Colistin-resistant Enterobacteriaceae are widely identified in clinical infection settings and animal husbandry, and the status of antimicrobial resistance is a concern (Shen et al., 2019). As a transcription regulator commonly found in E. coli, K. pneumoniae, and other members of Enterobacteriaceae, marR played an essential role in regulating efflux pumps. Furthermore, marR mediated the inhibition of marA, while salicylic acid, antibiotic pressure, and endogenous amino acid replacement could release the disinhibition and activate marA expression under physiological conditions, thus upregulating the MDR efflux pump AcrAB-TolC (Grkovic et al., 2002). Activated marA could also upregulate the expression of gene encoding lipopolysaccharide core heptose (II) kinase, known as waay. This upregulation increases the negative charge of the bacterial outer membrane, subsequently enhancing its ability to bind cationic antimicrobial peptides (collateral sensitivity) (Lázár et al., 2018). Sundaramoorthy et al. (2019b) found that the negative charge of the outer membrane of colistin-resistant E. coli increased in the presence of the marR inhibitor salicylic acid, thereby restoring susceptibility to colistin. Another study found that the decreased polymyxin B susceptibility due to the marAB upregulation and marR mutant was observed, mainly attributed to the action of AcrAB and other TolC-dependent efflux pumps (Warner and Levy, 2010). These findings demonstrated the different pathways through which marRA is involved in colistin resistance. Another soxRS system, common in E. coli and Salmonella, plays an essential role in coping with superoxide, nitric oxide, and antibiotic stress. Increased soxS expression induced multidrug-resistant efflux pump AcrAB expression, decreased membrane permeability, and promoted the formation of multidrug-resistant phenotypes (Koutsolioutsou et al., 2005). The same binding sites exist in the promoters regulating Sox, Rob, and Mar systems (mar/sox box), resulting in a high degree of overlap between the genes (micF, acrAB, ompF, fumC, etc.) of soxRS and mar regulons (Figure 1) (Martin et al., 1999). Both soxRS and waaY could be activated by superoxide induction, but waaY transcription was SoxRS dependent (Figure 1) (Lee et al., 2009). Although the absence or overexpression of soxS in E. coli harboring the soxR mutation did not significantly affect CAMP susceptibility, the overexpression of SoxS in the tolC mutant contributed to increased susceptibility to polymyxin B (Warner and Levy, 2010). A recent study showed that not only the collateral susceptibility of antimicrobial resistance development to cationic polypeptides was associated with the changes in the regulation of LPS-related genes, but also a variety of physiological metabolism-related genes such as iron ion-binding proteins and transmembrane transport proteins played an important role (Grézal et al., 2023). This also reflected the pleiotropy of these global regulators involved in regulating multiple links of physiological metabolism. The disruption of tolC directly affected outer membrane integrity, and the intracellular accumulation of toxic substances activated marA, soxS, and rob to upregulate the efflux pumps (Rosner and Martin, 2009; Zgurskaya et al., 2011). Additionally, Rob might directly or indirectly increase marRAB expression by upregulating micF in response to colistin pressure, although the rob deficiency had little effect on CAMP susceptibility (Oh et al., 2000; Warner and Levy, 2010).

Heteroresistance may be an intermediate stage in the transition of susceptible bacteria to antimicrobial resistance, which is an essential reason for the failure of clinical anti-infective therapy (Falagas et al., 2008). Telke et al. found that soxRS-induced AcrAB-TolC efflux pump mediated heterogeneous resistance to colistin and could restore colistin susceptibility through PAβN in Enterobacter spp. (Telke et al., 2017). Unlike CCCP and DNP, PAβN could act directly as an inhibitor of AcrAB and AcrEF efflux pumps. Therefore, these results also suggested that AcrAB efflux pump was involved in colistin resistance (Misra et al., 2015). Colistin could mediate bactericidal effects by producing hydroxyl radicals or causing oxidative stress reactions as a class of bactericidal antibiotics (Bialvaei and Samadi Kafil, 2015; Yu et al., 2019). However, this bactericidal effect did not appear to influence the soxRS transcriptional levels in heterogeneous colistin-resistant Enterobacter isolates (Telke et al., 2017). The necessity of colistin heteroresistance by tolC (but not acrB) was further confirmed in E. cloacae, and tolC could activate the PhoQ-PhoP system by affecting the ecr (Figure 1) (Huang et al., 2019). The deletion of acrB is not sufficient to completely reverse colistin heterologous resistance in E. cloacae (Huang et al., 2019). Previous findings suggested efflux pumps are the primary mechanism for generating stable heterogeneous subpopulations (Manjunath et al., 2021). The fitness costs of these resistance mechanisms remain to be evaluated. Besides the co-regulation of tolC with Mar, Rob, and Sox, the expression of tolC, which carried multiple promoters, was also affected by EvgAS and PhoPQ, indicating that AcrAB-TolC had the potential to cope with different environments and responded quickly (Zhang et al., 2008; Pérez et al., 2012).

