The general guidelines for reporting novel SCCmec/SCC elements
The defining feature of MRSA is the staphylococcal cassette chromosome mec (SCCmec). This is a mobile genetic element that carries the central determinant for broad-spectrum beta-lactam resistance encoded by the mecA gene. The emergence of methicillin-resistant staphylococcal lineages is due to the acquisition and insertion of the SCCmec element into the chromosome of susceptible strains. SCCmec elements are highly diverse in their structural organization and genetic content and have been classified into types and subtypes.
Many types, sub-types, and variants of SCCmec elements and SCC elements lacking mecA have been reported without following any standardized, internationally agreed rules of nomenclature. Consequently, there are ambiguities and inconsistencies in the classification of SCC elements in the published literature to date. Herein, we propose guidelines for the classification of SCCmec and other SCC elements.
I. Characteristics of SCCmec elements
MRSA strains produce an additional penicillin-binding protein, known either as PBP2a or PBP2’, which has low affinity for most of the semi-synthetic penicillins, e.g., methicillin, nafcillin, and oxacillin, as well as most cephem agents. PBP2a or PBP2’ is encoded by an acquired gene, mecA, which has been cloned and sequenced along with the genes that control its expression, mecR1 (encoding the signal transducer protein MecR1) and mecI (encoding the repressor protein MecI). When it became apparent that mecA was widely disseminated among multiple staphylococcal species, it was hypothesized that it could be carried on a mobile genetic element that could be easily transferred among staphylococcal species.
A genetic element that encoded methicillin resistance and carried unique site-specific recombinases designated as cassette chromosome recombinases (ccr) was subsequently identified and designated as the staphylococcal cassette chromosome mec (SCCmec)(Ito et al., 1999; Katayama et al., 2000). Soon after the initial description of SCCmec, several structurally different SCCmec elements were described. These elements shared several characteristics: (1) carriage of mecA in a mec gene complex, (2) carriage of ccr gene(s) (ccrAB or ccrC) in a ccr gene complex, (3) integration at a specific site in the staphylococcal chromosome, referred to as the integration site sequence for SCC (ISS), which serves as a target for ccr-mediated recombination, and (4) the presence of flanking direct repeat sequences containing the ISS.
II. Basic concepts for the classification of SCCmec elements
SCCmec elements are classified by a hierarchical system into “types” and “subtypes”. “Types” are defined by the combination of (1) the type of ccr gene complex, which is represented by ccr gene allotype, and (2) the class of the mec gene complex. These are the key elements of the cassette responsible for integration and excision of SCCmec, and the beta-lactam resistance phenotype, respectively.
The ccr gene complex.
The ccr gene complex is composed of the ccr gene(s) and surrounding open reading frames (ORFs) several of which have unknown functions. Currently, three phylogenetically distinct ccr genes, ccrA, ccrB, and ccrC, have been identified in S. aureus with DNA sequence similarities below 50%. To date, the ccrA and ccrB genes that have been identified in S. aureus have been classified into four allotypes. These allotypes are also found in other staphylococcal species as well as other allotypes have been described for these species only. In general, ccr genes with nucleotide identities of more than 85% are assigned to the same allotype, whereas, ccr genes that belong to different allotypes have lower nucleotide identities of between 60% and 82%, each other. All ccrC variants identified to date in staphylococcal strains have shown ≥87% similarity; thus, there is only one ccrC allotype. We suggest describing their differences as alleles by using previously used numbers, e.g., ccrC1 allele 2 or ccrC1 allele 8.
In the proposed nomenclature, novel ccr genes should be defined based on DNA sequence similarities of <50% and novel allotypes of ccr genes should be designated if their DNA sequence identities are between 50% and 85%. This convention should be followed for naming novel ccr genes. In the future, it may be necessary to define additional allotypes, including those of ccrC.
The phylogenetic relationships and DNA sequence similarities of representative ccr genes, including those identified in staphylococci other than S. arueus, are illustrated in Fig. 1. A few extra ccr allotypes have been identified in staphylococci other than S. aureus, as follows: ccrA5 is the ccrA of S. pseudintermedius KM241; ccrB6 is the ccrB of S. saprophyticus ATCC15305; and ccrB7 is the ccrB of S. saprophyticus TSU33.
