Heavy Metal Toxicity in Armed Conflicts Potentiates AMR in A. baumannii by Selecting for Antibiotic and Heavy Metal Co-resistance Mechanisms

Abstract

Acinetobacter baumannii has become increasingly resistant to leading antimicrobial agents since the 1970s. Increased resistance appears linked to armed conflicts, notably since widespread media stories amplified clinical reports in the wake of the American invasion of Iraq in 2003. Antimicrobial resistance is usually assumed to arise through selection pressure exerted by antimicrobial treatment, particularly where treatment is inadequate, as in the case of low dosing, substandard antimicrobial agents, or shortened treatment course. Recently attention has focused on an emerging pathogen, multi-drug resistant A. baumannii (MDRAb). MDRAb gained media attention after being identified in American soldiers returning from Iraq and treated in US military facilities, where it was termed "Iraqibacter." However, MDRAb is strongly associated in the literature with war injuries that are heavily contaminated by both environmental debris and shrapnel from weapons. Both may harbor substantial amounts of toxic heavy metals. Interestingly, heavy metals are known to also select for antimicrobial resistance. In this review we highlight the potential causes of antimicrobial resistance by heavy metals, with a focus on its emergence in A. baumanni in war zones.

Keywords: Acinetobacter baumannii; antimicrobial resistance; bacteria; conflict; heavy metal tolerance; heavy metals; weapons.

FIGURE 1

Resistance to Copper via the Cue and Cus systems. The Cue system is activated at low Cu concentrations and under aerobic conditions. Cu¹⁺/Cu²⁺ enter the bacterial cells via non-specific porin proteins. CueR senses an increase in intracellular Cu concentrations and activates the expression of copA and cueO. Then, CopA translocate Cu¹⁺ ions into the periplasm thus, protecting Cu-sensitive cytoplasmic compartments. In the periplasm, CueO oxidizes Cu¹⁺ ions to the less toxic form Cu²⁺. The Cus efflux system is activated at high Cu concentrations, is strictly anaerobic, and pumps out Cu and Ag ions. Cu¹⁺/Ag¹⁺ ions enter the periplasm and induce the activation of CusS, which in turn phosphorylates and activates CusR. CusR induces the expression of cusCFBA operon. The protein products CusC, CusB, and CusA form a multi-Cu/Ag efflux pump (CusCBA) that pumps out Cu¹⁺/Ag¹⁺ ions after being transferred by the CusF metallochaperone (Franke et al., 2003; Delmar et al., 2015; Pal et al., 2017). Adapted and modified with permission from Pal et al. (2017).

FIGURE 2

Resistance to Copper via the Pco system. The Pco system consists of 2 operons, pcoGFE and pcoABCDRS in addition to a single gene pcoE. This system cannot function independently; it requires the activity of the Cue system and CopA in specific to induce resistance to Cu, which is heavily present in bullets, missiles, gun barrels, and in military vehicles. First, Cu¹⁺/Cu²⁺ enter the bacterial cell via non-specific porin proteins. PcoD transports Cu¹⁺ into the cytoplasm. Cu¹⁺ is toxic in the cytoplasm; Apo-PcoA transports Cu¹⁺ back to the periplasm and PcoR/PcoS senses an increase in Cu concentrations and in turn induces the expression of pcoGFE and pcoABCDRS. In addition to Apo-PcoA, the periplasmic Cu-chaperone PcoC transports Cu¹⁺ to the periplasm, where CopA from the Cue system and PcoA oxidize Cu¹⁺ to the less toxic form Cu²⁺. Cu²⁺ ions are expelled out via the PcoB efflux pump. Finally, PcoE is a metallochaperone, which is believed to provide initial bacterial resistance to Cu upon its entry through sequestering Cu¹⁺ ions until the activation of the Pco system is fulfilled (Bondarczuk and Piotrowska-Seget, 2013; Pal et al., 2017). Adapted and modified with permission from Pal et al. (2017).

