Resistance development in Escherichia coli to delafloxacin at pHs 6.0 and 7.3 compared to ciprofloxacin.

Understanding the resistance mechanisms of antibiotics in the micro-environment of the infection is important to assess their clinical applicability and potentially prevent resistance development. We compared the laboratory resistance evolution of Escherichia coli to delafloxacin (DLX) compared to ciprofloxacin (CIP), the co-resistance evolution, and underlying resistance mechanisms at different pHs. Three clones from each of the eight clinical E. coli isolates were subjected to subinhibitory concentrations of DLX or CIP in parallel at either pH 7.3 or 6.0. Minimum inhibitory concentrations (MICs) were regularly tested (at respective pHs), and the antibiotic concentration was adjusted accordingly. After 30 passages, MICs were determined in the presence of the efflux pump inhibitor phenylalanine-arginine-ß-naphthylamide. Whole genome sequencing of the parental isolates and their resistant derivatives (n = 54) was performed. Complementation assays were carried out for selected mutations. Quantitative PCR and efflux experiments were carried out for selected derivatives. For DLX-challenged strains, resistance to DLX evolved much slower in acidic than in neutral pH, whereas for CIP-challenged strains, the opposite was the case. Mutations in the quinolone resistance-determining region were mainly seen in CIP-challenged E. coli, whereas a multifactorial mechanism including mutations in efflux-related genes played a role in DLX resistance evolution (predominantly at pH 6.0). This work provides novel insights into the resistance mechanisms of E. coli to delafloxacin and highlights the importance of understanding micro-environmental conditions at the infection site that might affect the true clinical efficacy of antibiotics and challenges our current antibiotic susceptibility-testing paradigm.

AmpC hyperproduction in a Cedecea davisae implant-associated bone infection during treatment: a case report and therapeutic implications.

BACKGROUND: Data on antimicrobial resistance mechanisms are scanty for Cedecea spp., with very variable antibiotic resistance patterns documented. Here we report the first in vivo resistance evolution of a C. davisae clinical isolate in a patient with a complex hand trauma and provide insight in the resistance mechanism, leading to therapeutic implications for this pathogen. CASE PRESENTATION: Cedecea davisae was isolated from a patient with hand trauma during a first surgical debridement. Six days after primary surgical treatment and under antimicrobial treatment with amoxicillin-clavulanic acid and later cefepime, follow up cultures yielded C. davisae which demonstrated a resistance development. The susceptible parental isolate and its resistant derivative were characterized by whole genome sequencing, ampC, ompC and ompF by RT- PCR. The resistant derivative demonstrated an A224G SNP in ampD, the transcriptional regulator of ampC, leading to a His75Arg change in the corresponding AmpD protein. AmpC transcription of the resistant derivative was 362-times higher than the susceptible isolate. Transcription levels of ompF and ompC were 8.5-fold and 1.3-fold lower, respectively, in the resistant derivative. Downregulation of OmpF putatively resulted from a mutation in the presumed promoter region upstream of the dusB-Fis operon, a proposed regulator for ompF. CONCLUSIONS: This case demonstrates the in vivo resistance development of C. davisae within 7 days similar to that of the members of the Enterobacter cloacae complex. Our findings add valuable information for future therapeutic management of these opportunistic pathogens as they warrant the same empirical treatment as AmpC producers.

Multifactorial resistance mechanisms associated with resistance to ceftazidime-avibactam in clinical Pseudomonas aeruginosa isolates from Switzerland.

Background: Increasing reports of multidrug resistance (MDR) in clinical Pseudomonas aeruginosa have led to a necessity for new antimicrobials. Ceftazidime-avibactam (CZA) is indicated for use against MDR P. aeruginosa across a broad range of infection types and particularly those that are carbapenem resistant. This study sought to determine the molecular mechanisms of CZA and imipenem (IPM)-resistance in clinical P. aeruginosa isolates obtained from Swiss hospitals. Methods: Clinical P. aeruginosa isolates were obtained from inpatients in three hospitals in Switzerland. Susceptibility was determined by either antibiotic disc testing or broth microdilution according to EUCAST methodology. AmpC activity was determined using cloxacillin and efflux activity was determined using phenylalanine-arginine ß-naphthylamide, in agar plates. Whole Genome Sequencing was performed on 18 clinical isolates. Sequence types (STs) and resistance genes were ascertained using the Centre for Genomic Epidemiology platform. Genes of interest were extracted from sequenced isolates and compared to reference strain P. aeruginosa PAO1. Results: Sixteen different STs were identified amongst the 18 isolates in this study indicating a high degree of genomic diversity. No carbapenemases were detected but one isolate did harbor the ESBL bla PER-1. Eight isolates were CZA-resistant with MICs ranging from 16-64 mg/L, and the remaining ten isolates had either low/wildtype MICs (n=6; 1-2 mg/L) or elevated, but still susceptible, MICs (n=4; 4-8 mg/L). Ten isolates were IPM-resistant, seven of which had mutations resulting in truncations of OprD, and the remaining nine IPM-susceptible isolates had intact oprD genes. Within CZA-R isolates, and those with reduced susceptibility, mutations resulting in ampC derepression, OprD loss, mexAB overexpression and ESBL (bla PER-1) carriage were observed in various combinations and one harbored a truncation of the PBP4 dacB gene. Within the six isolates with wildtype-resistance levels, five had no mutations that would affect any antimicrobial resistance (AMR) genes of interest when compared to PAO1. Conclusion: This preliminary study highlights that CZA-resistance in P. aeruginosa is multifactorial and could be caused by the interplay between different resistance mechanisms including ESBL carriage, increased efflux, loss of permeability and derepression of its intrinsic ampC.