Molecular Study: Nitroreductase Enzyme and Nitroimmidazole Resistance Genes (Nim Genes) Effect on AMR to Metronidazole in Clostridium difficile

Nitroreductase enzymes and Nim genes have been observed to be a factor predisposing to reduced susceptibility to metronidazole in anaerobic bacteria. Therefore, this study was determined the effect of nitroreductase enzymes and nim genes in metronidazole susceptibility in UK C. difficile isolates. Three UK C. difficile ribotypes 027, 001, 106 including metronidazole reduced susceptible (CDRM) and metronidazole susceptible (CDSM) C. difficile strains, and 2 control strains from ribotype 010 (CDRM) and 038 (CDSM) were used. Control strain C. perfringens ATCC 3626 previously identified to be nitroreductase positive, and positive control for nimB which was Bacteroides fragilis strain BF8 was also included in this research. These selected strains were observed for their nitroreductase activity and presence of Nim genes. C. perfringens ATCC 3626 demonstrated nitroreductase activity but was absent in C. difficile control. Three CDRM strains (001 and 106 ribotype) demonstrated nitoreductase activity. None of the CDSM strains analysed showed detectable nitroreductase activity. The nim gene PCR amplification was only observed in the nim positive control strain BF8 (about 400 bp). All samples tested including ribotype 001 027 and 106 CDSM and CDRM strains showed no nimPCR product. In conclusion, despite the nitroreductase result obtained, inconsistencies observed during the assay showed methodology issues, thus using radio labelled metronidazole and monitoring the decline in the radiolabel signal proportional to the level of nitroreduction, maybe a preferred method in correlating nitroreductase activity to the CDRM phenotype. Also, this research observed the absence of nim genes in C. difficile UK ribotypes irrespective of metronidazole susceptibility patterns, depicting the inability of the universal primers for nimA-NimE to amplify a potential nim gene in C. difficile UK ribotypes. The universal primers were previously reported incapable of amplifying nimJ due to a different nucleotide sequence coding for same proteins as the universal primers.