RESUMEN
Enterococcus faecium is a gut commensal bacterium which is gaining increasing relevance as an opportunistic, nosocomial pathogen. Its high level of intrinsic and acquired antimicrobial resistance is causing a lack of treatment options, particularly for infections with vancomycin-resistant strains, and prioritizes the identification and functional validation of novel druggable targets. Here, we use activity-based protein profiling (ABPP), a chemoproteomics approach using functionalized covalent inhibitors, to detect active serine hydrolases across 11 E. faecium and Enterococcus lactis strains. Serine hydrolases are a big and diverse enzyme family, that includes known drug targets such as penicillin-binding proteins (PBPs), whereas other subfamilies are underexplored. Comparative gel-based ABPP using Bocillin-FL revealed strain- and growth condition-dependent variations in PBP activities. Profiling with the broadly serine hydrolase-reactive fluorescent probe fluorophosphonate-TMR showed a high similarity across E. faecium clade A1 strains, but higher variation across A2 and E. lactis strains. To identify these serine hydrolases, we used a biotinylated probe analog allowing for enrichment and identification via liquid chromatography-mass spectrometry. We identified 11 largely uncharacterized targets (α,ß-hydrolases, SGNH-hydrolases, phospholipases, and amidases, peptidases) that are druggable and accessible in live vancomycin-resistant E. faecium E745 and may possess vital functions that are to be characterized in future studies.
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This work aimed to develop accurate, quick, and practical tools for the detection of residues of penicillin G antibiotic in biological and non-biological samples. The assays were developed based on the binding mechanism of ß-lactam to penicillin-binding proteins; samples of different concentrations of penicillin G were incubated with in vitro expressed 6X-Histidine-tagged soluble penicillin-binding protein (PBP2x*) of Streptococcus pneumoniae (S. pneumoniae), whereby penicillin G in samples specifically binds to PBP2x*. The fluorescent-labeled ß-lactam analogue Bocillin FL was used as a competent substrate, and two different routes estimated the amounts of the penicillin G. The first route was established based on the differences in the concentration of non-bounded Bocillin FL molecules within the reactions while using a real-time polymerase chain reaction (PCR)-based method for fluorescence detection. The second route depended on the amount of the relative intensity of Bocillin FL bounded to Soluble PBP-2x*, being run on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-page), visualized by a ChemiDoc-It®2 Imager, and quantified based on the fluorescence affinity of the competent substrate. While both of the methods gave a broad range of linearity and high sensitivity, the on column based real-time method is fast, non-time consuming, and highly sensitive. The method identified traces of antibiotic in the range 0.01-0.2 nM in addition to higher accuracy in comparison to the SDS-based detection method, while the sensitivity of the SDS-based method ranged between 0.015 and 2 µM). Thus, the on column based real time assay is a fast novel method, which was developed for the first time based on the binding inhibition of a fluorescence competitor material and it can be adapted to screen traces of penicillin G in any biological and environmental samples.
Asunto(s)
Antibacterianos/química , beta-Lactamas/química , Antibacterianos/farmacología , Proteínas Bacterianas/química , Técnicas Biosensibles/métodos , Compuestos de Boro/química , Pruebas de Sensibilidad Microbiana/métodos , Proteínas de Unión a las Penicilinas/química , Penicilinas/química , Penicilinas/farmacología , Streptococcus pneumoniae/efectos de los fármacosRESUMEN
Low proliferation rate of bacterial populations was recently assumed to be a reason for higher resistance to antibiotics and appearance of many chronic infections. Slowly growing populations, called 'small colony variants' (SCVs) have been described in many bacterial species to make from as low as 0·02% up to 46% of population. Thirty enterococcal strains from urine and faeces of renal transplant recipients with asymptomatic, insignificant bacteriuria were studied. Growth characteristics were estimated by microculture and OD reading after 1, 3 and 5 h of culture. At the same time, penicillin binding and changes of aggregation of the cells were analysed by flow cytometry. The results of our study showed high diversity of the proliferation rates among studied isolates. Based on proliferation rates and aggregation, six of studied strains (20%) could be considered as SCVs-like. Significantly lower binding of penicillin was also observed for these SCV-like strains. SIGNIFICANCE AND IMPACT OF THE STUDY: This study provides relevant information about prevalence of enterococcal strains with low proliferation rate (likely small colony variant (SCV)) among kidney transplant recipients. Percentage of such strains in this cohort was relatively high (20%). Additionally, penicillin binding of these strains measured even at the beginning of proliferation (after 1 and 3 h of incubation), was significantly lower than among other strains. Finally, all of them were determined as penicillin resistant, with minimal inhibitory concentration value above 256 µg ml-1 . As the risk of systemic infections caused by such strains is probably higher than in case of other strains, screening for the SCVs in this group of patients should be recommended.
Asunto(s)
Antibacterianos/metabolismo , Enterococcus/crecimiento & desarrollo , Enterococcus/aislamiento & purificación , Huésped Inmunocomprometido/inmunología , Penicilinas/metabolismo , Adhesión Bacteriana/fisiología , Bacteriuria/microbiología , Proliferación Celular/efectos de los fármacos , Enterococcus/clasificación , Humanos , Trasplante de Riñón , Pruebas de Sensibilidad Microbiana , Receptores de TrasplantesRESUMEN
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.
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OBJECTIVE: To investigate the potential roles of PBPs, efflux pumps and slow drug influx for imipenem heteroresistance in nontypeable Haemophilus influenzae (NTHi). METHODS: Fifty-nine NTHi clinical isolates examined in this study were collected at Geneva University Hospitals between 2009 and 2014. Alterations in PBPs were investigated by gene sequencing. To evaluate the affinities of the PBPs to imipenem, steady-state concentration-response experiments were carried out using imipenem in a competition assay with Bocillin-FL. The effect of the carbonyl cyanide m-chlorophenylhydrazone (CCCP) on imipenem susceptibility was assessed using broth dilution and viable cell counting. Using whole-genome sequencing, we explored the potential roles of outer membrane protein P2 (OmpP2), LytM proteins and the dcw gene cluster in imipenem heteroresistance. RESULTS: All 46 imipenem-heteroresistant isolates (IMIhR) harboured amino acid substitutions in the ftsI gene, which encodes PBP3, corresponding to 25 different mutation patterns that varied from the ftsI gene mutation patterns found in imipenem-susceptible isolates. Among all PBPs, the highest affinity to imipenem was documented for PBP3 (IC50, 0.004 µg/mL). Different amino acid substitutions and insertions were noted in OmpP2, suggesting a relationship with imipenem heteroresistance. The IMIhR isolates were affected by CCCP differently and displayed a higher percentage of killing by imipenem in CCCP-treated cells at concentrations ranging between 0.5 and 8 µg/mL. CONCLUSIONS: The present study provides robust evidence indicating that in combination with the altered PBP3, the slowed drug influx and its enhanced efflux due to the loss of regulation led to the development of imipenem heteroresistance in NTHi.