Nosocomial superinfections due to linezolid-resistant Enterococcus faecalis: evidence for a gene dosage effect on linezolid MICs

Resistance to linezolid among Enterococcus faecium and Enterococcus faecalis isolates has been reported in patients who receive a prolonged course of the drug. We report two cases of linezolid-resistant Enterococcus faecalis that occurred in patients who previously received linezolid for infections with vancomycin-resistant Enterococcus faecium. Both isolates had the G2576U mutation in the 23S rRNA previously reported in isolates of Enterococcus faecium. The number of gene copies mutated in the 23S rRNA correlated with the level of resistance.     

Clinical-use-associated decrease in susceptibility of vancomycin-resistant Enterococcus faecium to linezolid: a comparison with quinupristin-dalfopristin

The susceptibility of 135 vancomycin-resistant Enterococcus faecium bacteremic isolates to linezolid and quinupristin-dalfopristin was determined. All were susceptible to linezolid, while 88% were susceptible to quinupristin-dalfopristin prior to the clinical use of the drugs at our hospital. More than 6 months after their clinical use, a decrease in susceptibility was noted for only linezolid at 83%. This was related in part to a single G2576U gene mutation in domain V of the 23S rRNA gene.     

Reversion to susceptibility in a linezolid-resistant clinical isolate of Staphylococcus aureus

OBJECTIVES: Linezolid resistance in rare isolates of Staphylococcus aureus has been associated with G2576T mutations in domain V of the 23S rRNA gene. We report the analysis of a clinical S. aureus isolate that developed linezolid resistance (MIC of linezolid of 12 mg/L) after a 25 day course of the drug. Sequencing identified G2576T mutations in four of the five copies of the 23S rRNA gene. METHODS: To examine the stability of this resistance, we serially passaged this original isolate 60 times over a 75 day period on antimicrobial-free medium. RESULTS: After 30 passages, the MIC of linezolid had decreased to 8 mg/L and only two of the five copies of the 23S rRNA gene contained the G2576T mutation. After 60 passages, the MIC of linezolid fell to 2 mg/L and only one of the five 23S rRNA gene copies contained the mutation. The original and two passaged staphylococci were indistinguishable by pulsed-field gel electrophoresis. CONCLUSIONS: In the absence of antibiotic pressure, linezolid resistance was unstable in a clinical isolate that did not have all copies of the 23S rRNA gene mutated, although a single copy of mutant rDNA was maintained. Gene conversion was probably the mechanism for this reversion, using the wild-type 23S rRNA gene sequences to replace the G2576T mutation by homologous recombination.     

利奈唑胺体外诱导肠球菌耐药及耐药机制研究

目的 研究利奈唑胺对肠球菌耐药的体外诱导作用并探讨其耐药机制.方法 采用多步诱导法对9株临床分离肠球菌(5株粪肠球菌,4株屎肠球菌)以及质控菌株进行体外诱导耐药试验,采用琼脂平皿二倍稀释法测定诱导前、后的MIC,PCR法扩增耐药菌23s rRNA基因,DNA测序并进行序列分析比较.结果 10株肠球菌均诱导出稳定的利奈唑胺耐药株,MIC值与原菌株比较增加了8～64倍.所有诱导耐药菌株的23s rRNA基因中均出现点突变.测序结果 与NCBI Blast中E.faecium ATCC27273比较,核苷酸序列第2576位发生G→U突变(G2576U).结论 利奈唑胺可诱导肠球菌产生获得性耐药,其耐药机制与肠球菌23s rRNA基因的点突变相关.     

