Involvement of Acquired Tobramycin Resistance in the Shift to the Viable but Non-Culturable State in Pseudomonas aeruginosa.

Persistent and viable but non-culturable (VBNC) Pseudomonas aeruginosa cells are mainly responsible for the recurrence and non-responsiveness to antibiotics of cystic fibrosis (CF) lung infections. The sub-inhibitory antibiotic concentrations found in the CF lung in between successive therapeutic cycles can trigger the entry into the VBNC state, albeit with a strain-specific pattern. Here, we analyzed the VBNC cell induction in the biofilms of two CF P. aeruginosa isolates, exposed to starvation with/without antibiotics, and investigated the putative genetic determinants involved. Total viable bacterial cells were quantified by the validated ecfX-targeting qPCR protocol and the VBNC cells were estimated as the difference between qPCR and cultural counts. The isolates were both subjected to whole genome sequencing, with attention focused on their carriage of aminoglycoside resistance genes and on identifying mutated toxin-antitoxin and quorum sensing systems. The obtained results suggest the variable contribution of different antibiotic resistance mechanisms to VBNC cell abundance, identifying a major contribution from tobramycin efflux, mediated by MexXY efflux pump overexpression. The genome analysis evidenced putative mutation hotspots, which deserve further investigation. Therefore, drug efflux could represent a crucial mechanism through which the VBNC state is entered and a potential target for anti-persistence strategies.

Linezolid Resistance Genes in Enterococci Isolated from Sediment and Zooplankton in Two Italian Coastal Areas.

Linezolid is a last-resort antibiotic for the treatment of severe infections caused by multidrug-resistant Gram-positive organisms; although linezolid resistance remains uncommon, the number of linezolid-resistant enterococci has increased in recent years due to worldwide spread of acquired resistance genes (cfr, optrA, and poxtA) in clinical, animal, and environmental settings. In this study, we investigated the occurrence of linezolid-resistant enterococci in marine samples from two coastal areas in Italy. Isolates grown on florfenicol-supplemented Slanetz-Bartley agar plates were investigated for their carriage of optrA, poxtA, and cfr genes; optrA was found in one Enterococcus faecalis isolate, poxtA was found in three Enterococcus faecium isolates and two Enterococcus hirae isolates, and cfr was not found. Two of the three poxtA-carrying E. faecium isolates and the two E. hirae isolates showed related pulsed-field gel electrophoresis (PFGE) profiles. Two E. faecium isolates belonged to the new sequence type 1710, which clustered in clonal complex 94, encompassing nosocomial strains. S1 PFGE/hybridization assays showed a double (chromosome and plasmid) location of poxtA and a plasmid location of optrA Whole-genome sequencing revealed that poxtA was contained in a Tn6657-like element carried by two plasmids (pEfm-EF3 and pEh-GE2) of similar size, found in different species, and that poxtA was flanked by two copies of IS1216 in both plasmids. In mating experiments, all but one strain (E. faecalis EN3) were able to transfer the poxtA gene to E. faecium 64/3. The occurrence of linezolid resistance genes in enterococci from marine samples is of great concern and highlights the need to improve practices aimed at limiting the transmission of linezolid-resistant strains to humans from environmental reservoirs.IMPORTANCE Linezolid is one of the few antimicrobials available to treat severe infections due to drug-resistant Gram-positive bacteria; therefore, the emergence of linezolid-resistant enterococci carrying transferable resistance determinants is of great concern for public health. Linezolid resistance genes (cfr, optrA, and poxtA), often plasmid located, can be transmitted via horizontal gene transfer and have the potential to spread globally. This study highlights the detection of enterococci carrying linezolid resistance genes from sediment and zooplankton samples from two coastal urban areas in Italy. The presence of clinically relevant resistant bacteria, such as linezolid-resistant enterococci, in marine environments could reflect their spillover from human and/or animal reservoirs and could indicate that coastal seawaters also might represent a source of these resistance genes.

The Natural Alkaloid Berberine Can Reduce the Number of Pseudomonas aeruginosa Tolerant Cells.

The eradication of recurrent Pseudomonas aeruginosa (PA) lung infection in cystic fibrosis (CF) patients may be hampered by the development of persistent bacterial forms, which can tolerate antibiotics through efflux pump overexpression. After demonstrating the efflux pump inhibitory effect of the alkaloid berberine on the PA MexXY-OprM efflux pump, in this study, we tested its ability (80/320 µg/mL) to enhance tobramycin (20xMIC/1000xMIC) activity against PA planktonic/biofilm cultures. Preliminary investigations of the involvement of MexY in PA tolerance to tobramycin treatment, performed on the isogenic pair PA K767 (wild type)/K1525 (ΔmexY) growing in planktonic and biofilm cultures, demonstrated that the ΔmexY mutant K1525 produced a lower (100 and 10 000 times, respectively) amount of tolerant cells than that of the wild type. Next, we grew broth cultures of PAO1, PA14, and 20 PA clinical isolates (of which 13 were from CF patients) in the presence of 20xMIC tobramycin with and without berberine 80 µg/mL. Accordingly, most strains showed a greater (from 10- to 1000-fold) tolerance reduction in the presence of berberine. These findings highlight the involvement of the MexXY-OprM system in the tobramycin tolerance of PA and suggest that berberine may be used in new valuable therapeutic combinations to counteract persister survival.

