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The immunolimulus (IML) assay system uses solid-phase endotoxin antibodies to capture lipopolysaccharide (LPS), which is then quantified by a modification of the chromogenic limulus amebocyte lysate (CLAL) method. Monoclonal antibodies (MAbs) reactive with selected 0 antigen serotypes of Escherichia coli (O18) and Salmonella typhimurium (O-9,12), when used in the IML, were shown to be highly specific in detecting their respective endotoxins in purified form and in plasma samples from experimentally infected animals. A murine MAb that was broadly cross-reactive with E. coli, Salmonella, and Shigella endotoxins also proved to be highly effective in the IML assay for capturing LPS molecules from both E. coli and S. typhimurium strains. These results indicate that IML assays can detect smooth-type enterobacterial endotoxins in plasma and suggest that such assays have potential for use in the rapid diagnosis of sepsis and endotoxemia caused by different enterobacterial species.

Acute non-necrotizing cellulitis is a skin infection with a tendency to recur. Both general and local risk factors for erysipelas or cellulitis have been recognized in previous studies using hospitalized controls. The aim of this study was to identify risk factors for cellulitis using controls recruited from the general population. We also compared patients with a history of previous cellulitis with those suffering a single episode, with regard to the risk factors: length of stay in hospital, duration of fever, and inflammatory response as measured by C-reactive protein (CRP) level and leukocyte count. Ninety hospitalized cellulitis patients and 90 population controls matched for age and sex were interviewed and clinically examined during the period April 2004 to March 2005. In multivariate analysis, chronic oedema of the extremity, disruption of the cutaneous barrier and obesity were independently associated with acute cellulitis. Forty-four (49%) patients had a -positive history (PH) of at least one cellulitis episode before entering the study. Obesity and previous ipsilateral surgical procedure were statistically significantly more common in PH patients, whereas a recent (

We analyzed the performance of a selective enrichment broth combined with Taqman-based real-time duplex nuc-mecA-PCR to expedite the screening of methicillin-resistant Staphylococcus aureus (MRSA). We found the broth to be able to select MRSA strains (oxacillin MIC range 4-256 microg/ml) from MSSA strains. A total of 31 MRSA strains were found from 1250 clinical samples screened. The nuc-mecA-PCR was positive from all enrichment broths containing MRSA. From the remaining 1219 samples negative for MRSA on culture/subculture, 138 samples were nuc+/mecA+ in PCR. The sensitivity of the test was 93.5%, specificity 88.6%, positive predictive value 17.3%, and negative predictive value 99.8% as compared to culture. Thus, with this method, the negative MRSA results can be reliably reported within 24-48 h from sampling. The method is a practical additional alternative to those already described for the same purpose.

During the late 1990s, increases in referrals to the national reference laboratory of Streptococcus pyogenes isolates from injecting drug users (IDUs) with severe soft tissue infection indicated an emerging problem in the UK, later confirmed during the 2003–2004 European enhanced surveillance (Strep-EURO) programme. In light of these findings, further analyses were undertaken in an attempt to understand the reasons behind this increase in referrals. Single and multivariable analyses were undertaken to compare clinical, microbiological and demographic characteristics of IDUs diagnosed with severe S. pyogenes infection during the 2003–2004 enhanced surveillance study with those of other cases arising during this same period. Temporal and spatial analyses were undertaken for IDUs to identify clustering, as a means of understanding the transmission dynamics underpinning this increase. Infections in IDUs were spread across the UK, with some concentration in northern England and London. IDUs presented with a wide range of clinical manifestations, including pneumonia, which was found to be significantly more common in IDUs (OR 3.00) than in other cases. Marked differences in type distributions were found between IDUs and other cases, in particular the concentration of emm/M83 (22% of IDUs, 2% of non-IDUs). These findings indicate that an epidemic of severe S. pyogenes infections in IDUs occurred in the UK, peaking in 2003. The explanation for this rise remains unclear.

The main virulence factor of group A streptococcus (GAS), M protein, binds plasma complement regulators factor H (FH) and FH-like protein 1 (FHL-1) leading to decreased opsonization. The M protein binding site on FH is within domain 7 in which also the age-related macular degeneration (AMD)-associated polymorphism Y402H is located. We studied if FH allotypes 402H and 402Y have different binding affinities to GAS. Plasma-derived FH allotype 402H and its recombinant fragment FH5-7(402H) showed decreased binding to several GAS strains. Growth of GAS in human blood taken from FH(402H) homozygous individuals was decreased when compared with blood taken from FH(402Y) homozygous individuals. The effect of the allotype 402H can be explained by combining the previous M protein mutagenesis data and the recently published crystal structure of FH6-8. In conclusion the data indicate that the AMD-associated allotype 402H leads to diminished binding of FH to GAS and increased opsonophagocytosis of the bacteria in blood. These results suggest that the homozygous presence of the allele 402H could be associated with decreased risk for severe GAS infections offering an explanation for the high frequency of the allele despite its association with visual impairment.

