Recognition of two groups of methicillin-resistant Staphylococcus aureus strains based on epidemiology, antimicrobial susceptibility, hypervariable-region type, and ribotype in Finland

Epidemiological evidence suggests that some methicillin-resistant Staphylococcus aureus (MRSA) strains are more prone to dissemination than others. We studied 72 MRSA strains, collected through nationwide MRSA surveillance in 1992 through 1999 and known to be either (i) sporadic, (ii) local outbreak strains spread within one hospital, or (iii) epidemic strains spread among hospitals, by antimicrobial susceptibility testing, hybridization of the mec hypervariable region (HVR), and ribotyping. Our results show that two main groups can be identified among these strains. The first group includes mainly nonepidemic, nonmultiresistant MRSA strains showing a specific mec HVR hybridization pattern, A, in combination with a variety of ribotypes. The other group includes multiresistant strains with mec HVR hybridization pattern B or C in association with closely related ribotype a or b. Sixty-four percent (9 of 14) of Finnish epidemic MRSA strains belong to the latter group. These findings support the existence of differences in epidemic potential among MRSA strains.     

Comparison of protein A gene sequencing with pulsed-field gel electrophoresis and epidemiologic data for molecular typing of methicillin-resistant Staphylococcus aureus

The epidemiologic relatedness of methicillin-resistant Staphylococcus aureus (MRSA) isolates is currently determined by analysis of chromosomal DNA restriction patterns by pulsed-field gel electrophoresis (PFGE). We have evaluated an alternative typing system (MicroSeq StaphTrack Kit; Perkin-Elmer Biosystems) based on the sequence analysis of the chromosomally encoded polymorphic repeat X region of the S. aureus protein A (spa) gene. A total of 69 clinical MRSA isolates were divided into 18 groups according to the number and nucleotide sequences of the spa repeats. Molecular typing results obtained both by spa sequencing and from the PFGE patterns were concordant except for one group, which contained 20 isolates recovered over a 2-year period from hospitalized patients at the Mayo Clinic. Although the spa typing patterns were indistinguishable for those isolates, PFGE analysis yielded seven related but distinguishable patterns. Further coagulase gene sequence analysis subtyped those 20 strains into four groups which followed distinct temporal and geographic distributions. During a 2-year epidemic period there were up to 7 fragment changes in PFGE patterns among epidemiologically related isolates, suggesting that PFGE may be unsuitable for long-term typing of strains involved in epidemics. Although more limited than PFGE in discriminatory power, spa sequencing analysis could be used as a screening method for typing of MRSA strains because of the shorter turnaround time, ease of use, and the inherent advantages of sequence analysis, storage, and sharing of information.     

Sequence analysis of dru regions from methicillin-resistant Staphylococcus aureus and coagulase-negative staphylococcal isolates

Methicillin-resistance in staphylococci results from expression of mecA, which occurs in a larger region of DNA (the mec region) lacking counterpart in susceptible cells. The mec region harbors in addition a highly polymorphic element, the dru (direct repeat unit) segment, which in an early S. aureus strain, BB270, was found to contain 10 imperfect 40 base-pair repeats. We have explored the utility of direct sequencing of dru segments for discriminating among strains of methicillin-resistant S. aureus (MRSA) and coagulase-negative staphylococci (MRCNS). We sequenced dru segments of 24 clinical isolates of MRSA, and 15 of MRCNS, and reexamined strain BB270. Six S. aureus and 2 S. epidermidis isolates were found to have deletions which removed all drus. The other strains were found to have multiple contiguous dru repeats of precisely 40 bp. Analysis of these strains plus dru segment sequence from 4 recent reports yielded 18 unique dru segment sequences (designated "dru types") differing in numbers of repeats and/or sequences of particular repeats. Dru typing was more discriminating than sequencing of non-mec region genes, including a repeat-containing segment (spa Xr) of the S. aureus protein A gene. Yet dru type was sufficiently stable to register epidemiological clusters. Dru sequencing is a useful tool for tracking methicillin-resistant lineages of S. aureus and CNS.     

