Antigoni Foka

Biofilm development by clinical isolates of Staphylococcus spp. from retrieved orthopedic prostheses.

Background Biofilms are considered the key factor in the development of implant-related infections. However, only a few reports have dealt with the ability of organisms isolated from such infections to develop biofilms in vitro. Methods We evaluated different phenotypic techniques (2 microtiter plate assays and confocal laser scanning microscopy (CLSM) and genotypic techniques (detection of the ica operon) related to biofilm development by clinical isolates of Staphylococcus spp. Results All 26 strains tested (from 23 specimens) were biofilm producers. Stepanovic test detected biofilm formation in 85% of the strains, microtiter plate assay in 65%, and CLSM in 39%. The ica operon was detected in 73% of all strains (all 13 S. aureus strains and 6 of the 13 coagulase-negative Staphylococcus strains). 7 ica-negative strains were biofilm-positive by phenotypic methods. Interpretation The detection of ica genes could not be related to the phenotypic ability of the strains to develop a biofilm in vitro, so both studies (genetic and phenotypic) are required for a better evaluation of the biofilm-producing ability of clinical strains of Staphylococcus isolated from orthopedic infections.

Absolute and relative real-time PCR in the quantification of tst gene expression among methicillin-resistant Staphylococcus aureus: evaluation by two mathematical models

Aim:  Absolute and relative quantitative real‐time reverse transcriptase polymerase chain reaction (RT‐PCR) by the use of two mathematical models were applied in order to study the expression of tst gene encoding the toxic shock syndrome toxin‐1 (TSST‐1), among methicillin‐resistant Staphylococcus aureus (MRSA).

Emergence of a new clone carrying Panton-Valentine leukocidin genes and staphylococcal cassette chromosome mec type V among methicillin-resistant Staphylococcus aureus in Greece

Clonal analysis and PCR screening for the presence of Panton-Valentine leukocidin (PVL) genes was performed among 694 methicillin-resistant Staphylococcus aureus (MRSA) cases collected during a 2-y period in Greece. The detection rate of PVL-positive MRSA is high, both in the community and in hospital. Clonal analysis revealed the predominance among the PVL-positive strains of the clonal complex CC80 (ST80-IV) and the emergence of ST377 clone carrying agr1 allele and SCCmec type V.

Clinical and microbiological profile of persistent coagulase-negative staphylococcal bacteraemia in neonates

An atypical pattern of coagulase-negative staphylococcal (CoNS) sepsis, characterized by persistence despite aggressive antibiotic therapy, has been described in neonates cared for in neonatal intensive-care units. Our aim was to analyse the clinical, microbiological and molecular determinants of this persistent CoNS bacteraemia. Neonates with late-onset CoNS bacteraemia were studied for a 2-year period. Demographic, clinical, laboratory, microbiological and molecular data were compared between neonates with persistent (≥3 consecutive positive blood cultures) and non-persistent CoNS bacteraemia. Twenty-nine infants with persistent and 43 with non-persistent bacteraemia were identified, with no significant differences regarding demographic and clinical characteristics between the two groups. Of a total of 170 CoNS isolates, 80 showed biofilm production (54 persistent and 26 non-persistent; p 0.013), whereas 127 were positive for the icaA and icaD genes (74 persistent and 53 non-persistent; p 0.598). Sixty ica-positive isolates did not produce slime, whereas 13 ica-negative isolates showed biofilm production. Endotracheal intubation and the presence of central vascular catheters were significant risk factors for persistent bacteraemia, but, in a logistic regression model, only biofilm production was significantly related to the persistent form of the disease (p 0.005). In this study, persistent CoNS sepsis in neonates requiring intensive care was not related to most of the known clinical risk factors, and it was associated with severe thrombocytopenia. Isolates associated with persistent bacteraemia were more likely to produce biofilm, independently of the presence of the ica operon.

