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Phenotypic and genotypic characterization of linezolid-resistant Enterococcus faecium from the USA and Pakistan.

ABSTRACT: OBJECTIVES:Linezolid is an important therapeutic option for the treatment of infections caused by VRE. Linezolid is a synthetic antimicrobial and resistance to this antimicrobial agent remains relatively rare. As a result, data on the comparative genomics of linezolid resistance determinants in Enterococcus faecium are relatively sparse. METHODS:To address this knowledge gap in E. faecium, we deployed phenotypic antibiotic susceptibility testing and Illumina WGS on hospital surface (environmental) and clinical isolates from the USA and Pakistan. RESULTS:We found complete concordance between isolate source country and mechanism of linezolid resistance, with all the US isolates possessing a 23S rRNA gene mutation and the Pakistan isolates harbouring two to three acquired antibiotic resistance genes. These resistance genes include the recently elucidated efflux-pump genes optrA and poxtA and a novel cfr-like variant. Although there was no difference in the linezolid MIC between the US and Pakistan isolates, there was a significant difference in the geometric mean of the MIC between the Pakistan isolates that had two versus three of the acquired antibiotic resistance genes. In five of the Pakistan E. faecium that possessed all three of the resistance genes, we found no difference in the local genetic context of poxtA and the cfr-like gene, but we identified different genetic contexts surrounding optrA. CONCLUSIONS:These results demonstrate that E. faecium from different geographical regions employ alternative strategies to counter selective pressure of increasing clinical linezolid use.

SUBMITTER: Wardenburg KE

PROVIDER: S-EPMC6857194 | biostudies-literature | 2019 Dec

REPOSITORIES: biostudies-literature

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Phenotypic and genotypic characterization of linezolid-resistant Enterococcus faecium from the USA and Pakistan.

Wardenburg Kate E KE Potter Robert F RF D'Souza Alaric W AW Hussain Tahir T Wallace Meghan A MA Andleeb Saadia S Burnham Carey-Ann D CD Dantas Gautam G

The Journal of antimicrobial chemotherapy 20191201 12

<h4>Objectives</h4>Linezolid is an important therapeutic option for the treatment of infections caused by VRE. Linezolid is a synthetic antimicrobial and resistance to this antimicrobial agent remains relatively rare. As a result, data on the comparative genomics of linezolid resistance determinants in Enterococcus faecium are relatively sparse.<h4>Methods</h4>To address this knowledge gap in E. faecium, we deployed phenotypic antibiotic susceptibility testing and Illumina WGS on hospital surfac ...[more]

PMID: 31504566

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Project description:Background & objectivesEnterococci have emerged as important nosocomial pathogens and emergence of resistance to many of the antimicrobials used for Gram-positive organisms has made the management of infections due to Enterococcus species difficult. Resistance to glycopeptide antibiotics, especially vancomycin is of special concern. This study was undertaken to perform a phenotypic and genotypic characterization of vancomycin resistant Enterococcus (VRE) isolates obtained from clinical samples in a tertiary care hospital in southern India.MethodsSusceptibility testing was performed for Enterococcus isolates collected over a period of one year (November 2008-October 2009). Minimum inhibitory concentrations (MIC) of vancomycin and teicoplanin were determined for the isolates by the agar dilution method. Genotypic characterization of VRE isolates was done by performing multiplex polymerase chain reaction (PCR) for detecting the various vancomycin resistance genes.ResultsOf the 367 isolates of Enterococcus species isolated, 32 were found to be resistant to vancomycin after MIC testing. VanA was the commonest phenotype of vancomycin resistance and the commonest genotype was vanA. Among the other important findings of the study was the presence of heterogeneity in isolates of VRE with the vanA gene cluster with regards to resistance to teicoplanin and the coexistence of vanA and vanC1 gene clusters in an isolate of E. gallinarum which conferred high level glycopeptide resistance to the isolate.Interpretation & conclusionsEnterococcus species have emerged as important nosocomial pathogens in our patients with a capacity to cause a variety of infections. The vancomycin resistance among Enterococcus isolates was 8.7 per cent in our study which was high compared to other Indian studies. VanA was the commonest phenotype of glycopeptide resistance and vanA was the commonest vancomycin resistance gene. The study also demonstrates phenotypic as well as genotypic heterogeneity among isolates of VRE from clinical specimens.