Transcriptome and proteomics analyses revealed that the expression of efflux pump AcrAB in E. coli and K. pneumoniae significantly increased after exposure to polymyxin B under experimental conditions (Ramos et al., 2016; Liu et al., 2020). The overexpression of cpxR in the absence of acrB affected the expression of TCS genes (phoP, phoQ, pmrB, pmrC, pmrH, and pmrD) and restored colistin susceptibility in Salmonella (Zhai et al., 2018). The variation in the acrB 620 site in Salmonella contributed to the restoration of colistin susceptibility, which could be explained by the defective macromolecular transport due to the altered conformation of the switch loop in this site (Cha et al., 2014; Kapach et al., 2020). Either tolC deletion alone or dual deletion of cpxR and tolC increased the susceptibility of S. enterica to colistin to varying degrees. The inactivation of tolC impaired the function of the entire RND efflux family and other tolC-dependent efflux pumps, not only the AcrAB pump (Zhang M. K. et al., 2021). These results highlighted the different contributions of acrB, soxRS, and marRA to colistin resistance in different backgrounds, the role of tolC mutations in regulating colistin resistance, and the complex regulatory network among the efflux pump, two-component system, and LPS modification.

Multicenter studies from China have shown that the insertion inactivation of mgrB is the primary mechanism of colistin resistance in K. pneumoniae (Li et al., 2023). However, acrR insertion by IS26 is widespread in ST11-bla_KPC-2-producing PR-CRKP (Polymyxin-resistant carbapenem-resistant K. pneumonia), which may be related to the low level of colistin resistance in these high-risk clones (Yang et al., 2021; Li et al., 2023). ramRA is a widely described global regulator in K. pneumoniae compared with E. coli and Salmonella, which mediates resistance to antibiotics such as tigecycline, nitrofurantoin, and beta-lactams by regulating AcrAB and OqxAB (Hao et al., 2022). The expression of soxS, ramA, and acrAB-tolC significantly upregulated in mcr-negative colistin-resistant K. pneumoniae lacking specific TCS-related gene variants, suggesting the role of soxS, ramA, and efflux pumps in colistin resistance (Naha et al., 2020). Similar to marA and soxS, ramA can also affect LPS modification. ramA directly binds to and activates the genes involved in lipid A biosynthesis: lpxC, lpxL-2, and lpxO, thereby modifying lipid A and resulting in decreased colistin susceptibility and increased anti-serum phagocytosis under the condition of increased ramA expression (Figure 2) (De Majumdar et al., 2015). Li et al. demonstrated in vitro that ramR variants can mediate polymyxin resistance, possibly related to derepressed ramA-mediated LPS alterations and multidrug resistance efflux pump overexpression (AcrAB) (Li et al., 2023). Another survey in China showed that the transcription level of ramA in colistin-resistant K. pneumoniae was not statistically significantly different from that in non-colistin-resistant strains, but the study lacked the main antimicrobial resistant and hypervirulent lineages ST11 and ST23 prevalent in China (Wang et al., 2017). However, ramA overexpression and variation were common in some colistin-resistant K. pneumoniae ST11 and ST147 isolates (Naha et al., 2020; Lv et al., 2021; Bolourchi et al., 2021a,b). Cationic antimicrobial peptides are also common in the human environment. Thus, whether these high-risk clones contribute to the co-evolution of hypervirulence and antimicrobial resistance by activating the efflux pump is not clear. A previous study showed that ΔacrB K. pneumoniae mutant had increased susceptibility to colistin and that LPS and CPS production were not significantly affected, revealing a role for AcrAB in the antimicrobial peptides resistance (Padilla et al., 2010). A less-described class of multidrug efflux RND transporter, KexD, is also present in K. pneumoniae and can be expressed constitutively with E. coli TolC or K. pneumoniae KocC (Ogawa et al., 2012). KexD has been proven to contribute to colistin resistance, and both of them are more conducive to high-level colistin resistance in K. pneumoniae under the induction of its neighbor gene crrB (Figure 2) (Cheng et al., 2018; Pantel et al., 2023). The specific diversity of the crrBAC-kexD cluster in the K. pneumoniae ST11 group suggests that ST11, a closely related lineage with carbapenem resistance and hypervirulence, is at risk of further developing colistin resistance (Kim et al., 2022). Overall, the colistin resistance mechanisms in mcr-negative colistin-resistant pathogens may be accumulated, highlighting the synergistic involvement of multiple mechanisms. Notably, the homolog of mexCD-oprJ efflux, tmexCD-toprJ, can be plasmid-mediated to acquire resistance to tigecycline in Enterobacteriaceae rapidly (Lv et al., 2020). In recent years, it has been identified that tmexCD-toprJ and mcr coexist in the same host of mobile elements, declaring the failure of last-line antibiotics (tigecycline and colistin) treatment and suggesting the threat of the rapid emergence of superbugs (Sun S. et al., 2020; Dong et al., 2022).

