Dissemination of nitroimidazole resistance determinants among Gram-negative anaerobic bacteria in Greece

Abstract number: 1134_03_45

Katsandri A., Papaparaskevas J., Pantazatou A., Tzouvelekis L.S., Petrikkos G.L., Legakis N.J., Avlamis A.

Objectives:  

The surveillance of the incidence of nitroimidazole resistance (nim) determinants among Gram-negative anaerobic clinical isolates in Greece.

Materials and methods:  

A total of 185 Gram-negative anaerobic bacteria collected from eight hospitals in Athens, Greece, were tested for metronidazole resistance using the Etest method on brucella blood agar plates. Incubation in a ChelLab Anaerobic Chamber was performed for 48 hours. Interpretation of the results was according to NCCLS guidelines. For quality control the strains B. fragilis ATCC25285 and B. thetaiotaomicron ATCC29741 were used. Strains having an MIC > 1 mg/L were screened for nim genes by PCR using the nim³/nim⁵ set of primers. PCR products were stained with ethidium bromide and documented under UV illumination. Sequencing was performed in an ABI 3100 genetic analyzer using the BigDye terminator kit. The Blast search programme of the National Center for Biotechnology Information was used to search the Gene Bank for significant alignments.

Results:  

A total of 16 isolates having an MIC > 1 mg/L were detected (four Bacteroides fragilis group, eight Prevotella spp., one Bacteroides spp. non-fragilis and three miscellaneous). Three, three, two, one and seven strains had MICs of 2, 4, 8, 16 and 32 mg/L or higher, respectively. Eight strains were positive by PCR for the 458 bp amplicon, of which three had a metronidazole MIC of 4 to 16 mg/L (lower than the current NCCLS breakpoint). DNA sequencing revealed that one, two, two and three strains harboured nimA, nimC, nimD and nimE genes, respectively. No strain was detected harbouring the nimB gene. No particular relationship was detected regarding species or MIC distribution and nim gene class.

Conclusions:  

Nitroimidazole resistance determinants were detected among both metronidazole resistant and susceptible Gram-negative anaerobic bacteria in Greece. This study confirms the widespread occurrence of nim genes and demonstrates that ‘silent’ nim genes can be detected by PCR in metronidazole susceptible isolates.

Members of The Hellenic Study Group on Gram-Negative Anaerobic Bacteria are: A. Avlamis, C. Koutsia-Karouzou, C. Kontou-Kastelanou, A. Pangalis, E. Papafrangas, E. Trika-Grafakos, H. Malamou-Ladas and A. Vogiatzi.