Although the inappropriate use of linezolid in clinical settings has contributed to the selection and spread of resistance in Staphylococcus, it is important to note that the resistance mechanisms themselves are not novel but rather represent the utilization of pre-existing genetic elements in response to the selective pressure exerted by antibiotic use [13]. Currently, the resistance situation is quite severe, and the main identified resistance mechanisms include the following six types: (1) mutations in the domain V of 23S rRNA; (2) mutations in the rplC, rplD, and rplV genes; (3) acquisition of plasmids carrying the cfr resistance gene through horizontal or vertical transmission; (4) methylation of the 23S rRNA to protect the ribosome from binding with linezolid; (5) ribosome protection by the OptrA and PoxtA proteins in the ABC-F family, preventing linezolid from binding to the ribosome; (6) increased efflux of linezolid by the LmrS multidrug efflux pump. The interaction of these complex mechanisms ultimately leads to the resistance of S. aureus to linezolid [14].

Mutations in the domain V of 23S rRNA

Given that the domain V of 23S rRNA harbors the linezolid action site, any mutations or structural alterations within this region could influence bacterial resistance to linezolid [15]. The first clinical strain of linezolid-resistant S. aureus, identified in 2001, exhibited the G2576 mutation in the domain V of 23S rRNA, reducing the drug’s affinity for its target site [16]. Follow-up studies confirmed that mutations at this locus were predominant, occurring in 63.5% of linezolid-resistant Staphylococcus aureus (LRSA), 60.2% of linezolid-resistant coagulase-negative staphylococci (LRCoNS), and 57.5% of linezolid-resistant enterococci [14].

In a study investigating how oxazolidinone-class antibiotics interfere with the ribosomal peptidyl transferase center and affect tRNA positioning, it was revealed that G2576 is located at a position where it directly stacks onto one of the universally conserved residues, G2505, within the oxazolidinone binding site. In the crystals of the native Deinococcus radiodurans 50S ribosomal subunit (D50S)-oxazolidinone structure, the oxazolidine ring, a primary component of the drug, appears to stack against the base of U2504. Consequently, mutations at G2576 may confer resistance allosterically by altering the positioning of U2504. This is distinct from other mutation sites, which are primarily clustered around G2504 and its base-pairing partner C2452, indirectly affecting oxazolidinone binding by influencing the adjacent bases at these locations [17].

Additionally, research has established that staphylococci possess 5–6 copies of rRNA (rrn) operons, with resistance levels increasing alongside the number of mutant 23S rRNA alleles, while growth rates decrease due to mutation-mediated resistance in the 23S rRNA [18]. In an in vitro experiment, mutants exhibiting increased MIC of linezolid were generated by progressively exposing linezolid-sensitive cells to media containing escalating concentrations of the drug [19]. These mutants accumulated G2576 mutations across multiple copies of the 23S rRNA gene, a phenomenon that closely correlated with the degree of drug resistance [20, 21]. Locke et al. demonstrated, through serial passage of methicillin-susceptible S. aureus (MSSA) and MRSA in linezolid, that the MICs for MRSA and MSSA increased 32-fold and 64-fold, respectively, over 30 generations, indicating that mutations conferring linezolid resistance in the 23S rRNA can persist even without antibiotic pressure [22]. Most linezolid resistance mutations are centrally located between nucleotides 2400 and 2600 of the 23S rRNA [15, 23,24,25,26]. Additionally, specific point mutations in the 23S rRNA genes, including T2500, C2534, G2766, T2504, and G2447, have been identified as associated with resistance in clinical strains (Table 1) [22, 27, 28].

Table 1 Mutation sites on 23 s rRNA, L3, and L4 proteins were detected in linezolid-resistant Staphylococcus aureusMutations in the rplC, rplD, and rplV genes

Linezolid targets the 50S large ribosomal subunit, specifically the binding sites closely associated with ribosomal proteins uL3, uL4, and uL22 [28]. Studies have shown that mutations in the rplC, rplD, and rplV genes on the chromosome, which encode these ribosomal proteins, alter the structure and stability of 23S rRNA [14], thereby affecting bacterial sensitivity to linezolid. However, resistance to linezolid in S. aureus caused by these mutations is relatively rare [29].

