{"id":34042,"date":"2020-06-25T10:06:16","date_gmt":"2020-06-25T10:06:16","guid":{"rendered":"https:\/\/biomedpharmajournal.org\/?p=34042"},"modified":"2021-11-10T05:43:40","modified_gmt":"2021-11-10T05:43:40","slug":"sequencing-analysis-of-pyelonephritis-associated-pili-gene-of-uro-pathogenic-e-coli-isolated-in-from-patients-baghdad-iraq","status":"publish","type":"post","link":"https:\/\/biomedpharmajournal.org\/staging\/vol13no2\/sequencing-analysis-of-pyelonephritis-associated-pili-gene-of-uro-pathogenic-e-coli-isolated-in-from-patients-baghdad-iraq\/","title":{"rendered":"Sequencing Analysis of Pyelonephritis-Associated Pili Gene of Uro-Pathogenic E. Coli Isolated in From Patients-Baghdad, Iraq"},"content":{"rendered":"

\u00a0Introduction <\/strong><\/p>\n

Uropathogenic Escherichia coli <\/em>(UPEC) is the most abundant causative bacteria of urinary tract infections (UTIs), worldwide accounting for 80\u201390% of all infections.1 <\/sup>There are differences in prevalence of virulence factors among UPEC including the adhesins (Type 1, P, S, and F1C fimbriae), pore-forming hemolysin, toxins (RTX and CNF-1),sideophore and aerobactin,which exist\u00a0 in clusters, small virulence cassettes, or large blocks of genes in UPEC strains but, mainly, not observed in the genome of fecal isolates.2,3 <\/sup>Their expression and co-regulation depend on host response and environmental signals.4<\/sup>Iraqi studies from different cities\u00a0 referred to that the frequency of isolation of E. coli<\/em> from urine of patients with UTI were ranged from 39%-73.6%.\u00a05,6,7,8<\/sup><\/p>\n

The crucial step for starting of UTI is by adhesion of UPEC to mucosal cells that intermediated\u00a0 by P fimbriae and Type-1 fimbriae. Mannose-resistant pyelonephritis-associated pili fimbriae expressed from pap<\/em> in about 80% of UPEC cause pyelonephritis.9 <\/sup>Genotyping of pap<\/em> operon referred to its involve fimbriae structural subunits (pap A, C, D,E, F and G) to finish fimbrial growth and attach mature fimbriae to surface of host cell, pap H; and papB<\/em> and papI<\/em> ,the divergently encoded regulatory genes, within which the main promoter is located.10<\/sup><\/p>\n

These promoters expressed depending on the DNA methylation of the sequences GATC that located within the intergenic region. DNA methylation is essential epigenetic, post-replicative modification that is catalyzed by a group of enzymes as the DNA methyltransferases (MTases). It is key regulation mechanism of many cellular processes in prokaryotes and eukaryotes. Bacterial DNA methyl-transferases generate N4-methyl-cytosine, C5-methyl-cytosine, or N6-methyl-adenosine in GATC sequences, in an S-adenosyl-methionine-dependent reaction.11,12<\/sup><\/p>\n

DNA adenine methylase (Dam) have important functions in DNA mismatch repair, regulation of transcription, and SOS response activation as\u00a0 part of the cell cycle.1<\/sup>3 <\/sup>Methyl-directed mismatch repair is a regulatory process whereby it recognizes the biosynthetic error during the occurrence of the replication fork. The hemi-methylated site differentiates of the DNA template strand and the DNA newly synthesized strand allowing\u00a0 protein MutS to bind to the site where the mismatch occurs and promotes the process of the recruitment of the addition repair proteins to form a ternary complex that initiate repair.13,14 <\/sup>Adherence is essential for UPEC strains, as it avoid removal of bacteria through micturition, and multiple phase-variable adhesins are exists to permit binding to different host tissues. The pap <\/em>epigenetic switch activates through establishment of differential methylation patterns that regulate the expression phase (on or off) of the pap<\/em> operon.15,16 <\/sup>Novel antibiotics drug that targets Dam<\/em> can be intriguing as the enzymatic activity is a lack in human. Inhibiting Dam<\/em> by DNA methyl-transferase inhibitors (DNMTi) can be detrimental to the bacterium. The inhibitors will reversely modify the deviating pattern of the DNA methylation by interfering the enzymatic activity of the DNMTs.17,18 <\/sup>The current study aimed to investigate if there is a relationship between sequence variation in pap <\/em>E , presence of Dam<\/em> and susceptibility to antibiotics in UPEC.<\/p>\n

Material and Methods <\/strong><\/p>\n

Bacterial Isolation<\/strong><\/p>\n

Sixty Five isolates of E. coli<\/em> from urine specimens were obtained from hospitalized and out-patients suffering from signs and symptoms of UTI recruited to Department of Microbiology Lab,AL-Kadiymia Teaching Hospital, Baghdad, Iraq, from March 2018 to October 2018. Also, 2 E. coli<\/em> isolates from wound infections and 2 E. coli<\/em> isolates from chest fluid were obtained. These isolates were cultured on MacConkey agar and identified using API20E Epi 20 system and\/or VITEK2 Vitic system. Information such as age, gender, antibiotic susceptibility of included patients were obtained from consent form of each patient.<\/p>\n