The past 2 decades have brought worrying increases in severe Streptococcus pyogenes diseases globally. To investigate and compare the epidemiological patterns of these diseases within Europe, data were collected through a European Union FP-5-funded program (Strep-EURO). Prospective population-based surveillance of severe S. pyogenes infection diagnosed during 2003 and 2004 was undertaken in 11 countries across Europe (Cyprus, the Czech Republic, Denmark, Finland, France, Germany, Greece, Italy, Romania, Sweden, and the United Kingdom) using a standardized case definition. A total of 5,522 cases were identified across the 11 countries during this period. Rates of reported infection varied, reaching 3/100,000 population in the northern European countries. Seasonal patterns of infection showed remarkable congruence between countries. The risk of infection was highest among the elderly, and rates were higher in males than in females in most countries. Skin lesions/wounds were the most common predisposing factor, reported in 25% of cases; 21% had no predisposing factors reported. Skin and soft tissue were the most common foci of infection, with 32% of patients having cellulitis and 8% necrotizing fasciitis. The overall 7-day case fatality rate was 19%; it was 44% among patients who developed streptococcal toxic shock syndrome. The findings from Strep-EURO confirm a high incidence of severe S. pyogenes disease in Europe. Furthermore, these results have identified targets for public health intervention, as well as raising awareness of severe S. pyogenes disease across Europe.

Background Bacterial, nonnecrotizing cellulitis is a localized and often recurrent infection of the skin. The aim of this study was to identify the beta-hemolytic streptococci that cause acute nonnecrotizing cellulitis infection in Finland. Methods A case-control study of 90 patients hospitalized for acute cellulitis and 90 control subjects was conducted during the period of April 2004-March 2005. Bacterial swab samples were obtained from skin lesions or any abrasion or fissured toe web. Blood culture samples were taken for detection of bacteremia. The patients, their household members, and control subjects were assessed for pharyngeal carrier status. beta-Hemolytic streptococci and Staphylococcus aureus were isolated and identified, and group A and G streptococcal isolates were further analyzed by T serotyping and emm and pulsed-field gel electrophoresis typing. Results beta-Hemolytic streptococci were isolated from 26 (29%) of 90 patients, 2 isolates of which were blood-culture positive for group G streptococci, and 24 patients had culture-positive skin lesions. Group G Streptococcus (Streptococcus dysgalactiae subsp. equisimilis) was found most often and was isolated from 22% of patient samples of either skin lesions or blood, followed by group A Streptococcus, which was found in 7% of patients. Group G streptococci were also carried in the pharynx of 7% of patients and 13% of household members but was missing from control subjects. Several emm and pulsed-field gel electrophoresis types were present among the isolates. Six patients (7%) had recurrent infections during the study. In 2 patients, the group G streptococcal isolates recovered from skin lesions during 2 consecutive episodes had identical emm and pulsed-field gel electrophoresis types. Conclusions Group G streptococci, instead of group A streptococci, predominated in bacterial cellulitis. No clear predominance of a specific emm type was seen. The recurrent nature of cellulitis became evident during this study.

In Finland, the incidence of methicillin-resistant Staphylococcus aureus (MRSA) strains has increased ten fold within the last decade. In order to follow the changing epidemiology of MRSA, accurate typing of S. aureus strains is important. The purpose of this study was to reanalyse 44 previously recognised Finnish epidemic MRSA strains (EMRSA) by several molecular typing methods and to revise their nomenclature. The 44 EMRSA strains were grouped into 26 pulsed-field gel electrophoresis (PFGE) clusters, 20 multi locus sequence typing (MLST) sequence types (ST) belonging to 12 clonal complexes (CC) of which CC8 was the most prevalent, and 27 spa types belonging to four clonal complexes. The staphylococcal cassette chromosome mec (SCCmec) type IV was predominant, and 48% of the strains were nonmultiresistant to antibiotics. The discriminatory power of PFGE clusters, MLST, and spa typing was high. The overall concordance values of typing methods differed when assessed by two different methods. Adjusted Rand coefficient provided fairly low correlations for all comparisons. However, spa type was able to efficiently predict types and clonal complexes of most of the other methods with high probability (or =80%).

An in-house PCR was compared with the GenoType® MRSA and the MRSA EVIGENETM tests, both of which corresponded 100% with the results of in-house PCR. The cefoxitin disk diffusion method was superior to both the oxacillin disk diffusion and minimum inhibitory concentration tests for predicting the mecA status.

The implementation of routine whole-genome sequencing (WGS) promises to transform our ability to monitor the emergence and spread of bacterial pathogens. Here we combined WGS data from 308 invasive Staphylococcus aureus isolates corresponding to a pan-European population snapshot, with epidemiological and resistance data. Geospatial visualization of the data is made possible by a generic software tool designed for public health purposes that is available at the project URL (http:// www.microreact.org/project/EkUvg9uY?tt=rc). Our analysis demonstrates that high-risk clones can be identified on the basis of population level properties such as clonal relatedness, abundance, and spatial structuring and by inferring virulence and resistance properties on the basis of gene content. We also show that in silico predictions of antibiotic resistance profiles are at least as reliable as phenotypic testing. We argue that this work provides a comprehensive road map illustrating the three vital components for future molecular epidemiological surveillance: (i) large-scale structured surveys, (ii) WGS, and (iii) communityoriented database infrastructure and analysis tools. IMPORTANCE The spread of antibiotic-resistant bacteria is a public health emergency of global concern, threatening medical intervention at every level of health care delivery. Several recent studies have demonstrated the promise of routine wholegenome sequencing (WGS) of bacterial pathogens for epidemiological surveillance, outbreak detection, and infection control. However, as this technology becomes more widely adopted, the key challenges of generating representative national and international data sets and the development of bioinformatic tools to manage and interpret the data become increasingly pertinent. This study provides a road map for the integration of WGS data into routine pathogen surveillance. We emphasize the importance of large-scale routine surveys to provide the population context for more targeted or localized investigation and the development of open-access bioinformatic tools to provide the means to combine and compare independently generated data with publicly available data sets., 0, SCOPUS: ar.j, info:eu-repo/semantics/published

