Hindawi

BioMed Research International

Volume 2023, Article ID 7278070, 12 pages
https://doi.org/10.1155/2023/7278070

Research Article
Virulence Factors and Antimicrobial Resistance of Uropathogenic
Escherichia coli EQ101 UPEC Isolated from UTI Patient in
Quetta, Balochistan, Pakistan

Sareen Fatima,1 Ali Akbar ,1,2 Muhammad Irfan ,3 Muhammad Shafee,4 Amjad Ali,5
Zaara Ishaq,5 Syed Kashif Raza,6 Abdul Samad,4 Mohammad Y. Alshahrani,7
and Syed Shah Hassan 3
1
Department of Microbiology, University of Balochistan, Quetta, Balochistan, Pakistan
2
Centre for Biotechnology and Microbiology, University of Swat, Charbagh, 19120 Khyber Pakhtunkhwa, Pakistan
3
Jamil-ur-Rahman Center for Genome Research, International Center for Chemical and Biological Sciences (ICCBS),
University of Karachi, Karachi, Pakistan
4
Center for Advanced Studies in Vaccinology & Biotechnology (CASVAB), University of Balochistan, Quetta, Balochistan, Pakistan
5
Department of Industrial Biotechnology, Atta-ur-Rahman School of Applied Biosciences (ASAB), National University of Sciences
and Technology (NUST), Islamabad 44000, Pakistan
6
Riphah International University Faisalabad, Pakistan
7
Department of Clinical Laboratory Sciences, College of Applied Medical Sciences, King Khalid University, P.O. Box 61413,
Abha 9088, Saudi Arabia

Correspondence should be addressed to Ali Akbar; [email protected] and Syed Shah Hassan; [email protected]

Received 21 September 2022; Revised 31 March 2023; Accepted 11 July 2023; Published 11 September 2023

Academic Editor: Nikhil Agrawal

Copyright © 2023 Sareen Fatima et al. This is an open access article distributed under the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Infectious diseases have been tremendously increasing as the organisms of even normal flora become opportunistic and cause an
infection, and Escherichia coli (E. coli EQ101) is one of them. Urinary tract infections are caused by various microorganisms, but
Escherichia coli is the primary cause of almost 70%–90% of all UTIs. It has multiple strains, possessing diverse virulence factors,
contributing to its pathogenicity. Furthermore, these virulent strains also can cause overlapping pathogenesis by sharing resistance
and virulence factors among each other. The current study is aimed at analyzing the genetic variants associated with multi-drug-resistant
(MDR) E. coli using the whole genome sequencing platform. The study includes 100 uropathogenic Escherichia coli (UPEC)
microorganisms obtained from urine samples out of which 44% were multi-drug-resistant (MDR) E. coli. Bacteria have been isolated
and antimicrobial susceptibility test (AST) was determined by disk diffusion method on the Mueller-Hinton agar plate as
recommended by the Clinical and Laboratory Standards Institute (CLSI) 2020, and one isolate has been selected which shows
resistance to most of the antibiotics, and that isolate has been analyzed by whole genome sequencing (WGS), accompanied by data
and phylogenetic analysis, respectively. Organisms were showing resistance against ampicillin (10 μg), cefixime (5 μg), ceftriaxone
(30 μg), nalidixic acid (30 μg), ciprofloxacin (5 μg), and ofloxacin (5 μg) on antimicrobial susceptibility test. WGS were done on
selected isolate which identified 25 virulence genes (air, astA, chuA, fyuA, gad, hra, iha, irp2, iss, iucC, iutA, kpsE, kpsMII_K1, lpfA,
mchF, ompT, papA_F43, sat, senB, sitA, terC, traT, usp, vat, and yfcV) and seven housekeeping genes (adk, fumC, gyrB, icd, mdh,
purA, and recA). Among resistance genes, seven genes (TolC, emrR, evgA, qacEdelta1, H-NS, cpxA, and mdtM) were identified to be
involved in antibiotic efflux, three AMR genes (aadA5, mphA, and CTX-M-15) were involved in antibiotic inactivation, and two
genes (sul1 and dfrA14) were found to be involved in antibiotic drug replacement. Our data identified antibiotic resistance and
virulence genes of the isolate. We suggest further research work to establish region-based resistance profile in comparison with the
global resistance pattern.
2 BioMed Research International