Antimicrobial Susceptibility Testing<\/strong><\/p>\n

The antibiotic susceptibility test of included isolates were done using disk diffusion method according to the Clinical and Laboratory Standards Institute (CLSI) guidelines.19 <\/sup>The following antibiotics were included: amoxicillin-clavulanic acid (AMC),\u00a0 ciprofloxacin (CIP), gentamicin (GE), nitrofurantoin (NIT), trimethoprim-sulfamethoxazole (SXT), cefpodoxime (CPD),cephalothin (CLT), tetracycline (TE), rifampin (RP)and ticarcillin(TIM).The definitions of multidrug resistant (MDR),extended drug resistant (XDR) and pan drug resistant (PDR) isolates were as per standardized by European Centre for Disease Control (ECDC) and Centre for Disease Control & Prevention (CDC).20<\/sup><\/p>\n

Molecular Identification of E. Coli<\/em> <\/strong><\/p>\n

Genomic DNA was extracted from E. coli<\/em> isolates using WIZARD Genomic DNA Extraction Kit following manufacturer instructions (Promega,USA). Concentration and purity of extracted DNA of each sample were measured using Nanodrop (AcT <\/em>Gene NAS-99, USA). Primers specific for the lac<\/em>Y (lactose permease gene) and pho<\/em>A (bacterial alkaline phosphatase gene) were used for molecular identification of E. coli<\/em> following Das Mitra et al., 2015with modifications.21 <\/sup><\/p>\n

The reaction was prepared in 25\u03bcl total volume. The concentration of primer was 0.15\u03bcM for lac<\/em>Y, <\/em>and 0.6\u03bcM for pho<\/em>A(Alpha DNA,Canada),1X PCR buffer (Promega,USA), 1.5 U of Taq <\/em>DNA polymerase(Promega,USA), 200 mM dNTPs(Promega,USA). Then, DNA(100 ng) was added. The cycling conditions were as followings: 94 \u00baC for 5 min followed by 35 cycles of 94 \u00baC for 30 sec, 52\u00baC for 1min and 72 \u00baC for 1 min and final extension at 72o<\/sup>C for 10 min using Thermal Cycler (Eppendorf master cycler,Germany). The amplified products were analyzed using agarose gel 1.5%. Extracted DNA from Pseudomonas aeroginosa<\/em> identified using VITEK2 VITIC system and molecularly from previous study,\u00a022 <\/sup>and extracted DNA from Klebsiella pneumonia<\/em>e (identified using VITIC system in this study) were used as negative control to evaluate primer specificity. Two bands with molecular size 289bp and 468bp will be seen if the DNA was from\u00a0 E. coli<\/em>. The appearance of amplicon with 468bp only or no amplified products will be refereed to bacteria other than E. coli<\/em>.<\/p>\n

Detection of pap E <\/em><\/strong><\/p>\n

The presence of\u00a0 pap<\/em> E in UPEC isolates was detected using conventional PCR.23 <\/sup>Master mix was prepared\u00a0 in 25\u03bcL total volume\u00a0 containing 1X PCR buffer,10 pmol of forward and reverse primers, 200\u03bcM of dNTP, 1.25U of Taq DNA polymerase. Then, 100ng DNA was added. Two extracted DNA samples from each of E. coli<\/em> isolated from wound infection and chest fluid, respectively, were used as negative control for the presence of pap E<\/em>.\u00a0 PCR conditions were as following: 94o<\/sup>C for 2min followed by 35 cycles of 94\u00b0C for 1 min,\u00a0 52\u00b0C for 1.5min, 72o<\/sup>C for 3min\u00a0 and final extension at 72o<\/sup>C for 10 minutes using Thermal Cycler (Eppendorf master cycler,Germany). PCR products were electrophoresed on agarose gels1.5%. Appearance of amplicon with molecular size 336bp referred to presence of pap E<\/em>.<\/p>\n

DNA Sequencing of pap<\/em> E<\/strong><\/p>\n

The amplicons of\u00a0 pap<\/em> E\u00a0 from 21 selected strains of UPEC were send for Sanger sequencing using automated DNA sequencer\u00a0 ABI 3730XL (Macrogen Corporation, Korea). The results were analyzed using Genious software. The sequences of each fragment were trimmed to a uniform length that corresponded with the region used to identify the target gene. Sequences were compared with standard strain MH455215 using online BLAST software (http: \/\/www.ncbi.nlm.nih.gov\/BLAST\/). Partial sequencing tree of fimbrial adaptor papE<\/em> and fimbrial adaptor papF<\/em> of 13 E. coli<\/em> isolates was constructed.<\/p>\n

Nucleotide Sequence Accession numbers<\/strong><\/p>\n

The partial sequencing alignment of pap E<\/em>\u00a0 of 5 included strains were compared with the sequences of previously published strains from DNA GenBank sequences using Basic Local Alignment Search Tool (BLAST) search and deposited in GeneBank under accession numbers: LC479519.1 of strain MR-1,LC479520.1 of strain MR-2, LC479521.1 of strain MR-14, LC479522.1 of strain MR-18 and LC 479523.1 of strain MR-21.<\/p>\n

Detection of DNA Adenine Methyl-Transferase Gene in UPEC Isolates<\/strong><\/p>\n