Resistance to Linezolid: Characterization of Mutations in rRNA and Comparison of Their Occurrences in Vancomycin-Resistant Enterococci

To assess the potential for emergence of resistance during the use of linezolid, we tested 10 clinical isolates of vancomycin-resistant enterococci (VRE) (four Enterococcus faecalis, five Enterococcus faecium, and one Enterococcus gallinarum) as well as a vancomycin-susceptible control (ATCC 29212) strain of E. faecalis. The enterococci were exposed to doubling dilutions of linezolid for 12 passes. After the final passage, the linezolid plate growing VRE contained a higher drug concentration with E. faecalis than with E. faecium. DNA sequencing of the 23S rRNA genes revealed that linezolid resistance in three E. faecalis isolates was associated with a guanine to uracil transversion at bp 2576, while the one E. faecium isolate for which the MIC was 16 microg/ml contained a guanine to adenine transition at bp 2505.     

Linezolid-resistant Enterococcus faecium isolated from a patient without prior exposure to an oxazolidinone: report from the SENTRY Antimicrobial Surveillance Program

A patient case report describes an Enterococcus faecium strain isolated from a blood culture that was resistant to linezolid (MIC, 8 microg/mL; G2576U mutation of 23S rRNA). Co-resistances were identified for vancomycin, ampicillin, macrolides, fluoroquinolones, chloramphenicol, rifampin, gentamicin (high-level), nitrofurantoin and trimethoprim/sulfamethoxazole. Etest (AB BIODISK, Solna, Sweden) and disk diffusion results also detected the oxazolidinone resistance pattern. Laboratories should be aware of the rare possibility of these strains occurring during linezolid therapy or spontaneously (this case) in contemporary practice, and have in vitro susceptibility methods available capable of detecting oxazolidinone resistance.     

Increasing incidence of linezolid-intermediate or -resistant, vancomycin-resistant Enterococcus faecium strains parallels increasing linezolid consumption

Clinical enterococcal resistance to linezolid is defined by the presence of the G2576T mutation. We evaluated the incidence of genetically proven linezolid resistance among vancomycin-resistant Enterococcus faecium strains and linezolid consumption for a possible association. A relationship was found (r(2) = 0.73, P = 0.03) and predicts increasing resistance with current trends of linezolid use.     

Gene dosage and linezolid resistance in Enterococcus faecium and Enterococcus faecalis

Resistance to linezolid has been associated with a G2576U mutation in domain V of the 23S rRNA. We analyzed nine clinical isolates of linezolid-resistant enterococci and showed a clear association between the number of 23S rRNA genes containing this mutation and the level of linezolid resistance expressed.     

Linezolid-resistant enterococci: report of the first isolates in the United Kingdom

Linezolid, the first oxazolidinone antibacterial agent to be developed for clinical use, was licensed in the UK in early 2001. We report the first three examples of resistant enterococci (two isolates of Enterococcus faecium and one Enterococcus faecalis) isolated in the UK, which were obtained from patients who had received linezolid. The linezolid MICs for the resistant isolates were 64 mg/L. Pulsed-field gel electrophoresis (PFGE) analysis of the linezolid-susceptible and -resistant isolates from two of the patients, combined with sequence analysis of rRNA, indicated that resistance developed in previously susceptible strains, most probably via a point mutation in the 23S rRNA.     

Trends in linezolid susceptibility patterns in 2002: report from the worldwide Zyvox Annual Appraisal of Potency and Spectrum Program

