Current types of staphylococcal cassette chromosome mec (SCCmec) in clinically relevant coagulase-negative staphylococcal (CoNS) species

Abstract

Coagulase-negative staphylococci (CoNS) colonize human skin and mucosal membranes, which is why they are considered harmless commensal bacteria. Two species, Staphylococcus epidermidis and Staphylococcus haemolyticus belong to the group of CoNS species and are most frequently isolated from nosocomial infections, including device-associated healthcare-associated infections (DA-HAIs) and local or systemic body-related infections (FBRIs). Methicillin resistance, initially described in Staphylococcus aureus, has also been reported in CoNS species. It is mediated by the mecA gene within the staphylococcal cassette chromosome (SCCmec). SCCmec typing, primarily using PCR-based methods, has been employed as a molecular epidemiological tool. However, the introduction of whole genome sequencing (WGS) and next-generation sequencing (NGS) has enabled the identification and verification of new SCCmec types. This review describes the current distribution of SCCmec types, subtypes, and variants among CoNS species, including S. epidermidis, S. haemolyticus, and S. capitis. The literature review focuses on recent research articles from the past decade that discuss new combinations of SCCmec in coagulase-negative Staphylococcus. The high genetic diversity and gaps in CoNS SCCmec annotation rules underscore the need for an efficient typing system. Typing SCCmec cassettes in CoNS strains is crucial to continuously updating databases and developing a unified classification system.

Keywords:

• Methicillin-resistant CoNS
• epidemiology
• molecular typing
• NGS
• Staphylococcus

1. Introduction

1.1. Epidemiology of coagulase-negative staphylococci (CoNS)

Staphylococcus bacteria are known opportunistic pathogens. Staphylococci constitute a large heterogeneous group of 85 described species and 30 subspecies as of 2023 (List of Prokaryotic names with Standing in Nomenclature (LPSN) accessed 10th March 2023; Parte et al. 2020). These bacteria can be classified on the basis of many enzymatic characteristics, one of which is the ability to produce coagulase. Therefore, generally, the whole Staphylococcus genus is divided into two groups. The first group comprises coagulase-positive staphylococci (CoPS), with the most common being Staphylococcus aureus, which is also considered highly pathogenic and is a priority 2 (HIGH) pathogen on the World Health Organization (WHO) confirmed list since 2017. The second group comprises CoNS, which for many years were considered completely nonpathogenic or moderately commensal organisms. CoNS constitute more than half of the staphylococcal species and play an important role in the natural human and animal microbiota (Becker et al. 2020). In recent years, there has been an observed increase in infections, particularly bacteremia and skin and soft tissue infections, associated with CoNS (Michalik et al. 2020). For example, Staphylococcus epidermidis is the most frequently recovered staphylococcal species from humans and colonizes the skin of the axillae, inguinal and perineal areas, anterior nares, conjunctiva, and toe webs (Becker et al. 2014). In clinical laboratory practice, CoNS are frequently considered contaminants of blood cultures. It is very important to distinguish contamination and infections to prevent antibiotic resistance transmission (Elzi et al. 2012). According to the standard diagnostic procedure, a minimum of two blood culture sets have to be drawn from different peripheral venous sites. A single source of contamination for CoNS grown in blood culture is not sufficient and may be misleading if the second set is sterile. If CoNS strains are isolated from two sets, the result is interpreted as a blood infection (Yamamoto et al. 2021). In the past decade, an increase in infections caused by CoNS was observed, and a few of them have been regularly associated with human infections (Rupp and Archer 1994; Pujol et al. 2007; Azih and Enabulele 2013). Staphylococcus epidermidis is the first CoNS to be described in clinical infections, followed by Staphylococcus hominis, Staphylococcus haemolyticus, and Staphylococcus capitis (Spanu et al. 2003; Huang et al. 2005; Shin et al. 2011). These groups of bacteria cause systemic infections, including bloodstream, skin and soft tissue, endocarditis, prosthetic joint infections (PJIs), and urinary tract infections (Becker et al. 2014). Bacterial genomes are characterized by high plasticity and variability. The evolution of genomes is relatively fast due to horizontal gene transfer (HGT), which enables bacteria to incorporate exogenous genetic material into their genomes. HGT plays an important role in the evolution of all bacteria, including Staphylococcus species, such as via the acquisition of antibiotic resistance or virulence genes associated with pathogenicity. An example is the well-established role of the staphylococcal cassette chromosomal mec gene (SCCmec) in the genomes of S. aureus and S. epidermidis (Méric et al. 2018; Zapotoczna et al. 2018). This suggests that the stepwise evolution of the cassette chromosomal elements and the mecA gene is critical to the spread of β-lactam resistance among staphylococci, thus leading to methicillin-resistant S. aureus (MRSA) and methicillin-resistant coagulase-negative staphylococci (MRCoNS) epidemics.

