Molecular Characterization of Escherichia coli Causing Urinary Tract Infections Through Next-Generation Sequencing: A Comprehensive Analysis of Serotypes, Sequence Types, and Antimicrobial and Virulence Genes

Introduction An enormous increase in antimicrobial resistance (AMR) among bacteria isolated from human clinical specimens contributed to treatment failures. Increased surveillance through next-generation sequencing (NGS) or whole genome sequencing (WGS) could facilitate the study of the epidemiology of drug-resistant bacterial strains, resistance genes, and other virulence determinants they are potentially carrying. Methods This study included 30 Escherichia coli (E. coli) isolates obtained from patients suffering from urinary tract infections (UTIs) attending Prathima Institute of Medical Sciences, Karimnagar, India. All bacterial isolates were identified, and antimicrobial susceptibility patterns were determined through conventional microbiological techniques and confirmed by automated systems. All the isolates were investigated using NGS to identify genes coding for resistance, such as extended-spectrum beta-lactamases (ESBLs), metallo-beta-lactamases, and virulence genes. Multilocus sequence typing (MLST) was used to understand the prevalent strain types, and serotyping was carried out to evaluate the type of O (cell wall antigen) and H (flagellar antigen) serotypes carried by the isolates. Results The conventional antimicrobial susceptibility testing revealed that 15 (50%) isolates were resistant to imipenem (IPM), 10 (33.33%) were resistant to amikacin (AK), 13 (43.33%) were resistant to piperacillin-tazobactam (PTZ), 17 (56.66%) were resistant to cephalosporins, and 14 (46.66%) were resistant to nitrofurantoin (NIT). Among the isolates, 26 (86.66%) had revealed the presence of multiple antibiotic-resistant genes with evidence of at least one gene coding for beta-lactamase resistance. There was a high prevalence of blaCTX-M (19/30, 63.33%) genes, followed by blaTEM and blaOXA-1. The blaNDM-5 gene was found in three isolates (3/30, 10%). The virulence genes identified in the present study were iutA, sat, iss, and papC, among others. The E. coli serotype found predominantly belonged to O25:H4 (5, 16.66%), followed by O102:H6 (4, 13.33%). A total of 16 MLST variants were identified among the examined samples. Of the MLST-based sequence types (STs) identified, ST-131 (7, 23.33%) was the predominant one, followed by ST-167 (3, 10%) and ST-12 (3, 10%). Conclusions The study results demonstrated that the E. coli strains isolated from patients suffering from UTIs potentially carried antimicrobial resistance and virulence genes and belonged to different strain types based on MLST. Careful evaluation of bacterial strains using molecular analyses such as NGS could facilitate an improved understanding of bacterial antibiotic resistance and its virulence potential. This could enable physicians to choose appropriate antimicrobial agents and contribute to better patient management, thereby preventing the emergence and spread of drug-resistant bacteria.


