The Great ESKAPE: Exploring the Crossroads of Bile and Antibiotic Resistance in Bacterial Pathogens

Throughout the course of infection, many pathogens encounter bactericidal conditions that threaten the viability of the bacteria and impede the establishment of infection. Bile is one of the most innately bactericidal compounds present in humans, functioning to reduce the bacterial burden in the gastrointestinal tract while also aiding in digestion. It is becoming increasingly apparent that pathogens successfully resist the bactericidal conditions of bile, including bacteria that do not normally cause gastrointestinal infections.

ABSTRACT

Throughout the course of infection, many pathogens encounter bactericidal conditions that threaten the viability of the bacteria and impede the establishment of infection. Bile is one of the most innately bactericidal compounds present in humans, functioning to reduce the bacterial burden in the gastrointestinal tract while also aiding in digestion. It is becoming increasingly apparent that pathogens successfully resist the bactericidal conditions of bile, including bacteria that do not normally cause gastrointestinal infections. This review highlights the ability of Enterococcus, Staphylococcus, Klebsiella, Acinetobacter, Pseudomonas, Enterobacter (ESKAPE), and other enteric pathogens to resist bile and how these interactions can impact the sensitivity of bacteria to various antimicrobial agents. Given that pathogen exposure to bile is an essential component to gastrointestinal transit that cannot be avoided, understanding how bile resistance mechanisms align with antimicrobial resistance is vital to our ability to develop new, successful therapeutics in an age of widespread and increasing antimicrobial resistance.

INTRODUCTION

For bacterial pathogens, resistance to multiple host factors is paramount for establishing infection. Many pathogens are exposed to harsh conditions and bactericidal compounds during gastrointestinal (GI) transit. Bacterial pathogens have evolved to expertly overcome each challenge encountered in the GI tract through use of efflux pumps, outer membrane remodeling, and/or other resistance mechanisms. One particularly bactericidal compound encountered in the small intestine is bile, an essential component of digestion that is composed of bile acids, phospholipids, cholesterol, bilirubin, inorganic salts, and trace metals (1). Primary bile acids are synthesized by liver hepatocytes and further modified to reduce toxicity and increase solubility. At physiological pH, bile acids are almost fully ionized and are termed bile salts. Bile salts are maintained in the small intestine to solubilize and digest lipids and lipid-soluble vitamins (1, 2) and are further metabolized by commensal bacteria in the intestines, resulting in production of secondary bile salts. In the distal ileum of the small intestine, the majority of bile is recycled through reabsorption by the epithelium and does not enter the colon (2, 3). Bile salt concentrations in the small intestine range from 0.2% to 2% (wt/vol) depending on time of day, diet, and the individual (4).

Bile is also associated with the “aerodigestive” tract, a term that reflects the interconnectedness of the digestive system with the airway. Classically, bile was not considered present in the airway since it is released into the duodenum of the small intestine distal to the pyloric sphincter, which prevents intestinal contents from entering the stomach. However, bile can enter the stomach and can be further refluxed into the esophagus and oropharynx (5). Gastroesophageal reflux (GER) is highly prevalent in pediatric and adult cystic fibrosis (CF) patients (6–15), where high concentrations of bile acids have been detected in sputum and saliva (16), bronchoalveolar lavage (BAL) fluid samples (6), and the explanted lungs or new allografts of transplant recipients (17, 18). While some studies have identified important limitations in measuring bile salts (19, 20), detection is increased with use of sensitive methodology like mass spectrometry (17). Bile acids detected in CF patient sputum have been inversely correlated with lung function (16), while the detergent effect of bile has also been shown to disrupt the intrinsic barrier of mucus and/or surfactant present throughout the airways (21).

Bile is bactericidal (22, 23); however, many pathogens are known to resist the bactericidal activity of bile and utilize this host component as a localization signal to regulate virulence gene expression and enhance infection (24). Furthermore, strategies employed by pathogens to resist bile align with antibiotic resistance mechanisms. In this review, we discuss the crossroads of bile and antibiotic resistance strategies utilized by bacterial pathogens, with an emphasis on efflux pumps, and highlight implications for multidrug resistance.

EFFLUX PUMPS

Efflux pumps are multiprotein transmembrane complexes that expel toxic materials out of bacterial cells. The six major classes of efflux pumps include the ATP-binding cassette (ABC) superfamily, proteobacterial antimicrobial compound efflux (PACE) superfamily, small multidrug resistance (SMR) family, major facilitator superfamily (MFS), multidrug and toxic compound extrusion (MATE) superfamily, and resistance-nodulation-division (RND) family (25). Nearly all efflux pump families are found in both Gram-positive and Gram-negative bacteria (25, 26), with the exception of the newly described PACE transporter first identified in Acinetobacter baumannii (25) and additional homologs identified in other Gram-negative pathogens (27). Efflux pumps are organized into families based on size, transmembrane domains, and substrate specificity (Table 1). While bacterial efflux across the membrane is sufficient to remove toxic compounds in Gram-positive bacteria, Gram-negative bacteria must transport substances across both the inner and outer membranes. Thus, efflux pump proteins work in coordination with the outer membrane protein TolC to complete multimembrane channels. TolC is often stabilized by additional periplasmic proteins spanning the inner and outer membrane pores (25, 26, 28). The ABC and PACE families use primary active transport to expel compounds and require ATP hydrolysis to function (25, 26). The MFS, SMR, MATE, and RND families use secondary active transport, such as proton motive force and electrochemical gradients, to efflux substances either by symport, antiport, or uniport (25). The AcrAB efflux pump functions by adopting different structural conformations to allow transport (29–31).

TABLE 1.

Bacterial efflux pumps

Pump family (abbreviation)	Description (reference[s])	Mechanism (25)	Membrane location	Example(s) (25, 26, 175)
				Protein(s)	Organism(s) (reference)
ATP-binding cassette (ABC)	Single-component efflux pump comprised of a multidomain protein with at least two nucleotide-binding domains and transmembrane domains	Actively transports compounds from the cytoplasm, dependent on ATP	Inner membranea	MacAB	Escherichia coli
				LmrA	Lactococcus lactis
Proteobacterial antimicrobial compound efflux (PACE)	Single-component efflux pump comprised of 150 amino acids with two tandem transmembrane domains (25, 176)	Moves compounds from the cytoplasm to periplasm using an electrochemical gradient	Inner membrane	AceI	Acinetobacter baumanniib
Small multidrug resistance (SMR)	Single-component efflux pump comprised of 100–120 amino acids with four transmembrane helices (25)	Moves compounds from the cytoplasm using an electrochemical gradient	Inner membrane	NepAB	Arthrobacter nicotinovorans
				EmrE, SugE	Escherichia coli
Major facilitator superfamily (MFS)	Single-component efflux pump comprised of 400–600 amino acids with 12–14 transmembrane alpha-helices (25)	Moves compounds from the cytoplasm using an electrochemical gradient	Inner membranea	MdfA, MdtM	Escherichia coli
				NorA, NorC, QacA, QacB	Staphylococcus aureus
				LmrP	Lactococcus lactis
Multidrug and toxic compound extrusion (MATE)	Single-component efflux pump comprised of 400–700 amino acids spanning 12 helical transmembrane domains	Moves compounds from the cytoplasm using an electrochemical gradient	Inner membrane	NorM	Vibrio parahaemolyticusc
				PfMATE	Pyrococcus furiosus
				DinF	Bacillus halodurans
				MepA	Staphylococcus aureus
				YdhE	Escherichia coli
				AbeM	Acinetobacter baumannii
				CdeA	Clostridium difficile
Resistance-nodulation-division (RND)	Multicomponent transmembrane pump	Exports compounds from the cytoplasm, resulting in complete expulsion of compounds outside the bacterium	Spans both inner and outer membranes	TtgABC	Pseudomonas putida
				EmhABC	Pseudomonas fluorescens cLP6a
				CmeABC	Campylobacter jejuni
				AcrAB-TolC	Multispecies
				CusCFBA	Escherichia coli (177)
				MexAB-OprM	Pseudomonas aeruginosa (25)

^aGram-negative bacteria utilize TolC in the outer membrane to fully expel substances.