1.2. Clinical importance of CoNS infections

S. epidermidis is the most common opportunistic pathogen that primarily causes device-associated healthcare-associated infections (DA-HAIs) and systemic body-related infections (FBRIs), (Becker et al. 2014). This species is responsible for 30–40% of nosocomial bloodstream infections (BSIs), (Severn and Horswill 2023). Most cases of neonatal sepsis are caused by S. epidermidis, which is also associated with retinopathy of prematurity, necrotizing enterocolitis, white matter injury, and bronchopulmonary dysplasia (Burke et al. 2023). Similar to S. aureus, which is more virulent, S. epidermidis also produces toxins and factors that contribute to biofilm formation (Rohde et al. 2005). Although a component of the physiological skin microbiota, S. epidermidis is one of the CoNS in which the rate and type of infection led to molecular and genomic investigations in search of associated virulence factors (Méric et al. 2018). The next species, S. haemolyticus, is also a part of the skin microbiota and the second most frequently isolated species from clinical infections (10–20%), especially nosocomial infections (Eltwisy et al. 2022). S. haemolyticus strains cause BSIs and are highly resistant to antibiotics. Furthermore, these strains have one of the highest degrees of resistance to methicillin among CoNS (Szczuka et al. 2015). Moreover, S. haemolyticus may cause severe infections, including skin infections, prosthetic joint infections, and urinary tract infections (Schuenck et al. 2008). S. haemolyticus not only infects humans but can also infect animals, and can then be transmitted to humans (Marsilio et al. 2018). In the literature, most S. haemolyticus strains disseminated resistance genes contributing to the emergence of epidemic clones of S. aureus, which is a more virulent nosocomial pathogen (Fluit et al. 2013; Cavanagh et al. 2014). Furthermore, a recent worldwide increase in the number of multidrug-resistant S. haemolyticus isolates that cause hospital infections has been associated with unusual genome plasticity generated by a high number of insertion sequences (Czekaj et al. 2015). The next important coagulase-negative staphylococcal species is endemic S. capitis, which occupies a specific niche on the skin of the head and accounts for approximately 5% of CoNS invasive clinical isolates (Thakker et al. 2021). S. capitis was recovered from neonatal intensive care units (NICUs) and may be responsible for bloodstream infections and endocarditis (Butin et al. 2017; Pinheiro-Hubinger et al. 2021; Thakker et al. 2021). Moreover, S. capitis has been reported as a cause of nosocomial late-onset sepsis (LOS) in several studies (Van Der Zwet et al. 2002; Rasigade et al. 2012; Ben Said et al. 2016; Butin et al. 2017). S. capitis is also recovered from nasal or wound samples of adults (Pinheiro-Hubinger et al. 2021). Although this species has occasionally been reported, it is most often considered a blood culture contaminant (Ruhe et al. 2004; Pinheiro-Hubinger et al. 2021).

2. Resistance to methicillin and mechanism of resistance to β-lactam antibiotics

Antibiotics of the β-lactam group, such as penicillin, were in the past the medicines of choice in the treatment of S. aureus infections. After penicillin-resistant staphylococcal isolates emerged due to the acquisition of mobile genetic elements (MGEs), new effective antibiotics or chemotherapeutics were examined and introduced. Methicillin was the first semisynthetic penicillin derivative antibiotic effective as an S. aureus infection therapy. This antibiotic was introduced in 1959 to treat infections caused by penicillin-resistant strains of S. aureus (Fishovitz et al. 2014). However, in 1961, the first hospital-acquired methicillin-resistant S. aureus strain (HA-MRSA) was isolated shortly after the introduction of methicillin to clinical practice (Jevons 1961). β-lactam antibiotics demonstrate a common mechanism of action that blocks the biosynthesis of the bacterial cell wall. Another point of view of this mechanism is based on the activity of the penicillin-binding proteins (PBPs) present on the bacterial surface. β-lactam antibiotics inhibit the transpeptidation step of cell-wall biosynthesis by acting as the substrate analog of the D-Ala-D-Ala peptidoglycan side chain, which is affected by PBPs (Figure 1), (Tipper and Strominger 1965). Bacterial strains have evolved numerous strategies to counteract the effect of antibiotics. Staphylococcal resistance to β-lactam antibiotics is mediated by one of the two following mechanisms: (i) production of β-lactamases, which are encoded by the blaZ gene, and (ii) production of an altered target penicillin binding-protein, PBP2a, which is a synthetic bacterial cell wall PBP that functions as a transpeptidase and is encoded by the mecA gene (Peacock and Paterson 2015).

Figure 1. Mechanism of target and resistance of β-lactam antibiotics. (A) Mechanism of β-lactam antibiotic action. (B) Mechanism of induction of staphylococcal β-lactamase synthesis in the presence of the β-lactam antibiotic penicillin. (1) The DNA-binding protein BlaI binds to the operator region (repressed transcription of RNA from blaZ and blaR1-blaI). In the absence of penicillin, β-lactamase is present at low levels. (2) The binding of penicillin to BlaR1 induces autocatalytic activation of BlaR1. (3–4) Active BlaR1 either directly or indirectly (by BlaR2) cleaves BlaI into inactive fragments (transcription of blaZ and blaR1-blaI). (5–7) β-Lactamase, (encoded by blaZ (5)), hydrolyzes the β-lactam ring of penicillin (6), rendering it inactive (7). (C) Synthesis of PBP2a proceeds similarly to that described for β-lactamase. Exposure of MecR1 to a β-lactam antibiotic stimulates MecR1 synthesis. MecR1 inactivates MecI (synthesis of PBP2a is allowed). MecI and BlaI are coregulators that affect the expression of PBP2a and β-lactamase.