Project description:One of the most pressing problems of enterococci infections is occurring resistance to linezolid, which is an antibiotic used in the treatment of infections caused by vancomycin-resistant strains (VRE). The main objective of our research was to investigate the relationship of 19 linezolid-resistant E. faecium isolates from 18 patients hospitalized at Clinical Hospital in Gdansk (Poland). One of the LZDREF was isolated in 2003 (K2003), and another 18 were collected from 2013 to 2017. Genotyping with PCR MP method indicated 14 main unrelated genetic profiles and no association with K2003 strain. Two isolates with the same genotype and genetically closely related two sub-types (2 isolates for each sub-type) were hospital-derived colonizations of patients. The other unrelated genotypes were discussed in the context of colonization, nosocomial infections, and commensal origin, taking into account prior exposure to linezolid. We determined the presence of a point mutation G2576T in six loci of 23S rDNA. There was also a significant correlation (p<0.0015) between the presence of MIC>32 value and the presence of G2576T point mutation on the sixth rrn. We also detected 5 virulence genes for all isolates: gelE, cylA, asa1, hyl, esp. Correlation (p?0.0001) was observed between the presence of gelE gene encoding gelatinase and two other genes: cylA and asa1 encoding cytolysin and collagen binding protein responsible for aggregation of bacterial cells, respectively. Significant correlation was also observed between asa1 and cfr genes encoding 23S rRNA rybonuclease responsible for resistance to PhLOPSA antibiotics (p = 0.0004). The multidimensional analysis has also shown the correlation between cfr gene and GI-tract (p = 0, 0491), which suggests horizontal gene transfer inside the gut microbiota and the risk of colonization with linezolid-resistant strains without previously being treated with the antibiotic. The patient could have been colonized with LZDRVREF strains which in the absence of competitive microbiota quickly settle in ecological niches favourable for them and pose a risk for the patient.

Project description:Enterococcus faecium clinical isolates A902 and BM4538, which were resistant to relatively high levels of vancomycin (128 and 64 microg/ml, respectively) and to low levels of teicoplanin (4 microg/ml), and Enterococcus faecalis clinical isolates BM4539 and BM4540, which were resistant to moderate levels of vancomycin (16 microg/ml) and susceptible to teicoplanin (0.25 microg/ml), were studied. They were constitutively resistant by synthesis of peptidoglycan precursors ending with d-alanyl-d-lactate and harbored a chromosomal vanD gene cluster which was not transferable by conjugation to other enterococci. VanX(D) activity, which is not required in the absence of d-Ala-d-Ala, was low in the four strains, although none of the conserved residues was mutated; and the constitutive VanY(D) activity in the membrane fractions was inhibited by penicillin G. The mutations E(13)G in the region of d-alanine:d-alanine ligase (which is implicated in d-Ala1 binding in A902) and S(319)N of the serine involved in ATP binding in BM4538 and a 7-bp insertion at different locations in BM4539 and BM4540 (which led to putative truncated proteins) led to the production of an impaired enzyme and accounted for the lack of d-Ala-d-Ala-containing peptidoglycan precursors. The same 7-bp insertion in vanS(D) of BM4539 and BM4540 and a 1-bp deletion in vanS(D) of A902, which in each case led to a putative truncated and presumably nonfunctional protein, could account for the constitutive resistance. Strain BM4538, with a functional VanS(D), had a G(140)E mutation in VanR(D) that could be responsible for constitutive glycopeptide resistance. This would represent the first example of constitutive van gene expression due to a mutation in the structural gene for a VanR transcriptional activator. Study of these four additional strains that could be distinguished on the basis of their various assortments of mutations confirmed that all VanD-type strains isolated so far have mutations in the ddl housekeeping gene and in the acquired vanS(D) or vanR(D) gene that lead to constitutive resistance to vancomycin.

Dataset Information

Phenotypic and genotypic characterization of linezolid-resistant Enterococcus faecium from the USA and Pakistan.

Publications

Phenotypic and genotypic characterization of linezolid-resistant Enterococcus faecium from the USA and Pakistan.

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