Methicillin-resistant Staphylococcus aureus: phylogenetic relatedness between European epidemic clones and Swiss sporadic strains

We have compared the phylogenetic diversity of methicillin-resistant Staphylococcus aureus (MRSA) strains from Switzerland and their phylogenetic relationships with European epidemic clones, using multiprimer random amplification polymorphic DNA (RAPD). Strains included 24 European epidemic clones (59 strains), 66 sporadic strains isolated in Switzerland in 1996-1997, and 15 reference strains of five other Staphylococcus species. Similarity and clustering analysis with the Jaccard's coefficient showed that the maximum genetic distance between MRSA strains was 0.43, whereas the minimum genetic distance between the six Staphylococcus species was 0.97, indicating that the method permits phylogenetic hierarchization. The 24 MRSA clones reported to be epidemic in European countries during the 1990s were distributed into seven different genetic clusters with a maximum distance of 0.29 among them. This clustering pattern was confirmed by the analysis of a subset of MRSA strains by multilocus enzyme electrophoresis at 12 loci. Most of the sporadic Swiss strains were distributed into these seven different genetic clusters, together with the epidemic MRSA clones. This suggests that there is no phylogenetic cluster specific to epidemic clones of MRSA.     

Evolutionary relationships between sporadic and epidemic strains of healthcare-associated methicillin-resistant Staphylococcus aureus

National surveillance of healthcare-associated methicillin-resistant Staphylococcus aureus (HA-MRSA) isolates by pulsed-field gel electrophoresis (PFGE) typing allowed identification of rarely occurring 'sporadic' isolates with patterns significantly distinct from those of major epidemic clones of methicillin-resistant S. aureus (MRSA) circulating in Belgian hospitals. The aim of the present study was to compare the genetic background, antibiotic susceptibility profile and in vitro growth rates of 36 MRSA isolates with either 'epidemic' or 'sporadic' PFGE profiles to identify factors that could be involved in the epidemic behaviour of S. aureus . Sequence analysis of seven housekeeping genes (multilocus sequence typing) and seven surface-associated genes, combined with staphylococcal cassette chromosome mec (SCC mec ) typing and spa typing results, segregated sporadic isolates into four groups: (1) isolates phylogenetically distant from epidemic HA-MRSA clones that possessed several properties of community-acquired MRSA strains; (2) isolates derived from the same methicillin-susceptible S. aureus ancestor as epidemic isolates but possessing a distinct type of SCC mec ; and (3) and (4) isolates that were closely related to epidemic strains, either as recent descendants of these or as intermediate evolutionary steps between epidemic HA-MRSA strains and their putative ancestors. Sporadic isolates did not show slower growth in vitro than epidemic isolates. These findings suggest that the SCC mec type and insertion/deletion of other mobile genetic elements may be involved in modulating the epidemic behaviour of MRSA strains of similar genetic background, independently of fitness cost.     

Protein A and coagulase expression in epidemic and non-epidemic Staphylococcus aureus.

Strains of Staphylococcus aureus were divided into groups on the basis of antimicrobial sensitivity and epidemiology and tested for protein A expression in a simple microtitre test, which detected the non-immunological binding of immunoglobulin to protein A on whole cells of S aureus. Isolates of the methicillin resistant strain prevalent in south east England (EMRSA) showed a low expression of protein A compared with the other strains of methicillin resistant S aureus (MRSA), other multiple resistant strains, and sensitive strains. Protein A and coagulase expression in 27 strains of MRSA from 15 countries associated with hospital outbreaks were compared with 27 strains of MRSA from 11 countries reported to be sporadic isolates. Twenty four of the 27 outbreak associated MRSA showed low expression of protein A and high expression of coagulase. Conversely, sporadic strains generally gave higher levels of protein A and a wide variety of coagulase reactions. The results suggest that many epidemic strains of MRSA may have phenotypic characteristics that distinguish them from sporadic strains.     

