nimE Gene in a Metronidazole-Susceptible Veillonella sp. Strain

Metronidazole (MTZ) is widely used for the prophylaxis and the treatment of infections caused by anaerobes. These infections are mainly due to Bacteroides fragilis, but several reports identified Veillonella spp. as pathogens in severe infections (5, 7). Despite the extensive use of MTZ, resistance was maintained at a very low level for a long time and was first described for B. fragilis in 1978 (4). In contrast, an increasing level (3 to 5%) of B. fragilis group strains with decreased susceptibility to MTZ was observed (2). The presence of nitroimidazole (nim) resistance genes was first reported in 1989 (3), and five resistance determinants, nimA to nimE, were characterized either on plasmids or chromosomes (9). Only the location of nimE was not reported when the gene was described for B. fragilis group strains (9). More recently, the nimF gene, sharing 78% identity in sequence with nimD, has been reported in a B. fragilis group strain (S. Behrendtz, H. Fang, M. Hedberg, and C. Edlund, Abstr. 3rd World Congr. Anaerob. Bact. Infect., abstr. 6.003, 2003).

We have studied MTZ susceptibility and presence of nim genes for 116 clinical isolates of Veillonella spp. These strains were recovered either in pure culture or in aerobic-anaerobic polymicrobial cultures from various samples in 106 different patients hospitalized or consulting at the Montpellier University Hospital between March 1999 and July 2001. MTZ MICs were determined by the NCCLS reference agar dilution method (8). According to the NCCLS breakpoints, all the Veillonella sp. strains were susceptible to MTZ (Table 1). A PCR method using universal primers was used to detect nim genes after extraction of total genomic DNA according to the rapid procedure proposed by Trinh and Reysset (10). An amplification product was obtained for one isolate for which the MTZ MIC was 4 mg/liter. The 458-bp amplified fragment obtained was sequenced and gave the maximum identity (99.8%) with the nimE gene sequence of B. fragilis ARU 6881 (GenBank accession number AJ244018). To search for a possible chromosomal location of this nimE gene, DNA electrophoresis of I-CeuI restriction fragments, Southern blotting, and hybridization with a nim digoxigenin-labeled probe were performed (1). None of the I-CeuI-generated fragments hybridized with the probe. A Bacteroides strain known to harbor a chromosomal copy of the nim gene was used as a positive control. On the other hand, plasmid purification was performed by using a Quantum Prep plasmid miniprep kit (Bio-Rad Laboratories). Plasmid DNA was apparently not detectable when analyzed by agarose gel electrophoresis, but plasmid extraction yielded positive nim amplification. Taken together, these results suggested that nimE was probably located on a plasmid present in low copy number in the Veillonella strain, and this could be related to the low MTZ MIC observed for this strain.

TABLE 1.

Repartition of the MTZ MICs obtained by the agar dilution method for the 116 Veillonella sp. clinical isolates

MTZ MIC (mg/liter)	No. of Veillonella sp. strains for each MIC value
8	1
4	8a
2	70
1	35
0.5	1
0.25	1

^aIncludes the nimE gene-harboring strain isolated from a pressure ulcer of the sacrum in mixed aerobic-anaerobic cultures.

nim genes were mainly described in B. fragilis group isolates, and nimA is the only one presently recovered in other anaerobic bacteria: Propionibacterium spp., Actinomyces odontolyticus, Prevotella bivia, and Clostridium bifermentans (6). We report the first detection of a nim gene in a Veillonella sp. It is also the first description of the nimE gene outside the B. fragilis group. As previously described for the genus Bacteroides, the presence of nim genes did not lead systematically to the expression of MTZ resistance, but the isolation of this Veillonella sp. strain extends the list of nim gene-harboring bacteria.

Nucleotide sequence accession number.

A partial sequence of 448 bp for the Veillonella sp. nitroimidazole resistance protein (nimE) gene has been deposited in GenBank under accession number AY575922.