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Characterization of non-beta-lactamase-mediated ampicillin resistance in Haemophilus influenzae
Abstract:
Ampicillin resistance in Haemophilus influenzae is most often due to the plasmid-mediated production of TEM beta-lactamase. We studied four strains with high-level ampicillin resistance (MIC of 32 micrograms/ml with an inoculum of 10(5) CFU on solid media) which did not produce detectable beta-lactamase activity with two different detection methods. Two of the four strains contained extrachromosomal DNA by agarose gel electrophoresis. Conjugation failed to transfer ampicillin resistance; in contrast, transformation yielded ampicillin-resistant transformants in three of the four strains. These transformants did not contain detectable extrachromosomal DNA. In addition, mobilization of the resistance determinant by transformation to, or conjugation with, recombination-deficient strains was unsuccessful. DNA-DNA hybridization experiments revealed no homology of the DNA of these strains with two R plasmids (one coding for ampicillin resistance, the other for chloramphenicol and tetracycline resistance). We conclude that the genetic basis of the non-beta-lactamase ampicillin resistance in these strains appears to be chromosomally mediated. We investigated the mechanism of resistance in these strains. Enzymatic modification of penicillin was not detected by autoradiography of a thin-layer chromatogram of cell sonic extracts of three ampicillin-resistant transformant strains incubated with [14C]penicillin. To assess changes in permeability of the cell envelope, a plasmid coding for beta-lactamase was conjugated into these strains, and the hydrolysis of penicillin by intact cells and cell sonic extracts was compared. Only one of three transformant strains had significantly diminished permeability. Outer membrane proteins of these strains analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed apparent differences in comparison with the isogenic ampicillin-susceptible recipient strain. Autofluorography of a sodium dodecyl sulfate-polyacrylamide gel electrophoresis of Sarkosyl-solubilized crude membrane (the putative inner membranes) from these ampicillin-resistant transformant strains incubated with [3H]penicillin compared with the isogenic ampicillin-susceptible recipient strain revealed reduced binding to PBP 3 and 6, 3 and 4, or 4. In addition, affinity binding studies revealed decreased affinity of PBP 4 for ampicillin of all four transformants tested. We conclude that the major mechanism of resistance in these strains is altered penicillin-binding proteins; however, other mechanisms, including permeability, may also play a role.
Insights
This study investigates ampicillin resistance in Haemophilus influenzae strains lacking beta-lactamase. Researchers found that altered penicillin-binding proteins, not plasmids, are the primary cause of this resistance.
Area of Science:
- Microbiology
- Molecular Biology
- Genetics
Background:
- Ampicillin resistance in Haemophilus influenzae is typically plasmid-mediated via TEM beta-lactamase.
- This study focuses on ampicillin-resistant strains that do not produce detectable beta-lactamase.
Purpose of the Study:
- To determine the genetic basis of ampicillin resistance in beta-lactamase-negative Haemophilus influenzae strains.
- To elucidate the specific mechanisms conferring high-level ampicillin resistance in these strains.
Main Methods:
- Agarose gel electrophoresis to detect extrachromosomal DNA.
- Conjugation and transformation experiments to transfer ampicillin resistance.
- DNA-DNA hybridization to assess genetic homology with known R plasmids.
- Enzymatic assays and permeability studies to investigate resistance mechanisms.
- Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and affinity binding to analyze penicillin-binding proteins (PBPs).
Main Results:
- Three of four ampicillin-resistant strains were successfully transformed, indicating a chromosomal basis for resistance.
- No homology was found with known ampicillin or multi-drug resistance plasmids.
- Altered penicillin-binding proteins (PBPs), specifically reduced binding or affinity to ampicillin for PBP 3, 4, and 6, were identified as the major resistance mechanism.
- Diminished cell envelope permeability was observed in one transformant strain.
Conclusions:
- Ampicillin resistance in these beta-lactamase-negative Haemophilus influenzae strains is primarily chromosomally mediated.
- Altered penicillin-binding proteins are the main mechanism of resistance.
- Other factors, such as changes in cell envelope permeability, may also contribute to ampicillin resistance.