Linezolid Dependence in Staphylococcus epidermidis Bloodstream Isolates

Spyros Pournaras

, Eleni Ntokou, Olympia Zarkotou, Kyriaki Ranellou, Katerina Themeli-Digalaki, Constantinos Stathopoulos, and Athanassios Tsakris

Author affiliations: Author affiliations: University of Thessaly Medical School, Larissa, Greece (S. Pournaras, E. Ntokou); Tzaneio General Hospital, Piraeus, Greece (O. Zarkotou, K. Themeli-Digalaki); University of Athens Medical School, Athens, Greece (K. Ranellou, A. Tsakris); University of Patras School of Medicine, Patras, Greece (C. Stathopoulos)

Abstract

We document linezolid dependence among 5 highly linezolid-resistant (LRSE) Staphylococcus epidermidis bloodstream isolates that grew substantially faster at 32 µg/mL linezolid presence. These isolates carried the mutations T2504A and C2534T in multiple 23S rRNA copies and 2 mutations leading to relevant amino acid substitutions in L3 protein. Linezolid dependence could account for increasing LRSE emergence.

Linezolid is highly effective against Staphylococcus epidermidis (1). Linezolid-resistant S. epidermidis (LRSE) isolates are limited worldwide (2), and few LRSE outbreaks have occurred (3,4). Linezolid resistance in S. epidermidis has been attributed to specific 23S rRNA mutations (G2576U, G2447U, U2504A, C2534U, and G2631U) (5,6), cfr gene (7), or mutations in ribosomal proteins L3, L4, and L22 (7).
Dependence on linezolid for bacterial growth has not been reported but has been described for other antimicrobial drugs (8–10). We report the characteristics of partially linezolid-dependent LRSE causing bloodstream infections (BSIs).

The Study

Twenty-seven LRSE isolates were randomly selected for study among the 46 single-patient LRSE isolates recovered from BSIs in Tzaneio General Hospital (Piraeus, Greece) during 2008–2010. Isolates were identified by Vitek 2 (bioMérieux, Marcy l’Etoile, France). Chloramphenicol and clindamycin MIC was determined by E-test (bioMerieux) and linezolid MIC by using broth microdilution (11).
The 27 LRSE isolates were tested by pulsed-field gel electrophoresis (PFGE) as described (12) and screened for cfr gene (7). Mutations in the peptidyl-transferase center were identified for each separate 23S rRNA copy as described (13).
In 8 LRSE isolates representing all PFGE types, genes encoding the L3, L4, and L22 ribosomal proteins that factor in ribosome assembly were sequenced to identify mutations conferring linezolid resistance (6). Nucleotide and amino acid sequences were analyzed by using Lasergene software (DNASTAR, Madison, WI, USA) and compared with those of the linezolid-susceptible S. epidermidis (LSSE) strain ATCC12228 (GenBank accession no. AE015929).
Growth curves were conducted in the presence and absence of linezolid for the above 8 LRSE isolates, 1 clinical LSSE isolate (A1521, linezolid MIC 2 μg/mL), and the ATCC 29213 S. aureus strain (linezolid MIC 0.5 μg/mL) as controls. Linezolid concentrations tested were half-MIC for controls and 3 LRSE isolates with low MIC (16–32 μg/mL) and 8, 16, 32, 64, and 128 μg/mL for 5 LRSE isolates with MIC >256 μg/mL. Growth curves were performed in triplicate by diluting 20 μL Mueller-Hinton broth culture in 2 mL broth, followed by incubation at 37°C under constant shaking; turbidity of cultures (McFarland scale) was measured every 6 h for 36 h. We statistically compared isolate growth at each time point using the paired t test and Minitab software version 13.31 (www.minitab. com); p<0.05 indicated statistical significance.
We retrospectively examined medical records (anonymized demographic data, clinical characteristics, comorbidities, prior linezolid treatment for >3 days, and in-hospital deaths) of the 27 patients harboring LRSE to ascertain factors influencing resistance acquisition and outbreak persistence. Each of the 27 patients yielding LRSE had prolonged hospitalization and carried a central venous catheter. Twenty-one were mechanically ventilated, and 25 received linezolid treatment (Table 1).
Linezolid MICs were >256 μg/mL for 23 LRSE isolates and 8–32 μg/mL for 4 LRSE isolates. All isolates were co-resistant to clindamycin and chloramphenicol, but the cfr gene was not detected by PCR in any isolate (7). Three PFGE types were identified. PFGE type I comprised the 23 highly LRSE isolates, which all carried mutations T2504A and C2534T; 3 LRSE isolates were related to each other (type II) and carried the mutations G2576T and C2534T; and 1 LRSE isolate was unique (type III) and carried G2576T along with novel mutations C2356T or T2334C in different 23S rRNA copies each. All isolates had mutations in 3–6 copies of 23S rRNA. The cfr gene was not detected in any isolate.