The Evolution and Dissemination of Methicillin Resistance Determinant in Staphylococcus aureus The Evolution and Dissemination of Methicillin Resistance Determinant in Staphylococcus aureus

Staphylococcus aureus is an opportunistic pathogen and is frequently associated with the antimicrobial resistance. There has been horizontal gene transfer of Staphylococcus chromosome cassette mec (SCC mec ) among the staphylococcal species that colonize a similar colonization niche, which eventually results in emergence of new variant with enhanced survival ability in terms of antimicrobial resistance and virulence level in S. aureus . Evolution and dissemination of SCC mec structure resulted in the emergence of methicillin-resistant S. aureus (MRSA) clones around the world covering hospital, community, and livestock settings. MRSA also has the ability to resist different antibiotic profiles known as multidrug-resistant S. aureus (MDR S. aureus ).


Introduction
Staphylococcus aureus is an opportunistic pathogen and lives as part of the animal normal flora of skin and nasopharynx. Favorably, it resides in the nasal mucosal environment posing infection threat to human as well as in domestic animals [1,2]. In human, it is the leading agent of infection involving bloodstream, skin, and soft tissue to the lower respiratory tract [3][4][5]. S. aureus can easily colonize certain part of the body, especially the exposed area on skin due to ulcers, burns, and surgical wounds [6].
Immunocompromised patients with S. aureus infection may suffer several diseases such as bacteremia, ventilator-assisted pneumonia (VAP), endocarditis, and osteomyelitis, especially when the patients are frequently exposed to injections and catheter insertions [20,21]. S. aureus can also cause toxin-mediated disease such as toxic shock syndrome, scalded skin syndrome, and Staphylococcal foodborne diseases (SFD) [21]. Frequently, S. aureus is the main cause of skin and soft tissue infection (SSTI) in human [22].

Methicillin-resistant S. aureus (MRSA)
MRSA has the ability to resist almost all available β-lactam antibiotics. Statistics showed about 40-70% of S. aureus nosocomial infections worldwide are caused by MRSA. MRSA was first reported in a hospital in the United Kingdom in 1961 after the introduction of methicillin to treat patient with penicillin-resistant Staphylococcus infection [23].
Generally, MRSA can be categorized into two major groups known as hospital-associated MRSA (HA-MRSA) and community-associated MRSA (CA-MRSA). Globally, the majority of MRSA infections are HA-MRSA that are acquired from healthcare facilities. Currently, MRSA isolates are subdivided into three major groups known as hospital-associated MRSA (HA-MRSA), community-associated MRSA (CA-MRSA), and livestock-associated MRSA (LA-MRSA). Previous reports revealed that both HA-MRSA and CA-MRSA isolates differ distinctly from each other, with HA-MRSA showing high antimicrobial resistance but less virulence and lack of capabilities as colonizers [23][24][25][26]. Meanwhile, CA-MRSA isolates exhibit a low antimicrobial resistance but a high virulence harboring PVL gene and numerous pathogenicity factors, as well as good colonizers [26][27][28][29].
MRSA spread in population since 1990 and become the major cause of community-associated infection [27]. The scenario worsens when multidrug MRSA emerges, in which it can resist more than two antibiotics of different classes that reduce the option for available treatment of Staphylococcal infection [30,31].
Methicillin resistance characteristic in S. aureus is due to the presence of altered penicillinbinding protein (PBP2a) in the cell wall that has a reduced binding affinity to β-lactam antibiotics. PBP2a is encoded by mecA gene that is located in the large chromosomal cassette called staphylococcal chromosome cassette mec element (SCCmec) [32][33][34][35]. The mecA gene expression is controlled by mecI-mecRI regulatory genes encoding repressor and inducer protein, respectively [36].

