Saara Salmenlinna

Evaluation of Molecular Typing Methods in Characterizing a European Collection of Epidemic Methicillin-Resistant Staphylococcus aureus Strains: the HARMONY Collection

ABSTRACT We 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 spa and clonal designations based on multilocus sequence typing (MLST), combined with PCR analysis of the staphylococcal chromosome cassette containing the mec genes 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/SCCmec typing distinguished 10 groups containing at least two isolates, and these correspond to the majority of nosocomial MRSA clones described in the literature. PFGE and spa typing 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/SCCmec typing—and these alternative methods can therefore be used as frontline typing systems for multicenter surveillance of MRSA.

Community-acquired methicillin-resistant Staphylococcus aureus, Finland.

Methicillin-resistant Staphylococcus aureus (MRSA) is no longer only hospital acquired. MRSA is defined as community acquired if the MRSA-positive specimen was obtained outside hospital settings or within 2 days of hospital admission, and if it was from a person who had not been hospitalized within 2 years before the date of MRSA isolation. To estimate the proportion of community-acquired MRSA, we analyzed previous hospitalizations for all MRSA-positive persons in Finland from1997 to 1999 by using data from the National Hospital Discharge Register. Of 526 MRSA-positive persons, 21% had community-acquired MRSA. Three MRSA strains identified by phage typing, pulsed-field gel electrophoresis, and ribotyping were associated with community acquisition. None of the strains were multiresistant, and all showed an mec hypervariable region hybridization pattern A (HVR type A). None of the epidemic multiresistant hospital strains were prevalent in nonhospitalized persons. Our population-based data suggest that community-acquired MRSA may also arise de novo, through horizontal acquisition of the mecA gene.

Elimination of Epidemic Methicillin- Resistant Staphylococcus aureus from a University Hospital and District Institutions, Finland

From August 1991 to October 1992, two successive outbreaks of methicillin-resistant Staphylococcus aureus (MRSA) occurred at a hospital in Finland. During and after these outbreaks, MRSA was diagnosed in 202 persons in our medical district; >100 cases involved epidemic MRSA. When control policies failed to stop the epidemic, more aggressive measures were taken, including continuous staff education, contact isolation for MRSA-positive patients, systematic screening for persons exposed to MRSA, cohort nursing of MRSA-positive and MRSA-exposed patients in epidemic situations, and perception of the 30 medical institutions in that district as one epidemiologic entity brought under surveillance and control of the infection control team of Turku University Hospital. Two major epidemic strains, as well as eight additional strains, were eliminated; we were also able to prevent nosocomial spread of other MRSA strains. Our data show that controlling MRSA is possible if strict measures are taken before the organism becomes endemic. Similar control policies may be successful for dealing with new strains of multiresistant bacteria, such as vancomycin-resistant strains of S. aureus.

Molecular Epidemiology of Methicillin-Resistant Staphylococcus aureus in Finland

Abstract This study reports the recent trends in the occurrence of methicillin-resistant Staphylococcus aureus in Finland, with special focus on characterization of the strains linked to interhospital epidemics and local outbreaks. Between 1981 and 1997, the annual number of methicillin-resistant Staphylococcus aureus isolations ranged from 89 to 272. Of all blood isolates of Staphylococcus aureus reported to the National Infectious Disease Register during the period 1995–97 (n=2049), only six were resistant to methicillin. Between 1992 and 1997, typing analysis by various methods (i.e., antibiogram, phage typing, ribotyping, and pulsed-field gel electrophoresis) identified 18 different strains capable of causing intrahospital outbreaks or interhospital epidemics. These 18 strains were separated into 13 different ribotypes and 14 major pulsed-field gel electrophoresis types. Multiresistance was investigated as a possible marker for epidemicity. Eight of the ten interhospitally spread strains were multiresistant compared to only three of the eight intrahospitally spread outbreak strains. More than one-third of the epidemic and local outbreak strains were suspected to be of foreign origin. The majority (6 of 10) of the epidemics were localized in southern and western Finland, and the largest epidemic, which occurred in the Helsinki metropolitan area, involved over 200 persons. Thus far, the epidemics have remained primarily intracity problems, and only two strains have become endemic.