Diffusion and Characterization of Pseudomonas aeruginosa Aminoglycoside Resistance in an Italian Regional Cystic Fibrosis Centre.

AIMS: Extensively-drug-resistant Pseudomonas aeruginosa constitutes a serious threat to patients suffering from Cystic Fibrosis (CF). In these patients, P. aeruginosa lung infection is commonly treated with aminoglycosides, but treatments are largely unsuccessful due a variety of resistance mechanisms. Here we investigate the prevalence of resistance to gentamicin, amikacin and tobramycin and the main aminoglycoside resistance genes found in P. aeruginosa strains isolated at a regional CF centre. RESULTS: A total number of 147 randomly selected P. aeruginosa strains isolated from respiratory samples sent by the Marche regional Cystic Fibrosis Centre to the Microbiology lab, were included in this study. Of these, 78 (53%) were resistant to at least one of the three aminoglycosides tested and 27% were resistant to all three antibiotics, suggesting a major involvement of a chromosome-encoded mechanism, likely MexXY-OprM efflux pump overexpression. A specific pathogenic clone (found in 7/78 of the aminoglycoside resistant strains) carrying ant(2″)-Ia was isolated over time from the same patient, suggesting a role for this additional resistance gene in the antibiotic unresponsiveness of CF patients. CONCLUSIONS: The MexXY-OprM efflux pump is confirmed as the resistance determinant involved most frequently in P. aeruginosa aminoglycoside resistance of CF lung infections, followed by the ant(2″)-Ia-encoded adenylyltransferase. The latter may prove to be a novel target for new antimicrobial combinations against P. aeruginosa.

Contribution of Drugs Interfering with Protein and Cell Wall Synthesis to the Persistence of Pseudomonas aeruginosa Biofilms: An In Vitro Model.

The occurrence of Pseudomonas aeruginosa (PA) persisters, including viable but non-culturable (VBNC) forms, subpopulations of tolerant cells that can survive high antibiotic doses, is the main reason for PA lung infections failed eradication and recurrence in Cystic Fibrosis (CF) patients, subjected to life-long, cyclic antibiotic treatments. In this paper, we investigated the role of subinhibitory concentrations of different anti-pseudomonas antibiotics in the maintenance of persistent (including VBNC) PA cells in in vitro biofilms. Persisters were firstly selected by exposure to high doses of antibiotics and their abundance over time evaluated, using a combination of cultural, qPCR and flow cytometry assays. Two engineered GFP-producing PA strains were used. The obtained results demonstrated a major involvement of tobramycin and bacterial cell wall-targeting antibiotics in the resilience to starvation of VBNC forms, while the presence of ciprofloxacin and ceftazidime/avibactam lead to their complete loss. Moreover, a positive correlation between tobramycin exposure, biofilm production and c-di-GMP levels was observed. The presented data could allow a deeper understanding of bacterial population dynamics during the treatment of recurrent PA infections and provide a reliable evaluation of the real efficacy of the antibiotic treatments against the bacterial population within the CF lung.

Berberine Derivatives as Pseudomonas aeruginosa MexXY-OprM Inhibitors: Activity and In Silico Insights.

The natural alkaloid berberine has been demonstrated to inhibit the Pseudomonas aeruginosa multidrug efflux system MexXY-OprM, which is responsible for tobramycin extrusion by binding the inner membrane transporter MexY. To find a structure with improved inhibitory activity, we compared by molecular dynamics investigations the binding affinity of berberine and three aromatic substituents towards the three polymorphic sequences of MexY found in P. aeruginosa (PAO1, PA7, and PA14). The synergy of the combinations of berberine or berberine derivatives/tobramycin against the same strains was then evaluated by checkerboard and time-kill assays. The in silico analysis evidenced different binding modes depending on both the structure of the berberine derivative and the specific MexY polymorphism. In vitro assays showed an evident MIC reduction (32-fold and 16-fold, respectively) and a 2-3 log greater killing effect after 2 h of exposure to the combinations of 13-(2-methylbenzyl)- and 13-(4-methylbenzyl)-berberine with tobramycin against the tobramycin-resistant strain PA7, a milder synergy (a 4-fold MIC reduction) against PAO1 and PA14, and no synergy against the ΔmexXY strain K1525, confirming the MexY-specific binding and the computational results. These berberine derivatives could thus be considered new hit compounds to select more effective berberine substitutions and their common path of interaction with MexY as the starting point for the rational design of novel MexXY-OprM inhibitors.