Typing of methicillin-resistant Staphylococcus aureus in a university hospital setting by using novel software for spa repeat determination and database management

The spa gene of Staphylococcus aureus encodes protein A and is used for typing of methicillin-resistant Staphylococcus aureus (MRSA). We used sequence typing of the spa gene repeat region to study the epidemiology of MRSA at a German university hospital. One hundred seven and 84 strains were studied during two periods of 10 and 4 months, respectively. Repeats and spa types were determined by Ridom StaphType, a novel software tool allowing rapid repeat determination, data management and retrieval, and Internet-based assignment of new spa types following automatic quality control of DNA sequence chromatograms. Isolates representative of the most abundant spa types were subjected to multilocus sequence typing and pulsed-field gel electrophoresis. One of two predominant spa types was replaced by a clonally related variant in the second study period. Ten unique spa types, which were equally distributed in both study periods, were recovered. The data show a rapid dynamics of clone circulation in a university hospital setting. spa typing was valuable for tracking of epidemic isolates. The data show that disproval of epidemiologically suggested transmissions of MRSA is one of the main objectives of spa typing in departments with a high incidence of MRSA.     

耐甲氧西林金黄色葡萄球菌的脉冲场凝胶电泳分型研究

目的　探讨耐甲氧西林金黄色葡萄球菌 (MRSA)的分子流行病学特点。方法　采用脉冲场凝胶电泳 (PFGE)技术对我院在 2 0 0 1年 6月～ 2 0 0 2年 4月临床分离的 5 0株MRSA作同源性分析。结果　 5 0株MRSA的PFGE图谱分为 6个组 (A～F型 )。以A型 (2 7株 )、B型 (10株 )、C型 (10株 )流行为主。 5 0株MRSA中共有 17株和重症监护室 (ICU)相关 ,占 34%。流行菌株在各病房之间传播。结论　ICU是MRSA的高发区域和疫源地 ,神经外科、肝胆胰外科和干部病房中MRSA的分离率也较高。在同一病人不同时间不同部位所采集的菌株同源性不尽相同     

Increasing incidence and widespread dissemination of methicillin-resistant Staphylococcus aureus (MRSA) in hospitals in central Europe, with special reference to German hospitals

Objective: to present data on prevalence and interregional spread of methicillin-resistant Staphylococcus aureus (MRSA) in Germany.
Methods: A nationwide collection of MRSA isolates from nosocomial infections in 143 hospitals was established from isolates ( n =4368) sent to a microbiological reference center during 1993–95. As chosen by distinguishable resistance phenotypes at each time of occurrence during the study period, 1830 isolates were subjected to molecular typing by means of Sma l macrorestriction patterns, PCR for RNA gene spacer patterns, and PCR for patterns of DNA stretches flanked by the ERIC-2 sequence and flanked by Tn 976 and ribosomal binding site. In addition, data from a multicenter study on the incidence of antibiotic resistance have been analyzed (32 centers, 637 S. aureus isolates).
Results: In 1995 the prevalence of MRSA among S. aureus isolates was 8.7% overall in central Europe (including Germany), in comparison to 1.7% in 1990. From 1993 until now, a continuous interregional dissemination of six epidemic strains, which were identified by molecular typing, was recorded. Besides these epidemic strains, 15 MRSA strains were identified which could not be allocated to the epidemic MRSA or to the known clonal groups of the species S. aureus. MRSA from three cases of sporadic nosocomial infections exhibited characteristics of the clonal group of S. aureus with the capacity for toxic shock syndrome formation. The pattern of one MRSA corresponded to those of the S. aureus group exhibiting phage pattern 94,96.
Conclusions: The prevalence of MRSA has increased in central Europe (and Germany) during the last 5 years, to 8.7%. The main source of infection with MRSA is obviously interregional dissemination of epidemic strains. At the same time, the mecA gene has been acquired by strains previously sensitive to methicillin.     

