Correlation of <i>Escherichia coli</i> Strains Isolated from Wild Bird Feces and Human Urinary Tract Infections from Phylogenetic Point of View

Hamid Staji; Elham Shahaboddin; Khatereh Kafshdouzan

doi:10.5812/ajcmi.12653

Correlation of Escherichia coli Strains Isolated from Wild Bird Feces and Human Urinary Tract Infections from Phylogenetic Point of View

Hamid Staji ^{1 , *} , Elham Shahaboddin ² and Khatereh Kafshdouzan ¹

Authors Information

¹ Department of Pathobiology, Faculty of Veterinary Medicine, Semnan University, Semnan, Iran

² Graduated Student in DVM Degree, Faculty of Veterinary Medicine, Semnan University, Semnan, Iran

Article Notes and Dates

Avicenna Journal of Clinical Microbiology and Infection: August 2017, 4 (3); e12653.Published Online: August 31, 2017

Article Type: Research Article; Received : May 6, 2017; Revised : May 14, 2017; Accepted : July 17, 2017
DOI: http://dx.doi.org/10.5812/ajcmi.12653

To Cite : Staji H, Shahaboddin E, Kafshdouzan K. Correlation of Escherichia coli Strains Isolated from Wild Bird Feces and Human Urinary Tract Infections from Phylogenetic Point of View, Avicenna J Clin Microb Infec. 2017 ;4(3):e12653. doi: 10.5812/ajcmi.12653.

Abstract

Copyright: Copyright © 2017, Avicenna Journal of Clinical Microbiology and Infection. This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial 4.0 International License (http://creativecommons.org/licenses/by-nc/4.0/) which permits copy and redistribute the material just in noncommercial usages, provided the original work is properly cited.

1. Background

Few microorganisms act as Escherichia coli, an important representative of the enteric microbiota of humans and other animals, because it can be a highly all-around pathogen from beneficial to lethal forms (1). Pathogenic E. coli can cause a wide spectrum of human diseases that extent from the enteric sites to extraintestinal areas such as the urinary tract, bloodstream, and central nervous system (2). The prompt source of E. coli that is implicated in extraintestinal infections such as urinary tract infections (UTIs) is the enteric lumen of the person and characterization of such strains suggests that environmental sources, perhaps contaminated foods especially with animal origin, can play a role in the local spread of intently associated E. coli strains (3, 4). Through the ages, wildlife has been a significant source of infectious diseases transmissible to humans (5). Currently, zoonosis with a wildlife source constitute can act as a remarkable public health problem all over the world. E. coli strains exist commonly in the gastrointestinal tract of wild birds and such animals can achieve the mentioned pathogens from infected surroundings and expand it directly to humans or indirectly by contaminating livestock farms (5, 6). E. coli strains based on the presence of three genetic markers (chuA, yjaA, and TspE4.C2) are divided in four phylogroups, namely A, B₁, B₂, and D, as follows: subgroup A0 (group A), lacking chuA, yjaA, and TspE4.C2; subgroup A1 (group A), lacking chuA and TspE4.C2 and having yjaA; subgroup B22 (group B₂), having chuA and yjaA and lacking TspE4.C2; subgroup B23 (group B₂), having chuA, yjaA, and TspE4.C2; subgroup D1 (group D), having chuA and lacking yjaA and TspE4.C2; and subgroup D2 (group D), having chuA and TspE4.C2 and lacking yjaA. Group B₂ and, to a lesser degree, group D, harbor the most of virulent extraintestinal E. coli, while groups A and B₁ mainly perform commensal, low-pathogenic E. coli or animal enteric pathogen E. coli (7). Based on these observations, we determine the phylogenetic groups of E. coli strains isolated from the feces of wild birds and human UTI cases to assess the correlation of such strains from phylogenetic point of view and evaluate whether wild birds can be involved in human urinary tract infections as pathogen sources or not.