1. Introduction and economic issues even in the first world countries, like the
UK, Norway, and Georgia [16–19].
The World Health Organization (WHO) has issued a report Despite the knowledge of such a wide range of virulence
highlighting common healthcare-associated bacterial organ- genes of E. coli, antibacterial resistance is still a major con-
isms showing increased rates of antimicrobial resistance cern around the globe [20]. The treatment for an organism
globally [1]. It was revealed in a study that the mortality has become progressively complicated because of the pres-
rates can reach to around ten million per year by 2050 in ence of resistance to usually the 1st-line antimicrobial drugs
the absence of any action taken against antimicrobial resis- [21]. Antimicrobial resistance in UPEC has remarkably
tance [2]. increased in the last few years and has become a tremendous
Therefore, among the various resistance-causing organ- public health issue [14, 22, 23]. UPEC strains were observed
isms, E. coli has gained great attention globally after being to be resistant mainly to trimethoprim-sulfamethoxazole,
a serious pathogen and with its diverse virulence capabili- ampicillin, and ampicillin-sulbactam [21, 24]. E. coli can
ties [3]. acquire AMR genes through mobile genetic elements
Urinary tract infections (UTIs) are one of the most prev- (MGE), which include plasmids, integrons, gene cassettes,
alent bacterial outpatient infections and frequently occur in insertion sequences, and transposons [25]. A large number
women accounting for almost 50-60% infections in adult of resistance-encoding MGE, specific plasmids, are shared
women and affecting 150 million people per annum globally between different members of the Enterobacteriaceae and
[4, 5]. This high prevalence has been linked to several risk thus further promote the spread of resistance genes [26].
factors, including catheterization, surgical manipulation, MGE can also indicate virulence factors and the interplay
and disruption of the urinary tract, mainly among diabetic between virulence and antimicrobial resistance [23]. There-
and immunosuppressive patients, along with recurrent hos- fore, it imposes the need to identify the genes which are
pitalizations and other comorbidities [6]. UTIs are caused by responsible for the resistance to cater and improve the treat-
various microorganisms but uropathogenic Escherichia coli ment outcome.
(UPEC) being the primary cause (70%–90% of all UTIs) Most E. coli strains are isolated and identified by their O-
[7, 8]. In developing countries like Pakistan, it is a serious antigen (lipopolysaccharide), H-antigen (flagellar), and K-
threat to the public health due to its association with high antigen (capsular). The detailed structure of its genome
morbidity and mortality rate [9]. and the responsible resistant genes can only be discovered
E. coli is a gram-negative organism and usually resides in through specialized molecular testing techniques, like whole
the intestine of humans [10]. Structurally, the genome of E. genome sequencing [27, 28]. With the advent of sequencing
coli strain varies in size between extragenetic elements, path- technologies, it has become easier to get better insight into
ogenic variants, and commensal strains [11]. bacterial pathogenesis and to identify alternative therapeu-
Animals and humans both have Escherichia coli in their tics [29]. Therefore, the current study is aimed at character-
intestines. Numerous strains include enteropathogenic Escher- izing a local MDR E. coli strain specifically associated with
ichia coli (EPEC), Shiga toxin-producing Escherichia coli UTIs and at analyzing the genetic variants associated with
(STEC), enterohaemorrhagic Escherichia coli (EHEC), enter- it using the whole genome sequencing approach. In this
oaggregative Escherichia coli (EAEC), enterotoxigenic Escheri- study, we aim to identify the virulence and AMR profile of
chia coli (ETEC), enteroinvasive Escherichia coli (EIEC), and the local UPEC strain. Eventually, the sequenced genome
diffusely adherent Escherichia coli(DAEC) [12]. will be used to get an insight into epidemiological studies
Each strain possesses different virulence genes, including of local E. coli strains which will help to get the global pat-
eae, tir, and bfpA by EPEC, and apAA by EAEC. Two differ- tern using pangenomic approach as there is very less
ent toxins are reported in EHEC that include Stx1/Stx2 genome sequencing data available from Pakistan (especially
(Shiga-like toxin) and ehxA (enterohaemolysin), whereas, from Balochistan). Moreover, the global burden of UTIs
an invasion plasmid (vir regulon) is reported in EIEC. There suggests detailed in silico analyses of UPEC, thus identifying
are two enterotoxins heat-stable (S.T.) and labile-stable possible therapeutic strategies.
(L.T.) enterotoxins, by ETEC [12]. The World Health Organization (WHO) has issued a
UPEC contains many genes that encode different virulence report highlighting common healthcare-associated bacterial
factors taking part to its pathogenesis including toxins, capsule, organisms showing increased rates of antimicrobial resistance
serum resistance, adhesive properties, and its iron uptake sys- globally [1]. It was revealed in a study that the mortality rates
tems [13, 14]. Major virulence genes possessed by UPEC strains can reach to around ten million per year by 2050 in the absence
are aerobactin (aer), P fimbriae (pap), hemolysin (hly), type 1 of any action taken against antimicrobial resistance [2].
fimbriae, afimbrial adhesin I (afa I), cytotoxic necrotizing fac- Therefore, among the various resistance-causing organ-
tor 1 (cnf 1), S fimbriae (sfa), adhesins, and fimbriae. The other isms, E. coli has gained great attention globally after being a
virulence genes are kpsMT, ompT, usp, iroN, iha, set 1, and serious pathogen and with its diverse virulence capabilities [3].
astA, group II capsule synthesis; sfa/foc, S, and F1Cfimbriae;
iutA and traT, serum resistance; and fimH [14, 15]. These vir- 2. Materials and Methods
ulence genes/factors possess crucial properties that allow
microorganisms to establish its virulence properties effectively 2.1. Bacterial Isolates and Antibiotic Susceptibility Testing. A
on its host to induce disease and also induce resistance, total of 100 UPEC samples were collected from pyelonephritis
enabling them to cause pathogenesis impacting health, social, patients at Bolan medical complex Hospital and Sandeman
BioMed Research International 3