A study showed that several mcr-negative colistin-resistant K. pneumoniae strains isolated from CC15 and CC101 had different degrees of overexpression of acrAB, ramA, kpnEF (SMR pump), and kpnGH (MFS pump) (Naha et al., 2022). KpnEF efflux pumps belong to the small MDR family. The ΔKpnEF mutants showed increased susceptibility to various cationic antimicrobial peptides such as colistin, whereas KpnEF expression differs in the cpxAR mutant background (Figure 2) (Srinivasan and Rajamohan, 2013). The kpnGH is homologous to emrAB and belongs to the MFS efflux pump. In vitro, ΔkpnGH mutant susceptibility to cephalosporins, imipenem, polymyxin B, chlorhexidine, and other antimicrobial agents increased (Srinivasan et al., 2014).

The antimicrobial resistance surveillance from China showed that the colistin resistance rate of A. baumannii was still relatively low, but more than half of the isolates showed carbapenem-resistant A. baumannii (CRAB) (Gao et al., 2017; Zhang et al., 2020). A. baumannii had intrinsic resistance to multiple antibiotics. Hence, colistin has become one of the few options for treating CRAB. In the clinical application of colistin in anti-infection therapy, it is easy to induce A. baumannii to lead to colistin resistance by modifying phosphoethanolamine mediated by the two-component regulatory system PmrAB (Qureshi et al., 2015). In A. baumannii, three significant efflux pumps, AdeAB, AdeIJK, and AdeFGH, are involved in expelling antibiotics such as tetracycline, beta-lactam, and quinolones (Ayoub Moubareck and Hammoudi Halat, 2020). After A. baumannii was exposed to colistin, the upregulation of Ade cluster encoding genes was common; emrB and macAB overexpression were also observed (Cheah et al., 2016; Hua et al., 2017; Boinett et al., 2019). In E. coli, MacA demonstrated high affinity and specificity for the core LPS, suggesting that MacAB-TolC could be involved in LPS transport (Lu and Zgurskaya, 2013). phoP inhibited macAB transcription, and macAB deletion attenuated Salmonella’s virulence more than the tolC mutant (Nishino et al., 2006). Despite the upregulation of MDR efflux pumps, the laboratory-induced colistin-resistant isolates showed restoration of susceptibility to cefepime, azithromycin, and teicoplanin compared with the parental isolates (Moffatt et al., 2010; Li et al., 2015). Hua et al. also found that colistin induced partial restoration of antibiotic susceptibility in resistant strains but they mainly focused on β-lactams (Hua et al., 2017). Increased susceptibility to bacitracin, vancomycin, and beta-lactams was also observed in A. baumannii with high permeability of the outer membrane (Leus et al., 2018). These phenomena suggested that efflux pumps might be more involved in the transport of toxic compounds rather than in the efflux of the dominant antibiotics in colistin-resistant strains with increased outer membrane permeability due to LPS loss (Henry et al., 2012; Henry et al., 2015). Such collateral sensitivity could be masked by multiple β-lactamases and other plasmid-mediated resistance determinants in some clinical isolates of colistin-resistant A. baumannii.