^bNew homologs have been identified in other Gram-negative pathogens (27).

^cHomologs are found in Vibrio cholerae and Neisseria gonorrhoeae.

Expression of efflux pumps occurs in response to exposure to antimicrobial or toxic compounds. In a recent study, adaptation of bactericide-sensitive strains isolated from organically produced food was achieved by exposing bacteria to increasing concentrations of bactericidal compounds. The efflux pump genes acrAB, sugE, norC, qacE, and qacH and several other antibiotic resistance genes were detected in the compound-adapted strains (32). This observation demonstrates maintenance of efflux pump genes in environments without constant exposure to toxic compounds. Furthermore, efflux pumps are the most ubiquitous type of resistance element in bacteria, and some efflux pump homologs are found in multiple species of bacteria, suggesting either horizontal gene transfer of operons or convergent evolution to resist toxic compounds. Indeed, a repeating theme in the Enterobacteriaceae family is the use of AcrAB, found in Escherichia coli, Salmonella, Shigella, Klebsiella, and other pathogens, to resist both bile salts (24) and antibiotics (30, 31), thereby making it essential for survival under extreme environmental conditions.

ESKAPE PATHOGENS

A previous review from our group highlighted resistance mechanisms and virulence regulation following bile exposure in the enteric pathogens Escherichia coli, Shigella, Vibrio, Salmonella, Campylobacter, Clostridium, and Listeria (24). Updates to information about these pathogens are provided briefly below. Given the clinical importance of multidrug resistance, this review will focus on the Enterococcus, Staphylococcus, Klebsiella, Acinetobacter, Pseudomonas, and Enterobacter (ESKAPE) pathogens and discuss the relationship between bile and antibiotic resistance mechanisms, with summaries presented in Table 2. The ESKAPE group of pathogens represents a significant public health threat as antibiotic resistance rates rise from the acquisition of multiple resistance mechanisms (33). Interestingly, many ESKAPE pathogens are not known to cause infection in the GI tract; however, isolation from bile, the gallbladder, pancreatic or biliary stent biofilms, and bile duct infections have been reported and antibiotic resistance is often detected (34–46). The observations are not limited to hospital-based infections, given a recent study that identified positive bile cultures in 22.2% of patients undergoing elective gallbladder removal surgery. The most common bacterial isolate present in the bile samples was Enterococcus, followed by E. coli, Klebsiella, Enterobacter, and Pseudomonas; of these isolates, 22.7% displayed antibiotic resistance (47). Furthermore, bile exposure in the lungs of CF patients has been shown to affect Staphylococcus aureus, Acinetobacter baumannii, and especially Pseudomonas aeruginosa infection (48–50). Finally, ESKAPE pathogen-related infections are not limited to humans, as highlighted by a recent veterinary study that identified antibiotic-resistant pathogens in bile and other body site cultures from canines and felines (51). Since ESKAPE pathogens can be exposed to bile at some point during an infection cycle, understanding the association between bile and antibiotic resistance mechanisms (Fig. 1) is important to our ability to develop novel therapeutics and treat infections caused by these pathogens.

TABLE 2.

Summary of the effects of bile exposure on the ESKAPE pathogens^a

Genus and species	Mechanism(s)	Description and/or resistance function (reference[s])
Enterococcus
E. faecalis	BSH	Deconjugates bile acids to neutralize the antimicrobial activity (56, 57)
	Bile resistance	DNA repair, oxidative response, transcriptional regulation, and cell wall synthesis genes in bile salt resistance (58); alters bacterial outer membrane (68–70)
	Gls24, GlsB	General stress response proteins in bile resistance (61–63, 65)
	EmrB/QacA	Drug resistance transporter genes induced in bile (66)
	PrkC	One-component signaling protein that helps to maintain cell wall integrity for bile resistance (67)
	Biofilm	Glycotransferases involved in biofilm-associated antibiotic resistance and resistance to bile salts (72)
E. faecium	BSH	Deconjugates bile acids to neutralize the antimicrobial activity (56)
	Bile resistance	75 total genes, including a glutamate/aspartate transport system permease and a phospholipid synthetase linked to daptomycin resistance (59)
	Gls24, GlsB	General stress response proteins in bile resistance (64)
	LiaFSR, BsrXRS	Two-component systems that contribute to bile salt resistance (71)
	Biofilm	VRE forms long chains and biofilms in bile acids; mutations in two-component kinase yycG (walK) gene and the three-component response regulator liaR gene prevented the phenotype (73)
Staphylococcus
S. aureus	WTA	A mutant in cell WTA displayed increased sensitivity to bile salts (86)
	MnhF	Efflux pump expels cholate, important for bile resistance (87)
	Biofilm	Bile-induced biofilm formation utilizes WTA biosynthesis and transport genes (88)
Klebsiella
K. pneumoniae	Urease	Induces epithelial cell adherence following bile salt exposure (102)
	OxyR	Mutation leads to higher sensitivity to bile (103) and increases susceptibility to other antibiotics, with acrB expression significantly reduced in an oxyR mutant (104)
	CpxAR	Two-component signal transduction system important for bile and antibiotic resistance (105)
	KpnO	Outer membrane protein in which a mutation causes increased susceptibility to bile; expression dependent on the PhoBR two-component system (106)
	KpnEF	Efflux pump facilitates bile and antibiotic resistance (107)
	CadC, TdcA	Transcriptional activators important for the bile response (108)
	GalET	Galactose utilization and LPS modifications for bile resistance (109)
	pgaABCD	Synthesizes PNAG for the bile salt-induced biofilm matrix (110)
Acinetobacter
A. baumannii	Bile response	Following bile salt exposure in strains lacking the AdeABC efflux pump, antibiotic MICs, motility, and biofilm formation increase; gene expression analyses identified a glutamate/aspartate transporter, motility, biofilm formation, and quorum sensing genes for the bile salt response (121)
Pseudomonas
P. aeruginosa	Bile response	Genes associated with metabolism, redox control, biofilm formation, antibiotic tolerance, and cell envelope biogenesis upregulated in bile salts (48, 134, 135, 140)
	Mex efflux pumps	Strains deficient in the Mex efflux pumps have decreased viability in medium containing bile; efflux pump inhibitor also decreases viability (137)
	Biofilm	Both bile salts and human-derived bile induce biofilm formation, with the wspF gene associated with exopolysaccharide production (141)
	pqsABCDE	Bile induces quorum sensing signaling, with the alkyl quinolone operon generating the PQS signal important for biofilm formation (48)
	ExoU	Toxin expression decreased in bile salts (48)
	T6SS	Increased activity of the type VI secretion system (48)
	Motility	Reduced swarming motility (48, 135, 178)
	SNPs	Identified following long- term bile exposure affecting quorum sensing and biosynthetic pathways (135)
Enterobacter	Bile response	Studies lacking; Cronobacter, formerly E. sakazakii, uses efflux pumps for bile resistance (153, 154)

^aBSH, bile salt hydrolase; VRE, vancomycin-resistant Enterococcus; WTA, wall teichoic acid; PNAG, poly-β-linked N-acetylglucosamine; MIC, minimal inhibitory concentration.