We studied colonization with methicillin-resistant and -sensitive Staphylococcus aureus (MRSA, MSSA) in the second largest nursing home in Finland, in which the residents volunteered had their nostrils, throats, perineums, skin lesions, and catheter exit sites swabbed, and catheter urines cultured. The specimens were cultured onto non-selective and selective agar, with or without enrichment in salt-containing trypticase soy broth (TSB). S. aureus was identified by routine methods, methicillin resistance was detected by oxacillin and cefoxitin disk diffusion and MIC E-tests, and GenoType MRSA -test was used for mecA gene confirmation. A total of 663 cultures were obtained from 213 residents. Of those, 165 specimens (25%) from 94 residents (44%) were positive for S. aureus, and 3 specimens (0.4%) from 2 (0.9%) residents were positive for MRSA. Of the 165 S. aureus isolates, 31 (19%) from 25 (27%) residents were found only from sites other than nostrils (30 MSSA and 1 MRSA). TSB enrichment detected additional 33 (5%) S. aureus isolates (32 MSSA and 1 MRSA), resulting in 8 (5%) additional residents. None of the MRSA strains would have been found if only nostrils and throat had been screened, and no enrichment broth had been used.

To better understand the molecular events involved in the origin of new pathogenic bacteria, we studied the evolution of a highly virulent clone of serotype M1 group A Streptococcus (GAS). Genomic, DNA-DNA microarray, and single-nucleotide polymorphism analyses indicated that this clone evolved through a series of horizontal gene transfer events that involved (1) the acquisition of prophages encoding streptococcal pyrogenic exotoxin A and extracellular DNases and (2) the reciprocal recombination of a 36-kb chromosomal region encoding the extracellular toxins NAD+-glycohydrolase (NADase) and streptolysin O (SLO). These gene transfer events were associated with significantly increased production of SLO and NADase. Virtual identity in the 36-kb region present in contemporary serotype M1 and M12 isolates suggests that a serotype M12 strain served as the donor of this region. Multiple horizontal gene transfer events were a crucial factor in the evolutionary origin and emergence of a very abundant contemporary clone of serotype M1 GAS.

Genome sequencing of group A Streptococcus (GAS) has revealed that prophages account for the vast majority of gene content differences between strains. Serotype M28 strains are a leading cause of pharyngitis and invasive infections, but little is known about genetic diversity present in natural populations of these organisms. To study this issue, population-based samples of 568 strains from Ontario, Canada; Finland; and Houston, Texas, were analyzed. Special attention was given to analysis of variation in prophage-encoded virulence gene content by a PCR-based method. Thirty and 29 distinct prophage-encoded virulence gene profiles were identified among pharyngitis and invasive infection isolates. Thirteen profiles, representing the majority of the strains, were shared between these two classes of isolates. Significant differences were observed in the frequency of occurrence of certain prophage toxin gene profiles and infection type. M28 strains are highly diverse in prophage-encoded virulence gene content and integration site, supporting the key concept that prophages are critical contributors to GAS genetic diversity and population biology. Nucleotide sequence variation in the emm gene (encodes M protein) was also examined. Only three allelic variants were identified in the hypervariable portion of the emm28 gene. All but one strain had the same inferred amino acid sequence in the first 100 amino acids of the mature M28 protein. In contrast, size differences in the emm28 gene and inferred protein due to variable numbers of C-terminal repeats were common. The presence of macrolide resistance genes ( mefA , ermB , and ermTR ) was analyzed by PCR, and less than 2% of the strains were positive.

pls , a gene found in type I staphylococcal cassette chromosome mec (SCC mec ) regions of methicillin-resistant Staphylococcus aureus strains, was present in 12 of the 15 human clinical Staphylococcus sciuri isolates studied. Pls was expressed in the S. sciuri isolates, although at a lower level than in S. aureus . Other parts of SCC mec could also be found in the S. sciuri genome.

Data on methicillin-resistant Staphylococcus aureus (MRSA) cases notified to the National Infectious Disease Register (NIDR) and antibiotic resistance profiles of MRSA isolates sent to the national reference laboratory between 1997 and 2002 were analysed. In addition, the diagnostic methods used for MRSA identification in Finnish microbiology laboratories, the number of MRSA screening specimens studied, and the MRSA situation in long-term care facilities in 2001 were reviewed. MRSA cases notified to the NIDR rose from 120 in 1997 to 597 in 2002 (from 2.3 to 11.5 cases per 100,000 population). The increase was greatest in elderly people and outside Helsinki metropolitan area, in the districts where the proportion of non-multiresistant strains was most prominent. The National Committee for Clinical Laboratory Standard's guidelines for the oxacillin disk diffusion test were followed, except for the incubation temperature and time, which may have hindered detection of some MRSA strains. There was a wide geographic variation in the rates of MRSA, but this was not related to screening activity. MRSA isolates from long-term facilities accounted for more than half of the notifications to the NIDR in 2001.

