The impact of multiple resistance mechanisms in the success of A. baumannii as a notorious pathogen has led to confined treatment regimens for this tenacious microorganism . In the present study, the majority of isolates showed an increased rate of resistance to all the first-line antibiotics in comparison to a previous study from Tehran, Iran . Although carbapenems are the mainstay of treatment, all of our XDR isolates were resistant to carbapenems. Since carbapenem-resistant A. baumannii is listed as the “top priority” pathogen in the summit of critical resistant bacteria by WHO, its emergency for research and discovery to explore novel therapeutic options has been recently highlighted [33, 34].
Hitherto, there has been no consensus for the optimal treatment of A. baumannii-associated infections, in particular hospital-acquired pneumonia and bloodstream infections, caused by XDR isolates that are often carbapenem-resistant. Whereas treatment regimen should be made on the case-by-case basis of antimicrobial susceptibility; but considering the importance of early appropriate action, combination therapy is beneficial to target several resistance determinants. Despite dosage limitation due to the colistin nephrotoxicity, this agent is used as a backbone for salvage therapy of the carbapenem-resistant XDR-AB isolates. Colistin-based therapy in combination with sulbactam and tigecycline, in case of susceptibility, responds better than monotherapy. Fortunately, all of our isolates were susceptible to colistin. However, only 13% and 26% of the isolates were susceptible to ampicillin-sulbactam and tigecycline, respectively. The inconsistency in outcomes of colistin-based treatment have made it impotent. As an alternative, tigecycline as a drug of last resort for treatment of XDR-AB isolates confers a lower cure rate in cases with bloodstream infections due to low serum concentration, and high rates of non-susceptibility in the studies like ours [2, 33, 35, 36]. Consequently, approaching an evidence-based therapy is still controversial. Furthermore, it has been alarming that A. baumannii can develop drug resistance under the selective pressure both in vitro and through exposure with different antibiotics especially imipenem [5, 18, 37, 38]. Thus, with attention to concerns over rapidly growing number of resistant organisms, some strategies have been brought up to survey novel effective therapeutic trajectories to restore the efficacy of approved antibiotics. For instance, EPIs can hamper efflux activity and have the potential to be used in combination therapy. RND efflux systems have a major role in resistance to multiple categories of antibiotics, and has been verified via inactivation of efflux pump encoding genes [8–10]. Our results were in contrast to another study in which PAβN reduced the MIC of imipenem in 66% of imipenem-resistant A. baumannii isolates . It is suggested that higher concentration of PAβN inhibitor can bypass efflux activity against imipenem. Considering that almost all of our isolates were resistant to imipenem with MIC of ≥ 16 µg/ml, resistance determinants such as reduced permeability of the outer membrane or production of carbapenemases could be involved in the carbapenem resistance in addition to efflux systems . In our previous study, we detected OXA-type encoding genes in these isolates . PAβN also affected the gentamicin-resistant phenotype of eight isolates. Moreover, this EPI reduced the MIC of cefepime- and levofloxacin-resistant isolates, but had no impact on their susceptibility patterns. Thus, the contribution of other resistance mechanisms can be deduced . To be noted, PAβN remarkably restored tigecycline susceptibility (from 26–61%); accordingly, active multidrug efflux pumps conferred tigecycline non-susceptibility in our isolates but the type of the pump is not clarified yet . Tigecycline non-susceptibility has been associated with three RND systems as already mentioned [2, 7, 36]. According to previous studies, AdeABC is the predominant pump conferring acquired resistance to a wide range of antibiotics. It is the only RND pump that extrudes aminoglycosides [8, 13, 14, 37, 41]. Although the role of this pump in carbapenem resistance is controversial, efflux activity was associated with reduced susceptibility to carbapenems under imipenem-selected stress . AdeRS in the adjacent of AdeABC operon regulates it by transcribing in the opposite direction. Some putative mutations in AdeR are responsible for adeB overexpression: A91V and A136V in the signal receiver domain [3, 14], D20N in the phosphorylation site , and P116L at the first residue of the helix α5 . Among these, A136V polymorphism in the signal receiver of AdeR regulator was detected in two adeB overexpressed isolates (M9 and M24) in our study. In AdeS, which is more prone to mutation, numerous point mutations can boost adeB expression: G30D located in the periplasmic loop , G103D alterations in the histidine kinase, adenylyl cyclase, methyl-accepting chemotaxis protein and phosphatase (HAMP) linker domain , G186V in the α-helix of the dimerization and histidine phosphotransfer (DHp) domain , and T153M in the histidine box . In our study, two isolates (M9 and M24) with overexpression of adeB had G186V mutation, which can alter the conformation of AdeS DHp domain, and then stimulates overexpression of the AdeABC efflux pump . Four of our isolates harbored H189Y located at the C-terminal of the DHp domain of AdeS, which can affect HK autokinase activity or RR phosphorylation . Furthermore, the coexistence of A136V and G186V polymorphisms respectively in AdeR and AdeS components of two tigecycline-resistant isolates (M9 and M24) is noteworthy. M9 with a 10.70-fold increase in adeB expression level showed efflux pump activity for levofloxacin, cefepime and gentamicin antibiotics, and M24 with a 5.27-fold increase in adeB expression level showed efflux activity for tigecycline in the phenotypic assay. In line with our results, the coexistence of these two amino acid substitutions have been detected in both tigecycline-resistant and -susceptible isolates; hence, their detailed impact is a matter of controversy [3, 4, 6, 14]. The highest expression level of adeB was detected in M40 (19.69-fold) and M42 (16.44-fold) isolates; two XDR isolates without any reduction in MIC after PAβN addition. Additionally, these isolates had a few identical point mutations in each of AdeS, BaeR and BaeS regulators, and had no mutations in AdeR. They harbored none of the renowned mutations and all the detected substitutions were also found in isolates with no pump overexpression. Furthermore, disrupted adeS by ISAba1can lead to tigecycline non-susceptibility and even other antibiotics by enhancing the AdeABC overexpression [6, 11, 12, 41]. However, this insertion was not detected in our studied isolates.
