Dissemination of blaVIM in Greece at the peak of the epidemic of 2005-2006: clonal expansion of Klebsiella pneumoniae clonal complex 147.

VIM-producing Klebsiella pneumoniae (n = 21) isolated from ten Greek hospitals during 2003-2007 were characterized with multilocus sequence typing (MLST), semi-automated repetitive sequence-based PCR (rep-PCR) (Diversilab), plasmid replicon typing, serotyping and screening for multiple resistance determinants. The isolates were selected to represent different strain clusters (defined by >80% similarity) according to pulsed-field gel electrophoresis. MLST identified three major clonal complexes (CCs); CC147 (n = 8), CC18 (n = 5) and CC14 (n = 3). Plasmid replicon typing showed that IncA/C and/or IncFIIK replicons were detected among isolates in each of the major CCs. Good concordance was observed between semi-automated-rep PCR genotyping and MLST.

Characterization of pKP1433, a novel KPC-2-encoding plasmid from Klebsiella pneumoniae sequence type 340.

The nucleotide sequence of pKP1433 (55,417 bp), a blaKPC-2-carrying plasmid from Klebsiella pneumoniae sequence type 340, was determined. pKP1433 displayed extensive sequence and structural similarities with the IncN plasmids possessing the KPC-2-encoding Tn4401b isoform. However, the replication, partitioning, and stability of pKP1433 were determined by sequences related to diverse non-IncN plasmids.

Characterization of pKP1780, a novel IncR plasmid from the emerging Klebsiella pneumoniae ST147, encoding the VIM-1 metallo-ß-lactamase.

OBJECTIVES: To determine the complete nucleotide sequence of the VIM-1-encoding plasmid pKP1780 from Klebsiella pneumoniae ST147 representing a distinct group of IncR replicons. METHODS: The plasmid pKP1780 was from a K. pneumoniae clinical strain (KP-1780) isolated in Greece in 2009. Plasmid DNA was extracted from an Escherichia coli DH5α transformant and sequenced using the 454 Genome Sequencer GS FLX procedure on a standard fragment DNA library. Contig gaps were filled by sequencing of PCR-produced fragments. Annotation and comparative analysis were performed using software available on the Internet. RESULTS: Plasmid pKP1780 (49 770 bp) consisted of an IncR-related sequence (12 083 bp) including replication and stability systems, and a multidrug resistance (MDR) mosaic region (37 687 bp). blaVIM-1 along with the aacA7, dfrA1 and aadA1 cassettes comprised the variable region of an integron similar to In-e541 from pNL194. The mosaic structure also included the strA, strB, aphA1 and mphA resistance genes as well as intact (n = 10) or defective (n = 3) insertion sequences and fragments of various transposons. CONCLUSIONS: The mosaic structure of pKP1780 exhibited high similarity with the acquired region of the IncN plasmid pNL194, indicating the acquisition of the VIM-1-encoding MDR region from pNL194 by an IncR-type plasmid.

Antimicrobial activity of prulifloxacin in comparison with other fluoroquinolones against community-acquired urinary and respiratory pathogens isolated in Greece.

Prulifloxacin, the prodrug of ulifloxacin, is a broad-spectrum fluoroquinolone rather recently introduced in certain European countries. We compared the antimicrobial potency of ulifloxacin with that of other fluoroquinolones against common urinary and respiratory bacterial pathogens. The microbial isolates were prospectively collected between January 2007 and May 2008 from patients with community-acquired infections in Greece. Minimum inhibitory concentrations (MICs) were determined for ciprofloxacin, levofloxacin, moxifloxacin (for respiratory isolates only), and ulifloxacin using the E-test method. The binary logarithms of the MICs [log2(MICs)] were compared by using the Wilcoxon signed-ranks test. A total of 409 isolates were studied. Ulifloxacin had the lowest geometric mean MIC for the 161 Escherichia coli, 59 Proteus mirabilis, and 22 Staphylococcus saprophyticus urinary isolates, the second lowest geometric mean MIC for the 38 Streptococcus pyogenes respiratory isolates (after moxifloxacin), and the third lowest geometric mean MIC for the 114 Haemophilus influenzae and the 15 Moraxella catarrhalis respiratory isolates (after ciprofloxacin and moxifloxacin). Compared with levofloxacin, ulifloxacin had lower log2(MICs) against E. coli (p < 0.001), P. mirabilis (p < 0.001), S. saprophyticus (p < 0.001), and S. pyogenes (p < 0.001). Compared with ciprofloxacin, ulifloxacin had lower log2(MICs) against P. mirabilis (p < 0.001), S. saprophyticus (p = 0.008), and S. pyogenes (p < 0.001), but higher log2(MICs) against H. influenzae (p < 0.001) and M. catarrhalis (p = 0.001). In comparison with other clinically relevant fluoroquinolones, ulifloxacin had the most potent antimicrobial activity against the community-acquired urinary isolates studied and very good activity against the respiratory isolates.