Objectives:To study clusters of infections caused bySerratia marcescensin a neonatal intensive care unit (NICU) and to determine risk factors forS. marcescensinfection or colonization.Design:Genotyping ofS. marcescensisolates was performed by pulsed-field gel electrophoresis (PFGE). A retrospective case-control study was conducted.Setting:A tertiary-care pediatric hospital with a 16-bed NICU.Patients:All neonates with at least one culture positive forS. marcescensin the NICU during December 1999 to July 2002. Case-patients (n = 11) treated in the NICU during December 1999 to February 2000 were included in the case-control study. Neonates treated in the NICU for at least 72 hours during the same period with cultures negative forS. marcescenswere used as control-patients (n = 27).Results:S. marcescenswas cultured from 19 neonates; 9 were infected and 10 were colonized. PFGE analysis identified three epidemic strains; each cluster consisted of identical isolates, except one isolate in the first cluster that was different. The risk factors identified were low birth weight, prematurity, prolonged respiratory therapy, prolonged use of antibiotics, and maternal infection prior to delivery. Overcrowding and understaffing were recorded simultaneously with the clusters.Conclusions:PFGE analysis showed three independent clusters. Several factors contributed to spread of the epidemic strains: (1) there were many severely premature and susceptible neonates, (2) the NICU was overcrowded during the clusters, and (3) transmission was likely to occur via the hands of staff. Cohorting and improvement of routine infection control measures led to the cessation of each cluster.

Pulsed-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.

Streptococcal inhibitor of complement (Sic) is a highly polymorphic extracellular protein made by serotype M1 group A Streptococcus strains that contributes to bacterial persistence in the mammalian upper respiratory tract. New variants of the Sic protein arise very rapidly by positive selection in human populations during M1 epidemics. The human antibody response to Sic was analyzed. Of 636 persons living in diverse localities, 43% had anti-Sic serum antibodies, but only 16.4% had anti-M1 protein serum antibody. Anti-Sic antibody was also present in nasal wash specimens in high frequency. Linear B cell epitope mapping showed that serum antibodies recognized epitopes located in structurally variable regions of Sic and the amino terminal hypervariable region of the M1 protein. Phage display analyses confirmed that the polymorphic regions of Sic are primary targets of host antibodies. These results support the hypothesis that selection of Sic variants occurs on mucosal surfaces by a mechanism that involves acquired host antibody.

Despite the success of mass immunization in many countries, diphtheria continues to play a major role as a potentially lethal resurgent infectious disease. Early, accurate diagnosis is imperative since delay in specific therapy may result in death. The microbiologic diagnosis of the disease, the identification of contacts and carriers, and the appropriate clinical management of these patients are therefore crucial. The epidemiology of diseases caused by Corynebacterium diphtheriae has changed dramatically over the decades, a situation that is highlighted by the resurgence of infections in the European region. These factors have strengthened the need for laboratories to screen for C. diphtheriae. Many modified and new methodologies are now used widely within laboratories for diphtheria diagnosis. Recent developments have focused upon methods for detection of the lethal and potent exotoxin produced by the causative organism, C. diphtheriae; this detection is the definitive test for the microbiologic diagnosis of diphtheria.

Clonal spread and horizontal transfer in the spread of vancomycin resistance genes were investigated. Multiplex PCR, pulsed-field gel electrophoresis (PFGE), hybridization of enterococcal plasmids with the vanA and vanB probes, and sequencing of a fragment of vanB were used in the analysis. Before May 1996, 12 vancomycin-resistant Enterococcus faecium (VRE) isolates were found in Finland. Between May 1996 and October 1997, 156 VRE isolates were found in the Helsinki area. Between December 1997 and April 1998, fecal samples from 359 patients were cultured for VRE. One new case of colonization with VRE was found. During the outbreak period, 88% (137 of 155) of the VRE isolates belonged to two strains (VRE types I and II), as determined by PFGE. Each VRE type I isolate possessed vanB , and five isolates also had vanA . Of the 34 VRE type II isolates, 27 possessed vanA and 7 possessed vanB . Fifteen of 21 (71%) ampicillin-resistant, vancomycin-sensitive E. faecium (VSE) isolates found during and after the outbreak period in one ward were also of type II. Two VSE type II isolates were found in the hospital before the outbreak in 1995. By PFGE, the three groups ( vanA , vanB , or no van gene) of type II shared the same band differences with the main type of VRE type II with vanA . None of the differences was specific to or determinative for any of the groups. Our material suggests that vanA and vanB incorporate into an endemic ampicillin-resistant VSE strain.

Background Group B Streptococcus (GBS) is the most common cause of invasive infections in newborns. GBS bacteria are typed on the basis of capsular polysaccharides or surface-localized proteins. Both polysaccharides and protein antigens have been suggested as potential vaccine candidates. Methods A prospective nationwide laboratory-based study of invasive GBS infections in children younger than 3 months of age was conducted in 1985 through 1994. Isolates were serotyped by immunodiffusion in agar gel with HCl extracts and rabbit antisera. Clinical diagnoses and case fatalities were verified from the patient records or the national hospital discharge register. Results There were 485 cases registered during the 10-year period. The incidence of disease was 0.76/1000 live births. The case fatality rate was 8.0%. Of the 485 cases 398 (83%) were early onset and 87 (17%) late onset infections. The most common clinical diagnosis was bacteremia (77%) without an identified focus of infection. Other diagnoses included meningitis (17%), pneumonia (3%), osteomyelitis or septic arthritis (2%), pyelonephritis or cellulitis. Serotyping of 395 isolates revealed that 47% were of serotype III or III/R, 23% of Ia/c, 11% of Ib, 6% of II/R, 8% of IV, 1% of V and 7% were nontypable. Conclusions The clinical picture of GBS disease and serotype distribution are similar to what has been reported from other countries. Serotypes III and III/R dominated (47% of all infections), especially in late onset disease. On the basis of these results a GBS vaccine including at least the Ia, Ib, II and III components would provide coverage against 88% of GBS serotypes causing neonatal disease in Finland.

