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

Abstract

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 pBFTM10 and the 5-Ni resistance plasmid pIP419 but not to that of the 5-Ni resistance plasmid pIP421.The dissemination of drug resistance determinants among bacteria of medical importance is currently a major problem for the prescription of efficient antibiotic regimens. This is particularly true for Bacteroides spp., a prevalent anaerobic bacterial genus causing human infections, since the majority of the strains are now frequently resistant to commonly used antibiotics. The resistance determinants of tetracycline (TET) and macrolides-lincosamides-streptogramin B (MLS), which were first described in 1979 (24, 41, 45), have been disseminated by specific efficient conjugal transfer systems. Conjugal genetic exchange of these resistance markers in Bacteroides spp. is now well documented (16,(29)(30)(31)(32). Various genetic elements have been shown to be involved, including (i) large conjugative plasmids like pBF4, a 41-kb plasmid from Bacteroides fragilis, or pBI136, an 80-kb plasmid from Bacteroides ovatus; (ii) small mobilizable plasmids like pBFTM10, a 15-kb plasmid from B. fragilis; and (iii) conjugative transposons (Tc r / Tc r Em r elements). Bacteroides conjugative transposons are large chromosomal self-transmissible elements, ranging in size from 65 to more than 150 kb, which can also drive the mobilization of other elements, such as small coresident plasmids, either in trans or in cis. Three distinct families have been described: the Tc r Em r DOT family that usually carries both tetQ and ermF genes but includes at least one cryptic element, XBU4422; the Tc r Em r 12256 element (also called Tn5030) that consists...

Sequential Inactivation of rdxA (HP0954) and frxA (HP0642) Nitroreductase Genes Causes Moderate and High-Level Metronidazole Resistance in Helicobacter pylori

“…In principle, resistance might also result from (i) diminished Mtz uptake or its active export (26,40), (ii) more efficient DNA damage repair (6,43), or (iii) enhanced scavenging of oxygen radicals that are produced according to certain models of Mtz activation (23,41). Of particular note are plasmid-and transposon-borne nim genes in certain Mtz r strains of Bacteroides fragilis that promote conversion of nitroimidazoles from prodrug to harmless amino derivatives, rather than to toxic nitroso radicals, and that thus confer resistance without loss of chromosomal nitroreductase gene function (5,46). DNA fingerprint and sequence analyses have indicated that each H. pylori clinical isolate differs genetically from most other independent isolates (1, 2).…”

Identification and DNA sequence of the mobilization region of the 5-nitroimidazole resistance plasmid pIP421 from Bacteroides fragilis

“…Pairwise comparisons revealed that the unique mobilization polypeptide of pIP421 is definitively unrelated to the mobilization proteins of both plasmids pIP417 (25) and pBFTM10 (8) and also to those of plasmid pLV22a (15) and the transposon Tn4399 (13,14).…”

“…The spread of the resistance among these significant opportunistic pathogens, which are responsible for various infectious diseases, could be explained by the fact that nim genes are usually found on mobilizable genetic elements, either in non-self-transferable plasmids or on the chromosome. Three such 5-Ni r plasmids (pIP417, pIP419, and pIP421) have already been characterized, and the mobilization properties of one of them, pIP417, has been fully analyzed (25). The mobilization region of plasmid pIP417 is closely related to that of both the erythromycin resistance plasmid pBFTM10 from Bacteroides fragilis (8) and pIP419 but not to that of pIP421.…”

“…Thus, strain BF-F239 may contain a chromosomal conjugative transposon required for efficient pIP421 transfer, probably regulated by the products of the rteB and/or rteC genes (21). To test this hypothesis, the conjugal transfer of the plasmid pIP421 was studied through mating experiments with strain BF-2, containing a Tc r Em r DOT conjugative-type transposon (25), as the donor and strain BF-638R (5-Ni s Rf r ) of B. fragilis as the recipient. The transfer of pIP421 was effectively enhanced up to 500-fold in the presence of TET, and the transfer frequency of the plasmid was in the same range as that of the chromosomal Tc r determinant of strain BF-2 (1 ϫ 10 Ϫ5 versus 1.3 ϫ 10…”

“…We therefore examined the transfer properties of pIP421 and compared them with those of mobilizable genetic elements previously identified in Bacteroides species. The experimental procedures used in the present study have been previously described (25).…”