Antibiotic resistant Staphylococcus aureus: a paradigm of adaptive power

https://doi.org/10.1016/j.mib.2007.08.003Get rights and content

Nothing documents better the spectacular adaptive capacity of Staphylococcus aureus than the response of this important human and animal pathogen to the introduction of antimicrobial agents into the clinical environment. The effectiveness of penicillin introduced in the early 1940s was virtually annulled within a decade because of the plasmid epidemics that spread the ß-lactamase gene through the entire species of S. aureus. In 1960 within one to two years of the introduction of penicillinase resistant ß-lactams (methicillin), methicillin resistant S. aureus (MRSA) strains were identified in clinical specimens. By the 1980s, epidemic clones of MRSA acquired multidrug resistant traits and spread worldwide to become one of the most important causative agents of hospital acquired infections. In the early 2000s, MRSA strains carrying the Tn1546 transposon-based enterococcal vancomycin resistant mechanism were identified in clinical specimens, bringing the specter of a totally resistant bacterial pathogen closer to reality. Then, in the late 1990s, just as effective hygienic and antibiotic use policies managed to bring down the frequency of MRSA in hospitals of several countries, MRSA strains began to show up in the community.

Introduction

The purpose of this brief overview is to generate interest among readers in MRSA—a fascinating and dangerous human pathogen continually evolving under our eyes to cope with a constantly changing human environment. In this overview, a special emphasis was paid to the unique staphylococcal chromosome cassettes the structure of which plays a critical role in the epidemiology of both the hospital associated and community acquired MRSA.

The globally spread epidemic clones of MRSA are the product of convergence of several unusual evolutionary processes [1].

  • (i)

    The resistance mechanism itself is unusual: it involves acquisition of the mecA gene, the determinant of a unique penicillin binding protein PBP2A which has low affinity for ß-lactam antibiotics and which can function as a surrogate of the native staphylococcal PBPs – cell wall synthetic enzymes – that are inactivated by ß-lactam antibiotics [2].

  • (ii)

    The resistance gene mecA appears to have evolved from a domestic gene of the fully ß-lactam susceptible Staphylococcus sciuri, a frequent colonizer of the skin of both wild and domestic animals and one of the most abundant staphylococcal species on this planet [3].

  • (iii)

    Before ‘arriving’ to S. aureus the mecA gene has to be incorporated into a unique molecular vector called the staphylococcal chromosome cassette (SCC) which has its own independent evolutionary history and which is capable of delivering a variety of different resistance or virulence determinants to S. aureus, including mecA.

  • (iv)

    Successful ‘grafting’ of the SCCmec into an S. aureus cell requires still additional factors: a unique and apparently rare genetic background that allows maintenance and expression of mecA. The relatively few successful SCCmec acquisition events that give rise to an MRSA strain appear to require the absence of a genetic ‘barrier’ that is frequent in the genetic background of many S. aureus lineages [4].

  • (v)

    Of the MRSA strains that have managed to maintain and express the SCCmec, only a handful of lineages seem to be able to spread globally. Genetic determinants that define the superior epidemicity of these clones are not known at the present time but may be associated with genes that are responsible for the effective colonization of the host [5].

For the first three decades after their appearance, MRSA strains typically have remained hospital associated pathogens (HA-MRSA). Then, in an unexpected epidemiological ‘move’, MRSA strains also began to appear in the community among people who had none of the usual risk factors for such infections. Such community acquired MRSA (CA-MRSA) has since been recognized as a major and disturbing reality in many countries, but, the precise scenario that has led to its appearance is still a matter of speculation.

Screening of isolates from different staphylococcal species with a DNA probe internal to the mecA of MRSA has led to a staphylococcal species, S. sciuri in which each one of up to 200 epidemiologically unrelated isolates gave strong hybridization signal [3]. The large majority of these isolates were fully susceptible to all ß-lactam antibiotics including penicillin. The gene homologue of mecA was identified as a determinant showing linear structure and conserved motifs typical of a bacterial penicillin binding protein [6], with a transpeptidase (TPase) domain that showed 92% identity with the aminoacid sequence of the TPase domain of mecA from MRSA strains. Recent work identified the mecA homologue as the genetic determinant of an 84 kDa PBP in S. sciuri. Upregulation of the promoter of this gene was shown to provide wide spectrum ß-lactam resistance to the S. sciuri strain and transfer of the upregulated S. sciuri mecA homologue on a plasmid to a fully ß-lactam susceptible S. aureus caused substantial increase in the oxacillin MIC value of the S. aureus recipient (Zhou et al., in preparation). It was also possible to show that the plasmid-born S. sciuri gene was able to participate in the synthesis of peptidoglycan of the S. aureus transductant growing in the presence of ß-lactam antibiotics and under these conditions, the S. sciuri mecA homologue produced a peptidoglycan the composition of which was typical of the host strain S. aureus and was completely different from the peptidoglycan of S. sciuri [7].