The ccr gene complexes should be numbered according to the timing of their description. To date, two distinct groups have been reported, one carrying two adjacent ccr genes, ccrA and ccrB, and the other carrying ccrC. Based on allelic variations in ccr, a series of allotypes have been defined. The ccr gene complex identified in S. aureus include type 1 (carrying ccrA1B1), type 2 (carrying ccrA2B2), type 3 (carrying ccrA3B3), type 4 (carrying ccrA4B4), and type 5 (carrying ccrC), which can be detected by conventional PCR with pairs of specific primers.
To see currently identified ccr gene complexes in staphylococci, please visit SCCmec up to date.
The mec gene complex.
The mec gene complex is composed of mecA, its regulatory genes, and associated insertion sequences. The class A mec gene complex (class A mec) is the prototype complex, which contains mecA, the complete mecR1 and mecI regulatory genes upstream of mecA, and the hyper-variable region (HVR) and insertion sequence IS431 downstream of mecA. The class B mec gene complex (class B mec) is composed of mecA, a truncated mecR1 resulting from the insertion of IS1272 upstream of mecA, and HVR and IS431 downstream of mecA. The class C mec gene complex (class C mec) contains mecA and truncated mecR1 by the insertion of IS431 upstream of mecA, and HVR and IS431 downstream of mecA. There are two distinct class C mec gene complexes; in the class C1 mec gene complex, the IS431 upstream of mecA has the same orientation as the IS431 downstream of mecA (next to HVR), while in the class C2 mec gene complex, the orientation of IS431 upstream of mecA is reversed. C1 and C2 are regarded as different mec gene complexes since they have likely evolved independently. The class D mec gene complex (class D mec) is composed of mecA and ΔmecR1, but does not carry an insertion sequence downstream of ΔmecR1 (as determined by PCR) (Katayama et al., 2001).
Several variants within the major classes of the mec gene complex have been described including insertions of IS431 or IS1182 upstream of mecA for class A mec gene complex, or insertion of Tn4001 upstream of mecA in the class B mec complex. These variants are indicated by a numerical string following the class (e.g., classB2 indicated in Fig 2).
To see currently identified mec gene complexes in staphylococci , please visit SCCmec up to date.
The J regions: regions other than mec and ccr gene complexes.
Besides the mec and ccr gene complexes, the SCCmec element also contains three so-called J regions, which constitute nonessential components of the cassette. These regions may carry additional antimicrobial resistance determinants. They were first designated as the L-C, C-M, and I-R regions, but were later changed to J regions. We propose that the term “J region” refers to “joining-region”, rather than the previously used “junkyard region”.
J1 (formerly L-C) is the region between the right chromosomal junction and the ccr gene complex; J2 (C-M) is between the ccr gene complex and the mec gene complex; and J3 (I-R) is between the mec gene complex and the left chromosomal junction. Variations in the J regions within the same mec-ccr gene complex are used for defining SCCmec subtypes.
The SCCmec types
By the time the Guidelines for the classification of SCCmec elements were prepared (2009), eight SCCmec types have been described for S. aureus using the above criteria (Fig 2).
To see currently established SCCmec types, please visit SCCmec up to date.
The first three SCCmec elements were designated as types I, II, or III (Ito et al., 1999; Ito et al., 2001). These were followed by reports of SCCmec types IV-VIII (Berglund et al., 2008; Ito et al., 2004; Ma et al., 2002; Oliveira et al., 2006; Zhang et al., 2009). This nomenclature should be retained, but an additional (more informative) system for naming the novel SCCmec elements based on the type of ccr gene complex and class of mec gene complex present, is proposed. For example, type I (1B) SCCmec indicates an SCCmec harboring a type 1 ccr gene complex and a class B mec gene complex. The other known SCCmec types would be designated type II (2A), type III (3A), type IV (2B), type V (5C2), type VI (4B), type VII (5C1) and type VIII (4A). Thus, SCCmec types should be designated by Roman numerals in the order in which they are reported, followed by the ccr gene complex and the mec gene complex together in parentheses.
