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.
Highlights
Non-typeable Haemophilus influenzae (NTHi) commonly resides in the human nasopharynx, from which it can disseminate to other organs and cause two types of infections, differing in their epidemiology and severity
To gain insights into the effect of heat stress on imipenem resistance in H. influenzae, we measured the interaction between controlled heat stress and imipenem resistance in NTHi by four mutually supportive approaches: (i) we simultaneously measured growth and cell viability at either 37 or 42◦C in NTHi cells exposed to increasing concentrations of imipenem; (ii) we investigated penicillin binding protein (PBP) by using Bocillin-FL and bacterial cells growth at either 37 or 42◦C; (iii) we monitored transcriptome changes by RNA-seq after pre-incubation of bacterial cells at either 37 or 42◦C; and (iv) we confirmed by real-time quantitative reverse transcription-PCR the transcriptional changes of different key genes that can be implicated in the resistance of H. influenzae to β-lactam antibiotics
To determine whether colonies of NTHi cells growing in the presence of high concentrations of imipenem were originating from populations of cells with a different resistant phenotypes, one colony of each of the two NTHi strains was picked from HTM agar containing 16 or 32 μg of imipenem/mL and passaged in imipenem free HTM agar
Summary
Non-typeable Haemophilus influenzae (NTHi) commonly resides in the human nasopharynx, from which it can disseminate to other organs and cause two types of infections, differing in their epidemiology and severity. Finding alternatives to extended-spectrum cephalosporins for the treatment of invasive infections caused by resistant strains was considered important. This holds especially true in the context of the global spread of extendedspectrum-β-lactamases (ESBLs) in Enterobacteriaceae, as well as of the recent reports of ESBL (blaTEM−15) in the closely related Haemophilus parainfluenzae (Tristram et al, 2008). It is noticeable that the functions of PBPs are multiple and may differ according to growth conditions and physiological status As it has been shown previously, β-lactams resistance in H. influenzae is often multi-factorial (Tristram et al, 2007; Cherkaoui et al, 2016)
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