Cloning and hybridization analysis of ermP, a macrolide-lincosamide-streptogramin B resistance determinant from Clostridium perfringens

Abstract

The erythromycin resistance determinant from Clostridium perfringens CP592 was cloned and shown to be expressed in Escherichia coli. The resultant plasmid, pJIR122 (7.9 kilobase pairs [kb]), was unstable since in both recA+ and recA E. coli hosts spontaneous deletion of 2.7 kb, including the erythromycin resistance determinant, was observed. Subcloning, as well as deletion analysis with BAL 31, localized the erythromycin resistance gene (ermP) to within a 1.0-kb region of pjIR122. A 0.5-kb fragment internal to ermP was 32p labeled and used as an ermP-specific probe in DNA hybridization experiments which used target DNA prepared from representatives of each of the known erm classes and also from erythromycin-resistant isolates of a variety of clostridial species. Hybridizing sequences were detected in DNA from several Clostridium difficile isolates and a Clostridium paraputrificum strain; however, ermP was not widespread in erythromycin-resistant C.perfringens isolates. The ermP determinant hybridized to, and shared significant restriction identity with, the ermB class gene from the streptococcal plasmid pAM,ll. No hybridization was detected with the other six hybridization classes of erm determinants.

The closely related ermB-ermAM genes from Clostridium perfringens, Enterococcus faecalis (pAM beta 1), and Streptococcus agalactiae (pIP501) are flanked by variants of a directly repeated sequence

“…Streptococcal isolates were from France (P. Courvalin, Institut Pasteur). E. coli and clostridial strains were cultured as described before (4). Streptococcal strains were grown in nutrient broth no.…”

“…The recombinant plasmid pJIR122 carries the ermBP gene, which belongs to the ermB-ermAM hybridization class (4). Genes which hybridize to ermBP are also present in MLS-resistant isolates of Clostridium difficile and Clostridium paraputrificum (4). The second gene, ermQ, has been sequenced and shown to be the most common erythromycin resistance determinant in C. perfringens (3).…”

“…The recombinant plasmid pJIR122 carries the ermBP gene, which belongs to the ermB-ermAM hybridization class (4). Genes which hybridize to ermBP are also present in MLS-resistant isolates of Clostridium difficile and Clostridium paraputrificum (4).…”

“…Restriction and hybridization analysis revealed that the ermBP gene was closely related to the determinant from pAM␤1. The similarity extended into the regions flanking the erm structural genes; however, its precise extent was not determined (4). To further characterize the relationship between these determinants, the sequence of the ermBP gene and its flanking regions was determined and comparative sequence analysis was carried out.…”

tIS Cpe8 , an IS 1595 -Family Lincomycin Resistance Element Located on a Conjugative Plasmid in Clostridium perfringens

“…Lincomycin resistance in C. perfringens is relatively common, but it is usually conferred as MLS resistance by erm(B) or erm(Q) genes (10,11). Recent studies have shown that there has been an increase in lincomycin resistance in C. perfringens strains isolated from chickens in Belgium (28).…”