Rescuing Tetracycline Class Antibiotics for the Treatment of Multidrug-Resistant Acinetobacter baumannii Pulmonary Infection

Infections caused by carbapenem-resistant Acinetobacter baumannii are disseminated globally and are associated typically with high rates of morbidity and mortality (1). Habitually occurring in patients with significant health care system contact, A. baumannii is a major causative agent of bloodstream infections (BSIs), acute bacterial skin and skin structure infections (ABSSI), urinary tract infections, and ventilator-associated pneumonia (VAP). Within intensive care unit (ICU) settings, A. baumannii is one of the most common causative agents of VAP and is associated frequently with poor patient outcomes (2, 3). During the ongoing COVID-19 pandemic, challenges faced by health care professionals have been further exacerbated by multidrug-resistant (MDR) and extensively drug resistant (XDR) A. baumannii outbreaks (4–7). Recent reports suggest that up to 80% of ICU-admitted COVID-19 patients required invasive mechanical ventilation (8). Among these patients, MDR A. baumannii has been identified as a frequent cause of secondary bacterial infection, associated with a 2-fold increase in COVID-19-related mortality (9).

The persistence of A. baumannii in nosocomial settings has been attributed largely to the pathogen’s intrinsic ability to survive desiccation and exposure to disinfectants (10, 11). Moreover, A. baumannii demonstrates a high capability to develop resistance to multiple classes of antibiotics. In 2019, European resistance rates for invasive isolates resistant to three antimicrobial groups (fluoroquinolones, aminoglycosides, and carbapenems) exceeded 43% (12). In the United States during 2017, over 60% of isolates were resistant to all fluoroquinolones and extended-spectrum β-lactam class antibiotics, ampicillin-sulbactam combinations, and trimethoprim-sulfamethoxazole combinations (13). Treatment regimens encompassing broad-spectrum cephalosporins, β-lactam-β-lactamase inhibitor combinations, carbapenems, and tetracycline class antibiotics are common approaches used to treat antibiotic-susceptible A. baumannii infections (14–16). In the increasingly frequent scenario of resistance to the aforementioned agents, combination therapy incorporating tigecycline with the highly nephrotoxic antibiotic colistin remains a last-resort treatment option, to which resistance is now being reported (12, 17). The inability to provide patients with an adequate treatment strategy reduces the efficacy of patient care (13, 18).

We have reported previously that dysregulation of bacterial metal ion homeostasis breaks antibiotic resistance in select bacterial pathogens (19–21). Here, using the 8-hydroxyquinoline analog PBT2 [2-(dimethylamino) methyl-5,7-dichloro-8-hydroxyquinoline] (22, 23), we demonstrate that the disruption of metal homeostasis breaks resistance to tetracycline class antibiotics in carbapenem-resistant, MDR, and XDR A. baumannii clinical isolates in vivo, using murine wound and pulmonary infection models. PBT2 is an orally bioavailable hydroxyquinoline ionophore which mediates the transfer of zinc across biological membranes. Phase 2 clinical trials on PBT2 have been completed for neurodegenerative conditions in which once-daily oral dosing of 250 mg was generally safe and well tolerated when administered for up to 12 months (EURO, REACH, and IMAGINE clinical trials) (23–25). This study demonstrates the rescue of tetracycline class antibiotics, including tigecycline, which are commonly used to treat carbapenem-resistant A. baumannii infections.

Monotherapy or combination therapy with next-generation tetracycline class antibiotics (e.g., tigecycline) is often employed as a last-resort measure to treat MDR and XDR A. baumannii infections. Unfortunately, resistance to these antibiotics is now being reported, which significantly reduces patient treatment options (26, 27). To address this challenge, in accordance with the Clinical and Laboratory Standards Institute (CLSI) and the European Committee on Antimicrobial Susceptibility Testing (EUCAST) guidelines (28, 29), the combination of PBT2 ± zinc to disrupt resistance to tetracycline class antibiotics in the clinical MDR A. baumannii strains MS14413 (30), AB5075 (31), and PGC-204089 (clinical isolate from the Royal Brisbane Women’s Hospital) and the XDR A. baumannii strain AB0057 (32) was investigated. All A. baumannii strains demonstrated resistance to either tetracycline, doxycycline, or tigecycline (Table 1). Upon addition of PBT2, susceptibility to tigecycline was observed for MS14413, AB0057, and PGC-204089; susceptibility to doxycycline was observed for MS14413 and PGC-204089; and susceptibility to tetracycline was observed for AB5075. Moreover, the addition of both PBT2 and zinc potentiated susceptibility, according to EUCAST breakpoints, to tetracycline, doxycycline, and tigecycline for MS14413, AB0057, PGC-204089, and AB5075 (Table 1).

