Phylogenetic Grouping and Assessment of Virulence Genotypes , With Antibiotic Resistance Patterns , of Escherichia Coli Strains Implicated in Female Urinary Tract Infections

Background: Escherichia coli strains are common pathogens that can cause urinary tract infections (UTIs). They are classified into phylogroups based on three genetic markers: chuA, yjaA, and TspE4.C2. The E. coli strains that cause UTIs possess several genes that encode urovirulent factors and antimicrobial-resistance phenotypes. We determined the phylogenetic groups of E. coli isolates from UTI cases in Sabzevar, Iran, the prevalence of certain virulence genes, and the antibiotic-resistance phenotypes in these strains. Objectives: The aim of this study was to assess the correlation of detected E. coli phylogroups in female UTI patients with the antibioticresistance pattern and the distribution of certain virulence factors among the phylogroups. Materials and Methods: Ninety-three E. coli isolates from 150 women with UTI were studied. Three genetic markers were detected for phylogenetic grouping of strains, and four virulence determinants were analyzed with multiplex-PCR, including the genes for hemolysin (hly), aerobactin (iucD), P fimbriae (pap), and S/F1C fimbriae (sfa/focDE). The antibiotic-resistance phenotypes were also determined. Results: The isolates from UTI cases were distributed within phylogroups A (31%), B1 (10%), B2 (28%), and D (31%). The prevalence of iucD, hly, pap, and sfa/focDE virulence genes was significantly associated with groups B2 and D. The most-resisted antibiotics were cefazolin (93%) and co-trimoxazole (68%), while the isolates were most sensitive to nitrofurantoin (1%) and imipenem (2%). Conclusions: The phylogroups of E. coli isolates from UTI cases showed that groups D, B2, and A are prevalent in women in Sabzevar, as the dominant pathogenic phylogroups. The comparison showed that there was no significant difference in the occurrence of virulence factors or in the distribution of antibiotic resistance between urinary E. coli isolates, but the virulence genes were distributed more into groups B2 and D, respectively. Our study showed that the highest sensitivity was to nitrofurantoin and imipenem, but the decision on a treatment strategy remains based on the physician’s diagnosis and the antimicrobial-resistance tests.


Background
Escherichia coli is a particularly complex species that is grouped into pathotypes of partly zoonotic intestinal pathogenic E. coli and extraintestinal pathogenic E. coli (ExPEC) (1,2).ExPEC strains can cause serious infections of certain organs and systems, including the urinary, central nervous, and blood circulatory systems and the reproductive tract (3)(4)(5).
Every year, 130 -175 million cases of uncomplicated urinary tract infections (UTI) occur globally, and > 80% are associated with E. coli.UTIs alone are responsible for an estimated $1 -$2 billion annually in direct healthcare costs in some regions, such as the United States.Antimicrobial drug resistance is further adding to the cost of treating these infections because they often require more compli-cated treatment regimens, and result in more treatment failures (5)(6)(7).
An important step in the onset and expansion of UTI is adhesion of E. coli to uroepithelial cells by P fimbriae.S fimbriae has also been shown to attach efficiently to the Int J Enteric Pathog.2016;4(1):e31609 epithelial and endothelial cells of the lower human urinary tract.Studies have shown that papEF and sfa/focDE are essential for cystitis and/or pyelonephritis (10)(11)(12).Hemolysin, encoded by the hly gene, stimulates sloughing of the uroepithelial cells and bladder hemorrhage.The aerobactin system, which is encoded by a five-gene operon (iucA, iucB, iucC, iucD, and iutA), is an expression of the iron-acquisition system, with an important role in utilizing siderophores for scavenging iron from the environment, giving UPEC the ability to colonize and persist in iron-poor niches in the host, such as the urinary tract (13,14).
During the last decade, the European antimicrobial resistance surveillance network has reported a steady increase in the rates of invasive E. coli isolates resistant to common and choice antibiotics, and some strains have expressed multidrug-resistant phenotypes, leaving only limited therapeutic options (17).

Objectives
In the present study, the correlations between the antibiotic-resistance pattern, the presence of virulence factor genes, and the phylogroup of the E. coli strains from female UTI cases in Sabzevar, Iran, were evaluated, as phylogenetic typing has proved useful in predicting the pathogenic potential of extraintestinal E. coli (18).It has been demonstrated that distribution of E. coli phylogroups and their antimicrobial-resistance patterns in hosts are different in various regions due to the hosts' physiological status and the environment (15).