Oxazolidinones have become reliable clinical and candidate antimicrobial agents to be utilized for infections caused by multidrug-resistant Gram-positive cocci, especially vancomycin-resistant enterococci and methicillin-resistant staphylococci. However, mutational resistance of the ribosomal target has been described for several species. Longitudinal surveillance remains necessary to monitor for this evolving linezolid resistance pattern. A survey of linezolid and several comparison Gram-positive focused agents was initiated in 2002 (7971 strains, >99.0% compliance) for 54 participating sites in the United States, Canada, Europe (6 nations), Latin America (2 nations), and the Asia Pacific (2 nations). The 5 and 25 sites in Canada and the United States, respectively, submitted 200 strains each to a central laboratory for organism identification/confirmation and reference MIC processing. The 10 remaining nations had 200 strain samples from 1 to 4 separate institutions. Linezolid resistance (MIC >/= 8 microg/mL) was confirmed by alternative susceptibility testing methods (Etest, AB BIO Disk, Solna, Sweden; disk diffusion method) and target mutation characterization by PCR and sequence analysis. Linezolid activity against the 6 major organism groups did not vary between geographic areas. A total of 98.1% of linezolid MIC values were between 0.5 and 2 microg/mL, and only 0.5% of results were at 4 microg/mL, which included 32 Staphylococcus aureus (0.9%) and 5 (0.5%) enterococcal isolates. Linezolid resistance was detected in only 4 isolates (0.05%): 1 each Staphylococcus aureus, Staphylococcus epidermidis, Enterococcus faecium, and a viridans group Streptococcus. All 4 isolates had a G2576U mutation in the 23S rRNA target. Linezolid activity as outlined by these Zyvox Annual Appraisal of Potency and Spectrum (ZAAPS) Program results demonstrate sustained, near complete activity against contemporary Gram-positive isolates on 4 monitored continents and in centers utilizing oxazolidinones. Rare linezolid-resistant strains were identified in the United States only (0.05% resistance overall).     

Persistence of rRNA operon mutated copies and rapid re-emergence of linezolid resistance in Staphylococcus aureus

OBJECTIVES: The G2576T mutation in domain V of 23S rRNA has been most often associated with the rare cases of linezolid resistance in Staphylococcus aureus. In a linezolid-susceptible S. aureus (A8761B) possessing a single mutated (G2576T) copy, originally derived from a resistant clinical isolate, we assessed the persistence of the mutation on further passage on antibiotic-free medium and the selection of resistance upon re-exposure of the susceptible strain to linezolid. METHODS: The stability of the mutant rRNA copy was tested through 40 serial passages on antibiotic-free medium. The re-emergence of linezolid-resistant mutants was examined after serial passage on successively increasing linezolid concentrations. The efficacy of novobiocin, at subinhibitory concentrations, to prevent or delay the emergence of resistant mutants was examined. Strain relatedness was confirmed by PFGE and domain V of individual rRNA copies was sequenced. RESULTS: After 40 passages in antibiotic-free medium, the linezolid MIC of derived strain A9584 remained stable at 2 mg/L and the G2576T mutation persisted in one 23S rRNA gene copy (copy number 2). Upon re-exposure of the strain to increasing concentrations of linezolid, linezolid resistance (MIC of 64 mg/L) emerged rapidly. In a representative derivative (A9753), the G2576T mutation was found in four of the five rRNA copies. All laboratory derivates were closely related by PFGE. When A9584 was applied to plates containing linezolid at 4 x MIC, resistant colonies emerged at a frequency of 8 x 10(-6). Novobiocin at 1/4 x MIC prevented the emergence of resistant colonies. CONCLUSIONS: The persistence of the G2576T mutation in one rRNA operon copy in the absence of selective pressure suggests that the mutation has a minimal impact on the organism's fitness in vitro. Resistance to linezolid, associated with acquisition of multiple mutant copies, emerges rapidly upon re-exposure to linezolid. Novobiocin, predicted to interfere with gene conversion, may reduce the likelihood of rapid development of linezolid resistance.     