3. Characteristics of SCCmec types

In 1999, Ito et al. found and described the mecA gene and announced that it was carried by a novel genetic element. Designated staphylococcal cassette chromosome mec inserted into the chromosome (Ito et al. 1999). The mecA gene is located on the SCCmec, which is a MGEs characterized by the presence of terminal inverted and direct repeats and has also been called a genomic island (International Working Group on the Classification of Staphylococcal Cassette Chromosome Elements (IWG-SCC) 2009; Katayama et al. 2003). SCCmec contains the following two components: the mec complex and the ccr complex (Figure 2). The first complex is composed of IS431mec, mecA, and the regulatory genes – mecI (encoded repressor protein) and mecR1 (encoded signal transducer protein). The region between IS431mec and mecA is highly homogeneous, with the exception of the hypervariable subregion (Ito et al. 2014). The six different classes of the mec gene complex have been classified, including class A, B, C1, C2, D, and E. In all class complexes, IS431 was located behind mecA. Furthermore, SCCmec possesses insertion sequences (IS) that flank the mecA gene, as well as expression regulatory genes. The overall structure of mecI-mecR1-mecA-IS431mec is a prototypic class A mec complex (Katayama et al. 2001). In the class B mec complex, the mecR1 gene is partially deleted by insertion of IS1272 (IS431-mecA-ΔmecR1-IS1272). The class C mec complex is divided into two variants, C1 and C2, depending on the orientation of IS431 (IS431-mecA-ΔmecR1-IS431 (two IS431 in the same direction); IS431-mecA-ΔmecR1-IS431 (two IS431 in the opposite direction)). Additionally, in C1 and C2, the mecR1 gene is partially deleted by the insertion of IS431. The class D mec complex presents the deleted gene mecR1 but without an associated insertion sequence (IS431-mecA-ΔmecR1). The class E mec complex is similar to that of class D, but with a higher rate of mecR1 deletion (blaZ-mecA_LGA251-mecR1 LGA251-mecI_LGA251) (Martínez-Meléndez et al. 2015). The second component of the whole SCCmec cassette is the ccr complex, which encodes the ccr recombinases (ccrA, ccrB and ccrC) that mediate the integration of SCCmec into and its excision from the recipient chromosome and are involved in its mobility (Miragaia 2018]. Additionally, ccrA and ccrB were classified into subgroups ccrA1–7 and ccrB1–6, respectively (Ito et al. 2014). The third type of ccr – ccrC contains two subgroups – ccrC1 and ccrC2 (Ito et al. 2004; Wu et al. 2015). Furthermore, the SCCmec element also contains three J (junkyard) regions (J1, J2, and J3) that constitute nonessential components of the chromosome cassette (Zhang et al. 2005). The current types of SCCmec are presented in Table 1 and Figure 3.

Figure 2. SCCmec cassette structure in S. aureus (J1-J3 – joining region).

Figure 3. Structures of current SCCmec types. The mec and ccr gene complexes are shaded pink and blue, respectively. The figure is adapted from IWG-SCC commentary, 2009, ASM and Baig et al. (2018) with authors permission.

Table 1. Current SCCmec types.

Type of SCCmec	Type of ccr complex	Class of mec complex	References	Current methods which can be used for detection
I (1B)^a	1 (A1B1)	B (IS431-mecA-ΔmecR1-IS1272)	Ito et al. 2001	Oliveira and de Lencastre 2002; Milheiriço et al. 2007a; Kondo et al. 2007; Zhang et al. 2012; WGS
II (2A)	2 (A2B2)	A (IS431-mecA-mecR1-mecI)	Katayama et al. 2000; Ito et al. 2001	Oliveira and de Lencastre 2002; Milheiriço et al. 2007a; Kondo et al. 2007; Zhang et al. 2012; WGS
III (3 A)	3 (A3B3)	A (IS431-mecA-mecR1-mecI)	Ito et al. 2001	Oliveira and de Lencastre 2002; Milheiriço et al. 2007a; Kondo et al. 2007; Zhang et al. 2012; WGS
IV (2B)	2 (A2B2)	B (IS431-mecA-ΔmecR1-IS1272)	Ma et al. 2002	Oliveira and de Lencastre 2002; Milheiriço et al. 2007a; Kondo et al. 2007; Zhang et al. 2012; WGS
V (5C2)	5 (C1)	C2 (IS431-mecA-ΔmecR1-IS431)	Ito et al. 2004	Milheiriço et al. 2007a; Kondo et al. 2007; WGS
VI (4B)	4 (A4B4)	B (IS431-mecA-ΔmecR1-IS1272)	Oliveira, Milheiriço, de Lencastre 2006	Milheiriço et al. 2007a; Kondo et al. 2007; WGS
VII (5C1)	5 (C1)	C1 (IS431-mecA-ΔmecR1-IS431)	Berglund et al. 2008	only WGS
VIII (4A)	4 (A4B4)	A (IS431-mecA-mecR1-mecI)	Zhang et al. 2009	only WGS
IX (1C2)	1 (A1B1)	C2 (IS431-mecA-ΔmecR1-IS431)	Li et al. 2011	only WGS
X (7C1)	7 (A1B6)	C1 (IS431-mecA-ΔmecR1-IS431)	Li et al. 2011	only WGS
XI (8E)	8 (A1B3)	E (blaZ-mecALGA251-mecR1LGA251-mecILGA251)	Garcia-Alvarez et al. 2011	only WGS
XII (9C2)	9 (C2)	C2 (IS431-mecA-ΔmecR1-IS431)	Wu et al. 2015	only WGS
XIII (9A)	9 (C2)	A (IS431-mecA-mecR1-mecI)	Baig et al. 2018	only WGS
XIV (5A)	5 (C1)	A (IS431-mecA-mecR1-mecI)	Urushibara et al. 2020	only WGS
XV (7A)	7 (A1B6)	A (mecI-mecR1-mecA- IS431)	Wang et al. 2022a	only WGS

^a Description of SCCmec – example: Type I (1B), where 1 is the number of ccr gene complex; B is the class of mec gene complex.

4. Association of SCCmec with the molecular characteristics and pathogenicity of S. epidermidis, S. haemolyticus, and S. capitis