FIG 1.

Bile and antibiotic resistance: a chicken-versus-egg scenario. The complicated, cyclical relationship between bile (or other bactericidal compounds) and antibiotic exposures leads to enhanced resistance patterns. Efflux pump expression and other changes occur after exposure to bactericidal compounds. Consequently, antibiotic resistance is enhanced as bacteria utilize efflux pumps as an immediate defense strategy. To counter antibiotic-resistant bacteria, higher concentrations or additional antibiotics are administered in the clinical setting. The bacteria respond with higher mutation rates, increasing rates of antibiotic resistance.

Enterococcus.

The Enterococcus species are Gram-positive, catalase-negative, facultative anaerobic cocci that are resistant to harsh environmental conditions such as temperature, pH, and high salt concentrations. Enterococcus represents a small portion of the intestinal microbiota, with Enterococcus faecalis and Enterococcus faecium commonly found (52). Infections typically arise in hospitalized patients following antibiotic administration and outgrowth of hospital-associated clones (53) and include urinary tract, hepatobiliary sepsis, endocarditis, surgical wounds, bacteremia, and neonatal sepsis. Adherence, biofilm formation, toxin production, and quorum sensing are key virulence mechanisms associated with pathogenicity (52). Resistance to vancomycin, aminoglycosides, and ampicillin is routinely encountered (33, 52, 53). Multiple clinical reports have associated Enterococcus with bile-related infections. For example, two studies demonstrated that Enterococcus is the most frequently isolated pathogen from bile and related biliary infections (34, 35), while a German study identified Enterococcus bacteria with 33% vancomycin resistance rates as the predominant isolates from patients with pyogenic liver abscesses, wherein the most common cause was due to bile duct stenosis or obstruction (54). More recently, E. faecalis has been detected in the bile and pancreatic juice and tissue samples of patients with chronic pancreatitis and pancreatic cancer (55).

Several studies have investigated bile resistance in Enterococcus and related effects on virulence. Given the commensal nature of the genus, multiple species produce bile salt hydrolase (BSH) to deconjugate bile acids and neutralize antimicrobial activity (56). The BSH enzyme from E. faecalis has unique features compared to other BSH homologs, which likely contribute to higher activity (57). In addition to BSH, other mechanisms are needed to resist bile. Proteomic analyses identified 45 proteins induced following bile salt treatment in E. faecalis, with subsequent mutagenesis experiments confirming the importance for DNA repair, oxidative response, transcriptional regulation, and cell wall synthesis genes in bile salt resistance (58). In E. faecium, 75 genes have been associated with bile resistance, including genes encoding a glutamate/aspartate transport system permease and a phospholipid synthetase linked to daptomycin resistance (59). Even Enterococcus avium, a common bird isolate that causes infection in humans, harbors bsh and additional genes to facilitate bile resistance and adaptation to bile-rich environments (60).

More specific mechanisms of bile resistance have been identified, some of which affect antibiotic susceptibility. The general stress response proteins Gls24 and GlsB are important for bile salt resistance and virulence in both E. faecalis and E. faecium (61–65), while transcriptomic response analyses following 7.5% bovine bile exposure in E. faecalis identified induction of genes encoding two EmrB/QacA family drug resistance transporters and a vacuolar-type ATPase important for energy generation (66). Enterococcus faecalis encodes PrkC, a one-component signaling protein that modulates resistance to bile by helping to maintain cell wall integrity. Due to the presence of this protein, E. faecalis is naturally resistant to antibiotics that target the cell wall, particularly broad-spectrum cephalosporins (67). Moreover, E. faecalis can incorporate fatty acids from bile and other host components to alter the bacterial membrane and provide protection against membrane-damaging agents (68–70). In E. faecium, the LiaFSR and BsrXRS two-component systems contribute to bile salt resistance (71). Finally, Enterococcus is known to form biofilms; and glycosyltransferases (GTF), which contribute to bile salt resistance, and quorum sensing genes are important for antibiotic resistance in E. faecalis biofilms. It is hypothesized that polysaccharides in the biofilm extracellular matrix prevent antibiotics from reaching the bacterial targets (72). Interestingly, a recent study demonstrated that the secondary bile acid lithocholic acid impaired the separation of growing vancomycin-resistant Enterococcus (VRE) diplococci, resulting in long chains and increased biofilm formation (73). Mutations in the two-component kinase yycG (walK) and the three-component response regulator liaR prevented the long-chain phenotype and biofilm formation, and increased susceptibility of the pathogen to daptomycin. Biofilm formation in the presence of bile and related suppression of antimicrobial activity are recurrent themes in pathogens (24, 74; also see below). In all, the bile response in Enterococcus affects both fitness and virulence, with multiple strategies employed to ensure survival in the presence of bile. Understanding the implications of enterococcal bile response mechanisms to antibiotic resistance phenotypes should be considered for effective therapeutic development.

Staphylococcus.

Staphylococcus is a genus comprised of Gram-positive, aerobic to facultative anaerobic cocci that represent over 40 species and that include members of the commensal population of the skin, GI tract, and nasal epithelium. The most clinically important species is the opportunistic pathogen Staphylococcus aureus. Virulence is mediated through adherence factors, toxins, capsule production, and resistance to innate antimicrobial defenses such as the β-defensins and cathelicidin (75, 76). Staphylococcus aureus is frequently associated with multidrug resistance, often due to difficult and prolonged antibiotic treatment of skin and wound infections or to incidental exposure to antibiotics during commensal colonization (75, 77). Additionally, several groups of patients are exposed to suboptimal levels of antibiotics throughout treatment due to the timing of doses or the stability of drugs. For example, burn, CF, obese, critically ill, and pediatric patients process antistaphylococcal antibiotics differently than their healthy counterparts, thus reducing the antibiotic half-life and exposing S. aureus to antibiotics without beneficial clinical outcomes (77). To further facilitate antibiotic resistance, S. aureus encodes several efflux pumps, including NorA, NorB, NorC, and Tet38, that confer resistance to quinolones and tetracyclines (78). Interestingly, NorA regulation is mediated by ArlRS, a two-component signal transduction system that also regulates capsule formation (79). Tetracycline exposure controls expression of the Tet38 efflux pump and an additional efflux pump, LmrS, demonstrating acquisition of redundant mechanisms for extrusion of harmful antibiotics to promote bacterial survival (80). Finally, methicillin resistance is mediated through the mecA gene that encodes a penicillin binding protein to resist β-lactam antibiotics (81).

