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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.

The susceptibility to beta -lactams and the beta -lactamase content of 110 Salmonella spp. blood isolates collected during 1997-98 in 19 European centers participating in the SENTRY Surveillance Program were studied. Thirty-one isolates (28%) were resistant to penicillins, due to production of TEM-1 (27 isolates), OXA-1 (three isolates) or TEM-1 + OXA-1 (one isolate). All OXA-1 producers and 10 TEM-1-producing isolates were also resistant to penicillin-clavulanic acid combinations. In the latter isolates, this phenotype was associated with increased production of TEM-1. Sixteen TEM-1-producing Salmonella Enteritidis isolates and one OXA-1-producing S. Typhimurium isolate were able to transfer beta -lactam resistance by conjugation.

Pulsed-field gel electrophoresis (PFGE) is the most common genotypic method used in reference and clinical laboratories for typing methicillin-resistant Staphylococcus aureus (MRSA). Many different protocols have been developed in laboratories that have extensive experience with the technique and have established national databases. However, the comparabilities of the different European PFGE protocols for MRSA and of the various national MRSA clones themselves had not been addressed until now. This multinational European Union (EU) project has established for the first time a European database of representative epidemic MRSA (EMRSA) strains and has compared them by using a new “harmonized” PFGE protocol developed by a consensus approach that has demonstrated sufficient reproducibility to allow the successful comparison of pulsed-field gels between laboratories and the tracking of strains around the EU. In-house protocols from 10 laboratories in eight European countries were compared by each center with a “gold standard” or initial harmonized protocol in which many of the parameters had been standardized. The group found that it was not important to standardize some elements of the protocol, such as the type of agarose, DNA block preparation, and plug digestion. Other elements were shown to be critical, namely, a standard gel volume and concentration of agarose, the DNA concentration in the plug, the ionic strength and volume of running buffer used, the running temperature, the voltage, and the switching times of electrophoresis. A new harmonized protocol was agreed on, further modified in a pilot study in two laboratories, and finally tested by all others. Seven laboratories’ gels were found to be of sufficiently good quality to allow comparison of the strains by using a computer software program, while two gels could not be analyzed because of inadequate destaining and DNA overloading. Good-quality gels and inclusion of an internal quality control strain are essential before attempting intercenter PFGE comparisons. A number of clonally related strains have been shown to be present in multiple countries throughout Europe. The well-known Iberian clone has been demonstrated in Belgium, Finland, France, Germany, and Spain (and from the wider HARMONY collection in Portugal, Slovenia, and Sweden). Strains from the United Kingdom (EMRSA-15 and -16) have been identified in several other countries, and other clonally related strains have also been identified. This highlights the need for closer international collaboration to monitor the spread of current epidemic strains as well as the emergence of new ones.

The presence of erm genes conferring constitutive and inducible resistance, as well as that of the mefA gene conferring only constitutive resistance, was investigated using PCR in 70 erythromycin resistant (MIC greater than or equal to 1 mg/l) strains of viridans group streptococci (VGS) (18 Streptococcus mitis biotype 1, 16 S. mitis biotype 2, 15 S. oralis, 12 S. salivarius and nine S. sanguis) isolated from the oropharynx of healthy Greek children. All of the 56 isolates belonging to resistance phenotype M harbored the mefA gene. All of the 14 isolates constitutively resistant to macrolides and lincosamides (phenotype CR) harbored the ermB gene. Co-presence of both genes was not observed, whereas class A erm gene (previously known as ermTR) was not detected. Our results are consistent with a possible role of VGS as a reservoir of resistance genes now prevalent in pathogenic species of streptococci. (C) 2003 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved.

One hundred and twenty-one clinical isolates of Acinetobacter baumannii recovered from the intensive care units (ICUs) of nine tertiary-care hospitals in Athens, Greece were studied in order to determine whether the increasing appearance of resistant acinetobacters is due to the spread of epidemic strains. The majority of the isolates exhibited resistance to ampicillin-sulbactam, and the most common antibiotic resistance profiles comprised resistance to nine and eight of the 11 potentially active antibiotics tested, respectively. Pulsed-field gel electrophoresis showed that 68% of the isolates, recovered from all ICUs, belonged to two clonal groups, indicating inter-hospital dissemination of multiresistant A. baumannii in our region.