As of 2023, June 26, 179 complete genomes of S. epidermidis were available in GenBank. In the cases of S. haemolyticus and S. capitis, there were 28 and 11 complete genomes, respectively. The genomes of S. epidermidis strains exhibit high genetic diversity which is influenced by the origin of the strains and the mechanisms of HGT. Therefore, S. epidermidis has an open pan genome, and/or high potential to acquire new genetic traits especially through MGEs. Although the size of databases containing complete genomes of CoNS species increases every day, there are still too few of these genomes deposited in GenBank when compared to the 1538 complete S. aureus genomes. On the other hand, genomes in contigs or fragments can be found on a larger scale in publicly available databases, but genomes deposited as incomplete sequences lack many gene descriptions and/or appropriate annotations. Thus, improving this situation will require advanced bioinformatic analysis and corrections in international databases. These changes will facilitate the design of experiments and the optimization of methods to identify genes encoding virulence factors. The genes associated with virulence factors such as adhesins, hemolysins, toxins, and enzymes are acquired by CoNS from hospital S. aureus strains and then included in their genomes (Międzobrodzki et al. 1984; Lisowska-Łysiak et al. 2021). Most CoNS species can produce biofilms related to polysaccharide intercellular adhesin (PIA) encoded in the ica gene operon. Moreover, CoNS species with high genetic diversity have been described. Especially, S. epidermidis and S. haemolyticus have high recombination rates and an enhanced capacity to adapt faster to exogenous genetic material, or these elements could have been acquired earlier by these species than by S. aureus (Miragaia et al. 2007). CoNS, similar to S. aureus, have the ability to develop antibiotic resistance, increase the cost of treatment, and contribute significantly to morbidity and mortality (Bassetti et al. 2012). Resistance to commonly used antibiotic classes such as macrolides and aminoglycosides and to last resort antibiotics such as glycopeptides has been increasingly reported in CoNS species (Bourgeois et al. 2007; Ma et al. 2011; May et al. 2014). CoNS have particularly high rates of resistance to methicillin (70%–80%), as indicated by various reports in Canada, the United States, and Latin America (Diekema et al. 2001; Agvald-Ohman et al. 2004). An important feature of Staphylococcus, also including CoNS, is the SCCmec. Exposure to β-lactam antibiotics or other antibiotics can induce stress on bacterial populations. Some strains may respond to this stress by acquiring resistance genes or SCCmec elements, as a survival strategy. The important aspect is that in healthcare settings, S. epidermidis can be transmitted from patient to patient. Various patients carry different strains, some of which are methicillin-resistant or methicillin-susceptible. Cross-transmission in healthcare settings can contribute to the presence of both types of isolates. In research on healthcare-associated methicillin-resistant S. epidermidis (HA-MRSE) strains by Rolo et al. it was determined that the increased presence of some SCCmec elements, such as ccr genes was as a result of exposure to antibiotics. Moreover, hospitals serve as a large reservoir of SCCmec types that also exist in other CoNS species (Rolo et al. 2012). The presence of SCCmec also contributes to resistance to antibiotics other than β-lactams, leading to the emergence of multidrug-resistant strains. Antibiotic resistance genes are inserted into the J1-3 (junkyard) regions in the form of integrated copies of plasmids or transposons. For example, plasmid pUB110 codes for kanamycin, tobramycin (ant(4′)-1), bleomycin (ble) resistance, pI258 codes for penicillin resistance, and pT181 codes for tetracycline (tetK) resistance. In the case of transposons, one of them is TN554, which is encodes resistance to erythromycin (ermA) and spectinomycin (spc) (Martínez-Meléndez et al. 2015). Moreover, not only resistance genes are contained in chromosome cassettes. Heavy metal resistance genes were also found in SCCmec elements encoded by plasmid pI258. One of them is resistance to cadmium, which is conditioned by the presence of the cadDX operon. Apart from the resistance to cadmium, resistances to other heavy metals such as arsenic (ars) and copper (cop) are encoded within the SCCmec cassettes. In the literature, the SCCmec-SCCcad/ars/cop composite island that has likely emerged from two independent acquisition events has also been described (Martins-Simões et al. 2013). Furthermore, the authors described that the arginine catabolic mobile element (ACME) shows characteristics similar to those of the SCCmec element that integrates into the staphylococcal chromosome at the attachment site attB, which is flanked by direct repeat sequences and is mobilized by the SCCmec encoded ccrAB genes in S. aureus and S. epidermidis (Diep et al. 2008; Miragaia et al. 2009). It was reported that S. capitis also carried ACME type I and II cassettes (Asadollahi et al. 2021). The evolution of ACME in S. epidermidis could have important consequences for the epidemiology of S. aureus. S. epidermidis serves as a genetic reservoir for S. aureus (O’Connor et al. 2018). The presence of the arcA gene (encoding arginine deiminase) in the ACME cassette was also documented for S. haemolyticus (Yu et al. 2014). Another element related to SCCmec cassettes is the psm-mec gene. PSM-mec is a secreted virulence factor that belongs to the phenol-soluble modulin (PSM) family of amphipathic alpha-helical peptide toxins produced by Staphylococcus species. This element is found in the J2 region adjacent to the mecI gene of the mecA gene complex characteristic of SCCmec types II or III. These toxins play an important role as pathogenic determinants in the pathogenesis of both S. aureus and S. epidermidis (Schuster et al. 2018). PSM peptides have a significant influence on the formation of bacterial biofilms. The presence of psm-mec has been reported to increase biofilm formation by staphylococci, including S. aureus and S. epidermidis (Kaito et al. 2013; Yang et al. 2016). Furthermore, cassette transfer is facilitated in the bacterial biofilm due to the very high concentration of bacterial cells in this structure (Szczuka and Kaznowski 2014).

5. Main types of SCCmec

5.1. Staphylococcus aureus

The SCCmec structure in S. aureus has been shown to be relatively stable and exhibit low variability in contrast to that in CoNS. This low diversity has led to the identification and official approval of 15 types of SCCmec by the IWG-SCC. Described types of SCCmec correspond to different combinations of mec complex class, ccr allotypes, and the presence of regulatory genes and insertion sequences (Table 1 and Figure 3). Furthermore, several SCCmec subtypes, such as Ia and Ib (Oliveira, Milheirico and Vinga, et al. 2006; Shore et al. 2005), IIa to IIe (Shore et al. 2005), and IVa to IVo (Uehara 2022), and the SCCmec subtypes Va (5C2), Vb (5C2&5) and Vc (5C2&5) (Ito et al. 2004; Hisata et al. 2011; Li et al. 2011) have been reported to date.