Introduction
Antimicrobial resistance (AMR) is the ability of bacteria to resist its clearance from the hosts despite treatment.Bacteria resistant to various antimicrobial agents are labeled multidrug-resistant (MDR) bacteria.There are several mechanisms by which bacteria develop AMR, including mutations, drug efflux mechanisms, and alteration of drug-binding proteins, among others.Additionally, bacteria employ different virulence mechanisms, such as adherence and biofilm formation, which enable them to counteract the activities of drugs.Moreover, AMR is coded by genes on the chromosomes and plasmids, which could be transmissible from one bacterium to another bacterium.This can be achieved through intraspecies (vertical gene transfer) and interspecies (horizontal gene transfer) genetic transfer mechanisms that occur naturally among bacteria.The excessive use of antimicrobial agents, especially among hospitalized patients, facilitates the development of resistant bacterial species.Besides, people resort to self-medication by using over-the-counter drugs, which results in resistance among the bacterial species prevailing in individuals and communities.Subsequently, these resistant bacterial species could be accountable for hospital-acquired infections (HAIs) and community-acquired infections (CAIs).There is an increased probability of the spread of such bacterial species from one person to another in the community, from patient to patient, and from healthcare workers (HCWs) to patients in hospital settings.Infections with drug-resistant bacteria result in treatment failures [1].
The emergence of AMR cannot be predicted and probably is difficult to prevent, as observed by a recent case study of a patient suffering from pancreatitis.This case study emphasizes the role of clinicians in discussing and evaluating antibiotic treatment, which otherwise would result in AMR and treatment failure.It was suggested that patients must be assessed for any preexisting AMR determinants in colonized bacteria.Screening for AMR among bacteria in the hospital environment and evidence of de novo resistance that may be responsible for horizontal gene transfer was also recommended [2].
Escherichia coli (E.coli) is a commensal bacteria present in the intestines of humans and animals.Apart from being a normal flora of the intestines, E. coli has been identified as the most predominant bacteria to cause urinary tract infections (UTIs).Escherichia coli strains with pathogenic potential are responsible for other infections such as toxin-mediated diarrhea, bloodstream infections, and wound infections.Further, neonates and other individuals with suppressed immune systems could be predisposed to various infections caused by E. coli [3].Considering the complexity of this bacterium, as evidenced by its existence as a commensal, its presence in the environment, and its potential to cause mild to severe infections, it is essential to understand its epidemiological features, virulence determinants such as antimicrobial resistance capabilities, and invasive potential, among others.This study, therefore, is carried out to evaluate the presence of resistance and virulence genes in E. coli isolated from patients suffering from UTIs.Additionally, they were classified into serotypes and sequence types (STs) based on molecular characterization utilizing next-generation sequencing (NGS) or whole genome sequencing (WGS).

Materials And Methods
An observational, analytical, cross-sectional study was conducted among patients attending Prathima Institute of Medical Sciences, Karimnagar, Telangana, India.This study included 30 E. coli isolates obtained from patients suffering from urinary tract infections from April 2018 to April 2020.All bacterial isolates were identified, and antimicrobial susceptibility patterns were determined through conventional microbiological techniques and confirmed by automated systems [4][5][6].Further, all the isolates were analyzed using NGS/WGS to identify the genes coding for drug resistance and virulence.Multilocus sequence typing (MLST) was used to understand the prevalent strain types, and serotyping was carried out to evaluate the type of O (cell wall antigen) and H (flagellar antigen) serotypes expressed by the isolates.

Processing of urine samples
A wide-mouthed sterile container was provided to the patient.The patient was advised to clean the genital areas with soap and water.Later, the patient was asked to collect mid-stream urine after passing out the initial urine.The patient was advised not to touch the inside of the container, and the urine should not overflow from the container.The sample container was appropriately labeled and was immediately transported to the laboratory for processing.When delays in transporting specimens to the laboratory are expected, the urine samples were refrigerated between 2°C and 8°C.The sample was processed by inoculating 0.001 milliliters (mL) of urine in blood agar.The method used here was the semiquantitative counting technique.In this method, the urine sample is plated on the culture media after dividing the plate into four quadrants, as shown in Figure 1.

FIGURE 1: Growth obtained on blood agar from urine sample for semiquantitative estimation of bacteria
After overnight incubation of the inoculated plates at 37°C, the growth was recorded by counting the number of colonies or presence of growth in different quadrants of blood agar.The patients whose urine contained ≥10 5 colony-forming units (CFUs) per mL of urine were considered to be suffering from UTI.The CFUs are nothing but bacteria, i.e., each colony corresponds to a bacterium [7].

Interpretation
Growth in the first quadrant corresponds to 25,000 colonies, both in the first and second quadrants corresponds to 50,000 colonies, growth in the three quadrants corresponds to 75,000 colonies, and growth in all the quadrants corresponds to 100,000 or 10 5 CFU/mL of urine (Figure 1).