FIGURE 3

Bacterial resistance to mercury. The mer operon consists of a cluster up to 8 genes merTPCAGBDE. Upon the entry of the inorganic form of Hg (Hg²⁺) via non-specific porin proteins, the first protein to bind it is MerP, a small periplasmic chaperone. MerP transports Hg²⁺ to MerT, MerC or MerF, which are inner membranous mercuric ions-binding proteins that in turn transport Hg²⁺ to the cytoplasm. merT is the most prevalent gene within the mer operon as compared to merC and merF. In the cytoplasm, MerA detoxifies Hg²⁺ ions through reduction-catalyzed volatilization process to a non-toxic elemental form Hg⁰. This form is volatile at room temperature; it diffuses outside the membranes allowing the bacterial cell to escape Hg toxicity (Nies, 1999; Silver and Phung le, 2005; Boyd and Barkay, 2012; Hobman and Crossman, 2014). MerE is an inner membranous protein of unknown function (Silver and Phung le, 2005). In Gram-negative bacteria, the mer operon is regulated by MerR, which is in turn activated by increased Hg²⁺ levels in the cytoplasm. This induces the expression of the whole merTPCAGBDE operon (Boyd and Barkay, 2012; Hobman and Crossman, 2014). Resistance to the organic form of Hg (CH₃-Hg⁺) is achieved by merB, which encodes an Organomercurcial Lyase (MerB) located in the cytoplasm. When CH₃-Hg⁺ enter the cytoplasm via non-specific porin proteins, MerB cleaves the Mercury-Carbon bond and releases Hg²⁺ in the cytoplasm. At this point, Hg²⁺ is reduced to Hg⁰ that diffuse outside the bacterial cell (Nies, 1999; Boyd and Barkay, 2012; Hobman and Crossman, 2014; Pal et al., 2017). Adapted and modified with permission from Silver and Phung le (2005), Boyd and Barkay (2012) and Pal et al. (2017).

FIGURE 4

Bacterial resistance to arsenic. (As) enter the bacterial cell using Phosphate-Specific Transporters (Pst) and Type III Transporters (PiT) in the case of As⁵⁺ and Aquaglycerolporins in the case of As³⁺ (Paez-Espino et al., 2009). The ars operon harbors 3 co-transcribed core genes that confer resistance not only to As³⁺ and As⁵⁺, but also to Antimony (Sb³⁺). arsR encodes a Transcriptional Repressor, arsC encodes a Cytoplasmic Arsenate Reductase, and arsB encodes a membrane bound Arsenite Efflux Pump. Two additional genes may be present within the ars operon, arsA and arsD. The former encodes an intracellular ATPase which binds ArsB to form an ArsA-ArsB ATPase Efflux Pump, while the latter is a metallochaperone that binds and delivers As³⁺ and Sb³⁺ to ArsA-ArsB complex for efflux, in addition to its role as a trans-activating co-repressor of the ars operon along with ArsR (Silver and Phung le, 2005; Paez-Espino et al., 2009; Hobman and Crossman, 2014). Moreover, some microorganisms escape As toxicity by methylation thus, leading to the production of less toxic and volatile derivatives that diffuse outside the bacterial cell (Paez-Espino et al., 2009). Besides As toxicity, bacteria belonging to the Shewanella spp., Sulfurospirillum spp., Clostridium spp., and Bacillus spp., use As⁵⁺ as a final electron acceptor during anaerobic respiration by reducing it to As³⁺, while other bacteria use As³⁺ as an electron donor and oxidize it to As⁵⁺ during aerobic oxidation (Paez-Espino et al., 2009). The oxidation/reduction processes are mediated by the Respiratory Arsenate Reductase and Respiratory Arsenite Oxidase that are encoded by the arrAB operon and asoAB genes respectively (Silver and Phung le, 2005; Paez-Espino et al., 2009). Adapted and modified with permission from Paez-Espino et al. (2009).

