Comparison of Virulence Factors Distribution in Uropathogenic E . coli Isolates From Phylogenetic Groups B 2 and D

Background: Urinary tract infections are a significant health problem, with Escherichia coli as a primary pathogen in approximately 80% of cases. The pathogenesis of E. coli in urinary tract infections is attributed to the production of virulence factors and phylogenetic background groups. Objectives: The aim of this study was to determine differences in prevalence of virulence factors of E. coli isolates from phylogenetic groups B2 and D, collected from patients with urinary tract infections. Materials and Methods: A total of 100 E. coli isolates were identified by conventional biochemical tests from patients with urinary tracts infections (UTIs) in teaching hospitals of Zabol, Iran. DNA was extracted using the boiling method. Analysis of phylogenetic groups, along with detection of virulence factor genes was performed by the multiplex-PCR method. Associations were assessed between type 1 fimberiaencoding gene, siderophore receptor encoding genes and hemolysin encoding gene among 55 B2 group E. coli isolates and 22 D group E. coli isolates. Statistical analysis was performed using the Fisher exact test. Results: Phylogenetic analysis showed that 55 and 22 of 100 isolates belonged to the B2 and D phylogenetic groups, respectively. The hlyA, iroN, iucD and fimH genes were present in 29 (52.72%), 22 (40%), 46 (83.63%) and 55 (100%) isolates belonging to the phylogenetic group B2, whereas in 2 (9.09%), 2 (9.09%), 10 (45.45%) and 22 (100%) isolates belonging to the phylogenetic group D, respectively. The comparison showed that there was a significant difference between the presence of hlyA and iroN genes in isolates belonging to the phylogenetic group B2 and D (P ≤ 0.05). Conclusions: This study determined that strains belonging to group B2 are the most important and abundant E. coli strains causing urinary tract infections.


Background
Urinary tract infections (UTIs) are the most frequent bacterial infections among outpatients and inpatients.Acute uncomplicated UTIs are mainly due to extra intestinal pathogenic Escherichia coli (ExPEC) (1).However, management of patients with UTIs has become a particular problem in the recent decades due to the presence of various virulence factors among uropathogenic E. coli (UPEC) strains (2,3).The severity of a UTI is amplified by the existence of a diverse range of virulence factors such as type 1 fimberia encoding genes, siderophore receptors encoding genes or iron intake system and hemolysin encoding gene conferring the pathogenicity of UPEC strains (4)(5)(6).The generally accepted hypothesis today is that UPEC strains evolved from commensal strains by acquiring new virulence factors from accessory DNA horizontal transfer at the chromosomal or plasmid level (7,8).Many of these properties contribute to UPEC colonization and invasion (9).Uropathogenic E. coli strains, like other pathogenic forms of E. coli do not have very di-verse origins (10, 11) and fall into four major phylogenetic groups.Phylogenetic analysis of E. coli strains separates them into four major phylogenetic groups (A, B1, B2 and D) (12).Most ExPEC are derived from group B2 and to a lesser extent from group D, while the other pathogenic E. coli are consistently distributed in all classes with the non-pathogenic strains belonging to group A and B1 (12).Results from molecular studies on the recognized ExPEC virulence factors are compatible with this classification, where B2 group strains are highly pathogenic with numerous virulence determinants responsible for extra intestinal infections and strains of phylogenetic group D seem to have fewer virulence determinants than B2 group strains, whereas strains of the group A and B1, are most often devoid of extra intestinal virulence factors (8).There is clear evidence that the association of virulence properties and phylogenetic background of E. coli is a complex phenomenon, resulting from their different interplays.Therefore, we thought that these may not be mutually exclusive.To clarify whether the present virulence factors (VFs) are directly associated with phylogenetic background, we compared VFs distribution difference in two major ExPEC phylogenetic groups, including B2 and D in a collection of 100 E. coli strains.

Objectives
The aim of this study was to compare virulence factors distribution between two major ExPEC phylogenetic groups B2 and D in a collection of 100 E. coli strains using multiplex-PCR.

Bacterial Isolates
Urine samples were collected from patients with UTIs from two major hospitals in Zabol, Iran.Specimens were taken from clean-catch sample, midstream urine and uri-nary catheters.One loop of specimens (10 µL) was seeded on MacConkey and Eosin-Methylene Blue agar.The isolates were identified based on standard methods (13, 14) and UTI was defined as cultures yielding greater than or equal to 105 cfu/mL.

