Investigation of Delafloxacin Resistance in Multidrug-Resistant Escherichia coli Strains and the Detection of E. coli ST43 International High-Risk Clone.

Delafloxacin is a novel fluoroquinolone agent that is approved for clinical application. In this study, we analyzed the antibacterial efficacy of delafloxacin in a collection of 47 Escherichia coli strains. Antimicrobial susceptibility testing was performed by the broth microdilution method and minimum inhibitory concentration (MIC) values were determined for delafloxacin, ciprofloxacin, levofloxacin, moxifloxacin, ceftazidime, cefotaxime, and imipenem. Two multidrug-resistant E. coli strains, which exhibited delafloxacin and ciprofloxacin resistance as well as extended-spectrum beta-lactamase (ESBL) phenotype, were selected for whole-genome sequencing (WGS). In our study, delafloxacin and ciprofloxacin resistance rates were 47% (22/47) and 51% (24/47), respectively. In the strain collection, 46 E. coli were associated with ESBL production. The MIC50 value for delafloxacin was 0.125 mg/L, while all other fluoroquinolones had an MIC50 value of 0.25 mg/L in our collection. Delafloxacin susceptibility was detected in 20 ESBL positive and ciprofloxacin resistant E. coli strains; by contrast, E. coli strains that exhibited a ciprofloxacin MIC value above 1 mg/L were delafloxacin-resistant. WGS analysis on the two selected E. coli strains (920/1 and 951/2) demonstrated that delafloxacin resistance is mediated by multiple chromosomal mutations, namely, five mutations in E. coli 920/1 (gyrA S83L, D87N, parC S80I, E84V, and parE I529L) and four mutations in E. coli 951/2 (gyrA S83L, D87N, parC S80I, and E84V). Both strains carried an ESBL gene, blaCTX-M-1 in E. coli 920/1 and blaCTX-M-15 in E. coli 951/2. Based on multilocus sequence typing, both strains belong to the E. coli sequence type 43 (ST43). In this paper, we report a remarkable high rate (47%) of delafloxacin resistance among multidrug-resistant E. coli as well as the E. coli ST43 international high-risk clone in Hungary.

Emergence and Dissemination of Extraintestinal Pathogenic High-Risk International Clones of Escherichia coli.

Multiresistant Escherichia coli has been disseminated worldwide, and it is one of the major causative agents of nosocomial infections. E. coli has a remarkable and complex genomic plasticity for taking up and accumulating genetic elements; thus, multiresistant high-risk clones can evolve. In this review, we summarise all available data about internationally disseminated extraintestinal pathogenic high-risk E. coli clones based on whole-genome sequence (WGS) data and confirmed outbreaks. Based on genetic markers, E. coli is clustered into eight phylogenetic groups. Nowadays, the E. coli ST131 clone from phylogenetic group B2 is the predominant high-risk clone worldwide. Currently, strains of the C1-M27 subclade within clade C of ST131 are circulating and becoming prominent in Canada, China, Germany, Hungary and Japan. The C1-M27 subclade is characterised by blaCTX-M-27. Recently, the ST1193 clone has been reported as an emerging high-risk clone from phylogenetic group B2. ST38 clone carrying blaOXA-244 (a blaOXA-48-like carbapenemase gene) caused several outbreaks in Germany and Switzerland. Further high-risk international E. coli clones include ST10, ST69, ST73, ST405, ST410, ST457. High-risk E. coli strains are present in different niches, in the human intestinal tract and in animals, and persist in environment. These strains can be transmitted easily within the community as well as in hospital settings. WGS analysis is a useful tool for tracking the dissemination of resistance determinants, the emergence of high-risk mulitresistant E. coli clones and to analyse changes in the E. coli population on a genomic level.

Delafloxacin, Finafloxacin, and Zabofloxacin: Novel Fluoroquinolones in the Antibiotic Pipeline.

