Insights into Emergence of Antibiotic Resistance in Acid-Adapted Enterohaemorrhagic Escherichia coli

The emergence of multidrug-resistant pathogens presents a global challenge for treating and preventing disease spread through zoonotic transmission. The water and foodborne Enterohaemorrhagic Escherichia coli (EHEC) are capable of causing intestinal and systemic diseases. The root cause of the emergence of these strains is their metabolic adaptation to environmental stressors, especially acidic pH. Acid treatment is desired to kill pathogens, but the protective mechanisms employed by EHECs cross-protect against antimicrobial peptides and thus facilitate opportunities for survival and pathogenesis. In this review, we have discussed the correlation between acid tolerance and antibiotic resistance, highlighting the identification of novel targets for potential production of antimicrobial therapeutics. We have also summarized the molecular mechanisms used by acid-adapted EHECs, such as the two-component response systems mediating structural modifications, competitive inhibition, and efflux activation that facilitate cross-protection against antimicrobial compounds. Moving beyond the descriptive studies, this review highlights low pH stress as an emerging player in the development of cross-protection against antimicrobial agents. We have also described potential gene targets for innovative therapeutic approaches to overcome the risk of multidrug-resistant diseases in healthcare and industry.


Introduction
Low, acid pH kills bacteria. Humans have evolved acid conditions to protect from food-borne pathogens, and pathogens have evolved ways to bypass those protections. Acid tolerance, the ability to survive acid conditions, is a clinically important phenotype of foodborne pathogens and an overwhelming issue for public health [1,2]. Most microorganisms, including pathogenic bacteria, prefer to grow at pH 6-7 [3]. Globally, the use of acid-based antimicrobial agents is widely practiced because it kills microbes at workplaces, in hospitals, on medical equipment, as food preservatives, in soil niches and during wastewater treatment [2,4]. Commercially, wastewater is treated with nitrous acid (disinfectant) while diluted acetic acid, hypochlorous acid, chlorhexidine, ethanol, acetate, hydrogen peroxide and boric acid are commonly used to treat wounds and infections in hospitals [5][6][7]. Acidified-chlorinated water (a blend of hydrochloric acid and mild organic acids) is often sprayed on meat and lettuce leaves to attain a pH of 2.5 [8,9]. This helps kill certain Enterohaemorrhagic Escherichia coli (EHEC), which causes severe poultry, bovine and human extra-intestinal diseases at a very low infectious dose (10-500 cells) [10,11].

Acid Tolerance Potential of EHECs
Extremely virulent serotypes of EHECs (O157:H7 strains) have regulatory aspects of acid tolerance that are not found in other E. coli strains [59]. Although EHECs can acquire antibiotic resistance by horizontal gene transfer, they can also develop de novo resistance during exposure to various environmental stresses, especially low pH. The mechanisms that initially allow the bacteria to survive stress subsequently result in resistance to even higher antibiotic concentrations measured by minimum inhibitory concentration (MIC) [60]. The set of genes involved in protecting against acid stress are also responsible for the acquisition Antibiotics 2021, 10, 522 4 of 27 of antibiotic resistance [51]. Thus, it is important to understand the regulation of several transcriptional regulators (GadE, H-NS) and two-component signal transduction kinases (EvgAS, PhoPQ, RcsB) that are activated in response to low pH. At the molecular level, key players involved in mediating acid tolerance include enzymatic cascades of specific decarboxylases and families of two-component signal-transduction kinases.
The signal-transduction kinases consist of a sensor kinase and a response regulator that controls acid-tolerance, pathogenicity, and antibiotic resistance [61][62][63][64]. These twocomponent kinases work in coordination with multiple global regulators, transcription factors, and several other local regulatory proteins [1,14,65,66]. This regulation further involves several regulatory proteins, chaperons, and periplasmic proteins that protect the EHECs against DNA damage and protein coagulation.
acquisition of antibiotic resistance [51]. Thus, it is important to understand the regulation of several transcriptional regulators (GadE, H-NS) and two-component signal transduction kinases (EvgAS, PhoPQ, RcsB) that are activated in response to low pH. At the molecular level, key players involved in mediating acid tolerance include enzymatic cascades of specific decarboxylases and families of two-component signal-transduction kinases.
The signal-transduction kinases consist of a sensor kinase and a response regulator that controls acid-tolerance, pathogenicity, and antibiotic resistance [61][62][63][64]. These twocomponent kinases work in coordination with multiple global regulators, transcription factors, and several other local regulatory proteins [1,14,65,66]. This regulation further involves several regulatory proteins, chaperons, and periplasmic proteins that protect the EHECs against DNA damage and protein coagulation.
Four main systems regulate acid tolerance: oxidative, glutamate-dependent, argi nine-dependent and lysine-dependent acid resistance systems. They work in tandem to protect cells from acid stress ( Figure 1; Table 1) [67][68][69][70][71][72]. These mechanisms exchange intracellular protons for an amino acid (glutamate, arginine, or lysine) and expel amines into the extracellular media in exchange for the corresponding amino acid [73]. The oxidative system does not involve an externally-derived amino acid; it is regulated by sigma factor RpoS and catabolite repressor protein (CRP). It provides the lowest level of protection at pH 2.5 at the expense of energy, as shown in Figure 1 [74]. The glutamate-dependent system is adapted to protect under extremely acidic conditions and is Figure 1. Representation of the oxidative, glutamate-dependent, arginine-dependent, and lysine-dependent acid resistance systems in Escherichia coli. All abbreviations are listed at the end of the manuscript.
The oxidative system does not involve an externally-derived amino acid; it is regulated by sigma factor RpoS and catabolite repressor protein (CRP). It provides the lowest level of protection at pH 2.5 at the expense of energy, as shown in Figure 1 [74]. The glutamatedependent system is adapted to protect under extremely acidic conditions and is highly efficient. The arginine-dependent system is only induced under anaerobic conditions. It requires arginine decarboxylase (AdiA) and an arginine:agmatne antiporter (AdiC) to provide a modest level of protection under mild acidic conditions (Table 1) [75]. The lysine-dependent system also works under slightly acidic environments; the efficiency of resistance is lower than seen with other systems [68,76]. This system involves a lysine decarboxylase (CadA) and a bifunctional lysine:cadaverine antiporter (CadB) ( Table 1). The pH inside certain compartments of the mammalian gastrointestinal tract drops below 2; which renders all acid resistance systems inactive except the glutamate-dependent acid one.  [75,79] Note: All abbreviations are defined at the end of the manuscript.