Observations on the resistance to drying of staphylococcal strains

Death rates have been determined for staphylococcal strains dried on cotton blanket material and stored at room temperature in the dark and in the light. Methicillin-resistant Staphylococcus aureus (MRSA) strains that produced a golden pigment and had a wide distribution within the hospital survived for longer periods than MRSA strains that produced little pigment and had a restricted local distribution. Death rates of methicillin-sensitive strains of S. aureus at day 7 were similar to those of the general epidemic MRSA strains, and there was no significant difference between the death rates at day 7 of the local epidemic MRSA strains and the coagulase-negative strains.     

Numerical analysis of electrophoretic protein patterns of methicillin-resistant strains of Staphylococcus aureus.

A total of 50 strains of Staphylococcus aureus, including 41 methicillin-resistant S. aureus (MRSA) strains, were characterized by one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis of whole-cell proteins. The protein patterns contained 40-50 discrete bands and were highly reproducible. Partial patterns were used as the basis of a computer-assisted numerical analysis. The MRSA strains clustered into four phenons at the 83% similarity level; and further division of phenon 1, at the 86% similarity level, resulted in a total of six clusters. All of the MRSA isolates from an MRSA epidemic in the United Kingdom were found to cluster in phenon 1 together with 9 of the 12 MRSA isolates from eastern Australia and 3 other MRSA isolates from the United Kingdom. The remaining three eastern Australian isolates clustered separately in phenon 2. Phenon 3 appeared to be exclusive to strains that were both susceptible and resistant to methicillin and that reacted with group V phages, and phenon 4 comprised 11 isolates, all of which were other MRSA isolates from the United Kingdom. We conclude that computer-assisted numerical analysis by high-resolution sodium dodecyl sulfate-polyacrylamide gel electrophoresis of whole-cell proteins provides additional criteria for the study of the epidemiology and the evolution of MRSA.     

Comparison of pulsed-field gel electrophoresis and PCR analysis of polymorphisms on the mec hypervariable region for typing methicillin-resistant Staphylococcus aureus

Two hundred fifty-four methicillin-resistant Staphylococcus aureus (MRSA) strains typed by pulsed-field gel electrophoresis (PFGE) were tested by PCR for the mec-associated hypervariable region (HVR-PCR) to determine their number of direct repeat units (DRUs). Eight different groups of repeats were found among the MRSA strains and compared to 28 pulsotypes classified by PFGE. Some MRSA strains belonging to the same pulsotype showed different numbers of DRUs. HVR-PCR was rapid, easy to perform, and reproducible and has the ability to obtain an unambiguous positive result for each isolate analyzed. However, this technique shows a discriminatory power inferior to that of PFGE. We conclude that PFGE is a more reliable method of typing MRSA than HVR-PCR.     

Two clones of methicillin-resistant Staphylococcus aureus in Poland

Objective: To evaluate relatedness among methicillin-resistant Staphylococcus aureus (MRSA) strains isolated in Poland.
Method: Ninety-three MRSA hospital isolates were collected from different regions in Poland from 1990 to 1992. Strains were analyzed with respect to heterogeneity of methicillin resistance, phage types, resistance patterns, crystal violet staining, chromosomal DNA Sma I restriction patterns by PFGE, ERIC1 and ERIC2 AP-PCR types and DNA repeat polymorphism within the protein A gene. Resistance to methicillin was confirmed by the detection of the mecA gene by PCR.
Results: The combined results of typing methods demonstrate that all MRSA strains analyzed could be easily divided into two distinct clones (clonally related strains). The first consisted of strains with clear heterogeneous expression of resistance to methicillin (34 isolates) and the second showed more homogeneous resistance (59 isolates). In this study the best method for epidemiologic analysis of MRSA was found to be PFGE. A good correlation between the epidemic behavior of MRSA and a high number of repetitive DNA units within the protein A gene was observed.
Conclusions: Results show that in Poland two distinct clones of epidemic MRSA have circulated in the past, easily discriminated by pheno and genotyping methods, and both could be found together in a single hospital.     

