Prevalence and Molecular Characterization of Fim H Gene in Escherichia Coli Isolates Recovered From Patients With Utis

Narmin Saeed Merza
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Keywords : E. coli, FimH, Single nucleotide polymorphisms, Phylogeny, Sequencing.
Medical Journal of Babylon  14:3 , 2018 doi:1812-156X-14-3
Published :06 January 2018

Abstract

In this study, the prevalence of fim H gene was studied among 105 E. coli isolates obtained from urine samples of patients attended Azadi hospital in Duhok City. The intended gene was detected in94.3% of the isolates. Triplex PCR assay was applied and according to which the studied isolates were assigned into four groups namely A, B1, B2, and D groups which constituted 20.95 %, 3.8 %, 54.28 %, 20.95 %, respectively. Ten randomly selected isolates were subjected to SNPs fimH analysis with 3 reference strains of E. coli. The results revealed that 44 SNPs observed at 42 polymorphic sites accounting for 5.59%. All mutations were of substitutions and 29.5 % of mutations were transversions while transition type mutations constituted of 70.5 %. Ten SNPs accounting for 22.7 % of mutations gave rise to amino-acid changes (sense mutation) while the rest 34 (77.3 %) resulted in silent mutations. Moreover, twelve SNPs were singletons and among them five were with amino acid replacements. Amino acid replacements due to SNPs accounted for 1.27% of whole sequenced fragment of fimH.It can be concluded that there is no relationship inferred between the isolates of E.coli when the two phylotyping techniques are compared but the results of both can serve the purpose of genotypic characterization of uropathogenic E. coli.

Introduction

The genotypic characterization of pathogens has become a major aim for searching the genetic relatedness between clinical pathogenic strains and standard strains and for epidemiological investigation and public health strategies [1] Clermont et al. [2] proposed a triplex PCR assay for phylotyping of E. coli isolates into one of the major phylo-groups, A, B1, B2 or D depending on presence or absence of three markers namely huA, yjaA genes and a DNA fragment TspE4.C2 which is later characterized as a putative lipase esterase gene [3] Single-nucleotide polymorphism (SNP) analysis of fimH is another cheap, screening tool that has been widely used for epidemiological typing and genotypic analyses of Uropathogeneic Escherichia coli (UPEC) [4]. It has been found that during the course of bacterial evolution, genomic changes take place as a result of point mutations or horizontal transfer of genetic information resulting in strains divergence [5]. SNP analysis takes advantage of these changes at multiple loci to differentiate isolates [6]. FimH is a unique adhesin found at the top of type 1 fimbriae that specifies mannose-sensitive binding of bacteria to eukaryotic cells [7]. The usefulness of fimH single-nucleotide polymorphisms (SNPs) for E. coli typing has been proven by many studies [4,8,9]. The aim of the present study is to apply SNP fimH analysis for characterization and genotyping of E. coli isolates assigned to different phylogroups, recovered from urine samples.

Materials and methods

Sample collection and Phylogenetic grouping
One hundred fiveE. coliisolates have been recovered from urine samples of individuals attending Azadi Hospital in Dohuk City. Phenotypic identification and characterization have been done by other work [10]. Lyophilized master mix (Bioneer/South Korea) was reconstituted by adding 12µl grade distal water, 1µl of each primer including forward and reverse in a concentration of 20 pmol/?l of chuA, yjaA and TspE4.C2 genes, and  2µl of genomic DNA (50-100ng/µl) [2]. Regarding the screening of fimH gene, the amplification reaction consisted of 16µl PCR-grade water along with 1 µl of each forward and reverse primers (10 pmol/µl) of fimH (FimH-F 5’-CGAGTTATTACCCTGTTTGCTG-3’) and FimH-R 5’-ACGCCAATAATCGATTGCAC-3’) and 2 µl  DNA template (50-100ng/µl) [9]. The PCR products were electro-phoresed by running through 2% (w/v) agarose in TBE Buffer [11].
Sequencing fimH gene
For sequencing reaction of fimH gene, 50ul of PCR product was prepared as follows;Illustra GFX PCR DNA and Gel Band Purification Kit(manufactured in UK) were used for amplicons purification.The Big Dye Terminator V3.1 cycle sequencing Kit was utilized for sequencing the intended DNA fragments; sequencing reaction consisted of 1µl of BigDye, 3µl of 5X Buffer reaction, 1µl of primers (3.2 pmol/µl) either forward or reverse  and 10 µl of distilled water. Sequencing program created as follows: 1 cycle at 96oC/1minute, 25 consecutive cycles of 96 oC /10 seconds, 50 oC / 5 seconds and 60 oC /4 minutes, holding at 4oC. Capillary electrophoresis sequencer (ABI 3130 DNA sequencer, Singapore) was used to determine sequences of the reaction products. The resulting raw sequences were visualized by Chromas3.5V software to create counting of each individual gene using forward and reverse sequences.  The sequences of each segment were trimmed to a specified length of 786bp correspondent to the region used to determine the target gene. All DNA sequences deposited into NCBI/Blastn to identify corresponding region of the gene of interest.
  SNP analysis was performed by multiple sequence alignments of the corresponding fimH gene for 10 E. coli isolates with3 reference stains including; CFT073, E. coli UTI89 and E coli MM_1856/ Iran with accession number AE014075.1, CP000243.1 and JX847135.1 respectively using BioEdit (Version 6). E. coli CFT037 was used as standard to compare with other E. coli stains.  MEGA4was used for phylogenetic analysis and construction of neighboring trees.




