The impact of multiple resistance mechanisms in the success of A. baumannii as a notorious pathogen has led to confined treatment regimen for this tenacious microorganism [1]. In the present study the majority of isolates showed increased rate of resistance to all the first-line antibiotics in comparison to a previous study from Tehran, Iran [32]. Albeit 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 highlighted in recent years [33,34].
There is hitherto no consensus for the optimal treatment of A. baumannii-associated infections, in particular importance hospital-acquired pneumonia and bloodstream infection, 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. In spite of dosage limitation due to colistin nephrotoxicity, it is used as a backbone for salvage therapy of 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 isolates were susceptible to ampicillin-sulbactam and tigecycline respectively. Inconsistent outcomes of colistin has made it impotent. As an alternative, tigecycline as the drug of last resort for treatment of XDR-AB isolates, confers lower cure rate in cases with bloodstream infections because of the low serum concentration and in the studies like ours high rates of non-susceptibility [2,33,35,36]. Consequently, approaching an evidence-based therapy is still controversial. Furthermore, it has been alarming that A. baumannii is able to develop drug resistance under selective pressure both in vitro and throughout medicament with different antibiotics especially imipenem [5,18,37,38]. Herein, with due attention to concerns and controversies over increasing resistant isolates and combating their causative infections, some strategies have been brought up to survey novel effective therapeutic trajectories to restore efficacy of approved antibiotics. For instance, EPIs can hamper efflux activity and have potential to be used in combination therapy. RND efflux systems have a major role in resistance to multiple categories of antibiotics has been verified via efflux pumps encoding genes inactivation [8-10]. Our results was in contrast to another study that PAβN reduced imipenem MIC in 66% of imipenem resistant isolates [39]. Bypassing efflux activity against imipenem can be feasible with higher concentration of PAβN. 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 carbapenem resistance in addition to efflux systems [33]. OXA-type encoding genes has been detected in these isolates in our previous study [40]. PAβN also affected the gentamicin resistant phenotype of eight isolates. Moreover, this EPI reduced MIC of cefepime and levofloxacin resistant isolates but had no impact on their susceptibility patterns. Thus, contribution of other resistance mechanisms can be deduced [33]. To be noted, PAβN remarkably restored tigecycline susceptibility (from 26% to 61%); accordingly, active multidrug efflux pumps conferred tigecycline non-susceptibility in our isolates but type of the pump is not clarified yet [4]. Tigecycline non-susceptibility have 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, but efflux activity was associated with reduced susceptibility to carbapenems under imipenem-selected stress [5]. 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 [5], and P116L at the first residue of the helix α5 [13]. Among these, A136V polymorphism in signal receiver of AdeR regulator was detected in two adeB overexpressed isolates (M9 and M24) in our study, which may affect the interaction between AdeR and AdeS. Similar to a previous study, we found H158L in effector domain of AdeR [6]. In nine isolates I120V substitution, which is another polymorphism was found in receiver domain. In AdeS, which has been showed to be more prone to mutation, numerous point mutations can boost adeB expression: G30D located in the periplasmic loop [42], G103D alterations in the histidine kinase, adenylyl cyclase, methyl-accepting chemotaxis protein and phosphatase (HAMP) linker domain [14], the G186V in the α-helix of the dimerization and histidine phosphotransfer (DHp) domain [3], and T153M in the histidine box [13]. In our study, two isolates (M9 and M24) with adeB overexpressing had six similar polymorphisms including; L172P, G186V, N268H, S280A, Q281D and Y303F. Among them, G186V can alter AdeS DHp domain conformation and then stimulates overexpression of the AdeABC efflux pump [43]. Four of our isolates harbored H189Y located at the C-terminal of DHp domain of AdeS, which can affect HK autokinase activity or RR phosphorylation [6]. In the present study, H158L substitution in AdeR accompanies H189Y in AdeS in four out of fifteen isolates (M2, M3, M4 and M31) with no significant increase in adeB expression and with tigecycline resistant phenotype, although efflux activity against cefepime and gentamicin was observed in phenotypic assay with EPI. Furthermore, 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 phenotypic assay. In previous investigations, coexistence of these two amino acid substitutions have been detected in both tigecycline resistant isolates, like our results, and tigecycline susceptible ones; hence, their detailed effect is in debate [3,4,6,14]. The highest expression level of adeB was detected in M40 (19.69-fold) and M42 (16.44-fold); 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 it is probable that some of the detected polymorphisms in AdeRS or mutations in BaeSR confer overexpression of AdeABC pump. Furthermore, disrupted adeS by ISAba1can lead to tigecycline non-susceptibility and even other antibiotics by enhancing AdeABC overexpression [6,11,12,41]. However, this insertion was not detected in our studied isolates.
Regarding strict regulation of resistance mechanisms under external pressures, another trajectory to overcome resistance and develop an optimal treatment was investigated by Trebosc et al.; transcriptional regulators as promising drug targets, in this case AdeR, can rejuvenate the efficacy of antibiotics. Nevertheless, the results revealed that there are AdeR-unrelated mechanisms mediating tigecycline resistance and made AdeR insufficient target for adjuvant therapy merely [17]. Tigecycline non-susceptibility can occur as a result of synergistic contribution of AdeIJK with AdeABC [10], while AdeABC has superior influence [18,38]. AdeIJK efflux pump is species-specific and contributes to intrinsic resistance to various antibiotics [10]. It is tightly regulated by adeN in ca. 800 kb away from the AdeIJK operon transcribing in the same direction [16,44]. Because 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 [16]. In another study, three types of insertions including ISAba1 leading to adeN inactivation were detected [11]. Overall, the expression level of adeJ was very slight in our isolates confirming its lethality for the host. M20 and M24 isolates with minor increased in expression of adeJ were tigecycline resistant with pump activity (Table 5). Therefore, role of other mechanisms in regulation of AdeIJK operon cannot be ruled out.
BaeSR is a global regulator and has been associated with tigecycline resistance by controlling AdeIJK and AdeABC pumps. It has been reported that function of BaeSR occurs through a cross-talk with AdeRS, suggesting overlapping of these two TCSs regulons [17,19-21]. Accordingly, we assessed BaeSR sequence for any mutation that might be effective in efflux pump expression. In two isolates (M9 and M2) with increased expression level in adeB and adeJ, we found S437T polymorphism in BaeS. Other amino acid substitutions were found as well. As far as we know, this is the first investigation on BaeSR and its probable role in resistance of A. baumannii isolates in Iran. Better understanding of the dynamic interaction between AdeRS and BaeSR in regulation of RND efflux pumps of A. baumannii merits further investigations.
AdeFGH contribution 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 signal recognition domain confer increased expression of adeG [9]. Only Q262R amino acid substitution was found in our isolates. M42 with the highest expression level (42.51-fold) displayed Q262R substitution in AdeL; but 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 tigecycline resistance, as previously described [11,17]. Among studied isolates, only M54 was tigecycline susceptible with no efflux activity and no increase in efflux pumps expression, but surprisingly a 5-fold reduction of cefepime MIC that displays efflux activity.