DNA sequence-based tandem repeat analysis of the clfB gene is less discriminatory than spa typing for methicillin-resistant Staphylococcus aureus

Methicillin-resistant Staphylococcus aureus (MRSA) are a major challenge for hospital hygiene. Typing by DNA sequence analysis of the repeat region of the protein A gene (spa) significantly improved typing of MRSA in the hospital setting. However, microevolution of spa repeats in epidemic clones appears to occur at a fairly slow clock rate. Therefore, DNA sequence-based methods providing additional resolution are desirable in some situations. We evaluated the use of the clfB repeat region proposed recently by others as a possible complementation to spa typing. Using epidemic MRSA isolates from two German university hospitals, we show that the clfB repeat region does not offer any additional discriminatory power.     

Susceptibility to antimicrobial agents and analysis of plasmids in gentamicin- and methicillin-resistant Staphylococcus aureus from Dublin hospitals

Methicillin- and gentamicin-resistant Staphylococcus aureus (MGRSA) strains isolated from Dublin Hospitals were classified into two groups (phenotypes). Phenotype-I strains expressed high level resistance to gentamicin and were susceptible to fusidic acid; strains resistant to tetracycline harboured a 3 X 10(6)-mol. wt plasmid. Strains in phenotype II usually expressed low level resistance to gentamicin, were resistant to fusidic acid and often harboured a (22-24) X 10(6)-mol. wt plasmid that specified resistance to ethidium bromide, tetracycline, kanamycin, neomycin and trimethoprim, or to combinations of these markers. A few phenotype-II strains expressed higher levels of resistance to gentamicin and other aminoglycosides. All MGRSA strains carried a 21 X 10(6)-mol. wt plasmid conferring resistance to penicillin, ethidium bromide, cadmium and mercury. Gentamicin resistance was invariably chromosomal and all strains carried chromosomal resistance to methicillin, erythromycin, streptomycin and spectinomycin. Several methicillin-resistant S. aureus (MRSA) strains isolated before the emergence of gentamicin resistance harboured a 21 X 10(6)-mol. wt penicillinase plasmid with the same restriction endonuclease profile as that from some MGRSA strains. Some MRSA strains carried other plasmids related to those found in MGRSA strains.     

An international multicenter study of antimicrobial resistance and typing of hospital Staphylococcus aureus isolates from 21 laboratories in 19 countries or states

During 1996, 4065 consecutive Staphylococcus aureus strains from different patients were collected in 21 worldwide hospital laboratories. The strains, their resistance pattern, and hospital demographic data were forwarded to Statens Serum Institut where the strains were typed and data analyzed. Resistance patterns varied by region and resistance to other antibiotics than methicillin were mainly related to the occurrence of methicillin resistance, except for mupirocin, rifampicin, and fusidic acid. Methicillin-resistant S. aureus (MRSA) occurred with low levels in hospitals in Northern Europe (<1%), increasing levels in middle-European countries, United States, New Zealand, and Australia (6-22%), and very high levels in Southern European countries as well as in parts of the United States, Asia, and South Africa (28-63%). MRSA found in large hospitals were more resistant to other antibiotics than MRSA found in smaller hospitals serviced by the same laboratory. No difference in resistance levels was seen for methicillin-susceptible S. aureus (MSSA) isolated in large or small hospitals. Intensive Care Units had the highest level of MRSA. Strains from the lower respiratory tract showed the highest resistance levels and blood isolates the lowest. A dominating MRSA clone was found in hospitals with an MRSA frequency of more than 10%. Pulsed-field gel electrophoresis (PFGE) typing recognized several of these clones as international epidemic MRSA (E-MRSA). All MSSA isolates were phage typed (typeability 85.4%) and divided in seven major phage patterns. Isolates of all patterns were found in all hospitals except one, indicating that the MSSA seldom represented the spread of clones within the hospital. The comparison should evaluate the prevalence of community-acquired MRSA and identify internationally E-MRSA. The present study gives a snapshot of the MRSA situation, but it is important to build up a continuous national and international surveillance, because MRSA is a global socioeconomic problem. Global infection control procedures, including rational antibiotic use, should be agreed on. The accompanying paper will address the issue of antibiotic consumption and MRSA.     