While the antibacterial activity of bile salts on S. aureus has recently been defined (82) and researchers are looking to utilize bile salts for new or improved therapeutics against the pathogen (82–84), S. aureus can survive GI transit and intestinal carriage, which has emerged as an important risk factor for infection (85). A few studies have defined essential genes that are required for bile resistance. Cell wall teichoic acid (WTA) is required for GI colonization in a mouse model since a WTA mutant displayed increased susceptibility to bile salts, proteases, and a GI defensin while also exhibiting reduced epithelial cell adherence (86). Additional research has demonstrated that the MnhF efflux pump is important for bile resistance by expelling cholate (87). Finally, bile rapidly induces biofilm formation in clinical isolates of S. aureus, and WTA biosynthesis and transport genes have been shown to be necessary for bile-induced biofilm formation (88). Biofilm-associated infections are very difficult to treat since most antibiotics are ineffective (89).

Staphylococcus aureus remains a considerable threat to human health. Given the recent recognition of intestinal carriage of S. aureus, future studies investigating the bile response are certainly warranted. Indeed, it is intriguing how S. aureus evolved to resist bile and colonize the GI tract. As highlighted in Fig. 1, it is unclear if this evolution occurred as a consequence of antibiotic resistance that enabled multidrug-resistant (MDR) isolates to survive GI transit. Alternatively, it is possible that MDR isolates arose from the adaptation required to resist bile exposure. Nevertheless, future therapeutic interventions must consider bile exposure and subtherapeutic antibiotic effects on bacterial adaptation, person-to-person variation in antibiotic half-lives, current Staphylococcus commensal populations, and disease status to design custom, multifaceted therapeutics to effectively treat and prevent further adaptation of this superbug.

Klebsiella.

The Klebsiella genus is comprised of Gram-negative, nonmotile, oxidase-negative bacilli that are members of the Enterobacteriaceae family and often found in the normal commensal population of the skin, respiratory, and GI systems (90). Of the four species of Klebsiella, Klebsiella pneumoniae (encompassing 72 serovars) and Klebsiella oxytoca are the prominent pathogens causing hospital-acquired infections that include pneumonia, bacteremia, wound infections, urinary tract infections, and diarrhea (90). Virulence factors associated with pathogenicity include several adhesins, production of a capsule, and the use of siderophores for iron acquisition (91). Additionally, biofilm formation enhances nosocomial infection through medical device colonization (92). Gastrointestinal colonization is considered the first stage of nosocomial infection, no matter where the eventual site of infection occurs (93, 94).

Antibiotic resistance in Klebsiella has significantly increased throughout the world, with concerning resistance patterns emerging for K. pneumoniae. Resistance to extended-spectrum cephalosporin through the production of plasmid-mediated β-lactamases was first reported in 1983. Strains producing these extended-spectrum β-lactamases (ESBLs) have since spread worldwide and have resulted in increased use of carbapenems to treat infections. Consequently, carbapenemase-producing isolates, in which the enzyme is encoded on a mobile transposon, emerged in the early 2000s. These resistance profiles have resulted in limited treatment options for infections and typically include polymyxins, tigecycline, and aminoglycosides (95). The relative ease at which β-lactamases are transferred to other bacteria, particularly within the Enterobacteriaceae family, has resulted in significant antimicrobial resistance and the emergence of highly resistant pathogens throughout the world (95). Finally, antibiotic resistance in K. pneumoniae is further facilitated by multiple efflux pumps, including AcrAB and KexD of the RND family, KdeA of the MATE family, and KmrA of the MFS family (96–99). Genome sequencing has identified additional putative novel efflux pumps utilized by Klebsiella (100). Alarmingly, sequencing of a tigecycline-resistant K. pneumoniae isolate obtained from bile samples of a patient with bile duct cancer (cholangiocarcinoma) identified 16 putative efflux pump genes and regulators based on homology to known efflux pumps (101).

As noted above, Klebsiella is often isolated from bile or from infections associated with gallstones, the gallbladder, or bile ducts. Multiple studies have evaluated K. pneumoniae fitness and survival under GI-like conditions. Urease activity that enhances assimilation to the GI environment also induces epithelial cell adherence following bile salt exposure (102). The transcriptional regulator OxyR is important for biofilm formation, fimbrial synthesis, intestinal colonization, and resistance to GI stresses including bile (103). A subsequent study confirmed the OxyR requirement for bile resistance and demonstrated that an oxyR mutation increased susceptibility to several antibiotics. Expression of the acrB gene encoding the inner membrane component of the AcrAB efflux pump was significantly reduced in the oxyR mutant (104).

A further significant link between bile and antibiotic resistance in K. pneumoniae has centered around two-component signal transduction systems. First, analysis revealed that the CpxAR system is important for resistance to bile as well as to β-lactam and chloramphenicol antibiotics. A ΔcpxAR mutant had reduced expression of the acrB, acrD, and eefB efflux genes (105). Given the antibiotic resistance profile and the induction of efflux pump mechanisms that often involve membrane proteins, the authors also evaluated whether CpxAR alters the expression of membrane proteins of K. pneumoniae. The expression of three outer membrane proteins was dependent upon CpxAR, including a homolog to OmpC in which promoter analyses revealed CpxR binding sites (105). A second study evaluated KpnO, another OmpC homolog, and found that a ΔkpnO mutant displayed increased susceptibility to bile and additional antibiotics, including tetracycline, tobramycin, and streptomycin (106). KpnO expression was dependent upon the PhoBR two-component signal transduction system, which is also important for antibiotic efflux. Interestingly, PhoBR affected the expression of the Klebsiella capsule that similarly enhances stress tolerances (106). Finally, a third study characterized the KpnEF proteins in K. pneumoniae, which are homologous to the SMR-type efflux pump EbrAB. Mutations in kpnEF resulted in increased susceptibility to bile, multiple antibiotics, and even clinically based disinfectants. Furthermore, KpnEF was dependent upon the CpxAR system (107). These studies highlight the link between efflux mechanisms and resistance to both bile and antibiotics in K. pneumoniae.

Additional factors important for the K. pneumoniae bile response include the transcriptional activators CadC and TdcA (108) and the GalET proteins for galactose utilization and lipopolysaccharide (LPS) modifications to resist bile (109). Additionally, bile salt-induced biofilm formation through the pgaABCD operon encodes components to synthesize and secrete poly-β-linked N-acetylglucosamine (PNAG), essential for the biofilm matrix (110). Indeed, Klebsiella serves as a prime example of the bile response and antibiotic resistance relationship that includes induction of efflux pumps, alterations to the outer membrane, and biofilm formation. Future therapeutic development must consider bile exposure and the induction of these resistance factors in order to develop effective treatments against this highly resistant pathogen.

Acinetobacter.

The Acinetobacter species are Gram-negative, aerobic, nonfermentative bacteria that are commonly found in the environment or as commensal bacteria (111). Acinetobacter baumannii has emerged as a severe pathogen acquired in hospital and long-term care settings, in the community, and among wounded military patients. Typical infections resulting from A. baumannii include bacteremia, wound (skin and soft tissue) infections, pneumonia, and urinary tract infections (112, 113). Multidrug resistance in A. baumannii has increased at alarming rates, and strains resistant to all clinical antibiotics have been isolated. Antibiotic resistance mechanisms include efflux pumps, altered outer membrane protein expression, and other target modifications against the various classes of antibiotics (111–113). In addition to the MDR phenotype, A. baumannii is able to form biofilms on both biotic and abiotic surfaces, further facilitating the transmission of this pathogen in the health care setting (114).

