Related Experiment Videos
Cloning and hybridization analysis of ermP, a macrolide-lincosamide-streptogramin B resistance determinant from
1Department of Microbiology, Monash University, Clayton, Victoria, Australia.
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 beta 1. No hybridization was detected with the other six hybridization classes of erm determinants.
Insights
The Clostridium perfringens erythromycin resistance gene (ermP) was cloned but found unstable in E. coli. While ermP is related to the ermB gene, it was not widespread in resistant Clostridium isolates.
Area of Science:
- Molecular biology
- Microbiology
- Genetics
Background:
- Antibiotic resistance is a growing public health concern.
- Clostridium species are significant pathogens, and understanding their resistance mechanisms is crucial.
- Erythromycin resistance determinants are important targets for studying antimicrobial resistance.
Purpose of the Study:
- To clone and characterize the erythromycin resistance determinant (ermP) from Clostridium perfringens.
- To investigate the prevalence and genetic relatedness of ermP in various clostridial species.
- To determine the relationship between ermP and other known erythromycin resistance genes.
Main Methods:
- Cloning of the erythromycin resistance determinant into Escherichia coli.
- Plasmid instability and deletion analysis using recA+ and recA E. coli hosts.
- Subcloning and BAL 31 deletion analysis to localize the ermP gene.
- DNA hybridization using a 32P-labeled ermP-specific probe against various clostridial DNA samples.
- Restriction analysis to compare ermP with other erm determinants.
Main Results:
- The erythromycin resistance determinant from C. perfringens CP592, designated ermP, was successfully cloned and expressed in E. coli.
- The plasmid containing ermP (pJIR122) exhibited instability with spontaneous deletions in E. coli hosts.
- ermP was localized to a 1.0-kb region, and a specific probe detected its presence in some Clostridium difficile and Clostridium paraputrificum isolates, but not widely in C. perfringens.
- ermP showed hybridization and significant restriction identity with the ermB class gene from streptococcal plasmid pAM beta 1, but not with other erm classes.
Conclusions:
- The ermP gene confers erythromycin resistance and is related to the ermB class of resistance genes.
- The instability of the plasmid carrying ermP in E. coli suggests potential mechanisms for its regulation or maintenance.
- While ermP is present in some other clostridial species, it is not a common determinant among erythromycin-resistant C. perfringens isolates, indicating diverse resistance mechanisms within this genus.