SEARCH

SEARCH BY CITATION

Keywords:

  • Evolution;
  • MLST;
  • MRSA;
  • multilocus sequence typing;
  • Staphylococcus aureus

Abstract

  1. Top of page
  2. Abstract
  3. Background
  4. Models of mrsa evolution
  5. Tools for investigating clonal spread
  6. Mlst
  7. SCCmec TYPING
  8. Mrsa nomenclature
  9. Origins of the first mrsa
  10. How many mrsa clones are there?
  11. Acknowledgements
  12. References

The prevalence of methicillin-resistant Staphylococcus aureus (MRSA) in many countries is increasing and, in hospitals in some areas, more than half of all S. aureus disease isolates are MRSA. MRSA strains are becoming increasingly multiresistant, and have recently developed resistance to vancomycin, used successfully to treat MRSA for more than 30 years. This review summarises recent studies that have elucidated the evolutionary history of MRSA. The first MRSA isolate evolved from a sensitive, epidemic strain prevalent in Europe, and its progeny—the first MRSA clone—quickly spread to other continents. Analyses of epidemic MRSA isolates from hospitals in different countries by molecular methods, including multilocus sequence typing (MLST) and DNA microarray analysis, reveal that MRSA strains have evolved separately within five distinct epidemic, sensitive lineages. However, resistance has been transferred to S. aureus on many more than five occasions, as some lineages have acquired different structural types of the element carrying the methicillin resistance gene. The emergence of MRSA as a community pathogen has been noted in several countries, and MLST and SCCmec typing have been used to demonstrate that community-acquired MRSA strains are typically related only distantly to hospital MRSA strains, and thus represent novel acquisitions of SCCmec.


Background

  1. Top of page
  2. Abstract
  3. Background
  4. Models of mrsa evolution
  5. Tools for investigating clonal spread
  6. Mlst
  7. SCCmec TYPING
  8. Mrsa nomenclature
  9. Origins of the first mrsa
  10. How many mrsa clones are there?
  11. Acknowledgements
  12. References

Nosocomial methicillin-resistant Staphylococcus aureus (MRSA) infections represent a major challenge to hospital microbiologists because of the emergence and spread of clones with decreased susceptibility to many antibiotic classes. Since the mid to late 1990s, hospital MRSA isolates have increased in prevalence in Europe, the USA and elsewhere [1,2]. In one European study of 25 university hospitals [3], one-quarter of 3051 S. aureus isolates collected were MRSA, with a geographical bias towards higher rates in southern countries such as Italy (50.5%) and Portugal (54%), and lower rates in northern European countries, including The Netherlands (2%), Austria (9%) and Switzerland (2%). MRSA infections are associated with increased morbidity, mortality and length of hospital stay, and represent a major financial burden on healthcare services [4,5].

The first strain of MRSA was isolated in 1961 [6], 2 years after the introduction of methicillin; this strain rapidly spread to other countries throughout the 1960s, and became a problem in the USA in the 1970s. The antibiotic of choice for treating MRSA infections is the glycopeptide vancomycin, but reports of vancomycin intermediately susceptible S. aureus (VISA), first isolated in Japan in 1997 [7,8], caused widespread alarm among physicians fearful of an era of untreatable MRSA infections. Reports of VISA isolates with an MIC ≥ 8 mg/L have so far been very rare, but two recent reports of fully vancomycin-resistant S. aureus (VRSA) from Michigan [9] and Pennsylvania [10] (MICs of 32 and 128 mg/L, respectively) in the USA have again caused alarm, and it is as yet unclear whether either VISA or VRSA isolates will become epidemic, leading to an exacerbation of the global MRSA problem.

In a climate of increasing S. aureus antibiotic resistance, the study of MRSA epidemiology has assumed new importance, because any strategies to contain the spread of MRSA at the local (hospital), national or international level require knowledge of how strains are spread and how MRSA epidemics occur. Epidemiological studies can be used to provide basic knowledge of the population biology of MRSA, and can help to answer fundamental questions such as: (1) how strains spread; (2) the number of major MRSA, VISA and VRSA clones circulating globally, and their relatedness to each other and to susceptible isolates; and (3) the ancestry of modern MRSA, VISA and VRSA strains. The answers to these questions have, until recently, been unclear, but several recent studies employing modern molecular typing technologies have now significantly increased our knowledge in these areas.