Both MDR and susceptible Acinetobacter species have been isolated from bile-related infections (37, 115–117). Gastrointestinal illness with Acinetobacter is typically not seen; however, an association with foodborne infections has been suggested (118). Additionally, A. baumannii was detected in the digestive tract of intensive care unit (ICU) patients (119, 120), which points to the ability of Acinetobacter to resist bile. Comprehensive analyses of the bile response in Acinetobacter have been limited. However, a recent analysis characterized the molecular and microbiological characteristics of the A. baumannii bile salt response in isogenic mutants and clinical isolates lacking the AdeABC efflux pump important for acquired antibiotic resistance (121). The authors found that both the antibiotic MICs and biofilm formation were increased following bile salt exposure while expression analyses identified induction of genes for a glutamate/aspartate transporter, surface motility, biofilm formation, and quorum sensing (121). This important first study will hopefully lay the foundation for future analyses of the bile response in A. baumannii. It will certainly be interesting to evaluate the role of the AdeABC efflux pump in bile resistance and to determine whether induced antimicrobial resistance occurs in Acinetobacter species following bile exposure.

Pseudomonas.

The Pseudomonas genus is comprised of a group of Gram-negative, motile, aerobic bacteria that are versatile and ubiquitous, capable of thriving in a variety of environments due in large part to the marked adaptive potential encoded within the genomes (122, 123). Pseudomonas species are found in water, soil, and vegetation and can be isolated from the skin, throat, and stool of healthy individuals (122). Often found robustly colonizing various hospital surfaces, Pseudomonas is considered an opportunistic pathogen and may be spread to hospitalized or immunocompromised patients via contaminated surfaces (122). In particular, Pseudomonas aeruginosa is involved in a wide array of human diseases, including ventilator-associated pneumonia, soft tissue infections and bacteremia in burn patients, and medical device-associated infections (124–127). Pseudomonas aeruginosa can thrive in bile-enriched environments, and infection of bile, the gallbladder, and biliary tract has been observed clinically for decades (128–130). Additionally, P. aeruginosa is an important pathogen in lung diseases wherein mucociliary clearance is disrupted, as in the autosomal recessive genetic disease CF that is caused by mutations of the chloride channel cystic fibrosis transmembrane conductance regulator (CFTR) gene. Cystic fibrosis-associated pulmonary disease is characterized by a progressive decline in lung function over time, frequently associated with the acquisition of the pathogens P. aeruginosa, S. aureus, and members of the Burkholderia genus (131). The pathophysiologic implications of CFTR dysfunction are broad, with affected patients commonly having severe pancreatic, GI, and pulmonary disease.

In light of the association of bile and P. aeruginosa infection in CF-related lung disease, researchers have begun to investigate the effect of bile exposure on P. aeruginosa (48, 50). It is important to note that prolonged bile exposure in these studies may be more reflective of GI colonization; nevertheless, bile salts and/or bile clearly has an effect on P. aeruginosa. Notably for antibiotic resistance, pseudomonal isolates from human bile samples display a bile-mediated MDR phenotype, suggesting that bile may be an external cue for P. aeruginosa to activate resistance mechanisms and survive in the gut (132). Antimicrobial drug resistance and the increasing prevalence of MDR P. aeruginosa clinical isolates are significant public health concerns (133). The ability of P. aeruginosa to display increased antibiotic tolerance in the presence of bile salts has been demonstrated in the laboratory setting (134, 135), in which increased tolerance to colistin, polymyxin B, and erythromycin was observed following growth in bile-containing medium (134). Furthermore, the transcriptomic profile analysis of P. aeruginosa in the presence of 0.3% (wt/vol) bile shows upregulation of genes associated with metabolism (siaAD) redox control, biofilm formation (psl), antibiotic tolerance (mexAB-oprM), and cell envelope biogenesis (134). As noted in Table 1, Mex efflux pumps are members of the resistance-nodulation-division (RND) superfamily of exporters that mediate multidrug resistance (136). Lamers and collaborators incubated a P. aeruginosa strain deficient in the four major Mex efflux pumps (MexAB-OprM, MexCD-OprJ, MexEF-OprN, and MexXY-OprM) in medium containing bile and found that bacterial viability was significantly decreased (137). Additionally, it was observed that the viability of strain PAO1 was significantly reduced in the presence of bile when the efflux pump inhibitor phenylalanine-arginine β-naphthylamide (PAβN) was added (137). These findings suggest that efflux pumps are partially responsible for the ability of P. aeruginosa to survive within bile-containing fluids.

Biofilm formation, which is commonly associated with the ability of P. aeruginosa to resist antibacterial or environmental stressors and results in chronic infections that are difficult to treat (138, 139), is also impacted by bile. Physiologic concentrations of both a synthetic bile salt mixture and individual human-derived bile salts increased biofilm formation in P. aeruginosa, while exogenous bile significantly increased P. aeruginosa pellicle formation and thus attachment of bacteria to abiotic surfaces (48). Similarly, the human bile salt taurolithocholic acid (TLCA) modulated biofilm formation in strain PA14 as well as metabolism and virulence (140). Recent work has demonstrated that a ΔwspF mutant of strain PAO1, which has an increased biofilm formation phenotype due to increased production of cyclic-di-GMP, displayed a significant decrease in biofilm biomass during growth in TLCA (141). Confocal microscopy revealed increased dispersion of preformed ΔwspF biofilms following treatment with TLCA. Since the wspF gene is associated with exopolysaccharide production of the biofilm matrix, the data suggest that various bile acid derivatives and bile components have different effects on the ability of P. aeruginosa to form and maintain biofilms (141). Bile exposure also induces P. aeruginosa quorum sensing signaling, a cell-cell communication mechanism that regulates gene expression based on bacterial cell density (122), by increasing pqsABCDE alkyl quinolone operon activity to generate the Pseudomonas quinolone signal (PQS) important for biofilm formation (48). Expression of genes associated with chronic-stage lung infections, such as pqsA, which is part of the quinolone signal biosynthetic pathway; lasI, a quorum sensing gene; and psl, an exopolysaccharide-related gene involved in biofilm development, is also induced by bile (134). Thus, bile salts appear to exert pleiotropic effects on P. aeruginosa biofilm formation, including the modulation of quorum sensing that can impact various states of bacterial growth and lifestyle, likely contributing to an MDR phenotype.

Data indicate that P. aeruginosa virulence is also impacted by the presence of bile or bile salts. First, promoter activity and transcript levels of the exotoxin ExoU, an important virulence factor that augments inflammation and induces host cell lysis, are markedly diminished in the presence of bile (48). ExoU is expressed by multiple clinically relevant strains of P. aeruginosa and delivered into mammalian cells via the type III secretion system (T3SS) (125, 142). This observation supports the hypothesis that bile salts promote biofilm formation in P. aeruginosa while suppressing the expression of virulence-related genes and is consistent with previous data showing that T3SS genes are downregulated during biofilm development (134, 143). Second, there is increased activity of the type VI secretion system (T6SS) tssA promoter in the presence of bile salts (48). The T6SS inhibits the growth of neighboring bacterial cells by secreting effectors through a contact-mediated delivery system, so this finding indicates that bile can enhance the ability of P. aeruginosa to survive polymicrobial environments such as the lungs of CF patients, the GI tract, and in the biliary tract of patients with gallstones. Finally, swarming motility, the coordinated translocation of bacterial populations across solid or semisolid surfaces, was decreased in the P. aeruginosa clinical isolate PA14 and two CF isolates when exposed to bile, thus promoting biofilm development (48).