Provincial Hospital, Quetta. 44 clinical samples with positive sequencing using the MiSeq Illumina platform. The paired-
MDR species of E. coli were transported to the Microbiology end sequencing was carried out by 2 × 151 cycles using V3
Laboratory, University of Balochistan. Samples were then cul- flow cell.
tured on the MacConkey agar and cystine-lactose-electrolyte-
deficient agar (CLED). Gram-staining characteristics and 2.4. In Silico Genome Characterization and Resistome Analysis.
standard biochemical tests, sulfide indole motility [30], The paired-end sequencing data was obtained in the fastq for-
triple sugar iron (TSI), urease, and citrate, were performed mat containing short sequencing reads. The sequenced reads
following the procedures described by Akbar et al. [31] were assembled using Unicycler v0.4.9, which uses information
and Ishaq et al. [32]. by SPAdes v3.13.0 for assembly followed by polishing the
Antimicrobial susceptibility test (AST) was determined aligned reads using Pilon v1.8 with a minimum threshold of
by disk diffusion method on the Mueller-Hinton agar plate 300 bases of contig length. Contigs having a length of fewer than
as recommended by the Clinical and Laboratory Standards 500 bases were filtered out. The serotype of the sequenced isolate
Institute (CLSI) 2019. was confirmed by depositing the sequenced data in the Center
Amoxicillin and clavulanate (30 μg), ampicillin (10 μg), for Genomic Epidemiology (http://www.genomicepidemiology
amikacin (30 μg), aztreonam (30 μg), gentamicin (10 μg), .org) for E. coli using the web-based SerotypeFinder 2.0, sero-
ceftriaxone (30 μg), nitrofurantoin (300 μg), nalidixic acid typing tool by applying default parameters. FimH was identi-
(30 μg), trimethoprim (25 μg), cefixime (5 μg), fosfomycin fied using FimTyper 1.0, and virulence genes were identified
(50 μg), ciprofloxacin (5 μg), piperacillin/tazobactam (40 μg), using the VirulenceFinder 2.0 database with the following
ertapenem (10 μg), and ofloxacin (5 μg) antibiotics were tested parameters: threshold for ID 90% and minimum length 60%.
for AST. All those isolates which were found resistant to four The assembled genome was annotated using RAST tool kit.
or more than four antibiotics were considered MDR and The comprehensive antibiotic resistance database [36] pack-
included in the study for detailed analysis. Similarly, the E. coli age was used to predict the antimicrobial resistance genes
strain resistant to more than five antibiotics was used in the [37]. Resistance gene identifier criteria were set to predict,
study to be processed for whole genome sequencing. strict, and complete genes only. VirulenceFinder 2.0 was used
Tables 1–4 are shown. to find out the genes responsible for virulent mechanisms.
2.2. DNA Extraction. Bacterial genomic DNA was extracted
from the cultured isolate using CTAB method with few 2.5. Pangenome Analysis. Here, 63 UPEC genomes were
modifications [33]. The isolated bacterial colonies were retrieved from the NCBI database (40 complete, 22 draft,
mixed with CTAB buffer containing 0.2% β-mercaptoetha- and E101) and were annotated by Prokka using the default
nol and proteinase K (20 mg/mL) into the 1.5 mL safe lock parameters [38]. The pangenome analysis and the core-
tube. The mixture was incubated at 60°C for 30 minutes, genome SNP-based phylogenetic analysis were performed
and after that, chloroform-isoamyl alcohol (24 : 1) was added by PanRV [39] which uses Roary [40] for the pangenome
into the tube. The tube was centrifuged, and the aqueous estimation.
phase was collected into the new tube. Isopropanol (equal
volume) was added and incubated at 4°C for 30 minutes 3. Results
and centrifuged. DNA was collected as a pellet while the
supernatant was discarded. The precipitated DNA was All samples showed the presence of E. coli after being proc-
washed two times with 70% ethanol, and the DNA pellet essed through culture and sensitivity. Organisms showing
was air-dried. The DNA pellet was dissolved in 1× TE buffer resistance against a list of antibiotics were selected as
and stored at 4°C till further processing. The quality of geno- multi-drug-resistant.
mic DNA was assessed using 1% agarose gel electrophoresis The de novo whole genome assembly of E. coli EQ101
while the quantity was estimated by dsDNA high sensitivity was done by Unicycler using SPAdes. The assembled
kit by Qubit fluorometer [34]. genome comprised 918 contigs covering a total length of
5,764,348 bases with an average G+C content found around
2.3. DNA Sequencing and Assembly. DNA library of MDR E. 50.89% and N50 was 9,699. The serotype of the sequenced
coli EQ101 was prepared using the Nextera XT kit (Illumina, isolate was identified as H18 with 100% identity of H type
San Diego, CA, US) according to the manufacturer’s guide- (fliC gene) with GenBank accession number AY250001,
lines. High molecular weight genomic DNA (5 ng) was frag- while no hit was identified for O-type genes. FimH64 was
mented (~300 bases) by transposomes. The adapters and identified in the sequenced isolate, and the threshold was
indexes were ligated to both DNA ends, and then, amplifica- 95% using FimTyper 1.0.
tion of the adapter-ligated library was performed by PCR. Genome annotation of the assembled whole genome was
The amplified library was purified by Agencourt AMPure carried out by PATRIC. The assembled genome consisted of
XP beads. The quantity of purified library was estimated 6,277 protein coding sequences (CDS), 53 transfer RNA
by dsDNA HS Qubit kit while the size distribution of the (tRNA) genes, and 2 ribosomal RNA (rRNA) genes. The
library was evaluated by 3% agarose gel electrophoresis. sequenced reads were aligned with the E. coli reference
The library was denatured and diluted to 16pM with a genome MG1655. The genome coverage of the sequenced
hybridization buffer (HT1) [35]. The diluted library was isolate was found to be around 93.7%, and the mean depth
loaded into the sequencing cartridge for high-throughput coverage was 60.82×.
4 BioMed Research International

