Phylogenetic Study of Escherichia coli Isolated from Clinical Samples in Hilla City, Iraq

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Extraintestinal pathogenic E. coli (Ex-PEC) represents a distinct group of pathogenic E. coli that causes most of the extraintestinal E.coli infections (urinary tract infection, prostatitis, bacteremia, septicemia, and neonatal meningitis, vaginosis) human infections ( Donnenberg et al., 2002;Gordon and Cowling, 2003 ;Al-Khaqani et al.,2016 ;Abdulla et al.,2016).ExPEC isolates are genetically distinct from commensal E. coli found in the intestinal flora.Phylogenetic analyses have revealed that E. coli isolates are composed of four main phylogenetic groups (A, B1, B2, and D) (Gordon , 2004 ;Al-Khaqani et al., 2016).Isolates of each of the four groups have different phenotypic features, causing their ability to exploit different sugars, antibiotic-resistance profiles and growth rate-temperature relationships .The distribution (presence/absence) of a variety of genes thought to enable a strain to cause extra-intestinal disease also varies among isolates of the four phylo-groups (Johnson et al., 2001).Several studies have shown the relation between phylogeny and pathogenicity of E.coli isolates ( Bashir et al., 2012 ;Escobar-Paramo et al., 2004).Bearing in mind that most commensal isolates belong to A and B1 groups (Duriez et al., 2001), Phylogenetically and epidemiologically ExPEC are potentially different from those of intestinal pathogenic and commensal isolates (Smith et al., 2007).Most of the ExPEC isolates phylogenetically belong to B2 and to a lesser extent D groups and are equipped with various virulence factors that help these isolates during different mode of infection mechanisms like adhesion, invasion of host tissues, escape host defence mechanisms, signaling and production of different toxins interfering host cellular functions thereby promoting extraintestinal infection in both normal and immune compromised hosts (Dobrindt and Hacker, 2008;Wiles et al., 2008;Al-Dahmoshi et al., 2016) The aim of this study was to investigate the phylo-genetic groups of E. coli isolated from different clinical samples which includes urine, vaginal , seminal fluid and wound swap in Hilla, Iraq using a molecular primer.

MATERIAL AND METHODS
A total of 200 various clinical samples represent by 50 urine samples from patients with urinary tract infection,50 high vaginal swap from pregnant and non-pregnant women suffering from vaginosis, 50 seminal fluid samples of male suffering from bacteriospermia and 50 wound swabs samples .All samples were obtained from patients or individuals who were admitted to Babylon Hospital for Maternal and Pediatrics, and to Al-Hilla Surgical Teaching Hospital in Babylon city (Iraq) during the period from May to August 2016.In order to isolate E. coli, samples were directly inoculated on MacConkey agar (Himedia/ India) plates.After overnight incubation at 37°C, lactose fermenting colonies, Gram-negative, oxidase negative bacilli transferred to UTI Chromogenic medium (Condalab/Spain) and Eosin methylene blue agar (Himedia/India) to confirm E. coli isolates.

DNA extraction for gram negative bacteria
Typical E. coli colonies (with metallic green color on Eosin methylene blue agar and pink color on UTI Chromogenic medium ) were grown in LB broth(Condalab/Spain) at 37ºC for 18 h, and following the protocols of Favor Prep Genomic DNA Mini Kit (Blood/Cultured Cell) (Favorgen/Taiwan).The extracted DNA checked using Agarose gel electrophoresis (0.7% in TBE buffer) (Condalab/Spain) and then visualized using and gel documentation (Vilber/France).

Detection of phylogeny groups by PCR
PCR was conducted to determine the phylogenetic grouping of the isolates by targeting three genes, chuA, yjaA and TspE4.C2 using 20ìL reaction mix (IntronBio/Korea) (Clermont et al., 2000).Thermal cycler conditions were as follows: 95°C for 4 min, 30 cycles of (denaturation at 94ºC for 30sec.),(annealing at 59 ºC for 30sec.),(extension at 72ºC for 30sec).and final extension at 72ºC for 5 min.Agarose gel electrophoresis (1.5% in TBE buffer) and gel documentation (Vilber/France) were used to visualized and document the PCR products.The amplicon sizes were 279 bp for chuA, 211 bp for yjaA and 152 bp for TspE4C2 were recorder using 100bp ladder (IntronBio/Korea).After electrophoresis the gel was photographed under UV light.The results allowed the classification of isolates into either one of the four major phylogroups (A, B1, B2, or D) (Abdallah et al., 2011;Gordon et al., .2008).

RESULTS
This study was carried out to expose the phylogeny of E. coli isolated from different clinical samples to investigate the source of these isolates whether they are intestinal or extraintestinal.However, the phylogenetic groups of E. coli  A total of 53 E. coli isolates represented by (12 vaginal, 17 UTI, 10 seminal fluid and 14 wound swap) samples and according to the presence or absence of these genes were assigned to one of four phylogenetic groups (i.e.A,B1, B2 and D) group A and B1 (intestinal groups); B2 and D (extraintestinal groups) and six subgroups (i.e.A0, A1, B2 2 , B2 3 , D1 and D2).According to PCR-based phylotyping the result showed that all vaginal samples ,14 urine samples, 8 of wound samples and 6 of seminal fluid samples were belong to B2 3 subgroup , 1 of urine samples and 3 of wound samples were belong to B2 2 subgroup , 2 of urine samples and 1 of seminal fluid samples were belong to D 2 subgroups , 2 of wound samples and 2 of seminal fluid samples were belong to A 1 subgroup , 1 of wound swap samples was belong to A 0 subgroup and 1 of seminal fluid samples was belong to D 1 subgroup.Also the results showed that the subgroup B2 3 contained the majority of the collected isolates ( 40 isolates, 75.47 %), followed by subgroups A1, B2 2 (4 isolates for each subgroup, 7.55 %) then subgroup D2 (3 isolates, 5.66% ) and A 0 , D 1 (1 isolate for each subgroup, 1.88 %).No isolates were found to belong to group B1 (Table 1and 2)

DISCUSSION
The niche of commensal E. coli is the mucous layer of the colon.However, there are E. coli clones that are distinct from the intestinal commensal E. coli, possessing specific fitness and virulence attributes which allow adaptation to other niches (e.g.urinary tract, central nervous system, blood) and confer enhanced ability to cause a broad 12% and 8% respectively).These differences in distribution of the phylogenetic groups among the isolates of geographically distinct populations in different studies may be due to the health status of the host, geographic climatic conditions, dietary factors, the use of antibiotics, or host genetic factors.( Duriez et al., 2001) .Based on the results of this study , the majority of isolates were extraintestinal pathotypes.Therefore, the role of E. coli in human infections including urinary tract infections, septicemia, vaginitis and meningitis should be considered for further researches.

Table 1 .
Percentage for isolation of extraintestinal E. coli

Table 2 .
Percentage of E. coli isolates for each phylogenetic subgroups