Future investigations should consider several factors when exploring the association of bile and P. aeruginosa, particularly in the context of CF. It is not clear if P. aeruginosa lung colonization occurs through bacteria embedded in a bile-induced biofilm from the gut or through planktonic cells entering the lung. Independent of the transmission route, P. aeruginosa present in the lungs appears to be exposed to bile upon reflux. Similarly, the duration of bile exposure in the lungs necessary to alter P. aeruginosa pathogenicity needs to be further elucidated. A recent study using an artificial sputum medium to model CF conditions in the lung demonstrated an adaptive response of the pathogen to long-term bile exposure, leading to the identification of single nucleotide polymorphisms (SNPs) in the genome that affected quorum sensing (lasR) and both the pyocyanin (phzS) and pyomelanin (hmgA) biosynthetic pathways (135). Interestingly, pigmented isolates adapted to bile were recovered from bile-containing cultures. These isolates retained reduced swarming motility and enhanced antibiotic tolerance, but the biofilm and redox responses were no longer present. The results highlight the adaptive response of P. aeruginosa to bile in CF-like medium and demonstrate the emergence of ecologically competitive subpopulations with increased survival in the presence of bile (135). In the future, potential new therapies and standards of care may emerge following studies that further define the P. aeruginosa response to bile exposure in the GI tract as well as reflux into the lung. Efflux pump inhibitor drugs could have a therapeutic benefit in this clinical context, particularly in light of the possible role that efflux pumps play in adaptation of P. aeruginosa to bile (134, 137). Inhaled bile acid sequestrants could similarly be deployed to mitigate the effect of this potent host signal (134). Surgical interventions for the lung, such as fundoplication and laryngeal cleft repair, might also be further considered as mechanical prophylaxis (145). Although therapies based on modulating the interactions of bile and P. aeruginosa within the aerodigestive tract represent a promising avenue to consider, further research is clearly necessary to translate current findings into effective interventions to mitigate P. aeruginosa in an era of high antibiotic resistance.

Enterobacter.

The Enterobacter species are a group of Gram-negative, facultative anaerobic, non-spore-forming bacilli of the Enterobacteriaceae family. Often seen as commensal organisms in the GI tract of humans and animals, Enterobacter aerogenes and Enterobacter cloacae have emerged as opportunistic nosocomial pathogens. Nosocomial infections include wound, respiratory, urinary, and GI infections and even meningitis, with sepsis and death as frequent outcomes to infection (146, 147). Virulence studies with Enterobacter appear limited; however, a recent study demonstrated E. cloacae adherence to, invasion of, and subsequent apoptosis induction in Hep-2 epithelial cells (148). Resistance to disinfectants and antimicrobial agents plays important roles in the increasing prevalence of Enterobacter nosocomial infections, often leading to outbreaks in hospitals (147, 149). Enterobacter aerogenes has acquired ESBL resistance, with reports of further resistance to the last-line antibiotics carbapenems and colistin. Enterobacter cloacae is resistant to multiple antibiotics including ampicillin, first-generation cephalosporins, and cefoxitin due to β-lactamase production (147). Both E. cloacae and E. aerogenes harbor several efflux pumps to resist antibiotics, including AcrAB-TolC (149, 150). Additional efflux pumps include EefABC, OqxAB, and members of the RND and MATE families, while outer membrane alterations also contribute to antibiotic resistance (147).

Like other ESKAPE pathogens, Enterobacter has been isolated from bile- or gallbladder-related infections. For example, a clinical study evaluating the emergence of antibiotic resistance in Enterobacter, Serratia, Citrobacter, and Morganella species found that resistance to cephalosporins occurred more often in Enterobacter species and that biliary tract infection associated with malignant bile duct invasion was significantly associated with the emergence of resistance to broad-spectrum cephalosporins (151). Another example is a case report detailing the isolation of carbapenem-resistant E. aerogenes from a liver transplant recipient in which liver abscesses and a biliary fistula were noted (152). Meanwhile Cronobacter species, formerly named Enterobacter sakazakii, have been associated with infection in the elderly, in immunocompromised patients, and in infants in which contaminated formula has been linked to infections. Cronobacter causes the same wide-ranging infections as Enterobacter and has also been shown to adhere to and invade epithelial cells. High tolerance to harsh environmental conditions including osmotic stress and elevated temperatures has been described. Furthermore, an association of exopolysaccharide production has been linked to biofilm formation while efflux pumps have been shown to promote resistance to bile salts and disinfectants (153, 154). Unfortunately, there have not been extensive studies evaluating the Enterobacter bile response and the related effects on virulence and antibiotic resistance. Given the emergence of these pathogens, the presence of efflux pumps, and the association with bile-related infections, these studies are urgently needed to enhance our understanding of Enterobacter pathogenesis and to develop effective therapeutics in an era of rampant antibiotic resistance.

UPDATE ON ENTERIC PATHOGENS

As previously reviewed (24), most enteric pathogens utilize the AcrAB efflux pump and other mechanisms to resist bile. The induction of efflux pumps during GI transit appears to have important implications for subsequent drug resistance phenotypes. Given the difficulties of vaccine development in this group of pathogens that cause diseases ranging from mild gastroenteritis and watery diarrhea to bacillary dysentery, it is critical to understand bile and related drug resistance mechanisms to develop novel therapeutics to successfully target these pathogens. Table 3 summarizes new findings on the effects of bile on growth and/or resistance mechanisms in enteric pathogens, with interesting observations highlighted below. Finally, Table 4 provides a summary of bile resistance mechanisms in additional genera of bacteria, including members of the human microbiome.

TABLE 3.