Extracted DNA from 26 UPEC was screened for Dam<\/em> using conventional PCR.24 <\/sup>PCR condition was modified as the following: 95o<\/sup>C for 5min followed by 35 cycles of 95\u00b0C for 1 min,55\u00b0C for 1.5min, 72o<\/sup>C for 1.5min\u00a0 and final extension of 72o<\/sup>C for 10 min using Thermal Cycler (Eppendorf master cycler,Germany). Products of PCR were electrophoresed on agarose gels0.8%. Appearance of amplicon with molecular size of 1071bp referred to presence of Dam<\/em>.<\/p>\n

Results<\/strong><\/p>\n

Patients Demography<\/strong><\/p>\n

The mean age of included patients was 38.11 \u00b122.02 ranging from (1-78) years with median 42.5 (36.5) years. Male 24\/64 (37.5 %) to female 40\/64 (62.5 %) ratio 1:1.7.<\/p>\n

Prevalence of pap E<\/em> among UPEC Isolates<\/strong><\/p>\n

Of the obtained 65 isolates, 64(98.4%) were approved to be E. coli <\/em>depending\u00a0 on molecular identification of lacY<\/em> and pho<\/em>A using PCR. Detection of papE<\/em> in E. coli<\/em> strains using PCR revealed that 26\/64(42.6%) were UPEC (Fig.1).Prevalence of UPEC was showed to be the highest among the female patients within age group (10-20) years and (50-60) years (Table1).<\/p>\n\n\n\n
\"Figure<\/td>\nFigure 1: <\/strong>Detection of pap E<\/em> in E. coli <\/em>isolates from urine specimens.<\/strong><\/span><\/p>\n

Click here to View Figure<\/span><\/a><\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

Table 1:<\/strong> Age distribution of patients infected with E. coli<\/em> positive to<\/strong> papE<\/em><\/strong>\u00a0 UPEC.<\/strong><\/p>\n\n\n\n\n\n\n\n\n\n\n\n\n
Age group (year)<\/strong><\/td>\n\u00a0<\/strong><\/td>\n\u00a0Female<\/strong><\/td>\n<\/tr>\n
Male<\/strong><\/td>\n<\/tr>\n
10-Jan<\/td>\n1<\/td>\n–<\/td>\n<\/tr>\n
20-Oct<\/td>\n–<\/td>\n5<\/td>\n<\/tr>\n
20-30<\/td>\n–<\/td>\n1<\/td>\n<\/tr>\n
30-40<\/td>\n2<\/td>\n2<\/td>\n<\/tr>\n
40-50<\/td>\n1<\/td>\n3<\/td>\n<\/tr>\n
50-60<\/td>\n–<\/td>\n5<\/td>\n<\/tr>\n
60 and more<\/td>\n3<\/td>\n3<\/td>\n<\/tr>\n
Total<\/td>\n7<\/td>\n19<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

Analysis of pap E <\/em>Sequencing in Studied Strains<\/strong><\/p>\n

PCR amplified products of papE <\/em>from 21strains were partially sequenced, only 13 strains gave successful sequencing analysis of target gene which reviled three clades labeled as A,B and C (Fig. 2). The correlation between studied strains according analysis of genetic distance based on partial sequencing \u00a0of pap E<\/em> showed that\u00a0 strain 17(clade A) have 100% similarity with standard strain, while clade B, which included strains (1,2,5,14,18,21) and clade C which included strains (4,7,8,10,12,13.16) have\u00a0 (99%) similarity with standard strain.<\/p>\n

From clade B, strains 1, 2 and 21 considered as sister group. Also, strains 5, 14 and18 considered as sister group. From clade C, strains 12, 4, 7, 8,10,13,16 have similarity of 100%. The main clusters that were similar were conserved.<\/p>\n\n\n\n
\"Figure<\/td>\nFigure 2:<\/strong> Partial sequencing tree of pap E<\/em> .<\/strong><\/span><\/p>\n

Click here to View Figure<\/span><\/a><\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

Analysis of nucleotide sequence changes from partial sequencing of pap E<\/em> shown that UPEC included in clade B displayed the most nucleotide sequence changes as substitution result in synonymous and non-synonymous mutation (Table 2).<\/p>\n

Prevalence of Dam<\/em> among UPEC Isolates<\/strong><\/p>\n

Dam <\/em>was detected in 11\/64 (17.1%)E. coli<\/em> isolates (Fig. 3). The association between the presence of pap E<\/em> and Dam <\/em>in studied isolates shown that 10\/64(15.6%) isolates contain both genes (Table 3).<\/p>\n