Table 1: CLSI zone size.

CLSI zone sizes

Antibiotic Sensitive ≥ Intermediate Resistant ≤ EQ101
Amoxicillin and clavulanate (30 μg) 18 14-17 13 18
Ampicillin (10 μg) 17 14-16 13 0
Amikacin (30 μg) 17 15-16 14 21
Aztreonam (30 μg) 21 18-20 17 23
Gentamicin (10 μg) 15 13-14 12 15
Ceftriaxone (30 μg) 23 20-22 19 0
Nitrofurantoin (300 μg) 17 15-16 14 20
Nalidixic acid (30 μg) 19 14-18 13 6
Trimethoprim (25 μg) 16 11-15 10 17
Cefixime (5 μg) 19 16-18 15 0
Fosfomycin (50 μg) 16 13-15 12 22
Ciprofloxacin (5 μg) 31 21-30 20 0
Piperacillin/tazobactam (40 μg) 21 18-20 17 21
Ertapenem (10 μg) 22 19-21 18 22
Ofloxacin (5 μg) 16 13-15 12 8

Table 2: The phenotypic resistance profile of EQ101.

No. Antibiotics Sensitivity Resistant Intermediate

1 Ampicillin (10 μg) 35% 60% 5%
2 Amoxicillin/clavulanic acid (30 μg) 70% 26% 4%
3 Piperacillin/tazobactam (40 μg) 80% 19% 1%
4 Cefixime (5 μg) 60% 38% 2%
5 Ceftriaxone (30 μg) 78% 21% 1%
6 Ertapenem (10 μg) 55% 39% 6%
7 Amikacin (30 μg) 51% 39% 10%
8 Gentamycin (10 μg) 55% 39% 6%
9 Nalidixic acid (30 μg) 35% 60% 5%
10 Ciprofloxacin (5 μg) 45% 48% 7%
11 Ofloxacin (5 μg) 34% 66% 0%
12 Fosfomycin (50 μg) 60% 25% 15%
13 Nitrofurantoin (300 μg) 80% 11% 9%
14 Sulfamethoxazole-trimethoprim (25 μg) 37% 41% 22%
15 Aztreonam (30 μg) 58% 30% 12%

Table 3: The genomic features and characteristics of the E coli Commission (EC) numbers, 1,304 with Gene Ontology (GO)
strain EQ101. assignments, and 1,104 proteins that were mapped to KEGG
pathways. PATRIC annotation included two types of protein
Characteristics EQ101
families, the genus-specific protein families (PLFams) which
Genome size 5,764,348 bp have 5,956 proteins sequenced genome and the cross-genus
Contigs 918 protein families (PGFams) involving 6,070 proteins.
G+C content 50.89% A subsystem is a collection of proteins that combinedly
N50 9,699 implement a targeted biological process or structural complex
Standard deviation of contig lengths 8531.109786558065 and PATRIC. Numerous genes are involved in metabolisms
followed by energy, protein processing, membrane transport,
stress response, defense, virulence, cellular process, etc.
The annotation included 737 hypothetical proteins and
5,540 proteins with functional assignments. The proteins with 3.1. Pangenome-Based Phylogenetic Analysis. The pangen-
functional assignments included 1,573 proteins with Enzyme ome of selected UPEC strains consists of 21585 genes, of
BioMed Research International 5

Table 4: Phenotypic and genotypic resistance profile of EQ101.