Glutamate-Dependent Acid Resistance System
The glutamate-dependent system provides the highest level of protection under extremely acidic conditions. This system involves two glutamate decarboxylases, GadA and GadB, that work in coordination with the gamma-aminobutyric acid (GABA) antiporter (GadC) and a set of these three genes known as GAD [83]. Extracellular glutamate is exchanged with intracellular GABA through the GABA antiporter GadC and subsequently decarboxylated by GAD [84]. During the decarboxylation of L-glutamate, the α-carboxylic group is released as carbon dioxide and a proton is incorporated into the GABA molecule, which is exported across the inner membrane in exchange for more glutamate through GadC [67]. In addition, the antiporter increases the availability of glutamate to the GAD enzymes, thereby, enhancing the efficiency of the system by acidifying the cytoplasm [85].
The functional side chain of glutamate imported by GadC has a pK a of 4.1. Before entering the cytoplasm, during acid stress (pH 2.5), this side chain gets more than 50% protonated and these protons dissociate to acidify the cytoplasm. Therefore, the cytoplasmic pH drops to 3.6, which is an optimal pH for glutamate decarboxylase while rendering arginine and lysine decarboxylases inactive, as their optimal pH is 5.25 and 5.5, respectively [86,87]. This defense strategy works by reversing the membrane potential to maintain more protons inside as compared to the external environment [76]. The inner membrane potential remains more positive and gradually slows the flow of protons into the cell, thereby maintaining homeostasis.

Control of Glutamate-Dependent System
The gap between an environmental stimulus and gene regulation is bridged by sensors and regulators of two-component systems. A two-component system typically consists of a sensory kinase that monitors the environmental conditions and modulates phosphorylation of the respective response regulator. The response regulator then regulates gene expression, which changes the behavior of the bacterial cell. To cope with acid stress in the gastrointestinal tract, several two-component systems play specific roles in maintaining homeostasis and cell integrity. The selection of resistance mechanism depends upon the energy source and extracellular environmental conditions. The protection conferred by the glutamate-dependent system is significantly higher than the other systems, allowing up to 80% survival. As a consequence, the glutamatedependent system is considered a key player in acid regulation. This system comprises a complex network of two-component regulators with a wide array of interactions to cope with mild to extreme acid stress. The key interacting regulators of this network are GadE, EvgAS, PhoPQ, YdeO, GadW, RcsB, and GadX, which regulate gene expression spatially and temporally ( Figure 2) [44,67,[87][88][89][90][91][92][93][94][95][96][97]. This system is activated either by mild acidic pH during the exponential growth phase or by entry into stationary phase. Twocomponent systems are regulated by the induction of mild acid shock, while the rpoS-gadX-gadY-gadW circuit is activated during stationary phase. Once triggered, this system activates a cascade of regulatory genes that then activate the central regulators GadE and YdeO ( Figure 2) [87,95,98,99]. The increased expression of central regulators results in the activation of several acid-resistance genes at different loci. This activation involves more than 20 proteins (CRP, Dps, EvgA/S, GadE, GadX, GadW, H-NS, Lon, PhoP/Q, RNaseE, sigma factor 70, sigma factor RpoS, SspA, TrmE, TopA, TorS/R and YdeO) and several non-coding RNAs (DsrA, GadY, and GcvB) ( Figure 2) [91,[99][100][101][102][103][104][105][106][107][108].
The sensor kinase EvgS detects the low pH signal and activates response regulato EvgA, which then starts a gene transcription cascade leading to activation of the ydeP safA-ydeO circuit. The activated YdeO increases the expression of GadE and other gene involved in the regulation of acid-fitness-island (AFI) genes, namely, slp-dctR-yhiD-hdeB hdeA-hdeD-yhiU-yhiV-gadW-gadY-gadX-gadA. It also activates glutamate-dependen acid-resistance genes, namely, gadA, gadB, and gadC ( Figure 2). Activation of these gene and gadE requires the heterodimerization of RcsB with GadE. Activation of GadX als stimulates the LEE (locus of enterocyte effacement) to mediate acid-induced regulation o pathogenic traits including biofilm formation, multidrug resistance, and enhanced colo nization, as shown in Figure 2.  The sensor kinase EvgS detects the low pH signal and activates response regulator EvgA, which then starts a gene transcription cascade leading to activation of the ydeP-safA-ydeO circuit. The activated YdeO increases the expression of GadE and other genes involved in the regulation of acid-fitness-island (AFI) genes, namely, slp-dctR-yhiD-hdeB-hdeA-hdeD-yhiU-yhiV-gadW-gadY-gadX-gadA. It also activates glutamate-dependent acid-resistance genes, namely, gadA, gadB, and gadC ( Figure 2). Activation of these genes and gadE requires the heterodimerization of RcsB with GadE. Activation of GadX also stimulates the LEE (locus of enterocyte effacement) to mediate acid-induced regulation of pathogenic traits including biofilm formation, multidrug resistance, and enhanced colonization, as shown in Figure 2.

