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Biomedical subjects

N J Legakis

Publications and source records attributed to N J Legakis.

At least 73 records · Page 4Linked to original sources
PubMed1999

Spread of a Salmonella typhimurium clone resistant to expanded-spectrum cephalosporins in three European countries.

Twelve Salmonella typhimurium strains resistant to broad-spectrum cephalosporins were isolated from cases of gastroenteritis during 1996 to 1998 in Russia, Hungary, and Greece. Resistance was due to the production of CTX-M-type extended-spectrum beta-lactamases encoded by similar 12-kb plasmids. By pulsed-field gel electrophoresis, all strains shared the same chromosomal type. These data suggest that an S. typhimurium clone resistant to broad-spectrum cephalosporins is present in at least three European countries.

Cephalosporin Resistance↗
PubMed1999

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.

Blood↗
PubMed1999

An electronic network for the surveillance of antimicrobial resistance in bacterial nosocomial isolates in Greece. The Greek Network for the Surveillance of Antimicrobial Resistance.

The present article reports an evaluation of the national electronic network for the continuous monitoring of antimicrobial resistance in Greece. The network employs a common electronic code and data format and uses WHONET software. Our four years' experience with the network confirms its practicality. A total of 22 hospitals in Greece are currently using the software, of which 19 participate in the network. Analysis of the information obtained has greatly helped in identifying the main factors responsible for the emergence of antimicrobial resistance in the participating hospitals. The data collected have also helped to identify priorities for further investigation of the genetic and molecular mechanisms responsible for the emergence of resistance and facilitated development of hospital-based empirical therapy of infections. In conclusion, the implementation of national networks for the surveillance of antimicrobial resistance should be regarded as a priority.

Computer Communication Networks↗
PubMed1998

Effect of substitution of Asn for Arg-276 in the cefotaxime-hydrolyzing class A beta-lactamase CTX-M-4.

The effect of substitution of asparagine for arginine at position 276 (Ambler's numbering) on the properties of the extended-spectrum beta-lactamase CTX-M-4 was studied. Compared with CTX-M-4, the mutant beta-lactamase CTX-M-4(R276N) conferred lower levels of resistance to cefotaxime, ceftriaxone and aztreonam while the levels of resistance to penicillins and penicillin-inhibitor combinations were similar. Arg-276-->Asn substitution rendered CTX-M-4 slightly less susceptible to inhibition by clavulanate and tazobactam. It also caused a three-fold reduction in the relative rate of hydrolysis of cefotaxime. These results indicate that Arg-276 in CTX-M-type beta-lactamases may be implicated in hydrolysis of oxyimino-beta-lactams; they do not, however, support the hypothesis that Arg-276 is the functional equivalent of Arg-244 found in other class A beta-lactamases.

Amino Acid Substitution↗
PubMed1998

Two novel plasmid-mediated cefotaxime-hydrolyzing beta-lactamases (CTX-M-5 and CTX-M-6) from Salmonella typhimurium.

Two novel plasmid-mediated beta-lactamases (CTX-M-5 and CTX-M-6) produced by Salmonella typhimurium clinical strains were characterized. The enzymes exhibited a pI of 8.4, hydrolyzed oxyimino-beta-lactams and were susceptible to mechanism-based beta-lactamase inhibitors. The respective bla genes were cloned and sequenced. The deduced amino acid sequences showed a high degree of homology with those of the previously described plasmid class A CTX-M-type enzymes and appeared related to the chromosomal beta-lactamases of Klebsiella oxytoca.

Amino Acid Sequence↗
PubMed1998

Substitution of Arg-244 by Cys or Ser in SHV-1 and SHV-5 beta-lactamases confers resistance to mechanism-based inhibitors and reduces catalytic efficiency of the enzymes.

The conserved residue Arg-244 was substituted by the smaller uncharged amino acids Cys and Ser in SHV-1 and SHV-5 beta-lactamases by a PCR-based site-specific mutagenesis procedure. The mutant beta-lactamases displayed decreased susceptibility to clavulanate and, to a lesser extent, to tazobactam and sulbactam. As shown in comparative MIC determinations, R244C and R244S enzymes retained a residual penicillinase activity while their activity towards cephalosporins was drastically diminished. The respective SHV-5 mutants were unable to hydrolyze oxyimino-beta-lactams except aztreonam. The impaired catalytic activity of the mutant beta-lactamases was mainly due to the lowering of affinity for beta-lactam substrates. The above alterations were more pronounced in the R244C mutants. These results provide information on the mode of involvement of Arg-244 in (a) inactivation by beta-lactamase inhibitors and (b) the proper positioning of beta-lactams in the active site of SHV enzymes.

