Molecular Mechanisms of Antibiotic Resistance in Uropathogenic Escherichia coli: A Narrative Review

1. Virulence Factors of UPEC

UPEC possesses various virulence factors that facilitate adhesion to host cells, nutrient acquisition, and immune system evasion, which contribute to bacterial survival and pathogenesis [3,18]. These include adhesins, toxins, and iron acquisition systems that act synergistically to allow bacterial survival in the urinary tract [14,19,20].

A critical step in UPEC pathogenesis is the adherence to the urothelium, which is mediated by adhesion factors that enable bacterial colonization and invasion. Type 1 fimbriae (FimH), in particular, provides a crucial role in the initial attachment to bladder epithelial cells, allowing for bacterial invasion and establishment of intracellular bacterial communities (IBCs). FimH binds to mannosylated glycoproteins on bladder epithelial cells, promoting invasion and biofilm formation, contributing to recurrent UTIs. Biofilm formation creates a physical barrier that impedes antibiotic penetration, leading to insufficient drug concentrations in the deeper layers and allowing subpopulations of bacteria to remain unaffected. In contrast, adhesion in kidneys is facilitated through P fimbriae (PapG adhesin). P fimbriae recognize Gal(α1-4)Gal receptors on kidney epithelial cells, facilitating upper UTIs and pyelonephritis [21,22].

In addition to adhesion, UPEC produces toxins that disrupt host cell integrity and modulate immune responses.

Hemolysin (HlyA), a pore-forming transmembrane toxin, induces lysis of host cell, and evasion of the immune system [23-25]. Cytotoxic necrotizing factor 1 interferes with host cell signaling pathways, enhancing bacterial invasion and intracellular survival [26,27]. These virulence factors contribute to host tissue damage and bacterial survival within the urinary tract.

Furthermore, to overcome iron limitations in the urinary tract, UPEC utilizes high-affinity iron acquisition systems to compete against the host for iron. Siderophores such as enterobactin and aerobactin scavenge iron from host proteins to provides iron to support bacterial growth and virulence [28]. The expression of these iron acquisition systems is tightly regulated in response to iron availability, allowing for UPEC to efficiently adapt to the host environment [29].

2. Infection Process

The infection mechanism of UPEC is a coordinated cascade of events that enable bacterial colonization, immune evasion, and long-term persistence [16]. Following initial adhesion via type 1 fimbriae, UPEC invades urothelial cells and forms IBCs, which develop biofilm-like aggregates that protect the bacterium from immune clearance and antibiotic treatment, leading to persistent and recurrent infections [30-32]. To further evade host immune responses, the modification of lipopolysaccharides reduces the efficacy of antimicrobial peptides and complement-mediated killing of bacteria, while the expression of a polysaccharide capsule inhibits phagocytosis by immune cells [33,34]. Additionally, UPEC secretes immune-modulating factors that interfere with neutrophil recruitment and cytokine signaling, further impairing host defenses [17,35].

As the infection progresses, some UPEC cells transition into a quiescent intracellular state, forming quiescent intracellular reservoirs within bladder epithelial cells. These dormant bacterial populations evade immune responses and antibiotic treatment, but reactivate under favorable conditions to cause recurrent infections [36]. This cycle of dormancy and reactivation poses a significant therapeutic challenge, necessitating targeted strategies against persistent UPEC populations [37,38].

1. TMP-SMX Resistance

UPEC employs diverse molecular mechanisms to resist commonly prescribed antibiotics. The major strategies include enzymatic inactivation, target site modification, reduced membrane permeability, and efflux pump overexpression. These mechanisms compromise the efficacy of key antibiotic classes used to treat urinary tract infection, such as TMP-SMX, β-lactams, and fluoroquinolones (Table 1).

Table 1.

Major antibiotic resistance mechanisms in uropathogenic Escherichia coli

TMP-SMX is a combination antibiotic that targets the bacterial folate synthesis pathway, which is essential for nucleotide biosynthesis and DNA replication (Fig. 1) [39]. Trimethoprim selectively inhibits dihydrofolate reductase (DHFR), preventing the reduction of dihydrofolate (DHF) to tetrahydrofolate (THF), a critical cofactor in DNA synthesis [40]. Sulfamethoxazole inhibits dihydropteroate synthase (DHPS), an enzyme required for the biosynthesis of DHF. By synergistically disrupting folate metabolism, TMP-SMX exerts a strong bactericidal effect against E. coli [41]. Despite its long-standing clinical use, resistance to TMP-SMX has become increasingly prevalent among UPEC strains, reducing its efficacy in UTI treatment. Epidemiological studies report substantial regional variations in TMP-SMX resistance rates, with resistance levels ranging from 14.6% to 60%, posing a major challenge to empirical treatment strategies [4]. In the United States, surveillance data from 2017 indicated that 32.1% of UPEC isolates were resistant to TMP-SMX, with resistance rates reaching up to 43.5% in certain regions [6]. Furthermore, patients with recurrent UTIs or recent exposure to TMP-SMX exhibited a significantly higher risk of resistance [42]. This increasing prevalence of TMP-SMX resistance has necessitated a reassessment of its role as a first-line treatment and stresses the urgent need for alternative therapeutic strategies.