Phylotyping
E. coli isolates were classified into phylogenetic biotypes (A, B1, B2, and D) according to the presence of the two virulence genes (chuA, encoding a hem transporter protein in E. coli O157: H7 and yjaA, initially identified in the genome of E. coli K-12) and one DNA fragment TspE4.C2 as described by Clermont et al. 2000 (12).The amplification of DNA was performed according to the methods provided by Clermont et al. 2000.Briefly, PCR was performed in a reaction mixture with total volume of 25 μL, containing 2 μL of bacterial DNA in a mix reaction containing 12.5 µL 2 × MasterMix Red Taq polymerase, and 30 pmol of each primer (Table 1).

Multiplex-PCR for Virulence Genes
A multiplex-PCR assay was designed for the simultaneous detection of four ExPEC-associated VFs, fimH, hlyA, iucD and iroN (encoding, respectively, type 1 fimbriae, hemolysin and the aerobactin iron transport system) in a total of 100 isolates.
Briefly, DNA was extracted from the logarithmic phase cultures by the boiling method.PCR was performed in a reaction mixture with total volume of 25 μL, containing 12.5 μL of 2 × MasterMix red Taq polymerase and 0.3 µL of each primer.The PCR procedure was as follows: initial denaturation step at 94°C for 5 minutes followed by 35 cycles consisting of denaturation (94°C for 30 seconds), annealing (59°C for 50 seconds ) and extension (72°C for 70 seconds), followed by a final extension step at 72°C for five minutes.The PCR products were evident after migration by gel electrophoresis on 2% agarose gel prepared in tris-acetate-EDTA (TAE) 1X, stained with ethidium bromide 0.5 μg/mL, visualized by UV and photographed with a Polaroid camera.The amplicon size of fimH-170bp, hlyA-1361 bp, iucD-534 bp and iroN-1084 bp genes were determined by comparison with a molecular size marker (Fermentas 100 bp) (Figure 1).Specific primers were designed to amplify sequences of the fimH, hlyA, iucD, and iroN using the MPprimer 2.0 software (Table 1).

Statistical Analysis
The Fisher exact test was used for statistical analysis.The threshold for statistical significance was a P value of ≥ 0.05.

Phylogenetic Groups Among Isolates
E. coli isolates causing UTIs in patients attending teaching hospitals in Zabol belonged to all four phylogenetic groups; however, the phylogenetic groups, B2 (55%) and D (22%), comprised the majority of all isolated strains (Table 2).

Distribution of Virulence Genes Among the Studied Uropathogenic E. coli
The studied virulence genes were widely distributed among isolates belonging to the two phylogenetic groups B2 and D. The fimH genes were found in 100% of isolates from the B2 and D groups.Differences in the prevalence of the four studied ExPEC-associated virulence genes was observed between isolates belonging to phylogenetic groups B2 and D (Table 2).Among the 55 isolates from group B2, iroN, iucD and hlyA were found in 22, 46 and 29 isolates, respectively while among 22 isolates from group D, iroN, iucD and hlyA were present in two, ten and two isolates, respectively (Table 2).

Phylogenetic Distribution of Virulence Factors
According to the phylogenetic classification, E. coli isolates belonging to the extraintestinal phylogenetic group B2 possess 55 genes, as compared to those belonging to the phylogenetic group D with 22 virulence genes.The majority of isolates belonging to the phylogenetic group B2 harbor the complete set of four virulence factors (10 strains) (Table 4).For groups B2 and D, fimH was distributed in all isolates, while hlyA and iroN genes were predominating present in group B2 strains (29 out of 31 strains for hlyA and 22 out of 24 for iroN), followed by D (4 strains); in group B2 only four isolates harbored these genes (Table 3).The three virulence genes were observed in 24 isolates belonging to group B2, while in group D only two isolates harbored the three virulence genes (Tables 4 and 5).Considering the total prevalence of the four studied virulence determinants among the UPEC isolates, the fimH gene was present in all of the tested isolates (100%) belonging to groups B2 and D (Table 3), iroN in 31.16%(28.57% for group B2 and 2.59% for group D), hlyA in 40.25% (37.66% for group B2 and 2.59% for group D) and iucD in 72.72% (59.74% for group B2 and 12.98% for group D) of the studied strains.The analysis of the association between the presence of different combinations of virulence genes among the two studied phylogenetic groups B2 and D, allowed us to devide the tested strains into seven virulence patterns noted Ec1 to Ec7 (Table 4) for phylogenetic group B2 and five virulence patterns noted Ec1 to Ec5 (Table 5) for phylogenetic group D. The twelve strains (two isolates for B2 group and 10 isolates for D group) with only one virulence marker were included in patterns Ec7 and Ec5, while 27 isolates harboring iucD and fimH genes were included in pattern Ec2 (Table 4 and  5).The pattern Ec3 included strains simultaneously positive for hlyA + , iucD + and fimH + (22 isolates).The two patterns Ec4 and Ec5 in group B2 included strains possessing a combination of three virulence genes; Ec4 with fimH + , iroN + and iucD + (five isolates) and Ec5 with fimH + , hlyA + and iroN + (six isolates).The two patterns Ec4 and Ec5 in group D included strains possessing a combination of different virulence genes; Ec4 with only fimH (10 isolates) and Ec5 with fimH + and iroN + (one isolate).

