The Continuing Threat of Methicillin-Resistant Staphylococcus aureus

Staphylococcus aureus has been an exceptionally successful pathogen, which is still relevant in modern age-medicine due to its adaptability and tenacity. This bacterium may be a causative agent in a plethora of infections, owing to its abundance (in the environment and in the normal flora) and the variety of virulence factors that it possesses. Methicillin-resistant S. aureus (MRSA) strains—first described in 1961—are characterized by an altered penicillin-binding protein (PBP2a/c) and resistance to all penicillins, cephalosporins, and carbapenems, which makes the β-lactam armamentarium clinically ineffective. The acquisition of additional resistance determinants further complicates their eradication; therefore, MRSA can be considered as the first representative of multidrug-resistant bacteria. Based on 230 references, the aim of this review is to recap the history, the emergence, and clinical features of various MRSA infections (hospital-, community-, and livestock-associated), and to summarize the current advances regarding MRSA screening, typing, and therapeutic options (including lipoglycopeptides, oxazolidinones, anti-MRSA cephalosporins, novel pleuromutilin-, tetracycline- and quinolone-derivatives, daptomycin, fusidic acid, in addition to drug candidates in the development phase), both for an audience of clinical microbiologists and infectious disease specialists.


Introduction
Taxonomically, the genus Staphylococcus is included in the Micrococcaceae family within the phylum Actinobacteria [1,2]. They are Gram-positive, catalase-positive, and bacitracin-resistant cocci [3]. Staphylococci are non-spore forming bacteria, nevertheless they are very common in nature and they can survive in a variety of harsh environments outside of the body, in addition to being resistant to many disinfecting agents [1,2,4,5]. Staphylococcus aureus is coagulase-positive, which is another important differentiating factor between this species and coagulase-negative staphylococci (CoNS; e.g., S. epidermidis) [1,6]. S. aureus (and its methicillin-resistant counterpart) may colonize various mucosal sites of the body: the nostrils (nares), throat, dedicated areas of the skin (including the axilla, groin, and perineum; these skin surfaces are usually moist), and rectum. S. aureus is an exceptionally successful pathogen, which is still relevant and dangerous in modern age-medicine [7]. Furthermore, small-colony variants of S. aureus (SCVs; a sub-population of bacteria that are naturally present in small quantities) allow for chronic, recurrent, and antibiotic-resistant infections to develop and persist in the host [4,8]. In this morphotype of S. aureus, mutations occur in the genes that regulate metabolic activity, resulting in the so-called "dwarf colony" phenotype on agar plates, while, in vivo, these bacteria can withstand otherwise lethal doses of antibiotics [9,10]. S. aureus is an important causative agent of bacteremia and, though hematogenic dissemination, additional infections, such as infective endocarditis, complicated skin and soft tissue infections (cSSTI), osteoarticular infections, prosthetic device infections (PDI), and pleuropulmonary infections may also occur. In addition, S. aureus

MRSA Colonization and Screening
In the current climate of the antibiotic-resistance crisis, it is important for laboratories to monitor the trends and mechanisms of resistance in S. aureus (especially methicillin/oxacillin-resistance), in addition to the spread of successful clones [85]. The detection of carriage is another important hallmark in infection control and the successful eradication of MRSA, which is among the responsibilities of clinical microbiology/public health laboratories [86]. This is further highlighted by the fact that, in most cases, colonization (lasting for periods of few months to a few years) precedes infection [87]. Colonization with MRSA is a well-known risk factor in developing an MRSA infection in adults and children; this is especially true for patients who acquire MRSA colonization in the nosocomial setting, where the risk of developing an MRSA infection as a result is around 30% [85,88]. MRSA is usually spread by direct skin-to-skin contact and this may occur during hospital admission, transfer, or other healthcare-related contact; however, the role of shared public spaces (e.g., dormitories, gym, barracks, etc.) was also noted [86]. A set of bacterial determinants influence carriage (adhesive proteins (SdrC, SdrD, and SdrE), clumping factor, fibronectin binding proteins (clfA, clfB, fnbA, fnbB), adhesive molecules (altA, eap), cell surface-remodeling enzymes (sceD, oatA, altA), and biofilm formation) and host-specific factors (the integrity of the skin barrier, chronic inflammation, toll-like receptor 2 (TLR-2) gene polymorphisms, antimicrobial peptides (AMPs), such as cathelicidins, defensins, proteolysis-inducing factor, lactoferrin, RNase 7; individual variation in cortisol and 25-OH-D-vitamin levels, HLA-DR3 allele) [86,88]. Based on the patterns of carriage, persistent, intermittent, and non-carriage has been described. The so-called "culture rule" was established for the appropriate detection of these carriage types: based on two nasal cultures taken a week apart, the number of positive cultures (two: persistent, one: intermittent and zero: non-carrier) was indicative of carriage status [89]. Screening should be performed from multiple sites of the body to ensure the adequate pickup rate of this pathogen. Additionally, it would be ideal to simultaneously process these samples for financial considerations [90].