Kirby-Bauer Disk Diffusion Method
Two to three pure and isolated colonies from overnight bacterial growth (inoculum) were picked up from the culture plate.The inoculum was mixed well into the peptone water/sterile saline.Later, it was incubated at 37°C for 1-2 hours.The test tube now shows growth in the form of turbidity.The turbidity is adjusted to match turbidity standards, as measured by the McFarland standards.The McFarland standards are used for measuring turbidity manually by comparing and adjusting the culture turbidity with a solution prepared by mixing 0.05 mL of 1% barium chloride and 9.95 mL of 1% sulfuric acid.
After adjusting to the desired McFarland standards, the test organisms were inoculated into Mueller-Hinton agar (MHA).A lawn culture/carpet culture was made with the help of sterile cotton swabs.Later, different antibiotic-impregnated filter paper disks were applied with the help of sterile forceps.The plates were then incubated overnight at 37°C for 12-18 hours.If an organism is not growing near the antibiotic discs, i.e., if organisms are sensitive/susceptible to the antibiotic, there is a zone of inhibition/clearance, measured in millimeters (mm).However, if the microorganisms are resistant to the antibiotic, growth will be noted even closer/toward the edge of the antibiotic-impregnated disk.The results were interpreted according to the Clinical and Laboratory Standards Institute (CLSI) guidelines [8].Isolates with phenotypic resistance, including both resistant (R) or intermediate (I) resistance, are considered resistant (R).An isolate was designated as MDR when it showed resistance to more than one agent in three or more antimicrobial categories.

Control Strains
Escherichia coli American Type Culture Collection (ATCC) 25922, Klebsiella pneumoniae ATCC 1706, Pseudomonas aeruginosa ATCC 27853, and Staphylococcus aureus ATCC 25923 were used as controls.

Whole genome sequencing and genomic analyses
The deoxyribonucleic acid (DNA) was extracted from bacterial isolates using the QIAamp DNA Mini Kit (Qiagen, Hilden, Germany) as mandated by the manufacturer's instructions.Double-stranded DNA libraries with 450 base pairs (bp) insert size were prepared and sequenced on the Illumina platform with 150 bp paired-end chemistry.The genomes that passed sequence quality control were assembled using Spades v3.14 to generate contigs and annotated with Prokka v1.5 [9,10].Species identification was carried out using a Bactinspector, and contamination was assessed using ConFindr [11].All the quality metrics were combined using MultiQC and qualifyr to generate web-based reports.MLST, AMR, and virulence factors were identified using ARIBA tool v2.14.4 with BIGSdb-Pasteur MLST database, National Center for Biotechnological Information (NCBI) AMR acquired gene, PointFinder databases, and VFDB, respectively [12][13][14].All bioinformatic analysis was performed using Nextflow pipelines developed as a part of the Global Health Research Unit (GHRU)-AMR.

Discussion
Among the several public health concerns encountered globally, resistance of microbes to antimicrobial agents has become a tough nut to crack.AMR could be attributed to factors such as irrational and indiscriminate use of antimicrobial agents and lack of newer and more efficient drugs being developed by pharmaceutical industries owing to the high cost associated with drug development.Antibiotic use began with penicillin during the Second World War (1940).However, slowly and gradually, bacterial species resistant to penicillin emerged.This led to using newer penicillins such as methicillin and other antibiotics such as tetracyclines, erythromycin, and aminoglycosides such as gentamicin.Later, cephalosporins, including ceftazidime, were prescribed to treat bacterial infections.However, due to irresponsible prescription practices, bacterial species resistant to most of antimicrobial agents started to emerge, and such bacterial species were labeled as MDR, pan-drug-resistant (PDR), and extensively drug-resistant (XDR) bacteria [15].
MDR strains are identified based on the resistance shown by bacteria to at least one drug under three or more antimicrobial agent categories.When a bacterium is found resistant to at least one antimicrobial agent in most categories, it is identified as an XDR bacterium.A bacterial species showing resistance to all the antibiotics under different categories is labeled PDR bacteria [16].
Increased AMR has resulted in the rise of morbidity and mortality among affected patients.Additionally, AMR could cause an economic burden on patients attributed to extended hospital stays and prolonged antibiotic use [17].