Serotype M1 group A Streptococcus strains cause epidemic waves of human infections long thought to be mono- or pauciclonal. The gene encoding an extracellular group A Streptococcus protein (streptococcal inhibitor of complement) that inhibits human complement was sequenced in 1,132 M1 strains recovered from population-based surveillance of infections in Canada, Finland and the United States. Epidemic waves are composed of strains expressing a remarkably heterogeneous array of variants of streptococcal inhibitor of complement that arise very rapidly by natural selection on mucosal surfaces. Thus, our results enhance the understanding of pathogen population dynamics in epidemic waves and infectious disease reemergence.

Twenty well-characterized isolates of methicillin-resistant Staphylococcus aureus were used to study the optimal resolution and interlaboratory reproducibility of pulsed-field gel electrophoresis (PFGE) of DNA macrorestriction fragments. Five identical isolates (one PFGE type), 5 isolates that produced related PFGE subtypes, and 10 isolates with unique PFGE patterns were analyzed blindly in 12 different laboratories by in-house protocols. In several laboratories a standardized PFGE protocol with a commercial kit was applied successfully as well. Eight of the centers correctly identified the genetic homogeneity of the identical isolates by both the in-house and standard protocols. Four of 12 laboratories failed to produce interpretable data by the standardized protocol, due to technical problems (primarily plug preparation). With the five related isolates, five of eight participants identified the same subtype interrelationships with both in-house and standard protocols. However, two participants identified multiple strain types in this group or classified some of the isolates as unrelated isolates rather than as subtypes. The remaining laboratory failed to distinguish differences between some of the related isolates by utilizing both the in-house and standardized protocols. There were large differences in the relative genome lengths of the isolates as calculated on the basis of the gel pictures. By visual inspection, the numbers of restriction fragments and overall banding pattern similarity in the three groups of isolates showed interlaboratory concordance, but centralized computer analysis of data from four laboratories yielded percent similarity values of only 85% for the group of identical isolates. The differences between the data sets obtained with in-house and standardized protocols could be the experimental parameters which differed with respect to the brand of equipment used, imaging software, running time (20 to 48 h), and pulsing conditions. In conclusion, it appears that the standardization of PFGE depends on controlling a variety of experimental intricacies, as is the case with other bacterial typing procedures.

The aim of this study was to evaluate the bacterial adherence to biodegradable self-reinforced polyglycolic acid (SR-PGA) and self-reinforced poly-DL-lactic acid (SR-PLA 96) spiral stents in vitro. They are used as temporary urethral stents in urology. Gold-plated metal wire, polyurethane and latex were used as controls. Materials were incubated up to 28 days in artificial urine, after which a bacterial suspension was added. After detaching by sonication the adhesive bacteria were analysed as colony forming units (CFUs) and by scanning electron microscopy (SEM) analysis. Adhesion was more significantly correlated to stent bacterial type than to the tested material in both assays. No encrustation was seen on SR-PGA or SR-PLA 96. SR-PGA and SR-PLA 96 had no effect on the bacterial growth. In conclusion, the bacterial properties are equally or more important than the material properties in the adhesion process.

In 1990, a new type of erythromycin resistance phenotype, designated NR, was found in group A streptococcus (GAS) in Finland. In the present study, the distribution of GAS isolates with this and other erythromycin-resistance phenotypes was surveyed in Finland, and the clonality of the isolates was explored. Of 4179 GAS isolates collected from all over Finland, 695 (17%) were resistant to erythromycin, and 82% of these had the NR phenotype. Of a group of 96 isolates with the NR phenotype from different areas, 91% was T4 serotype, opacity factor-positive. The majority of these isolates were studied further: All were M4 serotype and 88% were of one clonal origin in genetic analyses. Thus, one single clone predominates among erythromycin-resistant GAS in Finland. This clone is of T4M4 serotype and mediates the new type of erythromycin resistance, characterized by the NR phenotype.

Background In the early 1990s there was an increase in erythromycin resistance among group A streptococci in Finland. In response, policies regarding outpatient antibiotic therapy were changed, and nationwide recommendations were issued that called for reductions in the use of macrolide antibiotics for respiratory and skin infections in outpatients. We studied the effect of this policy on the pattern of erythromycin resistance throughout Finland. Methods From 1991 through 1996, a total of 39,247 group A streptococcal isolates from throat swabs (82 percent of the isolates) and pus samples (18 percent) and 290 isolates from blood cultures were studied in regional microbiology laboratories. The susceptibility of the isolates to erythromycin was tested by the disk-diffusion or the screening-plate method. Results Consumption of macrolide antibiotics decreased from 2.40 defined daily doses per 1000 inhabitants per day in 1991 to 1.38 in 1992 (P = 0.007) and remained near the lower level during the study period....