In vitro selection of mutants of Streptococcus pneumoniae resistant to macrolides and linezolid: relationship with susceptibility to penicillin G or macrolides

OBJECTIVES: To evaluate the rate of acquisition of resistance to linezolid and macrolides in Streptococcus pneumoniae isolates with different levels of susceptibility to penicillin and erythromycin. Materials and methods: Thirty strains of S. pneumoniae were tested by serial passages in subinhibitory concentrations of each antibiotic by the spiral method. The four copies of the 23S rRNA rrl gene of parent strains and linezolid-resistant mutants were amplified and sequenced. RESULTS: The rate of acquisition of macrolide resistance did not differ when C-14 and C-16 macrolides were tested. Resistance to linezolid in strains susceptible to penicillin and erythromycin was difficult to produce. For mutants with low-level resistance to linezolid the cut-off value of the MIC was between 6 and 8 mg/L depending on the strain. All linezolid-resistant mutants displayed a mutation in 2-4 copies of the 23S rRNA rrl gene, mainly the G2576U mutation (27/30) with an additional C2610U mutation observed in certain mutants. Two new mutations were also noted, namely C2612A and C2571G. In three linezolid-resistant mutants no mutation was identified within the studied domain, suggesting another mechanism of resistance. CONCLUSIONS: Linezolid resistance in pneumococcal strains susceptible to penicillin and macrolides was more difficult to obtain than with macrolides. Increased resistance to these agents may therefore influence the clinical use of linezolid.     

Increased copy number of 23S ribosomal RNA gene with point mutation in MRSA associated with linezolid resistance in a patient treated with long-term linezolid

BackgroundMethicillin-resistant Staphylococcus aureus (MRSA) infection is one of the most difficult infections we have to treat. Linezolid is one of the effective treatment options for refractory MRSA infections. There are cases where we are forced to use long-term linezolid treatment for refractory MRSA infections.ObjectiveTo discuss the evolution of Linezolid resistance factors in clinical isolates of MRSA.MethodsWe investigated 16 MRSA isolated from a patient treated with linezolid for a long period of 75 days. We performed antibiotic susceptibility test, 23S rRNA genes sequencing analysis, Pulsed-field gel electrophoresis.ResultsMRSA isolates were susceptible to linezolid before the start of treatment, but became less susceptible by prolonged treatment. The 23S rRNA sequencing analysis of linezolid-resistant strains that appeared 17 days after the start of treatment with linezolid revealed that all resistant MRSA had the G2576T substitution (Escherichia coli 23S rRNA gene number). The number of copies of this mutation increased with the use of linezolid.ConclusionLong-term use of linezolid in a patient or reuse of linezolid in a patient who has been previously treated with linezolid can lead to the emerging of linezolid-resistant MRSA in the host.     

Recombination proficiency influences frequency and locus of mutational resistance to linezolid in Enterococcus faecalis

In vitro linezolid resistance was selected at a higher frequency in Enterococcus faecalis JH2-2 than in recombination-deficient E. faecalis UV202. Resistance in JH2-2 was related to accumulated G2576T mutations in 23S rRNA genes, with the least resistance conferred by mutations in two of four copies. UV202 resistance was associated with a G2505A mutation present in a single copy in mutants with different MICs.     

Linezolid for the treatment of resistant gram-positive cocci

OBJECTIVE: To provide a comprehensive review of linezolid, the first of a new class of antibiotics, the oxazolidinones. Therapeutic issues regarding the emergence of multidrug-resistant bacteria and a brief history of the oxazolidinones are also discussed. DATA SOURCES: A MEDLINE search (1966-March 2001) was conducted to identify pertinent literature, including preclinical trials, clinical trials, and reviews. Unpublished clinical data, adverse effects, and dosing information were abstracted from product labeling. STUDY SELECTION: Clinical efficacy data were extracted from clinical trials, case reports, and abstracts that mentioned linezolid. Additional information concerning antibiotic resistance, the oxazolidinones, in vitro susceptibility and the pharmacokinetic profile of linezolid also was reviewed. DATA SYNTHESIS: Linezolid exhibits activity against many gram-positive organisms, including vancomycin-resistant Enterococcus faecium, methicillin-resistant Staphylococcus aureus, and penicillin-resistant Streptococcus pneumoniae. Linezolid inhibits bacterial protein synthesis at an early step in translation and is rapidly and completely absorbed from the gastrointestinal tract following oral administration. Efficacy has been demonstrated in a number of unpublished clinical trials in adults with pneumonia, skin and skin structure infections, and vancomycin-resistant E. faecium infections. The adverse effect profile is similar to that of comparator agents (beta-lactams, clarithrornycin, vancomycin). CONCLUSIONS: Linezolid is the first oral antimicrobial agent approved for the treatment of vancomycin-resistant enterococci. Since the oxazoildinones have a unique mechanism of action and expanded spectrum of activity against virulent and highly resistant gram positive pathogens, linezolid is a valuable alternative to currently available treatment options. Clinical trials evaluating linezolid and other oxazolidinones for antibiotic-resistant gram-positive infections, as well as comparator studies comparing linezolid with other candidate drugs, such as quinupristin/dalfopristin and choramphenicol, will further define the role of linezolid.     