Sample Collection
One hundred fifty urine samples were collected from female patients (25 -45 years old), suspected to have UTIs based on a urologist's diagnosis and who were referred to hospitals and independent diagnostic laboratories located in different regions of Sabzevar.The samples were cultured, and 93 E. coli strains were isolated from the UTIs according to the protocol described by Alonso et al. (19).The samples were directly streaked on MacConkey and EMB agar (HiMedia TM , India) for isolation of E. coli, then the lac+ colonies from the MacConkey agar and the lac+ colonies with metallic features from the EMB agar were selected for the biochemical identification procedure, which included indole (I) production, methyl red (MR), Voges-Proskauer (VP), and citrate utilization (C) tests.The colonies with indole + , MR + , VP -, and citrate-characteristics were biochemically diagnosed as E. coli, then stock cultures were prepared from the E. coli isolates and stored in Luria-Bertani broth with 15% (v/v) glycerol at -20°C until genotyping.

DNA Extraction and Phylogenetic Grouping
Genomic DNA was extracted from isolated strains with the rapid one-step extraction (ROSE) method (20), and isolates were assigned to one of the four major E. coli phylogenetic groups (A, B 1 , B 2 , and D) using an established triplex PCR-based assay for the chuA, yjaA, and tspE4.C2 genes according to the protocol of Gordon et al. (16).Briefly, the amplifications were modified and carried out in a total volume of 25 μl.Each reaction mixture contained 11.25 μl of distilled H2O, 2.5 μl of 10× buffer (supplied with Taq polymerase), 0.75 μl of MgCl2, 1 μl of dNTPs (each deoxynucleoside triphosphate at a concentration of 200 mM), 1 μl of each primer (20 pmol), 2.5 U of Taq polymerase (all from CinnaGen Co., Iran), and 3 μl of DNA template.Thermal cycler (BIO-RAD-USA) conditions were as follows: 4 minutes of initial denaturation at 94°C, followed by 30 cycles of 5-sec denaturation at 94°C, 10 seconds of annealing at 57°C, and a final extension step of 5 minutes at 72°C.A negative control (a reaction lacking the template DNA) was included in all amplifications performed, and strain ECOR62 was used as a positive control (Figure 1).

Virulence Genotyping
For the next step, the detection of virulence factor genes (hly, iucD, sfa/focDE, and pap) was performed according to the protocol of Adib et al. (10).The amplifications were modified and carried out in a total volume of 25 μl.Each reaction mixture contained 8.25 μl of distilled H2O, 2.5 μl of 10× buffer (supplied with Taq polymerase), 0.75 μl of MgCl2, 1 μl of dNTPs (each deoxynucleoside triphosphate at a concentration of 200 mM), 1 μl of each primer (20 pmol), 2.5 U of Taq polymerase (all from CinnaGen Co., Iran), and 4.5 μl of DNA template.Thermal cycler conditions were as follows: 3 minutes of initial denaturation at 95°C, followed by 35 cycles of 60 seconds denaturation at 95°C, 60 seconds of annealing at 53°C, 60 seconds of extension at 72°C, and a final extension step of 10 minutes at 72°C.A negative control (reactions lacking the template DNA) was included in all amplifications performed, and the reference strains 28C (hly+), J96 (sfa/focDE+, papEF+), and A30 (iucD+) were used as positive controls (Figure 2).The primers used in this study and their sequences are submitted in Table 1.

Results
In this study, 93 strains of E. coli from 150 women (62%) who had been referred to hospitals and diagnostic laboratories in Sabzevar with clinical signs of UTI, were isolated.The phylotyping of the E. coli strains showed 29 isolates (31%) in phylogroup A, 29 isolates (31%) in phylogroup D, 26 isolates (28%) in phylogroup B 2 , and nine isolates (10%) in phylogroup B 1 .

