Prevalence and characteristics of nim genes encoding 5-nitroimidazole resistance among Bacteroides strains isolated in Morocco.

We report here an evaluation of the dissemination of nim genes, encoding 5-nitroimidazoles resistance, among Bacteroides clinical strains isolated in Morocco. This study was done using a PCR method. Among 60 strains studied, nine contain a copy of a nim gene. The sequence determination of these genes showed that they are homologous to three nim genes previously characterized in strains isolated in France: nimB (five genes), nimC (three genes), and nimA (one gene). Although the nimA and nimC genes were previously identified on plasmids pIP417 and pIP419, respectively, we found here that they have a chromosomal location. The MICs of three 5-nitroimidazole antibiotics (metronidazole, ornidazole, and tinidazole) of the nim gene-containing strains were very low (0.5-2 microg/ml), indicating that the nim genes were not efficiently expressed in these clinical isolates.

Conjugal transfer of the 5-nitroimidazole resistance plasmid pIP417 from Bacteroides vulgatus BV-17: characterization and nucleotide sequence analysis of the mobilization region.

Three small 5-nitroimidazole (5-Ni) resistance plasmids (pIP417, pIP419, and pIP421) from Bacteroides clinical isolates are transferable by a conjugative process during homologous or heterologous matings. The mobilization properties of pIP417 originated from strain BV-17 of Bacteroides vulgatus were studied. The plasmid was successfully introduced by in vitro conjugation into different strains of Bacteroides and Prevotella species and could be transferred back from these various strains to a plasmid-free 5-Ni-sensitive Bacteroides fragilis strain, indicating that in vivo spread of the resistance gene may occur. The transfer of plasmid pIP417 harbored by the Tc(r) strain BF-2 of B. fragilis was stimulated by low concentrations of tetracycline or chlorotetracycline. This suggests a possible role for coresident conjugative transposons in the dissemination of 5-Ni resistance among gram-negative anaerobes. The nucleotide sequence of the 2.1-kb DNA mobilization region was determined. It contains a putative origin of transfer (oriT) in an A+T-rich-region, including three inverted repeats, and two integration host factor binding sites. The two identified mobilization genes (mobA and mobB) are organized in one operon and were both required for efficient transfer. Southern blotting indicated that the mobilization region of plasmid pIP417 is closely related to that of both the erythromycin resistance plasmid pBFTM1O and the 5-Ni resistance plasmid pIP419 but not to that of the 5-Ni resistance plasmid pIP421.

Genetic analysis of the minimal replicon of plasmid pIP417 and comparison with the other encoding 5-nitroimidazole resistance plasmids from Bacteroides spp.

The nucleotide sequence of the DNA replication origin region of a Bacteroides vulgatus plasmid, pIP417, encoding 5-nitroimidazole resistance has been determined. This region of 1934 bp presents some characteristics similar to those of other replication protein-dependent origins. It contains a large open reading frame which could encode a basic Rep protein (RepA) of 36.8 kDa. Upstream of this ORF exist an AT-rich region, three direct repeats (iterons) of 21 bp, multiple DnaA binding sites, and sites, and sites for the integration host factor (IHF). Moreover, the amino acid sequence of the pIP417 RepA protein shows similarities with those of other Rep proteins encoded by plasmids of gram-negative bacteria: pRO1600 from Pseudomonas aeruginosa; pPS10 from Pseudomonas syringae; pFA3 from Neisseria gonorrhoeae; and two cryptic plasmids from Campylobacter hyointestinalis and Butyrivibrio fibrisolvens. Although RepA can be expressed in an Escherichia coli in vitro transcription-translation assay, vectors containing the pIP417 replication origin did not replicate in E. coli. The homology of the pIP417 replication region with the corresponding regions of other Bacteroides spp, plasmids was also studied by Southern blot hybridization. The results indicated that the repA gene of plasmid pIP417 is homologous to that of plasmid pIP421, but not of plasmid pIP419. The replication region of plasmid pIP421 was sequenced and showed about 80% identity at the nucleotide level with that of pIP417. A small (3634-bp) cloning vector (pFK12) of entirely defined nucleotide sequence was constructed for Bacteroides spp.

Plasmids pIP419 and pIP421 from Bacteroides: 5-nitroimidazole resistance genes and their upstream insertion sequence elements.