5.2. Coagulase-negative staphylococci

Coagulase-negative staphylococci have been recognized as reservoirs of SCCmec based on the evidence of horizontal gene transfers of the SCCmec elements from CoNS to S. aureus (Forbes and Schaberg 1983; Martínez-Meléndez et al. 2015). According to research by Gill et al. and Sabat et al. integration of SCCmec is sequence-specific at a unique site (attB gene) in CoNS genomes and is found downstream of an open reading frame of unknown function, designated orfX, that is well conserved, similarly to S. aureus strains (Gill et al. 2005; Sabat et al. 2021). Additionally, the mecA gene was found to be more widely distributed among CoNS than among S. aureus, as confirmed by the diversity of SCCmec elements (Ibrahem et al. 2009). Moreover, Berglund et al. described the transfer of SCCmec from a methicillin-resistant S. haemolyticus strain to a methicillin-sensitive S. aureus (MSSA) strain (Berglund and Soderquist 2008). Similarly, Argudin et al. and Price et al. described that transfer from CoNS species into S. aureus could frequently occur among staphylococci inhabiting animals (Argudin et al. 2010; Price et al. 2013). In comparison with S. aureus, for CoNS, SCCmec types I-VII were identified and reported (Table 1; Figure 3), (Saber et al. 2017). Diversity of SCCmec types among CoNS strains may be related to the increase in human and animals migration, leading to the spread of antibiotic resistance and CoNS isolates carrying MGEs, including various SCCmec types. In research performed by the de Lencastre group, the authors demonstrated that the distribution of different types of SCCmec in MRCoNS varied depending on the host species and possibly on the geographical location (Miragaia et al. 2007). For S. epidermidis, SCCmec types I, II, III, IV, V, and VI were described, with the most common type being IV (Ruppé et al. 2009; Ibrahem et al. 2009; Garza-González et al. 2010; Szczuka et al. 2016). Various reports conducted on S. epidermidis strains showed that there was also an association between the SCCmec cassette and the sequence type (ST). Among S. epidermidis isolates, the most common type was ST2, which was identified in strains isolated in 13 different countries across four continents (Denmark, Italy, Iceland, Argentina, Mexico, Greece, Cape Verde, Spain, Hungary, Colombia, Uruguay, Japan, Bulgaria). ST2 is associated with II, III, and IV SCCmec types (Miragaia et al. 2007). Another study also confirmed that ST2 is typical for HA-MRSE isolates (Cherifi et al. 2014; Martínez-Santos et al. 2022). So both geographic region and type of MRCoNS infection is of importance for the detection of particular SCCmec type and more detailed research is needed. In the case of S. haemolyticus isolated from hospital infections, SCCmec type V was detected most frequently (Ruppé et al. 2009; Ito et al. 2004). However, SCCmec types III, II and IV also appeared in S. haemolyticus isolated from bloodstream infections (Dier-Pereira et al. 2021). Takeuchi et al. described that the whole genome sequencing of S. haemolyticus strain JCSC1435 revealed the existence of SCCmec type V in tandem with several pseudo-SCCs, lacking the ccr complex (Takeuchi et al. 2005). In addition, many authors indicate that in the genome, S. haemolyticus has multiple copies of the ccr recombinase genes, some of which cannot be classified as a particular allotype (Ruppé et al. 2009; Lebeaux et al. 2012). S. capitis was identified as SCCmec types I, II, III, IV, V and subtypes Ia, IVa, and Va (5C2&5) (Martins-Simões et al. 2013; Saber et al. 2017; Wang et al. 2022b). Many SCCmec cassettes found in coagulase-negative staphylococci cannot be classified as existing types, as they most likely contain yet undescribed allotypes of the chromosomal (mec) and ccr recombinase gene complexes. These include new combinations of the ccr and mec complexes. In many studies, variations in SCCmec and nontypeable SCCmec for S. epidermidis, S. haemolyticus, S. capitis, and other CoNS have been described, indicating that the 7 main types described thus far are not all present in the environment. The unclassified SCCmec types among CoNS are associated with (i) the presence of the mecA gene with classified mec Class and ccr gene complex, however those elements does not correspond to any of the so far described cassettes approved by the IWG-SCC; (ii) the presence of the mecA gene without classified mec Class and presence of ccr gene complex; (iii) the presence of only the mecA gene with mec Class and none ccr gene detected; (iv) the presence of multiple ccr gene complexes; and (v) the presence of only genes including J1-3 regions (Hanssen et al. 2004; Miragaia et al. 2008; Bouchami et al. 2012; Barros et al. 2012; Szemraj et al. 2020; Al-Bakri et al. 2021).

5.2.1. New SCCmec types, subtypes, and variants in CoNS

Among coagulase-negative staphylococci, the highest diversity of SCCmec cassettes is observed in S. epidermidis, S. haemolyticus, S. capitis and S. hominis, species that most frequently colonize and infect humans (Szczuka and Kaznowski 2014; Saber et al. 2017). The results of many analyses show the presence of new compositions of the mec complex and the chromosomal recombinase ccr gene in these cassettes. There is no doubt that the enormous diversity of SCCmec elements necessitates genome sequencing to detect and characterize them all. On the other hand, polymorphism occurs in the J regions within the same mec gene complex and in the combination of the ccr gene complex, leading to the emergence of subtypes of SCCmec elements (Lakhundi and Zhang 2018). Based on the sequence analyses performed, the ccr genes were found to be significantly more diverse than the mec complex. Moreover, in the genome, multiple copies of ccr genes may be present, and the different allotypes of these genes, in turn, indicate a high recombination capacity of these bacteria. In several studies where SCCmec with a nontypeable ccr complex was observed, mec complexes simultaneously exist even in the absence of known types of ccr. Most strains exhibited phenotypic resistance to methicillin (Hanssen et al. 2004; Bouchami et al. 2012). However, Hanssen et al. observed that the mecA gene was identified in a few strains despite their sensitivity to the antibiotic (Hanssen et al. 2004). The absence of phenotypic resistance in the presence of mecA is a concerning phenomenon in clinical settings due to the incorrect determination of antibiotic sensitivity and, consequently, the ineffectiveness of patient therapy. In 2021, Sabat et al. described a new subclass of class B mec (B4) originating from IS431-mediated integration of the plasmid pUB110 into SCCmec subtype IVc in S. epidermidis (Sabat et al. 2021). The coexistence of two types of SCCmec elements appears to be common in MRCoNS, which strongly suggests that new variants may be present in CoNS and may have a different impact on drug resistance (Zong et al. 2011; Machado et al. 2007; Garza-González et al. 2010; Saravanan et al. 2014; Murugesan et al. 2015). Furthermore, in several studies, two types of SCCmec elements were found in a single strain. Chen et al. showed the multiple ccr complex composition in CoNS strains. The authors showed the ccrAB3 and ccrAB4 genes in S. hominis and in S. capitis. This was the first report about the heterogeneous combination of ccr complexes in a single CoNS strain (Chen et al. 2017). Similarly, Kosecka-Strojek et al. described a new composition of SCCmec. Eleven linezolid-resistant S. epidermidis (LRSE) isolates recovered from 10 pediatric patients possessed class A mecA genes, two ccr complexes (ccrAB3 and ccrAB4) and a combination of SCCmec cassette II and III elements in all LRSE strains (Kosecka-Strojek et al. 2020).