More recently, the protein product of the S. sciuri mecA homologue was purified to homogeneity and compared to the properties of PBP2A, the product of the S. aureus mecA. The two proteins showed very similar properties which included inhibition by a wide range of ß-lactam antibiotics; the existence of an allosteric site for binding of S. aureus peptidoglycan precursors; both PBP2A and the S. sciuri protein contained a sheltered active site, the access of which to both substrates and ß-lactam inhibitors required a conformational change in the proteins [8].

The first MRSA type mechanism was identified in clinical specimens within a very short – one to two years – period after the introduction of the first penicillin resistant – oxazolidine type – ß-lactam into clinical use. Given the complexity of the mecA-based resistance mechanism, the notion that MRSA emerged under the selective pressure of these new ß-lactamase resistant antibiotics is unlikely. It was proposed recently that the evolution of mecA may have occurred over a much longer span of time in a staphylococcal species free of penicillinase under the selective pressure of penicillin [1] which began to be used extensively as a prophylactic agent in veterinary medicine in 1949, that is, shortly after the introduction of penicillin into human clinical practice [9]. The staphylococcal species involved may have been S. sciuri, which is free of the penicillinase plasmid and is a very frequent inhabitant of the skin of domestic animals. In this scenario, an upregulated version of the native S. sciuri mecA homologue may have emerged among colonizers of the skin of domestic animals under the selective pressure of prophylactic use of penicillin.

The mecA gene, is embedded in a large heterologous chromosomal island—staphylococcal cassette chromosome (SCCmec) [10], that integrates into the S. aureus chromosome at a site-specific location (attBscc), located near the origin of replication [11]. The genetic organization of the close mecA vicinity defines the mec gene complex and, in S. aureus, three major classes have been described: class A containing the complete mecA regulon (mecI-mecR1-mecA) and classes B and C containing the mecA regulatory genes disrupted by insertion sequences, ΨIS1272mecR1-mecA and IS431mecR1-mecA, respectively [12].

The mobility of SCCmec is in part because of the presence of fully functional recombinases of the invertase/resolvase family and encoded by the ccr gene complex [13]. The ccr gene complex may have two genes, ccrAB, with four known allotypes [14, 15], or a single gene not closely related to the ccrAB genes, ccrC [16].

SCCmec types are defined by combining the class of the mec gene complex with the ccr allotype [10, 14, 16, 17, 18] and on the basis of this definition a new nomenclature has been recently proposed [19], so that SCCmec type definitions are as follows (proposed new names in parenthesis): type I—mec class B and ccrAB allotype 1 (1B); type II—class A and ccrAB2 (2A); type III—class A and ccrAB3 (3A); type IV—class B and ccrAB2 (2B); type V—class C and ccrC (5C); and type VI—class B and ccrAB4 (4B).

The remaining parts of SCCmec are called junkyard (J) regions (J1, J2 and J3), which constitute nonessential components of SCCmec; although, in some cases, these regions carry additional antibiotic resistance determinants [20]. Starting clockwise from the origin of replication of S. aureus chromosome, the J3 is the region between the chromosomal left junction and the mec complex; J2 is the region between the mec complex and the ccr complex; and J1 is the region between the ccr complex and the chromosomal right junction. Therefore, SCCmec structural organization may be summarized as: J3-mec-J2-ccr-J1—see Figure 1. Variations in the J regions (within the same mec-ccr combination) are used for defining SCCmec subtypes or variants.