Fig. 1.

Mechanisms of antibiotics resistance (TMP-SMX, β-lactams, and fluoroquinolones) and multidrug resistance in UPEC. (A) TMP-SMX inhibits folate synthesis by blocking both DHPS and DHFR, preventing the conversion of PABA into DHF and ultimately inhibiting the reduction of DHF into THF, the active form of folate. (B) Beta-lactamases recognize and bind to the β-lactam ring of antibiotics, hydrolyzing the ring and converting it into a linear, inactive structure. (C) Point mutations in native pbp genes reduce β-lactam binding affinity, rendering antibiotics ineffective. Horizontal gene transfer of novel pbp genes can also decrease β-lactam efficacy. (D) Mutations in gyrA and gyrB alter fluoroquinolone-binding pockets in DNA gyrase, reducing drug affinity. Similarly, mutations in parC and parE modify binding sites in topoisomerase IV. (E) MarA and SoxS upregulate the AcrAB-TolC efflux system, actively pumping antibiotics out of the cell. They also downregulate expression of OmpF, a large outer membrane porin, thereby reducing antibiotic influx, while OmpC, which forms a smaller pore, is upregulated. Under normal conditions, MarR represses the marA operon; however, mutations can abolish this repression. SoxS is activated by SoxR, which may become constitutively active even in the absence of oxidative stress. TMP-SMX, trimethoprim-sulfamethoxazole; UPEC, uropathogenic Escherichia coli; DHPS, dihydropteroate synthase; DHFR, dihydrofolate reductase; PABA, para-aminobenzoic acid; DHF, dihydrofolate; THF, tetrahydrofolate; PBP, penicillin-binding protein.

The primary mechanism of TMP-SMX resistance involves the acquisition of plasmid-encoded resistance genes, encoding alternative forms of DHFR and DHPS with lowered affinities for TMP and SMX, respectively [43]. The dfrA and sul gene families play a central role in this resistance, as dfrA genes (e.g., dfrA1, dfrA5, dfrA12) encode trimethoprim-resistant DHFR variants that allow bacterial folate synthesis to continue despite TMP exposure, while sul genes (sul1, sul2, sul3) encode sulfamethoxazole-resistant DHPS enzymes, preventing SMX from inhibiting folate biosynthesis [44,45]. These genes are often carried on integrons and transposons, facilitating horizontal gene transfer and promoting their widespread dissemination among UPEC isolates [46].

In addition to plasmid-mediated resistance, chromosomal mutations in endogenous dhfr and dhps genes have also been identified in TMP-SMX-resistant UPEC isolates. These mutations alter the three-dimensional structures of DHFR and DHPS, reducing drug-binding affinity while preserving enzymatic activity [43,47]. The combination of these resistance mechanisms allows UPEC to survive despite antibiotic exposure, increasing the adaptability of this pathogen in response to selective pressure.

2. Beta-Lactam Resistance

Beta-lactam antibiotics inhibit penicillin-binding proteins (PBPs), which are important for bacterial cell wall synthesis. PBPs catalyze the cross-linking of peptidoglycan layers, a process necessary for maintaining cell wall integrity [48]. By irreversibly binding to PBPs, β-lactams disrupt peptidoglycan synthesis, leading to cell lysis and bacterial death [49]. However, UPEC has evolved multiple mechanisms to evade β-lactam activity, significantly reducing the efficiency of these antibiotics in UTI treatment.

A primary mechanism of β-lactam resistance in UPEC is the production of β-lactamases, enzymes that hydrolyze β-lactam antibiotics, rendering them ineffective. Among these, ESBLs, such as CTX-M type β-lactamase enzymes (named for their high level of activity against cefotaxime [CTX] and first observed in Münich [M]), TEM (named after a Greek patient, Temoneira, whose clinical samples yielded this gene), and SHV-type enzymes (originally reported in Klebsiella pneumoniae clinical isolate and exhibit an overall preference for hydrolyzing sulfhydryl-containing cephalosporins [hence the SHV name]), confer resistance to a broad range of β-lactams, including third-generation cephalosporins and monobactams [50-53].

MEGs play a crucial role in the rapid dissemination of β-lactam resistance in UPEC. Plasmids carrying bla_CTX-M, bla_TEM, and bla_SHV facilitate horizontal gene transfer, promoting the cotransfer of multiple resistance determinants via integrons and transposons [54]. This accumulation of resistance genes within a single UPEC strain reduces treatment efficacy and increases the risk of therapeutic failure. Additionally, AmpC β-lactamases, often plasmid-encoded, hydrolyze cephamycins and penicillins while evading inhibition by traditional β-lactamase inhibitors such as clavulanic acid [55].

