Detection of possible resistance mechanisms in uropathogenic escherichia coli strains isolated from kidney transplant recipients based on whole genome sequencing

Academic Editor: Chiara Scapoli Received: 8 January 2025 Revised: 4 February 2025 Accepted: 6 February 2025 Published: 11 February 2025 Citation: Herrera-Espejo, S.; Rubio, A.; Ceballos-Romero, L.; Pachón, J.; Cordero, E.; Pérez-Pulido, A.J.; Pachón-Ibáñez, M.E. Detection of Possible Resistance Mechanisms in Uropathogenic Escherichia coli Strains Isolated from Kidney Transplant Recipients Based on Whole Genome Sequencing. Biomolecules 2025,15, 260. https://doi.org/10.3390/ biom15020260 Copyright: © 2025 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/ licenses/by/4.0/). Article Detection of Possible Resistance Mechanisms in Uropathogenic Escherichia coli Strains Isolated from Kidney Transplant Recipients Based on Whole Genome Sequencing Soraya Herrera-Espejo 1,† , Alejandro Rubio 2,† , Lucía Ceballos-Romero 1, Jerónimo Pachón 3,4 , Elisa Cordero 1,4,5,* , Antonio J. Pérez-Pulido 2,* and María Eugenia Pachón-Ibáñez 1,5 1Clinical Unit of Infectious Diseases, Microbiology and Parasitology, Institute of Biomedicine of Seville (IBiS), Virgen del Rocio University Hospital/CSIC/University of Seville, 41013 Seville, Spain; [email protected] (S.H.-E.); [email protected] (L.C.-R.); [email protected] (M.E.P.-I.) 2Andalusian Centre for Developmental Biology (CABD, UPO-CSIC-JA), Faculty of Experimental Sciences (Genetics Area), University Pablo de Olavide, 41013 Seville, Spain; [email protected] 3Institute of Biomedicine of Seville (IBiS), Virgen del Rocio University Hospital/CSIC/University of Seville, 41013 Seville, Spain; [email protected] 4Department of Medicine, School of Medicine, University of Seville, 41004 Seville, Spain 5CIBER de Enfermedades Infecciosas (CIBERINFEC), Instituto de Salud Carlos III, 28029 Madrid, Spain *Correspondence: mcorder[email protected] (E.C.); ajper[email protected] (A.J.P.-P.) †These authors contributed equally to this work. Abstract: Background: Urinary tract infections are a global health concern, with uropathogenic Escherichia coli (UPEC) accounting for 80–90% of cases. Given the rise in antimicrobial resistance, our aim was to elucidate the genetic mechanisms behind low-level resistance to ciprofloxacin and fosfomycin (LLCR and LLFR) in UPEC strains, using whole-genome sequencing (WGS) to identify point mutations in chromosomal and plasmid genes. Methods: A cohort UPEC was collected from kidney transplant recipients at the Virgen del Rocío University Hospital, Spain. Minimum inhibitory concentrations were determined for ciprofloxacin and fosfomycin to categorize strains into LLCR and LLFR. Twenty strains were selected for WGS, with genome annotations. Point mutations were identified and analyzed using alignment tools, and protein stability changes were predicted. Results: LLCR strains exhibited mutations in key quinolone resistance-determining regions of the gyrA gene, in 83% of cases. The qnrS1 plasmid gene was found in 17% of LLCR strains. LLFR strains showed mutations in the glpT and cyaA genes. Mutations in the uhp gene family were linked to the fosfomycinresistant phenotype, suggesting a multi-step resistance evolution mechanism. Conclusions: This study highlights the complex interplay between chromosomal and plasmid genes in UPEC’s resistance to ciprofloxacin and fosfomycin. The findings contribute to understanding low-level resistance mechanisms and may guide the development of novel therapeutic strategies to combat multidrug-resistant strains. Keywords: whole-genome sequencing; antimicrobial resistance; uropathogenic Escherichia coli 1. Introduction Urinary tract infections (UTIs) are a common health problem that affects a significant portion of the global population. Every year, around 150 million people worldwide develop UTIs, with substantial social and economic costs [ 1 , 2 ]. UTIs are more common in women, with an estimated 40% developing at least one UTI during their lifetime, and Biomolecules 2025,15, 260 https://doi.org/10.3390/biom15020260 Biomolecules 2025,15, 260 2 of 15 11% experiencing an episode of UTI each year [ 2 ]. The mean cost of each UTI is estimated at EUR 5700 and EUR 6987 in Europe and USA, respectively [ 3 , 4 ]. UTIs are caused by the presence of bacteria ( ≥ 10 5 CFU/mL) in the urine with or without symptoms associated (asymptomatic bacteriuria). Symptomatic UTIs are classified according to severity as urosepsis syndrome, pyelonephritis or upper UTI (kidney infection), and cystitis or lower UTI (bladder infection) [ 5 ]. Moreover, UTI is a common and serious problem among kidney transplant recipients (KTR), ranging from 40% to 50% [ 6 , 7 ]. This prevalence depends on factors such as preventive strategies and transplant characteristics. Several factors can increase the susceptibility to develop a UTI [ 8 ]. Among the risks that UTI might cause in KTR include immunosuppression, bladder catheterization, ureteral stents, deceased donor transplantation, and acute rejection episodes [9]. Uropathogenic Escherichia coli (UPEC) is the primary cause of UTIs acquired in the community, accounting for 80–90% of cases [ 1 , 2 ]. UPEC can be classified into different groups based on the presence of genomic pathogenicity islands (PAIs) and virulence factors [ 10 ]. UPEC uses a wide variety of virulence factors to colonize the bladder, including lipopolysaccharide (LPS), polysaccharide capsule, flagella, pili, and TonB-dependent ironuptake receptors [ 8 ], all of which are potential targets for the development of new drugs and/or vaccines. At present, despite the increase in antimicrobial resistance, there are numerous options for the treatment of UTIs. The European Antimicrobial Resistance Report 2021 (EARS-Net) informed that approximately 50% of E. coli strains were resistant to at least one group of antimicrobials. Ciprofloxacin resistance rates have increased up to 50% in the global scenario [ 11 ]. Specifically, in Spain, resistance rates of up to 30% for ciprofloxacin and 17% for aminoglycosides have been reported [ 12 ]. One of the primary mechanisms of antibiotic resistance in bacteria is the acquisition of genetic mutations that confer resistance to one or more antibiotics. Point mutations, which involve changes in a single nucleotide within the genome, are a common type of genetic mutation that can lead to antibiotic resistance [ 13 ]. Moreover, point mutations can be classified as synonymous (mutations or alterations that do not change the amino acid sequence of the protein) or non-synonymous (mutations that change the amino acid sequence of the protein, resulting in substitutions, deletions . . . ), and occur spontaneously or can be induced by exposure to the antibiotics. These mutations can lead to changes in the target sites or efflux pumps of antibiotics, or alterations in the bacterial cell wall or membrane that reduce antibiotic uptake [ 14 ]. The study of antibiotic resistance in bacteria has traditionally relied on phenotypic methods, such as testing to determine the susceptibility/resistance profile of a given strain. However, with the arrival of next-generation sequencing technologies and bioinformatics tools, it is now possible to study the genetic basis of antibiotic resistance in more detail [ 15 ]. Currently, the most commonly used antibiotics are fosfomycin, ciprofloxacin, and amoxicillin-clavulanate for UTIs caused by UPEC [ 16 ]. Chromosomal and plasmid-mediated mechanisms on their own might confer low-level ciprofloxacin or fosfomycin resistance (LLCR or LLFR), which is clinically translated as a susceptible phenotype. However, the accumulation of multiple mutations could lead to clinical resistance [17]. These cumulative antimicrobial resistance genes could be acquired in nature from other bacteria [18] or even phages [19]. Thus, the aim of this study was to analyze chromosomal and plasmid genes involved in low-level resistance to ciprofloxacin and/or fosfomycin in UPEC strains collected from a well-characterized cohort of KTR, to identify point mutations that might be responsible for these. By characterizing these low-level genetic mechanisms of resistance, we seek to better understand the mechanisms underlying resistance of the mentioned antibiotics. Ultimately, the results may contribute to the development of new strategies to combat Biomolecules 2025,15, 260 3 of 15 infections caused by these strains, for example, by identifying new therapeutic targets or by optimizing treatment. 2. Materials and Methods 2.1. Antimicrobial Susceptibility, Whole-Genome Sequencing and Multilocus Sequence Typing (MLST) of Escherichia coli Strains One hundred and fifteen strains were collected in an observational cohort of adult KTRs with E. coli and UTI episodes (cystitis and asymptomatic bacteriuria [AB]), who signed the informed consent form (ethical approval numbers: FIS-CIP-2016-01 and FISFOS-2020-01) and attended as outpatients at the Virgen del Rocío University Hospital, Seville, Spain, from January 2017 to December 2019 [ 20 ]. Clinical strains were collected, and the hospital microbiology service identified the bacterial strains using a MicroScan WalkAway ® Plus system (Beckman Coulter, Nyon, Switzerland), and performed susceptibility testing with standard tests. Moreover, ciprofloxacin and fosfomycin Minimal Inhibitory Concentrations (MICs) were determined in duplicate, by broth microdilution or agar diffusion, respectively [ 20 ]. The results were interpreted according to the European Committee on Antimicrobial Susceptibility Testing (EUCAST) breakpoints for both antibiotics [ 21 ]. Antimicrobials to perform the MIC were purchased as standard powders (Sigma-Aldrich, Madrid, Spain) and E. coli ATCC 25922 was used as a quality control strain. MIC breakpoints used to designate susceptibility and resistance for ciprofloxacin were ≤ 0.001 µ g/mL and >0.50 µ g/mL and, in the case of fosfomycin, the breakpoint used to designate resistance was >8 µ g/mL. MIC values of >0.06 to 0.5 mg/L and 4 to 8 mg/L were considered to be classified as LLCR and LLFR, respectively. After MIC determinations, 20 UPEC strains were selected for in silico studies, 12 LLCR and 8 LLFR. Briefly, DNA from these strains was extracted (QIAamp ® DNA Mini Kit, Venlo, The Netherlands). Sequencing was performed using the MiSeq platform (Illumina), following standard protocols for WGS paired-end, producing 2 × 300 bp fragment reads. Unicycler was chosen for de novo assembly of the reads into contigs [ 22 ] (BioProject number: PRJNA1219036). Prodigal v2.6.3 was used to predict the protein-coding genes and translate them into their corresponding amino acid sequences [ 23 ]. The bacterial taxonomic division of the UniProt v2023_05 database was used for the functional annotation of the predicted proteins, using Sma3s v2 [24]. The contig files obtained from the assembly of each UPEC isolate were uploaded to the MLST web server (v. 2.0) of the Centre for Genomic Epidemiology (CGE) to perform MLST, following the schemes of Pasteur [ 25 ] and Achtman [ 26 ]. The settings for the analysis of these three web-based programs were established using a threshold of 90% identity and 80% for sequence coverage. 2.2. Quinolone and Fosfomycin Resistant Genes Sequence Alignments and Analysis For the study of ciprofloxacin resistance genes, a total of eleven plasmid genes (aac6’- 1b-cr,qnrA,qnrB,qnrS,qnrC,qnrD,qnrE,qepA,oqxA,oqxB, and crpP) and eight chromosomal genes (gyrA,gyrB,parC,parE,marR,acrR,norB, and rpoB) associated with quinolone resistance were examined. In the case of fosfomycin, eight plasmid genes (fosA3,fosA4,fosA5, fosB,fosX,fosC,fomA, and fomB) and nine chromosomal genes (murA,uhpA,uhpB,uhpC, uhpT,glpT,ptsl,cyaA, and crp) were analyzed. To examine these genes, the amino acid sequences of the encoding proteins predicted by Prodigal were aligned, using MAFFT (v. 7.526), to the reference amino acid sequences of the proteins obtained from The Comprehensive Antibiotic Resistance (CARD, v. 5.0.2) with default settings [ 27 ]. The pheatmap library from the R language was used to create a heatmap with the results obtained [28]. Biomolecules 2025,15, 260 4 of 15 Subsequently, the structural stability of the mutated proteins was investigated using the Protein Variation Effect Analyzer (Provean v. 2.6.3) [ 29 ] and the CUPSAT [ 30 ] and DDMut [ 31 ] algorithms, which calculate Gibbs energy to predict changes in protein stability. To study the effect of mutations on the folding of the protein encoded by the uhpB gene, a 3D affine crystal structure was generated with the algorithm AlphaFold [ 32 ] and visualized using PyMol [ 33 ], representing the different mutations calculated in the previous steps of the study. 2.3. Identification of Unknown Resistance and Virulence Mechanisms The data assembled from the 20 strains were used to identify resistance mechanisms. For this, CGE’s ResFinder web server (v. 4.1) [ 34 , 35 ], CGE’s Plasmid Finder web server (v. 2.1) [ 36 , 37 ], and NCBI’s AMRFinderPlus (v. 3.11) [ 38 ] were used for the acquired antimicrobial resistance genes. The thresholds for the analysis in these three web-based programs were established as 90% identity and 80% sequence coverage. CGE’s VirulenceFinder (v. 2.0.5) [ 35 , 39 , 40 ] web server was used for the identification of genes encoding virulence factors, with thresholds of 90% identity and 80% sequence coverage. 3. Results 3.1. Antimicrobial Susceptibility and Multilocus Sequence Typing (MLST) of Escherichia coli Strains Selected for Sequencing MICs were determined for the 115 UPEC strains from the patients included in the cohort. Of these, 20 strains presented low-level resistance: 12 were categorized as LLCR and 8 as LLFR (Tables 1and 2). Demographics and characteristics of KTRs with UTI by LLCR or LLFR E. coli strains are described in Supplementary Table S1. LLCR strains exhibited a range of MIC values from 0.12 to 0.50 µ g/mL and LLFR from 4 to 8 µ g/mL. Two ciprofloxacin-susceptible strains (strains 5 and 44, MIC ≤ 0.015 mg/L) and two fosfomycinsusceptible strains (strains 145 and 139, MIC = 1 and 0.5 mg/L, respectively) were included as susceptibility controls. Table 1. Multilocus sequence type and minimum inhibitory concentration of Escherichia coli strains with low resistance to ciprofloxacin. Strains MLST MIC (µg/mL) Pasteur Achtman 5 * ST509 ST625 <0.015 (S) 44 * Unknown (nearest 74) Unknown (nearest 12,985) <0.015 (S) 57 ST831 ST1136 0.12 149 ST2 ST548 0.06 14 ST14 ST68 0.25 64 ST3 ST5021 0.12 17 Unknown (nearest 132) ST48 0.12 126 ST14 ST68 0.50 18 Unknown (nearest 35) Unknown (nearest 1439) 0.12 164 ST634 ST429 0.25 140 Unknown (nearest 24) Unknown (nearest 889) 0.50 167 ST634 ST429 0.25 26 Unknown (nearest 14) ST68 0.25 168 ST35 ST12 0.12 *: Clinical strains 5 and 44 were added as control susceptible (S) strains. Biomolecules 2025,15, 260 5 of 15 Table 2. Multi-locus sequence type and minimum inhibitory concentration of Escherichia coli strains with low resistance to fosfomycin. Strains MLST MIC (µg/mL) Pasteur Achtman 145 * ST7 ST23 1 (S) 139 * ST3 ST69 0.50 (S) 44 Unknown (nearest 74) Unknown (nearest 12,985) 2 149 ST2 ST548 8 112 Unknown (nearest 966) Unknown (nearest 4195) 2 8 ST83 ST93 2 156 ST471 ST410 2 160 ST2 ST10 2 90 ST32 ST127 2 142 Unknown (nearest 14) ST68 2 *: Clinical strains 145 and 139 were added as control susceptible (S) strains. Including the susceptibility controls, Achtman’s MLST scheme appeared to be more accurate assigning ST to 11/14 (78.6%) and 8/10 (80.0%) of the studied strains, for ciprofloxacin and fosfomycin, respectively. Independently of the scheme used, the strains presented a varied clonality without any single clone standing out (Tables 1and 2). Pasteur MLST scheme assigned a ST to 9 out of 14 (64.3%), and 7 out of 10 (70.0%) of the strains for ciprofloxacin and fosfomycin, respectively. 3.2. Identification of Point Mutations in Chromosomal or Plasmid Genes Involved in Ciprofloxacin or Fosfomycin Resistance Additional analyses were performed on point mutations in the chromosomal and plasmid sequences to compare low-level resistance versus susceptible strains. For LLCR strains, 11 plasmid (aac6 ′ -1b-cr,qnrA,qnrB,qnrS,qnrC,qnrD,qnrE,qepA,oqxA,oqxB, and crpP) and 8 chromosomal (gyrA,gyrB,parC,parE,marR,acrR,sosX, and rpoB) genes associated with quinolone resistance were analyzed. We found that most of the point mutations we detected had been previously described in the Comprehensive Antibiotic Resistance Database (CARD) as conferring quinolone resistance (Supplementary Table S2). Regarding chromosomal genes, 83% (10/12) of the strains presented a point mutation in the quinolone-resistance-determining region of DNA gyrase (gyrA) (Figure 1A), a target enzyme for quinolones [ 41 ]. Specifically, Ser83Leu and Asp87Leu point mutations were observed (Figure 2and Supplementary Figure S1). Fifty percent (6/12) of the strains carried a non-previously described substitution mutation in parC and parE genes, encoding topoisomerase implied in bacterial replication [ 42 ], 33% also had a substitution in gyrB and acrR. While isolate 26 had a deletion in the gyrB gene, isolate 140 had a substitution plus deletion in the gyrB and parC genes. More than 91% (11/12) of the strains presented a point mutation in the marR gene, a repressor of the marRAB operon which is involved in the activation of both antibiotic resistance and oxidative stress genes [ 43 ]. Interestingly, all point mutations found in the already described genes involved in ciprofloxacin resistance were only present in LLCR strains and no point mutations were found in control strains. In addition, in plasmid mutations analysis, two strains (17%) carried a substitution in the qnrS1 gene (Supplementary Table S2). Biomolecules 2025,15, 260 6 of 15 Biomolecules 2025, 15, x FOR PEER REVIEW 6 of 15 While none of the LLFR strains carried point mutations in the plasmidic genes tested, 75% (6/8) of the LLFR strains carried a point mutation in cyaA, glpT, and ptsI genes. Conversely to that observed in LLCR strains, these genes were mutated in one of the control susceptible strains tested (Figure 1B). However, the strains used as controls, since they present a susceptible phenotype in the antibiotics analyzed, are clinical strains isolated from UTI in KTR patients, and it is expected to find point mutations in them. In addition, 50% (4/8) of the strains carried substitutions in the uhp genes family, specifically 25% (2/8) of the LLFR strains carried a substitution affecting the coding region of hexose-phosphate transporter (UhpT) gene involved in the fosfomycin transportation into the bacteria [44]. Also, substitutions in the uhp genes family were observed in control susceptible strains, and only 3/9 (33%) chromosomic genes analyzed showed no mutations in control strains. Specifically, for the uhpB gene, some of the amino acidic substitutions carried by LLFR strains were found among positions from 311 to 499, the histidine kinase domain (Figure 3). Finally, isolate 160 carried a point mutation in the murA and crp genes. Additionally, no mutations were observed in the analysis of plasmid mutations (Figure 1B and Supplementary Table S3). Figure 1. Heatmap of the accumulation of point mutations in the low-level ciprofloxacin resistance (LLCR) and low-level fosfomycin resistance (LLFR) strains. Abundance of point mutation in genes (axis Y) involved in (A) low-level quinolone resistance (LLCR, axis X) strains and (B) low-level fosfomycin resistance (LLFR, axis X) strains. Blue: Susceptible strains; Salmon: LLCR strains; Gray: LLFR strains; White: LLCR or LLFR strains with any point mutation in the studied genes involved in resistance. Range of colors from brown to blue: LLCR or LLFR strains with an accumulation from 1 to 8 point mutations in the studied genes involved in resistance. Figure 1. Heatmap of the accumulation of point mutations in the low-level ciprofloxacin resistance (LLCR) and low-level fosfomycin resistance (LLFR) strains. Abundance of point mutation in genes (axis Y) involved in (A) low-level quinolone resistance (LLCR, axis X) strains and (B) low-level fosfomycin resistance (LLFR, axis X) strains. Blue: Susceptible strains; Salmon: LLCR strains; Gray: LLFR strains; White: LLCR or LLFR strains with any point mutation in the studied genes involved in resistance. Range of colors from brown to blue: LLCR or LLFR strains with an accumulation from 1 to 8 point mutations in the studied genes involved in resistance. Biomolecules 2025, 15, x FOR PEER REVIEW 6 of 15 While none of the LLFR strains carried point mutations in the plasmidic genes tested, 75% (6/8) of the LLFR strains carried a point mutation in cyaA, glpT, and ptsI genes. Conversely to that observed in LLCR strains, these genes were mutated in one of the control susceptible strains tested (Figure 1B). However, the strains used as controls, since they present a susceptible phenotype in the antibiotics analyzed, are clinical strains isolated from UTI in KTR patients, and it is expected to find point mutations in them. In addition, 50% (4/8) of the strains carried substitutions in the uhp genes family, specifically 25% (2/8) of the LLFR strains carried a substitution affecting the coding region of hexose-phosphate transporter (UhpT) gene involved in the fosfomycin transportation into the bacteria [44]. Also, substitutions in the uhp genes family were observed in control susceptible strains, and only 3/9 (33%) chromosomic genes analyzed showed no mutations in control strains. Specifically, for the uhpB gene, some of the amino acidic substitutions carried by LLFR strains were found among positions from 311 to 499, the histidine kinase domain (Figure 3). Finally, isolate 160 carried a point mutation in the murA and crp genes. Additionally, no mutations were observed in the analysis of plasmid mutations (Figure 1B and Supplementary Table S3). Figure 1. Heatmap of the accumulation of point mutations in the low-level ciprofloxacin resistance (LLCR) and low-level fosfomycin resistance (LLFR) strains. Abundance of point mutation in genes (axis Y) involved in (A) low-level quinolone resistance (LLCR, axis X) strains and (B) low-level fosfomycin resistance (LLFR, axis X) strains. Blue: Susceptible strains; Salmon: LLCR strains; Gray: LLFR strains; White: LLCR or LLFR strains with any point mutation in the studied genes involved in resistance. Range of colors from brown to blue: LLCR or LLFR strains with an accumulation from 1 to 8 point mutations in the studied genes involved in resistance. Figure 2. Logo of the amino acid sequence encoded by the gyrA gene in low-level ciprofloxacin resistance (LLCR) strains. Sequence conservation of amino acids encoding GyrA created from the aligned sequences of the 12 LLCR E. coli strains. Complete logo sequence is shown in Supplementary Figure S1. Letters depict the consensus amino acid of each position; Blue: Amino acids with side chain charge positively; Red: Amino acids with side chain charge negatively; Gray: No-polar amino acids; Green: Polar amino acids without changes; Blue star depict differences; The ggseqlogo library was used to create logo graph from the multiple sequence alignment. On the other hand, in LLFR strains, eight plasmid (fosA3,fosA4,fosA5,fosB,fosX,fosC, fomA, and fomB) and nine chromosomal (murA,uhpA,uhpB,uhpC,uhpT,glpT,ptsl,cyaA, and crp) genes were analyzed. Most of the point mutations found were previously described in the curated CARD database that confers resistance to fosfomycin (Supplementary Table S3). While none of the LLFR strains carried point mutations in the plasmidic genes tested, 75% (6/8) of the LLFR strains carried a point mutation in cyaA,glpT, and ptsI genes. Conversely to that observed in LLCR strains, these genes were mutated in one of the control susceptible strains tested (Figure 1B). However, the strains used as controls, since they present a susceptible phenotype in the antibiotics analyzed, are clinical strains isolated from UTI in KTR patients, and it is expected to find point mutations in them. In addition, 50% (4/8) of the strains carried substitutions in the uhp genes family, specifically Biomolecules 2025,15, 260 7 of 15 25% (2/8) of the LLFR strains carried a substitution affecting the coding region of hexosephosphate transporter (UhpT) gene involved in the fosfomycin transportation into the bacteria [ 44 ]. Also, substitutions in the uhp genes family were observed in control susceptible strains, and only 3/9 (33%) chromosomic genes analyzed showed no mutations in control strains. Specifically, for the uhpB gene, some of the amino acidic substitutions carried by LLFR strains were found among positions from 311 to 499, the histidine kinase domain (Figure 3). Biomolecules 2025, 15, x FOR PEER REVIEW 7 of 15 Figure 2. Logo of the amino acid sequence encoded by the gyrA gene in low-level ciprofloxacin resistance (LLCR) strains. Sequence conservation of amino acids encoding GyrA created from the aligned sequences of the 12 LLCR E. coli strains. Complete logo sequence is shown in Supplementary Figure S1. Letters depict the consensus amino acid of each position; Blue: Amino acids with side chain charge positively; Red: Amino acids with side chain charge negatively; Gray: No-polar amino acids; Green: Polar amino acids without changes; Blue star depict differences; The ggseqlogo library was used to create logo graph from the multiple sequence alignment. Figure 3. Presence of point mutation in UhpB protein. (A) Rendered structure and point mutation outside/within the functional domain and (B) abundance of point mutation in uhpB gene in the lowlevel fosfomycin resistance (LLFR) strains. In (A), green mutations: carried by LLFR strains outside of the functional domain (red square (IPR005467—Histidine kinase domain); blue mutations: carried by control strains outside or inside of the functional domain (red square); red mutation: protein due to this single amino acid variation is not stable; purple mutation: carried by LLFR strains outside of the functional domain (red square). In (B), orange dots: presence/absence of the mutation. Analysis for plasmid resistance genes found that all strains, both LLCR and LLFR, carried at least one plasmid (Supplementary Tables S2 and S3). 3.3. Identification of Unknown Resistance Chromosomal or Plasmid Genes and Virulence Factors in LLCR and LLFR Strains Both the LLCR and LLFR strains showed at least one gene involved in Antimicrobial Resistance (AMR). Regarding the analysis of the presence of antimicrobial resistance genes in the selected strains exhibiting low-level resistance, 83% (10/12) of the LLCR strains carried mutations in gyrA, and 13% (1/8) of the LLFR strains carried fosA. This gene encodes metalloenzymes which hydrolyze fosfomycin [41]. Furthermore, most of these strains carried a wide variety of genes associated with resistance to aminoglycosides, βlactams, sulfonamides, and tetracyclines (Tables 3 and 4). However, no clear association was detected among the presence of these AMR genes and the strainsʹ MIC phenotype. Only two LLCR strains presented the aminoglycoside modifying enzymes (AMEs), aac(3′)-IIa (encoding tobramycin and gentamycin resistance) [45]. Similarly, AME was rarely present in LLFR strains, with only one strain presenting an aac(3′)-Ib involved in streptomycin resistance [46]. With respect to β-lactam resistance genes, both genes encoding class A and D β-lactamases were identified in all LLCR strains, especially, the bla TEM-1 gene, responsible for ampicillin, piperacillin, amoxicillin, and ticarcillin resistance [47]. Moreover, 62% (5/8) of the LLFR strains also carried the bla TEM-1 gene. However, 75% (9/12) and 37% (3/8) of the LLCR and LLFR strains, respectively, carried genes involved in folate Figure 3. Presence of point mutation in UhpB protein. (A) Rendered structure and point mutation outside/within the functional domain and (B) abundance of point mutation in uhpB gene in the lowlevel fosfomycin resistance (LLFR) strains. In (A), green mutations: carried by LLFR strains outside of the functional domain (red square (IPR005467—Histidine kinase domain); blue mutations: carried by control strains outside or inside of the functional domain (red square); red mutation: protein due to this single amino acid variation is not stable; purple mutation: carried by LLFR strains outside of the functional domain (red square). In (B), orange dots: presence/absence of the mutation. Finally, isolate 160 carried a point mutation in the murA and crp genes. Additionally, no mutations were observed in the analysis of plasmid mutations (Figure 1B and Supplementary Table S3). Analysis for plasmid resistance genes found that all strains, both LLCR and LLFR, carried at least one plasmid (Supplementary Tables S2 and S3). 3.3. Identification of Unknown Resistance Chromosomal or Plasmid Genes and Virulence Factors in LLCR and LLFR Strains Both the LLCR and LLFR strains showed at least one gene involved in Antimicrobial Resistance (AMR). Regarding the analysis of the presence of antimicrobial resistance genes in the selected strains exhibiting low-level resistance, 83% (10/12) of the LLCR strains carried mutations in gyrA, and 13% (1/8) of the LLFR strains carried fosA. This gene encodes metalloenzymes which hydrolyze fosfomycin [ 41 ]. Furthermore, most of these strains carried a wide variety of genes associated with resistance to aminoglycosides, β-lactams , sulfonamides, and tetracyclines (Tables 3and 4). However, no clear association was detected among the presence of these AMR genes and the strains’ MIC phenotype. Only two LLCR strains presented the aminoglycoside modifying enzymes (AMEs), aac(3 ′ )-IIa (encoding tobramycin and gentamycin resistance) [ 45 ]. Similarly, AME was rarely present in LLFR strains, with only one strain presenting an aac(3 ′ )-Ib involved in streptomycin resistance [ 46 ]. With respect to β -lactam resistance genes, both genes encoding class A and D β -lactamases were identified in all LLCR strains, especially, the bla TEM-1 gene, responsible for ampicillin, piperacillin, amoxicillin, and ticarcillin resistance [ 47 ]. Moreover, 62% (5/8) Biomolecules 2025,15, 260 8 of 15 of the LLFR strains also carried the bla TEM-1 gene. However, 75% (9/12) and 37% (3/8) of the LLCR and LLFR strains, respectively, carried genes involved in folate pathway antagonists, conferring resistance to trimethoprim [ 48 ]. Furthermore, and regarding the sulfonamide resistance, genes encoding sul1 and sul2, target enzyme dihydropteroate synthase [ 49 ], were found in 75% (9/12) and 50% (4/8) of LLCR and LLFR strains, respectively. Finally, 83% (10/12) of LLCR and 62% (5/8) of LLFR strains carried tetA or tetB genes, which encodes an efflux protein of the membrane that confers resistance to antibiotics of the tetracycline family, such as tetracycline, minocycline, and doxycycline [50]. Table 3. Analysis of the presence of antimicrobial resistance genes in the selected strains exhibiting low-level ciprofloxacin resistance. Antibiotic Family Affected Gene Abundance in LLCR (%) Specific Antimicrobials Affected Aminoglycoside aac(3)-II 25% Tobramycin, sisomicin, apramycin, dibekacin, netilmicin aph(3′′)-I 75% Streptomycin aph(6)-I 75% Streptomycin aadA1 42% Streptomycin, spectinomycin aadA5 25% Streptomycin, spectinomycin Amphenicol catA1 33% Chloramphenicol cmlA1 8% Chloramphenicol floR 33% Florfenicol, chloramphenicol β-lactam ampC 8% Cefoxitin, ampicillin, ampicillin/clavulanic acid, cefotaxime, ceftazidime blaCARB-2 8% Ampicillin, piperacillin, amoxicillin blaEC 100% Class C blaOXA-1 8% Ampicillin, amoxicillin/clavulanic acid, piperacillin, amoxicillin, cefepime, piperacillin/tazobactam blaOXA-10 8% Ampicillin, piperacillin/tazobactam, amoxicillin, piperacillin, aztreonam blaTEM-1A 83% Ampicillin, cephalothin, piperacillin, amoxicillin, ticarcillin blaTEM-1B 83% Ampicillin, cephalothin, piperacillin, amoxicillin, ticarcillin Folate pathway antagonist dfrA1 42% Trimethoprim dfrA8 8% Trimethoprim dfrA14 8% Trimethoprim dfrA17 25% Trimethoprim dfrA36 8% Trimethoprim sul1 58% Sulfamethoxazole sul2 67% Sulfamethoxazole sul3 8% Sulfamethoxazole Fosfomycin cyaA 8% Fosfomycin uhpT 42% Fosfomycin Macrolide mph(A) 42% Erythromycin, telithromycin, azithromycin, spiramycin Peroxide sitABCD 58% (hydrogen peroxide) Biomolecules 2025,15, 260 9 of 15 Table 3. Cont. Antibiotic Family Affected Gene Abundance in LLCR (%) Specific Antimicrobials Affected Quaternary ammonium compound qacE 58% Cetylpyridinium chloride, ethidium bromide, benzylkonium chloride, chlorhexidine qacL 8% Cetylpyridinium chloride, ethidium bromide, benzylkonium chloride, chlorhexidine Quinolone acr 33% Ciprofloxacin gyrA 83% Ciprofloxacin and nalidixic acid gyrB 33% Ciprofloxacin and nalidixic acid parC 50% Ciprofloxacin and nalidixic acid parE 50% Ciprofloxacin and nalidixic acid qnrS1 17% Ciprofloxacin Rifamycin ARR-2 8% Rifampicin Tetracycline tet(A) 58% Doxycycline, tetracycline tet(B) 17% Tetracycline, doxycycline, minocycline Various erm(B) 8% Streptogramin B, lincosamide (clindamycin, lincomycin), and macrolide (erythromycin) Table 4. Analysis of the presence of antimicrobial resistance genes in the selected strains exhibiting low-level fosfomycin resistance. Antibiotic Family Affected Gene Abundance in LLFR (%) Specific Antimicrobials Affected Aminoglycoside aph(3′′)-I 50% Streptomycin aph(6)-I 50% Streptomycin aadA1 38% Streptomycin, spectinomycin Amphenicol catA1 13% Chloramphenicol oqxB 13% Florfenicol, chloramphenicol β-lactam blaEC 88% Class C blaMIR-6 13% Ampicillin, amoxicillin/clavulanic acid, ticarcillin/clavulanic acid, cefoxitin, piperacillin, amoxicillin, piperacillin/tazobactam, ceftazidime, ticarcillin, cefotaxime, ampicillin/clavulanic acid blaTEM-1B 50% Ampicillin, cephalothin, piperacillin, amoxicillin, ticarcillin Folate pathway antagonist dfrA1 42% Trimethoprim dfrA5 8% Trimethoprim sul1 25% Sulfamethoxazole sul2 38% Sulfamethoxazole Fosfomycin cyaA 13% Fosfomycin fosA 13% Fosfomycin uhpT 25% Fosfomycin Peroxide sitABCD 38% Hydrogen peroxide Quaternary ammonium compound qacE 25% Cetylpyridinium chloride, ethidium bromide, benzylkonium chloride, chlorhexidine