The most frequently used methods for MRSA screening are still culture-based [91]. Generally, all of these methods include a preliminary step of selective enrichment in a broth medium, followed by culturing on selective solid media (containing oxacillin/cefoxitin) (Figure 1.) [92]. Several studies have highlighted the relevance of enrichment, showing that the direct plating of the sample onto MRSA-selective agars has inadequate selectivity and sensitivity [93]. Instead of methicillin (nowadays only having theoretical importance) or oxacillin (the use of which is not recommended anymore, because it is affected by other resistance mechanisms that are related to β-lactams), the use of cefoxitin disks, as a surrogate agent, is recommended, both by the Clinical Laboratory Standards Institute (CLSI) and European Committee on Antimicrobial Susceptibility Testing (EUCAST) guidelines [94][95][96]. Clearly, any anomaly that was detected around the cefoxitin (<22 mm zone diameter; together with the knowledge of local epidemiological data) during routine antimicrobial susceptibility testing (AST) should be investigated if the patient's MRSA carriage is unknown [94][95][96]. Around 48 hours are needed for identification (considering enrichment and growth on selective solid media) and the reporting of results using culture-based methods [91]. If cefoxitin susceptibility testing is not performed in parallel, another day may be required to perform AST. Alternative methods, such as latex agglutination, can also be used: these tests detect the product of the mecA gene (the PBP2a protein, found in the cell membrane of MRSA). The disadvantage of these methods is that the product of the mecC gene (see Section 3.) is not detected, which may lead to false-negative results. Additionally, selective enrichment prior to detection with latex agglutination is still recommended.
used for the detection of MRSA-colonization are usually determined by the settings of the healthcare institution (i.e., number of beds and patient characteristics) and the facilities of the clinical microbiology laboratory (i.e., the expected sample number), the required TAT for detection and monetary constraints [91,92,97]. Currently, there is no study confirming that the cost/benefit ratio and the overall benefits of PCR screening could surpass the culture-based techniques. Besides clinical microbiology laboratories, the abovementioned techniques are also used in the MRSA screening in livestock, based on the same principles. However, for national-level surveillance purposes, or during the investigation and follow-up of an outbreak, molecular methods (PCR) are the most frequently used, being complemented with genotyping (see Section 3.).

Genetics of MRSA, Typing Methods
By definition, the MRSA strains are S. aureus isolates that possess an altered penicillin-binding protein (namely PBP2a or PBP2c, encoded by the mecA/mecC genes; the expression of which is regulated by mecI and mecR1), a protein that is essential to bacterial cell wall synthesis, resulting in non-susceptibility to all β-lactam antibiotics, with the exception of anti-MRSA cephalosporins (ceftaroline and ceftobiprole) [53,99]. The resistance that is caused by modifications in the PBPs is not a unique phenomenon to S. aureus, as similar mechanisms are responsible for ampicillin-resistance in Enterococcus faecium (PBP5) [100] and the penicillin non-susceptibility in Streptococcus pneumoniae (PBP1x mosaic genes) [101]. In addition, these microorganisms can easily acquire resistance to With the advent of molecular methods (polymerase chain reaction, PCR), the reporting of results on the same day has become possible, although this means that the samples must reach the laboratory in time for batch processing [91]. These methods may include in-house PCRs (with the design of target-specific primers for the relevant genes) or commercially available systems (e.g., the Cepheid Xpert MRSA assay) [91,97]. Interestingly, several reports have also indicated the usefulness of matrix-assisted laser desorption-ionization time-of-flight mass spectrometry (MALDI-TOF MS) in the detection of MRSA, together with the selective enrichment and/or PBP2a' latex agglutination, which reduces the turnaround-time (TAT) for result reporting [92,98]. Appropriate logistics and infrastructure are needed to support the effectiveness of MRSA screening: including swift transport of the specimens from the ward to the microbiology laboratory, reporting of results to the clinicians in a clinically-relevant timeframe, and prompting action from the part of the infection control unit (including isolation and/or decolonization of the patient) [91,97]. In summary, the methods that are used for the detection of MRSA-colonization are usually determined by the settings of the healthcare institution (i.e., number of beds and patient characteristics) and the facilities of the clinical microbiology laboratory (i.e., the expected sample number), the required TAT for detection and monetary constraints [91,92,97]. Currently, there is no study confirming that the cost/benefit ratio and the overall benefits of PCR screening could surpass the culture-based techniques. Besides clinical microbiology laboratories, the abovementioned techniques are also used in the MRSA screening in livestock, based on the same principles. However, for national-level surveillance purposes, or during the investigation and follow-up of an outbreak, molecular methods (PCR) are the most frequently used, being complemented with genotyping (see Section 3.).

