JAC Advance Access originally published online on July 24, 2006
Journal of Antimicrobial Chemotherapy 2006 58(3):705-706; doi:10.1093/jac/dkl285
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Correspondence |
Dissemination of nim-class genes, encoding nitroimidazole resistance, among different species of Gram-negative anaerobic bacteria isolated in Athens, Greece
1 Department of Microbiology, Laikon General Hospital Athens, Greece 2 Department of Microbiology, Medical School, National and Kapodistrian University of Athens Greece
*Corresponding author. Dr Joseph Papaparaskevas, Lecturer, Department of Microbiology, Medical School, National and Kapodistrian University of Athens, Mikras Asias 75, 11527, Athens, Greece. Tel: +30-210-7462142; Fax: +30-210-7462143; Email: ipapapar{at}med.uoa.gr
Keywords: Resistance mechanisms , anaerobes , resistance epidemiology
Sir,
Metronidazole remains the cornerstone for treatment of anaerobic infections1 and, despite its widespread use, isolation of resistant strains is uncommon.1,2 Nevertheless, enzymatic modification mediated by nim-class genes is a well-characterized resistance mechanism, and a total of seven different nim genes have been described.3,4
The aim of the present study was to investigate the prevalence and to identify the nim gene types among Gram-negative anaerobic clinical strains belonging to different species, isolated from hospitalized patients, in Athens, Greece.
Since 2003, Gram-negative anaerobic bacteria have been referred from neighbouring hospitals to Laikon General Hospital, for confirmation of species identification and/or susceptibility testing. Clinical specimen and culture processing was performed in the microbiology departments of the respective hospitals. Species identification was confirmed in the microbiology department of Laikon General Hospital using standard methodology.1 Metronidazole MICs were determined using the Etest method (AB Biodisk, Solna, Sweden). Susceptibility plates were incubated in a Bactron Anaerobic Chamber (Cheldon Manufacturing, Cornelius, OR, USA) for 48 h. CLSI guidelines were followed for interpretation of the results and quality control perfomance.5 All isolates having an MIC 
2 mg/L were investigated by PCR for the presence of nim-class genes, followed by characterization of the positive result using DNA sequencing.3 All nim-positive strains were additionally investigated by PCR for the presence of elements IS1168, IS1169 and IS1170.3
A total of 315 non-duplicate Gram-negative anaerobic clinical strains were collected during the period 2003–2005, comprising 191 Bacteroides fragilis group, 34 other Bacteroides non-fragilis, 57 Prevotella, 17 Fusobacterium, 6 Porphyromonas and 5 Veillonella spp.
As shown in Table 1, 21 isolates (6.6 %) exhibited metronidazole MIC 2 mg/L (12 were resistant, 1 was intermediate and 8 exhibited reduced susceptibility), of which 8 were positive for the presence of nim-class genes (3, 2, 2 and 1 strains harboured nimE, nimD, nimC and nimA genes, respectively). Only three of the eight nim-positive strains were Bacteroides spp. Elements IS1169 and IS1168 were detected each in a single nim-positive Bacteroides spp. isolate.
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The present study demonstrated that metronidazole resistance may be an emerging problem in Greece, as a rate of 4.2% was recorded, mainly among Prevotella and Bacteroides spp. isolates. Metronidazole resistance is not frequent in European countries,2 although rates up to 7% have been detected among Bacteroides spp.3
Previous studies reporting the presence of nim genes in both resistant as well as susceptible Gram-negative anaerobic bacteria were confirmed here as well;3 three of the eight nim-positive isolates exhibited a susceptible or intermediate metronidazole MIC of 4–16 mg/L, whereas the remaining five were resistant. Nevertheless, it was also demonstrated that this is not the most prevalent metronidazole resistance mechanism among anaerobic bacteria in Greece, as the respective genes were not detected in a number of metronidazole non-susceptible isolates. The possible existence of alternative resistance mechanisms has been described,4 and this may be the situation regarding the nim-negative metronidazole-resistant isolates reported here.
