Efi Petinaki

A T2504A mutation in the 23S rRNA gene responsible for high-level resistance to linezolid of Staphylococcus epidermidis

Department of Microbiology, Medical School, University of Thessalia, Larissa, Greece; Department of Molecular Microbiology, Institute of BioMedical Research and Technology, Larissa, Greece; Department of Biochemistry and Biotechnology, University of Thessalia, Larissa, Greece; Department of Microbiology, ‘Sismanoglion’ General Hospital of Athens, Athens, Greece; Department of Microbiology, School of Medicine, University of Patras, Patras, Greece

Whole-blood interferon-γ assay for the diagnosis of tuberculosis infection in an unselected Greek population

Background and objective:  Although QuantiFERON‐TB Gold (QFT‐G) has been approved for the diagnosis of latent tuberculosis infection (LTBI), there are limited data regarding its performance in routine clinical practice. The aim of this study was to compare QFT‐G ‘In Tube’ results, based on stimulation with Mycobacterium tuberculosis‐specific antigens, with tuberculin skin test (TST) results in an unselected hospital‐based Greek population.

Linezolid-resistant Staphylococcus cohnii, Greece.

To the Editor: Since 2003, linezolid has typically been used to treat infections caused by multidrug-resistant gram-positive cocci such as vancomycin-resistant Enterococcus faecium and methicillin-resistant Staphylococcus aureus (1). In Greece, a major problem is nosocomial dissemination of vancomycin-resistant enterococci. Use of linezolid for the treatment of such infections led to the emergence of linezolid–vancomycin resistant E. faecium; however, linezolid resistance of staphylococci is still relatively low in this country (2). We describe an outbreak caused by a linezolid-resistant S. cohnii in an intensive care unit (ICU) in Greece. From July through October 2007, nonrepetitive coagulase-negative staphylococci that exhibited resistance to linezolid were isolated from blood cultures from 4 separate patients hospitalized in the ICU at Sismanoglion General Hospital of Athens, a 450-bed tertiary care hospital. The ICU is a 10-bed, level II unit, comprising 2 rooms with 1 bed each and 2 rooms with 4 beds each. Each isolate was recovered in 2 of 2 blood culture sets per patient, indicating true bacteremia. The demographic and clinical information for the patients is described in the Table. The mean duration of time preceding linezolid therapy was 22 days. Table Clinical characteristics of 4 patients from whom linezolid-resistant Staphylococcus cohnii was isolated, Greece, 2007* Isolates were first identified to the species level by using an API Staph system (bioMerieux, la Balme les Grottes, France) and a molecular method based on the tuf gene followed by sequencing analysis (3). Susceptibility testing for various antimicrobial agents was performed by disk diffusion and using Clinical Laboratory Standards Institute criteria; susceptibilities were interpreted according to Institute guidelines (4). In addition, MICs to oxacillin, vancomycin, teicoplanin, quinupristin-dalfopristin, linezolid, daptomycin, and tigecycline were determined by Etest (AB Biodisk, Solna, Sweden) according to manufacturer’s instructions. Resistance genes mecA, vat, vga, erm, aac(6´)-Ie+aph(2″), ant(4′)-Ia, and aph(3′)-IIIa, as markers for resistance to β-lactams, dalfopristin, macrolides, and aminoglycosides, were identified by PCR as previously reported (5,6). The presence of G2576T in domain V of the 23S rRNA, which is mainly associated with linezolid resistance in clinical isolates, was detected by using PCR and digestion of the product with NheI (2). The number of mutated versus nonmutated alleles was determined as described by Pillai et al. (7). In addition, isolates were examined for the presence of the cfr gene, which was found to be correlated with linezolid resistance in some coagulase-negative staphylococci and for mutations of ribosomal protein L4, L22 genes (8,9). Clonality of isolates was assessed by pulsed-field gel electrophoresis (PFGE) after digestion of chromosomal DNA with SmaI (2). The molecular method identified the isolates as S. cohnii subsp. ureolyticus. The API Staph system has correctly identified 2 of them; the remaining 2 isolates were falsely characterized as S. xylosus. According to disc diffusion test results, the isolates were resistant to cefoxitin, oxacillin, penicillin, rifampin, quinupristin-dalfopristin, erythromycin, clindamycin, fusidic acid, tobramycin, gentamicin, and linezolid. MICs were linezolid 32, oxacillin 256, quinupristin-dalfopristin 8, vancomycin 2, teicoplanin 2, tigecycline 0.125, and daptomycin 0.5 mg/L. Molecular methods detected the following resistance genes: mecA, ermA, aac(6´)-Ie+aph(2″), and aph(3´)-IIIa. The isolates, despite their resistance to streptogramins, were negative for vat and vgaA genes. In addition, all isolates carried the G2576T mutation and had 4 of 5 mutated alleles. No isolate carried the cfr gene or any mutation on ribosomal protein L4 and L22 genes. PFGE results indicated that all isolates were clonally related, belonging to the same clone. Outbreaks caused by linezolid-resistant staphylococci are rare worldwide (10); in Sismanoglion Hospital, before the outbreak period, no linezolid-resistant staphylococci and enterococci had been isolated. However, in the ICU, a statistically significant increase in the use of linezolid was observed in 2004 and in 2007 (0.58 vs. 1.34 defined daily doses/100 patient-days, respectively); heavy use of linezolid may have created substantial selection pressure in favor of the linezolid-resistant isolates. The 4 patients were treated in the same room by the same personnel; thus, a potential explanation for this outbreak is patient-to-patient transmission of linezolid-resistant strains on the hands of healthcare personnel. However, cultures of ICU personnel (nasal cavity and hands) grew only methicillin-resistant S. aureus and methicilllin-resistant S. epidermidis. In addition, environmental samples taken from the beds and the equipment of these patients were negative for S. cohnii. Strict control measures were taken (e.g., isolation of infected patients, increased environmental cleaning, and reinforcement of proper glove and gown use and hand disinfection with alcohol gel), and the outbreak strain was not recovered from other patients in the ICU or in other departments of the hospital after the initial outbreak. In conclusion, to avoid spread of staphylococcal resistance in ICUs, measures such as hand hygiene and adequate central venous catheter handling should be taken, and policies regarding antimicrobial drug use must be applied.

The first case of Staphylococcus aureus ST398 causing bacteremia in an immunocompromised patient in Greece

We describe a case of catheter-related bloodstream infection, in a patient with colon cancer, caused by a methicillin-sensitive Staphylococcus aureus strain, nontypeable by pulsed field gel electrophoresis of SmaI macrorestriction fragment analysis, belonging to ST398. The patient recovered after daptomycin therapy. This is the first report that documents the emergence of ST398 in Greece.

Direct detection of Clostridium sordellii in pleural fluid of a patient with pneumonic empyema by a broad-range 16S rRNA PCR

We report the case of a 56-y-old male admitted with a left-sided post-pneumonic empyema. Clostridium sordellii DNA was directly detected in its pleural fluid by a broad-range 16S rRNA PCR, after 24 h of specimen collection. This is the third case of pleural infection caused by C. sordellii in the literature.