Beta-lactamase resistance genes
Enterobacteriaceae members, including E. coli, are noted to develop intrinsic resistance to antibiotics and transferable resistance (chromosomes and plasmids).This forces us to monitor the presence of AMR genes among bacterial isolates.Intrinsic resistance was attributed to AmpC-beta-lactamase and broad-spectrum beta-lactamases such as bla TEM-1 , bla TEM-2 , bla SHV-1 , and bla OXA-1 [18].
The beta-lactam group of antibiotics are those antibiotics that interfere with cell wall synthesis.Penicillin, ampicillin, and amoxicillin are a few examples of antibiotics that possess a beta-lactam ring.However, bacterial species have been able to counter the activities of beta-lactam antibiotics by producing betalactamase enzymes that catalyze beta-lactam antibiotics, thereby inactivating them.Considering this, scientists have developed beta-lactamase inhibitors that could be combined with beta-lactam antibiotics, which enables them to counter the activities of beta-lactamases produced by bacteria.Amoxycillin is a betalactam antibiotic when combined with beta-lactamase inhibitors such as clavulanic acid to counter the betalactamases produced by bacteria.
Over time, bacteria have acquired the ability to survive the action of beta-lactam antibiotics.Infections due to these bacteria were treated by antibiotics such as cephalosporins.Further, a few bacterial species started showing resistance to the penicillin group and the narrow and broad-spectrum cephalosporin group of antibiotics.Such bacteria were labeled as extended-spectrum beta-lactamase (ESBL)-producing bacterial species.It was identified that bacteria develop resistance to lower cephalosporins such as cefazolin and cephalothin consisting of genes such as Temoniera-1 (TEM-1) and TEM-2.Following the availability of higher cephalosporins such as cefotaxime, ceftriaxone, and ceftazidime, among others, with broad-spectrum activities, bacteria with TEM-1 and TEM-2 were inhibited.Later, bacteria with lowered susceptibility to broad-spectrum cephalosporins with oxyimino side chain started to appear, which were found to possess the sulfhydryl variable (SHV) gene [19].
A high prevalence of bla CTX-M (19, 63.33%) type ESBL was witnessed in the current study.A similar finding was reported from Bangladesh wherein E. coli isolated in extraintestinal specimens demonstrated bla CTX-M prevalence of 52% [24].The study noticed a lower prevalence of bla TEM (20%) and bla OXA-1 (17%) compared to the present study results, wherein bla TEM and bla OXA-1 showed a higher prevalence of 36.66%(11/30).

ST-131 (O25:H4)
ST-131 clones were first identified in 2003.However, they became highly prevalent strains of ExPEC by the year 2008.These E. coli strains have become prominent due to their ability to carry the bla CTX-M-15 gene and demonstrate ESBL activities.Among extraintestinal infections, E. coli li is the primary cause of UTIs.It was also observed that most ST-131 E. coli demonstrate the same serotype (O25:H4).This finding was also confirmed by the results of the current study, wherein 85.71% (6 out of 7) of the ST-131 E. coli showed an O25:H4 serotype.There was one ST-131 that revealed the O16:H5 serotype, which was first identified in Japan in 2012 and later in other countries [29].
The ST-131 cone was also identified as being responsible for recurrent UTIs.Moreover, ST-131 was frequently associated with hospital-and community-acquired infections.Colonization in animals and birds and MDR were a few characteristic features established in ST-131 clones [30].

ST-410 (O8:H21)
This strain type was previously described as a high-risk variant that can develop antimicrobial resistance and high virulence capabilities such as E. coli ST-131.In this study, two isolates belonging to ST-410 (2/30, 6.66%) were recognized.Further, both these STs belonged to the O8:H21 serotype [38].
MLST of E. coli has been a familiar ST known to carry drug resistance genes that make it MDR.These strains can harbor the resistance genes and potentially transmit the drug resistance genes, intraspecies and interspecies.This is evident from a recent report that demonstrated the transfer of the bla KPC-2 gene from E.