To avoid confusion in giving Roman numerals, we would like to suggest that all researchers upon identification of a new SCCmec type contact the IWG-SCC and provide the information on the mec and ccr gene complexes and, if appropriate, the accession numbers.
IV. The classification of SCCmec types into subtypes
Many different structures, including ISs and transposons, have been identified among the major SCCmec types in regions other than mec gene complex and ccr gene complex; i.e. in the J regions. Each SCCmec type has therefore been further classified into subtypes based on the polymorphisms or variations in J regions within the same ccr gene complex and mec gene complex combination. To date, three methods have been used to describe sub-types of SCCmec elements:
(1) Expressing the J1 region differences as small letters, e.g., IVa, IVb, and IVc.
(2) Expressing the differences due to the presence or absence of mobile genetic elements as capital letters, e.g., IA, IIA, and IVA.
(3) Expressing the differences in each J1, J2, and J3 region in Arabic numbers, which are given in the order of discovery, e.g., II.1.1.1, II.1.1.2, and II.2.1.1.
Since the first and the second nomenclature systems have been used as markers for particular epidemic clonal lineages, the designations have been retained as generic names.
However, to cope with the increasing diversity of J regions of SCCmec elements being reported, the number of alphabetic letters will be limited. Therefore, we are developing a computer system to identify or specify the differences in J regions based on a binary system (i.e., the presence or absence of specific DNA regions) that has been developed by (Stephens et al., 2007).
Composites of two or more SCC elements
Recently, SCCmec elements carrying two ccr gene complexes have been identified (Fig. 1). For example, the SCCmec carried by S. aureus strain ZH47 is composed of an SCC with ccrC and an SCCmec with a class B2 mec gene complex (a subclass of class B mec gene complex into which transposon Tn4001 has integrated), type 2 ccr gene complex and a J1 region with homology to type IVc SCCmec. Although the two SCC elements are arranged in tandem, no characteristic directly repeated sequence corresponding to ISS was identified at the junction regions, but two DRs were identified at the extremities of the composite element, suggesting that it is a single SCCmec containing two ccr gene complexes. Other examples include the SCCmec elements carried by Taiwanese S. aureus strains, TSGH17 and PM1 (Boyle-Vavra et al., 2005; Takano et al., 2008). These SCCmec elements are composed of a SCC with ccrC1allele8 and an SCCmec with a class C2 mec gene complex, type 5 ccr gene complex carrying ccrC1allele2 and J1 regions specific to the SCCmec and demarcated by two DRs at the both extremities. When a composite SCC element carrying two ccrs is identified, the association of ccr genes, mec gene complexes, and J regions in the composite should be compared to those described previously in order to identify if it harbors any extant type of SCCmec. Following this, the association of the SCCmec element with the other ccr gene should be determined in order to establish whether the presence of the two ccrs is a result of two separately integrated SCC elements and/or the composite was generated by the fusion of the two elements following deletion of the original junction region containing the DR in ISS.
It is likely that many such composite elements will be discovered since these deletions of the original junction region seem to occur frequently. It is difficult to discriminate the presence of composite elements from the presence of structures carrying two elements in tandem using the current PCR strategies for SCCmec typing. Thus, we suggest novel elements carrying two ccr genes should be categorized as an SCCmec type variant based on the known type of SCCmec present in the composite element.
VI. Classification of SCC elements that do not carry mecA
SCC elements that do not carry mecA but contain other characteristic genes (e.g., capsule gene cluster, fusidic acid resistance, or the mercury resistance operon) have also been identified in staphylococci. These elements share characteristics in common with SCCmec: carriage of ccr gene(s) (ccrAB or ccrC) in a ccr gene complex; integration at ISS in the staphylococcal chromosome; and the presence of flanking direct repeat sequences containing the ISS.