In order to investigate the potential relationship between erythromycin resistance and specific M-serotypes among clinical isolates of Streptococcus pyogenes from children in Greece, we randomly selected a total of 49 erythromycin-resistant (EryR) and 21 erythromycin-susceptible (EryS) isolates from the 1158 S. pyogenes isolates from the two main children’s hospitals of Athens during the period October 1997 to October 1998. The isolates were further characterized by M-serotyping, examined for their susceptibility to penicillin, vancomycin and clindamycin, and categorized into resistance phenotypes. A total of 248 (21%) S. pyogenes isolates in the two main children’s hospitals of Athens during the study period were resistant to erythromycin. All 49 EryR and 21 EryS isolates were susceptible to penicillin and vancomycin. With respect to erythromycin and clindamycin resistance, phenotypes M and IR MLSB dominated, with 30 and 17 isolates, respectively, two isolates belonged to the CR MLSB phenotype. Among the erythromycin resistant isolates, two M serotypes were dominant: M22 (30%) and M84 (41%). More specifically, M22 and M84 were most prevalent in resistance phenotypes IR MLSB (65%) and M (63%), respectively. In the susceptible group, no isolate belonged to these two M-serotypes, nor was a predominant serotype found. In contrast to susceptible isolates, two distinct M-serotypes were highly represented among EryR S. pyogenes isolates and predominantly associated with two distinct phenotypes.

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.

Objective To investigate the possible genetic relationship among erythromycin-resistant Streptococcus pneumoniae strains isolated in Greece and the UK. Methods During 1995-97,140 S. pneumoniae strains were isolated from clinical specimens submitted to the microbiology departments of the two main children’s hospital in Athens. All erythromycin-resistant strains were further studied with respect to the presence of genes encoding for the two major mechanisms of macrolide resistance, their serotypes, and pulsed-field gel electrophoresis (PFGE) types, in comparison to a previously characterized UK erythromycin-resistant clone. Results Eleven of the 140 isolates (7.9%) were resistant to erythromycin; nine of these were susceptible to penicillin. Serotyping allocated seven, three and one isolates to serotypes 14, 19F and serogroup 6, respectively. The mefA gene was detected in seven isolates (five serotype 14 and two serotype 19F), ermB in two (one serotype 19F and the serogroup 6 isolate), whilst in the remaining two isolates no resistance gene could be detected by polymerase chain reaction (PCR). Pulsed-field gel electrophoresis of genomic DNA showed that five Greek serotype 14 isolates belonged to the same chromosomal type as the serotype 14 erythromycin-resistant UK clone. Conclusions The present study showed that erythromycin resistance among the S. pneumoniae isolates was mostly owing to the efflux mechanism and suggested a possible clonal spread of serotype 14 erythromycin-resistant S. pneumoniae strains between Greece and the UK.

Objective To study the possible distribution of metallo-beta-lactamases among nosocomial Pseudomonas isolates in a Greek hospital with a recent high prevalence of carbapenem-resistant Pseudomonas isolates. Methods All carbapenem-resistant (imipenem- and/or meropenem-resistant) (MICS > 8 mg/L) Pseudomonas non-replicate isolates recovered from clinical infections in the Microbiology Laboratory of Saint Demetrios Hospital, Thessaloniki, Greece, from April 1998 to November 2000 were studied for the presence of metallo-beta-lactamases. They were tested by a disk diffusion test, PCR analysis, and nucleotide sequencing. DNA fingerprints were obtained by pulsed-field gel electrophoresis (PFGE) of XbaI-digested chromosomal DNA. Results In total, 24 carbapenem-resistant isolates (23 P. aeruginosa and one P. putida) were recovered. The serotypes observed among the P. aeruginosa isolates were, in order of decreasing frequency, O:11 (52%), O:3 and O:12 (17% each), and O:6 (13%). PFGE grouped 17 of the P. aeruginosa isolates into four clusters, each containing from two to seven isolates while the remaining isolates exhibited unique genotypes. bla(VIM-2) was detected in the P. putida isolate and a P. aeruginosa serotype 0:3 isolate. The latter strain was genotypically distinct from other contemporaneous or older carbapenem-resistant P. aeruginosa Greek isolates. Conclusion These findings suggest that, although the prevalence of metallo-beta-lactamases is low, the integron-associated bla(VIM) genes can spread to P. aeruginosa serotypes that have not been previously associated with carbapenem resistance in our region, as well as to other pseudomonal species.