Rapid detection and estimation by pyrosequencing of 23S rRNA genes with a single nucleotide polymorphism conferring linezolid resistance in Enterococci

Pyrosequencing was used to detect rapidly and estimate the number of 23S rRNA genes with a G2576T mutation in 43 linezolid-resistant and -susceptible clinical isolates of enterococci. The method showed 100% concordance with PCR-restriction fragment length polymorphism for detecting isolates homozygous for either G2576 or T2576 or heterozygous for this mutation. A good correlation was found between linezolid MICs and the number of 23S rRNA gene copies carrying the mutation.     

Linezolid-resistant Staphylococcus aureus isolated from 2006 through 2008 at six hospitals in Japan

Limited use of linezolid for treating methicillin-resistant Staphylococcus aureus (MRSA) infection was approved in Japan in 2006. We report here the status of linezolid-resistant MRSAs in Japan. Eleven linezolid-resistant clinical isolates from 11 patients at six hospitals were collected from 2006 through 2008. The minimal inhibitory concentration (MIC) of linezolid in these strains varied from 8 to 64 μg/ml. All strains had at least one G2576T mutation in the chromosomal gene(s) encoding domain V of the 23S ribosomal RNA (rRNA). Chromosomal DNA encoding five copies of the domain V region was analyzed by polymerase chain reaction (PCR). Strains with the linezolid MICs of 64, 32, 16, and 8 μg/ml had the G2576T mutation(s) in four, three (or four), two, and one copy of the 23S rRNA genes, respectively. These results suggest that the level of linezolid resistance seems to be roughly correlated with the number of mutations in the genes encoding 23S rRNA. DNA samples from all 11 strains were subjected to pulsed-field gel electrophoresis and were classified into seven independent clones having >92% identity. Among the 11 patients, five had been treated with linezolid and the remainder, in two hospitals, had no history of prior linezolid use. The results suggested possible nosocomial infections by linezolid-resistant MRSA.     

LEADER Program results for 2009: an activity and spectrum analysis of linezolid using 6,414 clinical isolates from 56 medical centers in the United States

The LEADER Program monitors the in vitro activity of linezolid in sampled U.S. medical centers using reference broth microdilution methods with supporting molecular investigations in a central laboratory design. This report summarizes data obtained in 2009, the 6th consecutive year of this longitudinal study. A total of 6,414 isolates from 56 medical centers in all nine Census regions across the United States participated in 2009. For the six leading species/groups, the following linezolid MIC(90) values were observed: Staphylococcus aureus, 2 μg/ml; coagulase-negative staphylococci (CoNS), 1 μg/ml; Enterococcus spp., 2 μg/ml; Streptococcus pneumoniae, 1 μg/ml; viridans group streptococci, 1 μg/ml; and beta-hemolytic streptococci, 1 μg/ml. Linezolid resistance was only 0.34% overall, with no evidence of significant increase in the LEADER Program since 2006. The predominant linezolid resistant mechanism found was a G2576T mutation in the 23S rRNA. L3/L4 riboprotein mutations were also found. The mobile multidrug-resistant cfr gene was found in four strains (two S. aureus strains and one strain each of S. epidermidis and S. capitis) from four different states, suggesting persistence but a lack of dissemination. Linezolid continues to exhibit excellent activity and spectrum, and this study documents the need for continued monitoring of emerging mechanisms of resistance over a wide geographic area.     