Table 2: <\/strong>The sequencing analysis of partial<\/strong> sequencing tree of pap E<\/em><\/strong><\/p>\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n
Clade type of pap E<\/em><\/strong><\/td>\nStrain no.<\/strong><\/td>\nNucleotide change<\/strong><\/td>\nAmino-acid change<\/strong><\/td>\nType of nucleotides change<\/strong><\/td>\n<\/tr>\n
A<\/td>\n17<\/td>\nN<\/td>\nN<\/td>\nN<\/td>\n<\/tr>\n
<\/td>\n1<\/td>\nGTC<\/u>\/GTT<\/u><\/td>\nVal-Val<\/td>\nSubstitution (synonymous)<\/td>\n<\/tr>\n
<\/td>\nTGG\/TAA<\/u><\/td>\nVal-Val<\/td>\nSubstitution(synonymous)<\/td>\n<\/tr>\n
<\/td>\n<\/td>\n<\/td>\n<\/td>\n<\/tr>\n
<\/td>\n2<\/td>\nGTC\/GTT<\/u><\/td>\nVal-Val<\/td>\nSubstitution(synonymous)<\/td>\n<\/tr>\n
B<\/td>\nTGG\/TAT<\/u><\/td>\nTrp-Tyr<\/td>\nSubstitution(non-synonymous)<\/td>\n<\/tr>\n
<\/td>\n5<\/td>\nGAA\/GAG<\/td>\nGlu-Glu<\/td>\nSubstitution(synonymous)<\/td>\n<\/tr>\n
<\/td>\nATC\/GTA<\/td>\nLie-Val<\/td>\nSubstitution (non-synonymous)<\/td>\n<\/tr>\n
<\/td>\n<\/td>\n<\/td>\n<\/td>\n<\/tr>\n
<\/td>\n14<\/td>\nGAA\/GAG<\/td>\nGlu-Glu<\/td>\nSubstitution(synonymous)<\/td>\n<\/tr>\n
<\/td>\nATC\/GTA<\/td>\nLie-Val<\/td>\nSubstitution(non-synonymous)<\/td>\n<\/tr>\n
<\/td>\nTTC<\/td>\nPhe<\/td>\nInsertion-frame shift<\/td>\n<\/tr>\n
<\/td>\n18<\/td>\nGCT\/ATT<\/u><\/td>\nAla-Lie<\/td>\nSubstitution(non-synonymous)<\/td>\n<\/tr>\n
<\/td>\nGTC\/GTT<\/td>\nVal-Val<\/td>\nSubstitution(synonymous)<\/td>\n<\/tr>\n
<\/td>\nATC\/GTC<\/td>\nLie-Val<\/td>\nSubstitution(non-synonymous)<\/td>\n<\/tr>\n
<\/td>\n21<\/td>\nGTC\/GTT<\/td>\nVal-Val<\/td>\nSubstitution(synonymous)<\/td>\n<\/tr>\n
<\/td>\nTTC<\/td>\nPhe<\/td>\nInsertion-frame shift<\/td>\n<\/tr>\n
<\/td>\n4<\/td>\nN<\/td>\nN<\/td>\nN<\/td>\n<\/tr>\n
<\/td>\n7<\/td>\nN<\/td>\nN<\/td>\nN<\/td>\n<\/tr>\n
C<\/td>\n8<\/td>\nN<\/td>\nN<\/td>\nN<\/td>\n<\/tr>\n
<\/td>\n10<\/td>\nN<\/td>\nN<\/td>\nN<\/td>\n<\/tr>\n
<\/td>\n12<\/td>\nN<\/td>\nN<\/td>\nN<\/td>\n<\/tr>\n
<\/td>\n13<\/td>\nN<\/td>\nN<\/td>\nN<\/td>\n<\/tr>\n
<\/td>\n16<\/td>\nN<\/td>\nN<\/td>\nN<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

N: no change in nucleotides, no change in amino acid; Val: valine ; Trp: tryptophan ; Tyr: tyrosine; Glu: glutamine; Lei: leucine ; Phe: phenylalanine ; Ala: alanine.<\/p>\n\n\n\n
\"Figure<\/td>\nFigure 3: <\/strong>Detection of Dam<\/em> in E. coli<\/em> isolates from urine specimens.\u00a0<\/strong><\/span><\/p>\n

Click here to View Figure<\/a><\/span><\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

Table 3: <\/strong>Frequency distribution and prevalence rate of pap E<\/em> and Dam<\/em> <\/strong><\/p>\n\n\n\n\n\n\n\n\n\n
Characteristic<\/strong><\/td>\nN<\/em><\/strong>=64<\/strong><\/td>\n(%)<\/strong><\/td>\n<\/tr>\n
Pap E -\/ Dam –<\/td>\n37<\/td>\n57.8<\/td>\n<\/tr>\n
Pap E +\/ Dam –<\/td>\n16<\/td>\n25<\/td>\n<\/tr>\n
Pap E -\/ Dam +<\/td>\n1<\/td>\n1.6<\/td>\n<\/tr>\n
Pap E +\/ Dam +<\/td>\n10<\/td>\n15.6<\/td>\n<\/tr>\n
Pap E+<\/td>\n26<\/td>\n40.6<\/td>\n<\/tr>\n
Dam +<\/td>\n11<\/td>\n17.2<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

Antimicrobial Susceptibility Test<\/strong><\/p>\n

Of the UPEC isolates, 13\/26 (50%) isolates were considered as MDR, that acquired non-susceptibility to at least one antibiotic in three or more antimicrobial categories (Table S1). \u00a0\u00a0<\/strong><\/p>\n

Association between pap E<\/em> clades of UPEC and MDR <\/strong><\/p>\n

Studying the association between the pap E<\/em> clades of UPEC isolates and MDR was shown that there was statically not significant (Table 4).<\/p>\n

Table 4: <\/strong>UPEC pap E<\/em> clads in association with MDR.\u00a0\u00a0\u00a0\u00a0\u00a0 <\/strong><\/p>\n\n\n\n\n\n\n\n
Clad Type<\/strong><\/td>\nTotal<\/strong><\/td>\nMDR +<\/strong><\/td>\nMDR –<\/strong><\/td>\nP<\/em><\/strong><\/td>\n<\/tr>\n
A<\/strong><\/td>\n1<\/td>\n0<\/td>\n1<\/td>\n0.462 F<\/p>\n