From 1992 to 1997, only six sporadic isolates of Salmonella enterica serotype Enteritidis from patients with cases of gastroenteritis in southern Italy exhibited resistance to broad-spectrum cephalosporins. Five isolates produced SHV-12, and one isolate encoded a class C beta-lactamase. The bla(SHV-12) gene was located in at least two different self-transferable plasmids, one of which also carried a novel class 1 integron.

Extended-spectrum P-lactamase-producing Klebsiella pneumoniae (EPKP) strains are frequently implicated in outbreaks in neonatal intensive care units (NICUs). During the period from 1997 to 1998, 21 infections and 23 colonizations with EPKP were recorded in the NICU of a children’s hospital in Athens, Greece. Seventeen of the infected and 12 of the colonized neonates had been referred from other hospitals. The remaining infections and colonizations occurred during the current hospitalization. Pulsed-field gel electrophoresis typing showed that the latter cases were due to an outbreak strain that persisted in the unit, while the repeated introduction of EPKP carriers was mostly due to clonal outbreaks in two maternity hospitals.

A retrospective survey of the isolation rate of Enterococcus avium during the period March 1994-February 2000 conducted in Laikon General Hospital using the WHONET software, revealed a peak in the isolation rates of this species during March 1995-February 1996. The ten strains isolated during this time were studied further. No glycopeptide resistance was detected but resistance to ampicillin, ciprofloxacin, erythromycin, gentamicin (high-level) and streptomycin (high-level) was present in nine, ten, nine, three and seven of the isolates, respectively. The genes aac(6’)-Ie+aph(2”)-Ia and ant(6)-I, encoding for high-level gentamicin and streptomycin resistance, respectively, were detected only in the isolates with the corresponding phenotypes. Beta-lactamase production and haemolysis were not detected. There was evidence of ward-, floor- and building-specific distribution among the different aminoglycoside resistance phenotypes. DNA fingerprinting by PFGE grouped six of the ten isolates in a single cluster with 83% similarity, even though they expressed various resistance phenotypes. These results suggest dissemination of resistance genes among both genetically related and unrelated strains. (C) 2002 Elsevier Science B.V. and International Society of Chemotherapy. All rights reserved.

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. (C) 2001 Elsevier Science B.V. and International Society of Chemotherapy All rights reserved.

CTX-M-type beta-lactamases constitute a novel group of class A beta-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 beta-lactamases of Klebsiella oxytoca. They efficiently hydrolyze many newer broad-spectrum oximino-beta-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. (C) 2000 Published by Elsevier Science B.V. and International Society of Chemotherapy. All rights reserved.

Rapid and efficient epidemiologic typing systems would be useful to monitor transmission of methicillin-resistant Staphylococcus aureus (MRSA) at both local and interregional levels. To evaluate the intralaboratory performance and interlaboratory reproducibility of three recently developed repeat-element PCR (rep-PCR) methods for the typing of MRSA, 50 MRSA strains characterized by pulsed-field gel electrophoresis (PFGE) (SmaI) analysis and epidemiological data were blindly typed by inter-IS256, 16S-23S ribosomal DNA (rDNA), and MP3 PCR in 12 laboratories in eight countries using standard reagents and protocols. Performance of typing was defined by reproducibility (R), discriminatory power (D), and agreement with PFGE analysis. Interlaboratory reproducibility of pattern and type classification was assessed visually and using gel analysis software. Each typing method shelved a different performance level in each center. In the center performing best with each method, inter-IS256 PCR typing achieved R = 100% and D = 100%; 16S-23S rDNA PCR, R = 100% and D = 82%; and MP3 PCR, R = 80% and D = 83%. Concordance between rep-PCR type and PFGE type ranged by center: 70 to 90% for inter-IS256 PCR, 44 to 57% for 16S-23S rDNA PCR, and 53 to 54% for MP3 PCR analysis. In conclusion, the performance of inter-IS256 PCR typing was similar to that of PFGE analysis in some but not all centers, whereas other rep-PCR protocols showed lower discrimination and intralaboratory reproducibility. None of these assays, however, was sufficiently reproducible for interlaboratory exchange of data.