SCCmec structure
Staphylococcal cassette chromosome mec (SCCmec) has a size of about 20-60 kb. The structure is unique as it carries various mobile genetic elements that are integrated in it [37]. To date, more than 80 SCCmec elements have been identified in several staphylococci species [38].
SCCmec disseminates among Staphylococcal species by horizontal gene transfer and integrates at a specific site called attB or ISS (integration site sequence) at the 3′ end of orfX gene that encodes for unknown function [39].
A single SCCmec carries mec complex and cassette chromosome recombinase (ccr) flanked by direct inverted repeat (DR) and inverted repeat (IR) sequences; mec complex consists of mecA gene (methicillin resistance determinant), mecRI (sensor inducer), and mecI (mec repressor). Both mecRI and mecI are recognized as mec regulator elements, while ccr genes encode serine recombinases (ccrA, ccrB, ccrC) responsible for site and orientation of specific integration and excision of SCCmec. In addition, SCCmec also harbors other elements such as insertion sequences (IS), plasmids, and transposons [24, 40, 41].

Complete SCCmec
To date, the International Working Group-SCCmec (IWG-SCCmec) identified eleven SCCmec types based on complete nucleotide sequences in Staphylococcal databases, and each SCCmec type is named using a roman numeral based on the unique combination of ccr complex and mec complex [40][41][42]. A complete SCCmec structure in S. aureus contains a mec complex (mecA, mecRI and mecI), a ccr complex (ccrA, ccrB, ccrC), and a J region (region other than mec and ccr complexes) [40].
Furthermore, many different SCCmec subtypes have also been described containing the same ccr and mec gene combination but vary in the J regions [40]. Among the eleven SCCmec types (I-XI) that have been reported so far, five of them (SCCmec I, II, III IV, and V) are globally distributed, while others only distributed in certain countries [38,43]. Three (SCCmec IVa, SCCmec IVc, SCCmec V) from the 11 SCCmec types have been detected in MRSA isolated from animals called LA-MRSA [42]. In general, SCCmec type IV and V are more widely found among CA-MRSA, and the other three types (SCCmec I, II, III) are frequently found among HA-MRSA [44,45,46]. An early study by Ito et al. detected only three types of SCCmec structures (SCCmec type I, II, III) isolated from human [37], and a recent finding showed that MRSA with SCCmec type I, II, III is originated from animals [41].
Different types of SCCmec in MRSA are also observed to be geographically distributed. For example, SCCmec type III or IIIA was most commonly found in Asian countries, but Korea and Japan had more type II while Taiwan had more type IV [47]. SCCmec type IV was also commonly found in Latin and European countries [48,49]. Similarly, in African countries, SCCmec type III was also predominant with SCCmec types II, IV, and V found in selected countries such as Egypt, Niger, Nigeria, Algeria, Tunisia, and South Africa [50].

Pseudo-SCCmec
Pseudo-SCCmec is recognized as SCCmec that does not carry ccr complexes but has mecA gene. Although this element is different from the complete SCCmec in terms of gene or operon organization, it still has some similarities in certain parts in both pseudo-SCCmec and complete SCCmec structure. Deletion is the major event as inferred by the absence of certain genes or operon in pseudo-SCCmec structure. For example, regions within mec complex and J region are absent in both pseudo-SCCmec II.5 and pseudo-SCCmec16691. It was observed that pseudo-SCCmec16691 lacks J1, J2 regions, and ccr genes, whereas missing parts were detected in pseudo-SCCmec II.5 and replaced by transposable elements called Tn6012 [51,52].
However, certain pseudo-SCCmec does not carry both mec and ccr complexes. An example is the arginine catabolic mobile element (ACME) for having SCC-like elements but lack in mecA and ccr genes. This could be the remnant of SCCmec structure that had gone through multiple mutational events. Lindqvist et al. discovered first remnant of pseudo-SCCmec structure in methicillin susceptible S. aureus (MSSA) that caused clonal outbreak in Sweden. They suggested that this pseudo-SCCmec structure could be derived from SCCmec type II [52]. ACME is found in both MRSA and MSSA, especially with sequence type ST8 (ST8), and has been disseminated in virulent S. aureus by horizontal gene transfer [3,53]. Nevertheless, ACME was frequently associated with MRSA-IVa with sequence type 8 (ST8-MRSA-IVa), which was also known as CA-MRSA USA300 [3,53]. ACME has been associated with the ability of CA-MRSA to colonize on other parts of human body such as skin and mucosal membranes rather than limited to only nostril. The acquisition of ACME may enhance the ability of CA-MRSA to survive in acidic environment of human skin by driving production of polyamine-resistant enzyme that combats excess host polyamine (toxic compound on human skin for S. aureus) [54].