No. Antibiotics PR GR
1 Ampicillin (10 μg) R blaCTX-M-15
2 Amoxicillin/clavulanic acid (30 μg) S ND
3 Piperacillin/tazobactam (40 μg) S ND
4 Cefixime (5 μg) R TolC, H-NS, CTX-M-15, EC-5
5 Ceftriaxone (30 μg) R blaCTX-M-15
6 Ertapenem (10 μg) S marA, Escherichia coli soxS with mutation conferring antibiotic resistance
7 Amikacin (30 μg) S aadA5
8 Gentamycin (10 μg) S aadA5
9 Nalidixic acid (30 μg) R TolC, evgA, H-NS, mdtM, gadW, emrR
10 Ciprofloxacin (5 μg) R TolC, evgA, H-NS, mdtM, gadW, emrR
11 Ofloxacin (5 μg) R TolC, evgA, H-NS, mdtM, gadW, emrR
12 Fosfomycin (50 μg) S ND
13 Nitrofurantoin (300 μg) S ND
14 Sulfamethoxazole-trimethoprim (25 μg) S sul1, dfrA17, dfrA14
15 Aztreonam (30 μg) S marA, Escherichia coli soxS with mutation conferring antibiotic resistance
16 Tetracycline ND tet(B)
17 MLS (macrolide, lincosamide, and streptogramin B) ND mph(A)
18 Disinfectant ND qacE
PR = phenotypic resistance; GR = genotypic resistance; R = resistant; S = susceptible; ND = not determined.

which 2,926 (13.55%) were core genes, 3,393 (15.7%) were resistant sul (sul1), macrolide phosphotransferase [41] (mphA
accessory genes, and 15266 (70.7%) were unique genes and MrX), resistance-nodulation-cell division (RND) antibiotic
(Figure 1(a)). The stats confirm the highly diverse nature efflux pump (TolC, cpxA, evgA, h-NS, and gadW), major
of E. coli strains having an open pangenome. facilitator superfamily (MFS) antibiotic efflux pump (TolC,
The core-genome-based phylogenetic analysis grouped qacEdelta1, leuO, evgA, H-NS, mdtM, and emrR), and ATP-
the EQ101 with a Brazilian strain BR-14 DEC (Figures 2 binding cassette (ABC) antibiotic efflux pump (TolC) with
and 3). Both the strains have 99.95% identity. The EQ101 resistance to the following respective drug classes: diaminopyr-
genome did not contain any uniquely present or absent imidine antibiotic, lincosamide antibiotic, nucleoside antibiotic,
genes because of its contig level assembly. sulfonamide antibiotic, disinfecting agents and antiseptics,
penem, phenicol antibiotic, rifamycin antibiotic, aminocou-
3.2. Antibiotic Resistance Genes. We found a total of 58 AMR marin antibiotic, peptide antibiotic, tetracycline antibiotic,
genes in the whole genome data of E. coli EQ101. Resistance penam, cephamycin, glycylcycline, cephalosporin, carbapenem,
gene identifier criteria were set to predict, strict, and com- aminoglycoside antibiotic, fluoroquinolone antibiotic, and
plete genes only, which returned with 12 perfect hits and macrolide antibiotic.
46 strict hits with no lose hits. The resistance mechanism 14 gene families and their AMR genes were found to have
for the perfect RGI criteria includes seven genes TolC, emrR, “strict hits” (90-100% identity of matching region) which are
evgA, qacEdelta1, H-NS, cpxA, and mdtM, involved in anti- as follows: penicillin-binding protein mutations conferring
biotic efflux, and three AMR genes, i.e., aadA5, mphA, and resistance to beta-lactam antibiotics (Haemophilus influenzae
CTX-M-15, involved in antibiotic inactivation, while two PBP3 conferring resistance to beta-lactam antibiotics),
genes sul1 and dfrA14 involved in antibiotic drug replace- elfamycin-resistant EF-Tu (Escherichia coli EF-Tu mutants
ment. The resistance mechanism in strict hit includes 35 conferring resistance to pulvomycin), antibiotic-resistant
genes involved in antibiotic efflux, two have reduced perme- UhpT (Escherichia coli UhpT with mutation conferring resis-
ability to antibiotics, 3 involved in antibiotic inactivation, tance to fosfomycin), fluoroquinolone-resistant parC (Escher-
one involved in antibiotic target replacement, and 12 ichia coli parC conferring resistance to fluoroquinolones),
involved in antibiotic target alteration. The details of the general bacterial porin with reduced permeability to beta-
AMR gene, drug class, and resistance mechanism are shown lactams (marA and Escherichia coli soxS with mutation con-
in Figures 4(a) and 4(b). ferring antibiotic resistance), van ligase (vanG), glycopeptide
10 gene families and their respective AMR genes were resistance gene cluster (vanG), small multidrug resistance
found to have “perfect hits” (100% identity of matching (SMR) antibiotic efflux pump (Klebsiella pneumoniae KpnE,
region) including EC beta-lactamase (EC-5), trimethoprim- Klebsiella pneumoniae KpnF, and Escherichia coli emrE),
resistant dihydrofolate reductase dfr (dfrA14), CTX-M kdpDE (kdpE), trimethoprim-resistant dihydrofolate reduc-
beta-lactamase (CTX-M-15), undecaprenyl pyrophosphate- tase dfr (dfrA17), resistance-nodulation-cell division (RND)
related proteins (bacA), ANT(3″ ) (aadA5), sulfonamide- antibiotic efflux pump (CRP, AcrE, AcrS, rsmA, mdtC, mdtB,
6 BioMed Research International