Machado et al. (2018) identified several heterogeneous colistin-resistant strains that lacked TCS gene variants (lpxACD and pmrCAB). The EPIs CCCP, NMP, and PAβB were found to reverse polymyxin resistance to varying degrees in these strains. Furthermore, these strains’ efflux pump genes adeB, adeJ, adeG, craA, amvA, abeS, and abeM were overexpressed after colistin exposure (Machado et al., 2018). Colistin resistance mediated by the mutations in the pmr operon is generally expensive for A. baumannii (Geisinger and Isberg, 2017). Colistin heteroresistant A. baumannii isolates with only lpxACD mutations and overexpression of adeAB, adeG, and adeIJK were also observed in other studies, highlighting the role of efflux pumps in colistin heteroresistance (Chen et al., 2020). Besides participating in antibiotics efflux, the overexpression of these efflux pumps may be associated with fitness advantages mediated by these clinical isolates at specific sites of infection (Yoon et al., 2016). Another study also demonstrated that the resistance of A. baumannii to colistin could be reversed by NMP. AdeRS mutations mediating AdeAB or other RND-type efflux system overexpression were suggested to be the possible cause of colistin resistance in these isolates (Yilmaz et al., 2020). Additionally, increased expression of efflux transporter proteins (AdeABC and HlyD family) after colistin exposure was also observed in another study (Cheah et al., 2016). However, neither AdeRS nor AdeAB was shown to affect the colistin MIC in vitro (Richmond et al., 2016). These findings also suggested that efflux pump overexpression played a role in the excretion of toxic compounds and maintaining outer membrane integrity besides antibiotic efflux. The role of efflux pumps in antimicrobial resistance is also influenced by specific physiological settings (Leus et al., 2018). Additionally, different biological effects were found in AdeB deletion strains with different genetic backgrounds, suggesting that the function of the AdeAB efflux pump might be heterogeneous in other individuals (Richmond et al., 2016). These results indicated that the AdeAB pump could target different substrates under different living environments and selective pressures.

The two-component system and efflux pumps can be activated in bacteria by pH, nutrients, redox state, osmotic pressure, quorum signaling, and antibiotics (Worthington et al., 2013; Bazyleu and Kumar, 2014). One reported that the expression of AdeRS, AdeABC, and AdeFGH promoters did not show significant differences under sub-MIC colistin concentration and different growth conditions (Gil et al., 2021). However, the activation of the efflux pumps adeB, adeG, adeJ, adeH, and autoinducer synthase (abaI) by subinhibitory colistin has been observed in some clinical isolates (Sato et al., 2018; Shenkutie et al., 2022). Multiple biological effects on bacteria are observed at sublethal antibiotic concentrations, one of which can directly engage or interfere with quorum sensing systems (QS) (Figure 3) (Andersson and Hughes, 2014). As a signal molecule in the QS system, abaI is essential in regulating biofilms and can be used as a substrate for the AdeFGH efflux pump (He et al., 2015). A. baumannii isolates in a biofilm state has higher minimal biofilm inhibition concentrations (MBICs), which helps reduce the therapeutic effect of colistin (Kim et al., 2015). However, AdeRS did not always exist in A. baumannii isolates (Montaña et al., 2015). The restoration of colistin susceptibility by EPIs was observed in some colistin-resistant isolates without adeRS, suggesting the existence of other regulatory pathways and efflux pumps involved in colistin resistance (Yilmaz et al., 2020). Lin et al. observed that the transcription of emrB and several emrB- like genes were upregulated in colistin resistance-induced A. baumannii. Furthermore, ΔemrB mutants had increased susceptibility to colistin, demonstrating the contribution of the EmrAB pump to colistin resistance in A. baumannii (Lin et al., 2017). EmrAB was previously described mainly in E. coli and could mediate increased resistance to nalidixic acid, thiolactamycin, nitroquinoline, and hydrophobic proton uncouplers, with relatively little information available in A. baumannii (Yousefian et al., 2021). In another study, significant transcriptional changes were observed in the MATE (ydhE), MFS (mdfA), and SMR (ynfA and sugE) efflux pumps of colistin-resistant A. baumannii upon exposure to subinhibitory colistin concentrations (Paul et al., 2020). The overexpression of the MATE family efflux pump and mdfA contributes to the efflux of cationic compounds, but their contribution to colistin resistance remains confirmed (He et al., 2011; Li et al., 2015). A putative solvent/toluene-tolerant efflux ABC transporter protein, Ttg2C, may be essential in high-level colistin resistance in A. baumannii (Thi Khanh Nhu et al., 2016).

