Overexpression of efflux pumps is a key mechanism responsible for drug resistance in A. baumannii. Efflux pump excretes antimicrobial drugs from cells, which leads to the decrease of drug concentration and drug resistance (Meyer et al., 2022). There are five superfamilies associated with drug resistance in A. baumannii: the resistance-nodulation-cell division (RND) family, multidrug and toxic compound extrusion (MATE) family, the ATP-binding cassette (ABC) transporters, the major facilitator superfamily (MFS) and the small multidrug resistance (SMR) family, of which the first four have been reported to mediate tigecycline resistance (Rafiei et al., 2022).

To prevent the entry of antimicrobial agents into cells, reducing the permeability of the outer membrane is another common mechanism of resistance.

PlsC, encoding 1-acyl-sn-glycero-3-phosphate acyltransferase, catalyzes the acylation of lysophosphatidic acid to form phosphatidic acid, thereby playing a role in maintaining epidermal permeability barrier function (Lu et al., 2005). Li et al. identified a strain of A. baumannii, 19606-M24, with high levels of resistance to tigecycline (MIC = 24 mg/L), and whole genome comparison revealed a mutation in plsC. Complementation studies revealed that strains introduced with the wild-type plsC gene restored susceptibility to tigecycline, indicating that the frameshift mutation of plsC gene is associated with tigecycline resistance. In subsequent flow cytometry, the highest membrane potential was observed for 19606-M24, while a decrease in membrane potential was also observed in complementary strains. These results convincingly demonstrate that plsC mediates resistance to tigecycline by affecting cell membrane permeability (Li et al., 2015).

Soon thereafter, Li et al. identified a new gene, abrp, encoding the peptidase C13 family, with a frameshift mutation. Compared to the abrp knockout strain, the wild-type strain showed reduced susceptibility to a variety of antibiotics, including tigecycline. Furthermore, they found that the abrp deletion strain increased cell membrane permeability accompanied by impaired bacterial growth. Thus, it can be concluded that abrp increases resistance to tigecycline by altering cell membrane permeability (Li et al., 2016).

Another gene, gnaA, has also been reported to be associated with tigecycline resistance (Hammerstrom et al., 2015; Lucassen et al., 2021a). GnaA encodes an enzyme involved in catalyzing the initial step of UDP-D-MANPNAC3NACA synthesis and was first identified in Escherichia coli (Wang et al., 2012). In 2019, Xu et al. identified tigecycline-resistant mutant strains with an insertion of ISAba16 in the gnaA gene of unknown function. The membrane potential of gnaA-deficient bacteria was significantly higher than that of the wild-type strain, and complementation with the wild-type gnaA gene resulted in a reduced membrane potential, indicating that the barrier function of the cell membrane was restored. Moreover, the researchers revealed that the gnaA gene affects lipooligosaccharide (LOS) and capsular polysaccharide (CPS) synthesis and that disruption of gnaA also affects the virulence, morphology, and susceptibility of A. baumannii to other classes of antibiotics. Unfortunately, no changes in tigecycline MIC were seen in gnaA complementary experiments, this could still suggest that the gnaA gene may be involved in tigecycline resistance by altering cell membrane permeability (Xu et al., 2019). In conclusion, the resistance of gnaA to tigecycline needs to be supported by more studies.

Outer membrane protein (OMP) is a specific channel protein present in the lipid bilayer structure of gram-negative bacteria, located on the surface of the cell membrane or embedded in it, which can serve as a channel for antibiotics to enter the cell (Tokuda, 2009). Vijaya et al. uncovered a putative OMP, abuO, associated with antimicrobial and oxidative stress resistance in A. baumannii. Compared with the wild-type strain, the abuO knockout strain showed increased susceptibility to different types of antibiotics, including tigecycline, with MIC values changing from 2 mg/L to 0.75 mg/L. Meanwhile, the MIC of the complementary abuO isolate again returned to the same level as the wild-type strain. This may indicate a role for abuO in tigecycline resistance (Srinivasan et al., 2015). In addition, RT−PCR results showed that the abuO-deficient strain not only increased the expression levels of efflux pump-related genes but also changed the expression of other membrane proteins, which implies that abuO plays an integral role in A. baumannii and that its relationship with tigecycline resistance mechanisms should still be investigated in depth.

