Related Experiment Video
Updated: Aug 17, 2025
Functional Complementation Analysis FCA: A Laboratory Exercise Designed and Implemented to Supplement the Teaching of Biochemical Pathways
Published on: June 24, 2016
Use of trans-complementation method to determine the effects of various ftsI mutations on β-lactamase-negative
Melis Yalçın1, Stephen Tristram2, Bülent Bozdoğan3,2,4
1Recombinant DNA and Recombinant Protein Research Center, Adnan Menderes University, Aydın, Turkey. mels.yalcn@gmail.com.
Abstract:
Haemophilus influenzae is a causative agent of serious infections, especially among children. β-lactam antibiotics are commonly used for the treatment of these infections. Among H. influenzae isolates, β-lactam resistance is due to the presence of β-lactamase, or to mutations in the ftsI gene that generate altered PBP3 (penicillin-binding protein 3) with reduced affinity for β-lactams (BLNAR-β-lactamase-negative, ampicillin-resistant). Wild-type ftsI gene encoding for PBP3 was amplified in whole from β-lactam susceptible H. influenzae Rd and cloned in pLS88 plasmid to obtain pADUTAS17, which was then used to transform known BLNAR strains, susceptible strains, and a strain (CF55) with wild-type ftsI but unexplained reduced β-lactam susceptibility. Ampicillin and cefotaxime MICs (minimum inhibitory concentration) were determined after transformation with pLS88 and pADUTAS17 plasmids. The results showed that antibiotic susceptibilities were not affected by trans-complementation for isolates carrying wild-type ftsI gene. However, trans-complementation for all BLNAR strains showed decreases between - 0.957 and 0.5-fold for ampicillin and cefotaxime, confirming the role of the PBP3 substitutions in the BLNAR phenotype of these isolates. The first article showed that trans-complementation might be a useful tool in the investigation of decreased β-lactam susceptibility in H. influenzae.
Insights
Trans-complementation confirmed that altered penicillin-binding protein 3 (PBP3) is responsible for beta-lactam resistance in Haemophilus influenzae. This method helps investigate reduced antibiotic susceptibility in bacterial infections.
Area of Science:
- Microbiology
- Molecular Biology
- Antimicrobial Resistance
Background:
- * *Haemophilus influenzae* causes serious infections, particularly in children.
- * Beta-lactam antibiotics are primary treatments, but resistance is a growing concern.
- * Resistance mechanisms include beta-lactamase production or *ftsI* gene mutations affecting PBP3.
Purpose of the Study:
- * To investigate the role of PBP3 alterations in beta-lactam resistance in *H. influenzae*.
- * To evaluate the utility of trans-complementation as a tool for studying beta-lactam susceptibility.
Main Methods:
- * Amplification and cloning of wild-type *ftsI* gene into pLS88 plasmid.
- * Transformation of *H. influenzae* strains (BLNAR, susceptible, and CF55) with engineered plasmids.
- * Determination of ampicillin and cefotaxime Minimum Inhibitory Concentrations (MICs) post-transformation.
Main Results:
- * Trans-complementation did not affect antibiotic susceptibility in strains with wild-type *ftsI*.
- * BLNAR strains showed reduced susceptibility to ampicillin and cefotaxime after trans-complementation.
- * Results confirmed that PBP3 substitutions are critical for the BLNAR phenotype.
Conclusions:
- * Trans-complementation is a valuable method for investigating decreased beta-lactam susceptibility in *H. influenzae*.
- * This study validates the role of PBP3 mutations in conferring beta-lactam resistance.
- * Findings contribute to understanding antimicrobial resistance mechanisms in *H. influenzae*.
More Related Videos
05:06Author Spotlight: Advancing Antibiotic Resistance Research Using an Efflux-Deficient Bacterial Strain and a Single-Copy Gene Expression System
Published on: January 5, 2024
11:36A Protocol for Functional Assessment of Whole-Protein Saturation Mutagenesis Libraries Utilizing High-Throughput Sequencing
Published on: July 3, 2016