The genetic organization of two different 5-nitroimidazole (5-Ni) resistance genes was investigated: nimC and nimD from Bacteroides plasmids pIP419 and pIP421, respectively. The nimC gene (492 bp) and the nimD gene (495 bp) directed the synthesis of polypeptides with deduced molecular masses of 18.37 kDa and 18.48 kDa, respectively. The predicted proteins showed 67-83% identity and 78-91% similarity with the products of two other nimA and nimB genes previously described and could be derived from a common ancestral gene. An insertion sequence element (IS1170) was identified upstream of the nimC gene. IS1170 is 1604 bp in length and is flanked by imperfect inverted repeats (15 bp). IS1170 is similar to the Bacteroides insertion sequence element IS942 with an identity of 70% at the nucleotide level. The single copy of IS1170 present on plasmid pIP419 is integrated 24 bp upstream of the initiation codon of nimC. Similar genetic organization was found on plasmid pIP421. One copy of another insertion sequence (IS1169) was found 4 bp upstream of the first ATG codon of the nimD gene. This element (1325 bp) shows a strong homology at the nucleotide level (70% identity) with IS1186 and IS1168 found to be associated with the Bacteroides carbapenem resistance gene cfiA, and the 5-Nirgenes nimA and nimB, respectively. There is strong evidence that, as in the case of the cfiA gene, the transcription of the four nim genes so far studied is directed by outward-oriented promoters, carried on the right ends of the different insertion sequence elements.

Nucleotide sequence analysis of two 5-nitroimidazole resistance determinants from Bacteroides strains and of a new insertion sequence upstream of the two genes.

DNA sequence analysis of regions from plasmid pIP417 and chromosome BF8 which encode 5-nitroimidazole resistance in Bacteroides strains allowed the identification of two open reading frames corresponding to new genes, nimA (528 bp) and nimB (492 bp). Either gene may confer 5-nitroimidazole resistance to susceptible strains of Bacteroides. The encoded polypeptides have deduced molecular masses of 20.1 and 18.6 kDa, respectively, and share about 73% identity and 85% similarity. A new insertion sequence (IS) element named IS1168 lies 14 bases upstream of the nimA gene. The complete sequence of IS1168 was determined. A similar IS exists 12 bp upstream of the nimB gene. About 60% of the BF8 IS element was also sequenced and shown to be almost identical to IS1168.

Genetics of resistance of Bacteroides species to 5-nitroimidazole.

Thirty-three clinical isolates of Bacteroides strains with a moderate-to-high level of resistance to 5-nitroimidazole were compared with three strains previously studied by Southern blotting of their DNA. Whether plasmid-borne or chromosomally determined, all genetic determinants that are resistant to 5-nitroimidazole belong to two hybridization groups.

Cloning of a Bacteroides fragilis chromosomal determinant coding for 5-nitroimidazole resistance.

Strain BF8 is a plasmid-free Bacteroides fragilis, resistant to 5-nitroimidazole (5-Ni) antibiotics (metronidazole, ornidazole and tinidazole). The resistance was transferable by conjugation into Bacteroides fragilis BF638R. The total DNA of a Nir transconjugant was used for the construction of a Sau3A genomic library in a B. fragilis cloning vector pFK707 delta H1 (4.2 kb). By electrotransformation of strain BF638R, a recombinant plasmid containing an insert of 5.4 kb was obtained which conferred to the host strain the resistance to 5-Ni. The physical map of the insert was established. After deletion analysis of the insert, the Nir determinant was localized on a HpaII-HincII fragment of 1.6 kb in size. This Nir determinant has been compared by Southern-blot analysis with other Bacteroides Nir determinants of plasmid origin.

Genetic and molecular analysis of pIP417 and pIP419: Bacteroides plasmids encoding 5-nitroimidazole resistance.

This report describes a genetic and molecular analysis of two transferable Bacteroides plasmids, pIP417 and pIP419, which carry genetic determinants conferring low-level resistance to 5-nitroimidazoles. The restriction endonuclease cleavage sites for each plasmid were localized. The NiR genetic determinants of pIP417 and pIP419 plasmids have been cloned into the Bacteroides cloning vector pBI191 (C.J. Smith, J. Bacteriol. 164, 294-301, 1985) as PvuII and Sau3A fragments, respectively. Both inserts had different restriction sites and did not cross-hybridize by Southern blot analysis. Genetic data obtained by cloning into pBI191 clearly show that the PvuII-generated fragments A (Rep) and B (Mob) of pIP417 are involved in plasmid replication and transfer, respectively. Although encoding resistance to the same antibiotic, both plasmids appeared different with regard to the 5-nitroimidazole resistance and replication genetic determinants. However, they share a homology in a region involved, at least in one case, in plasmid transfer. Considering the spontaneous high level of resistance to 5-nitroimidazole in Escherichia coli, this work, based on direct gene cloning into Bacteroides, demonstrates the value of such an approach.