Origin of SCCmec structure
The origin of SCCmec in MRSA is still in debate; mecA gene was believed to be originated from Staphylococcus fleurettii due to a high sequence similarity (>99%) with mecA gene of a MRSA strain N315. It was proposed that SCCmec is a combination of SCC elements without mec complex, and the mec gene complex was derived from S. fleurettii since no evidence showed that S. fleurettii contained SCCmec structure in its chromosome [55].
Several studies described coagulase-negative staphylococci (CoNS) as the primary reservoir of the SCCmec structure in S. aureus, which was considered as the recipient strain due to some reasons; a very similar SCCmec structure and organization was observed in both S. aureus and CoNS [56,57], and the prevalence of methicillin-resistant coagulase-negative staphylococci (MRCoNS) in human is higher as compared to MRSA [35, [56][57][58][59]. Although a study discovered other non-staphylococci species called Macrococcus to also carry SCCmec-like elements, those were different with SCCmec in MRSA in terms of nucleotide sequences and genetic organization of the mec complex [55].

From coagulase-negative staphylococci species to MRSA
The existence of various forms of SCCmec in MRCoNS as compared to MRSA becomes the main argument why MRCoNS is suggested as the main reservoir of SCCmec for S. aureus leading to the emergence of MRSA [40,57]. In a rapid genetic typing, polymerase chain reaction (PCR) technique is used to characterize the SCCmec types instead of nucleotide sequencing analysis. Consequently, SCCmec from MRCoNS is frequently defined as non-typeable due to a diverse combination of ccr and mec complexes that could not be assigned based on current SCCmec structure databases used against S. aureus [7]. Nevertheless, Zong et al. successfully assigned 10 SCCmec elements with a new combination of ccr and mec complexes in various species of MRCoNS. They assigned these untypeable SCCmec elements as UT1-UT10 [35]. In addition, another study also described new SCCmec types in Staphylococcus hominis and described those as NT1till NT4 [60].
Staphylococcus epidermidis, Staphylococcus haemolyticus, and S. hominis were found to carry a diverse SCCmec structure among CoNS. SCCmec type IV is the common structure found in S. epidermidis, while other SCCmec types I, II, III, V, VI and non-typeable SCCmec were also detected at a lower rate [61,62]. For S. haemolyticus, SCCmec type V predominated in combination with other novel SCCmec types [60,63]. In S. hominis, SCCmec types contained a combination of novel non-typeable SCCmec, SCCmec types VI, VIII, III, and other elements [35,61].

From MSSA to MRSA
MRSA emerges when MSSA receives SCCmec structure elements from other MRSA or MRCoNS via horizontal gene transfer [64]. In a specific condition (high vancomycin concentration), SCCmec is unstable in certain MRSA that can lead to complete or partial deletion of SCCmec structure, which may result in the presence of certain SCCmec DNA fragment to remain in S. aureus chromosome [64][65][66][67].
Wong et al. [64] identified SCCmec type II with internal deletion in MSSA isolates from different geographical areas. This happened during in vitro exposure to vancomycin [64]. Furthermore, Vandendriessche et al. [67] described MSSA CC398 as the precursor for emergence of MRSA CC398 in livestock. They found non-SCCmec elements in MSSA CC398 harboring czrC and tet(K) genes generated during partial excision of SCCmec elements [67].

