Vancomycin-resistant Enterococcus (VRE) is a frequent cause of clinical outbreaks worldwide ^[253][254][253,254]. An example of this is the VRE clonal sequence type 796 (ST796) which was first detected in 2011 in Australia, then quickly spread both nationwide and internationally to New Zealand [255] before also causing outbreaks in European hospitals beginning in December 2017 [256].

Staphylococcus aureus is a Gram-positive, facultative anaerobic bacterium ^[257][294]. Both a commensal as well as a significant pathogen of humans ^[258][295], S. aureus is prevalent in community, healthcare ^[259][296] and agricultural settings ^[260][261][297,298], asymptomatically colonising up to 30% of the human population ^[262][299].

As one of the most versatile and successful opportunistic human pathogens ^[259][263][296,300], S. aureus possesses a large variety of virulence factors ^[264][301] that enable host colonisation, tissue damage, immune evasion and progression of disease ^[264][265][301,302]. Consequently, S. aureus infections can be grouped into three general categories: (i) toxinoses such as scalded skin syndrome, food poisoning and toxic shock syndrome; (ii) benign and self-limiting conditions such as superficial skin and soft tissue infections; and (iii) systemic, life-threatening complications such as brain abscesses, meningitis, pneumonia, osteomyelitis, endocarditis, bacteremia, multi-organ failure and sepsis which carry high rates of morbidity and mortality ^[266][267][303,304].

S. aureus infections can be either HA or CA. The characteristics and virulence profiles of CA S. aureus typically differ to those of HA S. aureus ^[268][269][305,306]. HA infections of methicillin-resistant S. aureus (MRSA) were first reported in the 1960s ^[270][307], but rarely affected non-hospitalised healthy people and failed to spread efficiently within the community. This was generally attributed to the fitness cost imposed upon HA-MRSA through acquisition of antibiotic resistance elements ^[271][308], and is consistent with studies that reported HA-MRSA being generally more drug-resistant ^[268][272][305,309] and have reduced fitness and virulence ^[273][310] than CA-MRSA. Nevertheless, HA-MRSA clones remain a major cause of nosocomial infections globally ^[270][307].

The global emergence of CA-MRSA ^{[274][275][276][277][278][279]}[311,312,313,314,315,316] began in the late 1980s ^[271][308], and was defined as a MRSA infection in the community whereby the infected persons exhibits no apparent nosocomial risk factors. This suggested that CA-MRSA evolved independently from lineages present in clinical settings. This hypothesis was further supported by the observation that CA-MRSA and HA-MRSA are epidemiologically, clinically, and microbiologically distinct ^[269][272][306,309]. Typically, CA-MRSA differ from HA-MRSA through the former exhibiting low-level susceptibility to non-β-lactam antibiotics, carriage of SCCmec types IV or V and production of Panton-Valentine leukocidins ^[271][308]. However, possible transmission between HA-MRSA to CA-MRSA may increase the overlap in similarities between the two MRSA sub-populations ^[269][306].

For HA-MRSA, ST239 was traditionally considered to be the dominant global hospital clone ^[280][317] and remains prevalent in Asia along with ST5 ^[281][282][318,319]. Elsewhere, the prevalence distribution of HA-MRSA clones will vary depending on geographical location: USA100 (North America) ^{[283][284][285][286]}[320,321,322,323], CC5 (Latin America) ^[287][288][324,325], ST22 (United Kingdom), ST225 (central Europe) ^[282][319], ST22-IV [2B] (Australia) ^[289][326] and ST5 and ST239/241 (Africa) ^[290][327].