EvgAS: An Acid-Resistance Regulator
EvgAS is indispensable for protecting against low pH through a range of interacting mechanisms that depend upon the conditions (stress in exponential phase or entry into stationary phase) [90]. Under acid stress, the sensory kinase EvgS phosphorylates the response regulator EvgA. The activated EvgA then phosphorylates a transcriptional regulator YdeO (the AraC/XylS super-family transcriptional regulator). This phosphorylation depends on a small membrane protein SafA (sensor-associating factor A) from the ydeO-safA operon. The phosphorylated YdeO ultimately activates GadE, which regulates various decarboxylases and provides resistance against acid stress. It also regulates several other cellular processes as shown in Figure 2 [109]. EvgA strongly binds to the promoter regions of several genes involved in the regulation of acid resistance, such as ydeP, safA, yfdX, frc, yegR, and gadE. The role of YdeP, YfdX and YegR needs to be investigated in the context of acid resistance.

PhoPQ: Role in Acid Regulation
PhoPQ is a two-component signaling system that responds to multiple environmental stimuli, including low pH, osmotic shock, low concentration of divalent cations and antimicrobial peptides (AMPs) [103,110,111]. It consists of a histidine kinase PhoQ that interacts with SafA and starts a phosphorylation cascade resulting in activation of the response-regulator PhoP. During the exponential phase, transcription factor PhoP activates IraM, which then interacts with RpoS [112]. RpoS is a central regulator of the stress that encodes sigma factor-38 and allows the cell to survive environmental challenges [113]. Due to this interaction, the level of RpoS increases and subsequently recruits RNA polymerase (RNAP) to RpoS-regulated promoters, including the gadE promoter [114,115]. Mg 2+ stress concurrently activates PhoPQ and other regulatory proteins, thereby enhancing pathogenesis by increasing pathogen survival [94].

RcsB: An Essential Activator/Repressor
RcsB is a response regulator that functions both as an activator and a repressor. It works in coordination with GadE to form a heterodimer on the GAD box that activates transcription of acid-resistance genes ( Figure 2). It is an essential element for gadA and gadB promoter activity [82][83][84][85]. All acid-resistance promoters activated by GadE are also dependent on RcsB for their activation; the regulation mechanism of RcsB is still unknown.

Cross-Protection in EHECs
Cross-protection is the defensive adaptability of a strain when exposed to certain environmental stresses, including acid stress. Cross-protection mechanisms are either non-specific for the choice of substrate (multidrug efflux pumps), share a few common regulatory sets of genes (glutamate-dependent pathway genes), or undergo structural modifications (lipopolysaccharide chain modification) [116,117]. Foodborne EHECs encounter several acidic treatments from farm to gut and gradually adapt. This exposure to acidic conditions helps them develop cross-protection against other environmental stresses, including antimicrobial agents [118]. In addition to EHECs, acid-adapted pathogens other than EHEC pathotypes' have also been reported in several major outbreaks all over the world, as shown in Table 2. This cross-protection poses serious concerns to the consumers (humans) and could lead to the emergence of new pathotypes.