Updates on enteric pathogens with a focus on bile resistance mechanisms

Genus and species or serovara	Mechanism(s)	Comment(s) in literature (reference[s])
Shigella
S. flexneri	AcrAB, GalU, MdtJI	acrAB genes induced in bile salts; ΔacrB and ΔgalU mutants did not grow in bile salts (74); expression of the efflux pump genes mdtJ and mdtI induced in bile salts (179)
S. flexneri, S. sonnei, and S. dysenteriae	Biofilm	Biofilm formation in the presence of bile salts (74, 155)
Escherichia
E. coli	MdtM	Drug/proton antiporter that protects laboratory E. coli from bile salts, antibiotics, and proline-rich antimicrobial compounds (180)
EHEC	acrAB, cvpA, other genes	Mutations resulted in reduced survival in the presence of deoxycholate (181)
E. coli strains and pathovars	Biofilm	Biofilm formation in the presence of bile salts (74, 155)
Various E. coli isolates	Bile resistance	95% of E. coli isolates from bile samples of patients with biliary tract disease displayed resistance to multiple classes of antibiotics (182)
Salmonella enterica
Serovar Typhi	Production of antioxidant enzymes	Bile induces production of reactive oxygen species during gallbladder colonization, which is countered by the production of superoxide dismutase and catalase; production of these enzymes dependent on quorum sensing machinery (183)
	marA, marR	Transcriptomic analysis of serovar Typhi biofilms formed under gallbladder-like conditions induced expression of genes for the multiple antibiotic resistance transcriptional activator and repressor proteins involved in MDR (184)
	Biofilm	Biofilm formation in the presence of bile salts (74, 155)
Serovar Typhimurium	PhoP-PhoQ, c-di-GMP	The two-component and cyclic di-GMP signaling systems used for bile resistance during biofilm formation (185)
	Tat system	Twin-arginine translocation (Tat) system amidase-dependent cell division involved in bile acid resistance (186)
	CspE, YciF	Cold shock protein CspE and the previously uncharacterized YciF protein regulate bile resistance through porin degradation to decrease membrane permeability (187)
	RamR	Cholic and chenodeoxycholic bile acids bind the transcriptional repressor RamR, which results in increased expression of ramA and acrAB-tolC genes required for bile resistance (188)
	acrAB	Ciprofloxacin-resistant serovar Typhimurium displayed higher induction of acrAB and other efflux-related genes and subsequently had better fitness in 0.1% bile (189)
Vibrio
V. cholerae	ToxR, ToxS	Periplasmic interaction of transcription factors ToxR and ToxS during bile exposure (190)
	LeuO, ToxR	Transcriptional regulators LeuO and ToxR increase sensitivity to polymyxin B by repressing the two-component signal transduction system CarRS during bile salt exposure (191), which contrasts with enhanced bile salts and antibiotic resistance through the VexGH efflux pump (192)
	VacJ/Yrb	Represses expression of the VacJ/Yrb transporter during early mammalian infection stimulates outer membrane vesicle formation, modifications to the lipopolysaccharide structure, and depletion of the outer membrane porin OmpT to confer resistance to bile (193)
	Bile response	Role of calcium in bile salt-induced virulence (194)
V. parahaemolyticus	VtrABC	Bile salts stimulate a sensor receptor complex comprised of VtrA and VtrC to activate the transcriptional regulator VtrB that activates the type III secretion system (195)
V. cholerae and V. parahaemolyticus	cvpA	Mutation increases sensitivity to deoxycholate (181)
V. fluvialis	Bile resistance genes	An emerging pathogen known to cause outbreaks of gastroenteritis; two strains isolated from the gallbladders of patients with acute cholecystitis identified genes important for bile resistance (196), but the isolates were not resistant to any of the antibiotics tested despite resistance documentation in the literature (196, 197); potentially useful in our understanding of pathogen evolution and the interplay between bile and antibiotic resistance in future analyses
Campylobacter
C. jejuni	Bile resistance, AddAB	Genes important for deoxycholate resistance, including a conserved survival response by altering genes important for survival to reactive oxygen stress (198); DNA repair proteins repair DNA damage in the presence of deoxycholate (199)
C. jejuni and C. coli	Bile resistance	RNA sequencing characterized the transcriptomic response of a highly virulent sheep abortion clone found in sheep gallbladders; identified important differences in transcription between bacteria grown in vitro with bile compared to that of bacteria isolated from gallbladders; identified noncoding RNAs that may facilitate adaptation to the gallbladder (200); tetracycline in the feed of sheep increased C. jejuni and C. coli titers in bile and gallbladders; antibiotic resistance profiles were not affected, but 95% of C. coli isolates were resistant to fluoroquinolone (201)
Clostridium
C. difficile	PrkC	Serine/threonine kinase that maintains cell wall homeostasis to resist bile salts and other antimicrobial compounds (165)
	Spore germination	Spore germination in mice only occurs following antibiotic treatment to removal the commensal population (166); gut microbiota-derived secondary bile acids inhibit spore germination and growth (167)
	Clinical FMT analyses	Fecal microbiota transplantation analyses identified higher levels of secondary bile acids and short-chain fatty acids by fecal microbiota inhibited the growth of C. difficile (168, 169)
	Growth inhibition	Commensal microbes that convert bile acids into secondary bile acids enhance the inhibitory activity of secreted tryptophan-derived antibiotics (170)
Listeria
L. monocytogenes	Bile resistance	Increased bile resistance under anaerobic conditions and fewer morphological changes to the bacterial cells; proteomic analyses revealed altered expression of cell morphology, DNA repair, invasion, and metabolism proteins (202)
	PrfA	Fatty acid components found in bile reduced virulence gene activation through the downregulation of the PrfA regulatory protein (203)

^aEHEC, enterohemorrhagic E. coli; serovar Typhi, S. enterica serovar Typhi; serovar Typhimurium, S. enterica serovar Typhimurium.

TABLE 4.

Summary of bile resistance mechanisms in additional genera

Genus and species	Mechanism(s)	Comment (reference[s])a
Lactobacillus
L. acidophilus	LBA0552, LBA1429, LBA1446, LBA1679	Deletion of transporters increase sensitivity to bile and antibiotics (204)
L. reuteri ATCC 55730	Lr1584	Gene mutation decreased capability of the strain to grow in the presence of bile and annulled its capacity to acquire bile-tolerant phenotypes (204)
L. plantarum	BSH	BSH can lead to bile salt deconjugation and is an inducible activity in Lactobacillus; expression of the bsh gene in L. plantarum was increased 6-fold after exposure to 2% bile (205)
L. brevis	Gtf27, Gtf28, Orf2	Three plasmid-encoded exopolysaccharide genes contribute to bile resistance; genes gtf27 and gtf28 belong to the membrane-bound glycosyltransferase family, while orf29 has homology to an ABC transporter (206)
Bifidobacterium
B. breve	Bbr_0838	If gene is inactivated, reduces growth in the presence of cholic acid (204)
B. longum	Cholate efflux transporter	Presence of the ctr gene enables active extrusion of labeled bile (207)
	BetA	Gene exhibits high levels of transcriptional induction after bile exposure (208)
	BmrRAB	BmrR, homologous to MarR, autoregulates the bmrRAB operon in which ox bile binds to repress BmrR that enables expression of the bmrAB efflux transporter genes (209)
B. animalis	BSH	BSH appears overrepresented in a bile-adapted B. animalis strain; displays higher hydrolyzing activity (210)
Bacteroides
B. fragilis	BmeB1-BmeB16	There was an association of 16 RND-family efflux pump genes (bmeB1-bmeB16) with B. fragilis bile salt adaptation by an increased expression of efflux pumps (211)
	Biofilm	Biofilm production was enhanced by bile salts supporting the possibility that B. fragilis could grow as a biofilm during colonization of the human intestine (211)
	OmpA	Most abundant outer membrane protein in B. fragilis, overexpressed in cells exposed to bile salts, likely as a compensatory adaptive response to replace any damaged membrane proteins and restore membrane structure (211)
Streptococcus
S. thermophilus LMD-9	SDPs, PrtS, MucBP	Lactic acid bacterium widely used in dairy industry as a starter due to its high capability of milk fermentation and flavor producing characteristics; based on the experimental conditions and bile salts used, it appears to be either very sensitive or resistant to bile salts; wild-type LMD-9 can survive in a bile salt mixture (taurocholate, cholate, and deoxycholate) of 3 mM; inactivation of cell surface sortase-dependent proteins (SDPs), PrtS, and MucBP increases cell membrane permeabilization to bile salts and affects survival (212)
Laribacter
L. hongkongensis	AcrAB	L. hongkongensis is a recently discovered Gram-negative bacillus of the Neisseriaceae family associated with freshwater fish-borne gastroenteritis and traveler’s diarrhea; harbors genes for bile resistance that include three copies of acrAB (LHK_01425–LHK_01426, LHK_02129–LHK_02130, and LHK_02929–LHK_02930) (213)
	EmrAB homologs	Harbors two pairs of genes homologous to emrAB (LHK_01373–LHK_01374 and LHK_03132–LHK_03133) (213)
	Tol proteins	Five genes (tolQ [LHK_00053], tolR [LHK_03174], tolA [LHK_03173], tolB [LHK_03172], and pal [LHK_03171]) encode Tol proteins that are crucial for bile resistance and outer membrane integrity (213)
Helicobacter
H. pylori	HefC	H. pylori uses two mechanisms for resistance to bile salts: HefC, a putative efflux pump homologous to AcrB, to efflux the bile salts it encounters in vivo before exerting antibacterial effects on the membrane; alternatively, incorporation of cholesterol into the membrane to counteract bile salts, possibly decreasing bile salt permeability (214)
Francisella
F. tularensis	AcrAB	A null mutation in the gene acrB exhibited increased sensitivity to multiple antibiotics and antimicrobial compounds, including bile salts (215)
Shewanella
S. algae	Candidate genes	Gram-negative marine bacterium and emerging human pathogen; whole-genome sequencing of an isolate from human bile revealed several candidate genes associated with bile adaptation, including htpB, exbBD, wecA, galU, adeFGH, and phoPQ (216)
Aeromonas
Several species	To be determined	Gram-negative marine bacteria in which multiple species have been associated with human diseases (217); isolates from the biliary tract, bile duct, or bile have been documented, some of which are resistant to antibiotics (218–225)