Six distinct clones were present among Greek multidrug-resistant Salmonella enterica serotype Typhimurium phage type DT104, since isolates belonging to resistance phenotypes including the ACSSuT (ampicillin, chloramphenicol, streptomycin, sulfonamides, and tetracycline) core could be distinguished with respect to their pulsed-field gel electrophoresis patterns, int1 integron structures, and presence or absence of antibiotic resistance genes ant(3”)-Ia, pse-1, and tem-1.

Of the 331 Neisseria gonorrhoeae strains isolated in Greece from 1996 to 1999, 39 (11.8%) exhibited decreased susceptibility to quinolones due to gyrA and parC mutations. Conventional typing and pulsed-field gel electrophoresis showed that 34 of these isolates were clonally related. Epidemiological data indicated that the epidemic clone was sustained in a group of high-frequency transmitters.

Objective To compare the chromosomal types of Mycobacterium avium strains infecting HIV-negative and AIDS patients in Greece. Methods In total, 41 Mycobacterium avium isolates, 23 from AIDS and 18 from HIV-negative patients, were compared by pulsed-field gel electrophoresis of genomic DNA after XbaI digestion. The majority (87%) of AIDS isolates were from disseminated infection, while the majority (61%) of HIV-negative isolates were from children with cervical lymphadenitis. Results Pulsed-field gel electrophoresis classified strains whose electrophoretic patterns were at least 85% similar into three clusters, A (four isolates), B (12 isolates), and C (15), while 10 isolates remained outside of these clusters. There was no statistically significant correlation of any PFGE cluster with a specific patient group. Within each patient group, no significant correlation of PFGE type with time, place of residence or, in the case of AIDS patients, hospital attended was observed. Conclusions Genotypic similarities between isolates responsible for disseminated infection in AIDS patients and lymphadenitis in HIV-negative children suggest that related strains, possibly from an environmental source, cause both types of infections.

The non-naturally occurring TEM-1 beta-lactamase mutant R164S:G238S, as well as the R164S (TEM-12) and G238S (TEM-19) beta-lactamases, were constructed and expressed in Escherichia coli under isogenic conditions. Comparison of susceptibilities to beta-lactam antibiotics and substrate profiles showed that the combination of R164S with G238S drastically reduced the extended-spectrum activity of the respective single mutants.

A total of 140 non-replicate Streptococcus pneumoniae community isolates from Greek children collected during the period 1995-1997 were studied. Combined intermediate and high penicillin resistance rates were 23% in 1995, 29% in 1996, and 27% in 1997. The proportion of highly resistant isolates steadily increased from 2% in 1995 to 12% in 1997. There was no significant difference in penicillin resistance rates among colonizing and infecting isolates (23 and 27%, respectively). Over the study period, a clear shift towards higher penicillin MIC was observed among both the susceptible and resistant groups. Thus, penicillin resistance rates were equally high among colonizing and infecting isolates and resistance levels appeared to be gradually increasing throughout the entire S. pneumoniae population. (C) 1999 Elsevier Science B.V. and International Society of Chemotherapy. All rights reserved.

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.

All 105 non-replicate consecutive Staphylococcus aureus strains isolated in 1997 from seven Creek hospitals, were found to be susceptible to vancomycin, teicoplanin and chloramphenicol, but only five (8%) were susceptible to all 16 antibiotics tested. Forty-three (41%) isolates were methicillin-resistant, 58% homogeneously (homMRSA) and 42% heterogeneously (hetMRSA). Resistance of homMRSA strains to other antibiotics was generally high (88-100%), although only one strain was resistant to netilmicin. Resistance in hetMRSA (6-39%) or in MSSA (5-11%) was significantly lower Consequently, the majority (76%) of homMRSA were multi-drug resistant, while the dominant phenotype of hetMRSA and MSSA was resistance to penicillin (50% and 76%, respectively). Comparison of these strains with isolates from 1994 showed higher resistance rates to erythromycin among MSSA, to erythromycin and amikacin among hetMRSA and to rifampicin among homMRSA strains. (C) 1999 Elsevier Science B.V. and International Society of Chemotherapy. All rights reserved.

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,vith multidrug resistance to antibiotics, has progressed to a multidrug-resistant state.

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.

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.

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 related. Homogeneously 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.

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.