCl for toxigenic C. difficile (n=3).…”

“…Experiments were performed in triplicates for each combination of the concentrations with BBRHCl. The inhibitory zone diameters (corresponding to the test concentrations of BBRHCl) were compared with EUCAST's epidemiological breakpoint diameters for metronidazole (23 mm), vancomycin (19 mm), and susceptibility breakpoint diameter of teicoplanin (15 mm), from published literature against C. difficile [14,13]. The disk potency of all the three drugs (metronidazole, vancomycin, and teicoplanin) was 5 µg as reported in literature [14,13].…”