Characterization of metallo-beta-lactamase VIM-27, an A57S mutant of VIM-1 associated with Klebsiella pneumoniae ST147.

VIM-27 metallo-ß-lactamase, an Ala(57) → Ser variant of VIM-1, was identified in three Klebsiella pneumoniae isolates belonging to sequence type 147. bla(VIM-27) was part of a class 1 integron carried by non-self-transferable plasmids. Kinetic parameters and MIC determinations indicated that VIM-27 hydrolyzed most ß-lactams, especially imipenem and cefoxitin, less effectively than VIM-1.

Panton-Valentine leukocidin-positive methicillin-resistant Staphylococcus aureus outbreak among healthcare workers in a long-term care facility.

BACKGROUND: We investigated an outbreak of community-acquired methicillin-resistant Staphylococcus aureus (MRSA) infections that occurred among healthcare workers (HCWs) but not among residents of a long-term care facility (LTCF). METHODS: Cases of S. aureus infection were sought by reviewing the medical records of residents and HCWs. In order to identify risk factors for the development of an S. aureus infection, an unmatched case-control study was conducted. Cases were all HCWs with a clinically compatible S. aureus infection; controls were HCWs with no history of a clinically compatible S. aureus infection. Cases and controls were interviewed and anterior nasal swabs were collected. RESULTS: Over a period of 14 months, a total of eight cases were identified among practice nurses, giving an attack rate of 10% for this category of profession. All isolates were identified as MRSA Panton-Valentine leukocidin (PVL)-producing SCCmec type IV. By multivariate analysis, working in a specific zone and being a practice nurse were found to be statistically significant risk factors for infection. CONCLUSIONS: The current outbreak indicates that HCWs may serve as vehicles for the entry of PVL-positive MRSA strains from the community into LTCFs, and that deficient hygiene practices and unrecognized carriage may facilitate spread. Given the increasing prevalence of PVL-positive MRSA infections worldwide, guidelines for the eradication of PVL-positive MRSA carriage within closed communities should be established and efforts to obtain cultures from compatible infections should be made.

Outbreak of infections due to KPC-2-producing Klebsiella pneumoniae in a hospital in Crete (Greece).

Starting in May 2007, an ongoing outbreak of infections due to carbapenem resistant KPC-2-producing Klebsiella pneumoniae occurred in a tertiary care hospital in Crete (Greece). The outbreak involved 22 patients, none of whom had travelled in a country with known high prevalence of such isolates. KPC-producing K. pneumoniae strains were mainly isolated from patients admitted in the Intensive Care Unit, on mechanical ventilation, with prolonged hospitalization, prolonged administration of antibiotics, and prolonged administration of carbapenems. Clinical diagnoses were: pneumonia (62% of cases), surgical site infection (19%), bacteremia (9.5%), urinary tract infection (4.7%), and peritonitis (4.7%). Overall, 61 KPC-producing K. pneumoniae isolates were recovered, mainly from the respiratory tract (59.1%), catheter tip (22.7%), surgical site (18.2%), and blood (18.2%). Among 16 patients for whom therapeutic data were available, 14 (87.5%) were treated with a combination of colistin and/or tigecycline and/or garamycin. Clinical failure was noted in 22.2% of 18 patients available for assessment of clinical outcome, and microbiologic failure in 87.5% of 8 patients available for assessment of microbiologic outcome. In conclusion, an outbreak of KPC-producing K. pneumoniae infections has occurred in a tertiary care hospital in Greece, with significant associated morbidity and mortality. Prospective studies are required to evaluate the available therapeutic options for these infections. Our efforts should focus on rational use of available antibiotics, enhancement of infection control measures, and implementation of active antibiotic resistance surveillance.

Trends in antimicrobial resistance of Acinetobacter baumannii clinical isolates from hospitalised patients in Greece and treatment implications.

This study analysed the proportions of Acinetobacter baumannii isolates resistant to various antibiotics that were recovered from patients hospitalised in Greek hospitals between 1996 and 2006. The microbiological data were derived from the ongoing WHONET Greek System for the Surveillance of Antibiotic Resistance. There were increases in the proportions of A. baumannii isolates resistant to imipenem from patients hospitalised in intensive care units, medical wards and surgical wards during the study period from 0% to 91%, 8% to 71%, and 5% to 71%, respectively.