Amino Acid Substitution↗
PubMed1998

Resistance pattern of Streptococcus pneumoniae in children during a four-year period in Greece.

The resistance pattern of 432 Streptococcus pneumoniae strains isolated from children with various infections over a 4-year period (1992-1995) was determined. The rates of resistance to penicillin, chloramphenicol, tetracycline, trimethoprim-sulfamethoxazole, erythromycin, clindamycin, cefotaxime, and vancomycin were 10%, 2.8%, 4.6%, 4.9%, 4.4%, 2.5%, 0.9%, and 0%, respectively. All strains not susceptible to penicillin were intermediately susceptible to penicillin-(MIC >0.06-< or = 1 microg/ml). Isolates not susceptible to penicillin were encountered significantly more often in children with localized infections than in those with invasive disease; these isolates displayed significantly lower susceptibility to non-beta-lactam agents as compared with their penicillin-susceptible counterparts.

Adolescent↗
PubMed1998

Properties of mutant SHV-5 beta-lactamases constructed by substitution of isoleucine or valine for methionine at position 69.

The effect of replacement of Met-69 by Ile or Val on the properties of the extended-spectrum beta-lactamase SHV-5 was studied. Mutant enzymes were constructed by site-specific mutagenesis and expressed under isogenic conditions in Escherichia coli DH5alpha cells. Compared with SHV-5, the mutant beta-lactamases conferred lower levels of beta-lactam resistance and were less efficient in hydrolyzing ampicillin, cephalothin, and cefotaxime. The substitutions rendered SHV-5 less susceptible to inhibition by clavulanate, sulbactam, and tazobactam; however, the MICs of penicillin-inhibitor combinations remained similar, suggesting an attenuation of penicillinase activity.

Anti-Bacterial Agents↗
PubMed1998

Sporadic emergence of Klebsiella pneumoniae strains resistant to cefepime and cefpirome in Greek hospitals.

The sporadic emergence of Klebsiella pneumoniae strains resistant to cefepime and cefpirome was observed in Greek hospitals during 1996. Examination of six epidemiologically distinct strains and clones selected in vitro provided indications that resistance is due to the cooperation of decreased outer membrane permeability and hydrolysis of the cephalosporins by SHV-5 beta-lactamase, which was produced in large amounts.

Cefepime↗
PubMed1998

Emergence of multidrug resistance in ubiquitous and dominant Pseudomonas aeruginosa serogroup O:11. The Greek Pseudomonas Aeruginosa Study Group.

The serotypes of 88 nonreplicate nosocomial Pseudomonas aeruginosa isolates from 11 Greek hospitals were studied in relation to their antibiotic susceptibilities. Rates of resistance to beta-lactams, aminoglycosides, and quinolones ranged from 31 to 65%, except for those to ceftazidime (15%) and imipenem (21%). Four serotypes were dominant: O:12 (25% of isolates), O:1 (17%), O:11 (16%), and O:6 (10%). Multidrug resistance rates in the major serogroups O:12 (91%) and O:11 (79%) were higher than those in serogroups O:1 (40%) and O:6 (43%). Further typing with respect to pulsed-field gel electrophoresis patterns following XbaI digestion of genomic DNA discriminated the isolates into 74 types. Pulsed-field gel electrophoresis revealed that the ubiquitous O:12 group was genetically homogeneous, since 95% of strains belonged to two clusters of genotypic similarity, while the O:11 strains, present in 8 of the 11 hospitals, were distributed among five such clusters. Therefore, apart from the already reported O:12 multidrug-resistant European clone, an O:11 population, characterized by a serotype known to be dominant in the environment and the hospital in several parts of the world, but previously not associated with multidrug resistance to antibiotics, has progressed to a multidrug-resistant state.

DNA Fingerprinting↗
PubMed1998

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.

AIDS-Related Opportunistic Infections↗
PubMed1997

Distinct genotypic clusters of heterogeneously and homogeneously methicillin-resistant Staphylococcus aureus from a Greek hospital.

