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ABSTRACTIn an attempt to compare the epidemiology of severe Streptococcus pyogenesinfection within Europe, prospective data were collected through the Strep-EURO program. Surveillance for severe cases of S. pyogenesinfection diagnosed during 2003 and 2004 was undertaken in 11 countries across Europe by using a standardized case definition and questionnaire. Patient data as well as bacterial isolates were collected and characterized by T and M/emmtyping, and selected strains were analyzed for the presence of superantigen genes. Data were analyzed to compare the clinical and microbiological patterns of the infections across the participating countries. A total of 4,353 isolates were collected from 5,521 cases with severe S. pyogenesinfections who were identified. A wide diversity of M/emmtypes (n= 104) was found among the S. pyogenesclinical isolates, but the M/emmtype distribution varied broadly between participating countries. The 10 most predominant M/emmtypes were M/emmtype 1 (M/emm1), M/emm28, M/emm3, M/emm89, M/emm87, M/emm12, M/emm4, M/emm83, M/emm81, and M/emm5, in descending order. A correlation was found between some specific disease manifestations, the age of the patients, and the emmtypes. Although streptococcal toxic shock syndrome and necrotizing fasciitis were caused by a large number of types, they were particularly associated with M/emm1 and M/emm3. The emmtypes included in the 26-valent vaccine under development were generally well represented in the present material; 16 of the vaccine types accounted for 69% of isolates. The Strep-EURO collaborative program has contributed to enhancement of the knowledge of the spread of invasive disease caused by S. pyogeneswithin Europe and encourages future surveillance by the notification of cases and the characterization of strains, which are important for vaccination strategies and other health care issues.

ABSTRACTWe analyzed a representative sample of methicillin-resistant Staphylococcus aureus(MRSA) from 11 European countries (referred to as the HARMONY collection) using three molecular typing methods used within the HARMONY group to examine their usefulness for large, multicenter MRSA surveillance networks that use these different laboratory methodologies. MRSA isolates were collected based on their prevalence in each center and their genetic diversity, assessed by pulsed-field gel electrophoresis (PFGE). PFGE groupings (=3 bands difference between patterns) were compared to those made by sequencing of the variable repeats in the protein A gene spaand clonal designations based on multilocus sequence typing (MLST), combined with PCR analysis of the staphylococcal chromosome cassette containing the mecgenes involved in methicillin resistance (SCCmec). A high level of discrimination was achieved using each of the three methodologies, with discriminatory indices between 89.5% and 91.9% with overlapping 95% confidence intervals. There was also a high level of concordance of groupings made using each method. MLST/SCCmectyping distinguished 10 groups containing at least two isolates, and these correspond to the majority of nosocomial MRSA clones described in the literature. PFGE and spatyping resolved 34 and 31 subtypes, respectively, within these 10 MRSA clones, with each subtype differing only slightly from the most common pattern using each method. The HARMONY group has found that the methods used in this study differ in their availability and affordability to European centers involved in MRSA surveillance. Here, we demonstrate that the integration of such technologies is achievable, although common protocols (such as we have developed for PFGE) may also be important, as is the use of centralized Internet sites to facilitate data analysis. PFGE and spa-typing data from analysis of MRSA isolates from the many centers that have access to the relevant equipment can be compared to reference patterns/sequences, and clonal designations can be made. In the majority of cases, these will correspond to those made by the (more expensive) method of choice—MLST/SCCmectyping—and these alternative methods can therefore be used as frontline typing systems for multicenter surveillance of MRSA.

ABSTRACTScreening of Greek nontyphoid salmonellae from 2000 to 2002 yielded three extended-spectrum ß-lactamase (ESBL)-producing human isolates. Salmonella entericaserotype Brandenburg harbored a multiresistant SHV-5 gene-carrying plasmid. S. entericaserotype Blockley and S. entericaserotype Hadar harbored a TEM-52 gene-carrying plasmid. An S. entericaserotype 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.

ABSTRACTWe report on the development of a scheme for the typing of Pseudomonas aeruginosa, multiple-locus variable number of tandem repeat (VNTR) analysis (MLVA). We first evaluated the polymorphisms of 201 tandem repeat loci selected from more than 3,000 such sequences present in strain PAO1 with a test collection of 12 genotypically distinct clinical strains. Seven VNTR loci which can be easily scored with the technology used here were identified and used to genotype a collection of 89 clinical isolates that had previously been classified into 46 ribotypes, including 2 widespread ribotypes. Seventy-one different MLVA genotypes could be distinguished. With only two exceptions, strains with identical ribotypes were grouped together upon cluster analysis of the MLVA data. The 27 isolates with the most frequent ribotype were divided into 14 MLVA types, and the 18 isolates with the second most frequent ribotype were divided into 15 MLVA types. Analysis of a subset of 17 strains belonging to the major ribotype by pulsed-field gel electrophoresis with the enzyme SpeI distinguished seven types, identical to the number of MLVA types in this subset. Our data show that MLVA typing of P. aeruginosabased on the first set of loci has a high discriminatory power. Because MLVA is highly reproducible and easily portable among laboratories, it represents a very promising tool for the molecular surveillance of P. aeruginosa. A free, online strain identification service based on the genotyping data produced herein has been developed.

ABSTRACTPulsed-fieldgel 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 othercountries, 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.

ABSTRACTFrom 1992 to 1997, only six sporadic isolates of Salmonella entericaserotype 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 ß-lactamase. The blaSHV-12gene was located in at least two different self-transferable plasmids, one of which also carried a novel class 1 integron.

Extended-spectrum beta-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.

ABSTRACTExtended-spectrum ß-lactamase-producing Klebsiellapneumoniae(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.

ABSTRACTRapid 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 showed a different performance level in each center. In the center performing best with each method, inter-IS256PCR 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-IS256PCR, 44 to 57% for 16S-23S rDNA PCR, and 53 to 54% for MP3 PCR analysis. In conclusion, the performance of inter-IS256PCR 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.

ABSTRACTTwelve Salmonella typhimuriumstrains 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 ß-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. typhimuriumclone resistant to broad-spectrum cephalosporins is present in at least three European countries.

ABSTRACTThe serotypes of 88 nonreplicate nosocomial Pseudomonas aeruginosaisolates from 11 Greek hospitals were studied in relation to their antibiotic susceptibilities. Rates of resistance to ß-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.

ABSTRACTOf the 331 Neisseria gonorrhoeaestrains isolated in Greece from 1996 to 1999, 39 (11.8%) exhibited decreased susceptibility to quinolones due to gyrAandparCmutations. 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.

ABSTRACTSix distinct clones were present among Greek multidrug-resistantSalmonella entericaserotype 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 genesant(3")-Ia, pse-1, and tem-1.