2926

16000

3393

No. of genes
8000

15226

Core genes 0 20 40 60
Accessory genes
No. of genomes
Unique genes
Key
Conserved genes
Total genes
(a) (b)

Figure 1: Pan-genome analysis of E. coli strains causing urinary tract infections in humans. (a) The pie chart shows a number of core,
accessory, and unique genes in 63 genomes of UPEC strains. (b) The pangenome vs. core-genome plot of UPEC genomes.

Core-genome based phylogenetic tree Core-genes Unique genes

CRE1
13KWH46
UFU EC98
BR07-DEC
BR03-DEC
BR02-DEC
MSHS 472
CHL5009T
BR25-DEC
13TMH22
EC24377A
MG655
CFT073 2
Sakai
EDL933
BR10-DEC
BR64-DEC
BR29-DEC
ST410
BR12-DEC
Combat13F7
KL53
BR32_DEC
5CRE51
Y5
APEC 01
Combat119
EC974
EC1515
118UI
FDAARGOS 448
28Eco12
IAI39
MS6198
CLSC36
AR bank 0349
M160133
H17
MSHS 133
EQ101
BR14-DEC
FDAARGOS 144
SA186
55989
MNCRE44
81009
20
O25bH4
40
UPEC132
ATCC 700415
ABU 83972
UPEC 26 1
CFT073
UTI89
NU14
53G
Combat2C1
BH100N substr MG2017
BH100L substr MG2017
BH100 substr MG2017
BH100 substr MG2014

Figure 2: Core-genome SNP-based phylogenetic tree and heatmap of gene presence and absence in 63 UPEC strains.
BioMed Research International 7

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EC (

)
DE

C
(Slov
CR

a)
KW

3F7

(
-DEC

B
29-

4-D

raz
13

C (Brazil)

akia)
BR

(Ho

il)
APEC_0
BR6

BR10

ng K
ong
)

Countries

Thailand UK
Pakistan Ghana
Brazil USA
Hong Kong Saudi Arabia
Canada UAE
New Zealand Sweden
China Unknown
Taiwan South Korea
Mexico Slovakia
Colombia

Figure 3: Circularized SNP tree of 63 global UPEC strains indicating same clade of EQ101 and BR-14 DEC. Most of the other Pakistani
strains shared similar clade. Color key indicating countries is provided alongside the circular tree.
8 BioMed Research International

hi...
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Escheric
i...

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ct
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erich 15
-M-

Pe
Esch
richi... CTX
Esche 4
dfrA1
emrK
emrA
emrY
ARO emrB
tet(B)
marA
mdtG
mdtH
Esch
erich Kleb
i... siell
acrB a...
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Str

siell
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ct

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CRP

acrS
mdtE

i...
E

(a)
emrR
gadW
emrA
dfrA14 mdtE TolC mdtN
Escherichia coli parC conferring resistance to fluoroquinolones
emrK mdtF YojI mdtP
EC–5 baeR mdto
emrY cpxA
Haemophilus influenzae PBP3 conferring resistance to beta-lactam antibiotics msbA
Escherichia coli EF-Tu mutants conferring resistance to Pulvomycin
Escherichia coli ampC1 beta-lactamase PmrF

5.5 Mbp CRP

mdtM 0.5 Mbp AcrS
5.0 Mbp AcrE
CTX-M-15 1.0 Mbp rsmA
4.5 Mbp

1.5 Mbp
Escherichia coli ampH beta-lactamase
4.0 Mbp
acrD
mdtB mphA
2.0 Mbp mdtC sul1
3.5 Mbp mdtA aadA5
2.5 Mbp evgS
3.0 Mbp
Escherichia coli uhpT with mutation conferring resistance to fosfomycin evgA
tet (B)
tetR ugd

mdtG mdtH
Escherichia coli gyra conferring resistance to fluoroquinolones kdpE
acrB
Escherichia coli mdfA Escherichia coli acrA
bacA
Escherichia coli acrR with mutation conferring multidrug antibiotic resistance

(b)