As shown by the treatment of A. baumannii strains MS14413 and AB0057, the combination PBT2 + zinc in the presence of either tetracycline, doxycycline, or tigecycline was observed to be bactericidal, indicated by a >3-log reduction in viable bacteria over 24 h (Fig. 1A). Furthermore, PBT2 alone and PBT2 + zinc visibly altered bacterial cell morphology, increasing the membrane permeability properties of both A. baumannii MS14413 and AB0057, respectively (Fig. 1B and C; see Fig. S1 and S2 in the supplemental material). As evidenced through visible membrane indentations and bacterial cell rupture, these effects were further augmented by the presence of either tetracycline, doxycycline, or tigecycline (Fig. 1B; Fig. S1).

We next investigated the likelihood of resistance development following treatment with tetracycline class antibiotics and PBT2 + zinc using the MDR A. baumannii strain MS14413. During serial passage for a period of 30 days in the presence of PBT2 + zinc and tetracycline class antibiotics, A. baumannii MS14413 demonstrated an appreciable increase in the MIC to tetracycline, doxycycline, and tigecycline, ranging from 31- to 127-fold (Fig. 2A). A 15-fold increase in the MIC of the control antibiotic rifampicin was also observed over the same time period (see Fig. S3 in the supplemental material). Compared with PBT2 in the presence of tetracycline or doxycycline, the reduced level of resistance for PBT2 in the presence tigecycline is possibly attributed to the presence of the N,N,-dimethylglycylamido side chain of tigecycline which has been associated with a decreased susceptibility to tigecycline resistance development (33). Notably, resistance to tetracycline, doxycycline, or tigecycline in the presence of PBT2 + zinc imposed a fitness cost, as evidenced by reduced growth in cation-adjusted Mueller-Hinton broth (CA-MHB) and lower survival in the lungs of mice compared with wild-type A. baumannii MS14413 (Fig. 2B; see Fig. S4 in the supplemental material). Whole-genome sequencing (WGS) of mutants resistant to tetracycline class antibiotics in the presence of PBT2 + zinc identified the presence of multiple chromosomal differences compared with the MS14413 reference genome (see Table S1 in the supplemental material). Notably, for mutants resistant to tetracycline in the presence of PBT2 + zinc, WGS identified the presence of insertion sequence (IS) ISAba125 in the TetR-family system regulator gene adeN which regulates the expression of the resistance-nodulation-cell division (RND) pathway AdeIJK (34, 35) (see Fig. S5 in the supplemental material; Table S1). Although the expression of AdeIJK contributes to the intrinsic resistance of A. baumannii to tetracycline class antibiotics, high levels of expression have been associated with toxicity to the bacterial cell (36). For mutants resistant to doxycycline in the presence of PBT2 + zinc, WGS identified appreciable stepwise mutations beginning in biofilm-associated cell-surface protein A (bapA) (37) followed by insertion of ISAba1 in adeS (encoding the AdeS sensor kinase of the two-component AdeRS regulatory system which regulates the RND AdeABC efflux pump system), proceeded by subsequent insertion of ISAba125 in adeN (Fig. S5; Table S1). Mutations in the two-component regulator AdeRS have been shown previously to result in an overexpression of the RND efflux system AdeABC, leading to moderate increases in resistance to tetracycline class antibiotics (38, 39). Observed changes in bapA are likely attributable to variation in the repetitive region. Although the impact of these changes has not been determined fully, they could potentially contribute to elevated MICs. For mutants resistant to tigecycline in the presence of PBT2 + zinc, WGS identified a stepwise insertion of ISAba125 in adeN followed by an insertion of ISAba1 in adeS (Fig. S5; Table S1). These mutations, combined with the previously noted chromosomal differences, may help explain the temporal stepwise development of resistance.

