Comparison of the Common Adhesin Coding Operons Distribution in Uropathogenic and Phylogenetic Group B2 and A Escherichia coli Isolates
Background: Escherichia coli is one of the most causative pathogen of urinary tract infection. Urinary tract infections (UTIs) are the second most common cause of morbidity and remain a serious health concern among the clinicians. The severity of UTI caused by uropathogenic E. coli (UPEC) is due to the expression of a wide spectrum of virulent factors such as adhesin coding operons. Little is known about the relationship between the E. coli genetic background and the acquisition of adhesin coding operons in UPEC isolates.
Objectives: The aim of this study was to determine the prevalence of adhesin coding operons in UPEC isolates belonged to phylogenetic group B2 and A collected from patients suffering from UTI.
Materials and Methods: A total 100 UPEC isolates were used for DNA extraction by the boiling lysis. The analysis of phylogenetic groups, along with detection of adhesin coding operons was performed by Multiplex-PCR method. Associations were assessed between afa, fim, foc, pap and sfa operons among to 55 B2 and 17 A groups E. coli isolates. Statistical analysis was performed using Fisher exact test.
Results: Phylogenetic analysis showed that 55 and 17 of 100 UPEC isolates belonged to the B2 and A phylogenetic groups, respectively. The afa, fim, foc, pap and sfa operons were present in five (9.09%), 55 (100%), 16 (29.09%), 46 (83.63%) and 46 (83.63%) of UPEC isolates belonged to phylogenetic group B2, and two (12.50%), 14 (87.50%), one (6.25%), two (12.5%) and 12 (75%) of isolates belonged to phylogenetic group A, respectively. Statistical analysis showed that pap gene was significantly more frequently detected in phylogenetic group B2 (P < 0.05).
Conclusions: The UPEC isolates belonging to group B2 harbored a greater number of adhesin coding operons than strains from phylogenetic groups A.
Keywords: Phylogenetic Analysis; Urinary Tract Infections (UTI); Uropathogenic Escherichia coli; Virulence Factors
Escherichia coli is one of the most extensively studied Gram-negative bacteria in microbiology (1, 2). This species is also a common cause of extraintestinal infections, such as urinary-tract infection and septicemia (1, 3). Phylogenetic studies have shown that UPEC has four main phylogenetic groups, designated as A, B1, B2, and D (4). Group B2 strains carry more virulence-factor genes than other strains (5-7). Furthermore, strains that cause extraintestinal infections belong mostly to group B2 and, to a lesser extent, group D (4). Extraintestinal pathogenic E. coli (ExPEC) strains differ widely in their capacity to localize in the human urinary tract (8). It has been reported that adhesins and other virulence factors may contribute to the colonization of E. coli strains in the human urinary tract. The UPEC strains express adhesin-encoding operons (pap, sfa and afa) (9, 10), and have genes for P fimbriae and type one fimbriae (11, 12). Genes encoding virulence factors are also enriched in UPEC strains. Up to now, there have been very few published studies on association of the common fimbriae virulence factors and phylogenetic background of UPEC isolates in Iran.
The objective of this study was to determine the distribution of adhesin-encoding operons between two phylogenetic groups of B2 and A in UPEC isolates collected from 100 patients suffering from UTI.
3. Materials and Methods
3.1. Bacterial Isolates
Urine samples were collected from patients with UTI from two major hospitals in Zabol, Iran. Specimens were taken from clean-catch sample, midstream urine and urinary catheters. One loop of specimens (10 µL) was seeded on Mac Conkey and Eosin-Methylene Blue agar (Biolife Laboratories, Milano, Italy). The isolates were identified based on standard methods (2, 13, 14) and UTI was defined as cultures yielding greater than or equal to 105 cfu/mL.
E. coli isolates were classified into phylogenetic biotypes (A, B1, B2 and D) according to present two virulence genes (chuA, encoding a heme 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. (4). The amplification of DNA was performed according to the methods provided by Clermont et al. Briefly, PCR was performed in a reaction mixture with total volume of 25 μL, containing 2 μL of bacterial DNA was used in a mix reaction containing 12.5 µL 2 × MasterMix Red Taq polymerase (Ampliqon, Pishgam Iran), and 30 pmol of each primer (Pishgam Iran) (Table 1). The PCR products were observed in an agarose gel 2% (Figure 1 A).
