Origin and evolution of European community-acquired methicillin-resistant Staphylococcus aureus.

UNLABELLED: Community-acquired methicillin-resistant Staphylococcus aureus (CA-MRSA) was recognized in Europe and worldwide in the late 1990s. Within a decade, several genetically and geographically distinct CA-MRSA lineages carrying the small SCCmec type IV and V genetic elements and the Panton-Valentine leukocidin (PVL) emerged around the world. In Europe, the predominant CA-MRSA strain belongs to clonal complex 80 (CC80) and is resistant to kanamycin/amikacin and fusidic acid. CC80 was first reported in 1993 but was relatively rare until the late 1990s. It has since been identified throughout North Africa, the Middle East, and Europe, with recent sporadic reports in sub-Saharan Africa. While strongly associated with skin and soft tissue infections, it is rarely found among asymptomatic carriers. Methicillin-sensitive S. aureus (MSSA) CC80 strains are extremely rare except in sub-Saharan Africa. In the current study, we applied whole-genome sequencing to a global collection of both MSSA and MRSA CC80 isolates. Phylogenetic analyses strongly suggest that the European epidemic CA-MRSA lineage is derived from a PVL-positive MSSA ancestor from sub-Saharan Africa. Moreover, the tree topology suggests a single acquisition of both the SCCmec element and a plasmid encoding the fusidic acid resistance determinant. Four canonical SNPs distinguish the derived CA-MRSA lineage and include a nonsynonymous mutation in accessory gene regulator C (agrC). These changes were associated with a star-like expansion into Europe, the Middle East, and North Africa in the early 1990s, including multiple cases of cross-continent imports likely driven by human migrations. IMPORTANCE: With increasing levels of CA-MRSA reported from most parts of the Western world, there is a great interest in understanding the origin and factors associated with the emergence of these epidemic lineages. To trace the origin, evolution, and dissemination pattern of the European CA-MRSA clone (CC80), we sequenced a global collection of strains of the S. aureus CC80 lineage. Our study determined that a single descendant of a PVL-positive methicillin-sensitive ancestor circulating in sub-Saharan Africa rose to become the dominant CA-MRSA clone in Europe, the Middle East, and North Africa. In the transition from a methicillin-susceptible lineage to a successful CA-MRSA clone, it simultaneously became resistant to fusidic acid, a widely used antibiotic for skin and soft tissue infections, thus demonstrating the importance of antibiotic selection in the success of this clone. This finding furthermore highlights the significance of horizontal gene acquisitions and underscores the combined importance of these factors for the success of CA-MRSA.

Livestock-associated methicillin-resistant Staphylococcus aureus in humans, Europe.

To estimate the proportion of methicillin-resistant Staphylococcus aureus (MRSA) isolates from humans that were sequence type (ST) 398, we surveyed 24 laboratories in 17 countries in Europe in 2007. Livestock-associated MRSA ST398 accounted for only a small proportion of MRSA isolates from humans; most were from the Netherlands, Belgium, Denmark, and Austria.

Streptococcus dysgalactiae subsp. equisimilis Bacteremia, Finland, 1995-2004.

We conducted a retrospective population-based study of 140 episodes of Streptococcus dysgalactiae subsp. equisimilis bacteremia occurring in Finland during 1995-2004. Rare emm types were associated with more severe disease and increased mortality rates. Skin and soft tissue infections were more frequent clinical signs among cases caused by common emm types.

A selective broth enrichment combined with real-time nuc-mecA-PCR in the exclusion of MRSA.

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.

Methicillin-resistant Staphylococcus aureus screening by online immunometric monitoring of bacterial growth under selective pressure.

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.

Predictors of death after severe Streptococcus pyogenes infection.

An evaluation of the relative importance of host and pathogen factors on the survival rate of patients with invasive Streptococcus pyogenes infection found a number of clinical and demographic factors to be associated with risk for death. Some evidence suggested a seasonal pattern to patient survival rate.

Community-associated methicillin-resistant Staphylococcus aureus isolated in Finland in 2004 to 2006.

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.

Predictors of mortality in beta-hemolytic streptococcal bacteremia: a population-based study.

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.

Clinical and microbiological characteristics of severe Streptococcus pyogenes disease in Europe.

