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ABSTRACTVIM-1-producing Klebsiella pneumoniae(VPKP) is an emerging pathogen. A prospective observational study was conducted to evaluate the importance of VIM production on outcome of patients with K. pneumoniaebloodstream infections (BSIs). Consecutive patients with K. pneumoniaeBSIs were identified and followed up until patient discharge or death. A total of 162 patients were included in the analysis; 67 (41.4%) were infected with VPKP, and 95 were infected with non-VPKP. Fourteen of the patients infected with VPKP were carbapenem resistant (Carbr) (MIC > 4 μg/ml), whereas none of the non-VPKP exhibited carbapenem resistance. The patients infected with a Carbrorganism were more likely (odds ratio, 4.08; 95% confidence interval [CI], 1.29 to 12.85; P= 0.02) to receive inappropriate empirical therapy. The all-cause 14-day mortality rates were 15.8% (15 of 95) for patients infected with VIM-negative organisms, 18.9% (10 of 53) for those infected with VIM-positive carbapenem-susceptible organisms, and 42.9% (6 of 14) for those infected with VIM-positive Carbrorganisms (P= 0.044). In Cox regression analysis, age (hazard ratio [HR], 1.03; 95% CI, 1.01 to 1.06; P= 0.021), rapidly fatal underlying disease (HR, 2.84; 95% CI, 1.26 to 6.39; P= 0.012), and carbapenem resistance (HR, 2.83; 95% CI, 1.08 to 7.41; P= 0.035) were independent predictors of death. After adjustment for inappropriate empirical or definitive therapy, the effect of carbapenem resistance on outcome was reduced to a level of nonsignificance. In patients with K. pneumoniaeBSIs, carbapenem resistance, advanced, age, and severity of underlying disease were independent predictors of outcome, whereas VIM production had no effect on mortality. The higher mortality associated with carbapenem resistance was probably mediated by the failure to provide effective therapy.

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.

ABSTRACTA prospective observational study was conducted to identify factors associated with bloodstream infections (BSIs) caused by integron-carrying Enterobacteriaceaeand to evaluate the clinical significance of integron carriage. Consecutive patients with EnterobacteriaceaeBSIs were identified and followed up until discharge or death. Identification of blood isolates and susceptibility testing were performed by the Wider I automated system. int-1-specific PCR, conserved-segment PCR, and DNA sequencing were used to determine the presence, length, and content of integrons. The relatedness among the isolates was examined by pulsed-field gel electrophoresis. Two hundred fifty episodes of EnterobacteriaceaeBSI occurred in 233 patients; 109 (43.6%) were nosocomial, 82 (32.8%) were community acquired, and 59 (23.6%) were health care associated. Integrons were detected in 11 (13.4%) community-acquired, 24 (40.7%) health care-associated, and 46 (42.2%) nosocomial isolates. Integron-carrying organisms were more likely to exhibit resistance to three or more classes of antimicrobials (odds ratio [OR], 9.84; 95% confidence interval [95% CI], 5.31 to 18.23; P< 0.001) or to produce extended-spectrum β-lactamases (OR, 5.75; 95% CI, 2.38 to 13.89; P< 0.001) or a VIM-type metallo-β-lactamase (P, 0.003). Inter- or intraspecies integron transfer and cross-transmission of integron-carrying clones were observed. Use of cotrimoxazole (OR, 4.77; 95% CI, 1.81 to 12.54; P< 0.001) and a nosocomial or other health care setting (OR, 3.07; 95% CI, 1.30 to 7.22; P, 0.01) were independently associated with BSIs caused by integron-carrying Enterobacteriaceae. Patients with a nonurinary source of bacteremia (OR, 9.46; 95% CI, 2.77 to 32.32; P< 0.001) and a Pitt bacteremia score of ≥4 (OR, 23.36; 95% CI, 7.97 to 68.44; P< 0.001) had a significantly higher 14-day mortality rate, whereas integron carriage did not affect clinical outcomes. These findings may have implications affecting antibiotic policies and infection control measures.

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.

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.

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.

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.

To determine whether 15 multiresistant Pseudomonas aeruginosaisolates from an intensive care unit (ICU) outbreak were related, were endemic, and belonged to the 0:12 European clone.

Objective:To study the mechanisms of antibiotic resistance in Salmonella typhiand Salmonella paratyphiB clinical isolates, and the clonality of resistant strains.

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.

Objective: To determine whether 15 multiresistant Pseudomonas aeruginosaisolates from an intensive care unit (ICU) outbreak were related, were endemic, and belonged to the Ο:12 European clone.

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.