Discussion
This study was conducted to determine the distribution of phylogroups in E. coli strains from female UTI cases in Sabzevar, the prevalence of certain virulence genes, and the antimicrobial-resistance genes in the related isolates.
E. coli strains can be assigned to one of four main phylogenetic groups: A, B 1 , B 2 , or D. These phylogroups apparently differ in certain characteristics, such as virulence factors, antibiotic-resistance profiles, and ecological niches (23)(24)(25)(26).It has been demonstrated that ExPEC strains usually belong to groups B 2 and D, and that the intestinal pathogenic strains belong to groups A, B 1 , and D, while the commensal strains belong to groups A and B 1 (18,(27)(28)(29).In the present study, after analyzing 93 strains of E. coli from UTI cases as a subgroup of ExPEC, we found that 60% of the isolates belonged to groups B 2 and D. This was in agreement with the results of Johnson et al. and Picard et al. (18,26,27), who concluded that extraintestinal pathogenic E. coli strains usually belong to groups B 2 and D, with more virulence properties compared to other phylogroups (18,26).Approximately 40% of the E. coli isolates from our study belonged to groups A and B 1 , in accordance with the studies of Bingen et al. and Pupo et al., demonstrating that the route of infection can be intestinal pathogenic E. coli and commensal strains (28,29), and that one of the most important causes of infection with commensal agents is poor observation of preventative criteria.It has also been demonstrated that the virulence factor properties of E. coli strains, the antibiotic pressure in each geographic region, and ecological differences can determine the phylogroup of isolates from UTIs in each region (30).
Extraintestinal pathogenic E. coli strains have multiple virulence factors that confer the potential for pathogenicity.Recently, extended virulence genotypes have been reported for ExPEC isolates, and similarities in virulence factors, phylogenetic backgrounds, and genetic profiles have been noted among E. coli strains from animals and humans (8,31).It has been stated that ExPEC strains possessing virulence factors for the invasion and colonization of extraintestinal sites typically belong to phylogenetic groups B 2 and D, while commensal E. coli strains usually derive from groups A and B 1 , lacking the specialized virulence factor genes associated with the B 2 and D strains (1,5).
In the present study, we monitored the distribution of certain virulence factor genes (iucD, hly, pap, and sfa/fo-cDE) in E. coli isolates from female UTI cases, and found that the iucD gene was present in 55% of the isolates, hly in 10%, pap in 10%, and sfa/focDE in 20%.When compared to commensal strains, aerobactin biosynthetic genes are more frequently detected in E. coli pathogenic strains, and their incidence correlates with highly pathogenic strains (32,33).We found that 55% of our isolates from female UTI cases harbored the iucD gene as a mechanism of iron uptake from the environment.Iron is an essential element for survival of E. coli.It facilitates numerous cellular activities, such as perox-ide reduction, electron transport, and nucleotide biosynthesis (34,35).As iron exists at low concentrations in extraintestinal sites of infection, the ExPEC strains have evolved multiple strategies for sequestering iron from the host (32).This explains why the other strains lack the iucD gene, as they probably have other strategies for iron uptake from the host organs.Comparing the prevalence of the iucD gene in our diagnosed phylogroups, we found that 72% of the iucD genes were present in the B 2 and D groups as the main pathogenic phylogroups of E. coli in the extraintestinal pathogenic strains (24% in group A and 4% in group B 1 ).These findings are in agreement with and confirm the previous studies (15,33).
As indicated above, the hly gene was detected in 10% (9 isolates) of E. coli strains from female UTI cases in Sabzevar, 55% of which were distributed in phylogroup D, 22% in group A, and 22% in group B 2 .The hemolysin enzyme, encoded by the hly gene, is secreted by uropathogenic E. coli strains.This enzyme causes tissue damage, facilitates bacterial distribution, and participates in bacterial pathogenesis (36,37).It is believed that hly is more common among invasive uropathogenic strains than in nonpathogenic or commensal isolates of E. coli (38).In the present study, 77% of the hly genes were detected in phylogroups B 2 and D. This confirms the results of previous research showing that these phylogroups are more pathogenic and have more virulence factors than others, although different studies have shown the prevalence of hly in UPEC isolates to range from 2% to 47% in different countries (10,39,40).