Pseudomonas aeruginosa is a common pathogen causing burn infection and cystic fibrosis, exhibiting intrinsic resistance to different antibiotics. MexAB-OprM, MexXY-OprM, and MexCD-OprJ are widely described RND family efflux pumps in P. aeruginosa with various efflux substrates: β-lactams, aminoglycosides, quinolones, tetracyclines, tigecycline, macrolides, amphenicols, novobiocin, sulfonamides, and trimethoprim (Li et al., 2015). MexXY is an induced efflux system, often characterized by concentration-dependent induction by ribosomal inhibitors (such as chloramphenicol, tetracyclines, macrolides, and aminoglycosides) (Jeannot et al., 2005). However, the overexpression of MexXY under exposure to ribosome-targeting antimicrobial agents was inversely correlated with colistin susceptibility and accompanied by the downregulation of the arn operon (Poole et al., 2015). Meanwhile, the heterogeneity of MexXY expression was observed in clinically isolated colistin-resistant P. aeruginosa isolates with different resistance levels, suggesting the possibility of intervention by other mechanisms (Goli et al., 2016). In an experiment on chlorhexidine (cationic polypeptide compounds) induced resistance, several mutants with decreased colistin susceptibility were observed, and these mutants exhibited MexY overexpression. While adding chlorpromazine significantly reduced the chlorhexidine MICs in the resistant mutants (Tag ElDein et al., 2021). Under colistin selective pressure, the two-component regulator ParR-ParS activated the arnBCADTEF operon, promoted mexY overexpression and inhibited oprD to reduce susceptibility to colistin (Fernández et al., 2010; Muller et al., 2011). mexXY deletion in ParRS-dependent pathways increased colistin tolerance by upregulating arnA and pmrA expression. The simultaneous overexpression of the arn opern and mexXY induced by the dual activation of PmrAB and ParRS contributed to the high-level resistance of the pmrB mutants to colistin, suggesting that the synergistic effect of efflux pumps and LPS modification promoted the development of colistin resistance (Puja et al., 2020) (Figure 3). In another study on the development of cross-resistance to colistin by exposing P. aeruginosa to chlorhexidine, proteomics revealed that the upregulation of MexA expression might be related to colistin resistance (Hashemi et al., 2019). Zhang W. et al. (2021) also observed significant upregulation of mexAB-oprM in laboratory-induced colistin-resistant P. aeruginosa. These results were similar to the previous findings of Pamp et al., who found that MexAB was involved in colistin tolerance, especially in adapting different subpopulations of bacteria to colistin in P. aeruginosa biofilms (Pamp et al., 2008). The efflux pump activator MerR induced mexAB-oprM and mexEF-oprN to participate in biofilm tolerance and acted as a repressor of phoPQ to participate in colistin resistance (Chambers and Sauer, 2013). In P. aeruginosa, the efflux pump is involved in the transport of signal molecules, and its expression can also be affected by the QS system. The genes related to QS were upregulated when P. aeruginosa was exposed to subinhibitory colistin concentration (Figure 3) (Cummins et al., 2009). However, whether this pathway can promote colistin resistance by activating efflux pumps or biofilms remains to be confirmed.

rsmA is a post-transcriptional regulatory protein involved in regulating various virulence-related genes, and its deletion causes overexpression of the MexEF-OprN pump and downregulation of type III secretion (Burrowes et al., 2006). However, the disruption of type III via secretion rsmA is associated with the overexpression of MexCD-OprJ or MexEF-OprN (Linares et al., 2005; Mlynarcik and Kolar, 2019). On exposure to different membrane-targeted drugs, these strains can mobilize various genetic determinants, such as pmr operons and efflux pumps, in response to environmental stresses (Chiang et al., 2012). The MexAB-oprM, MexCD-oprJ, and MuxABC-opmB efflux pumps all contribute to colistin-tolerant subpopulations (Chiang et al., 2012).

Stenotrophomonas maltophilia is widespread in the natural environment and exhibits natural resistance to numerous antibiotics, significantly limiting clinical use options. Despite colistin’s in vitro antimicrobial activity, the assessment of colistin susceptibility in S. maltophilia is influenced by various factors in vitro. (Martínez-Servat et al., 2018). Lin et al. found that MacABCsm had a broader substrate spectrum on macrolides, aminoglycosides, and polymyxin than its counterpart in E. coli, and the deletion of MacAB resulted in a significant decrease in the colistin MIC of S. maltophilia isolates (Lin et al., 2014). Meanwhile, MacABCsm was stably expressed in S. maltophilia, associated with intrinsic resistance (Lin et al., 2014). Although multiple genes encoding efflux pumps have been identified in colistin-resistant S. maltophilia, their specific contribution to colistin resistance remains elucidated (Li et al., 2019).