Genetics of MRSA, Typing Methods
By definition, the MRSA strains are S. aureus isolates that possess an altered penicillin-binding protein (namely PBP2a or PBP2c, encoded by the mecA/mecC genes; the expression of which is regulated by mecI and mecR1), a protein that is essential to bacterial cell wall synthesis, resulting in non-susceptibility to all β-lactam antibiotics, with the exception of anti-MRSA cephalosporins (ceftaroline and ceftobiprole) [53,99]. The resistance that is caused by modifications in the PBPs is not a unique phenomenon to S. aureus, as similar mechanisms are responsible for ampicillin-resistance in Enterococcus faecium (PBP5) [100] and the penicillin non-susceptibility in Streptococcus pneumoniae (PBP1x mosaic genes) [101]. In addition, these microorganisms can easily acquire resistance to additional antibiotic groups due to horizontal gene transfer [102]. The genes that encode for these proteins can be found in a chromosomal genetic element, called staphylococcal chromosomal cassette mec (SCCmec), which was found as a fundamental discovery by Hiramatsu et al. [103]. SCCmec is integrated into orfX (a staphylococcal gene of unknown function) and in addition to the mecA or mecC complex (which is a 30-60 kbp genetic element), it contains two recombinase genes (ccrA and ccrB), and is responsible for the integration/excision of this genetic element from the staphylococcal chromosome [104].
Currently, twelve allotypes of SCCmec (namely I-XII) have been defined, which are separated based on the type of ccr gene complex (responsible for site-specific excision and the insertion of the gene cassette) and the type of mec complex [102,105]. Presumably, this number is only going to increase (with more subtypes emerging), because the use of novel sequencing technologies (i.e., next-generation sequencing, NGS) with higher discriminatory power will also undoubtedly bring forth changes in this field, just like after the first time the S. aureus genome was first sequenced in 2001 [106,107]. In the 1980-1990s, CA-and HA-MRSA strains could be safely distinguished by phenotypic characteristics: the HA-MRSA strains were more frequently MDR strains (resistant to antibiotics other than β-lactams), while the CA-MRSA strains were predominantly susceptible to non-β-lactams [31]. However, this distinction between the two groups has eroded slowly over time [108]. Nowadays, a useful method for the differentiation of CA-and HA-MRSA strains is based on molecular methods. When compared to the community-associated strains, HA-MRSAs carry a larger SCCmec cassette and they usually belong to the I, II, or III allotypes [51,85,102]. In contrast, the CA-MRSA strains are associated with smaller genetic elements (carrying the mecA/C gene), which is thought to be influencing their mobility, belonging to the IV, V, or VII allotypes [57,61,85,102]. The detection of Panton-Valentine leukocidin (PVL) is another possible way for distinction, which is a characteristic toxin of the species [17,108,109]. Although the clinical role and significance of this toxin in the pathogenesis of the diseases that is caused by S. aureus is not clear, the presence of the toxin is much more frequent in the CA-MRSA strains [109][110][111].
When compared to mecA, mecC is a novel variant of the gene responsible for methicillin-resistance (encoded on a novel type XI SCCmec element [99]), which was first described in 2007 (although retrospective studies with old strain collections have found that these variants were probably around, ever since MRSAs were known) [112]. mecC shows around 70% nucleotide sequence homology with the classical mecA gene, resulting in false negative results in molecular detection systems [113]. Following 2010, mecC-positive MRSA strains are increasingly being reported in both humans and animal infections [113,114]. In fact, some studies suggest a zoonotic background and transmission for this allotype. There is limited data in the literature regarding the efficacy of detecting mecC-positive strains, using classical (i.e., phenotypic) AST methods. Another reason for the use of cefoxitin as a surrogate for MRSA-detection is that it was found to be more reliable in the detection of mecC-positive MRSAs (see Section 2.) [96]. Owing to the genetic nature of the SCCmec cassette (being a mobile genetic element), methicillin-resistant CoNS may also possess the mecA/mecC genes, which may result in dual colonization with methicillin-resistant CoNS and methicillin-susceptible S. aureus [112].