PBT2 facilitates the direct permeation of zinc ions across biological membranes, independent of membrane protein-dependent transport pathways (19). PBT2 treatment of A. baumannii MS14413 increased the cellular content of zinc and copper and reduced magnesium. Notably, iron was not affected significantly (Fig. 3A). The effect of PBT2 on cellular copper was amplified by the presence of exogenous zinc, indicating broad metal ion dyshomeostatic impacts. Treatment of MS14413 with tetracycline class antibiotics alone had no direct effect on cellular metal content (Fig. 3A). To gain insight into the potential mechanism of action of PBT2 and zinc treatment, a transcriptome analysis of A. baumannii MS14413 was undertaken upon treatment with subinhibitory concentrations of PBT2, zinc, and tetracycline. Treatment with PBT2 was associated with changes in iron-specific metal stress response systems and drug efflux systems (Fig. 3B; see Table S2 in the supplemental material), which was confirmed by quantitative real-time PCR (Fig. 3C). Specifically, PBT2 led to the upregulation of iron uptake systems, including Feo system genes (feoA and feoB), ferric acinetobactin ABC transporter system genes (bauC, bauD, and bauE), and TonB receptor genes (fpvA and pfeA). Additionally, PBT2 exposure resulted in the upregulation of multidrug efflux resistance nodulation division transport system genes (adeABC and adeFGH) and downregulation of the small multidrug resistance (SMR) family drug efflux system gene abeS. No PBT2-dependent changes in the primary zinc, copper, or magnesium transport systems of A. baumannii were observed (40, 41) (Table S2). Nevertheless, the putative zinc exporting cation diffusion facilitator (CDF) gene czcE was upregulated suggesting zinc intoxication may be sensed by the bacterium (Table S2). These transcriptional changes were PBT2 dependent and were not affected by the presence of zinc or tetracycline (see Fig. S6 in the supplemental material; Table S2).

A. baumannii is a common cause of nosocomial ABSSI and VAP infections. An increase in the abundance of endogenous zinc at the site of infection is an important marker of bacterial wounds and lung infections (42–45). As such, we investigated the therapeutic potential of PBT2, in the absence of exogenous zinc, to break the resistance of A. baumannii MS14413 to tetracycline class antibiotics in vivo. Using a murine model of ABSSI, treatment with PBT2, tetracycline, or tigecycline alone had no therapeutic effect. A significant therapeutic reduction in bacterial burden at the site of infection was observed only when PBT2 was used in combination with tetracycline or tigecycline (see Fig. S7 in the supplemental material). For lung infection, PBT2 treatment alone had no therapeutic effect, while tigecycline alone provided some therapeutic benefit. The combination treatment of PBT2 and tetracycline, or PBT2 and tigecycline, abrogated infection-associated weight loss in mice and resulted in significant 2.5-log and 4-log reductions in the bacterial burden in the lungs, respectively (Fig. S7; Fig. 4). Collectively, these data suggest that PBT2 may sequester zinc from the host niche, facilitating the rescue of tetracycline class antibiotics.

Carbapenem-resistant A. baumannii (CRAB) is a worldwide health concern, and CRAB resistance to last-resort therapies (i.e., polymyxin and tigecycline combination/monotherapy) has been reported (12, 17). Since 2018, only two new therapies have been approved by the U.S. Food and Drug Administration and/or the European Union European Medicines Agency for the treatment of CRAB infection, i.e., cefiderocol and eravacycline (1). Unfortunately, cefiderocol-nonsusceptible strains of CRAB emerged prior to initial U.S. FDA approval (46). Moreover, eravacycline was approved specifically for intra-abdominal infection and not serious BSI and VAP infections (1). The lack of financial drivers for the development and commercialization of new antibiotic therapies often outweighs the value to public health, and therefore, no investments are made for new therapies (47). As such, rescuing the efficacy of existing therapies for the treatment of serious bacterial infection represents a financially viable pathway, reducing time, cost, and risk associated with drug innovation.