Figure 4: (a) Classification of antimicrobial resistance genes. (b) Antimicrobial resistance genes in EQ101.
BioMed Research International 9

mdtA, evgS, acrB, Escherichia coli acrA, acrD, marA, baeR, ity than other characterized bacterial gyrases. Mdh (Uni-
mdtE, mdtF, Escherichia coli acrR with mutation conferring protKB:P61889) catalyzes the reversible oxidation of malate
multidrug antibiotic resistance, Escherichia coli soxR with to oxaloacetate, and recA (UniprotKB:P0A7G6) is required
mutation conferring antibiotic resistance, Escherichia coli soxS for homologous recombination, cellular response to DNA
with mutation conferring antibiotic resistance, and Escherichia damage stimulus, and the bypass of mutagenic DNA lesions
coli marR mutant conferring antibiotic resistance), pmr phos- by the SOS response. The SOS response controls an
phoethanolamine transferase (PmrF and ugd), major facilita- apoptotic-like death (Oliveira et al.) induced in response to
tor superfamily (MFS) antibiotic efflux pump (mdtN, mdtO, DNA damaging agents that is mediated by RecA and LexA.
mdtP, evgS, mdtH, Escherichia coli mdfA, mdtG, tet(B), emrY, Adk (UniprotKB:P69441), mdh (UniprotKB:P61889), and
emrK, emrA, Escherichia coli soxR with mutation conferring purA (UniprotKB:P0A7D4) take part in energy metabolism.
antibiotic resistance, Escherichia coli soxS with mutation con- TolC (UniprotKB:P02930) is an outer membrane chan-
ferring antibiotic resistance, and tetR), and ATP-binding cas- nel, which is required for the function of several efflux sys-
sette (ABC) antibiotic efflux pump (YojL, msbA, Escherichia tems such as AcrAB-TolC, AcrEF-TolC, EmrAB-TolC, and
coli soxR with mutation conferring antibiotic resistance, and MacAB-TolC. These systems are involved in the export of
Escherichia coli soxS with mutation conferring antibiotic antibiotics and other toxic compounds from the cell. evgA
resistance). The drug classes they show resistance against are seems to control the expression of multidrug efflux operon.
elfamycin antibiotic, penem, carbapenem, monobactam, H-NS (UniprotKB:P0ACF8) is involved in bacterial chromo-
glycopeptide antibiotic, phosphonic acid antibiotic, aminogly- some organization, compaction and binds to the upstream
coside antibiotic, aminocoumarin antibiotic, diaminopyrimi- and downstream regions of initiating RNA polymerase, trap-
dine antibiotic, phenicol antibiotic, rifamycin antibiotic, ping it in a loop and thereby, preventing the transcription pro-
tetracycline antibiotic, glycylcycline, cephamycin, cephalospo- cess. It can also increase translational efficiency of mRNA with
rin, penam, fluoroquinolone antibiotic, macrolide antibiotic, suboptimal Shine-Dalgarno sequences. Hydroxyisobutyryla-
nitroimidazole antibiotic, disinfecting agents and antiseptics, tion on Lys-121 decreases the DNA-binding activity of
nucleoside antibiotic, and peptide antibiotic. H-NS, promotes the expression of acid-resistant genes,
and enhances bacterial survival under extreme acid stress.
3.3. Virulence-Associated Genes. VirulenceFinder 2.0 identi- cpxA (UniprotKB:P0AE82) responds to envelope stress
fied 25 virulence genes (air, astA, chuA, fyuA, gad, hra, iha, response by activating the expression of downstream genes
irp2, iss, iucC, iutA, kpsE, kpsMII_K1, lpfA, mchF, ompT, and is involved in several diverse cellular processes, includ-
papA_F43, sat, senB, sitA, terC, traT, usp, vat, and yfcV). ing the functioning of acetohydroxyacid synthase I, the bio-
Air (UniprotKB:P50466), Iha (UniprotKB:A0A061YB93), synthesis of isoleucine and valine, the TraJ protein activation
and iutA (UniprotKB:P14542) act as transmembrane receptors, activity for tra gene expression in F plasmid, and the synthe-
and Gad system helps to maintain a near-neutral intracellular sis, translocation, or stability of cell envelope proteins. cpxA
pH when cells are exposed to extremely acidic conditions, so is also involved in cell adhesion, so it takes part in biofilm
the ability to survive transit through the acidic conditions of formation. mdtM (UniprotKB:P39386) plays in cellular
the stomach is essential for successful colonization of the transportation and confers resistance to acriflavine, chlor-
mammalian host by commensal and pathogenic bacteria. Sat amphenicol, and norfloxacin. emrR (UniprotKB:C3SY57)
(UniprotKB:P13018) is involved in resistance to streptothricin, and evgA (UniprotKB:P0ACZ4) play role in transcription.
a broad-spectrum antibiotic, via acetylation of the beta amino CTX-M-15 (UniprotKB:W8YE54) has beta-lactamase activ-
group of the first beta-lysyl moiety of streptothricin. traT ity and is involved in antibiotic resistance.
(UniprotKB:B1VCB1) is responsible for preventing unproduc- sul1 (UniprotKB:P0C002) is implicated in resistance to
tive conjugation between bacteria carrying like plasmids. lpfA sulfonamide. dfrA14 (UniprotKB:B6SCG1) is a key enzyme
(UniprotKB:Q8VPB9), yfcV (UniprotKB:P77288), and sitA in folate metabolism. The sul1 enzyme catalysed process is
(UniprotKB:Q6J5P0) play role in cell adhesion. iutA (Uni- an essential reaction for de novo glycine and purine synthe-
protKB:P14542), mchF (UniprotKB:Q9EXN5), kpsE (Uni- sis and DNA precursor synthesis.
protKB:P42501), chuA (UniprotKB:A0A0K3JCI0), fyuA
(UniprotKB:Q798T6), and irp2 (UniprotKB:A0A061JYT2) take 4. Discussion
part in cell transportation. ompT (UniprotKB:P09169) has
aspartic-type and serine-type endopeptidase activity and take Genes related to the resistance to a certain type of antibiotics
part in proteolysis. were the key aim of the present study. Therefore, the genes
MLSR analyzed seven E. coli housekeeping genes (adk, having roles in antimicrobial resistance were analyzed and
fumC, gyrB, icd, mdh, purA, and recA) through MLST 2.0 compared with the phenotype of the resistance pattern.
and identified adk_14, fumC_14, gyrB_10, mdh_17, purA_7, The identified isolates of the study showed resistance mainly
and recA_10 alleles while no hit was found for icd allele. Adk against amoxicillin, cefpirome, nalidixic acid, pipemidic acid,
(UniprotKB:P69441) plays an important role in cellular aztreonam, cefaclor, cefotaxime, ceftriaxone, ofloxacin, norflox-
energy homeostasis and in adenine nucleotide metabolism. acin, ciprofloxacin, cephradine, cefuroxime, cefixime, ceftazi-
Icd (UniprotKB:P08200) and fumC (UniprotKB:P05042) are dime, cefepime, and cotrimoxazole and showed genotype-
involved in the TCA cycle and response to oxidative stress. phenotype correlations as well according to the aim of the study.
gyrB (UniprotKB:P0AES6) maintain chromosomes in an While discussing the various genes, the isolated traT vir-
underwound state. E. coli gyrase has higher supercoiling activ- ulence gene was observed to be involved in antimicrobial
10 BioMed Research International