Although ribosomal protein uL3 is primarily located on the surface of the 50S subunit, it possesses two looped structures extending to the PTC. Since 2003, research has revealed that mutations in bacterial uL3 are associated with resistance to linezolid and other antibiotics. In this study, all detected uL3 mutations were concentrated in a central extension near the PTC. For instance, the G152D and G155R mutations are related to mycoplasma resistance [30,31,32]. The G152D mutation is located below a conserved mismatch site and may indirectly interfere with bases 2505 and 2506, reducing the affinity for oxazolidinones [33,34,35,36]. In S. aureus, mutations in the uL3 protein often occur simultaneously with the 23S rRNA G2576T mutation. Additionally, some studies have found that uL3 protein mutations (such as D145H146Y and M169G174) co-occur with the exogenously acquired cfr gene, which encodes an enzyme named chloramphenicol-florfenicol resistance protein, this enzyme can mediate resistance to linezolid [37].

In addition to the aforementioned mutations, other alterations in ribosomal protein uL3 are also associated with linezolid resistance in Staphylococcus species. When investigating the impact of G139D, L94V, D159, G152, T146Y and F84V mutations in uL3 on linezolid resistance, we observed that these changes structurally significantly affect the drug’s binding and mechanism of action. Through in silico analysis, we found that these mutations primarily influence the tertiary structure of uL3, particularly the α-helices and large coiled regions. Specifically, the mutations at D159 and G152 (transformed to Tyr and Asp, respectively) are located within a large coiled region. These amino acids, compared to the original ones, are larger and have different polarities. This alteration could lead to a significant deformation of the tertiary structure of uL3, thereby affecting its binding affinity with linezolid. Although the L94V substitution is also within a coiled region, it involves two amino acids of similar size and polarity, suggesting a smaller structural impact. Moreover, mutations such as T146Y and G139D may also influence the tertiary structure of uL3, thereby indirectly affecting its interaction with linezolid. These structural changes could result in alterations to the binding site of the drug to the ribosome or impact the ribosome’s function during synthesis, thereby affecting the sensitivity of bacteria to linezolid [38].

In summary, these mutations in uL3 protein may alter its structure, thereby influencing the interaction between linezolid and the ribosome, ultimately leading to linezolid resistance in bacteria. These findings provide important insights into the molecular mechanisms of linezolid resistance and offer a theoretical basis for the development of new strategies against drug resistance. However, these conclusions require experimental validation and further research to be fully substantiated.

The rplD gene encodes the uL4 protein located near the PTC, and mutations in this gene, including adjacent 6-bp deletions, may lead to a slight reduction in linezolid susceptibility. The extended tip of uL4 primarily interacts with 23S rRNA, stabilizing the key region of the PTC through charge neutralization and packaging interactions [39]. In S.aureus, the K68Q mutation in the uL4 protein can decrease the antibacterial effect. K68 interacts with the sugar-phosphate backbone of A2059 and G2061, collectively forming a pocket around A2503 [35]. The loss of compensatory charge due to the K68Q mutation can cause deformation of the pocket and subsequent movement of A2503 towards the PTC region, affecting the affinity for various types of antibiotics [35, 40]. Currently, clinical resistance to linezolid in S.aureus has been found in uL4 proteins containing G69A and T70P mutations [41].

Due to the proximity of the ribosomal protein uL22 gene to the linezolid binding site, mutations, deletions, or substitutions in uL22 amino acids may affect the spatial structure of the PTC, thereby being associated with linezolid resistance [35, 41, 42].