2. Methods

Bacterial isolation and identification: 159 E. coli isolates from patients (both sexes) aged 20-55 years with UTIs in Semnan, Iran, during 2015 were isolated according to the protocol described by Manges et al. (8) and UTI was defined via the presence of more than two specific clinical symptoms (9). Also 105 E. coli strains from wild birds were isolated through cloacal swabs and feces of the birds in Semnan suburbs (40 strains from captured wild birds and 65 strains from free living wild birds, mainly wild Mallard ducks) and bacterial isolation was carried out according to the protocol of Carrillo-Del Valle et al. (10). Isolated strains with biochemically E. coli characteristics were stocked in nutrient broth with 15% glycerol at -20°C until genotypic steps.

DNA Extraction and Multiplex PCR experiments: Before DNA extraction, isolated E. coli strains were grown up in Luria Bertani agar (LB) plates and incubated at 37°C for 24 hours. Next, genomic DNA was extracted from the isolated strains with alkaline lysis method using NaOH and Tris-Hcl reagents, as described by Staji et al. (11). Then, E. coli isolates were phylogrouped to A, B₁, B₂, and D, using an established Triplex-PCR based method by detection of the chuA, yjaA, and tspE4.C2 genetic markers as previously described by Gordon et al. (12).

2.1. Statistical Analysis

The present research is a Cross-Sectional study. Phylogenetic distribution within E. coli strains of a host and comparison of phylogroups prevalence between the hosts (human and wild birds) were carried out by SPSS 20 using the Chi square test at significance level of P < 0.05.

3. Results

The results of detection of phylogenetic markers are shown in Figure 1. Distribution of E. coli phylogroups are presented in Table 1. Based on the detection of phylogenetic markers via the mentioned Triplex-PCR, group B₂ was the most prevalent phylogroup within isolates from both human with UTIs and wild birds. In total, 75 isolates (46.9%) from human UTIs belonged to phylogenetic group B₂, followed by group D (48 isolates, 30%), group A (25 isolates, 15.6%), and group B₁ (12 isolates, 7.5%). 34 isolates (32.5%) from wild birds belonged to group B₁, followed by group A (29 isolates, 27.5%), group B₂ (23 isolates, 22%), and group D (19 isolates, 18%). In the case of E. coli isolates from human UTIs, the distribution of phylogroup B₂ was significantly higher than that of others groups (P = 0.00) within human strains. In addition, the sum of isolates belonging to groups B₂ and D was the most prevalent phylogroups (P < 0.05) in overall human isolates, significantly. There was no significant difference between distributions of E. coli phylogroups from feces of wild birds. Statistically, the binary correlation of phylogroup B₁ showed higher distribution of group B₁ among isolates of wild birds than among human UTI strains, significantly (P = 0.00).

Figure 1. Gel Electrophoresis of Multiplex-PCR Results for the Detection of E. coli Phylogroups

M, marker (50 bp); C+, positive control for three genes (ECOR62); 2 - 16, E. coli isolates harboring different phylogenetic markers (test strains); B, blank as negative control (reactions lacking DNA template).

4. Discussion

This study was planned to find the distribution of phylogroups in E. coli strains from human urinary tract infections in Semnan, Iran, and feces of captive and free-living wild birds in this region zoo and suburbs and compare the phylogroups distribution between these hosts. There is much evidence showing that Extraintestinal pathogenic E. coli (ExPEC) strains usually exist in groups B₂ and D, and the enteric pathogenic strains are related to groups A, B₁, and D, while the commensal strains exist in groups A and B₁ (11, 13). In the present study, we analyzed phylogenetic group distribution in 264 E. coli isolates (159 from human UTIs and 105 from feces of wild birds) and our results showed that in human strains, B₂ is the most prevalent group (47.2%) followed by group D (30.2%), and these two phylogroups covered 77.4% of human isolates (Table 1). Clermont et al. (2000) suggested that strains belonging to B₂ and to a lesser extent to D, mainly are related to ExPEC and according to the fact that uropathogenic E. coli strains are a subgroup of ExPEC, results of our work are in parallel with the results of these researchers (14). About E. coli strains from feces of wild birds, the A (27.5%) and B₁ (32.5%) phylogroups were significantly the most prevalent groups and B₂ (22%) and D (18%) phylogroups presented at lower prevalence rates. It has been concluded that A and B₁ phylogroups are commensal and low pathogenic E. coli strains in general; but in some non-human species especially in mammals, these strains can cause enteric infections (7, 14). Although the prevalence of B₂ and D groups in the gastrointestinal tract of wild birds of this area was not dominant, these strains covered 40% of the isolates, implying that these birds can play an important role as a source of these two phylogroups in the infection of other hosts like human (6), especially considering this fact that strains belonging to B₂ and D groups harbor the majority of virulence factors that are related to invasion of E. coli to extraintestinal organs and infections while commensal E. coli strains usually are derived from A and B₁ phylogroups, lacking specific virulence genes related to B₂ and D phylogenetic groups (13, 15, 16). Within the E. coli strains from human UTIs, we found that about 22.6% of these isolates belonged to groups A and B₁, in accordance with the studies of Bingen et al. and Pupo et al., demonstrating that the cause of infection can be intestinal pathogenic E. coli and commensal strains (17, 18) and one of the most important causes of infection with commensal agents is poor observation of preventative criteria in people. On the other hand, some investigations show that members of B₁ phylogroup can persist in environment (12) and sometimes these strains carry virulence factors such as shiga-like toxins (stx1 and stx2), which these toxins are implicated in hemolytic uremic syndrome (HUS) caused by some uropathogenic E. coli in human. Thus, the strains belonging to B₁ group cannot be ignored and presence and distribution of such bacteria in feces of wild birds can be a potential hazard for humans that are in contact with them (19).