In an attempt to compare the epidemiology of severe Streptococcus pyogenes infection within Europe, prospective data were collected through the Strep-EURO program. Surveillance for severe cases of S. pyogenes infection 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/emm typing, 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. pyogenes infections who were identified. A wide diversity of M/emm types (n = 104) was found among the S. pyogenes clinical isolates, but the M/emm type distribution varied broadly between participating countries. The 10 most predominant M/emm types were M/emm type 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 emm types. 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 emm types 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. pyogenes within 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.

Rapid emergence of emm84 among invasive Streptococcus pyogenes infections in Finland.

From 2005 to 2007, in Finland, the incidence of invasive Streptococcus pyogenes disease increased sharply, partly due to the uncommon emm84 gene becoming more prevalent from 2006 onwards. The overall case fatality rate of infections caused by strains carrying emm84 was not significantly different than that of infections caused by other types (7% versus 10%, respectively; P = 0.50).

Carriage of methicillin-resistant Staphylococci and their SCCmec types in a long-term-care facility.

Following an outbreak caused by staphylococcal cassette chromosome mec (SCCmec) type V methicillin (meticillin)-resistant Staphylococcus aureus (MRSA), a point-prevalence survey of the nasal carriage of staphylococci was conducted in a long-term-care facility in northern Finland in 2004. The focus was directed at methicillin-resistant coagulase-negative staphylococci (MR-CNS) and their SCCmec elements. A nasal swab was taken from 76 of the 80 residents 6 months after the onset of the outbreak. Staphylococcal isolates were identified by conventional methods and the GenoType Staphylococcus test, and their SCCmec elements were analyzed. Of the 76 individuals, 24 (32%) carried S. aureus and 67 (88%) CNS in their nostrils. Of the CNS carriers, 41 (61%) had at least one mecA-positive MR-CNS, and two individuals (3%) had both MRSA and methicillin-resistant Staphylococcus epidermidis (MRSE). Among the 61 MR-CNS isolates identified, 49 (80%) were MRSE. The distribution of the SCCmec types was diverse: 20 (33%) were of type IV, 11 (18%) of type V, 4 (6%) of type I or IA, 3 (4%) of type II, and 23 (38%) of new types (with six different combinations of ccr and other mec genes or only mecA). Both of the individuals with MRSA and MRSE shared SCCmec type V among their isolates. Nasal MR-CNS carriage was common among the residents of this long-term-care facility. A variety of SCCmec types, including many new types, were identified among the MR-CNS strains. The horizontal transfer of SCCmec elements is speculated based on the sharing of SCCmec type V between MRSA and MRSE.

Evaluation of phenotypic and molecular methods for screening and detection of methicillin-resistant Staphylococcus aureus.

An in-house PCR was compared with the GenoType MRSA and the MRSA EVIGENE 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.

Complement factor H allotype 402H is associated with increased C3b opsonization and phagocytosis of Streptococcus pyogenes.

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.

Binding of complement regulators factor H and C4b binding protein to group A streptococcal strains isolated from tonsillar tissue and blood.

Group A streptococcus (GAS) is the most common pathogen causing bacterial pharyngitis. We isolated streptococcal strains from tonsils removed from patients with tonsillar disease (n=202) and studied their ability to bind the complement regulators factor H (FH) and C4b binding protein (C4BP) using 125 I-labeled proteins. Blood isolates of GAS (n=10) were obtained from patients with bacteraemia. Streptococci were isolated from 21% of the tonsillitis patients. The emm and T types of the GAS strains were determined. Of the 26 GAS strains studied, only six could bind FH and/or C4BP above the threshold levels. The fraction of the offered radioactive protein bound ranged between 6-12% for FH and 19-56% for C4BP. The clinical course of the tonsillar disease was not related to the binding of FH or C4BP by GAS. The binding strains were mostly of the T4M4 or T28M28 type. From the invasive strains (n=10), three bound FH (binding level: 8-11%) and two C4BP (36-39%). The binding correlated only partially to M-protein (emm) type suggesting that the binding was not exclusively due to M-protein. The results indicate that complement regulator binding by GAS is only partially related to pathogenicity and not a universal property of all group A streptococci.

Proposal for common Nordic epidemiological terms and definitions for methicillin-resistant Staphylococcus aureus (MRSA).