The methylation of 23S rRNA by the Cfr methyltransferase encoded by the cfr gene

In the current arsenal of antibiotics for treating infectious diseases in humans, numerous drugs target the bacterial ribosome and exert their efficacy by inhibiting protein synthesis. Some of these drugs bind to the PTC region and function by competing with aminoacyl-transfer RNA (aa-tRNA) substrates at the PTC or its vicinity. However, there are also ribosome-targeting antibiotics that act on multiple other functional sites on the ribosome, such as the decoding center and the GTPase-associated center, among others [43, 44]. However, the natural evolutionary response of pathogenic bacteria to antibiotic treatment involves the acquisition and spread of resistance genes that are already present in microbial communities. Among these genes, the cfr gene is particularly common in S. aureus resistance to linezolid. Recent studies have identified new variants of the cfr gene, such as cfr(B), cfr(C), cfr(D), and cfr(E) [45,46,47].

The cfr methyltransferase encoded by the cfr gene confers resistance to a broad range of PTC-targeting antibiotics by performing C8-methylation on the universally conserved adenosine residue A2503 of the 23S ribosomal RNA [48, 49]. This modification is located at the center of the ribosome, near the A site of the PTC, affecting the binding of various antibiotics, including phenicols, lincosamides, oxazolidinones, pleuromutilins and streptogramins A (collectively known as PhLOPSA), as well as hygromycin A and 16-membered macrolides (16MMs) [50, 51]. The cfr gene was first identified in 2000 in Mammaliicoccus sciuri (formerly Staphylococcus sciuri) and has since been detected in a wide array of Gram-positive and Gram-negative bacteria [52].

The mechanism of cfr-mediated resistance involves two primary components: first, the direct steric hindrance of antibiotics by the m8A2503 group, and second, the cfr-induced rearrangement of nucleotide A2062, leading to an allosteric rearrangement of the drug-binding pocket. These mechanisms act in concert, preventing a variety of chemically unrelated antibiotic classes from binding to cfr-modified ribosomes. Structural studies indicate that the m8A2503 group spatially overlaps with the binding sites of PTC-targeting drugs, supporting the “direct spatial conflict” model. However, the degree of spatial overlap between the C8-methyl group and certain PTC-binding drugs is relatively small, suggesting the presence of additional resistance mechanisms [53].

In particular, nucleotide A2062 in cfr-modified ribosomes is unable to rotate, which prevents additional binding interactions with antibiotics and thereby promotes resistance through an allosteric mechanism. This mechanism plays a significant role in resistance to phenicols and oxazolidinone antibiotics. Compared to the A2062 mutation, the replacement of A2062 with a pyrimidine (U or C) results in strong resistance to most A site-targeting antibiotics, further underscoring the importance of A2062 in drug-ribosome binding [53].

In summary, structural information about cfr-modified ribosomes and their complexes with antibiotics provides an important starting point for the development of next-generation drugs against multidrug-resistant pathogens. Designing antibiotics that can form new interactions with the A site of cfr-methylated ribosomes may be an effective strategy to combat pathogens expressing the cfr gene.

23S rRNA modification protects ribosomes from binding to linezolid

Spr0333 is an hypothetical gene in Streptococcus pneumoniae that encodes a 385-amino acid protein containing a S-adenosylmethionine (SAM)-dependent methyltransferase (PFAM UPF0020) domain. The Spr0333 protein shares 32% sequence identity with a domain of the E. coli gene product RlmL, which encodes an rRNA methyltransferase responsible for the N2-methylation of G2445 in the 23S rRNA [54]. Mutations in Spr0333 are located within its conserved SAM methyltransferase domain, including non-synonymous mutations and frameshift deletions, which may lead to the inactivation of Spr0333 and are associated with linezolid resistance [55].

Methylation of ribosomal RNA is a common mechanism of acquired resistance to antibiotics such as erythromycin [55]. Endogenous RNA methylases may play a role in protecting 23S rRNA from natural xenobiotics [45]. Primer extension assays demonstrate that Spr0333 exhibits 23S rRNA methylation activity, and susceptibility testing confirms that different mutations in Spr0333 are directly linked to linezolid resistance [55].