Models of mrsa evolution

  1. Top of page
  2. Abstract
  3. Background
  4. Models of mrsa evolution
  5. Tools for investigating clonal spread
  6. Mlst
  7. SCCmec TYPING
  8. Mrsa nomenclature
  9. Origins of the first mrsa
  10. How many mrsa clones are there?
  11. Acknowledgements
  12. References

Two models of MRSA evolution were initially proposed in the early 1990s, based on studies using different typing techniques. The simplest model, described by Kreiswirth et al.[11], was based on analysis of restriction fragment length polymorphisms generated by ClaI digestion of chromosomal DNA, followed by hybridisation with Tn554 and mecA probes. The limited number of patterns observed in a geographical and temporally diverse sample of MRSA isolates was taken as evidence that S. aureus had acquired the methicillin resistance gene (mecA) on only one occasion, and the authors therefore hypothesised that all extant MRSA clones were recent descendants of this prototypical isolate.

Several months before the publication of this study, Musser and Kapur [12] described MRSA as being polyclonal in a multilocus enzyme electrophoresis analysis of 254 MRSA isolates. The association of mecA with divergent genetic backgrounds was taken as strong evidence that the gene is transferred horizontally between S. aureus isolates. The only alternative explanation for these results was that MRSA isolates had diversified so rapidly in the 31 years between 1961 and 1992 that they had lost any genetic similarity. The existence of modern MRSA lineages that are unrelated to the first MRSA strain by molecular typing methodologies supports the theory of Musser and Kapur, and this is further strengthened by evidence from microarray analysis [13] and multilocus sequence typing (MLST) [14] that conclusively demonstrate horizontal movement of the mecA gene.

Tools for investigating clonal spread

  1. Top of page
  2. Abstract
  3. Background
  4. Models of mrsa evolution
  5. Tools for investigating clonal spread
  6. Mlst
  7. SCCmec TYPING
  8. Mrsa nomenclature
  9. Origins of the first mrsa
  10. How many mrsa clones are there?
  11. Acknowledgements
  12. References

The recovery of isolates with identical bacteriophage types from different hospitals within and between countries was described in the 1950s in seminal studies by Rountree and Freeman [15] and Rountree and Beard [16], in which the existence of S. aureus types with increased epidemicity was demonstrated. However, bacteriophage typing has fallen out of favour as a means of characterising S. aureus, because of difficulties in typeability and reproducibility, as well as the cryptic genetic basis upon which characterisation relies [17,18].

Molecular typing techniques are commonly used to study the epidemiology of S. aureus. The international spread of epidemic clones, such as the Iberian [19], UK epidemic [20,21], New York–Japanese [22,23], Viennese [24] and German MRSA [24], has been investigated with a variety of techniques, the most popular of which has been pulsed-field gel electrophoresis (PFGE) [25,26]. PFGE is suitable for MRSA outbreak investigation because of the high level of discrimination attainable, which allows outbreak isolates to be separated from unrelated isolates. The main drawback of such simple and widely used ‘band-based’ technologies in studying longer-term, national or global epidemiology is the difficulty faced in making inter-laboratory comparisons of PFGE data. Standardisation of reagents and electrophoresis conditions increases the portability of PFGE, but subjective decisions about DNA banding pattern similarity still constitute a major barrier to establishing PFGE as a satisfactory method for characterising S. aureus clones.

Mlst

  1. Top of page
  2. Abstract
  3. Background
  4. Models of mrsa evolution
  5. Tools for investigating clonal spread
  6. Mlst
  7. SCCmec TYPING
  8. Mrsa nomenclature
  9. Origins of the first mrsa
  10. How many mrsa clones are there?
  11. Acknowledgements
  12. References

MLST [27] involves sequencing DNA fragments (typically c. 500 bp) of seven housekeeping genes. The sequences of these genes are compared to known alleles at each locus via the MLST website (http://www.mlst.net), where every isolate is described by a seven-integer allelic profile that defines a sequence type (ST). For example, isolates of the Iberian clone have the MLST profile 3-3-1-12-4-4-16, which defines ST247. MLST was first applied to S. aureus in a study published in 2000 [28], in which 155 invasive S. aureus isolates were typed. In addition to validating the method against PFGE, the study showed how epidemic clones of MRSA and methicillin-susceptible S. aureus (MSSA) could be unambiguously defined by their ST.

