In Vivo, In Vitro, and In Silico Characterization of Peptoids as Antimicrobial Agents

Bacterial resistance to conventional antibiotics is a global threat that has spurred the development of antimicrobial peptides (AMPs) and their mimetics as novel anti-infective agents. While the bioavailability of AMPs is often reduced due to protease activity, the non-natural structure of AMP mimetics renders them robust to proteolytic degradation, thus offering a distinct advantage for their clinical application. We explore the therapeutic potential of N-substituted glycines, or peptoids, as AMP mimics using a multi-faceted approach that includes in silico, in vitro, and in vivo techniques. We report a new QSAR model that we developed based on 27 diverse peptoid sequences, which accurately correlates antimicrobial peptoid structure with antimicrobial activity. We have identified a number of peptoids that have potent, broad-spectrum in vitro activity against multi-drug resistant bacterial strains. Lastly, using a murine model of invasive S. aureus infection, we demonstrate that one of the best candidate peptoids at 4 mg/kg significantly reduces with a two-log order the bacterial counts compared with saline-treated controls. Taken together, our results demonstrate the promising therapeutic potential of peptoids as antimicrobial agents

Phenotypic and Genome-Wide Analysis of an Antibiotic-Resistant Small Colony Variant (SCV) of Pseudomonas aeruginosa

Small colony variants (SCVs) are slow-growing bacteria, which often show increased resistance to antibiotics and cause latent or recurrent infections. It is therefore important to understand the mechanisms at the basis of this phenotypic switch.One SCV (termed PAO-SCV) was isolated, showing high resistance to gentamicin and to the cephalosporine cefotaxime. PAO-SCV was prone to reversion as evidenced by emergence of large colonies with a frequency of 10(-5) on media without antibiotics while it was stably maintained in presence of gentamicin. PAO-SCV showed a delayed growth, defective motility, and strongly reduced levels of the quorum sensing Pseudomonas quinolone signal (PQS). Whole genome expression analysis further suggested a multi-layered antibiotic resistance mechanism, including simultaneous over-expression of two drug efflux pumps (MexAB-OprM, MexXY-OprM), the LPS modification operon arnBCADTEF, and the PhoP-PhoQ two-component system. Conversely, the genes for the synthesis of PQS were strongly down-regulated in PAO-SCV. Finally, genomic analysis revealed the presence of mutations in phoP and phoQ genes as well as in the mexZ gene encoding a repressor of the mexXY and mexAB-oprM genes. Only one mutation occurred only in REV, at nucleotide 1020 of the tufA gene, a paralog of tufB, both encoding the elongation factor Tu, causing a change of the rarely used aspartic acid codon GAU to the more common GAC, possibly causing an increase of tufA mRNA translation. High expression of phoP and phoQ was confirmed for the SCV variant while the revertant showed expression levels reduced to wild-type levels.By combining data coming from phenotypic, gene expression and proteome analysis, we could demonstrate that resistance to aminoglycosides in one SCV mutant is multifactorial including overexpression of efflux mechanisms, LPS modification and is accompanied by a drastic down-regulation of the Pseudomonas quinolone signal quorum sensing system

Use of Faropenem as an Indicator of Carbapenemase Activity in the Enterobacteriaceae

The aim of this study was to determine the ability of a disc susceptibility test using faropenem (10 μg) to predict carbapenemase activity in Enterobacteriaceae. A collection of 166 isolates of carbapenemase-producing Enterobacteriaceae (CPE) and 82 isolates of Enterobacteriaceae that produced other β-lactamases was compiled from diverse sources. Disc susceptibility testing was performed using the CLSI/EUCAST methodology with discs of faropenem (10 μg), temocillin (30 μg), and four carbapenems (each 10 μg). A further prospective evaluation of the faropenem disc susceptibility test was performed using 205 consecutive isolates referred to a United Kingdom reference laboratory in parallel with molecular methods for carbapenemase detection. Of 166 isolates of CPE, 99% showed growth up to the edge of a 10-μg faropenem disc compared with only 6% of other β-lactamase producers (sensitivity, 99%; specificity, 94%). A “double zone” around 10-μg faropenem discs was frequently associated with OXA-48 producers. Of the carbapenems, the most useful agent was imipenem, where a zone diameter of ≤23 mm as a predictor of carbapenemase activity had a sensitivity of 99% and a specificity of 85%. The presence of no zone of inhibition around a 30-μg temocillin disc was a consistent feature of strains producing OXA-48 carbapenemase. For 205 isolates of Enterobacteriaceae referred to a United Kingdom reference laboratory, growth up to a 10-μg faropenem disc correctly identified 84 of 86 carbapenemase producers (98% sensitivity), with a specificity of 87%. Disc susceptibility testing using faropenem (10 μg) is a simple, convenient, and highly predictive screening test for carbapenemase-producing Enterobacteriaceae