Metabolic Adaptations
Once ingested, EHECs experience severe environmental challenges including extreme pH fluctuation and nitrosative stress (nitric acid) from volatile organic acids formed as a result of anaerobic fermentation in the gastrointestinal tract [30]. Mostly, EHECs favor pH 6-8 for their growth; to survive low pH stress they develop a transmembrane gradient to maintain homeostasis [2,126,127]. When grown at acidic pH, genes involved in metabolism, energy production and class I heat shock proteins are down-regulated to lower metabolic cost [128]. These strains consume intracellular protons through amino acid decarboxylation during acid stress, which highly acidifies the cytoplasm, resulting in increased acid tolerance [44,76]. During oxidative respiration, electron transport of membrane-bound systems, including the atp operon, is down-regulated to inhibit the import of protons [129]. While in the intestine, the enhanced expression of the Long Polar Fimbriae gene (lpf-2) mediates bacterial colonization in response to anaerobic nitrosative stress [126]. Genes involved in motility, type III secretion system (T3SS), bacterial chemotaxis, biofilm formation, adhesion, iron uptake and oxidative resistance are upregulated [1,127]. Cellular adhesion capacity (the intimin gene eae) of EHEC O157:H7 is enhanced by the histone-like, nucleoidassociated H-NS protein that regulates bacterial fitness and uncontrolled virulence [1,30]. In addition, the expression of fur, which is involved in iron uptake, is also up-regulated. In particular, low pH helps bacteria survive acid stress by enhancing motility, adhesion, and iron utilization, thereby assisting the pathogen in enhancing apoptosis of epithelial cells and become more virulent [2]. This mechanistic regulation helps the pathogen achieve homeostatic balance by modifying metabolic pathways at the cost of energy generated from redox or ATP-driven reactions.

Acid-Adaptive Antibiotic Resistance Strategies
Generally, growth-inhibiting stresses, such as low pH, high temperature, or nutritional deficiency, induce several metabolic rearrangements at the cellular and metabolic levels that influence differential regulation of more than 500 genes to ensure tight homeostasis. In response to extracellular acid stress, pathogens undergo several cellular and global transcriptional changes that alter their responsiveness to a wide array of antibiotics. These regulatory changes help the organism adapt to extreme environmental stresses and subsequently enable cross-protection consistent with the survival of the organism [44,60].
While passing through the gastrointestinal tract, EHECs experience anaerobic conditions and nitrosative stress that trigger enhanced expression of the multidrug efflux genes (mdtEF) and several two-component signaling systems including EvgAS, PhoPQ, RcsB, PmrAB, ArcAB, BaeSR, KdpA, and CpxAR [44,[130][131][132]. These two-component systems combat cell envelope disruption caused by proton imbalance and antibiotic-induced accumulation of mistranslated peptides that can cause cell damage by disturbing homeostasis [133]. Multidrug-resistance efflux pumps are essential for withstanding antibiotic challenges and other environmental toxins. During anaerobic conditions, the global transcription factor ArcA increases the expression of MdtEF (more than 20 fold), which dramatically enhances efflux activity leading to antibiotic resistance [130]. Incubation at low pH also aids the development of antibiotic resistance, which persists even after environmental conditions shift [16,60]. Overall, these stress-induced genetic alterations confer genetic plasticity that results in enhanced population diversity, strengthening of the envelope and resistance to a wide array of antibiotics.

Acid-Adaptive Structural Modifications
Gram-negative bacteria have a highly asymmetric outer membrane with phosphatidylethanolamine lipids at the inner side, while the external side is enriched with lipopolysaccharides [134][135][136]. The lipopolysaccharide membrane is composed of three vital parts, including a gel-like hydrophobic anchor (lipid A), branched oligosaccharides (core region), and polymer of repeating saccharide subunits (O-antigen) ( Figure 3) [135,136]. The structure of this membrane is enriched with many phosphoryl and carboxyl groups bridged with divalent cations that facilitate low permeability and antibiotic resistance [134,136].

Acid-Induced LPS Modification
PhoPQ upregulates the transcription of acid-resistance genes under acidic stress; the same set of genes also mediate LPS modifications in EHECs [103,137]. Several environmental changes, such as acidic pH, osmotic stress, low concentration of divalent cations (Mg 2+ ), and the presence of antimicrobial peptides (AMP), trigger this pathway [103,134]. EHECs have evolved this defensive strategy to remodel the outer membrane by adding a palmitoyl chain, a hydroxyl group, and a positively charged aminoarabinose sugar moiety to the lipid A anchor, acetylation of the O-antigen and hydroxylation of fatty acids through the PhoPQ two-component regulatory system ( Figure 3) [111,137]. These induced modifications help EHECs become more virulent by increasing cationic antimicrobial peptide resistance and suppressing TLR4 immune responses. They also increase permeability to large lipophilic agents [138]. EHEC serotype O157:H7 is reported to develop increased resistance to cationic antimicrobial peptides, especially Polymyxin B, in response to acid stress, bile salts, and ferric ions in the human gut (Table 3) [103,137].

Acid-Induced Antimicrobial Resistance by RcsB
The bacterial cell envelope comprises outer and inner membranes that act as a protective barrier. The outer membrane is an asymmetric bilayer of phospholipid and lipopolysaccharides (LPS). There is a thin peptidoglycan layer lying in the periplasmic space between the outer and inner membrane of the cell. EHECs are at high risk of losing cell envelope integrity and improper protein folding under extremely acidic conditions due to excessive osmotic pressure [139][140][141]. Penicillin-binding proteins (PBPs) keep adding new subunits to the outer membrane during cell growth and repair. Genetic profiling of EHECs confirmed that pH stress inhibits penicillin-binding proteins, which in turn activates the rcs phosphorelay to retain envelope integrity and develop resistance to amdinocillin (mecillinam) and cefsulodin (a member of the beta-lactam group of antibiotics) [141]. When acid stress is encountered, the expression of ugd is increased and incorporates a 4-amino acid modification to the lipid A anchor (Figure 3) [142]. The expression of PagP is also increased, which regulates lipid A palmitoylation, thereby limiting bacterial recognition by the host immune response. Activated PagP also triggers the expression of RcsB-GadE-regulated genes cpsB, rprA, gadA and gadB [96,142]. The Rcs phosphorelay cascade is widely distributed among EHECs; knockout mutants of rcsB are hyper-susceptible to beta-lactams, which suggests that RcsB is a global regulator of cell envelope integrity [139][140][141]. EVIEW 1 is widely distributed among EHECs; knockout mutants of rcsB are hyper-suscept beta-lactams, which suggests that RcsB is a global regulator of cell envelope integrit 141]. The Cpx-TCS (conjugative pilus expression) is a well-studied TCS that count