3.3. Multiplex-PCR for Virulence Genes
The assessed virulent factors were represented by five adhesin-encoding operons, afa, fim, foc, pap and sfa were also sought by using Multiplex-PCR. Briefly, genomic DNA was prepared from overnight cultures grown in 5 mL Luria Bertani (LB) broth (Invitrogen, Paisley, Scotland), by the boiling method. PCR was performed in a reaction mixture with total volume of 25 μL, containing 12.5 μL 2 × Master Mix red Taq polymerase (Ampliqon, pishgam Iran) and 0.3 µL of each primer. The PCR procedure was as follows: initial denaturation step at 94°C for five 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 min. The PCR products were observed in an agarose gel 2% (Figure 1 B). The amplicon size of afa-750 bp, fim-400 bp, foc-388 bp, pap-328 bp and sfa-100 bp operons were appreciated by comparison with a molecular size marker (Fermentase 100 bp) (Figure 1 B). Specific primers were design to amplify sequences of the fimH, hlyA, iucD, and iroN using MP primer 2.0 software (Table 1).
3.4. Statistical Analysis
The Fisher exact test was used for statistical analysis. The threshold for statistical significance was a Ρ value of ≤ 0.05.
4.1. 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 A (17%), comprised the majority of all isolated strains (Table 2).
4.2. Distribution of Virulence Genes Among the Studied Uropathogenic E. coli
The fim operon is widely distributed among isolates belonged to two phylogenetic groups B2 and A. The phylogenetic group B2 represents a 75%, higher than in group A. Differences in prevalence of five studied adhesin coding operons were observed between phylogenetic groups B2 and A isolates (Table 2). Among 55 B2 isolates, the prevalence of afa, foc, pap and sfa was five (9.09%), 16 (29.09%), 46 (83.63%) and 46 (83.63%) isolates, respectively; while among 17 type A isolates, the prevalence of afa, foc, pap and sfa was two (11.76%), one (5.88%), two (11.76%) and 12 (70.58%), respectively (Table 2).
4.3. Phylogenetic Distribution of VFs
According to the phylogenetic classification, E. coli isolates belonging to the extraintestinal phylogenetic B2 groups possess an accumulation of adhesin coding operons, as compared to those belonging to the phylogenetic group A. One of the isolates belonging to the phylogenetic B2 group harbor the complete set of five operons (Table 2). For B2 and A groups, the fim operon was distributed in 100% and 82.35% of isolates, respectively; while foc, pap and sfa operons are predominating in group B2 strains (16 out of 17 isolates for foc, 46 out of 48 isolates for pap and 46 out of 58 for sfa) (Table 2). Three adhesin coding operons were observed in 32 isolates belonging to B2 group; while in the A group, only one isolate existed with such operons (Tables 3 and 4). Concerning the total prevalence of five studied operons among the UPEC isolates, the fim operon was present in all of the tested isolates (100%) belonging to B2 and 82.35% of A group (Table 2); afa in 9.72% (6.94% for B2 group and 2.77% for A group), foc in 23.61% (22.22% for B2 group and 1.38% for A group), pap in 66.66% (63.88% for B2 group and 2.77% for A group) and sfa in 80.55% (63.88% for B2 group and 16. 66% for A group) of the studied isolates. The analysis of the association between the presence of different combinations of virulence genes among two studied phylogenetic group B2 and A, allowed us to include the tested isolates into 12 virulence patterns noted Ec1 to Ec12 (Table 3) for phylogenetic group B2 and eight virulent patterns noted Ec3-5 to Ec17 (Table 4) for phylogenetic group A. One isolate for phylogenetic group A with only one operon was included in Ec16, 33 isolates harboring fim, pap and sfa operons in Ec3 (Tables 3 and 4). The pattern Ec4 included the isolates simultaneously positive for fim+ and sfa+ (9 isolates; 1 isolate for B2 group and 8 isolates for A group). The pattern Ec2 in group B2 included the isolates simultaneously positive for fim+, pap+, foc+ and sfa+ (9 isolates). The two patterns Ec6 and Ec11 in B2 group were represented by the strains possessing a combination of three operon, Ec4 with fim+, foc+ and sfa+ (3 isolates) and Ec11 with fim+, pap+ and afa+ operon (1 isolate). The three patterns Ec12, Ec13 and Ec17 in A group were represented by the strains possessing a combination of different adhesin operons (Table 4).