To cure the long-term carriage of methicillin-resistant Staphylococcus aureus (MRSA), eradication treatment was given to 10 patients wearing complete dentures. In addition to multiple body sites, MRSA was cultured from the dentures of six patients. The contaminated dentures were rebased and sterilized with heat in order to prevent recolonization. The patients did not use their dentures during the decolonization therapy. Subsequently, MRSA was eradicated from three of these patients but three others remained MRSA-positive despite at least two courses of combined systemic and topical eradication treatment. These particular patients had persistent stomatitis and their dentures were a poor fit, in poor condition and repeatedly grew MRSA. Eradication treatment was successful in the remaining four patients whose dentures were MRSA-negative. These results confirm that dentures may function as foreign bodies and sustain persistent nasopharyngeal.MRSA colonization. Therefore, we suggest that whenever eradication of MRSA is deemed necessary in cases of nasal, oral or pharyngeal carriage, heat treatment of the dentures should be included. Further comparative studies with larger patient populations are needed to evaluate the contribution of dentures to the long-term carriage of MRSA, as well as to assess the value of denture sterilization during the eradication course.

Cell surface properties of 78 strains of Acinetobacter baumannii of different origin (lower respiratory tract, wound, blood and environment) were investigated. The bacterial adhesion to collagen, fibronectin, fibrinogen and vitronectin was detected by particle agglutination assays. Salt aggregation tests were used to determine the cell surface hydrophobicity of isolated A. baumannii strains. We found that A. baumannii strains originating from patients with wound infection and bacteraemia showed significantly lower aggregative properties compared to respiratory and environmental strains. Electron microscopic investigations revealed more fimbriated bacterial cells among the highly aggregative A. baumannii strains. This study demonstrates that the investigated A. baumannii strains can be divided into two different groups according to their cell surface properties and source of isolation, whereas the majority of strains, from the lower respiratory tract and the hospital environment expressed strong adhesive properties.

Ninety-nine penicillin-sensitive Streptococcus pneumoniae strains of common pediatric sero­ groups/types (6, 7, 14, 19, and 23) cultured from the blood of children with invasive disease (n = 49) or asymptomatic oropharyngeal carriage (n = 50) were analyzed by multilocus enzyme electrophoresis and ribotyping; 53 distinctive multilocus enzyme genotypes (ETs) and 53 ribotypes were identified. Multilocus enzyme electrophoresis showed good correlation with ribotyping. ETs and ribotypes among invasive and carriage isolates were similar. Within different S. pneumoniae serogroups/types, both clonal(7 and 14)and heterogenous(6,19, and 23)ET and rRNA hybridization patterns were observed. Greatest diversity was observed among serotypes 6A, 6B, and 19F. Use of 12 additional restriction enzymes besides Pvull and BglII did not increase ribotype discrimination within serotype 7 and 14 isolates. Serotype 7 and 14 strains, which appear clonal by subtyping, are rare among carriers but common causes of invasive disease. Characteristics associated with their clonality may represent an advantage for invasiveness. Streptococcus pneumoniae causes morbidity and mortality in children and adults worldwide. S. pneumoniaeis one of the most common causes of pneumonia, meningitis, bacteremia, and otitis media in children. In total, 83 serotypes (based on the capsular polysaccharide) of S. pneumoniaeare recognized, but the majority of infections are caused by only about one­ fourth of these [1]. The emergence of penicillin resistance or multiresistance among S.pneumoniaestrains and the spread ofresistant organ­ isms have become of major concern worldwide [2]. Motivated by the emergence of antimicrobial resistance, subtyping meth­ ods to differentiate S. pneumoniae strains of the same sero­ group/type have been developed and applied. These include both phenotypic and genotypic schemes: electrophoretic profil­ ing of penicillin-binding proteins [3-6], surface protein A typ­ ing [4], multilocus enzyme electrophoresis [3,4,6,7], chromo­ somal profiling [3, 8], ribotyping [9, 10], and pulsed-field gel electrophoresis of genomic DNA [6, 11]. All of these tech­ niques have shown additional discriminatory power compared with serotyping alone and have provided convincing evidence

The in-vitro activities of ten antibiotics against 83 toxigenic strains of Corynebacterium diphtheriae recently isolated in northwestern Russia and surrounding areas were determined by an agar dilution method. All of the strains were susceptible to erythromycin, penicillin, ampicillin, cefuroxime, chloramphenicol, ciprofloxacin, gentamicin and tetracycline. Trimethoprim and rifampicin were each active against 81 isolates, the two strains resistant to the latter agent having been isolated from two members of the same family.

The immunostimulatory properties of lactic acid bacteria which have been subjected to ecological conditions of the gut have not previously been studied. We analyzed the survival of three different Lactobacillus strains and of one Bifidobacterium strain in an in 6itro gastrointestinal model consisting of compartments corresponding to the human stomach and small intestine. Human peripheral blood mononuclear cells (PBMC) were stimulated with bacteria recovered from the stomach and the production of tumor necrosis factor alpha (TNF-α), interleukin-6 (IL-6) and IL-10 in PBMC was measured. While the viability of all the strains in the stomach was similar during the measured 3 h, there were considerable differences in the ileum delivery. Despite the poor viability of the strains in the stomach, they still had a detectable ability to induce production of TNF-α and IL-6. Our results suggest that lactobacilli and bifidobacteria not even viable after passage through the stomach can influence the immune system.Keywords: lactobacilli, bifidobacteria, gastrointestinal model, cytokine induction.

