Staphylococcus and Enterococcus are Gram-positive cocci that are recognized as globally important opportunistic pathogens that can cause serious infections in humans, especially in hospitalized patients (Otto, 2009; Gilmore et al., 2013; Lee A.S. et al., 2018). Over recent decades, there has been a significant increase in the rates of acquired antimicrobial resistance (AMR) in these species, either through the acquisition of resistance determinants by horizontal gene transfer of mobile genetic elements (MGEs), or through mutations that alter gene expression or binding sites in native genes. This has resulted in the emergence of polyclonal lineages that are resistant to front-line therapeutic agents. In staphylococci, this includes the development and global spread of methicillin-resistant Staphylococcus aureus (MRSA) (Enright et al., 2002) and the recent emergence of multidrug-resistant Staphylococcus epidermidis (MDRSE) (Lee J.Y.H. et al., 2018). In enterococci, the emergence and dissemination of vancomycin-resistant enterococci (VRE) (Arias and Murray, 2012), particularly in two healthcare-associated species, vancomycin-resistant Enterococcus faecalis (VREfs) and vancomycin-resistant Enterococcus faecium (VREfm), is of particular clinical concern.

As resistance to different antimicrobials increases in MRSA, MDRSE, and VRE, effective and appropriate treatment becomes increasingly difficult. Limited “last-line” therapeutic options, such as linezolid, daptomycin, and, in the case of staphylococci, vancomycin, remain to treat these infections. However, the high prevalence of these species in healthcare settings (Weiner et al., 2016; Weiner-Lastinger et al., 2020), the increasing clinical use of these last-resort antimicrobials, and the ability of these species to readily develop resistance, provides strong selective pressure for the emergence of clones that are resistant to these agents. Accordingly, resistance to these last-line agents with resultant treatment failure has been increasingly reported. Individual and multiple resistance to linezolid, daptomycin, and vancomycin have been reported in clinical MRSA and MDRSE. While combined resistance to linezolid and daptomycin has recently been reported in VREfm (Wardenburg et al., 2019).

Genomic data can be used to understand the evolution of resistance and investigate the underlying resistance mechanisms. Phylogenetic analyses based on whole-genome sequencing (WGS) can also identify the presence of clones associated with specific resistance determinants, and track their emergence and global spread. The utilization of WGS in hospital infection control to identify outbreaks and transmission events associated with resistant strains is increasingly common. In this mini-review we will provide an overview of the application of genomic analyses to provide critical insights into last-line AMR in MRSA, MDRSE, and VRE, with a particular focus on the emergence of resistant clones, the molecular mechanisms of resistance, and the importance of MGEs in the global spread of linezolid resistance.

Linezolid was the first oxazolidinone approved for clinical use due to its bacteriostatic activity against Gram-positive species. Its mechanism of action inhibits protein synthesis by preventing the formation of the ternary complex between tRNA^fMet, mRNA, and the ribosome (Hashemian et al., 2018). Although preliminary research indicated that resistance to linezolid should be rare, resistance in clinical enterococci and staphylococci have been increasingly reported since 2001 (Zurenko et al., 1996; Xiong et al., 2000; Gu et al., 2013; Bender et al., 2018a; Kosecka-Strojek et al., 2020). Early studies using PCR amplicon sequencing in clinical VRE and S. aureus isolates identified mutations in the 23S rRNA genes, that form the binding pocket of the ribosomal peptidyl transferase centre (PTC) to which linezolid binds (Howe et al., 2003; Meka et al., 2004; Livermore et al., 2007). A number of resistance-associated mutations, some specific to staphylococci, others conserved across staphylococci and enterococci, have been identified within and outside the PTC; the G2576T (E. coli nucleotide numbering) mutation is most common (Figure 1). Conferring high-level resistance in MRSA, MDRSE, and VRE, the G2576T 23S rRNA mutation is associated with linezolid treatment failure. Additional single nucleotide polymorphisms (SNPs) in the ribosomal proteins L3 and L4 have been identified through WGS of linezolid-resistant clinical staphylococci (Locke et al., 2009a,b, 2010; Endimiani et al., 2011). Mutations in the L3 and L4 proteins appear to be less common in enterococci, but several reports have identified SNPs in L3 (S133L) and L4 (T35A, N79D, I98V, and N130K) (Mendes et al., 2014; Hua et al., 2019). Further, mutations (A138G, C141T, and G166A) in ribosomal protein L22 have also been identified in linezolid-resistant staphylococci (Bender et al., 2015), as well as a S77T mutation identified in one linezolid-intermediate E. faecium (Lee et al., 2017). The L3, L4, and L22 proteins are in close proximity to the linezolid binding site in the ribosomal PTC, with identified mutations typically adjacent to the PTC, which may affect linezolid binding (Long and Vester, 2012). Using WGS, several studies have documented the local emergence of linezolid resistance in response to linezolid treatment during individual infections (Seedat et al., 2006; Wong et al., 2010; Chen et al., 2018). These studies identified common mutations in 23S rRNA and/or L3, L4, or L22 proteins that independently and repeatedly arise in different species of staphylococci and enterococci from different patients, demonstrating the conserved nature of mutational linezolid resistance and suggesting that convergent evolution is occurring.