6. Procedure to describe new types, subtypes, and variants of SCCmec

The International Working Group on Classification of Staphylococcal Cassette Chromosome Elements (IWG-SCC) was established in 2009. The main objectives of the group were to define consensus rules for a uniform nomenclature system for SCCmec elements, determine minimum requirements for the description of new SCCmec elements, and establish guidelines for the identification of SCCmec elements for epidemiological studies (IWG-SCC 2009). In the published guidelines, IWG-SCC decided to retain the SCCmec nomenclature with additional information about the combination of the ccr complex type, which is represented by the allotype of the ccr gene, and the class of mec complex present in the element. Novel SCCmec subtypes should be defined by the presence of specific DNA sequences located in J regions, including characteristic genes, pseudogenes or noncoding regions in J regions other than MGEs; for example, insertion sequences, plasmids or transposons, most of which encode antimicrobial resistance or other determinants. The nomenclature of new subtypes is important. The first description method refers to expressing the differences in the J1 region as small letters, for example IVa to IVo; the second is to express the differences due to the presence or absence of MGEs as capital letters, for example IA, IIA, and IVA. The third is to describe the differences in each J1, J2, and J3 region in Arabic numbers, which are given in the order of discovery, for example II.1.1.1 (IWG-SCC 2009). Due to the increasing diversity among SCCmec subtypes, IWG-SCC proposed preparing a computerized system and the available typing methods at http://www.staphylococcus.net and/or http://www.sccmec.org/, which will enable the characterization and assignment of certain SCCmec subtypes based on the occurrence of specific elements within the J regions.

7. Methods for SCCmec typing

7.1. PCR-based SCCmec typing methods

With reports about new SCCmec types, there is an increasing need for multiple methods to type SCCmec cassettes properly. Before 2000, SCCmec elements were typed using conventional molecular methods such as cloning and sequencing (Hiramatsu et al. 1992; Ito et al. 1999). To differentiate the types and subtypes of SCCmec, PCR-based methods have been widely used. To date, no single PCR method is available that can identify all types and subtypes. The first identification of mec and ccr complexes was amplification using long-range PCR with sets of primers. The first widely used SCCmec typing protocol for the identification of SCCmec types I, II, III, and IV was described in 2002 by Oliveira and de Lencastre. They developed a multiplex PCR strategy and optimized the rapid assignment of SCCmec types to MRSA strains for eight loci (I type: cifF2; II type: kdp; II and III types: mecI; I, II, and IV types: dcs; III type: rif4; to distinguish structural variants IA: IS431-pUB110; to distinguish structural variants IIIA: IS431-pT181), (Oliveira and de Lencastre 2002). Multiplex PCR seems to allow the identification of SCCmec types and subtypes efficiently in one analysis, although the sensitivity and specificity to detect each component are sometimes lower than that in a single PCR. The Oliveira and de Lencastre protocol for the typing of SCCmec was a useful tool for detecting multiple components of SCCmec, but due to difficulties in optimizing the assay, the Oliveira assay had limitations in detecting newly described SCCmec types. In comparison to the Oliveira and de Lencastre method, in 2005, the Zhang group designed eight sets with specific primers for the mec, ccr, and J1 regions. In 2012, the version of this method was updated (Zhang et al. 2005; Zhang et al. 2012). This multiplex PCR for SCCmec typing was based on the presence of a single band for each type or subtype and included three multiplex PCRs (M-PCRs). The first M-PCR allows for the classification of MRSA isolates into SCCmec types and subtypes I, II, III, IV (a–d), and V. The mec classes A and B can be determined by the second M-PCR. The third M-PCR allows for the identification of the 1–3 ccr complex. In 2007, the Oliveira group published an update on the method that was originally introduced in 2002. Milheiriço et al. formulated a protocol that allows for the identification of SCCmec types I, II, III, IV, V, and VI (Milheiriço et al. 2007a, 2007b). Furthermore, for the subtyping of SCCmec type IV, Milheiriço et al. developed a multiplex PCR strategy in which seven primer pairs were designed to simultaneously detect the ccrB allotype 2 and polymorphic J1 regions described for SCCmec type IV and a new J1 region. The Milheiriço et al. method also allowed for the identification of genes or gene fragments located in the J region. Furthermore, it was revealed that the SCCmec type I cassette contains the cifF2 element (J1) and the dcs gene (J3). In SCCmec type II, the dcs gene (J3), the kdpA-F (J1) operon, and the mecI gene (mec complex) were also identified. SCCmec type III includes the rif4 gene (J3), the SCCmec-III element (J1), and the mecI gene (mec complex). The dcs gene (J3) is present in the IV cassette, while the SCCmec-V element (J1) is present in SCCmec type V. The most significant advantage of this method is that it is a quick single-tube M-PCR for detecting all SCCmec types in both S. aureus and CoNS, providing versatility in its applications across numerous research groups till today. According to the research conducted by Augusto et al. clear results and SCCmec types were obtained using the Milheiriço method for S. aureus strains. However, for CoNS strains examined by Garbacz et al. the method was performed, but some isolates exhibited new combinations of genes, and for most CoNS strains, SCCmec types were not unambiguously identified (Weisser et al. 2010; Garbacz et al. 2021; Augusto et al. 2022). At the same time, Kondo et al. described a complex method for the identification of SCCmec types I to VI and J regions (Kondo et al. 2007). These methods include six multiplex PCRs in which M-PCRs 1 and 2 are used for the discrimination of the SCCmec type assignment; M-PCR 3 or M-PCR 4 is used for subtyping based on differences in the J1 region; and M-PCRs 5 and 6 are used for the identification of integrated copies of transposons (Tn554 or ΨTn554) and plasmids (pUB110 or pT181). Other SCCmec typing methods were developed in 2005 by Hisata et al. and in 2007 by Boye et al. (Hisata et al. 2005; Boye et al. 2007), but it was not possible to perform the analysis in a single M-PCR. Multiplex PCRs proposed by Milheiriço et al., Zhang et al. and Kondo et al. are by far the most commonly used methods for SCCmec typing. For confirmed results, it is worth using various methods. However, SCCmec typing by multiplex PCR is limited to SCCmec types I–VI. Often, PCR methods allow for the detection of additional genes that do not indicate a subtype but appear in cassette types in which they are not described. Therefore, other methods should applied to type the increasing number of SCCmec types, including types VII–XV. Table 1 presents the methods used for SCCmec cassette typing. In fact, analysis of the complete sequence of SCCmec by whole genome sequencing of these strains with cassettes that are not typable by traditional amplification techniques would provide important data for the study of novel SCCmec types and recombination events. On the other hand, next-generation sequencers are not basic equipment in every microbiological diagnostic laboratory, so the development of a new PCR-based method for the detection of all described types and unusual SCCmec compositions would also be highly appreciated by both clinicians and microbiologists.