Linezolid resistance in Enterococcus faecium isolated in Ontario,Canada

Recent studies have described linezolid-resistant MRSA and vancomycin-resistant enterococci (VRE) occurring worldwide, including an outbreak of linezolid-resistant MRSA. The objective of this study was to determine if linezolid-resistant enterococci are present in clinical isolates in Ontario, Canada. From January 2010 to June 2012, all enterococcal isolates submitted to the Public Health Ontario Laboratory (PHOL) for confirmation of VRE and susceptibility testing were included in this study. Of 2829 enterococcal isolates tested, 12 Enterococcus faecium were found to be resistant to linezolid. All linezolid-resistant isolates were also resistant to ampicillin, ciprofloxacin, and vancomycin. In addition, 33% of isolates were non-susceptible to daptomycin, whereas 41% were resistant to quinupristin/dalfopristin. Molecular characterization of these isolates showed that 8/12 isolates (66.7%) contained the mutation G2576T in 23S rRNA, which has been associated with linezolid resistance. Amplification and sequencing of L3- and L4-coding genes did not reveal mutations associated with linezolid resistance. One isolate contained the cfr gene, which is associated with linezolid resistance, and has been found in staphylococcal species and E. faecalis. These data show that occurrence of linezolid resistance is still rare among enterococcal isolates referred to PHOL though detection of cfr in E. faecium is concerning as it has the potential to disseminate among other enterococci.     

Linezolid resistance since 2001: SENTRY Antimicrobial Surveillance Program

BACKGROUND: The oxazolidinone class of antimicrobials has demonstrated remarkable activity against gram-positive cocci. Linezolid has proven to be a first-line therapeutic option for vancomycin-resistant strains. Linezolid clinical trial results and subsequent published case reports cite rare resistance emerging in patients receiving prolonged therapy. OBJECTIVE: To report the initial linezolid-resistant organisms from cases obtained through the SENTRY Antimicrobial Surveillance Program, after screening >40,000 gram-positive cocci without resistance between 1998 and 2000. METHODS: During 2001-2002, 8 resistant strains (from 8 different patients) located in 6 states from 7 different participating SENTRY institutions in the US were identified among bloodstream, respiratory, skin and soft tissue, and urinary tract infection isolates of Enterococcus faecalis, Enterococcus faecium, Staphylococcus epidermidis, and Streptococcus oralis. Resistance was detected by reference broth microdilution methods and confirmed by identical results using Etest (AB BIODISK, Solna, Sweden) and the standardized disk diffusion method. RESULTS: Minimum inhibitory concentration (MIC) and disk diffusion tests showed elevated MICs (> or =8 microg/mL) and small inhibitory zone diameters (< or =15 mm) for all strains to both linezolid and the investigational oxazolidinone AZD2563. Vancomycin resistance was detected in 2 of the 8 linezolid-resistant strains. All enterococci and the viridans-group streptococcus (S. oralis) strain showed resistance to erythromycin. E. faecium strains were resistant to penicillins, but susceptible to quinupristin/dalfopristin. Only 3 of the patients had previously received the drug. CONCLUSIONS: Linezolid resistance remains rare, with only 8 isolates among 9833 (0.08%) monitored isolates identified between January 1, 2001, and June 30, 2002. Resistance, however, was no longer limited to enterococci. Clinical laboratories should test linezolid more widely to detect emerging resistance, especially for patients receiving oxazolidinone therapy. Longitudinal surveillance programs are warranted to detect a trend in the development of resistance, determine the molecular mechanism of resistance, and recommend alternative therapies or epidemiologic interventions.