SCCmec TYPING

  1. Top of page
  2. Abstract
  3. Background
  4. Models of mrsa evolution
  5. Tools for investigating clonal spread
  6. Mlst
  7. SCCmec TYPING
  8. Mrsa nomenclature
  9. Origins of the first mrsa
  10. How many mrsa clones are there?
  11. Acknowledgements
  12. References

The methicillin resistance structural gene mecA is a small (2007 bp) part of a much larger genetic element which is inserted precisely into the S. aureus chromosome. This staphylococcal chromosomal cassette mec (SCCmec) varies in size from c. 20 to 68 kb, but always contains mecA and at least part of a regulatory gene mecR1 and chromosomal cassette recombinase genes (ccr). Four main types of SCCmec have been described [29,30], and although the same types are often associated with divergent lineages, particular MRSA clones are associated with single SCCmec elements. For example, all epidemic MRSA (EMRSA) clone 16 (ST36) isolates from the UK have SCCmec type II (http://www.mlst.net).

Mrsa nomenclature

  1. Top of page
  2. Abstract
  3. Background
  4. Models of mrsa evolution
  5. Tools for investigating clonal spread
  6. Mlst
  7. SCCmec TYPING
  8. Mrsa nomenclature
  9. Origins of the first mrsa
  10. How many mrsa clones are there?
  11. Acknowledgements
  12. References

A report on 912 MRSA and MSSA isolates from 20 different countries [14] contained a proposal that MRSA clones be named according to their MLST and SCCmec types in the form ST–resistance phenotype (i.e., MRSA, MSSA, VISA or VRSA)–SCCmec type (i.e., I, II, III or IV). For example, the Iberian clone would be known as ST247-MRSA-I. This was agreed by a subcommittee of the International Union of Microbiology Societies in Tokyo, 2002. It is hoped that this nomenclature will replace, or at least supplement, existing arbitrary designations of MRSA clones, based on geographical location or other less satisfactory typing methods, since MLST is systematic and objective, and provides a key for investigators to search for clones in the MLST website databases.

Origins of the first mrsa

  1. Top of page
  2. Abstract
  3. Background
  4. Models of mrsa evolution
  5. Tools for investigating clonal spread
  6. Mlst
  7. SCCmec TYPING
  8. Mrsa nomenclature
  9. Origins of the first mrsa
  10. How many mrsa clones are there?
  11. Acknowledgements
  12. References

The first study to examine the ancestry of the original MRSA isolates used MLST to compare MRSA isolates from different countries with a collection of MSSA isolates from the 1950s [31]. The study showed that the first MRSA clone belongs to ST250 (allelic profile 3-3-1-1-4-4-16), which is a genotype shared with epidemic MSSA isolates common in Denmark in the 1950s. This ‘archaic’ clone was found to be ancestral to the Iberian clone [19], as it shares six of seven housekeeping gene alleles. The Iberian clone is a modern pandemic MRSA that shares the high epidemicity of the ‘archaic’ clone, a genotype that spread extremely rapidly after its first emergence in 1961 and was isolated as recently as 1993 (http://www.mlst.net).

The origins of early MRSA isolates were further clarified in the larger-scale MLST study described above [14]. In this work, the authors used MLST and SCCmec typing to show the complexity of evolutionary events leading to the emergence of the first MRSA isolate and modern epidemic MRSA and MSSA clones from an ancestral isolate of ST8 (MSSA) (Fig. 1). The designation of ST8 as the ancestral genotype was achieved using the BURST algorithm (based upon related sequence types; http://155.198.40.150/new/data_analysis/burst/burst.htm), which separates MLST data sets into groups called clonal complexes (CCs). These share at least five housekeeping gene alleles in common with at least one other member. For each ST in the group, the ancestor is assumed to be the genotype with the largest number of variants differing at only one gene. The assignment of ST8-MSSA as the ancestor of ST250-MSSA is supported by the finding that ST8 and ST250 differ at a single locus whose alleles are identical except for a point mutation in yqiL, unique to ST250 and its descendants [14]. Further support for this hypothesis is the finding that all isolates of ST250 and its descendants are MRSA with SCCmec class I. Fig. 1 shows an evolutionary scenario that could have led to the emergence of major clones such as ST239-MRSA-III (named variously as UK EMRSA-1, UK EMRSA-4, Viennese clone and the Portuguese/Brazilian clone [32]) and ST247-MRSA-I (the Iberian clone, UK EMRSA-5 and UK EMRSA-17 [33]), among others.