Clonal dissemination of MRSA
Nowadays, the dissemination of MRSA has become a major global problem that threatens human health [27]. However, only limited clones of MRSAs could be inferred to disseminate in different countries and continents through genotypic analysis using several DNA typing methods such as SCCmec typing, PFGE, MLST, and spa typing [27, 68,69]. For example, more than 3000 MRSA isolates from certain continents (Europe, USA, and South America) were described to belong to only five major pandemic clones or clonal complexes (CC5, CC8, CC22, CC30, and CC45) [70]. To date, 11 clonal complexes (CC1, CC5, CC8, CC12, CC15, CC22, CC25, CC30, CC45, CC51, and CC121) have been detected in which 5 of them (CC8, CC15, CC22, CC30, and CC45) were isolated from human [71,72]. These successful clones may transmit their genetic elements into other S. aureus, which are well adapted to hospital environment [73].
MRSA strain COL was the first MRSA clone detected carrying SCCmec type I with sequence type 250 (ST 250) and belonged to clonal complex 8 (CC8). Then, other MRSA clones with SCCmec type II and III were reported and recognized as EMRSA-1 (ST239), EMRSA-5 (ST247), and New York/Japan clone (ST5, USA100) [74]. Certain MRSA clones were originated from community setting. For example, Wang and co-workers (2007) detected the spread of community-associated SCCmec type IV and V MRSA in hospital setting in Taiwan between 1999 and 2005. They concluded that SCCmec types IV and V are carried by both CA-MRSA and HA-MRSA [75][76][77].
The popular human MRSA pandemic clones, the EMRSA-15 and EMRSA-16, were identified in the United Kingdom (UK) around early 1990s. Since then, the clones become predominant healthcare-associated MRSA in UK [78,79] and several European countries such as Denmark [80], Sweden [81], Belgium [79], and Spain [82]. Studies in Kuwait [83] and USA [84] also reported the spread of EMRSA-15 and 16 clones in hospital setting in the countries. To date, these clones have already been widespread in 15 countries around the world [85]. Both MRSAs belong to SCCmec type IV with sequence type 22 (ST 22) for EMRSA-15 and sequence type 30 (ST 30) for EMRSA-16 and originated from hospital setting. EMRSA-15 and 16 have high surviving and spreading rate in hospital compared to other EMRSA in UK [78]. In 2013, MRSA clone with a rare sequence type, ST 779, was identified in eleven Irish hospitals from 2006 until 2011 harboring a novel pseudo (SCCmec)-SCC-SCC CRISPR composite element. This clone contained novel mec class region, a fusidic acid resistance gene (fusC), and two copper resistance genes (copB and copC) but lacking ccr genes [86].
CA-MRSA clones have also been observed to disseminate worldwide particularly with sequence types ST80 and ST30. MRSA clone with ST80 is the most common CA-MRSA clone in European countries and usually carries PVL genes. Moreover, ST80 clone also showed resistance toward fluoroquinolones, tetracyclines, and fusidic acid [87]. CA-MRSA clone with sequence type ST30 was observed to disseminate in Asian and Oceanic countries. An example is the multidrug USA300 clone, known as West Pacific clone. It was first identified in the USA and carried plasmid that encodes several antibiotic resistance genes [88]. Enany et al. identified novel clones with sequence types ST1010 (121)c and ST1009 (1153)c isolated from Egypt after they analyzed different genetic patterns of PVL+CA-MRSA isolates from different countries [89].
In certain countries, it was found that MRSA can also spread among livestock, known as LA-MRSA. LA-MRSA CC398 is the popular clonal complex among livestock and has already been reported to spread in several European farms in Netherland, Denmark, Germany, France, and Italy [90]. MRSA CC398 was originated from pigs and spread among dairy cattle and turkey [91,92]. In Netherlands, MRSA contamination on meat was reported after 2217 meat samples were analyzed covering 35.3% turkey, 15.2% beef, 15.2% veal, 10.7% pork, and 6.2% lamb meat [93]. LA-MRSA can be transmitted to human by physical contact with livestock contaminated with MRSA [94]. LA-MRSA may have equal virulence ability as compared to CA-MRSA and HA-MRSA toward human. Therefore, persons with continuous exposure to livestock carrying LA-MRSA are at high risk [95]. Other than meat, LA-MRSA can also be found in dairy milk. Recently, 11 sequence types were detected from LA-MRSA isolated from 15 Brazilian dairy farms (n = 552) with four of them contain novel sequence types (ST1622, ST1623, ST1624, and ST1625) [96].