Shortly after FDA approval of daptomycin for use in MRSA and in the context of off-label use in VRE, resistance, and corresponding clinical treatment failures were reported in both species (Skiest, 2006; Arias et al., 2007; Boyle-Vavra et al., 2011). The development of daptomycin resistance commonly coincides with prior patient exposure, indicating that therapeutic use of daptomycin is likely the main driver of resistance. In S. aureus, the emergence of daptomycin resistance has also been associated with the use of un-related antimicrobials, including vancomycin (Tran et al., 2015) and rifampicin (Guérillot et al., 2018b). Importantly, horizontally acquired and transferable resistance to daptomycin has not been reported to date. The worldwide prevalence of daptomycin resistance is poorly defined since few large-scale surveillance studies exist. A recent meta-analysis in S. aureus, suggested that only 0.1% of S. aureus and 0.1% of MRSA were daptomycin-resistant (Shariati et al., 2020). In enterococci, the prevalence of daptomycin resistance was estimated to be 2.6% in VREfm and 0.1% in VREfs, based on a systemic review of previous studies (Kelesidis et al., 2011). Little is known about which lineages of staphylococci and enterococci are associated with the development daptomycin resistance. In S. aureus, genomic analyses indicate that daptomycin resistance has arisen in ST5, ST398, and ST22 isolates, based on data from the United States and South America (Dabul and Camargo, 2014; Damasco et al., 2019). In S. epidermidis, no comprehensive phylogenetic analyses of daptomycin-resistant strains have been completed, but resistance has been reported in ST2, internationally (Lee J.Y.H. et al., 2018). Similarly, no phylogenetic studies have been completed for E. faecalis, but a novel ST736 clone associated with daptomycin resistance has been identified for E. faecium (Wang et al., 2014) in some United States hospitals (Wang et al., 2018), as has daptomycin-resistant ST80 E. faecium (Udaondo et al., 2020).

Daptomycin’s mode of action involves binding to the anionic phosphoglycerol and various undecaprenyl-coupled cell envelope precursors within the bacterial cell membrane in the presence of Ca²⁺, blocking cell wall synthesis and triggering delocalization of biosynthetic proteins from the membrane, resulting in bacterial cell death (Grein et al., 2020). Although the specific mechanisms leading to resistance are not fully understood, comparative genomic studies have been pivotal in identifying genetic determinants of daptomycin resistance. Genetically, a critical step toward developing a resistant phenotype appears to involve mutations in two groups of genes: (1) bacterial regulatory systems that function in cell wall homeostasis and (2) enzymes involved in membrane phospholipid metabolism.