In the community, the dominant CA-MRSA clone also varies by geographical location: USA300—ST8-IV (North America), USA1100 and USA300-Latin American variant (South America), ST80-IV (Europe), ST93-IV (Australia), high heterogeneity in Asia (no dominant clone) and insufficient data for Africa ^[291][328]. Although traditionally considered a HA pathogen, the burden of CA-MRSA disease has been on the rise since its global emergence in the 1990s ^[292][329] and it began to appear within HA facilities in the 2000s ^[271][308]. Since then, many countries such as Australia ^[293][330], China ^[282][319], India ^[294][331], Kuwait ^[295][332], South Korea ^[296][297][333,334] Switzerland ^[298][335] United Arab Emirates ^[299][336], United Kingdom ^[300][337] and the United States ^{[301][302][303]}[338,339,340] have reported the occurrences of persistence, dissemination, outbreaks and/or outright dominance of CA-MRSA clones within HA facilities which are attributed to the comparatively higher fitness of CA-MRSA through its carriage of smaller SCCmec variants and fewer, if any, other antibiotic resistance determinants ^[273][310].

The exact mechanisms driving the divergent evolution of MRSA clones, and reasons for the emergence and replacement or dominance of specific clones in different geographical locations remain unclear ^{[270][274][304][305][306][307]}[307,311,341,342,343,344]. RWesearchers hypothesise that factors such as the host population demographics, migration, environmental climate, presence of other microorganism communities (e.g., other bacteria and bacteriophages that can facilitate horizontal gene transfer), spontaneous gene mutations and level of antibiotic use and stewardship are all likely to play contributing roles. With such changing diversity in MRSA clones, rapid and accurate clinical diagnosis, combined with a tailored treatment regimen according to the resistance profile of the clonal type will be essential for effective patient care ^[291][328].

S. aureus has demonstrated a remarkable ability to rapidly acquire and develop antibiotic resistance (Figure 2), often achieved through horizontal gene transfer of mobile genetic elements (MGEs) and chromosomal mutations ^[306][343]. As a result, an extensive arsenal of resistance mechanisms has emerged in S. aureus that enables resistance to major antibiotic classes typically employed to treat infection (Table 2).

Like enterococci, the rapid emergence of resistance development in S. aureus has been attributed to the misuse and overuse of antibiotics in clinical and agricultural settings [9]. When combined with additional factors such as high rates of asymptomatic colonisation ^[262][310][299,347] and increased accessibility of international travel, S. aureus infections, particularly those caused by antibiotic resistant strains, have reached epidemic proportions in community and clinical settings worldwide ^[311][348]. Globally, S. aureus was responsible for more than 250,000 deaths associated with AMR in 2019 ^[312][268]. In the United States, S. aureus caused more than 119,000 bloodstream infections which led to nearly 20,000 deaths in 2017 ^[313][349]. As with VRE, antibiotic resistant S. aureus has also been designed as a “high priority” and “serious threat” pathogen by the WHO ^[314][274] and U.S. CDC [29] respectively.

2. Vancomycin

2.1. Discovery and History

Vancomycin is a tricyclic glycopeptide antibiotic first isolated in 1957 from the fungus Streptomyces orientalis. In vitro experiments showed that it had broad spectrum activity against Gram-positive bacteria, with no detected resistance in staphylococci following serial passages in media containing vancomycin. After showing promising efficacy and safety profiles in animal models, vancomycin (name derived from “vanquish”) entered human clinical trials ^[33][315][33,350]. During an initial clinical trial, vancomycin successfully treated 8 out of 9 patients with severe staphylococcal infection. Therapy failure occurred in one patient who was suffering from empyema, which prevented a therapeutic dose level of vancomycin from being administered ^[316][351]. In another human study, 5 out of 6 endocarditis patients who had already experienced antibiotic failure demonstrated resolution of disease indicators; the singular patient who experienced therapy failure had also presented with multiple conditions such as intractable heart failure and shock ^[317][352]. The culmination of positive data from these respective clinical trials subsequently resulted in the immediate approval of vancomycin by the U.S. Food and Drug Administration (FDA) in 1958. However, due to perceived nephrotoxicity ^{[33][318][319]}[33,353,354], vancomycin was originally categorized as a last resort medication reserved for patients who were infected with bacteria that were resistant to frontline drugs or those patients with serious allergies to standard therapy [33]. Today, vancomycin is used as a first-line treatment for MRSA ^{[320][321][322]}[355,356,357], and remains an important antibiotic used against serious Gram-positive bacterial infections ^[323][324][358,359].