Methicillin-resistant Staphylococcus aureus (MRSA) strains isolated over a one-year period from a Greek hospital were tested for their levels of resistance to methicillin by population analysis. Heterogeneously resistant strains belonged to classes I, II, and II/III, whereas homogeneously resistant ones belonged to class IV. Strains of all classes possessed the mecA gene. Pulsed-field gel electrophoresis (PFGE) of Smal-digested genomic DNA revealed that all heterogeneously resistant strains were also closely related, but in a cluster distinct from the heterogeneous one. The methicillin-sensitive strains displayed a greater variety of PFGE types compared to MRSA isolates.

Bacterial Proteins↗
PubMed1997

A molecular analysis of Greek and UK Haemophilus influenzae conjugative resistance plasmids.

Antibiotic resistance in Haemophilus influenzae has been associated with the presence of large, chromosomally integrated, conjugative plasmids. The plasmids of 10 beta-lactamase-positive, ampicillin-resistant strains, two from the UK and eight from Greece, were investigated. Plasmids were detected and isolated after transfer to a rec-deficient recipient. Purified whole plasmid was used as probe. In addition a 12 kb PstI fragment containing the putative point of recircularization in one plasmid, p1056, was cloned and used as a probe. All plasmids shared a high degree of sequence homology suggesting that plasmids of diverse geographical origin are highly related. All plasmids also shared sequence homology with the 12 kb PstI fragment containing the point of recircularization, suggesting that the sequences involved in excision and recircularization are conserved.

Ampicillin Resistance↗
PubMed1997

Comparative evaluation of the inhibitory activities of the novel penicillanic acid sulfone Ro 48-1220 against beta-lactamases that belong to groups 1, 2b, and 2be.

The inhibitory activity of the penicillanic acid sulfone Ro 48-1220 against group 1, 2b, and 2be beta-lactamases was evaluated. Ro 48-1220 inhibited TEM and SHV as effectively as clavulanate and tazobactam. It also inhibited group 1 beta-lactamases at lower concentrations than tazobactam. Ro 48-1220, at a concentration of 4 micrograms/ml, protected ceftriaxone and ceftazidime against strains producing group 1 and 2be beta-lactamases.

Bacteria↗
PubMed1997

Characterization to species level of Mycobacterium avium complex strains from human immunodeficiency virus-positive and -negative patients.

Forty human clinical Mycobacterium avium-M. intracellulare complex strains isolated in Greece were characterized to the species level by PCR with three sets of primers specific for one or both species. M. avium predominated in both human immunodeficiency virus-positive and -negative patients, but the frequency of M. intracellulare isolation appeared to be higher in the latter.

AIDS-Related Opportunistic Infections↗
PubMed1997

Molecular epidemiology of antibiotic resistance of Salmonella enteritidis during a 7-year period in Greece.

A significant increase in the frequency of isolation of Salmonella enteritidis has been observed during recent years in Greece, parallelled by an increasing rate of resistance of this organism to antibiotics. A substantial proportion of ampicillin- and doxycycline-resistant isolates exhibited cross-resistance to drugs of other classes, such as sulfonamides and streptomycin. Isolates of human origin were overall less resistant than those of animal or food-feed origin. Indeed, strains associated with animal infections were characterized by the highest rates of resistance to several antibiotics. These phenotypic data were correlated with genotypic information concerning two distinct populations: isolates from all sources that were resistant only to ampicillin, the drug toward which resistance rates were highest, and a control group of sensitive isolates. Ampicillin resistance was due to a 34-MDa conjugative plasmid. DNA fingerprinting by macrorestriction of genomic DNA revealed two types, A and B, common to both ampicillin-resistant and -sensitive strains, with 80 to 90% of strains being of type A. However, a third type, C, was specific for the sensitive population, representing 17% of those strains. Therefore, although the majority of resistant isolates were genetically related to sensitive ones, there existed a susceptible clone which had not acquired any resistance traits.

Ampicillin↗
PubMed1997

Rapid discrimination of Mycobacterium avium strains from AIDS patients by randomly amplified polymorphic DNA analysis.

A randomly amplified polymorphic DNA (RAPD) analysis was performed for the molecular typing of Mycobacterium avium strains. This method was applied to epidemiologically unrelated M. avium strains isolated from the blood of 10 different AIDS patients and to strains that were considered epidemiologically related, as they had been isolated from the same patient but from different body locations (4 patients, 10 strains). Three oligonucleotide primers among the six tested were found to generate RAPD profiles with DNA from all M. avium strains and to successfully type them. This method for the typing of M. avium strains is rapid and easy to perform.

AIDS-Related Opportunistic Infections↗