Occurrence of plasmid-mediated fosfomycin resistance (fos genes) among Escherichia coli isolates, Portugal.

OBJECTIVES: To evaluate the occurrence of plasmid-mediated fos genes among fosfomycin-resistant Escherichia coli isolates collected from patients in Lisbon, Portugal, and characterize the fos-positive strains. METHODS: A total of 19 186 E. coli isolates were prospectively collected between April 2022 and January 2023 from inpatients and outpatients at a private laboratory in Lisbon. Fosfomycin resistance was initially assessed by semi-automated systems and further confirmed by the disc diffusion method. Resistant isolates were investigated for plasmid-mediated fos genes (fosA1-fosA10, fosC and fosL1-fosL2) and extended-spectrum beta-lactamases (ESBLs) by PCR and sequencing. Multilocus sequence typing was performed to evaluate the clonal relationship among fos-carrying isolates. RESULTS: Out of the 19 186 E. coli isolates, 100 were fosfomycin-resistant (0.5%), out of which 15 carried a fosA-like gene (15%). The most prevalent fosfomycin-resistant determinant was fosA3 (n = 11), followed by fosA4 (n = 4). Among the 15 FosA-producing isolates, 10 co-produced an ESBL (67%), being either of CTX-M-15 (n = 8) or CTX-M-14 (n = 2) types. The fosA3 gene was carried on IncFIIA-, IncFIB-, and IncY-type plasmids, whereas fosA4 was always located on IncFIB-type plasmids. Most FosA4-producing isolates belonged to a single sequence type ST2161, whereas isolates carrying the fosA3 gene were distributed into nine distinct genetic backgrounds. CONCLUSION: The prevalence of fosfomycin-resistant E. coli isolates is still low in Portugal. Notably, 15% of fosfomycin-resistant isolates harbour a transferable fosA gene, among which there is a high rate of ESBL producers, turning traditional empirical therapeutical options used in Portugal (fosfomycin and amoxicillin-clavulanic acid) ineffective.

Characterization of FosL1, a Plasmid-Encoded Fosfomycin Resistance Protein Identified in Escherichia coli.

Fosfomycin is gaining renewed interest for treating urinary tract infections. Monitoring fosfomycin resistance is therefore important in order to detect the emergence of novel resistance mechanisms. Here, we used the Rapid Fosfomycin NP test to screen a collection of extended-spectrum-ß-lactamase-producing Escherichia coli isolates from Switzerland and found a fosfomycin-resistant isolate in which a novel plasmid-mediated fosfomycin resistance gene, named fosL1, was identified. The FosL1 protein is a putative glutathione S-transferase enzyme conferring high-level resistance to fosfomycin and sharing between 57% to 63% amino acid identity with other FosA-like family members. Genetic analyses showed that the fosL1 gene was embedded in a mobile insertion cassette and had likely been acquired by transposition through a Tn7-related mechanism. In silico analysis over GenBank databases identified the FosL1-encoding gene in addition to another variant (fosL1 and fosL2, respectively) in two Salmonella enterica isolates from the United States. Our study further highlights the necessity of monitoring fosfomycin resistance in Enterobacteriaceae to identify the emergence of novel mechanisms of resistance.

Characterization of BRPMBL, the Bleomycin Resistance Protein Associated with the Carbapenemase NDM.

The metallo-ß-lactamase NDM-1 is among the most worrisome resistance determinants and is spreading worldwide among Gram-negative bacilli. A bleomycin resistance gene, bleMBL, downstream of the blaNDM-1 gene has been associated with resistance almost systematically. Here, we characterized the corresponding protein, BRPMBL, conferring resistance to bleomycin, an antitumoral glycopeptide molecule. We have determined whether the expression of the blaNDM-1-bleMBL operon is inducible in the presence of carbapenems and/or bleomycin-like molecules using quantitative reverse transcription-PCR (qRT-PCR), determination of imipenem and zeocin MICs, and carbapenemase-specific activity assays. We showed that the blaNDM-1-bleMBL operon is constitutively expressed. Using electrophoretic mobility shift and DNA protection assays performed with purified glutathione S-transferase (GST)-BRPMBL, we demonstrated that BRPMBL is able to bind and sequester bleomycin-like molecules, thus preventing bleomycin-dependent DNA degradation. In silico modeling confirmed that the mechanism of action required the dimerization of the BRPMBL protein in order to sequester bleomycin and prevent DNA damage. BRPMBL acts specifically on bleomycin-like molecules since cloning and expression of bleMBL in Staphyloccoccus aureus did not confer cross-resistance to any other antimicrobial glycopeptides such as vancomycin and teicoplanin.

A Universal Culture Medium for Screening Polymyxin-Resistant Gram-Negative Isolates.

The colistin-containing SuperPolymyxin medium was developed for screening polymyxin-resistant Gram-negative bacteria. It was evaluated with 88 polymyxin-susceptible or polymyxin-resistant cultured Gram-negative isolates. Its sensitivity and specificity of detection were ca. 100%. The SuperPolymyxin medium is the first screening medium that is able to detect intrinsic and acquired polymyxin-resistant bacteria.

Detection of carbapenemase producers in Enterobacteriaceae by use of a novel screening medium.

A Drigalski agar-based culture medium containing an ertapenem, cloxacillin, and zinc sulfate (Supercarba medium) was tested for screening carbapenemase-producing members of the family Enterobacteriaceae. OXA-48 (n = 44), NDM (n = 25), VIM or IMP (n = 27), and KPC producers (n = 18) were detected with a low detection limit. Its overall sensitivity (95.6%) was higher than those of the currently available ChromID ESBL (bioMérieux) and CHROMagar KPC (CHROMagar) screening media. The Supercarba medium provides a significant improvement for detection of the most common types of carbapenemase producers.

Does broad-spectrum beta-lactam resistance due to NDM-1 herald the end of the antibiotic era for treatment of infections caused by Gram-negative bacteria?

The NDM-1 gene, first identified in Sweden in 2008 in Klebsiella pneumoniae from a patient hospitalized in New Delhi, encodes a metallo-ß-lactamase that inactivates all ß-lactams except aztreonam. This bla(NDM-1) gene has been identified in hospital-acquired bacterial species, such as K. pneumoniae, but also in the typical community-acquired species, Escherichia coli. This gene has been identified in strains that possess other resistance mechanisms contributing to their multidrug resistance patterns. It has been recently extensively reported from the UK, India and Pakistan and, albeit to a lesser extent, from a number of other countries worldwide. In most of the cases a link with the Indian subcontinent has also been established. To stem the onslaught of NDM producers, early identification of cases of NDM-related infections and prevention of their spread by implementing screening, hygiene measures and the isolation of carriers is needed.