Regarding the strict regulation of resistance mechanisms under external pressures, the transcriptional regulators were introduced as promising drug targets to overcome resistance. However, the results obtained by Trebosc et al. revealed that there are AdeR-unrelated mechanisms mediating tigecycline resistance, which made AdeR an insufficient target for adjuvant therapy . Tigecycline non-susceptibility can occur as a result of synergistic contribution of AdeIJK with AdeABC , while AdeABC has superior influence [18, 38]. The AdeIJK efflux pump is species-specific and contributes to intrinsic resistance to various antibiotics . It is tightly regulated by the product of adeN gene in ca. 800 kb away from the AdeIJK operon transcribing in the same direction [16, 44]. Since high-level expression of this pump is toxic for A. baumannii, AdeN represses AdeIJK and its disruption diminishes susceptibility following a tolerable expression level. A premature stop codon in the helix α9 sequence at position 211 within the dimerization domain inactivates AdeN . In another study, three types of insertions including ISAba1 leading to adeN inactivation were detected . Overall, the expression level of adeJ was very slight in our isolates confirming the theory of its lethality for the host. M20 and M24 isolates with minor increases in the expression of adeJ were tigecycline-resistant with pump activity (Table 5). Therefore, the role of other mechanisms in regulation of the AdeIJK operon cannot be ruled out.
The BaeSR is a global regulator and has been associated with tigecycline resistance by controlling AdeIJK and AdeABC pumps. It has been reported that the function of BaeSR occurs through a cross-talk with AdeRS, suggesting the overlap of these two TCS regulons [17, 19–21]. Accordingly, we assessed the BaeSR sequence for any mutation that might be effective in efflux pump expression. In two isolates (M9 and M24) with increased expression level in adeB and adeJ, we found N268H and S437T polymorphisms in AdeS and BaeS, respectively. These two polymorphisms were not found in the isolates with no increase in the expression of efflux pumps. So far as we know, this is the first investigation on BaeSR and its probable role in the resistance of A. baumannii isolates in Iran. A better understanding of the dynamic interaction between AdeRS and BaeSR in the regulation of RND efflux pumps of A. baumannii merits further investigations.
The contribution of AdeFGH to acquired resistance has been proved second to AdeABC. Its overexpression confers decreased susceptibility to several agents. The adeL regulates AdeFGH operon in the upstream of it transcribing in the opposite direction. Deletion of the 11 C-terminal residues, T319K and V139G in the signal recognition domain confer increased expression of adeG . Only Q262R amino acid substitution was found in our isolates. Isolate M42 with the highest expression level (42.51-fold) displayed Q262R substitution in AdeL; but isolate M9 with a 32.89-fold increase in adeG expression showed no alteration. Thus, involvement of other mechanisms is proposed.
The resultant findings of our study elucidated that tigecycline is a substrate for the three aforementioned RND pumps; however, some tigecycline-resistant isolates revealed no pump overexpression. Additional mechanisms contribute to the tigecycline resistance, as previously described [11, 17]. Among the studied isolates, only isolate M54 was tigecycline-susceptible with no efflux activity and no increase in efflux pump expression, but surprisingly showed a 5-fold reduction in the MIC of cefepime indicating efflux activity. Furthermore, two tigecycline-resistant isolates (M29 and M30) with efflux activity for tigecycline and no overexpression of the RND efflux pumps revealed the possibility that other efflux systems play a role in resistance to tigecycline .