Beginning from the 1980s (after the global spread of some successful MRSA clones), the need for the observation and characterization of the epidemic MRSA clones has become of pivotal importance [85]. Five different methods, with various advantages and disadvantages associated with each method, currently perform the typing of successful epidemic clones. Initially, phage typing was used for the differentiation of various MRSA strains, owing to their differential susceptibility of lytic phages (differentiating strains into phage types) [52]. Multi-locus sequence typing (MLST) is sequence based genotyping method, which is based on the single nucleotide polymorphisms of seven distinct S. aureus housekeeping genes (covering around 450 bps of genetic material) [115]. Using the allelic profile of various strains, a sequence type (e.g., ST44) can be assigned. If strains have identity at ≥5 housekeeping genes, they are assigned to the same clonal complex (e.g., CC78). Nowadays, the lineages of various epidemic strains are considered based on the CC identity [99]. The advantage of MLST is the ability to monitor variations over a longer time period to follow the evolution of the epidemic clones and its usefulness in scientific analysis; however, it may be too expensive for routine laboratories [85]. Pulse-field gel electrophoresis (PFGE) is more suitable for the evaluation of rapid changes over a shorter time period [116]. During this procedure, S. aureus genomic DNA is digested by SmaI restriction enzyme and the resulting fragments are then separated by pulsed-field electrophoresis in an agarose gel. The clustering of strains is performed based on >80% similarity, separating clones into various PFGE types (e.g., C1, D5, G10) [85,116]. Several national and international PFGE databases are available for the comparative analysis of epidemic MRSA strains [1,116,117]. Protein A typing (spa typing) is an inexpensive method, which is based on the sequence analysis of variable number tandem repeats (VNTRs) in the encoding gene of this protein (spa), while also taking the number of repeat variations and point mutations into account (e.g., t011, t899) [118][119][120]. As a novelty, some studies have reported in the use of MALDI-TOF MS for the rapid discrimination of epidemic clones of MRSA, based on the association of their measured protein peaks (for an excellent review on the background of mass spectrometry, see [121]) and their spa types [118,122]. Whole-genome sequencing (WGS) is currently considered to be the gold standard as a molecular typing tool for the investigation of MRSA outbreaks [123]. This method has the most discriminating profile, allowing for the analysis of core genome multi-locus sequence typing (cgMLST) data [106]. The price of the sequencing machines and the lack of the adequate bioinformatics pipeline hinders the extensive use of this method, therefore sequencing is usually limited to reference laboratories [106,123]. Table 1 presents the currently available drugs for the therapy of MRSA-infections. Following the emergence of MRSA in clinical practice, the significance of non-β-lactam-antibiotics (notably trimethoprim-sulfametoxazole [SMX/TMP] and doxycycline) has increased, especially in the therapy of cSSTI that is caused by CA-MRSA infections [45,124,125]. However, acquired resistance against the abovementioned drugs developed rapidly. At the end of the 1980s, MRSA strains with resistance against all other drugs except vancomycin were very common [108]. Resistance against SMX/TMP may occur due to alternative metabolic pathways of folate synthesis (due to point mutations in the dhfr gene) [126,127], while various tet efflux pumps and target modification (30S ribosomal RNA) mainly affected doxycycline [128]. Resistance to fluoroquinolones also frequently occurs in MRSA and is due to mutations in the quinolone-resistance-determining region (QRDR) of DNA gyrase (gyrA and gyrB) and topoisomerase IV (grlA and grlB in S. aureus) [59,60]. In addition, the overexpression of the Major Facilitator Superfamily (MFS) efflux pumps NorA and NorB contributes to high-level fluoroquinolone resistance (not to mention, the resistance to several antiseptics and disinfectants) in S. aureus [129][130][131][132]. For this reason, vancomycin (dosing generally includes a 25-30 mg/kg loading dose, followed by 15-20 mg/kg maintenance dose) was the "gold standard" of anti-MRSA-therapy for a very long time [133][134][135]. It exerts potent bactericidal activity in a concentration-and time-dependent manner against Gram-positive bacteria (including Flavobacterium spp. (which are Gram-negative) and excluding Erysipelotrix spp., Lactobacillus spp., Leuconostoc spp., and Nocardia spp. (due to intrinsic resistance to the drug)) [133][134][135]. However, the use of vancomycin also had pronounced drawbacks: it could only be parenterally used, as it is not absorbed in the gastrointestinal tract (which may even be useful in some cases; see therapy of C. difficile infections), its side effect profile (nephrotoxicity, ototoxicity, red man syndrome due to histamine liberation, etc.), and the subsequent need for therapeutic drug monitoring (TDM) [135,136]. The biggest concern is the phenotype of vancomycin-resistant S. aureus (VRSA; MIC ≥ 16 µg/mL), which has acquired the vanA gene from vancomycin-resistant Enterococcus spp. (where the prevalence of this gene is much higher) [100,137,138], while the vancomycin intermediate-resistant S. aureus (VISA; MIC, 4-8 µg/mL) strains are characterized by reduced susceptibility, owing to a thickened cell wall, which is capable of binding the drug and reducing its diffusion into the cell [139]. Resistance against teicoplanin characterizes a similar phenotype (Targocid ® [US/EU]; another member of the glycopeptide group of antibiotics; 800 mg/12 h), but not vancomycin, which is caused by the presence of the vanB gene [140]. It is important to note that the step-wise progression of VISA-to-VRSA does not occur, as the two resistance types have completely different mechanisms [139]. Luckily, the prevalence of VRSA remains very low, and most published reports detected strains that were colonizers, not the causative agents of infection [137,138]. An additional phenotypic group that warrants attention is the heterogeneous vancomycin intermediate-resistant S. aureus (hVISA; 1-4 µg/mL): these bacteria are described as being at a stage prior to the development of intermediate-level resistance [141]. With continuous selection pressure from vancomycin treatment, the environment favors the selection of VISA clones, which leads to a unanimous intermediate-resistant population. Based on the available experimental evidence, multiple, sequential mutations (involving various regulatory systems of cell wall homeostasis and remodeling) are required [141]. The role and clinical significance of hVISA is not yet understood, as there are no standardized methods to appropriately study this phenomenon in clinical microbiology laboratories [142].  [133,143]. Telavancin and oritavancin are semisynthetic derivatives of vancomycin with a hydrophobic side chain that is attached to the vancosamine sugar, while dalbavancin is a derivative of teicoplanin [133,143]. These drugs exhibit concentration-dependent antibacterial activity. They all show activity against hVISA/VISA, while oritavancin also exhibits antibacterial activity against vanA-positive S. aureus and Enterococcus spp. [144]. They are approved for complicated skin and skin structure infections (cSSTI), bone and joint infections, hospital-acquired (HAP), and ventilator-associated bacterial pneumonia (VAP) [133,143]. Their antimicrobial activity is attributed to a double mechanism: inhibition of cell wall synthesis (as seen previously in vancomycin) and the disruption of the integrity cell membrane barrier, which leads to permeabilization and cell death [133,143]. The antibacterial activity of these drugs is due to the novel combined action of inhibition of the cell wall synthesis and the disruption of bacterial cell membrane barrier function [145]. The additional advantage of dalbavancin and oritavancin in their long half-life (>300 h), therefore their half-life, coupled with their concentration-dependent activity, is appropriate for single-dose (once weekly) treatments, especially in OPAT settings. They do not require a loading dose or TDM [133,143]. However, when compared to vancomycin, they are much more expensive, which currently hinders their more widespread use in clinical practice. Another potential disadvantage of these drugs is that, due to their high levels of plasma protein binding (PPB > 90%), they are not removed by conventional dialysis. An increase in the mortality rate in patients that were treated with renal insufficiency was also described [79,133,143].