image

Figure 1. Evolutionary origins of notable CC8 genotypes. Arrows indicate either (1) acquisition of SCCmec, or (2) change of ST. For some epidemic MRSA clones, the name used previously in the literature is included in parentheses.

Download figure to PowerPoint

How many mrsa clones are there?

  1. Top of page
  2. Abstract
  3. Background
  4. Models of mrsa evolution
  5. Tools for investigating clonal spread
  6. Mlst
  7. SCCmec TYPING
  8. Mrsa nomenclature
  9. Origins of the first mrsa
  10. How many mrsa clones are there?
  11. Acknowledgements
  12. References

Analysis of a collection of 36 S. aureus isolates by DNA microarray analysis [13] showed that the mecA gene was associated with five genetically divergent groups of isolates, indicating that the gene for methicillin resistance has been horizontally transferred at least five times in S. aureus. This finding was confirmed by an MLST study [14] which showed that all epidemic hospital MRSA isolates that have been found in more than one country belong to five CCs. These CCs are named according to the ST of their proposed ancestor, and include CC8 (archaic MRSA), CC5, which contains most of the VISA isolates studied to date, and three clonal complexes (CC45, CC30 and CC22) that contain recently emerged international MRSA clones such as UK EMRSA-16 (CC30) [34], Berlin epidemic MRSA (CC45) [35], and UK EMRSA-15 or Barnim epidemic MRSA [36].

Major MRSA clones have emerged from some CCs on multiple occasions, resulting in isolates with the same MLST type that differ in SCCmec type. Enright et al.[14] described 11 major epidemic MRSA clones, defined as genotypes (same MLST and SCCmec type), found in more than one country and represented by at least ten isolates. This arbitrary designation contains all the epidemic MRSA isolates described in the literature, but the isolates examined are over-representative of European countries. What is clear from MLST studies is that a small number of ecologically successful genetic backgrounds can acquire the methicillin resistance gene and retain a high level of epidemicity. This is demonstrated by the fact that in each of the five MRSA-containing CCs, the ancestral genotype is represented by recent disease-causing MSSA isolates (http://www.mlst.net).

MRSA strains are emerging as a cause of community-acquired disease in some countries but, until recently, the genetic relatedness of such strains to each other and to hospital MRSA isolates was not known. Analysis of 47 community MRSA isolates from Australia and the USA [37] by MLST and SCCmec typing showed that some types are closely related to hospital MRSA, but that most isolates characterised had STs not found in studies of hospital MRSA to date. Isolates of ST1, the most common genotype in this study, and ST30 have been reported as common causes of community-acquired MSSA disease in the past [28], but the acquisition of SCCmec IV by these successful epidemic genotypes is worrying, especially as in this study isolates of these clones expressed Panton–Valentine leukocidin, which in one study was found to be associated with necrotising pneumonia in patients with a much younger average age than is usual for pneumonia [38]. This expansion of the host range of isolates causing MRSA disease to the community and to younger age groups is troubling at a time when the therapeutic options for the treatment of MRSA are becoming increasingly limited.