CA-MRSA are predominantly characterized by SCCmec type IV or by type V [21] (the later described only in 2004 [16]. SCCmec type IV is the smallest structural type of SCCmec and is believed to be the most mobile version [22]. Perhaps as a consequence of its enhanced mobility, SCCmec type IV is also more variable than other SCCmec types and eight subtypes (types IVa through IVh) differing mainly in the J1 region have been described so far [17, 20, 23, 24, 25]. Besides frequently associated to CA-MRSA, SCCmec type IV is also characteristic of some HA-MRSA clones, such as the EMRSA-15, an endemic clone in the UK, which is also spreading in several European countries [26].

For the sake of the extension of this review we will focus on SCCmec typing strategies based on multiplex PCR assays, which are very practical and, as consequence, the most commonly used. Multiplex PCR approaches have a limited number of interrogated loci which may restrict the resolution of the typing strategy but, as they came in different flavors, they can cover different discriminatory needs. Still, no single multiplex PCR reaction can include the 22 out of 34 binary targets required, according to Stephens et al. [27], to genotype properly all the 46 SCCmec variants.

In 2006, Kondo et al. have proposed a SCCmec typing scheme based on six multiplex PCR reactions [28]. This strategy is very complete and while being able to detect the great majority of SCCmec it also identifies new ones. Still, due the high number of required assays it may not be feasible for routine purposes.

The choice of the best SCCmec typing strategy depends on the purpose of the study and its setting, such as local versus global surveillance of MRSA clones, clinical or research laboratory, and so on. A flexible and feasible SCCmec typing scheme that could provide suitable discriminatory power for most study purposes would have three (sequential) stages and the following design.

  • (i)

    ccrB sequencing [29]. This tool, based on the DNA sequencing of an internal fragment of the ccrB gene amplified using a single pair of degenerate primers has the advantage to be easily integrated into the spa and MLST protocols, and, likewise, is amenable for data deposition and analysis in a central web-based database, which is currently being developed (http://www.ccrbtyping.net/). The sequence data generated may be used for tracing the dissemination of SCCmec elements within the MRSA population or even between S. aureus and coagulase-negative staphylococci (CoNS).

  • (ii)

    ‘SCCmec multiplex PCR’ [30]. This strategy is able to identify in a single multiplex PCR assay using 20 primers SCCmec types I–VI, which are assigned through the presence of a specific amplification pattern of 2–5 bands.

  • (iii)

    ‘SCCmec IV multiplex PCR’ [24]. This is a multiplex PCR assay for the subtyping of SCCmec type IV strains. SCCmec type IV is highly polymorphic and so far eight variants, differing mainly in the J1 region, have been described (IVa–IVh). Since type IV predominates among CA-MRSA, subtyping this SCCmec element may be critical to trace properly the dissemination of particular MRSA clones. Under this rationale, we have recently developed a single multiplex PCR assay based on the detection of subtype specific J1 sequences able to discriminate most subtypes of SCCmec IV—see Figure 2.

The SCC element, defined as a mobile chromosomal cassette with dedicated recombinase genes (ccr) and characteristic flanking short sequence repeats, is not restricted to the dissemination of the mecA gene, as several non-mec SCC and ψSCC (without or no functional ccr genes) elements have been described carrying other genetic determinants (for a recent review see [31]). Some of these non-mec SCC elements encode for traits that may contribute to the survival or pathogenic potential of the bacteria, such as resistance to heavy metals (SCC mer [19] and SCC CI [32]) or fusidic acid (SCC MSSA476 [33]), capsule biosynthesis (SCC cap1 [34] and SCC 15305cap [35]), potassium transport (ψSCC h1 [36]), cell wall cross-linking (pbb4) and teichoic acid biosynthesis (tagF) (SCCpbp4 [32]), DNA protection by restriction-modification systems (SCC CI [32]), or arginine deiminase and oligopeptide permease (arginine catabolic mobile element, ψSCC ACME [37]). Many of these non-mec SCC or ψSCC elements have been described in coagulase-negative staphylococci (CoNS), a finding which further implicates these species as responsible for the assembling and dissemination of SCC elements, including SCCmec.