7.2. Next generation sequencing (NGS) in SCCmec typing

The development of molecular diagnostic methods, which encompass sequence analysis, has facilitated the accurate identification and comprehensive characterization of microorganisms. This advancement is crucial for ensuring successful therapy and promoting patient recovery (Sabat et al. 2017; Kosecka-Strojek et al. 2019). Additionally, the typing of bacterial strains is essential to investigate transmission pathways and to support outbreak monitoring. NGS allows for the sequencing of whole genome(s) of pathogens isolated from patient samples and even with the detection of multiple species within a single sample. Moreover, NGS provides high-resolution genomic data, enabling detailed analysis of SCCmec elements, including subtype identification and characterization. Comprehensive characterization makes it possible to detect novel subtypes, reducing the likelihood of misclassifying SCCmec types. When compared to PCR-based methods that only detect the presence of mec genes and classic SCCmec cassettes, which are suitable for investigating hospital outbreaks, NGS, with its capability for not only identifying resistance genes but also identifying virulence factors and clonal relationships, is more appropriate for large-scale epidemiological studies (Tchamba et al. 2023). Despite the versatility of NGS, the critical point lies in library preparation, which is a time-consuming affair requiring careful attention to eliminate contamination and mistakes. Errors can lead to NGS platforms producing relatively short reads, which can complicate the assembly process and analysis of SCCmec cassettes. After sequencing, the sequenced fragments (reads) can be assembled de novo with appropriate software and/or pipelines. Then, contigs are aligned to a reference microorganism. After NGS, the genetic relationship between isolates can be investigated using a gene-by-gene comparison and multilocus sequencing typing (MLST) (Deurenberg et al. 2017). For a precise SCCmec analysis, the SCCmecFinder website (https://cge.cbs.dtu.dk/services/SCCmecFinder/) and the CLC Genomic Workbench software (Qiagen, Germany) can be used (Kaya et al. 2018). The first tool identifies SCCmec elements in sequenced S. aureus isolates. The analysis is based on the submission of the sequencing genome or contigs to the website of SCCmecFinder. Then, users obtain information on the prediction of the SCCmec types, including information about genes in the ccr complex, the mec complex, and J regions. This website has limitations because this analysis is not available for staphylococcal species other than S. aureus. For analysis with CLC Genomics Workbench software, genome or contig sequences are needed, and the SCCmec element is also downloaded from the NCBI online database (https://www.ncbi.nlm.nih.gov/). Next, the downloaded SCCmec element sequences undergo BLASTn searches against de novo contigs using CLC Genomics Workbench. NGS-based methods have been used and described in many studies on resistance profile identification, virulence gene identification, or molecular typing. Additionally, NGS has been used to delimit the transmission pathways and characterize outbreaks in hospital units by mostly MRSA rather than MRCoNS strains. Different studies have been published on next-generation sequencing of S. aureus isolates from outbreaks, and most of these studies determined relatedness by the number of SNPs between genomes. In 2008, Berglund et al. described a new MRSA clone that caused an outbreak in a neonatal ward (Berglund et al. 2008). Weterings et al. described the transfer of methicillin resistance to a methicillin-susceptible S. aureus that subsequently caused a methicillin-resistant S. aureus outbreak in the oncology ward. They found multiple variations in the compositions of MGEs within MRSA isolates and between individuals with MSSA strains (Weterings et al. 2017). Cavanagh et al. reported the population structure of a large collection of clinical European S. haemolyticus isolates in which several multiple antibiotic-resistant European clones were detected (Cavanagh et al. 2014). These cases confirm the ability to transfer the SCCmec cassette with antibiotic resistance genes between Staphylococcus strains, which confirms the occurrence of HGT between staphylococci.