The study additionally demonstrated the presence of nim genes among Gram-negative anaerobic species other than Bacteroides spp. and indicated differences between species, MIC distribution and nim gene positivity. Among fully resistant isolates (MIC 32 mg/L), nim genes were detected mainly in Prevotella spp., whereas among intermediate and susceptible isolates (MIC 
16 mg/L), nim genes were detected only among Bacteroides spp. Nevertheless, as only strains isolated in a particular area of Greece were investigated, further multicentre studies are needed in order to reach a definite conclusion regarding this association.
It has been reported previously that the different expression of resistance (MIC 2 to >32 mg/L) among nim-positive isolates may be attributed to the presence of IS elements.3,6 In our study, IS elements were detected in two nim-positive isolates, which were both metronidazole non-resistant (one intermediate and one susceptible). Overall, two of the three nim-positive Bacteroides spp. were IS-positive, whereas all five nim-positive strains belonging to species other than Bacteroides spp. were IS negative. In that respect a possible species- or genus-specific resistance mechanism may exist, as other studies also have reported the presence of IS elements only among Bacteroides spp. isolates.3,6 An unknown IS element, or involvement of a different nim gene activation pathway, as previously described,4 may explain the situation regarding the rest of the nim-positive IS-negative isolates. Notwithstanding the small sample size, IS specificity regarding nim gene type was according to previous reports, as IS1169 was found in a nimD-positive strain and IS1168 in a nimA-positive strain.7
In conclusion, the present study confirmed the dissemination of nim genes among both susceptible and resistant isolates belonging to a wide range of Gram-negative anaerobic bacterial species, other than Bacteroides spp., and also indicated possible differences between species, MIC distribution, nim gene and IS element positivity, differences that need further evaluation. In that respect, the importance of periodic susceptibility testing of anaerobic bacteria is emphasized, coupled with continuous surveillance of the respective molecular resistance mechanisms.
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None to declare.
Acknowledgements
Active contribution of the following members of the ‘Hellenic Study Group for Gram Negative Anaerobic Bacteria’ is acknowledged: Prof. N. J. Legakis (National and Kapodistrian University of Athens, Medical School), Dr E. Papafrangas and Dr M. Kanellopoulou (‘Sismanoglion’ General Hospital), Dr C. Koutsia-Karouzou and Dr D. Kairis (‘Asklepeion’ General Hospital), Dr C. Trikka-Graphakos (‘Thriassion’ General Hospital), Dr C. H. Kontou-Castellanou (‘Hippokration’ General Hospital), Dr H. Malamou-Ladas (‘G. Gennimatas’ General Hospital), Dr M. Foustoukou and Dr E. Lebessi (‘P. & A. Kyriakou’ Children Hospital), Dr A. Vogiatzi (‘Penteli’ Children Hospital) and Ass. Prof. L. Zerva (‘Attikon’ University General Hospital).
This study was co-funded by the European Social Fund and National Resources (grant EPEAEK II–PYTHAGORAS II).
References
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3
Gal M and Brazier JS. (2004) Metronidazole resistance in Bacteroides spp. carrying nim genes and the selection of slow-growing metronidazole-resistant mutants. J Antimicrob Chemother 54:109–16.
4
Diniz CG, Farias LM, Carvalho MA. (2004) Differential gene expression in a Bacteroides fragilis metronidazole-resistant mutant. J Antimicrob Chemother 54:100–8.
5 National Committee for linical Laboratory Standards. (2004) Methods for Antimicrobial Testing of Anaerobic Bacteria—Sixth Edition: Approved standard M11-A6 (NCCLS, Wayne, PA).
6
Löfmark S, Fang H, Hedberg M, et al. (2005) Inducible metronidazole resistance and nim genes in clinical Bacteroides fragilis group isolates. Antimicrob Agents Chemother 49:1253–6.
7
Soki J, Gal M, Brazier J, et al. (2006) Molecular investigation of genetic elements contributing to metronidazole resistance in Bacteroides strains. J Antimicrob Chemother 57:212–20.
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