The ACME element is a striking example of SCC dissemination routes and also how these elements may contribute to the phenotypic plasticity of S. aureus clones. In S. epidermidis, a ubiquitous commensal of the human skin, the ACME element is highly prevalent, whereas in S. aureus it is restricted to a single lineage circulating mainly in the USA community but also in Canada and Europe (CA-MRSA clone USA300, ST8-IVa) [37•, 38]. This clone has been implicated in several epidemiologically unassociated outbreaks of skin and soft tissue infections in otherwise healthy individuals and has also been associated with unusually invasive disease [37]. Although, all S. aureus carry a native arginine deiminase metabolic pathway (arc gene cluster), the acquisition of the ACME element was shown to increase the gene dosages, which was suggested to contribute to the unique virulent phenotype of USA300 [37]. The arginine deiminase has been established in Streptococcus pyogenes as a virulence factor involved in the invasion and intracellular survival of host cells [39]. Moreover, since the depletion of l-arginine by arginine deiminase inhibits nitric oxide production, the ACME element is likely to suppress both innate and adaptive host immune responses [37]. The fact that ACME has non-functional ccr genes and is inserted into the chromosome in tandem with SCCmec, suggests that its acquisition was mediated by the SCCmec resident recombinase genes. Since very few MSSA isolates positive for the ACME element have been detected [40], the USA300 MRSA clones may have originated in two sequential steps: first acquiring the SCCmec and then the ACME element.

While the primary mode of spread of MRSA is clearly clonal expansion, the ‘birth’ of each MRSA strain involves the acquisition of an SCCmec element. Only speculations exist concerning how these genetic elements enter an S. aureus cell. A favored model assumes transduction by one or the other of the enormous number of phages that have been identified in staphylococci. Using the e-BURST method, the types of SCCmec and the genetic backgrounds (sequence types) of MRSA strains, it has been estimated that the number of times SCCmec has been acquired by the species of S. aureus is around 20 [22]. The same method resulted in a substantially higher number of acquisition events (around 54) for the case of methicillin resistant S. epidermidis [41].

One should remember that such acquisition events would only be registered if they lead to the production of a MRSA lineage that can be detected in surveillance studies. Recent experiments by Katayama and Chambers suggest that not all S. aureus genetic backgrounds are hospitable to maintaining the mecA gene in an active form. It was proposed that many S. aureus genetic backgrounds have a ‘barrier’ against maintaining, transcribing and translating a plasmid born mecA gene and bypassing such a barrier appears to be the property of a select group of S. aureus genetic backgrounds [4]. The mechanism and genetic basis of the barrier effect is not known at the present time but its existence could certainly contribute to the relatively low number of MRSA lineages identified in epidemiological studies. A role for the restriction modification system in the spread of various SCCmec types have recently been proposed by Lindsay et al. [42].

One of the surprising findings of the first international molecular epidemiological study on MRSA was the recognition that a handful of MRSA lineages (initially defined by shared PFGE patterns) were responsible for MRSA infections in hospitals located in Europe, USA and the far East in as many sixteen different participating countries [43]. Subsequent and more extensive international surveillance studies confirmed this finding using additional molecular typing techniques, such as MLST and SCCmec typing [44]. These observations indicate the importance of yet another evolutionary process that MRSA clones need in order to successfully compete with drug susceptible lineages of the species. It is interesting that the globally spread and highly epidemic MRSA clones often share a common genetic background with epidemic lineages of MSSA suggesting that critical determinants for epidemicity reside in the genetic background of MRSA clones [45].

Just as the combination of strict antibiotic use and infection control procedures succeeded in reducing the level of MRSA infections in several hospitals in Nordic countries and the Netherlands, S. aureus ‘pulled’ another surprise: patients from the community without any of the usual risk factors for MRSA infections began to appear in emergency rooms of hospitals most often with skin lesions caused by methicillin resistant S. aureus strains. Initially, and by contrast with the MRSA clones spreading in hospitals (HA-MRSA), these community acquired MRSA strains (CA-MRSA) showed diverse genetic backgrounds; carried – invariably – the relatively small SCCmec type IV or V; were rarely multi-drug resistant and exhibited less defective ‘fitness’ (i.e. grew faster in vitro), than the typical HA-MRSA strains. Soon afterwards, unique clonal lineages of CA-MRSA began to take over, the most prominent ones being clones with the genetic backgrounds of ST30, ST80 and ST8 and often the CA-MRSA lineages also carried the genetic determinant of the PVL toxin which seems to be the critical factor for the overwhelmingly skin and soft tissue infections caused by CA-MRSA strains. (For a recent review see [46].)

