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Mechanism of transferable resistance to chloramphenicol in Haemophilus parainfluenzae
Abstract:
A clinical isolate of Haemophilus parainfluenzae resistant to chloramphenicol and tetracycline transferred both cam and tet determinants to Escherichia coli K-12 during mixed cultivation on solid media irrespective of the selection employed. The doubly resistant transconjugant was found to contain levels of the enzyme chloramphenicol acetyltransferase (CAT) comparable to those found in R plasmid-bearing chloramphenicol-resistant enteric bacteria. Purification of CAT from the transconjugant was achieved by affinity chromatography, and the electrophoretically homogeneous protein was compared with previously characterized CAT variants specified by R plasmids. Although the CAT associated with cam from H. parainfluenzae was found to be distinct from the three types described previously, its N-terminal peptide amino acid sequence was identical with that determined for a type II CAT. Attempts to demonstrate covalently closed circular deoxyribonucleic acid in the H. parainfluenzae donor and the E. coli transconjugant were unsuccessful. The cam and tet determinants were nontransmissible from E. coli but could be cotransferred following the introduction of a suitable conjugative plasmid.
Insights
Antibiotic resistance genes for chloramphenicol and tetracycline were transferred from Haemophilus parainfluenzae to E. coli. The new strain produced chloramphenicol acetyltransferase (CAT), distinct from known types.
Area of Science:
- Microbiology
- Molecular Biology
- Antimicrobial Resistance
Background:
- Haemophilus parainfluenzae is a species of Gram-negative coccobacillus.
- Antibiotic resistance is a growing global health concern.
- Mechanisms of antibiotic resistance gene transfer include conjugation, transformation, and transduction.
Purpose of the Study:
- To investigate the transfer of chloramphenicol (cam) and tetracycline (tet) resistance determinants from a clinical isolate of Haemophilus parainfluenzae to Escherichia coli K-12.
- To characterize the chloramphenicol acetyltransferase (CAT) enzyme produced by the transconjugant.
- To explore the transmissibility of these resistance determinants.
Main Methods:
- Mixed cultivation of H. parainfluenzae and E. coli K-12 on solid media.
- Selection of transconjugants.
- Enzyme assays for chloramphenicol acetyltransferase (CAT) activity.
- Protein purification using affinity chromatography.
- Electrophoresis and N-terminal peptide sequencing of CAT.
- Attempts to isolate plasmids from donor and transconjugant strains.
- Mobilization of resistance determinants using a conjugative plasmid.
Main Results:
- H. parainfluenzae successfully transferred both cam and tet resistance determinants to E. coli K-12.
- The resulting E. coli transconjugant exhibited chloramphenicol acetyltransferase (CAT) activity comparable to R plasmid-bearing strains.
- Purified CAT from the transconjugant was distinct from previously characterized types but shared an N-terminal sequence with type II CAT.
- No covalently closed circular DNA (plasmids) was detected in the donor or transconjugant.
- The cam and tet determinants were not initially self-transmissible from E. coli but became transferable after introduction of a conjugative plasmid.
Conclusions:
- H. parainfluenzae can act as a source of transferable antibiotic resistance genes.
- The CAT enzyme produced is a novel variant, suggesting diverse mechanisms of chloramphenicol resistance.
- The absence of detectable plasmids indicates potential alternative mechanisms for resistance gene carriage or transfer, possibly involving integrative elements.
- Co-transfer of resistance determinants is dependent on the presence of conjugative machinery.