resistance. Its role was also supported by a previous study Data Availability
where Rezatofighi et al. [42] reported the significant preva-
lence of pathogenicity-associated island, papAH, papEF, All short reads and assemblies associated with this study are
fimH, fyuA, and traT genes in UPEC isolates. Another study available at RAST, CARD, and PATRIC. The genomic data
revealed the presence of certain virulence genes including is present at NCBI https://www.ncbi.nlm.nih.gov/biosample/
iha, lpfA, aafC, nfaE, eilA, eae, and bfpA for adherence to SAMN26578128/.
host cells. It further identified virulence factors including
senB, astA, and pic that promote toxin production. Further- Conflicts of Interest
more, toxins that promote E. coli protease production
included sat and vat [43]. The authors declare that they have no conflict of interest.
The variability of the housekeeping genes has been sig-
nificantly identified and reported in a study that among Acknowledgments
the seven genes, fumC and gyrB presented the highest degree
of nucleotide diversity and the greatest number of polymor- The primary author of this work is thankful for the scholar-
phic nucleotide sites [3], suggesting to contribute to a high ship provided by the HEC Pakistan for this study. Integrative
degree resistance pattern as identified in this study as well. Biology and Genomics Lab of Atta-ur-Rahman School of
Among the identified resistance genes in the study, Applied Biosciences (ASAB), National University of Sciences
mdtM and acEdelta1 were found to be associated with anti- and Technology (NUST), Islamabad, and the JRC Genome
biotic efflux, which was further confirmed through a study Research, ICCBS, University of Karachi, have helped in the
and reported that the same genes were involved in causing analysis of the sequenced genome. The authors also express
multidrug resistance and helped the organism to tolerate their appreciation to the Deanship of Scientific Research at
higher concentrations of antibiotics, external pH, and stress King Khalid University, Saudi Arabia (Research Group Pro-
conditions involving alkaline environment [44, 45]. Further- gram under funding grant number: RGP. 2/524/44).
more, mphA gene, which was observed to involve in antibi-
otic inactivation, was found to be responsible for causing Supplementary Materials
resistance to azithromycin [46, 47]. Another sul1 gene was
observed to be responsible for antibiotic drug replacement Supplementary Results, Table 1 and Table 2. (Supplementary
in this study and was also found as a sulfonamide resistance Materials)
gene in some other study [46, 47].
The rest of the identified genes were found to be References
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