Discussion
In many UPEC strains, virulence is due to phylogenetic group background and to various virulence factors, such as hemolysins, adhesins and siderophore receptors (15).In this study, most of the uropathogenic E. coli isolates belonged to the phylogenetic group B2 and D. Previous research by Karimi Darehabi et al. (2013) (16) revealed a different result from that of the current research, identifying a higher prevalence of phylo-group A and D strains among E. coli isolates, which can be explained by the geographic effects of the E. coli population among hosts.Seventy-seven of the studied isolates were distributed between the phylogenetic groups (B2 and D), known for their higher virulence potential.The most virulent isolates belonged to the phylogenetic group B2, harboring the majority of tested virulence markers.We assumed that the virulence gene distribution pattern of phylogenetic group B2 would be similar to that of other ExPEC isolates belonging to the phylogenetic group D. Based on the distribution of the studied virulence genes, the studied isolates of phylogenetic group B2 exhibited seven virulence gene patterns, while group D isolates exhibited only five virulence gene patterns.Our results revealed the complexity of the association between phylogenetic groups and the properties of virulence markers in E. coli referred to as Ec (Tables 4 and 5).In the present study, the gene coding for type 1 fimbrial adhesion system (fimH) represented the most common factor for the virulence of E. coli isolates of both phylogenetic groups.The distribution of E. coli isolates possessing fimH gene, regardless of the phylogenetic group, was in good agreement with other published data (17-19).Our results illustrate a higher frequency of fimH compared with other virulence markers, which may suggest a crucial role of the virulence genes in E. coli causing UTIs.The distribution of the other studied virulence genes found among the studied isolates belonging to group B2 was also similar to other reported data (9, 20).The prevalence of hlyA gene encoding a toxin implicated in tissue damage and dysfunction of local immune responses was found more frequently in phylogenetic group B2.The distribution of this gene in our isolates is in agreement with other reports (21, 22).The prevalence of genes encoding the aerobactin and siderophore receptors (iucD and iroN), which confers the ability to take up iron, among isolates belonging to phy-Int J Enteric Pathog.2014;2(4):e21725 logenetic group B2 were fit with those published by other investigators (23, 24).Wereas we found a lower percent prevalence of iucD and iroN among isolates belonging to phylogenetic group D. In our study, phylogenetic distribution of virulence factors did allow the determination of a clear correlation between studied genes, phylogenetic background and the complexity of UTIs.
In conclusion, our study showed that: 1) a low number of E. coli virulence genes in isolates belonged to phylogenetic group D, 2) the characterization of E. coli isolates collected from patients with UTIs is of great interest to improve our knowledge regarding their virulence genetic markers and phylogenetic background, 3) further studies are needed to identify E. coli virulence factors responsible for UTIs and to determine the physiopathology of these infections to allow possible prevention measures and means.

Figure 1 .
Figure 1.Positive Multiplex-PCR Results for the Detection of E. coli Virulence Factors (A) and Phylogenetic Groups (B) Among E. coli isolates

Table 1 .
Primers Used for the Multiplex-PCR

Table 2 .
Distribution of Phylogenetic Groups Among 100 Escherichia coli Isolates From UTIs

Table 3 .
Association of Virulence Factor Genes with Phylogenetic Groups Among Escherichia Coli Isolates From UTIs

Table 4 .
Virulence Patterns Identified Among the Studied Isolates Belonged to Phylogenetic Group B2