^aThe bile resistance mechanisms of both commensal bacteria and additional pathogens are provided.

Conserved biofilm formation.

In addition to the conserved use of the AcrAB efflux pump, biofilm formation is another conserved response in enteric pathogens and much like what is observed for the ESKAPE pathogens. As part of our recent analyses (74, 155), we demonstrated robust biofilm formation in Shigella flexneri during bile salt exposure, which required glucose for production of the extracellular matrix. Biofilm formation occurred for multiple species of Shigella, Salmonella, and commensal and pathogenic E. coli (74, 155). Similar observations were previously demonstrated in Vibrio and Campylobacter (156, 157); and therefore, the combined data demonstrate a conserved enteric pathogen biofilm response to bile (24, 74, 155, 158). Biofilm dispersion analysis revealed a S. flexneri hypervirulent phenotype that related to observed transcriptional changes and confirmed previous observations of induced adherence and invasion following bile salt exposure (74, 158–161). We hypothesize that this biofilm formation is transient in nature to enable enteric pathogens to resist bile during transit of the jejunum of the small intestine. Upon bacterial translocation into the terminal ileum and colon, the reabsorption of bile enables biofilm dispersion for subsequent infection by the pathogens, thereby linking bile resistance and virulence.

Clostridium.

Analysis of the effects of bile on Clostridium (or Clostridioides) difficile have centered on germination and the greater abundance of primary bile acids in the colon to promote germination (24). Recent studies have significantly enhanced this understanding. First, calcium and amino acids help facilitate germination of C. difficile spores (162–164), while the C. difficile serine/threonine kinase PrkC helps maintain cell wall homeostasis to resist antimicrobial compounds, including bile salts and cephalosporins (165). Second, a comprehensive study used various antibiotic treatments to evaluate C. difficile germination and outgrowth in different commensal and bile acid environments in mice (166). Spore germination and outgrowth were detected in the small intestine under all conditions; however, germination occurred only following antibiotic treatment to remove the commensal population in the large intestine. A subsequent analysis confirmed these observations by demonstrating that gut microbiota-derived secondary bile acids inhibit spore germination, growth, and toxin activity (167).

More recently, two clinical analyses of fecal microbiota transplantation (FMT) to successfully treat C. difficile infection have provided further support for the effect of different bile salts on spore germination. The first study evaluated six patients with recurrent infection who received FMT therapy and found increased levels of short-chain fatty acids (SCFAs), variable recovery over time for secondary bile acids deoxycholate and lithocholate, and increased metabolites that corresponded to three microbiome families (168). The second study of 10 patients had similar findings of increased diversity of the fecal microbiota that correlated with genera known to inhibit C. difficile, higher levels of secondary bile acids, higher levels of SCFAs, and more importantly, lasting resolution of infection (169). Further insight into how the microbiome inhibits growth of C. difficile was provided by in vitro analysis of two commensal microbes, Clostridium scindens and Clostridium sordellii, which secrete tryptophan-derived antibiotics to inhibit the growth of C. difficile and other gut bacteria. Interestingly, these commensal strains can convert bile acids into secondary bile acids, and it was found that deoxycholate and lithocholate enhanced the inhibitory activity of the secreted antibiotics, which helps explain how gut-derived molecules can inhibit C. difficile growth (170). The studies have confirmed the mechanisms of induced C. difficile infection following antibiotic treatment, offer promising insight into FMT therapy, and have important implications for future therapeutic development. In fact, researchers have pursued using bile acid analogues to inhibit spore germination (171). Interestingly, Clostridium perfringens sporulates in the presence of deoxycholate, and the expression of the C. perfringens enterotoxin is also induced by bile salts (172, 173). Indeed, the different responses of two related species to various bile salts highlight an interesting divergence in pathogen evolution and could lead to future comparison studies to enhance our understanding of resistance and the Clostridium response to bile.

CONCLUDING REMARKS

The use of efflux pumps, outer membrane alterations, and biofilm formation are certainly conserved bacterial responses to bile. In addition, there is a clear link to enhanced antibiotic resistance phenotypes in bacterial pathogens following exposure to bile. To provide further evidence of this link, a recent study evaluated antimicrobial activity of antibiotics against E. faecalis and E. coli following exposure to pooled human bile in broth medium (174). Growth was either consistent or increased in the presence of human bile, respectively, for E. coli or E. faecalis. Furthermore, bile reduced the antimicrobial activity of ciprofloxacin, meropenem, and tigecycline for E. coli while linezolid and tigecycline had reduced activity against E. faecalis. The authors note that the stability of tigecycline was significantly reduced in bile (174), and investigations into the stability of antibiotics in bile are certainly warranted. As we move forward in the current era of rampant antibiotic resistance, novel therapeutic design is key to ensure our future success against ESKAPE and enteric pathogens. Paradigm-shifting, innovative approaches and solutions are urgently needed to counter the grim outlook in our ability to treat bacterial infections. The discovery of antibiotics that are more effective during bile exposure and the use of new alternatives, including bacteriophages, synthetic peptides, biofilm-targeting therapeutics, combination therapies, and of course vaccine development, offer promising strategies to ensure our future success against such elite pathogens. The extensive research highlighted in this review certainly provides an optimistic outlook on the ability of the scientific community to overcome this challenge.