Section snippets

Contrasting scenarios for the evolution of hospital acquired and community acquired MRSA

In the 1960s the first MRSA was ‘born’ in the hospital environment as an already multidrug resistant pathogen and for over 17 years spread extensively in hospitals as a single clonal type (Archaic clone) [47]. It was only after this period that new clonal types began to appear and spread globally. These epidemic HA-MRSA clones had different genetic backgrounds (defined by MLST) and carried a variety of different SCCmec types (mainly I, II and III).

By contrast, when MRSA was ‘born again’ in the

References and recommended reading

Papers of particular interest, published within the annual period of review, have been highlighted as:

  • • of special interest

  • •• of outstanding interest

Acknowledgements

Partial support for this study was provided by a grant (2 RO1 A1045738-08) from the National Institute of Health, US Public Health Service to Alexander Tomasz and by a contract from Fundação para a Ciência e a Tecnologia, Portugal: POCTI/BIA-MIC/58416/2004 to Hermínia de Lencastre.

References (57)

  • S. Wu et al.

    Tracking the evolutionary origin of the methicillin resistance gene: cloning and sequencing of a homologue of mecA from a methicillin susceptible strain of Staphylococcus sciuri

    Microb Drug Resist

    (1996)
  • A. Severin et al.

    High-level (beta)-lactam resistance and cell wall synthesis catalyzed by the mecA homologue of Staphylococcus sciuri introduced into Staphylococcus aureus

    J Bacteriol

    (2005)
  • C. Fuda et al.

    Shared functional attributes between the mecA gene product of Staphylococcus sciuri and penicillin-binding protein 2a of methicillin-resistant Staphylococcus aureus

    Biochemistry

    (2007)
  • F. Rasmussen

    Discovery, isolation, production and introduction of penicillin for veterinary use in Denmark during World War II

    Vet History

    (2007)
  • T. Ito et al.

    Cloning and nucleotide sequence determination of the entire mec DNA of pre-methicillin-resistant Staphylococcus aureus N315

    Antimicrob Agents Chemother

    (1999)
  • M. Kuroda et al.

    Whole genome sequencing of meticillin-resistant Staphylococcus aureus

    Lancet

    (2001)
  • Y. Katayama et al.

    A new class of genetic element, staphylococcus cassette chromosome mec, encodes methicillin resistance in Staphylococcus aureus

    Antimicrob Agents Chemother

    (2000)
  • T. Ito 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)
  • D.C. Oliveira et al.

    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)
  • T. Ito et al.

    Novel type V staphylococcal cassette chromosome mec driven by a novel cassette chromosome recombinase, ccrC

    Antimicrob Agents Chemother

    (2004)
  • X.X. Ma et al.

    Novel type of staphylococcal cassette chromosome mec identified in community-acquired methicillin-resistant Staphylococcus aureus strains

    Antimicrob Agents Chemother

    (2002)
  • D.C. Oliveira et al.

    Redefining a structural variant of staphylococcal cassette chromosome mec, SCCmec type VI

    Antimicrob Agents Chemother

    (2006)
  • P. Chongtrakool et al.

    Staphylococcal cassette chromosome mec (SCCmec) typing of methicillin-resistant Staphylococcus aureus strains isolated in 11 Asian countries: a proposal for a new nomenclature for SCCmec elements

    Antimicrob Agents Chemother

    (2006)
  • T. Ito et al.

    Insights on antibiotic resistance of Staphylococcus aureus from its whole genome: genomic island SCC

    Drug Resist Updat

    (2003)
  • N.H. Kwon et al.

    Staphylococcal cassette chromosome mec (SCCmec) characterization and molecular analysis for methicillin-resistant Staphylococcus aureus and novel SCCmec subtype IVg isolated from bovine milk in Korea

    J Antimicrob Chemother

    (2005)
  • C. Milheirico et al.

    Multiplex PCR strategy for subtyping the staphylococcal cassette chromosome mec type IV in methicillin-resistant Staphylococcus aureus: ‘SCCmec IV multiplex’

    J Antimicrob Chemother

    (2007)
  • A. Shore et al.

    Seven novel variants of the staphylococcal chromosomal cassette mec in methicillin-resistant Staphylococcus aureus isolates from Ireland

    Antimicrob Agents Chemother

    (2005)
  • A.P. Johnson et al.

    Surveillance and epidemiology of MRSA bacteraemia in the UK

    J Antimicrob Chemother

    (2005)
  • Cited by (0)

    View full text