In S. aureus and S. epidermidis, mutations in mprF (S295L, S337L, T345L, I420N, and L826F) and walKR (yycFG) (WalK^V500F and WalR^R216S) are associated with the development of daptomycin resistance in clinical isolates and laboratory-derived strains (Howden et al., 2011; Yang et al., 2009, 2013; Jiang et al., 2019). The MprF enzyme produces cationic phospholipid lysylphophatidyl-glyercol (LysPG) and flips it from the inner to outer leaflet of the cytoplasmic membrane, which decreases the negative cell surface charge (Oku et al., 2004). Although not characterized in S. epidermidis, the mutations in S. aureus likely increase the enzymatic activity of MprF since daptomycin-resistant isolates display increased levels of LysPG in their membrane, which variably leads to a more cationic cell surface that electrostatically repels the daptomycin-Ca²⁺ complex (Mishra et al., 2011). For mutations in walKR, the mechanism leading to resistance is distinct in S. aureus and S. epidermidis, since S. aureus strains carrying mutations in walKR display increased surface charge while S. epidermidis strains display decreased biofilm formation, compared to the wild-type strains (Jiang et al., 2019). Comparative genomic studies in S. aureus have also identified mutations in the cardiolipin synthase gene cls2 and RNA polymerase β and β’ subunits (rpoB and rpoC) that are associated with resistance to daptomycin, although these are less frequently observed than mutations in mprF and walKR (Miller et al., 2016). Transposon-sequencing in S. aureus has also identified multi-component sensing genes, specifically graRS and vraFG (which regulate cell envelope processes), as being associated with daptomycin resistance, with these genes being important for survival following daptomycin stress (Rajagopal et al., 2016; Coe et al., 2019). However, graRS and vraFG mutations have so far not been described in clinical daptomycin-resistant S. aureus isolates.

In enterococci, comparative genomic studies have shown that mutations in the three-component regulatory system liaFSR and cardiolipin synthase cls are associated with the development of clinical daptomycin resistance. Mutations within the LiaFSR three-component system, which regulates the cell envelope response to cationic host defense peptides and antimicrobial stress, have been frequently identified in daptomycin-resistant clinical isolates, with the LiaS^T120A and LiaR^W73C mutations being of particular importance (Arias et al., 2011). A genomic analysis of E. faecium clinical isolates found that isolates with higher daptomycin MICs (≥ 4 mg/L) usually contained mutations in the liaFSR system (Diaz et al., 2014). Mutations in Cls (H215R and R218Q), encoding a transmembrane protein that catalyzes the synthesis of cardiolipin from phosphoglycerol, are also frequently associated with daptomycin resistance (Arias et al., 2011; Palmer et al., 2011). However, molecular studies have thus far not demonstrated a causal relationship between cls and daptomycin resistance in E. faecium (Tran et al., 2013). Since cls mutations are often found in conjunction with substitutions in the liaFSR regulon, they likely contribute to the progression of an isolate developing a daptomycin-resistant phenotype rather than independently causing resistance (Davlieva et al., 2013). Recent genomic studies in enterococci have also identified daptomycin-resistant isolates that contain wild-type liaFSR and cls alleles, indicating that other unknown genetic determinants of daptomycin resistance exist that require further investigation (Wang et al., 2018; Prater et al., 2019). Indeed, putative associations with daptomycin resistance and mutations within mprF, cfa, pgsA, and dlt have been suggested, but have not yet been verified.

Vancomycin is a critical last-line antibiotic for treating infections caused by multidrug-resistant Staphylococcus. Thirty-nine years after FDA approval of vancomycin, the first instances of S. aureus with intermediate vancomycin resistance (MICs 4 to 8 mg/L) were reported (Sieradzki et al., 1999; Smith et al., 1999). Soon after, the first clinical case of a vancomycin-resistant S. aureus isolate (MIC 1024 mg/L), carrying the vanA vancomycin resistance operon, was reported (Chang et al., 2003). Subsequent molecular studies demonstrated that the vanA operon originated from a co-infecting VREfs strain, raising significant concerns about the potential dissemination of vancomycin resistance in S. aureus from VRE reservoirs (de Niederhäusern et al., 2011). Fortunately, this has not eventuated. Detailed genomic studies have demonstrated that the transfer of the van operon to S. aureus rarely occurs, with very few reports worldwide (McGuinness et al., 2017). The rare occurrence of VRSA may be due to the fitness cost associated with carriage of the van genes, as a longer lag phase was observed during in vitro growth with vancomycin, and the plasmid carrying the van genes was found to be genetically unstable. Further, a MRSA PBP2a strain was unable to utilize lipid II with the D-Alanyl-D-lactate alteration conferred by the van operon (Foster, 2017). A single study identified S. aureus clonal complex 5 (CC5) as the genetic background in which the van operon is most commonly acquired. This study found that CC5 vancomycin-resistant S. aureus (VRSA) strains were genetically distinct, indicating independent acquisition of the vanA operon had occurred (Kos et al., 2012). This suggests that the spread of van operons within S. aureus may occur should appropriate selective conditions develop. Continued vigilance is therefore paramount.