NS<\/td>\n

A versus others<\/td>\n<\/tr>\n
B<\/strong><\/td>\n6<\/td>\n4<\/td>\n2<\/td>\n0.592 F<\/p>\n

NS<\/td>\n

B versus others<\/td>\n<\/tr>\n
C<\/strong><\/td>\n6<\/td>\n3<\/td>\n3<\/td>\n1.000 F<\/p>\n

NS<\/td>\n

C versus others<\/td>\n<\/tr>\n
Total<\/strong><\/td>\n13<\/td>\n7<\/td>\n6<\/td>\n—<\/td>\n—<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

F: Fischer exact test; NS: not significant at p<\/em> \u2264 0.05\u00a0<\/strong><\/p>\n

Correlation between<\/strong> Antibiotic Susceptibility,<\/strong> MDR and the Existence of pap E <\/em>and Dam<\/em><\/strong><\/p>\n

The correlation between the presence of papE<\/em> and \/or Dam<\/em> and antibiotic susceptibility reviled that there was a statistically significant correlation between resistance to tetracycline and rifampin and the presence of both papE<\/em> and Dam<\/em>, a statistically significant correlation between resistance to tetracycline and the presence of pap E<\/em> and a high statistically significant correlation between resistance to tetracycline and the presence of Dam<\/em> (Table S2). The study of MDR risk in association with the presence of pap E<\/em> and Dam<\/em> in UPEC revealed that Dam<\/em> could be considered as etiological factored to developing MDR (Table 5).<\/p>\n

Table <\/strong>5: Multidrug resistance risk in association with pap E<\/em> and Da<\/em><\/strong>m<\/em><\/strong><\/p>\n\n\n\n\n\n\n
Gene<\/strong><\/td>\nMDR + <\/strong><\/p>\n

n<\/em><\/strong> = 28<\/strong><\/td>\n

MDR \u2013 <\/strong><\/p>\n

n<\/em><\/strong> = 36<\/strong><\/td>\n

P<\/em><\/strong><\/td>\nOR<\/strong><\/td>\n95 % CI<\/strong><\/td>\nEF<\/strong><\/td>\n<\/tr>\n
n<\/em><\/strong><\/td>\n%<\/strong><\/td>\nn<\/em><\/strong><\/td>\n%<\/strong><\/td>\n<\/tr>\n
pap E<\/em><\/td>\n13<\/td>\n46.4<\/td>\n13<\/td>\n36.1<\/td>\n0.404 C<\/p>\n

NS<\/td>\n

1.53<\/td>\n0.56 -4.20<\/td>\n0.17<\/td>\n<\/tr>\n
Dam<\/em><\/td>\n8<\/td>\n28.6<\/td>\n3<\/td>\n8.3<\/td>\n0.047 F<\/p>\n

S<\/td>\n

4.40<\/td>\n1.04 -18.54<\/td>\n0.56<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

n<\/em>: number of cases; OR: Odds ratio; CI: confidence interval; EF: etiologic fraction; C: Chi-square test; F: Fischer exact test; NS: not significant at P<\/em> \u2264 0.05\u00a0<\/strong><\/p>\n

Discussion<\/strong><\/p>\n

Colonization of E. coli <\/em>strains within the urinary tract and occurrence of UTI begin with binding of bacteria to epithelial surface.25,26<\/sup>In the current study, 26\/64(42.6%) of E.coli<\/em> were confirmed to be UPEC according to molecular detection of papE<\/em>. There is a diversity in the frequency of pap <\/em>gene among UPEC strains across the globe and within the same geographical region.<\/strong> Iraqi studies at 2015 and 2017 referred to that pap<\/em> was detected in 58\/112 (51.785%) E. coli<\/em> and 31\/43(72.09%) E. coli<\/em>, respectively, isolated from urine of patients with UTI.27,28 <\/sup>Iranian studies were revealed that there were variations in pap <\/em>gene prevalence in UPEC isolated from different locations including 83.63%, 16.6% and 20.5%.29,30,31 <\/sup>Indian study at 2018 found that the prevalence of pap <\/em>was 72\/350(20.5%) isolates.26 <\/sup>This variation result from UPEC use a variety of adhesions to attach to the bladder urothelium if the usual adhesions are not expressed. However, there is always the possibility of occurring mutations in pap<\/em> result in missing of its detection. A positive PCR product confirm the detection of the gene, but a negative result does not insure absence of the gene.<\/p>\n

Increased antibiotic resistance in bacteria causing UTI\u00a0 has complicated the treatment of such infections. In the current study, 13\/26(50%) UPEC isolates were MDR.Iraqi study referred to that of the 62 E. coli<\/em> isolated from urine, (69.4%) were showed MDR32<\/sup>.Iraqi stud was found that 44% of UPEC were resist to ciprofloxacin and these isolates have mutation in gyrA <\/em>that identified using sequencing of this gene.33 <\/sup>Other Iraqi study at 2018 found that of 42 UPEC, 37(88.09%) were found to be MDR34<\/sup>. Iranian study at 2015 found that 111\/150(74%) of UPEC strains showed MDR phenotype.31 <\/sup><\/p>\n

