Abstract
BackgroundLinezolid is an alternative treatment option for infections with multidrug-resistant Gram-positive bacteria including vancomycin-resistant enterococci. Some countries report an increasing number of isolates with resistance to linezolid. The recent publication of the Commission for Hospital Hygiene in Germany on enterococci/VRE recommends screening for linezolid-resistant enterococci (LRE). However, a suitable selective medium or a genetic test is not available. Our aim was to establish a selective screening agar for LRE detection and validate its application with a comprehensive collection of clinical LRE and linezolid-susceptible enterococci.MethodsWe decided to combine the selective power of an enterococcal screening agar with a supplementation of linezolid. Several rounds of analyses with reference, control and test strains and under varying linezolid concentrations of a wider and a smaller range were investigated and assessed. The collection of linezolid-resistant enterococcal control strains included isolates with different resistance mechanisms (23S rDNA mutations, cfr(B), optrA, poxtA). Finally, we validated our LRE screening agar with 400 samples sent to our National Reference Centre in 2019.ResultsSeveral rounds of pre-tests and confirmatory analyses favored Enterococcosel® Agar supplemented with a concentration of 2 mg/L linezolid. A 48 h incubation period was essential for accurate identification of LRE strains. Performance of the LRE screening agar revealed a sensitivity of 96.6% and a specificity of 94.4%.ConclusionsHere we describe preparation of a suitable screening agar and a procedure to identify LRE isolates with high accuracy.
Highlights
Linezolid is an alternative treatment option for infections with multidrug-resistant Gram-positive bacteria including vancomycin-resistant enterococci
Reference isolates E. faecalis ATCC 29212, E. faecium ATCC 19434, S. aureus ATCC 25923 and E. coli ATCC25922 as well as five E. faecium and three E. faecalis isolates with linezolid MICs of 4 to > 32 mg/L served as negative and positive control isolates, respectively (Table 1)
We repeated these experiments with the three agar brands supplemented with linezolid concentrations of 0, 1, 2, and 4 mg/L by streaking out the 10 μl of bacterial dilutions and by performing mixed culture growth experiments with (a) E. coli ATCC25922 / S. aureus ATCC25923 / E. faecium UW19369 and (b) E. coli ATCC25922 / S. aureus ATCC25923 / E. faecalis UW17810 in the same manner except for the E. coli and S. aureus isolates which were diluted 1:100 to reach similar colony counts as compared to the enterococcal strains
Summary
Linezolid is an alternative treatment option for infections with multidrug-resistant Gram-positive bacteria including vancomycin-resistant enterococci. The recent publication of the Commission for Hospital Hygiene in Germany on enterococci/VRE recommends screening for linezolid-resistant enterococci (LRE). In 2018, the German Commission for Hospital Hygiene and Infection Prevention (“Kommission für Krankenhaushygiene und Infektionsprävention” - KRINKO) released a recommendation for the prevention of infections with “enterococci harboring special resistances” [7]. This national directive focused on vancomycin resistance as the key resistance trait in clinical enterococci and addressed the growing problem of vancomycin-. The technical implementation of this recommendation is less clear, since commercial agar media for the detection of linezolid or other last resort-resistances in enterococci are not available yet. In the present study we tested different enterococcal nutrient agar media, supplemented with varying concentrations of linezolid, to determine the best media-antibiotic combination for reliable LRE screening
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