7.2.1. NGS to report new SCCmec types and subtypes

Despite the conventional PCR-based methods being used, a new approach based on NGS opened the field to whole genome sequencing. The NGS method was introduced to identify and verify the structures of new SCCmec types. According to the information made available on 2021, May 22 by IWG-SCC on the website https://www.sccmec.org/, to report the new SCCmec and SCCmec subtype, knowledge of the complete sequence of SCCmec is necessary. Before proceeding, researchers should contact the curator and obtain approval from IWG-SCC to avoid duplicate names and reporting structures that are not SCCmec. Due to the expression of a new element at different SCCmec sites, it is strongly recommended by IWG-SCC to use long-read sequencing technology, i.e. the Pacific Biosciences system or the Oxford Nanopore system to confirm the sequence of a new cassette. Furthermore, researchers are requested to send isolates, potentially candidates for a new SCCmec element, to two reference laboratories and to reference strain banks of SCCmec, such as the Statens Serum Institute (SSI) in Denmark and/or the National Institute of Infectious Diseases (NIID) in Japan. Subsequently, SSI and NIID will perform long-read whole genome sequencing and annotation to verify the nomenclature suggested by submitters. Furthermore, after strain deposition in reference laboratories, SSI and NIID will share strains when requested to contribute to SCCmec research worldwide. Researchers will be acknowledged and asked permission in order to share those strains. IWG-SCC announced that only complete structures of SCCmec present in S. aureus have been designated as new SCCmec type names, regardless of the host. Furthermore, the IWG-SCC has decided not to annotate new subtypes of SCCmec in species other than S. aureus due to the high complexity of the elements found in isolates other than S. aureus. However, the group has proposed an alternative nomenclature for non-aureus staphylococci as ‘SCCmec_{[NAME OF STRAIN]}’. However, the authors of this study argue that this may not be an optimal solution considering the global scale and the increasing identification of CoNS strains exhibiting atypical SCCmec cassette structures. We believe that establishing an independent database solely focused on SCCmec among CoNS would enhance the identification and monitoring of new SCCmec types or variants in CoNS. This approach would greatly aid scientists in their research and surveillance efforts.

8. Clinical aspects and significance of new types of SCCmec in coagulase-negative staphylococci

Staphylococcal infections generate serious questions in the diagnosis and epidemiology of infectious diseases. Molecular characteristics of isolated bacterial strains from nosocomial infections include very important data that will affect the diagnosis and choice of targeted therapy. Recent years of research have shown that CoNS staphylococci are reservoirs of antibiotic resistance genes, virulence genes, and SCCmec elements. Resistance to methicillin is a worldwide challenge that occurs in community-related infections, as well as in nosocomial infections. The increase in the frequency of infections caused by multiresistant microorganisms makes the study of the distribution and diversity of genetic elements that confer resistance to pharmaceuticals in bacteria of clinical importance necessary, as well as the study of the transmission mechanisms of such elements. The SCCmec element is a MGEs widely distributed among MRCoNS species, which varies depending on the host species, various environments, and geographical locations. SCCmec elements cannot be limited solely to being carriers of resistance to methicillin/β-lactam antibiotics, as they could also serve as carriers of other essential genes for the enhanced survival of staphylococci in diverse environments. The diversity of SCCmec between CoNS is a challenging phenomenon. Particularly in nosocomial infections where the SCCmec element can be transmitted to other staphylococcal species. Identification of MRCoNS species could help determine the contribution of each species to antibiotic resistance and SCCmec types in the community and help design effective surveillance and control strategies. An increase in antimicrobial resistance limits therapeutic options for infections caused by MRCoNS. SCCmec has a dynamic constitution prone to the accumulation of MGEs, leading to the further development of these elements and the genesis of new SCCmec types, which makes bacterial strains difficult to detect and identify. Antibiotic pressure in the environment could induce the insertion of other resistance genes into SCCmec elements. The IWG-SCC proposed the elaborate computerized system and the available typing methods at http://www.staphylococcus.net and/or http://www.sccmec.org/. There is a clear need to develop a unique and efficient operating typing system for CoNS because the IWG-SCC has decided not to annotate new SCCmec subtypes in species other than S. aureus due to the high complexity of the elements found in CoNS species. However, there are many reports of infections caused by CoNS strains carrying new SCCmec structure variants or compositions. Moreover, the number of such new, uncharacterized and various elements will increase in the near future. Unfortunately, the existing page is not complete. Compared to other international databases, e.g. https://pubmlst.org/, there is a lack of access and the deposit rules are too complicated. On the basis of data from the literature alone, it is difficult to control the emergence of new variants in CoNS strains. There are many publications, and there is an increase in infection rates caused by CoNS strains. Due to the strong need to monitor the transfer of MGEs between strains, the database would be a major improvement in the identification of bacteria and the recognition of the type of SCCmec, allowing for the introduction of a rational therapy for CoNS infections in terms of effectiveness.

9. Conclusions

This paper presents a review of the current distribution of SCCmec types, subtypes, and variants present among staphylococcal species with an in-depth review of CoNS species, including S. epidermidis, S. haemolyticus, and S. capitis. These species are the most common pathogens that primarily cause DA-HAIs and FBRIs. Resistance to methicillin/β-lactam antibiotics is a worldwide phenomenon that occurs in community-related infections as well as in nosocomial infections. Advanced phenotypic and genetic studies of CoNS isolates are decisive for their characterization and for understanding the transmission mechanisms of such elements because CoNS are also reservoirs of SCCmec elements for methicillin-susceptible S. aureus. Therefore, knowledge of SCCmec cassettes present in coagulase-negative staphylococci will allow us to improve our knowledge of the spread of antibiotic resistance not only among coagulase-negative staphylococcal strains but also among S. aureus strains. Many loopholes or a lack of CoNS SCCmec annotation rules and the high genetic diversity of genomes necessitate the development of a unique and efficient operating typing system for CoNS.

Authors contributions

MW-G and MK-S and conceived this study and investigated previous studies. MW-G and MK-S performed the scientific literature search. MW-G wrote the first draft of the manuscript and prepared figures. MK-S and JM provided significant feedback and edited the manuscript. All authors read and approved the final manuscript.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Additional information

Funding

Publication was co-funded by the Ph.D. Students’ Association of Jagiellonian University.