FIGURE 5

Bacterial resistance to chromium. Five Cr resistance mechanisms are reported (Camargo et al., 2005; Joutey et al., 2015). (1) Reduction of Cr⁶⁺ uptake. Cr⁶⁺ exists in the form of Oxyanions Chromate (CrO₄^2–) and Dichromate (Cr₂O₇^2–). Bacterial cells can reduce Cr⁶⁺ uptake via the sulfate transport system (Nies, 1999; Ahemad, 2014; Joutey et al., 2015). (2) Cr⁶⁺ efflux. Studies reveal that P. aeruginosa and Alcaligenes eutrophus can extrude Cr⁶⁺ by active efflux through ChrA (Chromate) pump (Collard et al., 1994; Alvarez et al., 1999). In 2008, a plasmid encoded operon (chrBACF) was identified in Ochrobactrum tritici responsible for Cr efflux, where chrB and chrA are the main genes involved (Branco et al., 2008). (3) Activation of oxidative stress related enzymes. When Cr⁶⁺ enter the bacterial cell, it interacts with reducing agents such as Nicotinamide Adenine Dinucleotide Phosphate (NADPH) and Ascorbic Acid to produce free radicals and unstable Cr intermediates (Cr⁴⁺ and Cr⁵⁺) that are further reduced to Cr³⁺. End products of these reactions cause oxidative stress leading to protein and DNA damage. This induces the up-regulation of antioxidants enzymes that scavenge ROS and protect cellular compartments (Ahemad, 2014; Joutey et al., 2015; Pradhan et al., 2016). (4) Repairing DNA damage induced by Cr⁶⁺ and its derivatives. This is achieved via SOS response activation. Several studies highlight the roles of RuvAB, RecA, and RecG (helicases) in mediating Cr resistance through repairing Cr⁶⁺ induced DNA damage (Miranda et al., 2005; Morais et al., 2011). (5) Cr⁶⁺ reduction. Cr⁶⁺ can be reduced aerobically or anaerobically to a less toxic form Cr³⁺. Aerobic reduction uses cytoplasmic soluble reductases and NADPH, while anaerobic reduction uses membrane reductases belonging to the electron transport chain (cytochromes b and c, and hydrogenases) (Morais et al., 2011; Ahemad, 2014; Joutey et al., 2015). Adapted and modified with permission from Ahemad (2014).

FIGURE 6

Bacterial resistance to lead. Bacterial species such as Pseudomonas spp., and Acinetobacter spp., developed Pb resistance mechanisms. (1) Adsorption of Pb on EPS and bacterial cell wall. Structures like cell wall and extracellular polymers can adsorb Pb²⁺ due to the presence of negatively charged functional groups [Carboxyl (C(= O)OH), Hydroxyl (R-OH), and Phosphate groups (PO^3–₄)] (Jaroslawiecka and Piotrowska-Seget, 2014). (2) Reducing Pb accumulation via intracellular and extracellular precipitation. S. aureus, Providencia spp., and Pseudomonas spp., can precipitate Pb intracellularly in the form of Lead(II) phosphate [Pb₃(PO₄)₂], while in Citrobacter freundii, extracellular Pb precipitation is mediated by phosphatase. In addition to intracellular and extracellular precipitation, periplasmic precipitation of Pb involves adsorption to polymers present in the cell wall (al-Aoukaty et al., 1991; Levinson et al., 1996; Jaroslawiecka and Piotrowska-Seget, 2014). (3) Pb sequestration via intracellular proteins. Pb binding-MTs were reported in Pb resistant P. aeruginosa strain WI-1 and Providencia vermicola strain SJ2A. This is mediated by a plasmid-borne MT encoding gene, bmtA responsible for Pb sequestration (Naik et al., 2012; Sharma et al., 2017). (4) Pb detoxification via methylation. Methylation of Pb is documented in Acinetobacter spp., Pseudomonas spp., Aeromonas spp., and others. Arctic marine bacteria convert inorganic Pb to tri-methyl-lead (C₃H₉Pb), while Acinetobacter spp., convert it to tetra-methyl derivatives (Wong et al., 1975; Jaroslawiecka and Piotrowska-Seget, 2014). (5) Pb extrusion via efflux pumps. Pb efflux is mediated by P-type ATPases such as, CadA of S. aureus, ZntA of E. coli, and PbrA of Cupriavidus metallidurans and to a lower extent by RND/CBA chemiosmotic transporters. CadA, ZntA, and PbrA are homologous P-type ATPases that can pump out Pb²⁺, Zn²⁺, and Cd²⁺ (Leedjärv et al., 2007; Jaroslawiecka and Piotrowska-Seget, 2014). Adapted and modified with permission from Jaroslawiecka and Piotrowska-Seget (2014).