Table 1. Phylogenetic Analysis of 164 E. coli Strains Isolated from Human UTI Cases and Wild Bird Feces^a

Phylogenetic Group	Frequency of E. coli Isolates
	Human UTIs	Wild bird feces	Both hosts
A	24 (15)	29 (27.5)	53 (20)
B1	12 (7.6)	34 (32.5)	46 (17.5)
B2	75 (47.2)	23 (22)	98 (37.2)
D	48 (30.2)	19 (18)	67 (25.3)
Total	159	105	264

^aValues are expressed as No. (%).

There is much documented evidence showing that extension of E. coli phylogroups and virulence genes are not accidental in different hosts, and frequency of phylogenetic groups in hosts is influenced by some factors including host characteristics and food regimen, the antibiotic pressure in each geographic region, ecological differences, body volume, and climate conditions (20, 21). Escobar-Paramo et al. (2006) analyzed E. coli strains from fecal samples of birds, human, and other mammals and observed the significant distribution of phylogroups D and B₁ in birds droplets and A and B₂ in human strains (22). These authors found out that domestication could act as a major force to shape the genetic arrangement of E. coli populations in a host. Therefore, observation of some differences in the percentage of phylogroups distribution in E. coli strains from wild birds in comparison with the mentioned studies may be related to geographical, domestication, or climate variations or host species between wild birds in the present study and the birds that had been monitored in other investigations. In our study, identification of phylogenetic groups showed that the distribution of B₁ phylogroup in wild bird isolates was significantly higher compared to human isolates (P = 0.00) and analysis of data by Phi test showed a negative significant correlation in the distribution of this phylogroup between human UTIs and wild birds isolates.

There are no data available on the distribution of E. coli phylogroups and distribution of virulence genes and antimicrobial resistance patterns within these phylogroups in different hosts and regions of Iran. As E. coli is one of the major pathogenic agents for human and animals and due to the influence of various environmental factors and host characteristics in shaping the genetic characteristics of E. coli population circulating in various hosts of an area, we decided to evaluate the phylogroups similarity in E. coli isolates from human UTIs and wild birds of Semnan suburb in Iran as the first step. We found out that distribution of phylogroups in human UTIs resembles to majority of other investigations but phylogroups circulating in wild bird populations are a little bit different from ones in human UTIs especially in the case of B₁ group. Moreover, considering the potential role of E. coli in the transmission from wild life to human via various routes, it is quite possible that this less frequent E. coli phylogroups (B₂ and D in wild birds of area) may be related to human infections. In conclusion, considering that most of E. coli isolates in wild birds of our study were commensal strains and due to their ability to transmit virulence factors vertically and horizontally, further research to comprehensively diagnose E. coli genetic pools in both hosts and their virulence factors in detail as epidemiological data by some high throughput molecular techniques like DNA microarrays seems to necessary to assess exact relationships of isolates from these hosts, and phylogrouping may not be a very good technique to evaluate the relationships of different isolates of E. coli.

Acknowledgements

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