Changing the target of antibiotic action is also one of the mechanisms by which bacteria develop resistance. Bacteria prevent antibiotic binding by altering the target structure, thereby creating resistance.

The S10 protein, which is closest to the tigecycline binding pocket, consists of 53-60 amino acid residues encoded by the rpsJ gene and helps to participate in maintaining the normal structure of the tigecycline binding site. Researchers found that rpsJ mutations emerged in a variety of pathogens under tigecycline exposure and conferred resistance to tigecycline. They believe that mutations in rpsJ caused a structural change in the S10 loop, which in turn changes the conformation of the 16S RNA, ultimately leading to reduced binding affinity for tigecycline and ribosomes. Another model is that reduced tigecycline susceptibility promotes entry of tRNA and increases binding to ribosomes (Beabout et al., 2015). Additionally, since rpsJ mutations have a low fitness cost and occur widely in a variety of pathogens, all tested strains had a mutation at amino acid position 57 of S10, while gram-positive bacteria have a higher variation at amino acid position 60 ( Figure 3 ). These results suggest that rspJ has promise as an important marker for detecting whether pathogens are resistant to tigecycline. Another result likewise reported that rspJ plays an important role in tigecycline resistance (Hammerstrom et al., 2015).

The gene trm (tigecycline-related-methyltransferase) encodes S-adenosyl-L-methionine-dependent methyltransferase. Chen et al. obtained the resistant strain 19606-T8 (MIC = 8 mg/L) with a deletion mutation in the trm gene and then transformed the wild-type trm gene into 19606-T8. Interestingly, it restored the sensitivity to tigecycline, MIC dropping from 8 mg/L to 1 mg/L, implying that trm plays an important role in reducing A. baumannii susceptibility to tigecycline (Chen et al., 2014). However, how methyltransferase mediates the reduction in sensitivity to tigecycline has not been investigated. Another study also reported diverse amino acid alterations in trm of tigecycline-resistant isolates, such as S94A, V121A, C285F, K291R, G310C, H312P, T321I, T323S, and M378K (Ghalavand et al., 2022). The mechanism by which trm mediates resistance to tigecycline is unclear. As a methyltransferase, trm may act as ArmA, RmtA and NpmA to methylate the ribosomal target, hinder the binding of tigecycline to its target, and mediate tigecycline resistance (Fritsche et al., 2008; Chen et al., 2014).

The rrf gene encodes the production of ribosome recycling factor (RRF). RRF is an essential component of protein synthesis and is involved in the release of polypeptides from the ribosome for a new round of translation (Ma et al., 2014). In an earlier study, Hammerstrom et al. identified mutations in the rrf gene in tigecycline-resistant A. baumannii. They proposed that mutations in rrf (M1 V, N3N) might reduce translation of mRNA and in return affect the binding of tigecycline to the ribosome, but they did not proceed to verify this hypothesis (Hammerstrom et al., 2015).

Subsequently, Hua et al. revealed the mechanism of rrf involvement in tigecycline resistance. They constructed recombinant mutants XH1457 (MDR-ZJ06 rrf^H33P) and XH1458 (MDR-ZJ06 rpoB^G136D), which showed only a slight increase in tigecycline tolerance compared to the parental strain, but Raman spectrometry provided a strong complement to the tigecycline resistance conferred by these two mutant genes. Furthermore, it was confirmed that mutations in rrf decreased the expression of RRF proteins, affecting the ribosome recycling process and ultimately exhibiting a tigecycline-resistant phenotype. In addition, the transcriptomic data also revealed that rpoB ^G136D could regulate the expression level of trm (Hua et al., 2021).

Resistance to antibiotics by degrading and modifying antibiotics to make them inactive is an important resistance mechanism for bacteria.