Aeromonas hydrophila is a common group of opportunistic pathogens in the Aeromonas genus associated with aquatic environments. The AheABC efflux pump regulates the efflux of cefoperazone, cefuroxime, erythromycin, pristinamycin, and trityltin in A. hydrophila and participates in its MDR phenotype (Hernould et al., 2008). A recent study showed that the expression levels of three putative RND efflux pump genes, AHA0021, AHA1320, and AheB, significantly increased in MDR A. hydrophila. Also, PAβN significantly reduced the MIC of piperacillin/tazobactam, imipenem, erythromycin, and polymyxin B, suggesting the contribution of the RND efflux pump to colistin resistance (Lo et al., 2022).

Proteus and S. marcescens are also common but easily overlooked opportunistic pathogens in clinical infections, often showing inherent resistance to colistin. LPS modification mediated by L-Ara4N in lipid A, Kdo residues, and arnBCADTEF operon contributes to the intrinsic resistance of P. mirabilis and S. marcescens to colistin (Olaitan et al., 2014). A recent study showed that the ABC transporter MacAB contributed to the intrinsic colistin resistance of S. marcescens, which was previously thought to be associated with the efflux of macrolide antibiotics and could be constitutively expressed with tolC (Shirshikova et al., 2021). In Salmonella, phoP inhibited macAB transcription, and macAB deletion attenuated the virulence of Salmonella more than the tolC mutant (Nishino et al., 2006). LPS played a role in colistin resistance and was a critical virulence factor in pathogenesis. However, whether macAB in S. marcescens is associated with PhoPQ or other TCSs remains to be investigated. The MFS efflux pump family SmvA was overexpressed in K. pneumoniae with increased resistance to multiple cationic biocides (chlorhexidine and octenidine) (Wand et al., 2019). Another study on P. mirabilis demonstrated that SmvA expression was insufficient to explain the differences in polymyxin B MIC of these intrinsically resistant isolates (Pelling et al., 2019).

Among Neisseria spp., N. meningitidis and N. gonorrhoeae are closely associated with clinical infections and causative agents of bacterial meningitis and gonorrhea, respectively. The MtrCDE efflux system in Neisseria is thought to be the leading cause of the low-level intrinsic resistance to colistin (Moffatt et al., 2019). MtrCDE is Neisseria’s most widely explored RND pump, contributing to resistance to β-lactams, macrolides, rifampicin, detergents, bile salts, and cationic polypeptides. The contribution of MtrCDE to colistin resistance has been demonstrated in vitro (Tzeng et al., 2005). The epistatic effects between mtrD and mtr promoter region promote the formation of the multidrug resistance phenotype (Wadsworth et al., 2018).

Yersinia comprises dozens of species, and only Y. pestis, Y. pseudotuberculosis, and Y. enterocolitica are closely related to clinical infection. Significantly different from other bacteria, the resistance of Yersinia to cationic peptides (including colistin) is affected by temperature as well as individual differences, which may be related to the successful adaptation of these pathogens at different sites (Bengoechea et al., 1996, 1998). Bengoechea et al. found that the rosAB locus encoded a temperature-regulated efflux pump and could participate in colistin efflux in response to antibiotic pressure (Bengoechea and Skurnik, 2000).

Like S. maltophilia, Burkholderia is widespread in the environment and exhibits inherent resistance to various antibiotics, including colistin. The primary mechanism of intrinsic resistance of Burkholderia to colistin is still Ara4N synthesis and Ara4N transfer to lipid A (Loutet and Valvano, 2011). A previously suggested norM belonging to MATE transporters contributes to colistin resistance, but it is mainly associated with the presence of tetracycline (Fehlner-Gardiner and Valvano, 2002). Another class of yej operons (yejA1, yejA2, yejB, yejE, and yejF) belonging to ABC transporters was also found to be directly activated by colistin and conferred colistin resistance. To further clarify the contribution of the efflux pump to colistin resistance, Zoaiter et al. (2023) found that CCCP restored susceptibility in Burkholderia isolates, while VRP, PAβN, and RSP did not. The genomic analysis showed that the efflux pump genes YejABEF, LolCDE, and NorM were widely present in these isolates (Zoaiter et al., 2023). This result suggested that multiple efflux pumps might be involved in the intrinsic colistin resistance of Burkholderia. Another study explored the interaction of the Amrab-OprA, BpeEF-OprC, and BpeAB-OprB efflux pumps with the outer membrane permeability and showed that only the simultaneous presence of Amrab-OprA inactivation and hyperporination contributed to the increased susceptibility to colistin (Krishnamoorthy et al., 2019).