Treatment Considerations, Emerging Concepts
The class of oxazolidinones is another important antibiotic group in the treatment of drug resistant Gram-positive infections. Oxazolidinones were first described in the 1970s, while the first drug linezolid was approved in 2000 by the FDA. Oxazolidinones were perceived as attractive drugs due to several features; firstly, the novel mechanism of action (inhibition of protein synthesis by binding to the peptidyl transferase A-site of the 50S subunit of ribosomal RNA) [146]; secondly, their spectrum of activity Gram-positives, including MDR strains), the option for intravenous-to-oral switch with excellent oral bioavailability, and the lack of cross-resistance with different antibiotics [147]. Linezolid (Zyvox ® [US], Zyvoxid ® [EU]; 600 mg/12 h) and tedizolid (Sivextro ® [US/EU]; 200 mg/24 h) was approved for the treatment of nosocomial and community-acquired pneumonia, complicated and non-complicated skin, and soft tissue infections (including MRSA) and bone and joint infections, while they are not recommended for the treatment of bacteremia and endocarditis. Tedizolid is a prodrug (phosphate), which is hydrolyzed by plasma or intestinal enzymes in vivo to produce the active form of the drug [147]. Tedizolid (the second-generation oxazolidinone) has more potent antibacterial activity, a longer half-life (12 h), and less pronounced toxicity. Due to its higher (75-80%) protein binding affinity, lower doses should to be administered (cf. linezolid). They have high in vitro activity against MRSA; however, there are few data regarding its efficacy against hVISA/VISA and vancomycin-resistant S. aureus (VRSA). The drawbacks of this antibiotic group are related to their pharmacodynamic properties: a. drug-drug interactions, which may have severe consequences (e.g., due to synergism with sympathomimetic drugs (various αand β-receptor agonists), their co-administration may result in an acute hypertensive episode; they increase the toxicity of opioid analgesics and fibrates (drugs against hyperlipidemia); they may also increase the pharmacological effects of insulin and oral antidiabetic drugs (like metformin and glimepiride)), b. serious side effects, such as serotonin-syndrome (because they can inhibit the monoamine-oxidase-A (MAO-A) enzyme) and myelosuppression (which may lead to hematological issues) [147,148]. Their clinical role in the treatment of VRSA infections is unknown due to the lack of data. The prevalence of oxazolidinone resistance is very low and causative mechanism includes changes in L3/L4 ribosomal proteins and methylation of the 23S rRNA, encoded by the cfr gene [149]. Bacteria carry multiple copies of the genes encoding 23S rRNA; therefore, for a pronounced increase in the MIC, the mutation needs to be present in several copies ("gene-dose" effect) [148]. These mutations frequently occur in CoNS strains. The carriage of cfr does not affect tedizolid (approved in 2014) susceptibility. Although the endemic areas have been reported, the carriage of cfr genes continue to be very rare, even though it is a transmissible determinant of (presenting with a risk of developing MDR/XDR infections) [149]. However, some reports suggest that increasing the dose of the antibiotic could overcome cfr-mediated resistance (unlike mutational resistance) [148,149]. Clinical trials are currently underway regarding contezolid (MRX-1), which is a third-generation oxazolidinone, for the treatment of cSSTI, complicated intra-abdominal infections (cIAI) and cUTIs [82].
Daptomycin (Cubicin ® [US/EU]; 6mg/kg over 24hrs) is a lipopeptide antibiotic, which showed pronounced activity against various Gram-positive bacteria, including MRSA [150]. It is bactericidal, damaging the cell membrane and the membrane potential in a calcium-dependent fashion. It has been postulated that the activity of this compound is most pronounced at the division septum of bacteria. Daptomycin is approved for the treatment of skin and skin structure infections, bloodstream infections, and infective endocarditis, while it is contraindicated in pneumonia, because of its interaction with pulmonary surfactant [150][151][152]. This drug is not available for oral use, only as a parenteral formulation. Electrostatic repulsion of the drug molecule is thought to mediate resistance to daptomycin, owing to the increased positive charge of the bacterial cell surface. The gene that is responsible for daptomycin resistant phenotype is mpfF, which results in the incorporation of lysine (a positively charged amino acid) in the peptidoglycan layer: this will increase the positive charge of the cell envelope, which inhibits the binding of daptomycin. In addition, genes encoding cardiolipin synthetases (pgsA, cls), which affect phospholipid metabolism, were also implicated [150,153].