CpxAR-Mediated Peptidoglycan Cross-Linking
The Cpx-TCS (conjugative pilus expression) is a well-studied TCS that counters cell envelope perturbations. Most induction stimuli for Cpx include misfolded proteins, alkaline pH, salt, changes in lipid composition and attachment to abiotic surfaces. Acidinduced activation of Cpx regulates proton influx and cell wall stability by influencing membrane porins and cross-linking between lipopolysaccharide and peptidoglycan in the outer membrane [143]. Under low pH stress, the activated CpxRA upregulates the expression of several proteins including CydAB, GadAC, CadA, and HdeABD [143]. Most of the activated genes are controlled by the glutamate-dependent acid resistance system that extends the function of Cpx acid-induced tolerance.
During acid stress, several Cpx-regulated proteases, multidrug efflux genes and peptidoglycan amidase genes also reduce susceptibility to cationic antimicrobial peptides (polymyxin B), aminoglycosides (kanamycin), novobiocin and beta-lactams [141,144,145]. Knockout mutants of the Cpx two-component system reduce susceptibility towards antimicrobial agents, as shown in Table 3 [141,145].

PhoPQ-and PmrAB-Mediated Competitive Inhibition
During stress, PhoP regulates the transcription of several stress-responsive and virulence pathway genes, including pagL, pagP, and pmrD, to induce modifications in the lipid A anchor (Figure 3). PmrD is a small regulatory RNA that triggers PhoPQ-mediated activation of PmrAB [111,146,147]. Under acid stress, histidine and glutamate residues of PmrB sense low pH and phosphorylate PmrA. Activated PmrA triggers the arn operon and eptA, which then modify the aminoarabinose and phosphoethanolamine residues in lipid A, respectively [111,148]. In parallel, PmrR blocks the regulatory domain of lipid A phosphotransferase LpxT (a competitive inhibitor of EptA) and facilitates EptA-mediated phosphoethanolamine modification. These PmrA-dependent modifications confer resistance to cationic antimicrobial peptides, including polymyxin. WD101 (a pmrA mutant strain) showed 40-fold lower susceptibility to polymyxin compared with its isogenic parent, W3110 [111,149].
DNA microarray studies confirmed that coupling of the above mentioned twocomponent systems results in significantly reduced susceptibility for polymyxin B and colistin (both are membrane-disrupting CAPs) [51,137]. Polymyxin B and colistin are last-resort antibiotics for multidrug-resistant EHECs [51]. The lipopolysaccharide membrane serves as the first site for interacting with, as well as combating against, cationic antimicrobial peptides. Thus, the two-component systems trigger genes responsible for structural modification that prevents binding of antimicrobial peptides.

Acid-Adaptive Activation of Drug Efflux Pumps
In Gram-negative bacteria, multidrug-resistant efflux pumps play an indispensable role in exporting toxins or harmful metabolites and antimicrobials of different families across the inner and outer membranes. Thus, efflux pumps decrease intracellular drug concentration.

Activation of EvgAS-Regulated Drug Efflux Genes
EvgAS regulates multiple regulatory mechanisms including acid tolerance, drug efflux transporters and bacterial drug-resistance pathways. Extracellular acid stress leads to cytoplasmic acidification that permits EvgA to activate emrKY, mdtEF, mdfA, tolC and acrAB drug efflux (TolC-dependent pumps) genes [150,151]. Low pH induces the expression of emrAB and emrKY multidrug-resistance efflux genes, which confer a growth advantage and facilitate multidrug resistance against extended-spectrum β-lactamases (ESBLs) ( Table 3) [150][151][152][153]. In addition to the development of resistance to ESBLs, the evgA-ydeO-gadE regulatory cascade also facilitates antimicrobial tolerance to other drugs, including gallium nitrate (GaNt) [91]. Gallium nitrate is an FDA-approved drug widely used for the treatment of carcinogenic hypercalcemia and is effective against several clinically significant MDR bacteria. Advanced genomic techniques confirmed the gain-of-function mutation in the EvgSA two-component system in these tolerant strains [91]. Deletion mutants of evgS and evgA failed to confer GaNt tolerance. The regulation of GaNt tolerance by EvgS substitutional mutant (E701G) depends on phosphor-transfer from EvgS to EvgA. The phosphorylated EvgA up-regulates the transcription of safA, ydeO, and gadE. Deletion of gadE in the E701G mutant failed to confer GaNt tolerance, while deletion mutants of ydeO and safA showed partial reversal of tolerance. Thus, GadE acts as a key regulator of EvgS mediated GaNt tolerance and is the central regulator of glutamate-dependent acid resistance system [91].