The recent increase in the incidence of methicillin-resistant Staphylococcus aureus in all the Nordic countries prompted the Scandinavian Society for Antimicrobial Chemotherapy (SSAC) to create the 'SSAC Working Party on MRSA' with the objective to identify methods to keep the invasive MRSA infections in the Nordic countries below 1%. The lack of common definitions was recognized as a major obstacle for a joint Nordic effort to combat MRSA. The aim of this publication is to present proposals for epidemiological definitions of individual cases, for how to report MRSA frequency per country, and for communication of MRSA strain characteristics between the countries.

Epidemiology of severe Streptococcus pyogenes disease in Europe.

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.

Methicillin-sensitive Staphylococcus aureus bacteraemia and endocarditis among injection drug users and nonaddicts: host factors, microbiological and serological characteristics.

BACKGROUND: Endocarditis has been associated with lower mortality and fewer complications among injection drug users (IDUs) than nonaddicts in Staphylococcus aureus bacteraemia (SAB). The better prognosis of IDUs has not been clarified but it has generally been explained by younger age and other host factors. In this study, bacterial strains, their virulence factors, and host immune responses were compared among IDUs and nonaddicts with SAB, including those with and without endocarditis. METHODS: A total of 430 consecutive adult patients with methicillin-sensitive SAB were followed prospectively for 3 months. All 44 IDUs were included, and 44 nonaddicts as controls for them. According to the modified Duke criteria, 20 patients in both groups had endocarditis. For each addict without endocarditis, an age and sex matched nonaddict was selected as a control. S. aureus isolates were assigned a genotype by PFGE, Panton-Valentine leukocidin (PVL), staphylokinase (SAK), protease, and haemolysin production. Acute and convalescent sera were tested for antibodies to alpha-haemolysin (ASTA) and teichoic acid (TAA). RESULTS: There were no differences between IDUs and nonaddicts with SAB in the proportion of patients with a deep infection (98% vs 86%, P=0.06) or a thromboembolic complication (30% vs 14%, P=0.12). Endocarditis among IDUs was not associated with any specific strains, and only the FIN-4 strain was observed more often in IDUs than in nonaddicts (21% vs 5%, P=0.03). The majority of isolates (98%) were PVL negative, and there were no differences in the numbers of SAK, protease and haemolysin production among strains between IDUs and nonaddicts. However, haemolytic properties were found more frequently in strains from IDUs without endocarditis than those with endocarditis (88% vs 47%, P=0.007). IDUs displayed more often elevated TAA titers than nonaddicts, especially in endocarditis at acute phase (33% vs 5%, P=0.04) and at convalescent phase (50% vs 10%, P=0.01). The ASTA titer was more frequently initially positive among IDUs without endocarditis than with endocarditis (44% vs 6%, P=0.01). CONCLUSIONS: Characterization of the bacterial strains and their virulence factors, and host immune responses did not reveal significant differences between IDUs and nonaddicts with similar clinical picture of SAB. Serological tests were not helpful in identifying patients with endocarditis.

Acute bacterial, nonnecrotizing cellulitis in Finland: microbiological findings.

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.

Severe Streptococcus pyogenes infections, United Kingdom, 2003-2004.

As part of a Europe-wide initiative to explore current epidemiologic patterns of severe disease caused by Streptococcus pyogenes, the United Kingdom undertook enhanced population-based surveillance during 2003-2004. A total of 3,775 confirmed cases of severe S. pyogenes infection were identified over 2 years, 3.33/100,000 population, substantially more than previously estimated. Skin/soft tissue infections were the most common manifestation (42%), followed by respiratory tract infections (17%). Injection drug use was identified as a risk factor for 20% of case-patients. One in 5 infected case-patients died within 7 days of diagnosis; the highest mortality rate was for cases of necrotizing fasciitis (34%). Nonsteroidal antiinflammatory drugs, alcoholism, young age, and infection with emm/M3 types were independently associated with increased risk for streptococcal toxic shock syndrome. Understanding the pattern of these diseases and predictors of poor patient outcome will help with identification and assessment of the potential effect of targeted interventions.

Staphylococcus aureus colonization among nursing home residents in a large Finnish nursing home.

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.