In the current study, the etiological factor of Dam<\/em> in MDR isolates was 0.56. That referred to the possibility of considered the presents of Dam<\/em> as one of predisposing factor for resistance. A study was done to determine the effect of the bacterial epigenome in antibiotic stress survival to describe genomic methylation kinetics using single-molecule real-time sequencing and they find that without adenine methylation at GATC sites, E. coli<\/em>\u00a0 growth under antibiotic stress is extremely\u00a0 reduced. The explanation for the role of Dam<\/em> in antibiotic resistance is that during drug stress, the adenine methylome persist stable but without GATC methylation, methyl-dependent mismatch repair is repressed and that cause toxic DNA breaks in bacteria. In drug-resistant and pathogenic E. coli<\/em> strains, Dam<\/em> insufficiency reduce responsiveness to antibiotics such as \u03b2-lactam and quinolone classes.35 <\/sup>A study referred to that there was an 8-fold decrease in resistance for amoxicillin\/clavulanic acid, gentamicin and trimethoprim\/sulfamethoxazole and 4-fold decrease in resistance for ciprofloxacin in Dam<\/em> deleted mutated UPEC strains.36<\/sup><\/p>\n

Current studies are focus on the detection of Dam,<\/em>17,37 <\/sup>but some E. coli <\/em>strains could be have DNA cytosine methyltransferase (Dcm) that methylates the second cytosine in the sequence 5\u2032CCWGG 3\u2032.13,38 <\/sup>Also, it was referred to that Dam could be recognized and methylate cytosine in sequences other than GATC.39<\/sup><\/p>\n

Conclusion<\/strong><\/p>\n

The study of multi-drug resistance(MDR) risk in association with the presence of pap E<\/em> and Dam<\/em> in UPEC revealed that Dam<\/em> may be considered as etiological factored to developing MDR. Dam<\/em> should be taken in consideration as one mechanism of MDR prediction development in UPEC. Farther studies included a large sample size required to sport the role of\u00a0 pap<\/em> and Dam<\/em> in responsiveness to antibiotic.<\/p>\n