2.2. Mechanism of Action

Vancomycin inhibits the cell wall synthesis of Gram-positive bacteria by binding to D-Ala-D-Ala dipeptide subunits of peptidoglycan monomers anchored to the sugar backbone of alternating N-acetylmuramic acid (MurNac) and N-acetylglucosamine (GlcNac) residues ^[325][326][360,361]. In susceptible bacteria, peptidoglycan monomers normally undergo transglycosylation and transpeptidation by the glycosyltransferase and transpeptidase activities of penicillin-binding proteins (PBPs), forming new peptidoglycan structures through pentaglycine cross-linkage ^[327][328][362,363]. Vancomycin, as a largely hydrophilic molecule, disrupts this process by forming hydrogen bonds to the D-Ala-D-Ala moiety through its aglycon subunit. As a result, this complex leads to a conformational change to the peptidoglycan which prevents subsequent transglycosylase and transpeptidase activity. Consequently, cell wall synthesis is inhibited as new peptidoglycan monomers are unable to be incorporated into the growing peptidoglycan skeleton, eventually leading to bacteriostasis in enterococci ^[315][350] or osmotic shock, cell lysis and death in S. aureus (Figure 3) ^{[23][315][329][330]}[23,350,364,365]. Although adverse effects are still observed from prolonged administration or high concentrations of use, vancomycin’s toxicity has been significantly reduced since its first introduction. This was most likely achieved due to the removal of impurities present in early batches ^[315][350]. The improvement in vancomycin’s safety profile, in addition to the emergence of methicillin-resistant bacteria in the 1970s, subsequently lead to its mainstream adoption and use [34]. Today, vancomycin’s utility and importance in modern medicine is highlighted by its inclusion on the WHO’s model list of essential medicines ^[331][366].

2.3. Vancomycin Resistance in Enterococcus

The widespread use of vancomycin has predictably resulted in the rapid emergence and spread of vancomycin resistance amongst various Gram-positive bacteria ^[315][350]. In 1988, Uttley and colleagues published the first clinical outbreak of highly resistant VRE, with some isolates having minimum inhibitory concentrations (MICs) greater than 2000 µg/mL ^[332][367]. Today, the Clinical Laboratory Standards Institute (CLSI) classifies complete vancomycin resistance in Enterococcus with a MIC of ≥32 µg/mL, intermediate resistance as 8–16 µg/mL and susceptible as ≤4 µg/mL using broth microdilution testing ^[333][368]. This is consistent with the breakpoints set by the European Committee on Antimicrobial Susceptibility Testing (EUCAST) which define the MIC of vancomycin susceptible enterococci to be ≤4 µg/mL and vancomycin-resistant enterococci to be>4 µg/mL ^[334][369]. Vancomycin resistance in enterococci is centered around the modification of the vancomycin target site i.e., modification of the D-Ala-D-Ala terminal amino acids of dipeptide monomer subunits into either D-Ala-D-Lac or D-Ala-D-Ser (Figure 4). These mutations confer high- and low-level vancomycin resistant phenotypes respectively. This is because the binding affinity of vancomycin for D-Ala-D-Lac is reduced 1000-fold due to loss of a single hydrogen bond ^[335][370] compared to its modestly (6-fold) reduced affinity for D-Ala-D-Ser due to steric hindrance by the D-Ser hydroxyl group ^[336][337][371,372]. As the mechanism of resistance (D-Ala-D-Lac or D-Ala-D-Ser) is determined by different van cassettes, the degree of vancomycin resistance in enterococci will be dependent upon which van operon they express ^[336][371]. The different van operons, their respective genes, proteins, and mechanisms of action responsible for these variable resistance levels are summarised in Table 3.