Ceftaroline  [154][155][156][157][158][159][160][161]. For this reason, they are sometimes termed anti-MRSA cephalosporins, or fifth generation cephalosporins [49,79]. It is important to note that these drugs are hydrolyzed by ESBL-enzymes, therefore they may not be appropriate for mixed infections involving Gram-positive and Gram-negative pathogens [156]. They have low minimum inhibitory concentrations for MRSA, which corresponds to their high affinity to the PBP2a/c proteins [154][155][156][157][158][159][160]. Ceftaroline and ceftobiprole are both approved for the treatment of cSSTI, CAP, and HAP (excluding ventilator-associated pneumonia), with ceftaroline being additionally approved for MRSA bacteremia [154][155][156][157][158][159][160]. Resistance to ceftaroline (with MIC values ≥4 µg/mL) was published, but the number of cases is very low, and the underlying mechanism is not yet understood [162]. An interesting therapeutic strategy involves the combination of these anti-MRSA cephalosporins and a glycopeptide or daptomycin (which is currently being evaluated in the CAMERA-2 trial) in the treatment of MRSA bacteremia, although this has been criticized for the increase in antibiotic use and therapeutic costs [163].
The role of tetracyclines in the therapy of MRSA infections has faded over the years, due to various mechanisms of acquired resistance [108]. There were high hopes for tigecycline (Tygacyl ® [US/EU]; 50 mg/12 h), a broad-spectrum agent and member of glycylcycline family of drugs, which was approved in 2005 for the therapy of cSSSI, cIAI, CAP [164][165][166]. Shortly after its approval, tigecycline received "black box" warning from the FDA, due to the significant increase in the mortality of patients that were treated with tigecycline, which is in contrast to the comparator drugs [167]. In addition, difficulties reaching the therapeutic serum levels for bacteremia can be considered to be drawbacks of this drug [164][165][166]. Eravacycline (Xerava ® [US]) and omadacycline (approved in the end of 2018; Nuzyra ® [US]; 100-150 mg/24, depending on the administration form) are novel drugs of the tetracycline group [168][169][170][171]. They were mainly developed for the treatment of MDR infections, and they are not affected by resistance mechanisms that are associated with tigecycline or other drugs of this family. Chemically, eravacycline is closely related to tigecycline, while omadacycline is an aminomethylcycline-derivative [168][169][170][171]. Omadacycline is licenced for the treatment of CAP and SSTIs. It has a broad spectrum of activity, including many multi-drug resistant strains of bacteria [170,171]. Phase III studies of eravacycline for cIAI and cUTI finished recently, where this drug presented inferiority to levofloxacin [168,169]. During the trials of both drugs, therapy had to be discontinued in some cases, due to severe nausea and vomiting [168][169][170][171]. It should be noted that these drugs do not cover Pseudomonas aeruginosa and Proteae (Proteus, Morganella, Providencia); therefore, they should not be used in mixed infections where these pathogens are suspected.
The clinical efficacy of some novel quinolones (avarofloxacin, finafloxacin, zaborfloxacin, nemonoxacin) is currently being assessed in a plethora of clinical trials for various indications, such as cSSTI, CAP, HAP, cUTI and sexually transmitted infections (mainly MDR gonorrhea), while delafloxacin (Baxdela ® [US]; 300 mg/8 h) has already been approved by the FDA for the therapy of cSSTI, with the evaluation of CAP and cUTI trials currently underway [172][173][174][175][176][177][178]. Unlike previous fluoroquinolones (which were zwitterionic), delafloxacin is anionic in character, which results in the accumulation of the drug in the intracellular space of bacteria, in phagocytes, urine, abscesses, and the gastic juice [173]. As these drugs are all representatives of the quinolone drug family (which has been a center of controversy lately, due to their side effect profile [179]), delafloxacin immediately received a "black box" warning by the FDA (for adverse events, such as peripheral neuropathy, tendinitis, C. difficile enterocolitis, and the worsening of myasthenia gravis, QTc-prolongation) and it is expected that the other novel drugs will receive similar designation [173]. Novel quinolones are broad-spectrum agents, active against fluoroquinolone (levofloxacin/moxifloxacin)-resistant bacteria, although some cases were reported where elevated delafloxacin MICs were observed [172][173][174][175][176][177][178].