Novel antimicrobial agents, approved for clinical use in past years, represent potential treatment options for various infections. In this review, we summarize the most important medical and microbiological features of three recently approved fluoroquinolones, namely delafloxacin, finafloxacin, and zabofloxacin. Delafloxacin possesses an anionic chemical structure, and represents broad-spectrum activity, as it targets both bacterial DNA gyrase and topoisomerase IV enzymes of gram-positive and gram-negative bacteria with equal affinity. Its molecular surface is larger than that of other fluoroquinolones, and it has enhanced antibacterial efficacy in acidic environments. Delafloxacin has been approved to treat acute bacterial skin and skin-structure infections, as well as community-acquired bacterial pneumonia. Finafloxacin has a zwitterionic chemical structure, and targets both DNA gyrase and topoisomerase IV enzymes. This enables a broad antibacterial spectrum; however, finafloxacin has so far only been approved in ear-drops to treat bacterial otitis externa. Zabofloxacin is also a broad-spectrum fluoroquinolone agent, and was first approved in South Korea to treat acute bacterial exacerbation of chronic obstructive pulmonary disease. The introduction of these novel fluoroquinolones into daily practice extends the possible indications of antibiotics into different bacterial infections, and provides treatment options in difficult-to-treat infections. However, some reports of delafloxacin resistance have already appeared, thus underlining the importance of the prudent use of antibiotics.

Diversity and Distribution of Resistance Markers in Pseudomonas aeruginosa International High-Risk Clones.

Pseudomonas aeruginosa high-risk clones are disseminated worldwide and they are common causative agents of hospital-acquired infections. In this review, we will summarize available data of high-risk P. aeruginosa clones from confirmed outbreaks and based on whole-genome sequence data. Common feature of high-risk clones is the production of beta-lactamases and among metallo-beta-lactamases NDM, VIM and IMP types are widely disseminated in different sequence types (STs), by contrast FIM type has been reported in ST235 in Italy, whereas GIM type in ST111 in Germany. In the case of ST277, it is most frequently detected in Brazil and it carries a resistome linked to blaSPM. Colistin resistance develops among P. aeruginosa clones in a lesser extent compared to other resistance mechanisms, as ST235 strains remain mainly susceptible to colistin however, some reports described mcr positive P. aeurigonsa ST235. Transferable quinolone resistance determinants are detected in P. aeruginosa high-risk clones and aac(6')-Ib-cr variant is the most frequently reported as this determinant is incorporated in integrons. Additionally, qnrVC1 was recently detected in ST773 in Hungary and in ST175 in Spain. Continuous monitoring and surveillance programs are mandatory to track high-risk clones and to analyze emergence of novel clones as well as novel resistance determinants.

Acquired qnrVC1 and blaNDM-1 resistance markers in an international high-risk Pseudomonas aeruginosa ST773 clone.

A multidrug-resistant Pseudomonas aeruginosa PS1 isolated from urine clinical sample was investigated in this study. The strain exhibited resistance to piperacillin/tazobactam, ciprofloxacin, imipenem, ceftazidime but it was susceptible to colistin. Analysis of whole-genome sequencing data by ResFinder detected various resistance determinants including qnrVC1 and blaNDM-1. The multiresistant P. aeruginosa isolate belonged to ST773 high-risk clone. The qnrVC1 and blaNDM-1 determinants were incorporated into different integrons. Expression of blaNDM-1 was fourfold and qnrVC1 was twofold increased, compared to that of rpsL housekeeping gene. Mutations in gyrA Thr83Leu and parC Ser87Leu were detected and additionally qnrVC1 expression indicates protective effect of QnrVC1 pentapeptid protein on gyrase and topoisomerase. High-risk P. aeruginosa clones integrate various carbapenemase and other resistance determinants into their genomes that facilitates further dissemination of multiresistance among clinical isolates. We report blaNDM-1 and qnrVC1 genes in P. aeruginosa ST773 international high-risk clone.

Plasmid copy number and qnr gene expression in selection of fluoroquinolone-resistant Escherichia coli.