Activation of the KdpA Proton Pump
Potassium ions are needed for a variety of cellular functions, including intracellular pH regulation and cross-membrane potential. KdpA is a part of the KdpFABC ion channel involved in the ATP-driven transport of potassium ions across the cytoplasm [154]. Recent comparative studies on acid-adapted and non-adapted EHEC strains revealed activation of the KdpA proton pump in response to low pH [16]. During acid stress, this system blocks the flow of protons across the cell to increase the survival rate by more than 100 hrs [16]. Transcriptomic studies revealed upregulation of KdpA, BhsA (outer membrane protein), and ArnA in acid-adapted E. coli O157:H7 strain [16,155]. The enhanced expression of ArnA confers resistance to polymyxin B and colistin in growth cultures (Table 3). BhsA renders the outer membrane hydrophobic by modifying the lipopolysaccharides in a way that renders the cell surface more hydrophobic than hydrophilic [155]. This outer-membrane modification helps by-pass disruptive damage from cationic antimicrobial peptides and also increases cell aggregation [155]. These findings confirm that the KdpFABC ion channel regulates the development of antibiotic resistance in acid-adapted EHEC strains. Intrinsic resistance [161] Aztreonam [162] Beta-lactams [161] Daptomycin [96,163,164]  1 Resistance is an increase in MIC above the breakpoint; tolerance is the loss of killing with no change in MIC.

Activation of TolC-Dependent Efflux Pumps
Nitrosative stress is a type of acid stress that is induced by the high concentration of nitric acid in the gastric fluid. This stress affects the transcription of several regulatory proteins [169]. EHECs activate several multidrug-resistance efflux pumps that contribute to both intrinsic and acquired antibiotic resistance [169,170].

AcrAB-TolC Regulation under Anaerobic Conditions
AcrAB-TolC is a resistance nodulation division (RND-type) efflux pump that contains an outer membrane channel (TolC), an inner membrane channel (AcrB), and a periplasmic protein (AcrA). As a housekeeping TCS, it is constitutively expressed to provide intrinsic resistance towards various toxins [130]. Under anaerobic conditions, AcrA (response regulator) triggers the upregulation of acid-induced efflux genes (gadE-mdtEF operon) by more than 20-fold (the activation of the gadE-mdtEF operon under aerobic conditions is controlled by EvgSA [130]). TolC is also reported to enhance GAD-EvgA acid tolerance, while bile salts and fatty acids present in the stomach trigger AcrAB-mediated activation of another global regulator rob [152,170]. This enhancing regulation of efflux genes results in increased drug resistance and survival of EHECs under nitrosative anaerobic environmental conditions in the human gut. Significantly reduced survival rate has been reported in knockout mutant strains of MdtEF and MdtABC (BaeSR regulated efflux pump) [130,152]. Further studies confirmed that AcrAB-TolC deletion mutants showed attenuated colonization in mice and chickens [170]. In contrast, under extremely acidic conditions, knockout mutants of tolC, emrB, mdtC, and mdtB showed extremely low survival rates (Table 3) [152]. These findings suggest a role for efflux pumps in the development of multidrug resistance and enhanced survival rate for EHECs while passing through the stomach.

Activation of Multiple Antibiotic Resistance Operon
Members of the enterobacteriaceae family have a locus called the multiple antibiotic resistance (marRAB operon), which can confer cross-resistance to several antibiotics including tetracycline, ampicillin, norfloxacin, chloramphenicol, nalidixic acid, and β-lactams [171]. The MarR transcriptional regulator belongs to the AraC/XylS regulatory family that is responsible for inducing adaptive changes in response to environmental stress. Antibiotic resistance induced by the Mar operon is influenced by low pH-mediated acidification of the cytoplasm [44]. Experimental studies show that norfloxacin-sensitive, wild-type EHECs display a significantly enhanced norfloxacin-resistant phenotype when subjected to low pH (Table 4) [44]. Acid-triggered mar regulation also upregulates the transcription of inaA, whereas deletion mutants of this gene showed increased chloramphenicol and nalidixic acid resistance. Several studies also show that inaA is located within the mar locus [73,[172][173][174][175][176].

BaeSR: Multidrug-Resistance Efflux Pump Regulator
BaeSR is one of the stress-triggered systems involved in the regulation of TolC-dependent multidrug efflux pumps (MdtABCD) and Spy (periplasmic chaperone) (Figure 3) [177][178][179]. As mentioned earlier, knockout mutants of mdtABC showed a significantly reduced survival rate under extremely acidic conditions (Table 3) [152]. As expected, Overexpression of the response regulator BaeR results in enhanced expression of MdtA and the AcrD efflux pump that mediates beta-lactam, cephalosporin and novobiocin resistance in large mammals (calves, pigs, and chickens) (Tables 3 and 4) [151,179].