Iclaprim, which is a diaminopyrimidine-type dihydrofolate-reductase (DHFR) inhibitor, is also being investigated for its efficacy against MRSA infections [180]. This drug-candidate was first developed for SSTIs; however, in the beginning 2010s, the FDA rejected further development plans due to QTc prolongation and the failure to show non-inferiority to the comparator drug. Following the subsequent acquiring of the license of iclaprim by another company, new clinical studies are underway. Iclaprim presented non-inferiority to vancomycin in two phase III studies targeting SSTIs, and a new indication for the drug (therapy of S. aureus infections in cystic fibrosis patients); nonetheless, the side effect profile, which is associated with prolonged use (liver toxicity), is concerning [127,181]. The dose used in these trials was 80 mg/12 hours. Iclaprim is not affected by the resistance mechanisms, which hindered the use of SMX/TMP previously [45,124,125].
Mupirocin (Bactroban ® [US/EU]) is used as a topical agent for the nasal decolonization of MRSA patients; in addition, it received a new indication for the topical therapy of impetigo [88,182]. The mechanism of action is through binding bacterial isoleucyl tRNA synthetase, resulting in the inhibition of protein synthesis. Resistance to mupirocin has been described: low-level resistance is characterized by point mutations in the ileS gene, in contrast, high-level resistance is plasmid-mediated (mupA gene), which code for a mutant isoleucyl tRNA synthetase, to which mupirocin is unable to bind [88,183].
Fusidic acid is another drug that was mainly used as a topical preparation for the treatment of skin infections (alone or in combination with topical steroids), atopic dermatitis, and in eye drops, in addition to MRSA decolonization (in combination with rifampicin) in the case of mupirocin resistance [49,88]. The safety and efficacy of fusidic acid orally or intravenously (as it is available in both forms) has also been demonstrated in combination with other antibiotics (e.g., vancomycin, gentamycin, levofloxacin) in the therapy of severe staphylococcal/MRSA-infections, typically in complicated SSTIs, osteomyelitis, and septic arthritis, owing to the excellent penetration of this drug into skin blisters, joints, and the bone tissue [184,185]. Unfortunately, resistance frequently emerges against this steroid-based antibiotic, particularly after prolonged therapy; the resistance levels are range between 0.3-64% worldwide, with significant variation in the prevalence of various resistance determinants in different geographical region [49,88]. This phenomenon is especially frequent in the CA-MRSA isolates, being mediated by the mutations in the chromosomal fusA (encoding elongation factor G) or rplF (or FusE, encoding ribosome protein L6) genes, or the acquision of the transferable fusB, fusC or fusD genes (protection of drug target site) [184,185]. When it comes to its antimicrobial mechanism of action, fusidic acid is a protein synthesis inhibitor, which interferes with ribosomal translation. Around 90% of the drug binds to the plasma proteins and the elimination half-life is around 8-10 hours [88]. Currently, there are Phase II studies underway evaluating its efficacy in cSSTI and bone-joint infection [184,185].
Pleuromutilin derivatives are well known drugs since the 1950s, with valnemulin and tiamulin being used routinely in veterinary medicine worldwide [186]. These drugs are inhibitors of protein synthesis, acting on the 50S ribosomal subunit (binding to peptidyl-transferase on the 23S rRNA). At the time of their discovery, azamulin was the primary drug candidate, but, due to its pronounced effect on the liver microsomal enzymes (CYP inhibitor), liver toxicity, and the availability of drugs with more advantageous properties, its development was discontinued [186]. Nevertheless, in the current age of MDR pathogens, there is a fresh interest in pleuromutilin-type antimicrobials for human use. Retapamulin (Altrabax ® , Altargo ® [US]) was the first class of these drugs to receive approval from the FDA, and it is currently used as a topical antibiotic (for the treatment of impetigo) [187][188][189]. There is interest in retapamulin to be used in decolonization regimens for MRSA as cross-resistance with mupirocin is unlikely, although clinical trials are needed to assess the value of this drug in the abovementioned indication [190]. Besides retapamilin, azamulin and lefamulin are too in the clinical (Phase II) phase of drug development in SSTIs and CAP that is caused by MDR Gram-positive bacteria [186].