Carriage of Methicillin-Resistant Staphylococci and Their SCCmec Types in a Long-Term-Care Facility

ABSTRACT 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.

Rapid differentiation between livestock-associated and livestock-independent Staphylococcus aureus CC398 clades.

Staphylococcus aureus clonal complex 398 (CC398) isolates cluster into two distinct phylogenetic clades based on single-nucleotide polymorphisms (SNPs) revealing a basal human clade and a more derived livestock clade. The scn and tet(M) genes are strongly associated with the human and the livestock clade, respectively, due to loss and acquisition of mobile genetic elements. We present canonical single-nucleotide polymorphism (canSNP) assays that differentiate the two major host-associated S. aureus CC398 clades and a duplex PCR assay for detection of scn and tet(M). The canSNP assays correctly placed 88 S. aureus CC398 isolates from a reference collection into the human and livestock clades and the duplex PCR assay correctly identified scn and tet(M). The assays were successfully applied to a geographically diverse collection of 272 human S. aureus CC398 isolates. The simple assays described here generate signals comparable to a whole-genome phylogeny for major clade assignment and are easily integrated into S. aureus CC398 surveillance programs and epidemiological studies.

Bacterial genotype affects the manifestation and persistence of bovine Staphylococcus aureus intramammary infection

ABSTRACT Two-hundred seventeen Staphylococcus aureus isolates from 116 dairy cows with intramammary infections were analyzed by pulsed-field gel electrophoresis to study the association between symptom severity, persistence of infection, and bacterial genotype. Among five main genotypes infecting 90% of the cows, one was associated with severe clinical symptoms but reduced persistence.

Clonality of epidemic methicillin-resistant Staphylococcus aureus strains in Finland as defined by several molecular methods

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 (≥80%).

Clustering of Serratia marcescens infections in a neonatal intensive care unit.

OBJECTIVES To study clusters of infections caused by Serratia marcescens in a neonatal intensive care unit (NICU) and to determine risk factors for S. marcescens infection or colonization. DESIGN Genotyping of S. marcescens isolates 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 for S. marcescens in 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 for S. marcescens were used as control-patients (n = 27). RESULTS S. marcescens was 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.

Nationwide trends in molecular epidemiology of methicillin-resistant Staphylococcus aureus, Finland, 1997–2004

BackgroundIn Finland, the annual number of MRSA notifications to the National Infectious Disease Register (NIDR) has constantly increased since 1995, and molecular typing has revealed numerous outbreak isolates of MRSA. We analyzed the data on MRSA notifications of the NIDR, and MRSA isolates were identified mainly by pulsed-field gel electrophoresis (PFGE) at the National Reference Laboratory (NRL) in Finland during 1997–2004. One isolate representative of each major PFGE type was further characterized by multilocus sequence (MLST)-, staphylococcal cassette chromosome mec (SCCmec)-, and Panton-Valentine leukocidin (PVL)-typing.ResultsThe annual number of MRSA notifications to the NIDR rose over ten-fold, from 120 in 1997 to 1458 in 2004, and the proportion of MRSA among S. aureus blood isolates tripled, from <1% during 1997–2003 to 2.8% in 2004. During the same period of time, 253 different strains among 4091 MRSA isolates were identified by PFGE: 215 were sporadic and 38 outbreak/epidemic strains, including 24 new strains. Two epidemic strains resembling internationally recognized MRSA clones accounted for most of the increase: FIN-16 (ST125:IA) from <1% in 1997 to 25% in 2004, and FIN-21 (ST228:I) from 6% in 2002 to 28% in 2004. Half of the ten most common strains carried SCCmec IV or V.ConclusionThe predominant MRSA strains seem to change over time, which encourages us to continue implementing active control measures with each new MRSA case.