Summary bfp, the structural gene of the major repeating bundle-forming pilus (BFP) subunit, was cloned from the enteroadherent factor (EAF) plasmid of enteropathogenic Escherichia coli (EPEC) strain B171 (0111:NM). The bfp open reading frame encoded a 193-amlno-acid protein; comparison of this sequence with the biochemically determined N-terminal amino acid sequence showed that the mature pilin protein is comprised of 180 amino acids, that this sequence is similar to other members of the type IV pilin family, and that it is preceded by a 13-amino-acid signal peptide. Expression of the cloned bfp structural gene in an EPEC strain that had been cured of the EAF plasmid yielded a 21000 dalton protein that co-migrated with the BFP precursor protein. Thus, other genes, probably carried by the EAF plasmid, are required for the maturation of the bfp product and for the production of extracellular pilus filaments. Use of bfp as a hybridization probe showed that homologous sequences are present in all tested EPEC strains and in 13 of 16 tested Salmonelia serotypes. Fifty per cent of these bfp probe-sensitive salmonellae exhibited the localized-adherence (LA) phenotype when incubated with tissue culture cell monolayers, a trait previously associated with EAF plasmid-containing EPEC strains. Scanning electron micrographs of a bfp probe-positive, LA-positive Salmonella dublin strain showed that it grows as adherent colonies on infected monolayers and that within these colonies, BFP-like fibres form inter-bacterial linkages. For EAF plasmid-containing EPEC strains and for severai Salmonella serotypes, BFP expression may lead to the development of adherent colonies on epithelial surfaces early in the infective process.

The molecular epidemiology of 33 Escherichia coli and 81 Klebsiella pneumoniae extended-spectrum β -lactamase-producing health-care-associated and community-acquired isolates collected in the Helsinki district during 2000–2004 was investigated. Clonality studies, antimicrobial susceptibility and genotyping of the isolates were performed. Newly emerging CTX-M-producing E. coli and bla SHV-12 -producing K. pneumoniae isolates were detected. Clonal clusters of both species persisted throughout the study period.

We analyzed the performance of a selective enrichment broth combined with Taqman-based real-time duplex nuc-mecA-PCR to expedite the screening of methicillin-resistant Staphylococcus aureus (MRSA). We found the broth to be able to select MRSA strains (oxacillin MIC range 4-256 microg/ml) from MSSA strains. A total of 31 MRSA strains were found from 1250 clinical samples screened. The nuc-mecA-PCR was positive from all enrichment broths containing MRSA. From the remaining 1219 samples negative for MRSA on culture/subculture, 138 samples were nuc+/mecA+ in PCR. The sensitivity of the test was 93.5%, specificity 88.6%, positive predictive value 17.3%, and negative predictive value 99.8% as compared to culture. Thus, with this method, the negative MRSA results can be reliably reported within 24-48 h from sampling. The method is a practical additional alternative to those already described for the same purpose.

Rapid, high-throughput screening tools are needed to contain the spread of hospital-acquired methicillin (meticillin)-resistant Staphylococcus aureus (MRSA) strains. Most techniques used in current clinical practice still require time-consuming culture for primary isolation of the microbe. We present a new phenotypic assay for MRSA screening. The technique employs a two-photon excited fluorescence (TPX) detection technology with S. aureus -specific antibodies that allows the online monitoring of bacterial growth in a single separation-free process. Different progressions of fluorescence signals are recorded for methicillin-susceptible and -resistant strains when the growth of S. aureus is monitored in the presence of cefoxitin. The performance of the new technique was evaluated with 20 MRSA strains, 6 methicillin-susceptible S. aureus strains, and 7 coagulase-negative staphylococcal strains and two different monoclonal S. aureus -specific antibodies. When either of these antibodies was used, the sensitivity and the specificity of the TPX assay were 100%. All strains were correctly classified within 8 to 12 h, and up to 70 samples were simultaneously analyzed on a single 96-well microtiter plate. As a phenotypic method, the TPX assay is suited for screening purposes. The final definition of methicillin resistance in any S. aureus strain should be based on the presence of the mecA gene. The main benefit afforded by the initial use of the TPX methodology lies in its low cost and applicability to high-throughput analysis.

A nationwide population-based study on community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) in Finland during 2004 to 2006 showed that both incidence (1.9/100,000 population) and strain variation increased in comparison to years 1997 to 1999. There were 7 community-associated epidemic and 25 sporadic MRSA strain types. Half of these had Panton-Valentine leukocidin genes.

Rapid and reliable diagnostic methods are needed to control methicillin-resistant Staphylococcus aureus (MRSA) transmission. We studied the BD GeneOhm™ MRSA Assay which is based on one specific amplification product at the junction of the right extremity sequence of the staphylococcal cassette chromosome mec (SCCmec) and the chromosomal sequence of orfX of S. aureus. The test was applied on 95 clinical isolates in Finland: 83% were positive. The isolates giving negative results represented several pulsed-field gel electrophoresis (PFGE) types and harboured SCCmec types IV, V, VI or were new types with different combinations of ccr genes.

In this population-based study, all 314 episodes of beta-haemolytic streptococcal bacteraemia in adult patients in the Pirkanmaa area, Finland, during the 10-year period 1995-2004 were retrospectively reviewed. Altogether, 92 cases of bacteraemia caused by Lancefield group A beta-haemolytic streptococci (GAS), 76 caused by group B beta-haemolytic streptococci (GBS), 18 caused by group C beta-haemolytic streptococci (GCS) and 128 caused by group G beta-haemolytic streptococci (GGS) were identified. The most important finding was that the incidence of GGS increased during the study period. Disruption of the cutaneous barrier was a very common predisposing factor in GAS and GGS bacteraemias. Skin infections were the presenting clinical manifestations in two-thirds of GAS and GGS bacteraemias.