References

  1. Top of page
  2. Abstract
  3. Background
  4. Models of mrsa evolution
  5. Tools for investigating clonal spread
  6. Mlst
  7. SCCmec TYPING
  8. Mrsa nomenclature
  9. Origins of the first mrsa
  10. How many mrsa clones are there?
  11. Acknowledgements
  12. References
  • 1
    Anonymous. European Antimicrobial Resistance Surveillance System (EARSS) annual report 2001. Bilthoven: National Institute of Public Health and the Environment, 2001.
  • 2
    Ayliffe GA. The progressive intercontinental spread of methicillin-resistant Staphylococcus aureus. Clin Infect Dis 1997; 24(suppl 1): S74S79.
  • 3
    Fluit AC, Wielders CL, Verhoef J, Schmitz FJ. Epidemiology and susceptibility of 3,051 Staphylococcus aureus isolates from 25 university hospitals participating in the European SENTRY study. J Clin Microbiol 2001; 39: 37273732.
  • 4
    Whitby M, McLaws ML, Berry G. Risk of death from methicillin-resistant Staphylococcus aureus bacteraemia: a meta-analysis. Med J Aust 2001; 175: 264267.
  • 5
    Ribner BS, Landry MN, Kidd K, Peninger M, Riddick J. Outbreak of multiply resistant Staphylococcus aureus in a pediatric intensive care unit after consolidation with a surgical intensive care unit. Am J Infect Control 1989; 17: 244249.
  • 6
    Jevons MP. Celbenin-resistant staphylococci. BMJ 1961; 1: 124125.
  • 7
    Hiramatsu K, Hanaki H, Ino T, Yabuta K, Oguri T, Tenover FC. Methicillin-resistant Staphylococcus aureus clinical strain with reduced vancomycin susceptibility. J Antimicrob Chemother 1997; 40: 135136.
  • 8
    Centers for Disease Control. Staphylococcus aureus with reduced susceptibility to vancomycin. MMWR 1997; 46: 765766.
  • 9
    Centers for Disease Control. Staphylococcus aureus resistant to vancomycin. MMWR 2002; 51: 565567.
  • 10
    Centers for Disease Control. Public health dispatch: vancomycin-resistant Staphylococcus aureus. MMWR 2002; 51: 902.
  • 11
    Kreiswirth B, Kornblum J, Arbeit RD et al. Evidence for a clonal origin of methicillin resistance in Staphylococcus aureus. Science 1993; 259: 227230.
  • 12
    Musser JM, Kapur V. Clonal analysis of methicillin-resistant Staphylococcus aureus strains from intercontinental sources: association of the mec gene with divergent phylogenetic lineages implies dissemination by horizontal transfer and recombination. J Clin Microbiol 1992; 30: 20582063.
  • 13
    Fitzgerald JR, Sturdevant DE, Mackie SM, Gill SR, Musser JM. Evolutionary genomics of Staphylococcus aureus: insights into the origin of methicillin-resistant strains and the toxic shock syndrome epidemic. Proc Natl Acad Sci USA 2001; 98: 88218826.
  • 14
    Enright MC, Robinson DA, Randle G, Feil EJ, Grundmann H, Spratt BG. The evolutionary history of methicillin-resistant Staphylococcus aureus (MRSA). Proc Natl Acad Sci USA 2002; 99: 76877692.
  • 15
    Rountree PM, Freeman BM. Infections caused by a particular phage type of Staphylococcus aureus. Med J Aust 1955; 2: 157.
  • 16
    Rountree PM, Beard MA. Further observations on infection with phage type 80 staphylococci in Australia. Med J Aust 1958; 2: 789795.
  • 17
    Bannerman TL, Hancock GA, Tenover FC, Miller JM. Pulsed-field gel electrophoresis as a replacement for bacteriophage typing of Staphylococcus aureus. J Clin Microbiol 1995; 33: 551555.
  • 18
    Weller TM. Methicillin-resistant Staphylococcus aureus typing methods: which should be the international standard? J Hosp Infect 2000; 44: 160172.
  • 19
    Sanches IS, Ramirez M, Troni H et al. Evidence for the geographic spread of a methicillin-resistant Staphylococcus aureus clone between Portugal and Spain. J Clin Microbiol 1995; 33: 12431246.
  • 20
    Marples RR, Cooke EM. Workshop on methicillin-resistant Staphylococcus aureus held at the headquarters of the Public Health Laboratory Service on 8 January 1985. J Hosp Infect 1985; 6: 342348.