Orthologs of Spr0333 are found in many bacterial species, including S.aureus. In a previous study of vancomycin intermediate MRSA (VISA) from the NARSA collection (www.narsa.net) [46], we confirmed that three isolates, NRS 119, 127, and 271, were resistant to linezolid. The ortholog of Spr0333 in S. aureus is SAV1444 (named according to the nomenclature of S. aureus Mu50) [46], which shares about 45% identity with the Streptococcus pneumoniae ortholog. In the isolate NRS119, which had the highest MIC against linezolid, the sequence of the SAV1444 gene revealed a 39-bp deletion in the SAM-methylation domain, No mutations were observed in the two other VISA isolates that were less resistant to linezolid. NRS271 and NRS127 were selected in vitro for increased linezolid resistance, and their SAV1444 gene was resequenced. A 60-bp deletion was observed in the in vitro linezolid-selected NRS271, indicating that the Spr0333 ortholog SAV1444 is directly related to the linezolid resistance of this S. aureus strain [55].

ABC-F protein through a ribosomal protection to prevent linezolid from binding to ribosomes

The ATP-binding cassette (ABC) protein superfamily is a class of proteins widely distributed across all life domains, primarily involved in the energy-dependent transport of molecules across biological membranes [47]. This superfamily comprises multiple subfamilies, the majority of which are composed of typical ABC transporters with two ABC domains and two transmembrane domains (TMDs) [56]. However, members of the ABC-E and ABC-F subfamilies are neither fused with TMDs nor genetically associated with TMDs in operons [56, 57]. The ABC-F subfamily is abundant in both eukaryotes and bacteria, and these members have been implicated in a variety of biological processes, including DNA repair [58, 59], translational control [60], and resistance to antibiotics targeting bacterial protein synthesis [57].

The classification of ABC-F proteins primarily includes the following types: (1) Antibiotic Resistance Expressers (AREs): These ABC-F proteins are closely related to the antibiotic resistance of bacteria, capable of protecting bacteria from the attack of specific antibiotics [60]. The expression of these proteins is typically inducible, meaning they are only activated when bacteria encounter specific antibiotics. For example, OptrA and PoxtA belong to this category of proteins. (2) House-keeping ABCFs: These ABC-F proteins are essential for maintaining the normal function of bacterial ribosomes and translational control. They play a fundamental role in the growth and metabolism of bacteria and are not directly involved in antibiotic resistance. For instance, EttA (Efflux Transporter) in Escherichia coli is a house-keeping protein [61]. (3) Ribosome Protection Proteins (RPPs): These ABC-F proteins can bind to ribosomes and protect them from certain antibiotics that typically target the bacterial protein synthesis mechanism [62]. (4) Translation regulatory proteins [63], and so on.

In pathogens, the interaction or binding of ABC-F ARE proteins with the 50S subunit is not always observed. This interaction is typically conditional, occurring when the pathogen is exposed to specific exogenous compounds (e.g., antibiotics) [60]. These antibiotics target the 50S ribosomal subunit of bacteria, thereby inhibiting protein synthesis. When antibiotics are absent, ABC-F ARE proteins may not bind to the 50S subunit, or their binding may not impact the normal function of bacteria. However, upon exposure to these antibiotics, the activity of ABC-F ARE proteins may be induced or enhanced, leading to their binding to the 50S subunit to prevent antibiotic binding or action, thus conferring antibiotic resistance. Therefore, the binding of ABC-F ARE proteins to the 50S subunit is a responsive mechanism aimed at protecting bacteria from antibiotics. This binding is dynamic and depends on whether the pathogen is exposed to specific antimicrobial drugs [64].

