Clinical outbreak of linezolid-resistant Staphylococcus aureus in an intensive care unit.

CONTEXT: Linezolid resistance is extremely uncommon in Staphylococcus aureus. OBJECTIVE: To report an outbreak with linezolid and methicillin-resistant S. aureus (LRSA) in an intensive care department and the effective control measures taken. DESIGN, SETTING, AND PATIENTS: Outbreak study of consecutive critically ill patients colonized and/or infected with LRSA at an intensive care department of a 1000-bed tertiary care university teaching hospital in Madrid, Spain. Patients were placed under strict contact isolation. Daily updates of outbreak data and recommendations for the use of linezolid were issued. Extensive environmental sampling and screening of the hands of health care workers were performed. MAIN OUTCOME MEASURES: Linezolid use and clinical and epidemiological characteristics and outcomes using minimal inhibitory concentrations, pulsed-field gel electrophoresis, and polymerase chain reaction of LRSA isolates. RESULTS: Between April 13 and June 26, 2008, 12 patients with LRSA were identified. In 6 patients, LRSA caused ventilator-associated pneumonia and in 3 patients it caused bacteremia. Isolates were susceptible to trimethoprim-sulfamethoxazole, glycopeptides, tigecycline, and daptomycin. Genotyping identified 1 predominant clone and 3 other types. Cfr-mediated linezolid resistance was demonstrated in all isolates. Potential hospital staff carriers and environmental samples were negative except for one. Six patients died, 5 of them in the intensive care unit, with 1 death attributed to LRSA infection. Linezolid use decreased from 202 defined daily doses in April 2008 to 25 defined daily doses in July 2008. Between July 2008 and April 2010, no new cases have been identified in the weekly surveillance cultures or diagnostic samples. CONCLUSIONS: The first clinical outbreak, to our knowledge, with LRSA mediated by the cfr gene developed at our center, was associated with nosocomial transmission and extensive usage of linezolid. Reduction of linezolid use and infection-control measures were associated with the termination of the outbreak.

Resistance to linezolid is mediated by the cfr gene in the first report of an outbreak of linezolid-resistant Staphylococcus aureus.

BACKGROUND: From April through June 2008, we identified 12 patients in the intensive care unit and 3 patients on other wards infected with methicillin-resistant Staphylococcus aureus that was also resistant to linezolid. We investigated the mechanism of resistance--point mutations in domain V of 23S ribosomal RNA (rRNA) or presence of the cfr gene--involved in the outbreak. METHODS: Strains for the study were obtained in the intensive care unit and other wards. Minimal inhibitory concentrations were determined using automated methods, the E-test, or dilution in Mueller-Hinton agar in accordance with Clinical and Laboratory Standards Institute guidelines. Strains were genotyped using pulsed-field gel electrophoresis and were sequenced to determine the presence of point mutations in 23S rRNA. The presence of the cfr gene was determined by specific polymerase chain reaction. RESULTS: The minimal inhibitory concentrations of linezolid ranged from 16 mg/L to 32 mg/L, and all the strains were susceptible to tigecycline, vancomycin, and daptomycin. Typing of strains sequentially isolated by pulsed-field gel electrophoresis showed that each patient carried only 1 clonal type of linezolid-resistant, methicillin-resistant S. aureus as detected by sequential isolations. The presence of the cfr gene was confirmed in all the isolates. Furthermore, sequencing of domain V of 23S rRNA showed that the most common mechanism of linezolid resistance reported to date, mutation G2576T, was not detected in any of the strains analyzed. CONCLUSIONS: We report the presence of the cfr gene underlying the resistance mechanism involved in a clinical outbreak of linezolid-resistant S. aureus.

Nuclear monothiol glutaredoxins of Saccharomyces cerevisiae can function as mitochondrial glutaredoxins.

Glutaredoxins are thiol oxidoreductases that regulate protein redox state. In Saccharomyces cerevisiae, Grx1 and Grx2 are cytosolic dithiol glutaredoxins, whereas Grx3, Grx4, and Grx5 are monothiol glutaredoxins. Grx5 locates at the mitochondrial matrix and is needed for iron/sulfur cluster biogenesis. Its absence causes phenotypes such as inactivation of iron/sulfur enzymes and sensitivity to oxidative stress. Whereas Grx5 contains a single glutaredoxin domain, in Grx3 and Grx4 a thioredoxin-like domain is fused to the glutaredoxin domain. Here we have shown that Grx3 locates at the nucleus and that the thioredoxin-like domain is required for such location. We have addressed the functional divergence among glutaredoxins by targeting Grx2/3/4 molecules to the mitochondrial matrix using the Grx5 targeting sequence. The mitochondrial forms of Grx3 and Grx4 partially rescue the defects of a grx5 null mutant. On the contrary, mitochondrially targeted Grx2 does not suppress the mutant phenotype. Both the thioredoxin-like and glutaredoxin domains are needed for the mitochondrial activity of Grx3, although none of the cysteine residues at the thioredoxin-like domain is required for rescue of the grx5 phenotypes. We have concluded that dithiol glutaredoxins are functionally divergent from monothiol ones, but the latter can interchange their biological activities when compartment barriers are surpassed.

Comparative genomics of yeast species: new insights into their biology.

The genomes of two hemiascomycetous yeasts (Saccharomyces cerevisiae and Candida albicans) and one archiascomycete (Schizosaccharomyces pombe) have been completely sequenced and the genes have been annotated. In addition, the genomes of 13 more Hemiascomycetes have been partially sequenced. The amount of data thus obtained provides information on the evolutionary relationships between yeast species. In addition, the differential genetic characteristics of the microorganisms explain a number of distinctive biological traits. Gene order conservation is observed between phylogenetically close species and is lost in distantly related species, probably due to rearrangements of short regions of DNA. However, gene function is much more conserved along evolution. Compared to S. cerevisiae and S. pombe, C. albicans has a larger number of specific genes, i.e., genes not found in other organisms, a fact that can account for the biological characteristics of this pathogenic dimorphic yeast which is able to colonize a large variety of environments.

Structure-function analysis of yeast Grx5 monothiol glutaredoxin defines essential amino acids for the function of the protein.

Grx5 defines a family of yeast monothiol glutaredoxins that also includes Grx3 and Grx4. All three proteins display significant sequence homology with proteins found from bacteria to humans. Grx5 is involved in iron/sulfur cluster assembly at the mitochondria, but the function of Grx3 and Grx4 is unknown. Three-dimensional modeling based on known dithiol glutaredoxin structures predicted a thioredoxin fold structure for Grx5. Positionally conserved amino acids in this glutaredoxin family were replaced in Grx5, and the effect on the biological function of the protein has been tested. For all changes studied, there was a correlation between the effects on several different phenotypes: sensitivity to oxidants, constitutive protein oxidation, ability for respiratory growth, auxotrophy for a number of amino acids, and iron accumulation. Cys(60) and Gly(61) are essential for Grx5 function, whereas other single or double substitutions in the same region had no phenotypic effects. Gly(115) and Gly(116) could be important for the formation of a glutathione cleft on the Grx5 surface, in contrast to adjacent Cys(117). Substitution of Phe(50) alters the beta-sheet in the thioredoxin fold structure and inhibits Grx5 function. None of the substitutions tested affect the structure at a significant enough level to reduce protein stability.