Transcriptional Modulation of Penicillin-Binding Protein 1b, Outer Membrane Protein P2 and Efflux Pump (AcrAB-TolC) during Heat Stress Is Correlated to Enhanced Bactericidal Action of Imipenem on Non-typeable Haemophilus influenzae

Abstract

Objective: The purpose of the present study was to investigate the penicillin binding proteins (PBPs), drug influx and efflux modulations during heat stress and their effects on the bactericidal action of imipenem on non-typeable Haemophilus influenzae (NTHi).Methods: The two NTHi clinical isolates (GE47 and GE88, imipenem MICs by E-test > 32 μg/mL) examined in this study were collected at Geneva University Hospitals. The imipenem killing activity was assessed after incubation of the NTHi strains at either 37 or 42°C for 3 h with increasing concentrations of imipenem. The detection of PBPs was carried out by Bocillin-FL. Global transcriptional changes were monitored by RNA-seq after pre-incubation of bacterial cells at either 37 or 42°C, and the expression levels of relevant target genes were confirmed by qRT-PCR.Results: Quantitation of NTHi viable cells after incubation with 0.25 μg/mL of imipenem for 3 h revealed more than a twofold decrease in GE47 and GE88 viable cells at 42°C as compared to 37°C. Transcriptome analysis showed that under heat stress conditions, there were 141 differentially expressed genes with a | log2(fold change)| > 1, including 67 up-regulated and 74 down-regulated genes. The expression levels of ponB (encoding PBP1b) and acrR (regulator of AcrAB-TolC efflux pump) were significantly increased at 42°C. In contrast, the transcript levels of ompP2 (encoding the outer membrane protein P2) and acrB gene (encoding AcrB) were significantly lower under heat stress condition.Conclusion: This study shows that the transcriptional modulation of ponB, ompP2, acrR, and acrB in the heat stress response is correlated to enhanced antimicrobial effects of imipenem on non-typeable H. influenzae.

Imipenem heteroresistance but not tolerance in Haemophilus influenzae during chronic lung infection associated with chronic obstructive pulmonary disease

“…To date, resistance of H. influenzae to imipenem remains low (García-Cobos et al, 2014;Lâm et al, 2020;Potts et al, 2022;Safari et al, 2022), and tolerance has not been reported. However, limitations of the broth microdilution method seem to underestimate the extent of imipenem heteroresistance in H. influenzae, as previously reported for invasive and respiratory isolates (Cerquetti et al, 2007;Cherkaoui et al, 2017Cherkaoui et al, , 2018aLâm et al, 2020). Although not formally demonstrated, imipenem heteroresistance may be linked to a combination of altered PBP3 and drug fluxes (Cherkaoui et al, 2017).…”

“…This opportunistic microorganism often develops resistance to β-lactam antibiotics by several mechanisms including β-lactamase production and polymorphisms in the penicillin-binding protein 3 (PBP3) ( Parr and Bryan, 1984 ). Cephalosporins have been used since the emergence of ampicillin-resistant strains, and carbapenems are currently used as an alternative to extended-spectrum cephalosporins for initial empiric treatment of severe infections ( Cherkaoui et al, 2018a , b ). To date, resistance of H. influenzae to imipenem remains low ( García-Cobos et al, 2014 ; Lâm et al, 2020 ; Potts et al, 2022 ; Safari et al, 2022 ), and tolerance has not been reported.…”

Inactivation of farR Causes High Rhodomyrtone Resistance and Increased Pathogenicity in Staphylococcus aureus

“…Oriented 50 bases single-read sequencing was performed on the Illumina HiSeq 4000 sequencer yielding a minimum of 8 million mapped reads per sample. Final RNA-seq analysis and data analysis were carried out using previous described procedures (Cherkaoui et al, 2017). The raw sequence data were filtered by removing reads containing adapter, reads containing poly-N, and low-quality reads.…”

Learning from –omics strategies applied to uncover Haemophilus influenzae host-pathogen interactions: Current status and perspectives

“…Such observations were further extended by differential gene expression profiling of H. influenzae wild-type, crp and sxy mutant strains, which also shed light on the competence-regulated toxin-antitoxin ToxTA system [83] . Moreover, analysis of global transcriptional changes has also been of use when assessing H. influenzae response to antibiotic pressure, showing increased ponB (encoding PBP1b) and acrR (negative regulator of AcrAB-TolC efflux pump) gene expression during heat stress, which may relate to the observed decrease in bacterial viability after incubation with imipenem at 42 °C as compared to 37 °C [84] . An effort to identify convergent molecular signatures related to lung adaptation led to comparative transcriptomic analysis of paired, isogenic NTHi strains, isolated from the NP and BAL of children with chronic lung disease, showing no convergence at the gene level, but a possible trend in terms of functional enrichment among genetically unrelated NTHi strains [24] .…”

“…Tn-seq based in vitro genetic screenings [69] , [70] , [71] , [72] , [73] C. Genome-wide gene expression profiling C1. Bacterial transcriptomic responses to changing environmental conditions [75] , [76] , [77] , [78] , [79] , [82] , [84] , [87] C2. Bacterial transcriptomic responses to gene inactivation [35] , [53] , [54] , [55] , [59] , [80] , [81] , [83] C3.…”