Multidrug-resistant (MDR) MRSA
Antibiotic or antimicrobial drugs are the most effective therapeutic agents used in treating microbial infections through either one or both bactericidal and bacteriostatic effects. Nevertheless, antibiotic or antimicrobial drug resistance has been a major problem worldwide, with incidence of MRSA reported in healthcare facilities in Asia to reach its peak in late 1990s, and stayed at plateau level during 2000s [97]. The heavy usage of drugs in treatment hastens the selection of bacteria that harbor multidrug resistance genes particularly S. aureus to proliferate and dominate [98,99]. Moreover, over-crowded community creates environment that is suitable for the rapid spread of numerous multidrug-resistant pathogens, particularly the airborne organisms such as S. aureus.
The emergence of multidrug-resistant S. aureus in both hospitals and community invokes a tremendous financial burden due to the persistence of hard-to-treat infections [97,[100][101][102]. Until present, it was reported that <90% of S. aureus strains are resistant to penicillin as well as ordinary antimicrobial agents such as drug from categories of aminoglycosides, ansamycins, anti-staphylococcal β-lactams (or cephamycins), chloramphenicols, fusidanases, fluoroquinolones, glycopeptides, lincosamides, macrolides, phenicols, and tetracyclines [103][104][105]. We are now observing the emergence of multidrug-resistant S. aureus and MDR-MRSA with broad spectrum of resistance with a distinct ability to survive and spread in the hospital environment, community setting, as well as livestock sectors.
There has been a dramatic increase in the incidence of nosocomial infections as well as community-associated MRSA and livestock-associated MRSA caused by strains of S. aureus that are resistant to multiple antibiotics [106]. At present, there have been reports that some strains demonstrate resistance to as many as 20 antimicrobial compound types, including antiseptics and disinfectants [107,108]. Central Asian surveillance studies found that the prevalence of MRSA infection in tertiary hospital was reported in 10 among 1000 hospital admissions [109] and incidence reported previously in Japan was between 0.7 and 0.8 per 100 admission from 1999 to 2003 with a total rate among hospitalized patients in the Asia-Pacific region at 45.9% [110,111]. Previous surveillance also reported that Asia is among the highest for the incidence of MRSA in the world, and interestingly a novel MRSA strain with glycopeptides resistance had spread in livestock animals making it as a potential human pathogen in this region [112].
Several studies attempted to profile all possible multidrug-resistant MRSA since 1987, encompassing samples from hospitals, community, as well as veterinary settings [113,114]. Lim et al. (2013) carried out temporal comparative surveillance of antibiograms from clinical samples in 2003-2008 and showed a significant increase in resistance rates (from 1 to 96%), as well as multidrug-resistant phenotypes (96%). This study also indicated the prevalence of multidrug-resistant MRSA with SCCmec type III and ST239 [99]. Another study also reported the prevalence of resistance against other important antibiotics such as mupirocin, whose resistance rate in Malaysia is still low, but still higher than previous reports in Malaysia [107]. Another cross-sectional studies at a few major medical centers in Malaysia found that the occurrence of MRSA infection increased gradually with years, from 25.7 to 28.7% in 1996, 27.9% in 1998, and 33% in 2000 [115,117,118]. Meanwhile, a study done at a single Malaysian hospital found a gradual reduction in MRSA prevalence from 2002 to 2006, most likely due to the improvement in the quality of healthcare systems [103, 109, 116 118].
The first international surveillance study on epidemiology of CA-MRSA in Asian countries revealed important findings with regard to the current epidemiology of MRSA infections in the community and hospitals within Asia with multidrug-resistance rates at 73.1 and 83.7% for CA-MRSA and HA-MRSA, respectively [119]. At least, 357 isolates of CA-MRSA were analyzed with resistance rates of gentamicin, ciprofloxacin, and trimethoprim/sulfamethoxazole being significantly lower than those of HA-MRSA isolates, whereas resistance rates of clindamycin, erythromycin, and tetracycline were similarly high in both CA-MRSA and HA-MRSA [119,120].

Conclusion
S. aureus and MRSA evolve and adapt the changing environment. Therefore, dissemination of MRSA should be continuously monitored for the antibiotic susceptibility pattern and molecular epidemiology comprising hospital, community, and livestock settings. The origin and dissemination of SCCmec are also important to be tracked in the diverse staphylococcal population. With the advancement in molecular methods such as next-generation sequencing, the pattern of the genetic evolution, spread of the bacteria, and the resistance determinants can be further explored and understood.