Fluoroquinolone resistance in Enterobacteriales is developed by chromosomal and plasmid-mediated mechanisms. Plasmids play an important role in dissemination of resistant genes and they carry genes that protect bacteria in different stress-induced situations. In this study, we studied Escherichia coli strains, each carried one plasmid-mediated quinolone resistance determinant namely, qnrA1, qnrB1, qnrC1, and qnrD1. We exposed 0.5 McFarland density of each strain to 0.5 mg/L ciprofloxacin from the period of 30, 60, 90, and 120 min over 24 h. All treated strains were further exposed to a constantly increasing 1, 2, 4, and 8 mg/L ciprofloxacin solution through 24, 48, and 120 h. In given timepoints, RNA was extracted from all treated strains. Expression of qnrA1, qnrB1, qnrC1, and qnrD1 was investigated by quantitative PCR. Mutations in gyrA and parC genes were analyzed by PCR and nucleic acid sequencing. In this study, during 0.5 mg/L ciprofloxacin exposition, the following expression levels were detected: 1.2 for qnrA1, 1.47 for qnrD1, 12.44 for qnrC1, and 80.63 for qnrB1. In case of long-term study, we selected a resistant strain in qnrB1-positive E. coli, and its expression increased from 105.91 to 212.31. On the contrary, plasmid copy number increased in time from 1 to 4.13. No mutations in gyrA or in parC chromosomal genes of treated strains were detected. Our results show that qnrB1-positive E. coli strain was able to develop fluoroquinolone resistance by upregulated qnrB1 expression that was linked to a minor increase in plasmid copy number but no mutations occurred in gyrA or parC.

Ciprofloxacin Promoted qnrD Expression and Phylogenetic Analysis of qnrD Harboring Plasmids.

Morganella morganii SE10MM harboring quinolone resistance determinant qnrD was investigated in our study. An entirely sequenced novel 2,662 bp qnrD-plasmid pSE10MM was identified and deposited at GenBank under accession number KU160530. Nucleic acid sequence of pSE10MM showed 94-97% similarity to previously detected qnrD-plasmids of Proteus mirabilis strains. Phylogenetic analysis by Geneious 9.0.5 showed clusters of plasmids with possible common origin. Initial expression of qnrD gene was found 12.5 normalized to rpoB housekeeping gene. Subsequently, a sub-minimum inhibitory concentration (1 mg/L) ciprofloxacin exposure resulted in a fold change of 30.06 at 24 hours. In contrast, qnrD-plasmid pSE10MM copy number increased in time from 1.1 to 6.63. Chromosomal mutations of gyrA with S83I, gyrB with S463A, and parC with S80I amino acid substitutions were detected, but no other mutations have occurred as a consequence of ciprofloxacin exposure. Elevated expression of qnrD correlated with that of recA in M. morganii during ciprofloxacin exposure, which indicates SOS-dependent regulation of qnrD. Protective effect of QnrD plays a role in fluoroquinolone-resistant strain even in the presence of chromosomal mutations in gyrase and topoisomerase IV.

Plasmid-mediated quinolone resistance determinants in Enterobacteriaceae from urine clinical samples.

Plasmid-mediated quinolone resistance (PMQR) determinants including, qnrA, qnrB, qnrC, qnrD, qnrS, aac(6')-Ib-cr, oqxAB, and qepA, were investigated in 214 Enterobacteriaceae strains from urine clinical samples. Antimicrobial susceptibility testing for ciprofloxacin, ceftriaxone, and imipenem was performed by broth microdilution method. All strains were screened for PMQR genes by PCR. Virulence determinants, namely afa, pap, pil, sfa/foc, and kpsMT of eight Escherichia coli strains proven positive for at least one qnr gene, were investigated by PCR. All of the eight investigated strains carried the pil gene, showing that P fimbria is a common virulence determinant among qnr positive E. coli. Out of 214 tested strains, 38 yielded any PMQR determinant, altogether 45 genes were detected namely, 6 qnrA, 1 qnrB, 2 qnrD and 8 qnrS, 9 aac(6')-Ib-cr, and 19 oqxAB; however, neither qepA nor qnrC were detected. Notably, 18 Klebsiella spp., harbored oqxAB, nine E. coli were positive for qnrS and two Morganella morganii yielded qnrD resistance determinant. In this study, we demonstrated 17.7% prevalence of PMQR-positive Enterobacteriaceae and first reported qnrD-resistance determinant in Hungary. Altogether, 25 PMQR-positive strains were susceptible or low-level resistant to ciprofloxacin with minimum inhibitory concentration (MIC) between 0.06 and 1 mg/L, suggesting that prevalence of PMQR determinants is underestimated and screening among clinical isolates exhibiting reduced susceptibility is necessary. Fluoroquinolone resistance breakpoints of Enterobacteriaceae were revised in 2017 by European Committee of Antimicrobial Susceptibility Testing indicating ciprofloxacin susceptibility only until 0.25 mg/L MIC value.