We recommend that SCC elements be described by adding the suffix describing the genes’ name or their function after SCC. For example, SCCcap1 carries the type-1 capsule gene cluster, SCCfur carries fusidic acid resistance, and SCCHg carries the mercury resistance operon. If no gene with inferable function is found in the SCC, we recommend describing the SCC elements by adding the name of strain, e.g. SCC476. In addition, staphylococci can also harbor SCC-like regions similar to SCC that are integrated at ISS and bracketed by ISS, but differ from SCC in that they do not harbor ccr gene(s). They are diverse in sizes from the shortest (0.1 kb) to the longest (34 kb), and have been described in different ways, e.g., SCC-like elements, the arginine catabolic mobile element (ACME), cassette chromosome (CC), or SCCmec insertion site genomic sequence. We suggest describing these elements as pseudo-SCC elements (ψSCC). We recommend that these SCC elements be designated by adding the suffix describing the genes’ name or their function or by adding the name of the strain similar to the case of SCC.
Berglund C., Ito T., Ikeda M., Ma X.X., Soderquist B., Hiramatsu K. (2008) Novel type of staphylococcal cassette chromosome mec in a methicillin-resistant Staphylococcus aureus strain isolated in Sweden. Antimicrob Agents Chemother 52:3512-6.
Boyle-Vavra S., Ereshefsky B., Wang C.C., Daum R.S. (2005) Successful multiresistant community-associated methicillin-resistant Staphylococcus aureus lineage from Taipei, Taiwan, that carries either the novel Staphylococcal chromosome cassette mec (SCCmec) type VT or SCCmec type IV. J Clin Microbiol 43:4719-30.
Ito T., Katayama Y., Hiramatsu K. (1999) Cloning and nucleotide sequence determination of the entire mec DNA of pre-methicillin-resistant Staphylococcus aureus N315. Antimicrob Agents Chemother 43:1449-58.
Ito T., Ma X.X., Takeuchi F., Okuma K., Yuzawa H., Hiramatsu K. (2004) Novel type V staphylococcal cassette chromosome mec driven by a novel cassette chromosome recombinase, ccrC. Antimicrob. Agents Chemother. 48:2637-2651.
Ito T., Katayama Y., Asada K., Mori N., Tsutsumimoto K., Tiensasitorn C., Hiramatsu K. (2001) Structural comparison of three types of staphylococcal cassette chromosome mec integrated in the chromosome in methicillin-resistant Staphylococcus aureus. Antimicrob Agents Chemother 45:1323-36. DOI: 10.1128/AAC.45.5.1323-1336.2001.
Katayama Y., Ito T., Hiramatsu K. (2000) A new class of genetic element, staphylococcal cassette chromosome mec, encodes methicillin resistance in Staphylococcus aureus. Antimicrob. Agents Chemother. 44:1549-1555.
Katayama Y., Ito T., Hiramatsu K. (2001) Genetic organization of the chromosome region surrounding mecA in clinical Staphylococcal strains: role of IS431-mediated mecI deletion in expression of resistance in mecA-carrying, low-level methicillin- resistant Staphylococcus haemolyticus. Antimicrob. Agents Chemother. 45:1955-1963.
Ma X.X., Ito T., Tiensasitorn C., Jamklang M., Chongtrakool P., Boyle-Vavra S., Daum R.S., Hiramatsu K. (2002) Novel type of staphylococcal cassette chromosome mec identified in community-acquired methicillin-resistant Staphylococcus aureus strains. Antimicrob. Agents Chemother. 46:1147-1152.
Takano T., Higuchi W., Otsuka T., Baranovich T., Enany S., Saito K., Isobe H., Dohmae S., Ozaki K., Takano M., Iwao Y., Shibuya M., Okubo T., Yabe S., Shi D., Reva I., Teng L.J., Yamamoto T. (2008) Novel characteristics of community-acquired methicillin-resistant Staphylococcus aureus strains belonging to multilocus sequence type 59 in Taiwan. Antimicrob Agents Chemother 52:837-45.
Zhang K., McClure J.A., Elsayed S., Conly J.M. (2009) Novel staphylococcal cassette chromosome mec type, tentatively designated type VIII, harboring class A mec and type 4 ccr gene complexes in a Canadian epidemic strain of methicillin-resistant Staphylococcus aureus. Antimicrob Agents Chemother 53:531-40.