In the ABC-F ARE subfamily, OptrA and PoxtA proteins are two significant members that play a crucial role in conferring resistance to linezolid in S.aureus [65]. The genes that encode these proteins are located on mobile genetic elements (MGEs), which can facilitate their transfer between different bacterial strains. This family of proteins provides resistance by directly protecting the target from the effects of antibiotics, rather than through drug efflux. OptrA gene is responsible for plasmid-mediated linezolid resistance, first identified in a human-derived Enterococcus faecium isolate and also found in MRSA, commonly co-existing with the cfr gene. The resistance mediated by OptrA primarily manifests as tolerance to oxazolidinone and phenylacetamide antibioticss [66, 67]. The catalytic domain of OptrA contains two glutamate (E) motifs that exhibit ATP hydrolysis activity, providing preliminary evidence for the resistance associated with OptrA [68]. The structure of the OptrA protein includes two nucleotide-binding domains (NBD1 and NBD2), which are connected by a spiral connector and do not contain known transmembrane domains [69]. These catalytic glutamate residues are located at the ATP-binding sites of the two NBD domains and are crucial for ATP hydrolysis.

The mechanism of linezolid resistance mediated by OptrA involves several key steps. OptrA binds to the 50S subunit and disrupts its structure, preventing antibiotics from binding to the ribosome. This mechanism directly affects the structure and function of the ribosome, particularly the alteration of the peptidyl transferase center, which is the site where antibiotics bind to inhibit protein synthesis. This structure allows OptrA to affect ribosomal function, especially at the drug-binding site. This mechanism differs from the traditional drug efflux mechanism, which involves ATP hydrolysis to drive antibiotics out of the bacterial cytoplasm [70].

The clinical significance of OptrA-mediated resistance lies in its ability to confer resistance to a range of important antibiotic classes, including oxazolidinones, lincomycins, macrolides, oxazolidinones, phenylacetamides, prulifloxacins, and streptogramin A and B groups [40, 71]. This resistance is prevalent in pathogens and has been observed in some clinical isolates mediated by the optrA gene against oxazolidinones and phenylacetamides. Therefore, the OptrA-mediated resistance mechanism is a complex biochemical process involving direct interactions between proteins and ribosomes, providing a scientific foundation for the development of new antimicrobial resistance strategies.

PoxtA is a resistance determinant belonging to the ABC-F ARE proteins [66]. It was initially detected in the genome of a linezolid-resistant MRSA strain isolated from a cystic fibrosis patient. The poxtA gene encodes a protein with 32% identity to the OptrA protein and possesses structural features for mediating antibiotic resistance through ribosome protection. The resistance mechanism of PoxtA protein involves perturbing the P-site tRNA, causing it to move approximately 4 Å towards the ribosome, equivalent to a shift of one amino acid during translation. This perturbation may interfere with the drug binding site by altering the conformation of the nascent peptide chain attached to the ribosome [72]. The structural studies provide crucial insights into how PoxtA mediates linezolid resistance. The specific mechanism of action of PoxtA involves the ATP-dependent regulation of the peptidyl transferase center, altering its stereochemical properties and global conformation, thereby influencing the binding geometry of the P-site tRNA. This mechanism enables PoxtA to protect bacteria from antibiotics targeting the ribosome.The poxtA gene has been shown to functionally contribute to the reduction of resistance to at least three classes of antiribosomal antibiotics, including phenylacetamides, oxazolidinones, and tetracyclines [66].

These two genes emergence and spread may gradually increase the level of resistance to linezolid and other drugs, posing challenges to clinical treatment. Therefore, monitoring the prevalence of these resistance genes in clinical and veterinary isolates and studying their interaction mechanisms of the encoded proteins with ribosomes are crucial for understanding and addressing the resistance of S.aureus to linezolid.

LmrS efflux pump facilitates linezolid resistance in S. aureus

A study characterized the LmrS efflux pump from S. aureus, a member of the major facilitator superfamily, as a secondary active multidrug efflux pump with 14 predicted transmembrane domains. The expression of the lmrS gene cloned into a multicopy plasmid within the host organism conferred resistance to various antimicrobials. Among the antibacterial agents tested, the MIC for the oxazolidinone drug linezolid increased 16-fold [73, 74]. In conclusion, LmrS represents a critical determinant of antimicrobial resistance in S. aureus. Further research into the distribution of this gene among clinical isolates and the levels of LmrS expression is required to elucidate its precise role in the physiology and pathogenesis of S. aureus, particularly its contribution to antimicrobial resistance [74].