N.J. Legakis

CTX-M-type β-lactamases: an emerging group of extended-spectrum enzymes

Abstract CTX-M-type β-lactamases constitute a novel group of class A β-lactamases with extended-spectrum properties. They are encoded by transferable plasmids and found in various enterobacteria, mostly Salmonella typhimurium , Escherichia coli , Klebsiella pneumoniae and Proteus mirabilis . CTX-M enzymes share extensive sequence similarity with the chromosomal β-lactamases of Klebsiella oxytoca . They efficiently hydrolyze many newer broad-spectrum oximino-β-lactams including cefotaxime, ceftriaxone and aztreonam and are readily inhibited by tazobactam and clavulanate. CTX-M-producing enterobacteria are endemic in Latin America and in some areas of North Eastern Europe. Data on their structure, properties and epidemiology are discussed.

Extended-spectrum β-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.

Antibiotic resistance rates and macrolide resistance phenotypes of viridans group streptococci from the oropharynx of healthy Greek children

A total of 200 isolates of viridans group streptococci isolated from the oropharynx of healthy Greek children were studied. Vancomycin, rifampicin, fluoroquinolones and dalfopristin/quinupristin were active against all tested isolates. High level resistance to gentamicin was not seen. Intermediate and high-level penicillin resistance was present in 28.5 and 14.5% isolates, respectively, with 41.3% of the latter group, being also resistant to cefotaxime. Resistance rates to other antimicrobials were as follows - erythromycin 38.5%, clarithromycin 33.5%, clindamycin 7.5% and tetracycline 23%. Penicillin resistance occurred more frequently in Streptococcus mitis isolates, while macrolide resistance was more frequent in S. oralis. MLSB resistance phenotype M was dominant (74%) among erythromycin resistant isolates, with phenotypes IR and CR being represented by 6 and 20% of isolates, respectively.

Diversity of resistance phenotypes and plasmid analysis in multi-resistant 0:12 Pseudomonas aeruginosa

Antibiotic resistance phenotypes, plasmid content and ability of conjugal transfer of antibiotic resistance genes of 35 multi-resistant Pseudomonas aeruginosa strains were examined. The strains were isolated in 12 Greek hospitals and the majority of them (80%) belonged to serotype 0:12. The isolates were distributed to a variety of different antibiotic resistance phenotypes. Plasmid analysis showed that 10 isolates harboured plasmids ranging in size from 20 to 100 Mda. Among these strains, four carried plasmids of 100 Mda, two strains had 60 Mda plasmid each while in three strains the plasmids detected were 65, 25 and 20 Mda, respectively. One strain harboured two plasmids of 100 and 60 Mda. All strains containing plasmids belonged to 0:12 serotype, except the one harbouring the 25 Mda plasmid, which belonged to serotype 0:6. Using a P. aeruginosa recipient resistant to rifampicin and ciprofloxacin, conjugal transfer was achieved in two occasions. These plasmids, 100 Mda in size, encoded high-level resistance to both gentamicin and tobramycin whereas resistance to other drugs was not transferable. Interestingly, all 100 Mda plasmids, including the self-tansferable ones, were found to share a certain degree of homology as judged by restriction analysis. It is suggested that both resistance phenotypes and analysis of plasmid content might be useful in subdividing 0:12 multi-resistant P. aeruginosa.

Multilocus sequence typing (and phylogenetic analysis) of Campylobacter jejuni and Campylobacter coli strains isolated from clinical cases in Greece

BackgroundThe molecular epidemiology of C. jejuni and C. coli clinical strains isolated from children with gastroenteritis, was investigated using the multilocus sequence typing method (MLST). This analysis establishes for the first time in Greece and constitutes an important tool for the epidemiological surveillance and control of Campylobacter infection in our country.MethodsThe MLST genotypes were compared with those gained by other typing methods (HS-typing, PFGE and FlaA typing) and were also phylogenetically analyzed, in order to uncover genetic relationships.ResultsAmong 68 C. jejuni strains, 41 different MLST-Sequence Types (MLST-STs) were found. Fifty six strains or 34 MLST-STs could be sorted into 15 different MLST-Sequence Type Complexes (MLST-STCs), while twelve strains or seven MLST-STs did not match any of the MLST-STCs of the database. Twenty C. coli strains belonged to 14 different MLST-STs. Eleven MLST-STs were classified in the same MLST-STC (828), and three were unclassifiable. There was no significant association between the MLST-STs and the results of the other typing methods.Phylogenetic analysis revealed that some strains, classified to the species of C. jejuni, formed a separate, phylogenetically distinct group. In eight strains some alleles belonging to the taxonomic cluster of C. jejuni, were also detected in C. coli and vice versa, a phenomenon caused by the genetic mosaic encountered inside the genus Campylobacter.ConclusionsThe MLST-ST determination proved to be a very useful tool for the typing as well as the identification of Campylobacter on the species level.

Prevalence of Four Virulence Genes in Campylobacter jejuni Determined by PCR and Sequence Analysis

AbstractIntroduction: The presence of four virulence genes (racR, wlaN, cgtB, virB11) in 356 Campylobacter jejuni strains isolated from confirmed clinical cases was examined by PCR and sequence analysis. The investigated genes were chosen on the basis of their variation in prevalence. Methods: The virulence genes were detected by PCR and the amplified products were submitted for sequence analysis. Results: The gene with the highest prevalence was racR (87.08%). virB was present in only 1.69% of the C. jejuni strains, and wlaN and cgtB were detected in 16.01% and 24.44%, respectively. Five strains associated with Guillain-Barré syndrome and Miller-Fischer syndrome out of the total of 356 (1.40%) were positive for cgtB.Conclusion: Our findings suggest that racR may encode factors necessary for bacterial pathogenicity in humans, while the roles of the other three genes remain ambiguous.

Heat-stable antigen serotyping of Campylobacter jejuni strains isolated from hospitalized children in Athens, Greece

A hundred and twentynine Campylobacter jejuni strains isolated from hospitalized children with gastroenteritis were serotyped by the heat-stable antigen scheme (HS, Penners method). Isolates belonged to two different periods. Group A contained strains isolated in 1987–1988 and group B contained strains which were isolated in 1998–2000. A variety of serotypes was found. Serotype HS:2 was predominant, followed by the HS:4 complex and HS:1,44. Many clinically important Guillain-Barré Syndrome associated serotypes – like HS:19 – were identified. There were no significant differences in the distribution of serotypes between the two periods. The present report provides reference data, as this is the first C. jejuni serotyping study ever made in Greece.

Penicillin resistant Streptococcus pneumoniae isolated from infected children in Athens, Greece: resistance patterns, serotypes and penicillin-binding protein 2B mutation characterization by PCR

Stylianos Chatzipanagiotou *, E. Papavasileiou , T. Panagea , A. Makri , I. Paraskaki , C. Nicolaou , A. Ioannidis , N.J. Legakis d a Department of Clinical Microbiology, Athens Medical School*/Aeginition Hospital, Vass. Sophias av. 72-74, 115 28 Athens, Greece b Department of Clinical Microbiology, Penteli Childrens’ Hospital, Athens, Greece c Children’s Hospital of Athens, P. and A. Kyriakou, Kallithea Annexe, Athens, Greece d Department of Microbiology, Athens Medical School, Athens, Greece