\u00a0<\/strong>Supplementary Material<\/strong><\/p>\n

Table S1: <\/strong>Antimicrobial susceptibility test of UPEC.<\/strong><\/p>\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n
Patient No.<\/strong><\/td>\nAge<\/strong><\/td>\nGender<\/strong><\/td>\nPap \/ Dam<\/em><\/strong><\/td>\nAntibiotics <\/strong><\/td>\nResistance type<\/strong><\/td>\n<\/td>\n<\/tr>\n
(Y) <\/strong><\/td>\nGE<\/strong><\/td>\nNIT<\/td>\nSXT<\/td>\nCPD<\/strong><\/td>\nAMC<\/strong><\/td>\nCLT<\/strong><\/td>\nTIM<\/strong><\/td>\n\u00a0<\/strong><\/td>\nRP<\/strong><\/td>\nTE<\/strong><\/td>\nCIP<\/strong><\/td>\n\u00a0<\/strong><\/td>\n<\/td>\n<\/tr>\n
1<\/td>\n48<\/td>\nM<\/td>\n+\/+<\/td>\nS<\/td>\nS<\/td>\nR<\/td>\nR<\/td>\nR<\/td>\nR<\/td>\nR<\/td>\n<\/td>\nS<\/td>\nR<\/td>\nR<\/td>\nMDR<\/td>\n<\/td>\n<\/tr>\n
2<\/td>\n35<\/td>\nF<\/td>\n+\/+<\/td>\nS<\/td>\nS<\/td>\nR<\/td>\nR<\/td>\nR<\/td>\nR<\/td>\nR<\/td>\n<\/td>\nS<\/td>\nR<\/td>\nS<\/td>\nMDR<\/td>\n<\/td>\n<\/tr>\n
3<\/td>\n78<\/td>\nM<\/td>\n+\/ –<\/td>\nS<\/td>\nS<\/td>\nR<\/td>\nR<\/td>\nR<\/td>\nR<\/td>\nS<\/td>\n<\/td>\nS<\/td>\nS<\/td>\nS<\/td>\n–<\/td>\n<\/td>\n<\/tr>\n
4<\/td>\n54<\/td>\nF<\/td>\n+\/ –<\/td>\nS<\/td>\nS<\/td>\nR<\/td>\nR<\/td>\nR<\/td>\nR<\/td>\nS<\/td>\n<\/td>\nR<\/td>\nS<\/td>\nS<\/td>\nMDR<\/td>\n<\/td>\n<\/tr>\n
<\/td>\n<\/tr>\n
11<\/td>\n14<\/td>\nF<\/td>\n+\/ –<\/td>\nS<\/td>\nS<\/td>\nS<\/td>\nS<\/td>\nS<\/td>\nS<\/td>\nR<\/td>\n<\/td>\nS<\/td>\nR<\/td>\nS<\/td>\n–<\/td>\n<\/td>\n<\/tr>\n
12<\/td>\n23<\/td>\nF<\/td>\n+\/ –<\/td>\nS<\/td>\nS<\/td>\nS<\/td>\nS<\/td>\nS<\/td>\nS<\/td>\nR<\/td>\nS<\/td>\nR<\/td>\nS<\/td>\n–<\/td>\n<\/td>\n<\/tr>\n
14<\/td>\n43<\/td>\nF<\/td>\n+\/ –<\/td>\nS<\/td>\nS<\/td>\nS<\/td>\nS<\/td>\nS<\/td>\nS<\/td>\nR<\/td>\nS<\/td>\nR<\/td>\nS<\/td>\n–<\/td>\n<\/td>\n<\/tr>\n
16<\/td>\n65<\/td>\nF<\/td>\n+\/ –<\/td>\nS<\/td>\nS<\/td>\nS<\/td>\nS<\/td>\nS<\/td>\nS<\/td>\nR<\/td>\nS<\/td>\nR<\/td>\nS<\/td>\n–<\/td>\n<\/td>\n<\/tr>\n
18<\/td>\n50<\/td>\nM<\/td>\n+\/ –<\/td>\nS<\/td>\nS<\/td>\nS<\/td>\nS<\/td>\nS<\/td>\nS<\/td>\nR<\/td>\nS<\/td>\nR<\/td>\nS<\/td>\n–<\/td>\n<\/td>\n<\/tr>\n
19<\/td>\n33<\/td>\nM<\/td>\n+\/+<\/td>\nS<\/td>\nS<\/td>\nS<\/td>\nS<\/td>\nS<\/td>\nS<\/td>\nR<\/td>\nS<\/td>\nR<\/td>\nS<\/td>\n–<\/td>\n<\/td>\n<\/tr>\n
20<\/td>\n46<\/td>\nM<\/td>\n-\/+<\/td>\nS<\/td>\nS<\/td>\nS<\/td>\nS<\/td>\nS<\/td>\nS<\/td>\nR<\/td>\nS<\/td>\nR<\/td>\nS<\/td>\n–<\/td>\n<\/td>\n<\/tr>\n
21<\/td>\n9<\/td>\nF<\/td>\n+\/+<\/td>\nR<\/td>\nS<\/td>\nS<\/td>\nS<\/td>\nS<\/td>\nS<\/td>\nR<\/td>\nR<\/td>\nR<\/td>\nS<\/td>\nMDR<\/td>\n<\/td>\n<\/tr>\n
22<\/td>\n64<\/td>\nM<\/td>\n+\/+<\/td>\nS<\/td>\nS<\/td>\nS<\/td>\nS<\/td>\nS<\/td>\nS<\/td>\nS<\/td>\nR<\/td>\nS<\/td>\nR<\/td>\nMDR<\/td>\n<\/td>\n<\/tr>\n
23<\/td>\n30<\/td>\nF<\/td>\n+\/+<\/td>\nS<\/td>\nS<\/td>\nR<\/td>\nR<\/td>\nR<\/td>\nR<\/td>\nR<\/td>\nR<\/td>\nR<\/td>\nR<\/td>\nMDR<\/td>\n<\/td>\n<\/tr>\n
24<\/td>\n36<\/td>\nM<\/td>\n+\/+<\/td>\nS<\/td>\nS<\/td>\nR<\/td>\nR<\/td>\nR<\/td>\nR<\/td>\nR<\/td>\nR<\/td>\nS<\/td>\nR<\/td>\nMDR<\/td>\n<\/td>\n<\/tr>\n
26<\/td>\n1<\/td>\nM<\/td>\n+\/ –<\/td>\nS<\/td>\nS<\/td>\nS<\/td>\nS<\/td>\nS<\/td>\nS<\/td>\nS<\/td>\n<\/td>\nR<\/td>\nS<\/td>\nS<\/td>\n–<\/td>\n<\/td>\n<\/tr>\n
31<\/td>\n76<\/td>\nM<\/td>\n+\/-<\/td>\nS<\/td>\nS<\/td>\nS<\/td>\nS<\/td>\nS<\/td>\nS<\/td>\nR<\/td>\nS<\/td>\nS<\/td>\nR<\/td>\n–<\/td>\n<\/td>\n<\/tr>\n
32<\/td>\n6<\/td>\nF<\/td>\n+\/+<\/td>\nS<\/td>\nS<\/td>\nS<\/td>\nS<\/td>\nS<\/td>\nS<\/td>\nR<\/td>\nS<\/td>\nS<\/td>\nR<\/td>\n–<\/td>\n<\/td>\n<\/tr>\n
34<\/td>\n11<\/td>\nF<\/td>\n+\/ –<\/td>\nS<\/td>\nS<\/td>\nS<\/td>\nS<\/td>\nS<\/td>\nS<\/td>\nR<\/td>\nR<\/td>\nR<\/td>\nS<\/td>\nMDR<\/td>\n<\/td>\n<\/tr>\n
35<\/td>\n10<\/td>\nF<\/td>\n+\/ –<\/td>\nR<\/td>\nR<\/td>\nS<\/td>\nS<\/td>\nS<\/td>\nS<\/td>\nR<\/td>\nR<\/td>\nR<\/td>\nS<\/td>\nMDR<\/td>\n<\/td>\n<\/tr>\n
36<\/td>\n44<\/td>\nF<\/td>\n+\/+<\/td>\nS<\/td>\nR<\/td>\nR<\/td>\nR<\/td>\nR<\/td>\nR<\/td>\nR<\/td>\nR<\/td>\nR<\/td>\nR<\/td>\nMDR<\/td>\n<\/td>\n<\/tr>\n
48<\/td>\n60<\/td>\nF<\/td>\n+\/+<\/td>\nR<\/td>\nS<\/td>\nS<\/td>\nS<\/td>\nS<\/td>\nS<\/td>\nR<\/td>\nR<\/td>\nR<\/td>\nR<\/td>\nMDR<\/td>\n<\/td>\n<\/tr>\n
53<\/td>\n56<\/td>\nF<\/td>\n+\/-<\/td>\nS<\/td>\nS<\/td>\nS<\/td>\nS<\/td>\nS<\/td>\nS<\/td>\nS<\/td>\nS<\/td>\nS<\/td>\nR<\/td>\n–<\/td>\n<\/td>\n<\/tr>\n
57<\/td>\n48<\/td>\nF<\/td>\n+\/-<\/td>\nS<\/td>\nS<\/td>\nR<\/td>\nS<\/td>\nS<\/td>\nS<\/td>\nS<\/td>\nS<\/td>\nS<\/td>\nR<\/td>\n–<\/td>\n<\/td>\n<\/tr>\n
58<\/td>\n62<\/td>\nF<\/td>\n+\/-<\/td>\nS<\/td>\nS<\/td>\nS<\/td>\nS<\/td>\nS<\/td>\nS<\/td>\nS<\/td>\nS<\/td>\nS<\/td>\nR<\/td>\n–<\/td>\n<\/td>\n<\/tr>\n
61<\/td>\n57<\/td>\nF<\/td>\n+\/-<\/td>\nS<\/td>\nS<\/td>\nR<\/td>\nR<\/td>\nR<\/td>\nR<\/td>\nR<\/td>\nS<\/td>\nS<\/td>\nR<\/td>\nMDR<\/td>\n<\/td>\n<\/tr>\n
63<\/td>\n55<\/td>\nF<\/td>\n+\/-<\/td>\nS<\/td>\nS<\/td>\nR<\/td>\nR<\/td>\nR<\/td>\nR<\/td>\nR<\/td>\nR<\/td>\nS<\/td>\nR<\/td>\nMDR<\/td>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