Prophage-Encoded AraC-Like Transcriptional Regulators
EHEC serotype O157:H7 strains possess a locus of enterocyte effacement pathogenicity island (LEE-PAI), which regulates virulence genes of T3SS, intimin and Tir (translocation receptor) [59,61]. These genes are required to colonize, adhere to, produce intestinal lesions, and destroy intestinal microvilli. When EHECs occupy favorable environmental niches in the host intestine, LEE-PAI causes many virulence factors to be expressed [59]. Expression of LEE-PAI genes is tightly regulated by a set of transcriptional regulators (GadE, QseA, H-NS, IHF (integration host factor), Ler, and GrlA). Further studies showed that prophageencoded loci of EHEC O157:H7 strains (specifically the EDL933 strain) carry a set of AraC-like transcriptional regulators PatE, PsrA, and PsrB. Mutational studies of these genes suggest that PatE and PsrB act as positive regulators of glutamate-dependent acidresistance genes and trigger several key virulence determinants in acidic environments [59].

Acquired Antibiotic Resistance among EHEC Serotypes
In addition to acid tolerance, the glutamate-dependent acid resistance pathway performs several extended cross-protective functions, such as strengthening the cell envelope, enhanced attachment, colonization, biofilm formation, multidrug resistance, and bacterial pathogenicity [180,181]. Recent studies on multidrug-resistant EHECs confirm that under low pH stress, different serotypes respond variably toward acid resistance. This variation is attributed to the expression of glutamate-dependent regulation of RpoS [182]. RpoS deletion mutants of EDL933 and other O157:H7 strains show down-regulation of GadA and acid fitness island genes [183,184]. In comparison to other serotypes, the EHEC O157:H7 and EHEC O26:H11 strains show enhanced resistance and improved survival in the mammalian gut [185]. Transcriptomic profiles of virulent EHEC serotypes confirm that the expression level of gadA, gadB, and gadE genes is significantly upregulated when exposed to low pH [185]. As expected, an O157:H7 knockout mutant of the central regulator GadE resulted in 40-fold decreased expression of GadA and enhanced susceptibility towards acid stress. Likewise, knockout mutagenesis of EHEC strains confirmed a non-colonizing phenotype for rcsB, arcA, cpxR, excluding evgS in mouse models [126,127]. Surprisingly, ArcA induced expression of GadE-MdtEF is highly dependent on the anaerobic environment provided by the human stomach [131]. These findings confirm that EHECs cannot survive in the human gut in the absence of the acid-adapted regulatory changes [65,98,186,187].
Interestingly, these adaptations not only help the pathogens survive but also facilitate high virulence and enhanced colonization in animal models. Although mutant studies provide information about the adaptation, colonization, and infection pattern of EHECs, appropriate animal models still need to be developed [188]. For example, mouse models have a gastric pH is less acidic than that of humans [189,190]. Nevertheless, several statements can be made from other systems. For example, GadC deletion mutants of serotype O157:H7, when grown in a calf model, show reduced survival [191]. Moreover, low pH-pretreated gadE and dctR transposon mutants of O157:H7 show strong adherence to human epithelial type 2 and human colorectal adenocarcinoma cell lines, increasing apoptosis [15]. Other studies confirm an acid-induced YadK adhesin that allows EHEC to adhere strongly to epithelial cells and facilitate bacterial-host attachment, resulting in increased colonization and pathogenesis [192]. Multiple studies with EHEC O157:H7 and other serotypes have also reported the importance of acid-induced development of other phenotypes that enhance survival and improve virulence [44,87,90,91,94,129,132,185,188,193,194]. EHECs use low pH stress to adapt and regulate a wide array of genes that enhance survival and increase pathogenicity. EHEC W3110 Acidic Chloramphenicol Increased [47] EHEC EV18 strain Acidic Norfloxacin Increased [44].

Effect on Pathogenicity and Biofilm Formation
The molecular mechanisms underlying the pathogenic regulation of infectious E. coli indicate that biofilm formation correlates significantly with pathogenicity. Approximately 42 genes are regulated within a biofilm matrix in response to acid stress [213][214][215][216], including differential expression of rpoS [217] gadAB, gadC, hdeABD and yjiD (anti-adapter protein iraD, which inhibits rpoS). Knockout mutants of the genes mentioned above, when grown in glutamate-rich medium, increased biofilm formation [218]. Transcriptomic analysis of another gene cluster, ymgABC, revealed a significant role in regulating acid stress, with the ymgB gene product being downregulated in biofilm-forming cells [219]. To confirm a role in acid regulation, ten isogenic mutants of E. coli strain K-12 (∆ymgB, ∆ymgA, ∆ymgC, ∆ycgZ, and ∆gadB, ∆gadA, ∆gadE, ∆hdeB, ∆hdeA, and ∆hdeD) were grown in glutamate enriched medium resulting in enhanced biofilm formation. These results highlight the importance of acid-resistance genes in biofilm formation [219].
Additionally, activation of several TCS response-regulators stimulates the expression of acid-fitness-island genes under acid stress that play an important role in pathogenesis regulation. In EHECs, NtrC, RcsB, and GadX are involved in the upregulation of the LEE (locus of enterocyte effacement) pathogenicity island, which indicates that nitrogen metabolism and glutamate-dependent-system genes play important roles in pathogenesis regulation [104,218,220]. Biofilm formation by another E. coli strain (MG1655) significantly increased at pH 5.5, while at lower pH the expression of flagellar synthesis genes and several virulence factors was strongly induced [1,221]. These studies highlight the biological relevance of acid stress in the regulation of pathogenesis in pathogenic E. coli [1,222,223].