Primers Used in This Study
Distribution of Virulence Genes in Phylogenetic Groups B2 and A
Virulence Patterns Identified Among the Studied Isolates Belonged to Phylogenetic Group B2
Virulence Patterns Identified Among the Studied Isolates Belonged to Phylogenetic Group A
Multiplex- PCR Profiles Specific for E. coli Phylogenetic Groups and Detection of Virulence Genes
Certain genotypic traits associated with E. coli extraintestinal diseases are frequently found in human UTI. Epidemiologic investigations have shown a good correlation between the occurrence of certain human diseases and the presence of adhesin operon in E. coli (18-20). Operons encoding P/Prs fimbriae, S/FIC fimbriae and Dr/AFA adhesins contribute to the pathophysiology of UTI (10, 19), whereas gene encoding for S fimbriae is correlated with the pathogenesis of neonatal meningitis (16). In the present study, we have identified the phylogenetic type of UPEC isolates collected from 100 patients who had UTIs. In phylogenetic analyses performed to date, it has been reported that UPEC strains mostly belong to group B2 (4, 21). As expected, in our study, similarly, the most common phylogenetic group in the UPEC isolates was B2. Seventy-two of studied isolates were distributed between the phylogenetic groups (B2 and A), known for their higher virulence potential. Previous research by Karimi Darehabi et al. (2013) (22) revealed a different result from that of the current research, identifying a higher prevalence of strains of phyla-group A and D among E. coli isolates, which can be usual because of geographic effects in the E. coli population among hosts. The most virulent isolates belonged to phylogenetic group B2, harboring the majority of tested adhesin operons. We assumed that the virulence gene distribution pattern of phylogenetic group B2 would be similar to that of other UPEC isolates belonged to phylogenetic group A. Our results, based on the distribution of the studied adhesin operons, the studied isolates of phylogenetic group B2 exhibited 12 virulent gene patterns; while in the A group only eight virulent gene patterns existed. Our results revealed the complexity of the association between phylogenetic groups and the properties of adhesin operons in UPEC referred to as Ec (Tables 3 and 4). In the present study, adhesin-encoding operons fim, foc, pap, and sfa are prevalent in UPEC isolates belonged to phylogenetic group B2. Group B2 strains have an accumulation of virulence-factor genes, as has been demonstrated both elsewhere (23) and in the present study. The distribution of E. coli isolates possessing fim, pap and sfa operons, regardless of phylogenetic group, showed a good agreement with other published data (12, 24). Our results illustrated a higher frequency of fim, pap and sfa operons compared with other virulence markers, which may suggest a crucial role of the adhesin-encoding operons in UPEC causing UTI. The distribution of the other studied virulent genes found among the studied isolates belonged to group B2 was also similar to other reported data (25, 26). We found a smaller proportions among isolates belonged to phylogenetic group A. In our study, phylogenetic distribution of virulent factors did allow the determination of a clear correlation between studied operons, phylogenetic background and the complexity of the UTI.
In conclusion, our study showed that a low number of UPEC adhesin-encoding operons in isolates belonged to phylogenetic group A. The characterization of UPEC isolates collected from patient with UTI is of great interest to improve our knowledge regarding their adhesin-encoding operons and phylogenetic background. Further studies are needed to identify UPEC virulent factors responsible for UTI and to determine the pathophysiology of these infections to consider possible prevention measures and means.
This work was performed in partial fulfillment of the requirements for MSc student’s thesis (Masoud Rahdar) and we wish to thank the staff of the laboratory of microbiology of faculty of vet-medicine.
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