Vancomycin-intermediate S. aureus (VISA) resulting from the progressive accumulation of mutations in key essential and regulatory genes are clinically more common than true vancomycin-resistant S. aureus. VISA isolates are commonly characterized by a thickened cell wall, slower growth, and increased autolysis (reviewed in Howden et al., 2010). Genetically, VISA strains typically contain mutations in two-component regulators vraRS (Cui et al., 2009), graRS (Cui et al., 2009), and walKR (Howden et al., 2011), which function in cell-wall synthesis, but also may contain mutations in the rifampicin resistance determining region of rpoB (Cui et al., 2010). A convergent evolution analysis of 7,099 S. aureus genomes suggested that clinical rifampicin use may promote the emergence of multidrug-resistant lineages of S. aureus that contain rifampicin resistance mutations in rpoB that also confer intermediate resistance to vancomycin (Guérillot et al., 2018a). Although VISA can be found in any genetic background, ST5 is most commonly associated with VISA (Howden et al., 2014), although ST239 isolates tend to display the highest vancomycin MICs (Holmes et al., 2014). Genome-wide association studies (GWAS) performed in ST239 S. aureus have identified mutations in rpoB (H481Y/L/N) (Alam et al., 2014) and the walKR genes (Baines et al., 2015) as being most strongly associated with the development of the VISA phenotype.

In S. epidermidis, the global dissemination of three nearly pan-drug-resistant lineages (two ST2 and one ST23) with heteroresistance to vancomycin was recently demonstrated (Lee J.Y.H. et al., 2018). Analyses of WGS data from 419 clinical isolates from 96 institutions in 24 countries, sampling at least 77 ST types, identified dual D471E and I527M RpoB mutations to be the most common cause of rifampicin resistance in S. epidermidis, accounting for 86.6% of mutations. Of note, the D471E and I527M combination occurred almost exclusively in isolates from the ST2 and ST23 lineages. Through site-specific mutagenesis, the dual RpoB mutation was shown to confer high-level rifampicin resistance as well as reduced susceptibility to vancomycin and teicoplanin. The presence of these same mutations in multiple genetically diverse backgrounds was consistent with their independent emergence with subsequent fixation, presumably due to their advantageous antimicrobial resistant phenotype. Furthermore, acquisition of additional resistance to linezolid (mediated by cfr carrying plasmids and/or mutations in 23S rRNA, L3 and L4) and daptomycin (substitutions in mprF) rendered some European S. epidermidis essentially untreatable. Further examples of convergent evolution include the presence of H481Y/L/N substitutions in RpoB that confer vancomycin resistance in both S. aureus (Guérillot et al., 2018a) and S. epidermidis (Gao et al., 2013; Lee J.Y.H. et al., 2018; Dao et al., 2020).

Collectively, MRSA, MDRSE, and VRE pose a significant clinical and economic healthcare burden. This has been compounded by increasing resistance to the key last-line agents daptomycin, linezolid and in the case of Staphylococcus, vancomycin. Genomic analyses have been pivotal in providing evolutionary insights into the underlying genetic mechanisms of resistance that have arisen in all three species. In particular, the identification of transferable and acquired linezolid resistance determinants through genomic studies, has been critical for understanding the global dissemination and genetic mobility of these genes. Genomic analyses using WGS data is therefore a powerful method for understanding last-line AMR in these and other clinically relevant multidrug-resistant species.

GC and AT conceptualized the mini-review topic. All authors contributed to writing the mini-review and editing the final version.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.