M: male; F:female;S: sensitive; R: resist; MDR: multi-drug resistance; AMC: amoxicillin-clavulanic acid, CIP: ciprofloxacin, GE: gentamicin, NIT:nitrofurantoin, SXT: trimethoprim-sulfamethoxazole (SXT),CPD: cefpodoxime, CLT:cephalothin, TE:tetracycline,RP: rifampin and TIM:ticarcillin.<\/strong><\/p>\n

Table S2: <\/strong>Correlation between papE<\/em> and\/or Dam<\/em> and antibiotic susceptibility.<\/strong><\/p>\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n
Variable<\/strong><\/td>\npap E<\/em><\/strong><\/td>\nDam<\/em><\/strong><\/td>\n<\/tr>\n
r<\/em><\/strong><\/td>\np<\/em><\/strong><\/td>\nr<\/em><\/strong><\/td>\np<\/em><\/strong><\/td>\n<\/tr>\n
Gentamycin<\/td>\n0.115<\/td>\n0.366<\/td>\n0.176<\/td>\n0.164<\/td>\n<\/tr>\n
\u00a0Nitrofurantion<\/td>\n-0.048<\/td>\n0.708<\/td>\n-0.004<\/td>\n0.972<\/td>\n<\/tr>\n
Trimethoprim.<\/td>\n-0.062<\/td>\n0.624<\/td>\n0.030<\/td>\n0.813<\/td>\n<\/tr>\n
\u00a0Cefpodoxime.<\/td>\n0.004<\/td>\n0.974<\/td>\n0.106<\/td>\n0.403<\/td>\n<\/tr>\n
\u00a0Amoxyclave<\/td>\n0.032<\/td>\n0.803<\/td>\n0.123<\/td>\n0.334<\/td>\n<\/tr>\n
\u00a0Cephalothin<\/td>\n0.004<\/td>\n0.974<\/td>\n0.106<\/td>\n0.403<\/td>\n<\/tr>\n
\u00a0Ticarcillin<\/td>\n0.281<\/td>\n0.025*<\/td>\n0.318<\/td>\n0.010*<\/td>\n<\/tr>\n
\u00a0Rifampien<\/td>\n0.261<\/td>\n0.037*<\/td>\n0.268<\/td>\n0.032*<\/td>\n<\/tr>\n
\u00a0Tetracycline<\/td>\n0.279<\/td>\n0.025*<\/td>\n0.331<\/td>\n0.007**<\/td>\n<\/tr>\n
\u00a0Ciprofloxacin<\/td>\n-0.078<\/td>\n0.541<\/td>\n0.082<\/td>\n0.520<\/td>\n<\/tr>\n
MDR<\/td>\n0.104<\/td>\n0.412<\/td>\n0.266<\/td>\n0.034*<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

*Significant at P<\/em> \u2264 0.05; **highly significant at P<\/em> \u2264 0.01.<\/p>\n

Acknowledgments<\/strong><\/p>\n

Not applicable.<\/p>\n

Ethics Statement<\/strong><\/p>\n

Not applicable\u00a0<\/strong><\/p>\n

Conflict of Interest<\/strong><\/p>\n

The author declare that there is no conflict of interest.<\/p>\n

Funding\u00a0Source<\/strong><\/p>\n

There is not funding source<\/p>\n

References<\/strong><\/p>\n

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