Antibiotic potentiator molecules represent a promising avenue for rescuing the efficacy of existing antimicrobial therapies (48). In complex with zinc, ionophore compounds have been shown previously to potentiate polymyxin and amikacin activity against A. baumannii in vitro (19, 49). Here, we demonstrate that the ionophore PBT2 breaks A. baumannii resistance to tetracycline class antibiotics in vivo. Our data indicate that PBT2 broadly perturbs A. baumannii metal ion homeostasis resulting in a redistribution of metal ions within the cell with elevated zinc and copper and decreased intracellular magnesium. This generalized dysregulation of metal homeostasis increases the efficacy of tetracycline class antibiotics. While zinc accumulation can be explained by PBT2 treatment, the mechanistic basis for the increased copper levels remains speculative. This increase could also be mediated by PBT2, but the upregulation of two cntO orthologs, the TonB receptors for secreted bacterial metallophores, may suggest an alternative pathway. Cnt-type systems function analogously with broad-spectrum metallophores to compete for essential metals during infection (50). Although the biosynthetic pathway for a broad-spectrum metallophore in A. baumannii has yet to be defined, copper accumulation could be facilitated by such a pathway. Irrespective of the precise mechanistic basis, zinc and copper accumulation did not result in upregulation of the primary efflux systems czcABC and copA, respectively (40, 41). However, an increased expression of the CDF transporter czcE, which has been shown to be highly upregulated in response to zinc intoxication (41), was observed. Taken together, these data suggest that while the bacterium may sense the increased abundance of zinc, the overall levels of both metals are within the buffering capacity of the cell and do not trigger derepression of the major efflux systems. An alternative explanation may be that the elevated zinc and copper levels are disrupting the metalloregulatory systems that control expression of the efflux pathways. The decrease in cellular magnesium also failed to activate a starvation response. Bacterial sensing of magnesium levels differs from that of zinc and copper, which is normally achieved by the concerted action of a two-component regulator to sense extracellular magnesium and riboswitches to sense cytoplasmic magnesium (51, 52). Here, the availability of magnesium in the CA-MHB medium and the increased abundance of other divalent cations within the bacterium may explain the failure of A. baumannii to accurately sense magnesium depletion. Magnesium is a requisite cofactor in a wide variety of enzymes, and its depletion or displacement in both proteins and lipid scaffolds of the outer membrane by the increased abundance of zinc or copper could severely impact cellular viability (53). Magnesium is known to play a key role in maintaining the structural integrity of the lipopolysaccharide outer membrane of Gram-negative bacteria (54, 55). Through electrostatic interaction, magnesium binds to anionic phosphate groups of the inner core, enabling structural integrity of the outer leaflet. Disruptions in these important electrostatic cross-link interactions have been shown to result in lipopolysaccharide (LPS) release and subsequent bacterial membrane rupture (54, 56). Here, we hypothesize the PBT2-mediated depletion of cellular magnesium content may contribute to increased membrane permeability and associated membrane defects.

Unexpectedly, iron import [the Fe(II) import system Feo and the biosynthetic pathways for the Fe(III) siderophore acinetobactin] was upregulated in response to PBT2 treatment. It is possible that the upregulation of iron acquisition systems may be caused by PBT2-mediated movement of iron out of the cell or possibly mediated by displacement from metalloprotein sites by zinc or copper. Alternatively, the elevated iron scavenging response may be attributed to inappropriate sensing by iron-responsive regulators due to zinc or copper mismetallation. Iron is a crucial component of heme and iron-sulfur clusters, which are critical for respiratory enzymes in the electron transport chain and is an essential cofactor in a number of other cellular processes (57). Since A. baumannii is a nonfermentative bacterium that relies on respiratory electron transport, perturbation of iron homeostasis would have profound effects on cell physiology and metabolism.

Clinical trials have demonstrated that PBT2 is safe and well tolerated during use in humans (24). Here, we provide evidence that PBT2 potentiates tetracycline-class antibiotics in vivo against MDR A. baumannii in a murine model of pulmonary infection. This study highlights the potential of PBT2 to be used in combination with tetracycline class antibiotics for the treatment of severe infection caused by drug-resistant A. baumannii.

We acknowledge the Australian Microscopy and Microanalysis Research Facility at the Centre for Microscopy and Microanalysis (CMM), University of Queensland, and in particular Richard Webb, Ron Rasch, and Thomas Jones for help with SEM sample preparation and image acquisition.

This article is a direct contribution from Mark J. Walker, a Fellow of the American Academy of Microbiology, who arranged for and secured reviews by William Schafer, Emory University School of Medicine, and Kimberly Kline, Nanyang Technological University.