C-2 phenyl replacements to obtain potent quinoline-based Staphylococcus aureus NorA inhibitors.

NorA is the most studied efflux pump of Staphylococcus aureus and is responsible for high level resistance towards fluoroquinolone drugs. Although along the years many NorA efflux pump inhibitors (EPIs) have been reported, poor information is available about structure-activity relationship (SAR) around their nuclei and reliability of data supported by robust assays proving NorA inhibition. In this regard, we focussed efforts on the 2-phenylquinoline as a promising chemotype to develop potent NorA EPIs. Herein, we report SAR studies about the introduction of different aryl moieties on the quinoline C-2 position. The new derivative 37a showed an improved EPI activity (16-fold) with respect to the starting hit 1. Moreover, compound 37a exhibited a high potential in time-kill curves when combined with ciprofloxacin against SA-1199B (norA+). Also, 37a exhibited poor non-specific effect on bacterial membrane polarisation and showed an improvement in terms of "selectivity index" in comparison to 1.

Structural Modifications of the Quinolin-4-yloxy Core to Obtain New Staphylococcus aureus NorA Inhibitors.

Tackling antimicrobial resistance (AMR) represents a social responsibility aimed at renewing the antimicrobial armamentarium and identifying novel therapeutical approaches. Among the possible strategies, efflux pumps inhibition offers the advantage to contrast the resistance against all drugs which can be extruded. Efflux pump inhibitors (EPIs) are molecules devoid of any antimicrobial activity, but synergizing with pumps-substrate antibiotics. Herein, we performed an in silico scaffold hopping approach starting from quinolin-4-yloxy-based Staphylococcus aureus NorA EPIs by using previously built pharmacophore models for NorA inhibition activity. Four scaffolds were identified, synthesized, and modified with appropriate substituents to obtain new compounds, that were evaluated for their ability to inhibit NorA and synergize with the fluoroquinolone ciprofloxacin against resistant S. aureus strains. The two quinoline-4-carboxamide derivatives 3a and 3b showed the best results being synergic (4-fold MIC reduction) with ciprofloxacin at concentrations as low as 3.13 and 1.56 µg/mL, respectively, which were nontoxic for human THP-1 and A549 cells. The NorA inhibition was confirmed by SA-1199B ethidium bromide efflux and checkerboard assays against the isogenic pair SA-K2378 (norA++)/SA-K1902 (norA-). These in vitro results indicate the two compounds as valuable structures for designing novel S. aureus NorA inhibitors to be used in association with fluoroquinolones.

Characterization of a new transferable MDR plasmid carrying the pbp5 gene from a clade B commensal Enterococcus faecium.

OBJECTIVES: To evaluate the transferability of antibiotic resistance from an MDR clade B Enterococcus faecium and to characterize the genetic elements involved. METHODS: The erm(B)-positive strain E. faecium 37BA (donor) and strains E. faecium 64/3 and Listeria welshimeri 11857RF (recipients) were used in mating experiments. Donors and transconjugants were characterized using MIC assays, PFGE, Southern blotting and hybridization, quantitative RT-PCR (RT-qPCR), next-generation sequencing and PCR mapping. RESULTS: One E. faecium and one L. welshimeri transconjugant were selected for in-depth investigation. Both acquired an ∼40 kb plasmid carrying erm(B). An additional plasmid of ∼200 kb, encoding the full conjugation machinery, was detected in the donor and in the E. faecium transconjugant. Next-generation sequencing revealed a new 40 396 bp plasmid that was designated pEf37BA; it contained 10 antibiotic resistance genes, tet(M), tet(L), erm(B), aadE, sat4, aphA, spw, lsa(E), lnu(B) and pbp5, resulting from the recombination of pM7M2 of E. faecium with an MDR chromosomal region of Erysipelothrix rhusiopathiae. A pbp5-carrying circular form was also detected. The PBP5 amino acid sequence differed from the C46 variant by two mutations (S39T and D644N). Its expression was documented in both transconjugants. pEf37BA persisted in the absence of selective pressure. CONCLUSIONS: The MDR clade B E. faecium plasmid, deriving from the recombination of two different resistance regions, carried a pbp5 element and was transferable to different bacterial species. This finding further documents the dissemination of ampicillin resistance among community-associated E. faecium and the key role of commensal strains in the spread of antibiotic resistance.