Comparison of epidemiological markers used in the investigation of an outbreak of methicillin-resistant Staphylococcus aureus infections.

An outbreak of nosocomial infections was caused by a single strain of methicillin-resistant (MR) Staphylococcus aureus. This strain was followed as it was transmitted from the index case to 17 patients, 3 hospital personnel, and 12 items in the hospital environment. The MR S. aureus strain was traced by using four specific epidemiological markers: antibiogram, phage type, production of aminoglycoside-inactivating enzymes, and plasmid pattern. These markers were assessed for their reliability in differentiating the epidemic S. aureus strain from resident nonepidemic strains and for the ease and rapidity with which they determined differences. The epidemic strain was resistant to beta-lactam antibiotics, gentamicin, erythromycin, clindamycin, and rifampin. Resistance to rifampin was the only unique marker in the antibiogram which distinguished the epidemic strain from the indigenous strains, and it was the easiest marker to use for screening isolates from culture surveys. Phage typing was poorly reproducible and did not yield results rapidly enough to be useful for ongoing epidemiology. The epidemic strain produced a unique aminoglycoside-inactivating enzyme (3'-phosphotransferase) which distinguished it from indigenous gentamicin-resistant staphylococci, but this marker was not easily identified, nor was identification helpful during the course of the investigation. Plasmid pattern analysis was rapidly performed (in less than 24 h), allowed many isolates to be examined at a time, was stable and reproducible, and yielded a unique fingerprint which distinguished the epidemic strain from all indigenous isolates. Plasmid pattern analysis is a promising epidemiological tool for MR S. aureus outbreaks in which epidemic strains lack unique antibiotic resistance markers.     

Emergence and spread of gentamicin-susceptible strains of methicillin-resistant Staphylococcus aureus in Belgian hospitals

The aim of this study was to follow the evolution of the clonal distribution and antimicrobial susceptibility of clinical strains of methicillin-resistant Staphylococcus aureus (MRSA) recovered from Belgian hospitals between 1995 and 1997-1998. MRSA strains were genotyped by inter-IS256 spacer length polymorphism PCR and SmaI macrorestriction analysis. MICs of 18 antimicrobials were determined by the agar dilution method. MRSA strains from the 1997-1998 survey were further tested by vancomycin screen agar, E-test, broth microdilution methods, and population analysis. Between 1995 and 1997-1998, epidemic group A strains decreased in proportion from 73% to 44%, whereas MRSA Group B and C strains increased from 17% to 38% and from 5% to 8%. The proportion of strains susceptible to gentamicin increased between the surveys from 22% to 48%. This was associated with a higher proportion of group B and C strains in the last survey. Heterogeneous vancomycin-intermediate S. aureus (hetero-VISA) strains were found in 2% isolates from 1997 to 1998. These hetero-VISA isolates were genotypically related to the MRSA group A strains and were resistant to gentamicin. In conclusion, two emerging epidemic MRSA genotypes, susceptible to gentamicin, have spread among Belgian hospitals during the 1990s. Hetero-VISA were present at low frequency among MRSA strains belonging to a widespread endemic genotype.     

Performance of CHROMagar MRSA medium for detection of methicillin-resistant Staphylococcus aureus

CHROMagar MRSA was evaluated for its ability to identify methicillin-resistant Staphylococcus aureus (MRSA). A well-defined collection consisting of 216 MRSA strains and 241 methicillin-susceptible Staphylococcus aureus isolates was used. The sensitivity of CHROMagar MRSA after 24 h of incubation was 95.4%, increasing to 100% after 48 h. The specificity was already 100% after 24 h.     

Molecular epidemiology of an outbreak caused by methicillin-resistant Staphylococcus aureus in a health care ward and associated nursing home

Our point-prevalence survey followed an outbreak of methicillin-resistant Staphylococcus aureus (MRSA) in a long-term care facility and identified five MRSA strains, of which two possessed an outbreak genotype not encountered previously and three had another profile. All of them possessed SCCmec type V. Six methicillin-sensitive S. aureus strains were genotypically related to the epidemic strains.