ST-648 (O1:H6)
In the current study, this ST was noted to carry bla CMY-42 , blaEC-19, bla OXA-1 , and bla CTX-M-15 .This ST was previously identified in birds, dogs, cats, and horses [40,41].ST-648 strain with O1:H6 serotype was also identified in UTI-causing E. coli isolates from Brazil [42].This ST was observed among E. coli isolates from wastewater pumps of the community.Similar to the results of this study, plasmid Col8282 and bla CTX-M-15 resistance genes were observed [43].ST-648 was the second most common ST identified with an O1:H6 serotype (5.6%) combination, second to ST-131 [44].ST-648 was among the predominant STs noted from E. coli isolated from human feces, sewage, and foodstuffs in England.Most of these isolates were noted to carry bla CTX-M-15 [45].
It was confirmed that ST-648 does not possess any specific host affliction.However, ST-648 was frequently associated with MDR and high virulence capabilities, including biofilm formation with the potential to cause invasive infections such as bacteremia [46].
Other STs identified in the present study that are scarcely reported in the literature

ST-156 (O54:H28)
In the present study, this ST revealed the presence of plasmid genes such as IncFIB and IncFII.Among the genes responsible for ESBL, this isolate showed the presence of bla CMY-42 , bla EC-15 , and bla OXA-181 genes, among others.

ST-827 (O4:H1)
In the present study, this ST revealed the presence of plasmid genes such as IncB/O/K/Z, IncFIB (AP001918), and IncFII.The genes responsible for ESBL were not identified in this isolate.

ST-38 (O11:H30)
In the present study, this ST revealed the presence of several plasmid genes such as IncFIB, IncFII, and IncX4.Among the genes responsible for ESBL, this isolate showed the presence of bla CTX-M-15 , blaEC-8, bla OXA-1 , and bla TEM-1 genes, among others.
Single Locus Variant of ST-14 (O75:H5) In the present study, this ST revealed the presence of several plasmid genes such as Col156, ColRNAI, ColBS512, IncFIA, IncFIB, IncFII, and IncI1.Among the genes responsible for ESBL, this isolate showed the presence of bla CMY-2 and blaEC-5 genes, among others.

Study limitations
This study analyzed a few E. coli strains isolated from patients with UTIs admitted to a tertiary care teaching hospital.The study also did not compare the phenotypic resistance characteristics of the individual isolates with the genes identified through WGS.Besides, the study did not carry out phylogenetic analysis and clade categorization/identification of the isolates.This study did not explore the carriage of resistance and virulence genes on specific plasmid replicons and their origins.

Conclusions
The results of this study demonstrate that E. coli isolated from people suffering from UTIs carry several genes coding for drug resistance and other genes for virulence.This could enable them to gain MDR and become invasive, thereby contributing to severe morbidity and mortality among the affected population.Additionally, the E. coli analyzed in this study demonstrated plasmid genes that have an increased potential to carry drug-resistant genes, which can be transmitted to other strains through horizontal or vertical routes.The results of this study emphasize the role of WGS in understanding the genetic characteristics of bacteria and predicting the emergence of antimicrobial resistance.Data generated from such studies require elaborate analysis before being applied to make decisions regarding the use of antimicrobial agents, thereby preventing future emergences of MDR strains.Further, these decisions can minimize treatment failures stemming from infections caused by the MDR bacterial strains.
following: Payment/services info: All authors have declared that no financial support was received from any organization for the submitted work.Financial relationships: All authors have declared that they have no financial relationships at present or within the previous three years with any organizations that might have an interest in the submitted work.Other relationships: Kempegowda Institute of Medical Sciences has made a memorandum of understanding (MOU) with Dr. Venkataramana Kandi, Prathima Institute of Medical Sciences, to collaborate under the project supported by the Global Health Research Unit (GHRU), United Kingdom. .

TABLE 2 : Details of antimicrobial resistance and virulence genes, and phenotypic susceptibility results of the isolates
EC: Escherichia coli, M: male, F: female, UTI: urinary tract infection, CKD: chronic kidney disease, I: imipenem, AG: aminoglycosides, PTZ: piperacillintazobactam, CE: cephalosporins, NIT: nitrofurantoin, S: sensitive, R: resistant Among the isolates, 26 (86.66%) had revealed the presence of multiple antibiotic-resistant genes with evidence of at least one gene coding for beta-lactamase resistance.The genes identified in this study were

TABLE 3 : Serotypes of the E. coli strains
EC: Escherichia coli, E. coli: Escherichia coliThe functions of the housekeeping genes based on which the MLST was carried out and the plasmid replicons identified among the isolates are shown inTable 4.