UPEC isolates, like enteric E. coli pathogens, are a genetically heterogeneous group and can vary significantly in their ability to colonize and persist within either the bladder or the kidneys (41).Bacteria assemble adhesins on their surface as monomers, simple oligomers, or components of supramolecular fibers, called fimbriae or pili.The adhesive organelles most commonly associated with UPEC include type 1, P, and S/F1C-related pili and the Dr family of adhesins (42,43).The pap and sfa/focDE genes monitored in our study are responsible for encoding the P pili and S/F1C pili, as important adhesins of UPEC in humans (42).We showed that the prevalence in our isolates of the pap gene was 10% (10 isolates) and of the sfa/focDE gene was 20% (19 isolates).After analyzing the relationships of these adhesion genes with our diagnosed phylogroups, it was revealed that 80% of the pap genes and 76% of the sfa/focDE genes were distributed in phylogroups B 2 and D, confirming the members of these groups to be more pathogenic strains.It seems that the other E. coli isolates in our study harbored genes for adhesins, such as type 1 fimbriae and members of the Dr adhesins, which can be found in some UPEC strains as the organelle used to attach and colonize in the host urinary tract.
Understanding the impact of drug susceptibility patterns is crucial, as the changing rate of antibiotic susceptibility has a large impact on the treatment of UTIs.
Int J Enteric Pathog.2016;4(1):e31609 Treatment failure, prolonged or repeated hospitalizations, increased costs of care, and increased mortality are some of the consequences of bacterial resistance in UTIs.Although several factors may play roles in antimicrobial resistance, the misuse of antibiotics in each geographic location directly affects the antibiotic-resistance pattern (44).Antibiotic-resistant E. coli strains pose a therapeutic challenge, leaving few suitable alternatives with unknown intrinsic virulence potential.Their multiple virulence factors may mediate colonization of host surfaces, injury to host tissues, and avoidance or subversion of host defense systems (45)(46)(47).In our study, we investigated the antimicrobial susceptibility pattern of UPEC strains isolated from UTI cases in our region, for better usage of empiric antimicrobial agents and to demonstrate the distribution of resistance patterns in the related phylogroups of E. coli bacteria.We monitored the antimicrobial resistance of 93 E. coli isolates from UTI cases in the Sabzevar region with the disk diffusion method.Nine antimicrobial agents, as described above, were applied for assessment of resistance patterns.These patterns showed that the most phenotypic resistance in our isolates was for cefazolin (93%), followed by co-trimoxazole (68%), nalidixic acid (52%), amikacin (40%), ciprofloxacin (25%), cefepime (19%), gentamycin (17%), imipenem (2%), and nitrofurantoin (1%), respectively.There was no significant relationship between specific phylogroups and antimicrobial-resistance patterns in the tested isolates.Characterization of antibiotic-resistant E. coli strains from UTIs in human cases resident in our region revealed a wide range of phenotypic resistance patterns between our isolates, and the most resistant strains were genotypically diverse across the phylogroups.
Several investigations have been conducted to determine the antibiotic-resistance patterns of UPEC strains from different regions of Iran, and different results have been obtained, demonstrating the diverse resistance patterns between isolates (48)(49)(50)(51)(52).In the present study, the antibiotics nitrofurantoin (with 1% resistance in all isolates) and imipenem (with 2% resistance) seem to be the best antimicrobials used in the region.
Monitoring of the emergence and spread of dominant antimicrobial-resistant strains within hospital surveillance programs may assist urologists in developing improved strategies for the treatment and prevention of infections for which the choice of antimicrobials is limited.The authors recommend that urologists review the antibioticresistance patterns in their region.The antibiogram test, the availability of antimicrobials for each UTI case, and the physician's decision based on clinical signs and limitations remain, of course, the most important guidelines for the initiation of an antimicrobial treatment strategy.

Figure 1 .
Figure 1.Positive Triplex PCR Results for the Detection of E. coli Phylogenetic Groups/Subgroups

Figure 2 .
Figure 2. Positive PCR Multiplex Results for the Detection of E. coli Virulence Genes

Table 1 .
The Sequences of Primers Used in This Study for Assigning E. coli Isolates in Phylogenetic Groups, and the Virulence Gene Distributions Between Them hly2 ACCATATAAGCGGTCATTCCCGTCA Int J Enteric Pathog.2016;4(1):e31609

Table 2 .
Frequency of Virulence Genes (VG) in Relation to Phylogenetic Group Among Urinary E. coli Strains Isolated From UTI Samples Collected in Sabzevar, Iran a a Source: E. coli strains from UTI cases.b Values are expressed as No.(%)