Results

Using PCR assay, fimH gene was detected in 99 isolates accounting for 94.3% of the studied E. coli isolates. These isolates were successfully amplified 878 bp fragment of fimH gene while 6 isolates (5.7%) lacked it (Figure 1).

Discussions

Urinary tract infection is one of the most common widely distributed hospital-acquired infections. The ability of Escherichia coli to expresses a number of different adhesive organelles as essential requirement for colonizing the urinary tract including P, type 1, S, and F1C fimbriae makes it to be the predominant pathogen responsible for urinary tract infections[13, 14]. Type 1 fimbriae are produced by more than 80% of all UPEC, it is very well established that the production of type 1 fimbriae by E. coli is a determining virulence trait for pathogenic strains [13].The adhesive subunit of type 1 fimbriae (FimH) is an essential determinant, which has increased tropism for urinary tract cell receptors; thus, FimH adhesin is critical for colonizing various sites by E. coli [4]. The prevalence of fimH gene was previously investigated by various researchers in different countries. The results of the present study revealed consistent observations regarding the prevalence of fimH gene among UPEC isolates with the results of others [15] who stated that 97.5% of the UPEC were positive of the existence of fimH gene. This high prevalence was also noted by another group of scientists[9]who showed that fimH gene was found in 316 (92%) among 345 UPEC isolates and others [4] who reported a percentage of 92.8 % of UPEC isolates which harbored fimH gene. Genotyping tests had improved our knowledge about the epidemiology of UTI due to UPEC, these tests greatly improve our abilities to characterize modes of transmission of a pathogen, determine the source and risk factors of an infection [8]. Phylogenetic grouping of E. coli based on Triplex-PCR assay has been used extensively worldwide as a simple and relatively inexpensive method for assigning E. coli isolates into different phylogroups including; A, B1, B2, and D and submitted further clues that strains of the phylogroups diverse in their phenotypic and genotypic traits, their ecological niche and potential capability to result in disease [16]. This strategy based on different combinations of the presence and/or absence of three genetic markers namely, chuA and yjaA genes and TSPE4.C2 fragment as proposed by group of researchers [2]. It have been demonstrated that virulent extra-intestinal E. coli strains belong mainly to phlogroups B2 and D while groups A and B1 represent the most commensal strains [17]. Interestingly, [18] gave conceivable interpretation behind the predominance of phylogroup B2 that this phylogroup is usually of human source and proposed that UTIs usually is attributed to contamination by feces. Furthermore, since most E. coli strains belonged to phylogroup A and B1 considered as commensal and usually originate from non-human sources such as soil, water or animals [8], the probability of contamination by these isolates is more likely accepted as a source of infection [10]. Sequence-based genotyping approaches are becoming increasingly popular in epidemiological investigations of infectious diseases such as UTIs. Since fimH gene is frequently associated with UPEC strains and it is more likely to be altered or modified due to selective pressure, the phenotypic variants of fimH gene is earnestly associated with genetic variations thus, SNPs may contribute to the ability of organisms to cause illness conferring epidemic distribution or long term evolution of virulence [15]. SNP fim H analysis has discriminating power for this locus and it may be accurate enough for investigating UTI caused by UPEC that occurs over limited time periods or in confined geographical settings [19]. This study may the first attempt as our knowledge for molecular characterization of E coli based on SNP of FimH analysis in Dohuk Province and involved a limited number of clinical samples. It s certain that the majority of studied clinical isolates of E. coli shared the same sense mutations which gave rise to amino acid replacements (Ser73Asn, Asn81Ser, Ala166Val), despite they belonged to different phylogroups according to Triplex PCR analysis. By comparing the results of both genotyping based on sequencing of fimH gene with phylogenetic grouping based on Triplex PCR analysis, it s obvious that the two types of results are irrelevant, for instance, although Eco/5, Eco6 and Eco7were assigned to one allelotype, Eco5, ECO6 belonged to Phylogroup D while Eco7/Duhok strain belonged to phylogroup B2. Moreover, most clinical isolates which belong to B2 Phylogroup had unique amino acid replacements, but all other groups lacked them, this observation led the way for suggesting an effect of these mutations on fimH gene related virulence among these isolates. Horizontal transmission of virulence genes like fimH among isolates regardless of phylogroups may explain such results. Interestingly, phylogroup B2 isolates are more virulent than others and are mostly predominant among UPEC strain thus, these isolates are supposedly exposed to selective pressure more than isolates which belong to other phylogroups. It s mentionable that most isolates shared reference strain E coli/MM-1865 isolated from Iran with most non-sense mutations in addition to three sense mutations (Ser73Asn, Asn81Ser, Ala166Val). This technique may has effectively role for finding the track of relevant strains in geographical studied area. The results of the current study were in line with these of others who emphasized that a combination of Triplex PCR and SNP fim H analysis being practiced with extra intestinal E. coli strains could serve as a fast, highly reproducible typing tool for epidemiological explorations of ExPEC [19,1]. Discrepancy between SNP and PCR typing maybe attributed to horizontal gene transfer that alter the SNP phylogeny, in particular when a difference of the two techniques showed among closely related groups, such as groups A and B1 or groups D and B2. Alternatively, as the PCR typing is founded on only three genomic DNA segments, Taken results from both techniques together may serve for epidemiological studies and evolution of ExPEC strains [1].

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