Dataset Information

Resensitization of Fosfomycin-Resistant Escherichia coli Using the CRISPR System.

ABSTRACT: Antimicrobial resistance is a public health burden with worldwide impacts and was recently identified as one of the major causes of death in 2019. Fosfomycin is an antibiotic commonly used to treat urinary tract infections, and resistance to it in Enterobacteriaceae is mainly due to the metalloenzyme FosA3 encoded by the fosA3 gene. In this work, we adapted a CRISPR-Cas9 system named pRE-FOSA3 to restore the sensitivity of a fosA3+ Escherichia coli strain. The fosA3+ E. coli strain was generated by transforming synthetic fosA3 into a nonpathogenic E. coli TOP10. To mediate the fosA3 disruption, two guide RNAs (gRNAs) were selected that used conserved regions within the fosA3 sequence of more than 700 fosA3+ E. coli isolates, and the resensitization plasmid pRE-FOSA3 was assembled by cloning the gRNA into pCas9. gRNA_195 exhibited 100% efficiency in resensitizing the bacteria to fosfomycin. Additionally, the edited strain lost the ampicillin resistance encoded in the same plasmid containing the synthetic fosA3 gene, despite not being the CRISPR-Cas9 target, indicating plasmid clearance. The in vitro analysis presented here points to a path that can be explored to assist the development of effective alternative methods of treatment against fosA3+ bacteria.

SUBMITTER: Walflor HSM

PROVIDER: S-EPMC9409345 | biostudies-literature | 2022 Aug

REPOSITORIES: biostudies-literature

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Resensitization of Fosfomycin-Resistant <i>Escherichia coli</i> Using the CRISPR System.

Walflor Haniel Siqueira Mortagua HSM Lucena Aline Rodrigues Castro ARC Tuon Felipe Francisco FF Medeiros Lia Carolina Soares LCS Faoro Helisson H

International journal of molecular sciences 20220816 16

Antimicrobial resistance is a public health burden with worldwide impacts and was recently identified as one of the major causes of death in 2019. Fosfomycin is an antibiotic commonly used to treat urinary tract infections, and resistance to it in Enterobacteriaceae is mainly due to the metalloenzyme FosA3 encoded by the fosA3 gene. In this work, we adapted a CRISPR-Cas9 system named pRE-FOSA3 to restore the sensitivity of a fosA3+ Escherichia coli strain. The fosA3+ E. coli strain was generated ...[more]

PMID: 36012441

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Project description:To investigate the efficacy of fosfomycin against extended-spectrum β-lactamases (ESBL) producing Escherichia coli in Taiwan and the resistance mechanisms and characterization of human and pig isolates, we analyzed 145 ESBL-producing isolates collected from two hospitals (n = 123) and five farms (n = 22) in Taiwan from February to May, 2013. Antimicrobial susceptibilities were determined. Clonal relatedness was determined by PFGE and multi-locus sequence typing. ESBLs, ampC, and fosfomycin resistant genes were detected by PCR, and their flanking regions were determined by PCR mapping and sequencing. The fosfomycin resistant mechanisms, including modification of the antibiotic target (MurA), functionless transporters (GlpT and UhpT) and their regulating genes such as uhpA, cyaA, and ptsI, and antibiotic inactivation by enzymes (FosA and FosC), were examined. The size and replicon type of plasmids carrying fosfomycin resistant genes were analyzed. Our results revealed the susceptibility rates of fosfomycin were 94% for human ESBL-producing E. coli isolates and 77% for pig isolates. The PFGE analysis revealed 79 pulsotypes. No pulsotype was found existing in both human and pig isolates. Three pulsotypes were distributed among isolates from two hospitals. ISEcp1 carrying blaCTX-M-group 9 was the predominant transposable elements of the ESBL genes. Among the thirteen fosfomycin resistant isolates, functionless transporters were identified in 9 isolates. Three isolates contained novel amino acid substitutions (Asn67Ile, Phe151Ser and Trp164Ser, Val146Ala and His159Tyr, respectively) in MurA (the target of fosfomycin). Four isolates had fosfomycin modified enzyme (fosA3) in their plasmids. The fosA3 gene was harboured in an IncN-type plasmid (101 kbp) in the three pig isolates and an IncB/O-type plasmid (113 kbp) in the human isolate. In conclusion, we identified that 6% and 23% of the ESBL-producing E. coli from human and pigs were resistant to fosfomycin, respectively, in Taiwan. No clonal spread was found between human and pig isolates. Functionless transporters were the major cause of fosfomycin resistance, and the fosA3-transferring plasmid between isolates warrants further monitoring.

Project description:ImportanceThe consumption of broad-spectrum drugs has increased as a consequence of the spread of multidrug-resistant (MDR) Escherichia coli. Finding alternatives for these infections is critical, for which some neglected drugs may be an option.ObjectiveTo determine whether fosfomycin is noninferior to ceftriaxone or meropenem in the targeted treatment of bacteremic urinary tract infections (bUTIs) due to MDR E coli.Design, setting, and participantsThis multicenter, randomized, pragmatic, open clinical trial was conducted at 22 Spanish hospitals from June 2014 to December 2018. Eligible participants were adult patients with bacteremic urinary tract infections due to MDR E coli; 161 of 1578 screened patients were randomized and followed up for 60 days. Data were analyzed in May 2021.InterventionsPatients were randomized 1 to 1 to receive intravenous fosfomycin disodium at 4 g every 6 hours (70 participants) or a comparator (ceftriaxone or meropenem if resistant; 73 participants) with the option to switch to oral fosfomycin trometamol for the fosfomycin group or an active oral drug or parenteral ertapenem for the comparator group after 4 days.Main outcomes and measuresThe primary outcome was clinical and microbiological cure (CMC) 5 to 7 days after finalization of treatment; a noninferiority margin of 7% was considered.ResultsAmong 143 patients in the modified intention-to-treat population (median [IQR] age, 72 [62-81] years; 73 [51.0%] women), 48 of 70 patients (68.6%) treated with fosfomycin and 57 of 73 patients (78.1%) treated with comparators reached CMC (risk difference, -9.4 percentage points; 1-sided 95% CI, -21.5 to ∞ percentage points; P = .10). While clinical or microbiological failure occurred among 10 patients (14.3%) treated with fosfomycin and 14 patients (19.7%) treated with comparators (risk difference, -5.4 percentage points; 1-sided 95% CI, -∞ to 4.9; percentage points; P = .19), an increased rate of adverse event-related discontinuations occurred with fosfomycin vs comparators (6 discontinuations [8.5%] vs 0 discontinuations; P = .006). In an exploratory analysis among a subset of 38 patients who underwent rectal colonization studies, patients treated with fosfomycin acquired a new ceftriaxone-resistant or meropenem-resistant gram-negative bacteria at a decreased rate compared with patients treated with comparators (0 of 21 patients vs 4 of 17 patients [23.5%]; 1-sided P = .01).Conclusions and relevanceThis study found that fosfomycin did not demonstrate noninferiority to comparators as targeted treatment of bUTI from MDR E coli; this was due to an increased rate of adverse event-related discontinuations. This finding suggests that fosfomycin may be considered for selected patients with these infections.Trial registrationClinicalTrials.gov Identifier: NCT02142751.

Dataset Information

Resensitization of Fosfomycin-Resistant Escherichia coli Using the CRISPR System.

Publications

Resensitization of Fosfomycin-Resistant <i>Escherichia coli</i> Using the CRISPR System.

Similar Datasets

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