Various emerging therapeutic approaches are currently in development for the treatment of MRSA and other related MDR infections, including antimicrobial peptides (AMPs, such as magainin, pexiganan) [191,192], quorum sensing-inhibitors (or quorum quenchers) [23], inhibitors of virulence factor-expression [193][194][195], efflux pump inhibitors [132,196,197], probiotics [193], repurposed natural compounds with antimicrobial activity (predominantly for topical use) [198,199], phage therapy [200,201], and the development of anti-staphylococcal vaccines [202,203]. There are numerous studies that indicate the promise of combating MRSA infections with AMPs; their mechanism of action mainly consists of creating pores on in the intact bacterial membrane, which leads to cell lysis [191,192,204]. However, there have been many difficulties (e.g., lack of stability in vivo, immunogenicity, toxicity to red blood cells, enzymatic degradation) in their translation to clinical use [191,192]. Peptidomimetics are de novo synthesized or modified peptide sequences with enhanced antibacterial potency and the lack of the disadvantages of the native peptides, which are currently in development [205][206][207]. Brilacidin is a defensin mimetic (defensins are antimicrobial peptides in vertebrates, which are predominantly found in neutrophil granulocytes; they have important roles in innate immunity [208]), which has broad-spectrum antibacterial activity [151,192,209]. This peptide affects the bacterial cell membrane in a similar fashion to daptomycin [151]. The main advantage of brilacidin is that it also exerts antimicrobial activity against the non-replicating, dormant forms (in case of S. aureus, small-colony variants) of bacteria [151,192,209]. Brilacidin is currently enrolled in Phase II. trials for the treatment of SSTIs and oral mucositis [192]. The therapeutic application of phages in the treatment of infectious diseases has been around since prior to the rise of antibiotics [200,201,210]. Due to the increase of MDR-infections, many people consider these bacteria-specific viruses to be potential therapeutic alternatives. There is an increasing number of case reports, where a seemingly untreatable infection (usually due to pan-resistant Enterobacteriaceae) was cured by the application of phages, therefore it would be valid to assume that they could also have significance in the treatment of MRSA infections. Although the regulatory environment is not yet defined, phages and artilysins (phage-derived lytic proteins) may be useful in MRSA-associated SSTIs. However, for their widespread use, an appropriate formulation for oral/parenteral use and the precise composition of effective phages/enzymes needs to be defined beforehand [200,201,210,211]. Novel drug targets include FabI inhibitors (FabI or enoyl-ACP-reductase is a key enzyme of unsaturated fatty acid synthesis in bacteria [212]), such as Debio 1452 and its prodrug Debio 1450 (or afabicin), which are in Phase II trials for the therapy of SSTIs that are caused by methicillin-susceptible and resistant S. aureus [11,213]. Many researchers are interested in the development of metal-based nanoparticles (NPs) with antimicrobial properties for use in MRSA infections [214]. Various NPs containing different metals (titanium, magnesium, gold, silver, zinc, copper, bismuth, etc.) showed promising activity against this pathogen, even though the penetration of S. aureus biofilms, while others (such as aluminum) were not recommended, based on experimental results. These studies predominantly administered these nanoparticles in some hydrogel form (i.e., for external use) or on the surface of an implanted medical device [215][216][217][218].
There were high hopes for the development of broad-spectrum, anti-MRSA/VRE carbapenems (e.g., tomopenem, razupenem), which bypassed the resistance that is caused by the modified PBPs and retaining their activity against Gram-negative organisms, however none of these agents thus passed onto clinical trials [219][220][221]. The closest thing in development with broad-spectrum activity is ceftaroline/avibactam, which is a combination of the anti-MRSA cephalosporin and a non-β-lactam-type β-lactamase-inhibitor [222,223].
New and emerging oligonucleotide-based antibacterial strategies, such as RNA-interference (RNAi), aptamers (single strand nucleic acids; ssRNA or ssDNA), and the use of CRISPR/Cas (clustered, regularly interspaced, short palindromic repeats, and CRISPR-associated protein 9, respectively) against MRSA and other MDR pathogens are also intensively studied, however, these are probably decades away from potential real-life applications [214,224,225]. The extremely high costs and risk-to-benefit ratio (which is especially high to antimicrobial research) is discouraging pharmaceutical companies to invest in studies targeting antibiotics, especially because, in addition to the physico-chemical characteristics, formulation, and adverse events of these compounds, companies have to keep in mind that microorganisms will develop resistance sooner or later, like in any facet of drug design and development [40,198,[226][227][228]. Unfortunately, this is a general concern involving all MDR bacteria, not only MRSA, where clinicians are facing worsening odds to provide their patients with adequate therapy [35,80,81]. Additionally, some of these compounds are protein-based, therefore the potential for hypersensitivity and parenteral-only administration are additional hindering factors [203,229].
The development of novel antibiotics may be further complicated by their potential to affect the normal intestinal microbiota (i.e., their "ecological impact"). Drugs that kill the gut microbiota may select for C. difficile, VRE, ESBL-, and carbapenemase-producing Enterobacteriaceae, leading to additional issues for the patients in the future [230].

Concluding Remarks
S. aureus is a fine example of a successful pathogen, which was able to morph and adapt with such tenacity to the changing landscape of modern medical interventions (e.g., implanted devices, catheters) and the available antimicrobial agents. Penicillin-, methicillin-, (and to a lesser extent) vancomycin-resistant strains of staphylococci appeared one after another due to the selection pressure, proving that the antibiotic "arms race" is very real, and humanity as a whole is still on the losing side. MRSA infections emerged in nosocomial settings; however, in the 21 st century, the infections that were acquired in the community setting are a more pressing concern. MRSA is still an important factor of mortality (especially as a causative agent of endocarditis and bacteremia) all around the globe. Vigilant screening may reduce the number of patients where carriage turns into infection, while typing is useful in obtaining information pertaining to the local and global epidemiology, and the spread of successful clones. Alternative therapeutic modalities for MRSA are, in fact, being developed, though with no time-frame or guarantee that they will be successful in a clinical evaluation. One would argue that the current therapeutic armamentarium (and the number of drugs in development) could provide us with temporary safety; however, this hubris could also lead to the demise of the healthcare system, as we know it.

Conflicts of Interest:
The author declares no conflict of interest, monetary or otherwise.