Risk of Acquired Resistance in Non-Pathogenic Bacteria
Under respiratory stress, expression of the GAD operon is equally essential for pathogenic and non-pathogenic E. coli [82,224,225]. In an acidic environment, GadC consumes protons to promote GABA production that generates a proton motive force along with ATP production. Specifically, commensal bacteria and lactic acid bacteria (LAB) harbor GAD to produce GABA and act as probiotics in the GIT [224,226]. GABA plays an important role in bacteria that helps in the fermentation of protein-rich foods, such as cheese, rice germ, kimchi, yogurt, green tea, and sourdough [82,225,227]. Recent studies have found that fermentation of grapes by Lactobacillus plantarum DSM 19463 results in the production of GAD-derived GABA, which plays an important role in inducing the expression of β-defensin-2, hyaluronan synthase, and filaggrin genes responsible for skin protection in humans [228]. These remarkable findings lead to novel cosmetic formulations to treat antimicrobial problems related to skin.
Non-pathogenic bacteria maintain long-term commensalism with the host by stimulating the host immune system and inhibiting the colonization of gut pathogens [229][230][231]. To survive pH fluctuations in different compartments of the gut, the commensal bacteria also undergo the same extent of outer-membrane lipopolysaccharide modifications that contribute to ampicillin resistance. These changes result in modification of lipid A by LpxF phosphatase in commensal isolates of Bacteroidetes thetaiotaomicron that show significantly high polymyxin B resistance and enhanced colonization [232]. These adaptations in gut microbiota occur in response to environmental change. Clinical studies also report acquired tetracycline resistance in 22-33% EHECs in the gastric fluid by horizontal gene transfer, indicating an alarming health concern [41]. In some cases, commensal bacteria are reported to cause diseases, such as Crohn's disease (CD), inflammatory bowel disease (IBD), and ulcerative colitis (UC) [233][234][235][236]. These adaptive pathogenic changes in gut microbiota occur in response to environmental factors, biodiversity, and genetic adaptability [230]. These findings indicate that modification of the lipid A anchor and other processes can promote a long-term commensal relationship between host and bacteria. On the other hand, horizontal gene transfer provides an open passage for the evolution of opportunistic pathogens having reduced antimicrobial susceptibility.

Conclusions and Future Perspective
EHECs have adapted to survive pre-and post-ingestion acid stress, thereby contributing to enhanced pathogenesis. Low pH positively regulates several metabolic pathways, such as motility, biofilm, chemotaxis, periplasmic secretory systems, and multidrug resistance that collectively regulate virulence. We highlighted the role of several signaltransduction cascades that enhance acid tolerance that results in the acquisition of antibiotic resistance. At present, almost all reported drugs are ineffective at controlling the spread of EHECs. Globally, the increasing resistance towards various classes of antibiotics, specifically cationic antimicrobial peptides and extended-spectrum β-lactamases, has become an overwhelming problem, making EHEC infections untreatable. EHECs have established complex regulatory mechanisms involving structural modification and efflux activation that provide an alarming condition for the emergence of new multidrug-resistant pathogens having improved colonization and infection capabilities.
Several factors influence the organism's choice of the resistance mechanism. Cytoplasmic acidification offers a baseline level of defense that can act in tandem with modifications/mutations to reduce antibiotic susceptibility. Bacterial exposure to low pH is associated with acquired antimicrobial resistance to various therapeutic antibiotics. Active efflux and structural modifications of the bacterial membrane are the best-documented mechanisms responsible for bacterial cross-protection to antibiotics. The judicious and ratio-nal use of acidic treatments is crucial to reduce the risk of selecting antimicrobial-resistant bacteria. Antibiotic resistance acquisition strategies are extremely diverse; knowledge of this phenomenon at the molecular level provides an understanding of the details and appreciation to scale this important health problem. An even deeper understanding of the defensive responses deployed by the pathogens may reveal novel targets for agents that will help overcome the spread of foodborne diseases.
We emphasize that rigorous hygiene measures must be followed and all available antimicrobial agents should be used wisely to control the spread of multidrug-resistant strains. The risk of acid-adapted cross-protection by subsequent antimicrobial inactivation necessitates the identification of novel determinants that can influence the future epidemiology and health impact of multidrug-resistant infections. More efforts should be placed to develop novel non-antibiotic approaches such as vaccines, immuno-stimulants, phage therapies, prebiotics, and probiotics to treat EHEC infections.

Acknowledgments:
The authors are grateful to Saad Sarfraz (Station de Neucfchateau, CIRAD, Sainte-Marie, Capesterre Belle Eau, Guadeloupe, France) and Muhammad Arslan (University of Alberta, Canada) for providing technical support.

Conflicts of Interest:
The authors declare no conflict of interest.

Abbreviations
The following abbreviations were used in the manuscript: