Genomic characterization of multidrug-resistant Escherichia coli strains identified from patients with urinary tract infection in Egypt

TL;DR

This study uses whole-genome sequencing to analyze two drug-resistant E. coli strains from Egyptian patients with urinary tract infections, revealing resistance genes and potential for spreading.

Contribution

The study provides genomic characterization of multidrug-resistant E. coli strains from Egypt, highlighting their resistance and virulence profiles.

Findings

Two E. coli isolates (UPE7 and UPE139) were found to carry multiple antibiotic resistance genes.

Mobile genetic elements near resistance genes suggest potential for horizontal gene transfer.

Serotyping identified UPE7 as O8:H9 and UPE139 as O9:H30.

Abstract

Extended-spectrum β-lactamases-producing Escherichia coli (ESBL-EC) pose a serious threat. Moreover, widespread antimicrobial use in Egypt increased the prevalence of antimicrobial resistance (AMR). In this study, whole-genome sequencing (WGS) using the Illumina NovaSeq 6000 was performed on two isolates (UPE7 and UPE139) recovered from participants with urinary tract infections to characterize their resistomes and virulomes. Antibiotic resistance and virulence genes of the two clinical E. coli strains were predicted using computational analysis tools. Several virulence traits and antibiotic resistance genes (ARGs) were identified. Strain UPE7 harbored blaTEM−1B, blaCTM−X−15,
blaCMY−2, and strain UPE139 revealed the presence of blaOXA−244, blaTEM−12, blaTEM−82, and blaCTM−X−15 rending the resistance phenotype. The presence of mobile genetic elements adjacent to ARGs thereby suggests their potential for dissemination through horizontal gene transfer. Furthermore, the serotyping in silico investigation revealed that E. coli UPE7 and UPE139 serotypes were O8:H9 and O9:H30, respectively. Notably, key mutations in the gyrA, parC, and parE genes were predicted, consistent with their confirmed resistance to levofloxacin. These findings emphasize the importance of genomic surveillance to guide antimicrobial therapy and monitor emerging high-risk clones, and they support the need for larger-scale genomic studies to improve epidemiological understanding and clinical relevance.

Figures

Antimicrobial resistance genes predicted from the whole-genome sequence data of (A) *E. coli* UPE7 and (B) *E. coli* UPE139 using CARD-RGI ([https://card.mcmaster.ca/analyze/rgi](https://card.mcmaster.ca/analyze/rgi)). ARO represents the Antibiotic Resistance Ontology, and the surrounding colored segments represent the detection confidence level of AMR genes. Green color (Perfect) indicates AMR genes perfectly matching reference sequences in the database. Yellow color (Strict) indicates that AMR genes are closely related to known AMR, with some variations. Red (Loose) indicated low-confidence

Schematic representation of ARGs and genetic mobile elements detected in (A) UPE7 and (B) UPE139. The colors represented: ARGs (Red), Transposase-related genes (green), Insertion sequence elements (Blue, Yellow, Orange).

Heatmap represents antibiotic resistance genes in *E. coli* UPE7, *E. coli* UPE139, and ten selected uropathogenic *E. coli* strains using the ARG detection method available through the BV-BRC web server. The purple and yellow colors represent present and absent genes, respectively.

Full text

Introduction

Antibiotic resistance (AMR) is a major concern in modern healthcare since it possesses the potential to spread worldwide and has limited treatment options^1^. Poverty, poor sanitation, easily available antibiotics without prescription, and ignorant clinical practice are factors that aid in the spread of multidrug-resistant (MDR) microbial strains in community and hospital settings^2^. In addition, antibiotics are extensively employed in agriculture to enhance crop yields and in animal husbandry, particularly in cattle farming, where they are administered for therapeutic, prophylactic, and metaphylactic purposes, as well as at controlled concentrations in animal feed as growth promoters to prevent disease and safeguard animal health^3,4^. Therefore, the extensive use of antibiotics outside human medicine has accelerated the emergence and dissemination of MDR bacteria, representing an escalating threat to both animal and public health^4^. New examples of AMR are regularly reported, and the time needed for bacteria to become resistant to newly introduced antibiotics is getting shorter^5^.

Escherichia coli was listed among the World Health Organization (WHO) priority organisms that cause critical infections and require the formulation of new treatments^6^. Remarkably, the resistance of E. coli strains to third-generation antibiotics is mainly associated with the production of extended-spectrum β-lactamases (ESBLs) globally^7^. In the last decade, ESBL-EC infections have become challenging because of the rapid pandemic spread of antimicrobial-resistant clones and limited treatment options that put the patient’s life in danger^8,9^. Furthermore, ESBL-EC is often linked to an increased incidence of nosocomial infections in hospitals^10^. E. coli isolates may carry multiple antibiotic resistance genes carried on mobile genetic elements (MGEs), including plasmids, transposons, integrons, and insertion sequences (IS), which can spread to compatible neighboring species via horizontal gene transfer (HGT)^11,12^. In addition, E. coli harbors diverse virulence factors (adhesion-associated fimbriae, hemolysin, siderophores, flagella, capsular polysaccharides, and outer membrane proteins) that contribute to distinct aspects of pathogenicity^13^. The combined action of these determinants enables the bacterium to establish infection, persist with the host, and evade defense mechanisms^14^.

Whole genome sequencing (WGS) has emerged as a feasible approach for detecting antibiotic resistance. Indeed, comprehensive genetic analysis via next-generation sequencing (NGS) enables high-resolution genotyping of clinically significant MDR strains and facilitates targeted antibiotic usage directly guided by genetic sequence^15^. In addition, NGS can study the evolutionary relationship of MDR strains from different geographical regions. Moreover, it is a helpful tool for tracking MGEs, identifying emerging resistance mechanisms, and uncovering chromosomal mutations^10,16^.

In this study, two MDR E. coli strains, UPE7 and UPE139, were isolated from patients with urinary tract infections (UTIs) at Mansoura University Hospital (Egypt). To characterize the two uropathogenic E. coli strains in-depth, WGS using Illumina sequencing was conducted. Additionally, MLST, serotyping, and phylotyping were used to investigate the clonal relatedness of the isolates. Genome assemblies of UPE7 and UPE139 were screened for the presence of ARGs, MGEs, and virulence factors on the genomic level.

Results

Antimicrobial resistance profile

The two isolates, UPE7 and UPE139, were identified from patients with UTI in Egypt and revealed extensive resistance patterns (Table 1). Among aminoglycosides, both isolates exhibited high-level resistance to gentamicin (≥ 32 µg/mL for UPE7; ≥16 µg/mL for UPE139) and amikacin (≥ 64 µg/mL for both). UPE7 and UPE139 strains were resistant to penicillins and β-lactams, including ampicillin-sulbactam, cefoperazone-sulbactam, and piperacillin-tazobactam, as well as extended-spectrum cephalosporins such as ceftazidime, cefotaxime, and cefepime. Carbapenems, including ertapenem, imipenem, and meropenem, also demonstrated elevated MIC values, confirming carbapenem resistance in both isolates. Resistance extended to fluoroquinolones, with high MIC values reported to levofloxacin (≥ 16 µg/mL for UPE7; ≥8 µg/mL for UPE139). Additionally, strain UPE139 revealed intermediate resistance to chloramphenicol, whereas polymyxin B exhibited resistance in UPE7 (8 µg/mL) but intermediate resistance in UPE139. Both strains were resistant to the sulfonamide antibiotic, trimethoprim-sulfamethoxazole.

Table 1. Minimal inhibitory concentrations (MICs) interpretation of Escherichia coli UPE7 and UPE139 as per CLSI 2021 guidelines.AntibioticsMIC values (µg/mL)UPE7UPE139Gentamicin≥ 32 (R)≥ 16 **(R)**Amikacin≥ 64 (R)≥ 64 **(R)**Ampicillin-sulbactam≥ 64/32 (R)≥ 32/16 **(R)**Ceftazidime-clavulanateND≥ 2/8 **(R)**Cefotaxime-clavulanateND≥ 2/8 **(R)**Cefoperazone-sulbactam≥ 128/64 (R)≥ 128/64 **(R)**Piperacillin-tazobactam≥ 128/4 (R)≥ 128/4 **(R)**Ertapenem≥ 32 (R)≥ 16 **(R)**Imipenem≥ 128 (R)= 16 **(R)**Meropenem≥ 128 (R)= 8 **(R)**CefazolinND≥ 32 **(R)**CefuroximeND≥ 64 **(R)**CefotaximeND≥ 64 **(R)**Ceftazidime≥ 64 (R)≥ 32 **(R)**Cefepime≥ 64 (R)≥ 32 **(R)**AmpicillinND≥ 32 **(R)**CefoxitinND≥ 64 **(R)**Levofloxacin≥ 16 (R)≥ 8 **(R)**AzithromycinND≥ 64 **(R)**ChloramphenicolND= 16 **(I)**Polymyxin B= 8 (R)= 1 **(I)**Minocycline≥ 32 (R)= 8 **(I)**Tigecycline= 8 (R)= 1 **(R)**Trimethoprim-sulfamethoxazole≥ 64/1216 (R)≥ 8/152 **(R)***R, Resistant; I, Intermediate; ND, not determined.

General genomic features of E. coli strains UPE7 and UPE139

According to VITEK biochemical identification and genomic characterization, the two isolates were identified as Escherichia coli. The predicted genome sizes were 4,828,053 bp and 5,096,432 bp for UPE7 and UPE139, respectively. The G + C contents for both isolates were about 50.6%. Moreover, WGS yielded 681,534 and 992,028 reads for UPE7 and UPE139, respectively. Genomic features, including coding sequence (CDS), rRNA, tRNA, and CRISPR array numbers, are listed in Table 2. The whole genome shotgun projects of UPE7 and UPE139 were submitted to the National Biotechnology Information Centre (NCBI) GenBank database with the accessions JBHWTZ000000000 and JBHWUA000000000, respectively.

Table 2. General genomic features of Escherichia coli UPE7 and UPE139 genome assemblies.Assembly metricsUPE7UPE139Length (bp)4,828,0535,096,432GC content (%)50.6150.64CDS4,7645,117tRNA8390rRNA1211CRISPR array23Plasmids45

Determination of serotypes, sequence type, and phylogroup

In silico serotyping identified E. coli UPE7 as O8:H9, based on the presence of wzt and wzy alleles (O8) and the fliC gene (H9). Similarly, UPE139 was assigned to O9:H30, carrying wzm and wzt alleles (O9) and the fliC gene (H30). Furthermore, MLST allelic analysis was performed based on the allelic combination of seven genes, and the results revealed no clonal relationship between the two isolates assigned to different sequence types. Based on the Pasteur (Achtman) scheme, the sequence type assigned the isolates UPE7 and UPE139 to ST692 (ST410) and ST650 (ST361), respectively. In addition, the Clermont E. coli phylotyping assigned UPE7 and UPE139 to phylogroup C (fdm, trpA,* gpC*, ybgD) and phylogroup A (fdm, cfaB, ybgD), respectively.

Detection of virulence factors

Screening the uropathogenic E. coli strains UPE7 and UPE139 genome sequences revealed the occurrence of a diverse array of virulence genes associated with adhesion, host cell invasion, iron acquisition, and persistence in the urinary tract. Strain UPE7 carried genes encoding fimbrial and adhesion factors, including fimH (type 1 fimbriae) and csgA (curlin major subunit CsgA), fdeC (intimin-like adhesion FdeC), IpfA (long polar fimbriae), and the yehABCD fimbrial cluster, which enhance adhesion and biofilm formation. Additionally, hlyE (hemolysin) and hha (hemolysin express modulator), nlpI (lipoprotein precursor), and terC (tellurium ion resistance protein). In comparison, UPE139 harbored several overlapping factors, including fimH, csgA, fdeC, nlpI, hylE, and the yehABC fimbrial cluster, but also possessed additional determinants, capU (hexosyltransferase homolog linked to capsule synthesis), shiA (associated with the Shigella SHI-2 pathogenicity island), sitABCD (iron transport), tia (Tia invasion determinant), and traT (outer membrane protein complement resistance).

Detection of antimicrobial resistance genes (ARGs)

Several ARGs conferring resistance to multiple antibiotic classes were identified in the UPE7 and UPE139 genome sequences, demonstrating a correlation between phenotypic resistance and underlying genetic determinants. The observed resistance to cephalosporins and β-lactams was confirmed by detection of ARGs, including blaTEM−1B, blaCMY−2, and blaCTM−X−15 in the UPE7 isolate, as well as blaTEM−12, blaTEM−82, and blaCTM−X−15 in UPE139. Moreover, high-level aminoglycoside resistance in both isolates was explained by the presence of aminoglycoside genes such as aph(3’’)-Ib, aph(6)-Id, and aadA5. Resistance to tetracyclines, quinolones, macrolides, sulfonamides, and trimethoprim was consistent with the detection of tetA, tetD, tetR, qnrS1, mphA, mdfA, sul1, sul2, and dfrA17 in both genome sequences. In addition, the resistance of the UPE139 strain to carbapenems was associated with the occurrence of blaOXA−244, a member of the OXA-48-like carbapenemase, whereas no carbapenemase resistance genes were detected in the UPE7 genome sequence. In particular, resistance extended to fluoroquinolones is related to mutations in quinolone resistance-determining regions (gyrA, parC, parE,* qnr*). In addition, both strains revealed regulatory and efflux-associated genes such as acrR, mdtK, and marR, which reduce intracellular quinolone concentrations through active efflux. The presence of the arnABCDTEF operon in both UPE7 and UPE139 was associated with resistance to polymyxin B (Fig. 1; Table 3).

Fig. 1. Antimicrobial resistance genes predicted from the whole-genome sequence data of (A) E. coli UPE7 and (B) E. coli UPE139 using CARD-RGI (https://card.mcmaster.ca/analyze/rgi). ARO represents the Antibiotic Resistance Ontology, and the surrounding colored segments represent the detection confidence level of AMR genes. Green color (Perfect) indicates AMR genes perfectly matching reference sequences in the database. Yellow color (Strict) indicates that AMR genes are closely related to known AMR, with some variations. Red (Loose) indicated low-confidence matches.

Table 3. Overview of resistome and mobilome in UPE7 and UPE139 strains.UPE7UPE139 MLST ST692^a^ – ST410^b^ST650^a^ – ST361^b^ pMLST IncF [F1:A1:B49]IncF [F31:A1:B49] Serotype O8:H9O9:H30 CH type 4–24 (fumC4, fimH24)99 − 54 (fumC99, fimH54) Antimicrobial Resistance Genes

β-lactamase bla CMY−2bla CTM−X−15bla TEM−12bla CTM−X−15 Aminoglycoside aph(3’’)-Ibaph(6)-Idaac(3)-IIaaac(3)-IId aadA5 aph(3’’)-Ibaph(6)-Id aadA5

Macrolide

mphA

mdfA

mphA

mdfA

Tetracycline

tetA

tetR

tetA

tetD

tetR

Quinolone

qnrS1

qnrS1

Polymyxin

pmrD

pmrG

arnABCDTEF

pmrD

pmrG

arnABCDTEF

Plasmids IncFIIIncFIAIncFIBCol(BS512)IncFIIIncFIAIncFIBIncQ1IncY Chromosomal mutations gyrA (S83L, D87N)parC (S80I)parE (S458A)gyrA (S83L, D87N)parC (S80I)parE (S458A)^a^MLST, Pasteur scheme; ^b^MLST, Achtman scheme.

The analysis of the UPE7 genome revealed the presence of ESBL genes near mobile genetic elements, such as insertion sequences and transposons. The investigation revealed that blaTEM−1B is located between the TnpA transposon. Moreover, the blaCMY−2-containing region revealed the occurrence of this gene chromosomally within the cassette comprising ISEcp1-blaCMY−2-orf-IS200-orf-orf-IS3. In addition, the blaCTX−M−15 gene was bracketed by transposon TnpA and the insertion sequence IS6 (Fig. 2A). Meanwhile, the analysis of ESBL-related genes in the UPE139 genome sequence revealed the presence of the blaCTX−M−15 gene associated with transposon TnpA and IS2. Notably, blaOXA−244 was positioned between two insertion sequences (IS1). Moreover, blaTEM−12 and blaTEM−82 were found to be associated with transposon Tn3 and insertion sequence IS6, respectively (Fig. 2B). Therefore, the WGS-based resistance profile confirmed that UPE7 and UPE139 were multidrug-resistant strains.

Fig. 2. Schematic representation of ARGs and genetic mobile elements detected in (A) UPE7 and (B) UPE139. The colors represented: ARGs (Red), Transposase-related genes (green), Insertion sequence elements (Blue, Yellow, Orange).

Investigating chromosomal mutations

In this study, the UPE7 and UPE139 isolates were phenotypically resistant to levofloxacin, a fluoroquinolone antibiotic. Therefore, the chromosomal mutations in DNA gyrase (gyrA) and topoisomerase IV (parC and parE) were investigated. Notably, two types of gyrA mutations (S83L and D87N), one parC mutation (S80I), and one parE mutation (S458A) were detected in UPE7 and UPE139 genome sequences.

Prediction of mobile genetic elements (MGEs)

Screening MGEs conferred the presence of numerous genes associated with the mobilization of ARGs in UPE7 and UPE139 strains. E. coli UPE7 genome sequence exhibited plasmid replicons, namely Col(BS512), IncFIA, IncFIB (AP001918), IncFII (pAMA1167-NDM-5) and IncQ1. Moreover, the UPE139 genome sequence revealed the presence of plasmid replicons: IncFII, IncFIA, IncFIB, IncQ1, and IncY. Plasmid-mediated quinolone resistance (PMQR) gene qnrS1 and ESBL gene blaCTX−M−15 were present in the IncFII plasmid in UPE7 and UPE139 genomes. In addition, several IS and transposable elements were detected in the two strains. UPE7 and UPE139 genome sequences revealed the presence of Insertion sequences ISEcp1, belonging to the IS1380 family transposase, and IS200/IS605. Furthermore, IS3 and IS6 were found at a relatively high frequency in the genomes of the two isolates.

Comparative analysis

A comparative genomic analysis involving UPE7, UPE139, and ten E. coli strains isolated from patients in Egypt was performed (Fig. 3). The genomic analyses of the annotated sequenced genomes revealed a variety of phylogroups, serotypes, and distributions of antibiotic resistance genes. The strains demonstrated combinations of ESBL genes, including the most predominant gene blaCTX−M15, and carbapenem genes such as blaNDM−1 and blaNDM−5. Notably, the four strains in this study harbored the blaCMY−2 gene, and one strain harbored the blaOXA−244 gene. The studied strains revealed the occurrence of one or more aminoglycoside genes. Most of the investigated strains revealed the presence of the macrolide resistance genes (mphA and mdfA), which confer macrolide resistance. Moreover, most of the isolates showed quinolone resistance-determining regions (gyrA, parC, parE), as well as the presence of qnrS1, which confers plasmid-mediated quinolone resistance.

Fig. 3. Heatmap represents antibiotic resistance genes in E. coli UPE7, E. coli UPE139, and ten selected uropathogenic E. coli strains using the ARG detection method available through the BV-BRC web server. The purple and yellow colors represent present and absent genes, respectively.

Pan-genome analysis revealed 5,367 orthologous gene clusters identified across the analyzed E. coli genome sequences, of which 3,352 shared orthologous gene clusters were identified among the E. coli strains under investigation (Fig. 4A). Notably, some gene families are involved in critical biological processes. Furthermore, the phylogenetic tree based on whole-genome sequencing revealed the close relation between UPE139 and HR135 (accession number QXNW00000000), a phylogroup A strain, which was previously isolated from UTI in Egypt. Moreover, UPE7 was clustered with SM_2771 (accession number JAJKQA000000000) and K12 strain 71 (accession number SSTS00000000), belonging to phylogroup C and sequence type ST692 (Fig. 4B). E. coli strains within the same phylogroup displayed varied levels of ARGs.

Fig. 4**(A)** Upset figure showing the common genes in UPE7, UPE139, and other selected E. coli genomes, indicating the number of shared and unique protein clusters and unique genes. (B) Whole genome-based phylogenetic tree of UPE7 and UPE139 isolates, constructed based on the presence/absence of orthologous gene clusters identified using the pan genome analysis tool.

Discussion

According to the WHO, ESBL-producing Enterobacteriaceae pose a significant risk to human health^17^. Therefore, pathogen surveillance using genomic approaches helps to understand the dynamics, and the results are important for controlling the spread of resistant bacteria^18^. Consequently, identifying resistance traits through WGS is significant in improving diagnosis and innovative regimes for treating patients with UTI^19,20^. In the current study, the resistance genotypes of UPE7 and UPE139 strains corresponded to the phenotypic resistance of antibiotics under investigation, reflecting the correlation between the high resistance rate and the frequent prescription of these antibiotics in the treatment of UTI in Egyptian healthcare^21^.

In Egypt, several findings have demonstrated a higher prevalence of AMR among carbapenem-resistant and ESBL-EC, highlighting the significance of understanding the molecular mechanisms of resistance^22^. The rapid emergence of MDR strains of E. coli is a serious concern in developing countries. These high observed rates in cephalosporins and carbapenemes are attributed to antibiotic misuse and extensive usage of third-generation antimicrobials, particularly in Egyptian hospitals^23,24^. Furthermore, various factors, including poor hygiene, antibiotics without prescription, and a lack of rapid diagnostic methods, promote the emergence of these ESBL-producing isolates^25^.

The sequence type UPE7 was ST410, according to Achtman’s scheme. This sequence type was previously identified in E. coli EC13655, isolated from a hospitalized patient in Egypt^21^. Furthermore, the uropathogenic E. coli NGCE33 strain (ST410 clone) containing an array of β-lactamase genes was previously detected in Bangladesh^26^. E. coli of the ST410 lineage is an emerging MDR high-risk clone that has shown an increasing worldwide spread and revealed non-susceptibility to carbapenems, cephalosporins, fluoroquinolones, aminoglycosides, and tetracyclines^27,28^. However, UPE139 was ST361, according to Achtman’s scheme. A previous study revealed that E. coli ST361 was a key player in the dissemination of carbapenem resistance in France, due to the chromosomal localization of blaOXA−244^29^. In addition, the clone ST361 was previously detected in seven patients in Denmark in May 2018^30^. Therefore, it is essential to understand the potential threats of these high-risk clones, use effective antibiotics, and take precautions against their spread.

Clermont phylotyping plays a crucial role in identifying the isolates and developing new therapeutic agents. Phylogenetic group A is usually associated with commensal strains, suggesting that the gastrointestinal tract is the primary source of E. coli strains that colonize the urinary tract^31,32^. Most E. coli isolates belong to phylogroup A and possess multiple ARGs and fewer virulence determinants^21^. Moreover, phylogroup C possesses a high rate of MDR E. coli with lower motility^33^.

Investigating the virulome of E. coli strains UPE7 and UPE139 more in depth through WGS revealed a diverse set of virulence determinants that underline their pathogenic potential and ability to persist in the urinary tract environment. Both strains carried fimbrial and adhesion-related genes, which are strongly associated with biofilm formation^34^. The presence of sitABCD, an iron uptake system that acquires iron to boost their pathogenicity potential, tends to restrict iron bioavailability, a traditional host defense mechanism against bacterial invasion^35^. Moreover, the presence of hemolysin genes in both isolates is associated with disease severity in UTI and indicates the cytotoxic activity against a broad range of cells^36^. The identification of shiA, typically associated with the Shigella SHI-2 pathogenicity island, points toward horizontal gene transfer events that may have expanded UPE139’s virulence repertoire^37^. These findings suggest that while both UPE7 and UPE139 exhibit core uropathogenic-associated virulence traits, UPE139 appears to possess a more complex virulence profile, which may confer increased adaptability and pathogenic potential in the urinary tract.

CTX-M-type ESBLs producing E. coli are associated with the international dissemination of MDR high-risk clones^38^. Several studies have revealed that blaCTX−M−15, one of the most prevalent genotypes, is widely found in E. coli and rapidly disseminates within patients with urinary tract infections^39,40^. A previous study reported that blaCTX−M−15 was predominant and present in 68.4% of urinary ESBL-producing E. coli isolates in Minia University Hospital (Minia, Egypt)^41^. In addition, the location of the blaCTX−M−15 gene adjacent to IS26 (which belongs to the IS6 family) might be involved in their mobilization^42^. Over the past two decades, CMY-2-like enzymes have been the most commonly observed in ESBL-EC^43^. In this study, the blaCMY−2 gene was flanked by a single copy of insertion sequence ISEcp1, which facilitates mobilization of the adjacent ARG sequence by a one-end transposition mechanism^21^. Studies have reported the occurrence of CMY-2 in E. coli (strain UEC59) isolated from urinary tract infection patients in Pakistan, and E. coli isolates in Mexico^44,45^. The blaCMY−2 gene was recently predicted in 10% of tested E. coli strains isolated from hospitalized patients in Alexandria Main University Hospital (Alexandria, Egypt)^21^.

OXA-244 carbapenemase was detected in the E. coli UPE139 genome sequence. It differs from OXA-48 by a single amino acid substitution, showing reduced activity against carbapenem antibiotics^46^. A previous study reported the presence of blaOXA−244 in the genome of MDR E. coli strain HR14_AS, recovered in December 2014 from a wound pus swab of a male patient in Egypt^47^. Similar to our results, blaOXA−244 is chromosomally integrated into an ISR1-related Tn6237 transposon, which is responsible for the dissemination of OXA-244-producing E. coli^48^. Despite the absence of known carbapenemase-encoding genes in the genome sequence of UPE7, the isolate revealed resistance to carbapenems. This result is in agreement with a previous study, which reported that imipenem-resistant E. coli ECV219 did not carry genes associated with carbapenem resistance^49^. This suggests the involvement of an alternative resistance mechanism, including inducible overexpression of chromosomal cephalosporinases, such as AmpC (e.g., CMY-2), combined with porin loss that can result in clinically significant carbapenem resistance^50,51^.

Healthcare-associated infections caused by isolates with mcr genes that contribute to colistin resistance are associated with increased morbidity and mortality rates, as these plasmid-borne genes facilitate horizontal transfer between bacterial species, promoting their rapid global dissemination^24^. In this study, although the genome sequences of UPE7 and UPE139 were mcr-negative, both isolates exhibited phenotypic resistance to polymyxin. This indicates that polymyxin B resistance is mediated by chromosomally encoded mechanisms. Polymyxins belong to a group of cationic antimicrobial peptides, and they owe their antimicrobial action to binding to lipid A, a component of the negatively charged lipopolysaccharide (LPS) in the outer membrane of Gram-negative strains, leading to cell lysis^52,53^. Moreover, resistance to colistin in E. coli can be acquired through chromosomal point mutation, particularly in genes encoding two-component regulatory systems, such as PhoPQ and PmrAB^54,55^. These mutations result in the expression of the arnBCDTEF operon, which leads to lipopolysaccharide modification via the addition of 4-amino-4-deoxy-l-arabinose (l-Ara4N) to lipid A, thereby reducing the net negative charge of the outer membrane and conferring resistance to polymyxins in E. coli^56^.

Notably, gyrA (S83L, D87N), parC (S80I), and parE (S458A) mutations were reported in UPE7 and UPE139 isolates. Fluoroquinolone resistance is mainly caused by point mutations in genes encoding DNA gyrase and topoisomerase IV, such as gyrA, gyrB, parC, and parE in quinolone resistance-determining regions^57^. These types of mutation are associated with high resistance levels to fluoroquinolones in MDR E. coli strains^58^. In addition, resistance to fluoroquinolones is dependent on the capacity of bacteria to reduce drug accumulation through different efflux systems^59^. A previous study in Thailand reported that E. coli urinary isolates have a high percentage of resistance against levofloxacin, greater than 90%^60^. Moreover, a recent study conducted in Egypt demonstrated that 58.7% of isolates of Gram-negative clinical pathogens have at least one quinolone resistance (qnr) gene^61^.

Studying MGEs is significant in understanding and suggesting the mobility dynamics of ARGs among pathogenic strains. The isolates UPE7 and UPE139 genome sequences harbor multiple plasmid replicons. Previous studies have denoted that IncF plasmids are associated with highly drug-resistant properties and may likely cause long-term resistance and pathogenicity in patients and hospital environments^10^. This agreed with a previous study from Egypt that demonstrated the presence of IncF replicon types in MDR E. coli isolates^62^. Similar to our results, previous studies have shown that plasmids expressing the ESBL phenotype also carry genes encoding resistance to antibiotics such as tetracyclines, fluoroquinolones, and aminoglycosides^63,64^. In agreement with a previous study, the Tn3 transposon harbors the β-lactamase TEM gene, which is transmitted between plasmids of different replicon types^39^.

This study was limited to only two E. coli isolates, which restricts the extent to which the findings can be generalized to the broader population of uropathogenic E. coli strains in Egypt. Although these isolates were selected based on distinct antimicrobial resistance profiles and prior epidemiological relevance, they do not encompass the full genetic and phenotypic diversity present in clinical settings. To address these limitations, future studies should include a larger and more representative collection of isolates, combined with complementary phenotypic and transcriptomic analyses, to provide a more comprehensive understanding of resistance mechanisms and their clinical implications.

Methods

Sample collection and characterization

A hundred clinical specimens were collected from females whose ages ranged from 20 to 40 years in a tertiary hospital (Mansoura, Egypt). The study was approved by the University Institutional Review Board of Mansoura University (IRB: R.25.01.3002). Ethical considerations, including personal data privacy, were considered during all study steps. Isolation was performed on MacConkey agar plates (MAC; SRL, India), followed by bacterial purification and conventional identification using the VITEK 2 system (BioMérieux, France) available in the Clinical Pathology Department, Faculty of Medicine. Pure cultures were stored at −20 °C in tryptic soy broth (TSB; SRL, India) supplemented with 50% (v/v) glycerol for further phenotypic and genotypic characterization.

Determination of minimum inhibitory concentration (MIC)

The minimum inhibitory concentration of the isolates was determined using the DL microbial AST system (Zhuhai DL Biotech, China; https://en.medicaldl.com). The results were obtained after 24 h against 24 antimicrobial agents, including gentamicin, amikacin, ampicillin-sulbactam, ceftazidime-clavulanate, cefotaxime-clavulanate, cefoperazone-sulbactam, piperacillin-tazobactam, ertapenem, imipenem, meropenem, cefazolin, cefuroxime, cefotaxime, ceftazidime, cefepime, ampicillin, cefoxitin, levofloxacin, azithromycin, chloramphenicol, polymyxin B, minocycline, tigecycline, and trimethoprim-sulfamethoxazole. The minimum inhibitory concentrations (MIC, µg/mL) of the isolates were interpreted as susceptible (S), intermediate (I), or resistant (R) according to Clinical and Laboratory Standards Institute (CLSI) guidelines^65^. UPE7 and UPE139 strains were categorized phenotypically as MDR and positive for ESBL based on the resistance profiles. Thus, the two strains were subjected to a comprehensive genomic investigation.

Whole-genome sequencing, assembly, and functional annotation

Genomic DNA from E. coli UPE7 and UPE139 was extracted using the GeneJET™ Genomic DNA Purification Kit (Thermo Fisher Scientific, UK) following the manufacturer’s instructions. Library preparation was carried out on a Hamilton Microlab STAR automated liquid handling system (Hamilton Bonaduz AG, Switzerland) using the Nextera XT Library Prep Kit (Illumina, UK). Genome sequencing was outsourced by MicrobesNG (Birmingham, UK; http://microbesng.uk), on the Illumina NovaSeq 6000 platform (Illumina, USA) using a 250 bp paired-end protocol with 30X sequence coverage. The sequencing adaptors and low-quality reads were trimmed using Trimmomatic (Version 0.30) with a sliding cut-off of Q15^66^. Subsequently, de novo genome assembly was performed using SPAdes software (Version 3.7.0)^67^. The newly sequenced genomes were annotated using Prokka software (Version 1.11) and the nucleotide Basic Local Alignment Search Tool (BLASTn) available through the NCBI Prokaryotic Genome Annotation Pipeline (PGAP)^68,69^.

Determination of serotypes, sequence type (MLST), and phylogroup

The serotypes of E. coli strains were determined with SeroTypeFinder 2.0 provided by the Centre for Genomic Epidemiology (CGE; https://cge.cbs.dtu.dk/services/SerotypeFinder/) for the identification of somatic (O) and flagellar (H) antigens. Multi-locus sequence type (MLST) was determined using the Institute Pasteur web server (https://bigsdb.pasteur.fr)^70^. The phylogroups of UPE7 and UPE139 were determined using Clermont typing^71^.

Identification of virulence, mutations, and antimicrobial resistance genes

Virulence factors in E. coli UPE7 and UPE139 genome sequences were investigated using the VirulenceFinder-2.0 server (https://cge.food.dtu.dk/services/VirulenceFinder/), available through CGE. Antimicrobial resistance genes (ARGs) were identified in the UPE7 and UPE139 genome sequences using the Bacterial and Viral Bioinformatics Resource Centre (BV-BRC; https://www.bv-brc.org/) webserver (Version 3.39.10)^72^ and Comprehensive Antibiotic Resistance Database (CARD-RGI; https://card.mcmaster.ca/) with 90% identity and ≥ 60% coverage thresholds^73^. Chromosomal mutations mediating AMR were analyzed using the ResFinder 4.6.0 server^74^ available through CGE.

Identification of plasmids and insertion sequences (IS)

The genome sequences of the two E. coli strains were investigated for plasmids using the PlasmidFinder (Version 2.0) online tool^75^. Plasmids of the IncF type were subtyped by the plasmid MLST webserver (https://pubmlst.org/organisms/plasmid-mlst). Insertion sequences (IS) were determined using ISfinder (https://www-is.biotoul.fr) using BLASTn (Version 2.2.31+)^76^.

Comparative genomic analyses

A total of ten genome sequences associated with urinary tract infections were selected based on their geographic relevance to Egypt, and the sequences were retrieved from the NCBI database (Table 4). Furthermore, comparative genomics analysis was performed using OrthoVenn3 (https://orthovenn3.bioinfotoolkits.net) and the Integrated Prokaryotes Genome and Pan-Genome Analysis service (IPGA Version 1.09; https://nmdc.cn/ipga/)^77,78^. A whole genome-based phylogenetic tree of UPE7 and UPE139 isolates, and other E. coli strains, was created using the IPGA web server^78^.

Table 4Escherichia coli strains were used in this study, including the GenBank accession number.StrainAccession NumberGeographical locationIsolation SourceYeartRNArRNAGC %^a^MLST^b^PhylogroupUPE7JBHWTZ000000000EgyptUrine Infection2022831250.61ST692CUPE139JBHWUA000000000EgyptUrine Infection2022901150.64ST650AHR135QXNW00000000EgyptUrine Infection201684850.41ST650AGCID_CRE_0002RYXW00000000EgyptUrine201876450.54ST1244B2GCID_CRE_0004RYXV00000000EgyptUrine Infection201875450.50ST1244B2GCID_CRE_00033RYXG00000000EgyptUrine201871450.78ST43B2SM_1442JAJKSZ000000000EgyptUrine2015832250.67ST43B2SM_2209JAJKRT000000000EgyptUrine201570450.75ST2ASM_2771JAJKQA000000000EgyptUrine201672350.50ST692CSM_2950JAJKPP000000000EgyptUrine201681650.50ST44DALPHECOLI2UJAENHQ000000000EgyptUrinary Catheter201984950.51ST641GK-12 strain 71SSTS00000000EgyptUrine201965150.55ST692C^a^MLST, Pasteur scheme; ^b^Phylogroup, Clermont typing.

Conclusions

This study offers insights into the prevalence of antibiotic resistance mechanisms and mobile genetic elements in two E. coli strains (UPE7 and UPE139) isolated from patients with urinary tract infections in Egypt. The clinical isolates were identified as multidrug-resistant, exhibiting resistance to multiple classes of antimicrobials. Genomic analysis of MGEs highlighted their role in the rapid dissemination of MDR traits across different clonal types of pathogenic E. coli. These findings underscore the importance of combining phenotypic testing with genomic approaches to guide the treatment of urinary tract infections and inform effective control strategies. However, the limited number of isolates analyzed, future studies involving larger and more diverse collections of clinical strains are essential to validate these results and capture broader resistance patterns. Moreover, subsequent investigations will include plasmid sequencing to further explore the presence and transferability of resistance genes. Additionally, expanded genomic surveillance across different geographic regions and healthcare settings will be critical for tracking the emergence and dissemination of high-risk clones, identifying resistance genes, and guiding antibiotic stewardship efforts.