Sources of diversity of carbapenem resistance levels in Klebsiella pneumoniae carrying blaVIM-1.

OBJECTIVES: To elucidate the mechanisms responsible for the diversity of beta-lactam resistance phenotypes among isolates of a VIM-1-producing Klebsiella pneumoniae (VPKP) strain that is endemic in Greek hospitals. METHODS: Five VPKP clinical isolates were studied. MICs of beta-lactams were determined by agar dilution. PFGE of XbaI-digested genomic DNA was used for typing. Profiles of outer membrane proteins (OMPs) were determined by SDS-PAGE. Selected isolates were transformed with a plasmid encoding the Omp36K porin. beta-Lactamase activities were analysed by IEF and imipenem hydrolysis was assessed by spectrophotometry. VIM-1-encoding, self-transmissible plasmids were characterized by replicon typing, RFLP and hybridization with bla(VIM)- and IS26-specific probes. Characterization of integrons was performed by PCR, cloning and sequencing. RESULTS: Isolates exhibited highly similar PFGE patterns. Imipenem MICs were 2, 4, 16, 32 and 64 mg/L. The isolate with the highest imipenem MIC (Vipm-64) lacked a 36 kDa OMP. Expression of a cloned OmpK36 in this isolate reduced the imipenem MIC to susceptibility levels. Imipenem-hydrolysing activity was significantly higher in Vipm-16 as compared with the other isolates that expressed similar amounts of VIM-1. All isolates transferred beta-lactam resistance to Escherichia coli through conjugative, IncN plasmids that exhibited differences in the RFLP and hybridization patterns with bla(VIM)- and IS26-specific probes. The Vipm-16 plasmid, mediating the higher imipenem MICs among transconjugants, carried two copies of bla(VIM-1). Cloning and sequencing showed In-e541-like integrons truncated at the 5'CS by insertion of IS26 elements at two different positions. CONCLUSIONS: A VIM-1-producing strain of K. pneumoniae has evolved through OMP alterations and rearrangements in the bla(VIM-1)-carrying plasmid probably mediated by IS26, generating isolates with imipenem MICs ranging from susceptibility to resistance.

Emergence of Proteus mirabilis carrying the bla metallo-beta-lactamase gene.

Seven genetically related Proteus mirabilis clinical isolates from a hospital in Thessaloniki, Greece, exhibited decreased susceptibility to imipenem and carried a bla(VIM-1) metallo-beta-lactamase gene. PCR mapping revealed that bla(VIM-1) was part of a class 1 integron that was probably located in the chromosome and also included the aacA7, dhfr and aadA genes. This is the first description of the bla(VIM-1) metallo-beta-lactamase gene in P. mirabilis.

Transferable DHA-1 cephalosporinase in Escherichia coli.

Three Escherichia coli isolates resistant to third-generation cephalosporins but negative for extended-spectrum beta-lactamase production were isolated from hospitalised patients in Zagreb, Croatia, during June 2003 to February 2004. Resistance was due to the inducible production of a DHA-1 cephalosporinase. Each isolate contained an integron-associated bla(DHA-1)-ampR sequence carried by similar-sized plasmids, of which one was self-transferable. Serotyping and polymerase chain reaction typing using ERIC2 primer indicated that the isolates were distinct. This is the first description of DHA beta-lactamase production in E. coli.

Repeated occurrence of diverse extended-spectrum beta-lactamases in minor serotypes of food-borne Salmonella enterica subsp. enterica.

Screening of Greek nontyphoid salmonellae from 2000 to 2002 yielded three extended-spectrum beta-lactamase (ESBL)-producing human isolates. Salmonella enterica serotype Brandenburg harbored a multiresistant SHV-5 gene-carrying plasmid. S. enterica serotype Blockley and S. enterica serotype Hadar harbored a TEM-52 gene-carrying plasmid. An S. enterica serotype Virchow strain producing plasmid-mediated CTX-M-32 was isolated twice from poultry end products. All ESBL plasmids were self-transferable and carried by clones currently common in Greece.

Discrepancies and interpretation problems in susceptibility testing of VIM-1-producing Klebsiella pneumoniae isolates.

Susceptibilities to beta-lactam antibiotics of five VIM-1-producing Klebsiella pneumoniae isolates were determined by broth microdilution, Etest, disk diffusion, and the automated systems Vitek 2, Phoenix, and MicroScan. Significant discrepancies were observed in the determination of susceptibility to imipenem and meropenem. Interpretation problems by the automated systems were also noted.