  • 21
    Kerr S, Kerr GE, Mackintosh CA, Marples RR. A survey of methicillin-resistant Staphylococcus aureus affecting patients in England and Wales. J Hosp Infect 1990; 16: 3548.
  • 22
    Ito T, Katayama Y, Hiramatsu K. Cloning and nucleotide sequence determination of the entire mec DNA of pre-methicillin-resistant Staphylococcus aureus N315. Antimicrob Agents Chemother 1999; 43: 14491458.
  • 23
    Oliveira DC, Tomasz A, De Lencastre H. The evolution of pandemic clones of methicillin-resistant Staphylococcus aureus: identification of two ancestral genetic backgrounds and the associated mec elements. Microb Drug Resist 2001; 7: 349361.
  • 24
    Witte W. Antibiotic resistance in gram-positive bacteria: epidemiological aspects. J Antimicrob Chemother 1999; 44(suppl A): 19.
  • 25
    Tenover FC, Arbeit RD, Goering RV et al. Interpreting chromosomal DNA restriction patterns produced by pulsed-field gel electrophoresis: criteria for bacterial strain typing. J Clin Microbiol 1995; 33: 22332239.
  • 26
    Chung M, De Lencastre H, Matthews P et al. Molecular typing of methicillin-resistant Staphylococcus aureus by pulsed-field gel electrophoresis: comparison of results obtained in a multilaboratory effort using identical protocols and MRSA strains. Microb Drug Resist 2000; 6: 189198.
  • 27
    Maiden MC, Bygraves JA, Feil E et al. Multilocus sequence typing: a portable approach to the identification of clones within populations of pathogenic microorganisms. Proc Natl Acad Sci USA 1998; 95: 31403145.
  • 28
    Enright MC, Day NP, Davies CE, Peacock SJ, Spratt BG. Multilocus sequence typing for characterization of methicillin-resistant and methicillin-susceptible clones of Staphylococcus aureus. J Clin Microbiol 2000; 38: 10081015.
  • 29
    Ito T, Katayama Y, Asada K et al. Structural comparison of three types of staphylococcal cassette chromosome mec integrated in the chromosome in methicillin-resistant Staphylococcus aureus. Antimicrob Agents Chemother 2001; 45: 13231336.
  • 30
    Ma XX, Ito T, Tiensasitorn C et al. Novel type of staphylococcal cassette chromosome mec identified in community-acquired methicillin-resistant Staphylococcus aureus strains. Antimicrob Agents Chemother 2002; 46: 11471152.
  • 31
    Crisostomo MI, Westh H, Tomasz A, Chung M, Oliveira DC, De Lencastre H. The evolution of methicillin resistance in Staphylococcus aureus: similarity of genetic backgrounds in historically early methicillin-susceptible and -resistant isolates and contemporary epidemic clones. Proc Natl Acad Sci USA 2001; 98: 98659870.
  • 32
    De Sousa MA, Sanches IS, Ferro ML et al. Intercontinental spread of a multidrug-resistant methicillin-resistant Staphylococcus aureus clone. J Clin Microbiol 1998; 36: 25902596.
  • 33
    Aucken HM, Ganner M, Murchan S, Cookson BD, Johnson AP. A new UK strain of epidemic methicillin-resistant Staphylococcus aureus (EMRSA-17) resistant to multiple antibiotics. J Antimicrob Chemother 2002; 50: 171175.
  • 34
    Anonymous. Epidemic methicillin resistant Staphylococcus aureus. Commun Dis Rep Wkly 1997; 7: 1.
  • 35
    Witte W, Klare I, Werner G. Selective pressure by antibiotics as feed additives. Infection 1999; 27(suppl 2): S35S38.
  • 36
    Witte W, Enright M, Schmitz FJ, Cuny C, Braulke C, Heuck D. Characteristics of a new epidemic MRSA in Germany ancestral to United Kingdom EMRSA 15. Int J Med Microbiol 2001; 290: 677682.
  • 37
    Okuma K, Iwakawa K, Turnidge JD et al. Dissemination of new methicillin-resistant Staphylococcus aureus clones in the community. J Clin Microbiol 2002; 40: 42894294.
  • 38
    Gillet Y, Issartel B, Vanhems P et al. Association between Staphylococcus aureus strains carrying gene for Panton–Valentine leukocidin and highly lethal necrotising pneumonia in young immunocompetent patients. Lancet 2002; 359: 753759.