Madeleine Sebald

Characterization and transferability of Clostridium perfringens plasmids.

Abstract Two strains of Clostridium perfringens resistant to clindamycin (Cl), chloramphenicol (Cm), erythromycin (Em), and tetracycline (Tc) were isolated in France in 1974 and 1975. For one of these strains, curing experiments and molecular characterization of the extrachromosomal DNA clearly demonstrate the existence of two plasmids, plP401 (54 kilobases) and plP402 (63 kilobases), which, respectively, code for Tc Cm and Em Cl resistance. With mixed cultures, the Tc Cm plasmid is transferable to sensitive strains of C. perfringens; a segregation of these markers is frequently observed during mating experiments. In contrast, the transfer of the naturally occurring plasmid Em Cl does not occur at a significant rate. In performing transfer experiments in axenic mice, we obtained a Clr Emr Tcr transcipient whose chromosomal properties are those of a hybrid. When used in mating as a parental strain, this strain promotes chromosomal gene exchange. The role of the plasmid in this phenomenon is discussed, these transcipients being generally Clr Emr Tcr. The plasmid transfer is not limited to antibiotic resistance plasmids, the transferability of a bacteriocinogenic plasmid, plP404, harbored by C. perfringens BP6K-N5 being shown also. The transfer mechanism remains to be proved; it might be a conjugation process, a cell-to-cell contact being necessary for the transfer.

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.

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.

Transferable 5-Nitroimidazole resistance in the Bacteroides fragilis group

We report the characterization of a strain of Bacteroides vulgatus, BV17, that exhibits a moderate resistance to 5-nitroimidazoles and carries plasmids of 4.5, 5, 7.7, and 56 kb. A genetic determinant involved in this resistance is carried by the 7.7 +/- 0.2-kb plasmid (pIP417). This plasmid can be introduced and replicated in a sensitive strain of B. fragilis 638R by transformation or by conjugation. In the latter case, the transfer may involve mobilization by the 56-kb conjugative plasmid (pIP418) regularly found in transconjugants but not in transformants.

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.

Common regulatory mechanism of expression and conjugative ability of a tetracycline resistance plasmid in Bacteroides fragilis

RESISTANCE to various antibiotics is common in the anaerobic gut bacterium Bacteroides fragilis. A transferable plasmid coding for resistance to antibiotics of the lincosamide/macrol-ide/synergistin groups has been found1,2 and transfer is most probably a conjugative process. This supports the observation of the recent emergence of resistance to antibiotics in these groups and its rapid spread in certain areas (ref. 3 and our unpublished data). However, in the case of tetracycline resistance the limited genetic and physical data were at variance with the epidemiological data. Although tetracycline resistance is common in B. fragilis4 (at present 60% of strains isolated in the USA5 and 30% of those isolated in France (our unpublished results) are resistant) intraspecies transfer of tetracycline resistance could not be demonstrated6. We report here that transfer of tetracycline resistance between B. fragilis strains is easily achieved if the donor bacteria are grown in the presence of tetracycline before mating. This suggests that tetracycline resistance is borne on a transferable plasmid whose conjugative transfer requires activation of the tetracycline resistance gene. This is supported by our finding that the transfer of tetracycline resistance is either inducible or constitutive, depending on whether tetracycline resistance itself is respectively inducible by tetracycline or constitutive.

Plasmid detection and isolation in strains of Clostridium acetobutylicum and related species

SummaryTwenty-one strains of Clostridium acetobutylicum, C. butylicum and Clostridium saccharoperbutylacetonicum were examined. Seven of them contained extrachromosomal DNA molecules, with a size ranging from 2.6 to more than 50 megadaltons. Strain M1 carries a small plasmid of 2.6 megadaltons, AB10 at least one plasmid of 2.6 megadaltons, AB12 one plasmid of 5.2 megadaltons, AB14 and AB16 a plasmid of about 7 megadaltons and a large one of more than 50 megadaltons, AB17 carries at least one plasmid of 6.7 megadaltons and AB18 two plasmids (4–6 megadaltons and 10–12 megadaltons). All of them are cryptic as at present no function can be correlated with their presence in a bacterial strain.

Protoplast Formation and Regeneration of Clostridium acetobutylicum Strain N1-4080

SUMMARY: A method for the production of protoplasts of Clostridium acetobutylicum strain N1-4080 utilizing lysozyme and penicillin G was optimized to yield about 100% protoplasts. Treatment of 109 cells left fewer than 102 that were viable and osmo-resistant. With a mutant strain deficient in autolytic activity, about 1% of the protoplasts regenerated walled cells, whereas most gave rise to stable L-form colonies. Addition of sodium polyanethole sulphonate to the medium improved the regeneration frequency by at least a factor of 10, probably by somehow inhibiting the residual autolytic activity of the strain.

Resistance to tetracycline, erythromycin, and clindamycin in the Bacteroides fragilis group: inducible versus constitutive tetracycline resistance.

The transferability of plasmid-mediated tetracycline, erythromycin, and clindamycin resistance was studied in 63 clinical isolates of the Bacteroides fragilis group. Of 48 strains which were tetracycline resistant (Tcr), the regulation of both the expression of Tcr and its transferability was shown to be under inducible control by tetracycline. In 29 of the strains, Tcr was transferable; in the majority of these (26 strains), transferability was inducible (Trai) and it was constitutive (Trac) in only 3 strains. All four possible phenotypes were found (Tci Trai, Tci Trac, Tcc Trai, and Tcc Trac), which indicates independent control of both Tcr expression and its transferability. Resistance to erythromycin and clindamycin was cotransferred with Tcr in 14 of the 48 Tcr strains and transferred independently of Tcr in only 1 strain.

Presence of Choline in Teichoic acid of Clostridium acetobutylicum N1-4 and Choline Inhibition of Autolytic Functions

Summary: Addition of choline to growing Clostridium acetobutylicum led to abnormal cell septation, lack of cell separation and the consequent formation of chains. Similar results were obtained with the wild-type strain N1-4 and its autolysin-deficient mutant N1-4081. With strain N1-4, addition of choline at 1 to 2 mg ml−1 resulted in inhibition of autolysis assessed as autoplast formation in 0.6 M-sucrose, lysis by 0.3 M-NaCl/0.03 M-sodium citrate, lysis by 0.1% Triton X-100 and lysis by penicillin G. In vitro data confirmed the inhibition by choline of wall-degrading activity, using N1-4 cell walls as substrate. Choline was shown to be a component of the teichoic acid of C. acetobutylicum N1-4.