CMY-13, a novel inducible cephalosporinase encoded by an Escherichia coli plasmid.

An IncN plasmid (p541) from Escherichia coli carried a Citrobacter freundii-derived sequence of 4,252 bp which included an ampC-ampR region and was bound by two directly repeated IS26 elements. ampC encoded a novel cephalosporinase (CMY-13) with activity similar to that of CMY-2. AmpR was likely functional as indicated in induction experiments.

Quinolone resistance among Escherichia coli strains from community-acquired urinary tract infections in Greece.

Susceptibility data for 10 049 Escherichia coli isolates derived from community-acquired urinary tract infections in Greece during the period January 2000 to June 2002 indicated 8.1% resistance to nalidixic acid and 36% resistance to ciprofloxacin. In a sample of 170 E. coli isolates, mutations in gyrA (25 isolates) and parC (15 isolates) were consistent with the levels of resistance to quinolones. Previous exposure to quinolones and underlying chronic disease were independent risk factors for infection by quinolone-resistant E. coli strains.

In vitro activity of telithromycin (HMR 3647) against Greek Streptococcus pyogenes and Streptococcus pneumoniae clinical isolates with different macrolide susceptibilities.

The susceptibilities to macrolides and telithromycin of 161 Streptococcus pyogenes and 145 Streptococcus pyogenes strains, consecutively isolated from five Greek hospitals, were determined by Etest. Moreover, mechanisms of resistance to macrolides were phenotypically and genetically determined by double disk induction test and PCR, respectively. Of the S. pneumoniae and S. pyogenes isolates, 42.8% and 30.8%, respectively, were found to be resistant to erythromycin. Of the erythromycin-resistant S. pneumoniae and S. pyogenes isolates, 57.5% and 59.5%, respectively, displayed the M phenotype and harbored the mefA/E gene. Telithromycin was found to be more active than both erythromycin and clarithromycin against both species, with considerably lower MIC50 and MIC90 values.

VIM-1 Metallo-beta-lactamase-producing Klebsiella pneumoniae strains in Greek hospitals.

Seventeen Klebsiella pneumoniae clinical isolates carrying the bla(VIM-1) metallo-beta-lactamase gene were collected in the intensive care units of three hospitals in Athens, Greece, in 2002. They exhibited various carbapenem resistance levels (Etest MICs of imipenem ranged from 4 to 32 microg/ml). All isolates gave positive results by the imipenem-EDTA synergy Etest. The isolates were classified into four main types by pulsed-field gel electrophoresis; the majority of the isolates (5 and 10 isolates) belonged to two types. The bla(VIM-1) gene cassette was part of the variable region of a class 1 integron that also included aac6, dhfrI, and aadA. This structure was carried by transferable plasmids.

Extended-spectrum beta-lactamase types in Klebsiella pneumoniae and Escherichia coli in two Greek hospitals.

Seventy-nine Klebsiella pneumoniae and 124 Escherichia coli clinical strains, isolated consecutively during August-October 2001 in two Greek hospitals, were examined for production of extended-spectrum beta-lactamases (ESBLs). Seventy-one (35%) isolates (46 K. pneumoniae and 25 E. coli) were ESBL-positive by phenotypic methods. Isoelectric focusing of beta-lactamases and PCR assays for bla genes showed that SHV-5-type ESBLs were the most frequent (45 isolates, 22%) followed by CTX-M (24 isolates, 12%) and IBC (three isolates, 1.5%). The latter two ESBL types may have been established recently in this setting.

Spread of integron-associated VIM-type metallo-beta-lactamase genes among imipenem-nonsusceptible Pseudomonas aeruginosa strains in Greek hospitals.

Fifty-eight imipenem-nonsusceptible (MIC >or= 8 microg/ml) Pseudomonas aeruginosa strains isolated during May 2001 in 15 Greek hospitals were studied. Thirty-six isolates derived from nine hospitals carried VIM-type metallo-beta-lactamase genes, as found by PCR. In 34 isolates, bla(VIM) was associated with class 1 integrons of various sizes. DNA sequencing indicated the presence of bla(VIM-2) gene cassettes in a variety of integron structures. Random amplified polymorphic DNA typing suggested diversity of the bla(VIM)-positive strains. Synergy between 2-mercaptoacetic acid and imipenem indicated carbapenemase activity in 26 bla(VIM)-positive strains.