Several studies have recently reported the widespread dissemination of Tet(X) and its variants through plasmid mediation in A. baumannii, giving rise to resistance to a variety of tetracyclines, which has drawn the attention of researchers. Tet(X) encodes flavin-dependent monooxygenase that modifies first- and second-generation tetracycline. Tigecycline is one of the substrates of Tet (X), which requires activation in the presence of FAD, NADPH, O₂, and Mg2⁺, Tet (X) modifies tigecycline to 11a-hydroxytigecycline, which alters the physical properties of tigecycline by weakening its binding to magnesium and decreasing its affinity to ribosomes, this inactivates tigecycline, thus allowing bacteria to develop resistance (Moore et al., 2005).

To date, eight genes associated with high levels of resistance to tigecycline have been reported in the Tet(X) family, namely Tet(X), Tet(X1), Tet(X2), Tet(X3), Tet(X4), Tet(X5), Tet(X6), Tet(X7), where Tet(X1) is a truncated variant with no enzymatic activity. These Tet(X) and its variants have been detected in various pathogens such as Klebsiella pneumoniae, Pseudomonas aeruginosa and Escherichia coli. Among them, Tet(X), Tet(X3), Tet(X5), and Tet(X6) have been reported to confer tigecycline resistance in A. baumannii ( Cui et al., 2021). In addition, the Tet(X) was originally identified in the obligate anaerobe Bacteroides fragilis ( Yang et al., 2004). As reported in the literature, compared to Tet(X) (M37699), Tet(X3) (MK134375), Tet(X5) (CP040912), Tet(X6) (CP044517) showed 86%, 90%, 84% amino acid identities, respectively (Cui et al., 2021). He et al. demonstrated that Tet(X3) catalyzes the oxygenation of tigecycline at C11a to form 11a-hydroxyltigecycline, resulting in tigecycline inactivation. Whereas the MIC of tigecycline was substantially increased (64-fold) in strains possessing the Tet(X3)-carrying plasmid, mouse infection models likewise revealed that Tet(X3) impaired the therapeutic efficacy of tigecycline in vivo and ultimately led to drug resistance (He et al., 2019). Wang et al. identified another Tet(X) variant, Tet(X5), which is structurally and functionally similar to other variants and hydroxylates tigecycline. Tet(X5) shows increased resistance to a wide range of tetracyclines (Wang et al., 2019). Not coincidentally, scientists have also found that Tet(X5) and Tet(X6) in A. baumannii are resistant to tigecycline through hydroxylation and that ISCR2-mediated transposition and recombination exacerbate the spread of Tet(X5) (Chen et al., 2021). Notably, ISCR2 is an insertion sequence type element capable of transposing neighboring DNA sequences, including antibiotic resistance genes, through a process of rolling-circle transposition (Chen et al., 2021). ISCR2 shares 65% amino acid identity with ISCR1, which is a mobile element involved in the spread of resistance genes (Xu et al., 2017).

Overall, the clonal transmission of Tet(X) and its variants should not be underestimated and deserves attention.

It has been demonstrated that DNA damage under antibiotic induction can mediate the killing of A. baumannii (Sampson et al., 2012), and recA and recBCD are important pathways for DNA repair. RecA is the major enzyme involved in gene homologous recombination and recombination repair (Aranda et al., 2011), and recBCD is involved in DNA double-stranded repair (Kuzminov, 1999). Taofeek et al. found increased susceptibility of recA- and recBCD-inactivated A. baumannii to various antibiotics, including tigecycline, suggesting that the recA- and recBCD-mediated repair pathway may protect A. baumannii from antibiotic killing, leading to drug resistance (Ajiboye et al., 2018).

In addition to the abovementioned mechanisms related to tigecycline resistance in A. baumannii, there are some potential resistance genes that need to be further investigated. Nogbou et al. found that A. baumannii with mutations in both the parC and gyrA genes had higher levels of resistance to tigecycline than strains without mutations (Nogbou et al., 2021). Hammerstrom et al. used a bioinformatics approach to infer that rnpA, rpoD, rppH, wzc, and pcaF are candidate tigecycline resistance genes (Hammerstrom et al., 2015). Whether these genes can indeed mediate tigecycline resistance and by what mechanism they cause resistance needs to be investigated more thoroughly in the future.