In addition to β-lactam degradation, mutations in PBPs also contribute to β-lactam resistance by reducing antibiotic binding affinity. Certain UPEC strains have mutations in both PBP2 and PBP3, leading to structural modifications that impair β-lactam binding, allowing for peptidoglycan synthesis even in the presence of antibiotics [16,56]. Another key mechanism involves the regulation of peptidoglycan remodeling, which enhances bacterial survival under β-lactam stress. Mutations in nlpD, a regulator of peptidoglycan hydrolases, have been linked to modifications in cell wall integrity, reducing the impact of β-lactam antibiotics without compromising cell viability [10,57]. This ability to alter cell wall synthesis pathways allows UPEC to tolerate β-lactam exposure while maintaining structural stability. The combination of β-lactamase production, PBP modifications, and cell wall remodeling emphasizes the complexity of β-lactam resistance in UPEC. These mechanisms allow UPEC to persist despite β-lactam treatment, contributing to recurrent infections and therapeutic challenges.

3. Fluoroquinolone Resistance

Fluoroquinolones are broad-spectrum antibiotics that target DNA gyrase (gyrA) and topoisomerase IV (parC), essential enzymes for maintaining DNA supercoiling during bacterial replication. By inhibiting these enzymes, fluoroquinolones disrupt DNA replication, ultimately leading to bacterial cell death [58]. However, UPEC has developed multiple resistance mechanisms that significantly reduce fluoroquinolone efficacy, contributing to treatment failure.

A primary mechanism of fluoroquinolone resistance is target site modification, which occurs via point mutations in the quinolone resistance-determining regions (QRDRs) of gyrA and parC. These mutations alter the structure of DNA gyrase and topoisomerase IV, reducing their binding affinity for fluoroquinolones. Among the most commonly observed mutations, S83L and D87N in gyrA and S80I and E84K in parC significantly impair fluoroquinolone binding, allowing bacterial replication to proceed despite antibiotic exposure [59,60]. Fluoroquinolone treatment can induce RecA-mediated activation of the LexA-controlled SOS response, increasing mutation rates in QRDRs and accelerating resistance development [61].

In addition to target site modifications, plasmid-mediated quinolone resistance (PMQR) genes further promote UPEC survival under fluoroquinolone exposure. Genes such as qnrA, qnrB, and qnrS encode proteins that bind to DNA gyrase and topoisomerase IV, shielding them from fluoroquinolone inhibition [62,63]. Unlike chromosomal QRDR mutations, which alter enzyme structure, PMQR genes do not modify target enzymes but instead prevent fluoroquinolones from binding effectively [64]. These genes are frequently located on conjugative plasmids, enabling horizontal gene transfer and facilitating the rapid dissemination of fluoroquinolone resistance among UPEC strains.

The presence of aminoglycoside acetyltransferase AAC(6′)-Ib-cr, a PMQR gene embedded within a gene cassette or an integron, further provides an additional resistance mechanism by modifying fluoroquinolones through acetylation. Originally identified for its role in aminoglycoside resistance, AAC(6′)-Ib-cr can also reduce the activity of ciprofloxacin and norfloxacin, further diminishing fluoroquinolone efficacy [65,66]. This dual-resistance function enhances UPEC adaptability under antibiotic pressure, contributing to multidrug resistance. The combination of QRDR mutations, plasmid-mediated protection, and enzymatic drug modification underlines the complexity of fluoroquinolone resistance in UPEC, allowing the bacterium to persist despite fluoroquinolone treatment, leading to recurrent infections and limited therapeutic options.

4. Multidrug Resistance

The emergence of MDR UPEC poses a significant challenge in UTI treatment, as these strains exhibit resistance to multiple antibiotic classes, including the aforementioned β-lactams, fluoroquinolones, and TMP-SMX. A recent experimental evolution studies on ampicillin-resistant UPEC has demonstrated that resistance to a single β-lactam antibiotic can rapidly extend to multiple drug classes, including ciprofloxacin, tetracycline, and chloramphenicol [10]. This phenomenon is primarily driven by mutations in global regulators such as marR (V66F), acrR, and envZ (T402M), which collectively lead to AcrAB-TolC efflux pump overexpression, differential regulation of porin proteins (OmpF loss and OmpC increase), and reduced membrane permeability, thus facilitating cross-resistance to unrelated antibiotics. Efflux pump overexpression and porin modifications play central roles in conferring MDR in UPEC strains [10,67,68]. Among these, the AcrAB-TolC efflux system is a key contributor to MDR phenotypes. This tripartite resistance-nodulation-division pump actively expels fluoroquinolones, β-lactams, and TMP-SMX, lowering intracellular antibiotic concentrations and reducing drug efficacy. Mutations in marR, acrR, and soxS disrupt efflux pump repression, leading to constitutive overexpression of AcrAB-TolC [10]. Additionally, overexpression of MdtK, a member of the major facilitator superfamily, has been linked to enhanced fluoroquinolone resistance in MDR UPEC isolates [69].

Alongside efflux-mediated drug extrusion, porin downregulation and structural modifications significantly contribute to MDR. The loss or mutation of outer membrane porins, particularly OmpF and OmpC, restricts antibiotic influx, thereby lowering intracellular drug concentrations [70]. These changes are often regulated by the EnvZ-OmpR 2-component system, which modulates porin expression in response to environmental stressors [10]. The combination of increased efflux activity and reduced membrane permeability creates an impermeable bacterial barrier, reinforcing MDR phenotypes and limiting the efficacy of multiple antibiotic classes [10].