Nicholas J. Legakis

VIM-1 Metallo-β-Lactamase-Producing Klebsiella pneumoniae Strains in Greek Hospitals

ABSTRACT Seventeen Klebsiella pneumoniae clinical isolates carrying the blaVIM-1 metallo-β-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 μg/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 blaVIM-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.

Frequent detection of cytomegalovirus in the intestine of patients with inflammatory bowel disease.

Background: Although a growing number of reports have described inflammatory bowel disease (IBD) complicated with cytomegalovirus (CMV) infection, there are limited molecular studies that investigate CMV genome in intestinal sections of patients with IBD. Methods: A cross‐sectional prospective study was conducted between September 2000 and June 2003 in a cohort of 85 patients diagnosed with IBD (58 with ulcerative colitis and 27 with Crohns disease) in two adult gastrointestinal referral centers in Athens, Greece. Prevalence of CMV infection was estimated by pathologic studies in intestinal sections and by molecular assays in blood and intestinal tissue samples and compared with a control group of 42 individuals with noninflammatory disease. Results: Immunohistochemical staining showed CMV antigen in 10 IBD patients (7 with ulcerative colitis; 9 with severe disease), whereas CMV antigen was not detected in any of the controls. CMV genome in both the intestinal tissue and blood was found by polymerase chain reaction in 23 (27.1%) of the total IBD patients, in 18 (31.0%) of those with ulcerative colitis, and in 5 (18.5%) of those with Crohns disease. In addition, five (5.9%) IBD patients (2 with ulcerative colitis and 3 with Crohns disease) had detectable CMV genome in their intestinal samples but not in their blood. In the control group, five (11.9%) individuals had detectable CMV genome in their blood, but only one (2.2%) in his intestine. Conclusion: Patients with ulcerative colitis had more often detectable CMV genome in their blood as well as in their intestinal tissue samples as compared with controls (P = 0.022 and P < 0.0001, respectively). However, patients with Crohns disease had more often detectable CMV genome only in their intestinal tissue samples as compared with controls (P = 0.001). Detection of CMV genome in blood or intestinal tissue was significantly associated with short duration of IBD (P = 0.0088 and 0.04, respectively) but not with age, sex, severity of the disease, activity at colonoscopy, pancolitis, administration of a specific treatment, and surgery. In this cross‐sectional prospective study, detection of CMV genome or antigen in the intestine was commonly associated with IBD.

First report of Cryptococcus laurentii meningitis and a fatal case of Cryptococcus albidus cryptococcaemia in AIDS patients.

We report the first case of Cryptococcus laurentii meningitis and a rare case of Cryptococcus albidus cryptococcaemia in AIDS patients. Both infections were treated with amphotericin B and flucytosine. The C. laurentii meningitis was controlled after 2 weeks of treatment with no evidence of infection 20 months later. The patient with C. albidus cryptococcaemia, despite the amphotericin B/flucytosine combination therapy, died on the 14th day of treatment. The minimum inhibitory concentrations (MICs) for C. laurentii, as determined by Etest on RPMI 1640 agar, were 0.25 microg ml(-1) of amphotericin B, 1.25 microg ml(-1) flucytosine, 4 microg ml(-1) fluconazole, 0.50 microg ml(-1) itraconazole and 1.0 microg ml(-1) of ketoconazole. The MIC of amphotericin B for C. albidus was 0.5 microg ml(-1), flucytosine 1.25 microg ml(-1), fluzonazole 4 microg ml(-1), itraconazole 0.5 microg ml(-1) and ketonazole 0.25 microg ml(-1). The agreement of the amphotericin B MIC values obtained in antibiotic medium 3 by the broth microdilution method, with those obtained on casitone medium by Etest, was within a two-dilution range for both isolates. C. laurentii may cause meningitis and may also involve the lungs in AIDS patients.

VIM-1 Metallo-β-lactamase in Acinetobacter baumannii

In 2004 and 2005, 5 metallo-β-lactamase (MBL)-positive Acinetobacter baumannii isolates were found in 2 Greek hospitals. Isolates were unrelated and carried blaVIM-1 in a class 1 integron; blaOXA-51- and blaOXA-58-like carbapenemase genes were also detected. VIM-1 MBL in Acinetobacter spp. causes concern, given the increasing resistance of this species.

SPREAD OF INTEGRON-ASSOCIATED VIM-TYPE METALLO-BETA-LACTAMASE GENES AMONG IMIPENEM-NONSUSCEPTIBLE PSEUDOMONAS AERUGINOSA STRAINS IN GREEK HOSPITALS

ABSTRACT Fifty-eight imipenem-nonsusceptible (MIC ≥ 8 μg/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-β-lactamase genes, as found by PCR. In 34 isolates, blaVIM was associated with class 1 integrons of various sizes. DNA sequencing indicated the presence of blaVIM-2 gene cassettes in a variety of integron structures. Random amplified polymorphic DNA typing suggested diversity of the blaVIM-positive strains. Synergy between 2-mercaptoacetic acid and imipenem indicated carbapenemase activity in 26 blaVIM-positive strains.

Discrepancies and Interpretation Problems in Susceptibility Testing of VIM-1-Producing Klebsiella pneumoniae Isolates

ABSTRACT Susceptibilities to β-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.

Repeated Occurrence of Diverse Extended-Spectrum β-Lactamases in Minor Serotypes of Food-Borne Salmonella enterica subsp. enterica

ABSTRACT Screening of Greek nontyphoid salmonellae from 2000 to 2002 yielded three extended-spectrum β-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.

Sequence of pNL194, a 79.3-Kilobase IncN plasmid carrying the blaVIM-1 metallo-β-lactamase gene in Klebsiella pneumoniae.

ABSTRACT The nucleotide sequence of pNL194, a VIM-1-encoding plasmid, is described in this study. pNL194 (79,307 bp) comprised an IncN-characteristic segment (38,940 bp) and a mosaic structure (40,367 bp) including blaVIM-1, aacA7, aadA1, aadA2, dfrA1, dfrA12, aphA1, strA, strB, and sul1. Tn1000 or Tn5501 insertion within fipA probably facilitated recruitment of additional mobile elements carrying resistance genes.

VIM-12, a Novel Plasmid-Mediated Metallo-β-Lactamase from Klebsiella pneumoniae That Resembles a VIM-1/VIM-2 Hybrid

ABSTRACT A transferable plasmid from Klebsiella pneumoniae carried a class 1 integron containing blaVIM-12, a novel blaVIM-type gene, flanked by two copies of aacA7. blaVIM-12 was clustered between blaVIM-1 and blaVIM-2 and differed from blaVIM-1 by 18 nucleotides that were all located at the 3′ end and matched the corresponding nucleotides in blaVIM-2. The blaVIM-12-associated 59-base element was identical to that described in blaVIM-2 alleles.

Rapid extraction of fungal DNA from clinical samples for PCR amplification

A hexadecyltrimethylammonium bromide (CTAB) method for isolating fungal DNA from clinical samples, suitable for PCR amplification is described. Yeast and filamentous fungi DNA from clinical samples was amplified with primers complementary to the genes coding for rRNA, amplifying a 105 bp fragment and internal transcribed spacer primers amplifying fragments between 242 and 622 bp. The level of sensitivity was 10 +/- 5 yeast and 28 Aspergillus fumigatus CFU ml-1 of biological fluid.