Coagulase-negative staphylococcal bloodstream and prosthetic-device-associated infections: the role of biofilm formation and distribution of adhesin and toxin genes

Coagulase-negative staphylococci (CNS), especially Staphylococcus epidermidis and Staphylococcus haemolyticus, have emerged as opportunistic pathogens in immunocompromised patients and those with indwelling medical devices. In this study, CNS recovered from patients with bloodstream infections (BSIs) or prosthetic-device-associated infections (PDAIs) were compared in terms of biofilm formation, antimicrobial resistance, clonal distribution, and carriage of adhesin and toxin genes. A total of 226 CNS isolates (168 S. epidermidis and 58 S. haemolyticus) recovered from hospital inpatients with BSIs (100 isolates) or PDAIs (126 isolates) were tested for biofilm formation, antimicrobial susceptibility, and mecA, ica operon, adhesin (aap, bap, fnbA, atlE, fbe) and toxin (tst, sea, sec) genes. The selected CNS were classified into pulsotypes by PFGE and assigned to sequence types by multilocus sequence typing. In total, 106/226 isolates (46.9%) produced biofilm, whereas 150 (66.4%) carried the ica operon. Most isolates carried mecA and were multidrug resistant (90.7%). CNS recovered from BSIs were significantly more likely to produce biofilm (P=0.003), be resistant to antimicrobials and carry mecA (P<0.001), as compared with isolates derived from PDAIs. CNS from PDAIs were more likely to carry the aap and bap genes (P=0.006 and P=0.045, respectively). No significant differences in the carriage of toxin genes were identified (P>0.05). Although PFGE revealed genetic diversity, especially among S. epidermidis, analysis of representative strains from the main PFGE types by multilocus sequence typing revealed three major clones (ST2, ST5 and ST16). A clonal relationship was found with respect to antimicrobial susceptibility and ica and aap gene carriage, reinforcing the premise of clonal expansion in hospital settings. The results of this study suggest that the pathogenesis of BSIs is associated with biofilm formation and high-level antimicrobial resistance, whereas PDAIs are related to the adhesion capabilities of S. epidermidis and S. haemolyticus strains.

Aminoglycoside-resistant staphylococci in Greece: prevalence and resistance mechanisms

Department of Biomathematics, School of Medicine, University of Thessaly, Larissa Corresponding author: Dr. E. Petinaki, MD Associate Professor, Department of Microbiology, Medic al School, u iopolis , Larissa, Greece Tel: +30 -241350 2517 Fax: +30 -241350 2535 e-mail: petinaki@ med.uth.gr peer-00657696, version 1 - 9 Jan 2012

Comparison of two commercial methods with PCR restriction fragment length polymorphism of the tuf gene in the identification of coagulase-negative staphylococci

Aims:  Two commercial methods for the identification of coagulase‐negative staphylococci (CNS) were compared with the restriction fragment length polymorphism (RFLP) of the amplified tuf gene, which served as the reference method.

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.

Dissemination of Methicillin-Susceptible CC398 Staphylococcus aureus strains in a rural Greek area.

A large collection of Staphylococcus aureus including a. 745 clinically significant isolates that were consecutively recovered from human infections during 2012–2013, b. 19 methicillin-susceptible (MSSA), randomly selected between 2006–2011 from our Staphylococcal Collection, c. 16 human colonizing isolates, and d. 10 strains from colonized animals was investigated for the presence and the molecular characteristics of CC398. The study was conducted in Thessaly, a rural region in Greece. The differentiation of livestock-associated clade from the human clade was based on canSNPs combined with the presence of the φ3 bacteriophage and the tetM, scn, sak, and chp genes. Among the 745 isolates, two MRSA (0.8% of total MRSA) and thirteen MSSA (2.65% of total MSSA) were found to belong to CC398, while, between MSSA of our Staphylococcal Collection, one CC398, isolated in 2010, was detected. One human individual, without prior contact with animals, was found to be colonized by a MSSA CC398. No CC398 was identified among the 10 S. aureus isolated from animals. Based on the molecular markers, the 17 CC398 strains were equally placed in the livestock-associated and in the human clades. This is the first report for the dissemination of S. aureus CC398 among humans in Greece.