In an attempt to compare the epidemiology of severeStreptococcus pyogenesinfection within Europe, prospective data were collected through the Strep-EURO program. Surveillance for severe cases ofS. 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 severeS. pyogenesinfections who were identified. A wide diversity of M/emmtypes (n= 104) was found among theS. 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 theemmtypes. 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. Theemmtypes 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 byS. 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.

Enteropathogenic Escherichia coli grow as discrete colonies on the mucous membranes of the small intestine. A similar pattern can be demonstrated in vitro; termed localized adherence (LA), it is characterized by the presence of circumscribed clusters of bacteria attached to the surfaces of cultured epithelial cells. The LA phenotype was studied using B171, an O111:NM enteropathogenic E. coli (EPEC) strain, and HEp-2 cell monolayers. LA could be detected 30-60 min after exposure of HEp-2 cells to B171. However, bacteria transferred from infected HEp-2 cells to fresh monolayers exhibited LA within 15 min, indicating that LA is an inducible phenotype. Induction of the LA phenotype was found to be associated with de novo protein synthesis and changes in the outer membrane proteins, including the production of a new 18.5-kD polypeptide. A partial NH2-terminal amino acid sequence of this polypeptide was obtained and showed it to be identical through residue 12 to the recently described bundle-forming pilus subunit of EPEC. Expression of the 18.5-kD polypeptide required the 57-megadalton enteropathogenic E. coli adherence plasmid previously shown to be required for the LA phenotype in vitro and full virulence in vivo. This observation, the correspondence of the 18.5-kD polypeptide to an EPEC-specific pilus protein, and the temporal correlation of its expression with the development of the LA phenotype suggest that it may contribute to the EPEC colonial mode of growth.

In order to study the clonality of clinical methicillin-resistant Staphylococcus epidermidis (MRSE) strains and their staphylococcal cassette chromosome mec (SCCmec) elements, 60 isolates of MRSE from bacteraemic patients in three units of the Helsinki University Hospital, Finland were selected, covering the periods 1990–1993 and 1997–1998. The MRSE strains were analysed by pulsed-field gel electrophoresis (PFGE), multilocus sequence typing and SCCmec typing. Eleven PFGE types (FIN-SE-1–11) with sequence type ST2 (clonal complex 2; CC2) were identified. The previously established methicillin-resistant Staphylococcus aureus SCCmec criteria were applied to name the MRSE SCCmec complexes, and it was found that 7% of the isolates carried SCCmec type IA (ccrA1, class B), whereas the majority (93%) yielded six non-typeable SCCmec PCR patterns (P1–P6). Within each SCCmec PCR pattern, two ccr recombinase genes (ccrA2 and ccr A3) and two mec gene complexes (class A and class B) were detected. In addition, the ccrC gene was associated with three of the six patterns. In conclusion, the MRSE strains were genetically related to each other (ST2) but their SCCmec complexes were unique combinations of elements previously recognized among SCCmec types III and IV.

Vancomycin-resistant enterococci (VRE) first appeared in the late 1980s in a few European countries. Nowadays, six types of acquired vancomycin resistance in enterococci are known; however, only VanA and to a lesser extent VanB are widely prevalent. Various genes encode acquired vancomycin resistance and these are typically associated with mobile genetic elements which allow resistance to spread clonally and laterally. The major reservoir of acquired vancomycin resistance is Enterococcus faecium; vancomycin-resistant Enterococcus faecalis are still rare. Population analysis of E. faecium has revealed a distinct subpopulation of hospital-acquired strain types, which can be differentiated by molecular typing methods (MLVA, MLST) from human commensal and animal strains. Hospital-acquired E. faecium have additional genomic content (accessory genome) including several factors known or supposed to be virulence-associated. Acquired ampicillin resistance is a major phenotypic marker of hospital-acquired E. faecium in Europe and experience has shown that it often precedes increasing rates of VRE with a delay of several years. Several factors are known to promote VRE colonisation and transmission; however, despite having populations with similar predispositions and preconditions, rates of VRE vary all over Europe.

Summary Objectives Several factors associated with mortality in Lancefield group A beta-hemolytic streptococcal bacteremia have been described in population-based surveillance studies, whereas such reports on group B, C, and G streptococcal are scant. Methods In this population-based study all 314 episodes of beta-hemolytic streptococcal bacteremia in adult patients in the Pirkanmaa area, Finland, during the 10-year period 1995–2004 were retrospectively reviewed. Results The 30-day case-fatality rate was 13%, being highest in group C (22%); in group A it was 15%, in group B 7%, and in group G 15%. Confusion, unconsciousness and dyspnea as the first sign or symptom were associated with increased case-fatality, while fever seemed to be a protecting factor for death. Alcoholism and ultimately or rapidly fatal underlying disease were significantly associated with increased case-fatality. Among infections of the skin and soft-tissues, necrotizing fasciitis had the highest risk of death (38%), while patients with cellulitis had a case-fatality of 8%. A history of previous cellulitis seemed to protect against death (case-fatality of 3% as compared to 16% among those without such a history ( p =0.014)). Conclusion A history of previous cellulitis seemed to be a protecting factor against death. Fever was also associated with a good prognosis.