|Year : 2017 | Volume
| Issue : 2 | Page : 305-310
Molecular characterisation of uropathogenic Escherichia coli isolates at a tertiary care hospital in South India
Arindam Chakraborty1, Prabha Adhikari2, Shalini Shenoy3, Vishwas Saralaya3
1 Department of Microbiology, Motilal Nehru Medical College, Allahabad, Uttar Pradesh, India
2 Department of Medicine, Kasturba Medical College, Manipal University, Mangalore, Karnataka, India
3 Department of Microbiology, Kasturba Medical College, Manipal University, Mangalore, Karnataka, India
|Date of Web Publication||5-Jul-2017|
Motilal Nehru Medical College, Allahabad, Uttar Pradesh
Source of Support: None, Conflict of Interest: None
Uropathogenic Escherichia coli (UPEC) express a multitude of virulence factors (VFs) to break the inertia of the mucosal barrier of the urinary tract. The aim of the present study was undertaken to characterised the UPEC strains and to correlate carriage of specific virulence markers with different phylogroups and also to correlate these findings with clinical outcome of patients. A total of 156 non-repeated, clinically significant UPEC isolates were studied. Virulent genes were determined by two set of multiplex polymerase chain reaction (PCR). Phylogenetic analysis was performed by triplex PCR methods. Antibiograms and patient's clinical outcomes were collected in a structured pro forma. Of the 156 patients infected by UPEC strains with significant bacterial counts the most common predisposing factors were diabetes (45.5%) followed by carcinoma (7%). On analysis of the VF genes of the isolates, a majority of strains (140; 90%) were possessing the fimH gene followed by iutA (98; 63%), papC (76; 49%), cnf1 (46; 29.5%), hlyA (45; 29%) and neuC (8; 5%), respectively. On phylogenetic analysis, 27 (17%) isolates were belong to phylogroup A, 16 (10%) strains to Group B1, 59 (38%) were from Group B2 and 54 (35%) were from Group D. High prevalence of antibiotic resistance was observed among the isolates. The incidence of papC, cnf1 and hlyA was significantly higher (P < 0.05) among the isolates from relapse patients. Our findings indicate that virulent as well as commensal strains are capable of causing urinary tract infection. Virulence genes as well as patients-related factors are equally responsible for the development of infections and also that virulence genes may help such isolates to persist even with appropriate chemotherapy and be responsible for recurrent infections.
Keywords: Outcome, phylogroup, polymerase chain reaction, uropathogenic Escherichia coli, urinary tract infection, virulence gene
|How to cite this article:|
Chakraborty A, Adhikari P, Shenoy S, Saralaya V. Molecular characterisation of uropathogenic Escherichia coli isolates at a tertiary care hospital in South India. Indian J Med Microbiol 2017;35:305-10
|How to cite this URL:|
Chakraborty A, Adhikari P, Shenoy S, Saralaya V. Molecular characterisation of uropathogenic Escherichia coli isolates at a tertiary care hospital in South India. Indian J Med Microbiol [serial online] 2017 [cited 2018 Feb 25];35:305-10. Available from: http://www.ijmm.org/text.asp?2017/35/2/305/209562
| ~ Introduction|| |
Escherichia More Details coli, one of the first enteric bacilli to be described and cultured, is a normal inhabitant of the intestinal tract of humans and animals. From a genetic and clinical perspective, E. coli strains of biological significance to humans may be broadly categorised as (1) commensal strains, (2) intestinal pathogenic strains and (3) extraintestinal pathogenic E. coli (ExPEC) strains. Among ExPEC, strains of uropathogenic E. coli (UPEC) are most commonly associated with human disease. These bacteria are the primary cause of community-acquired urinary tract infection (UTI) (70%–95%) and a large portion of nosocomial UTIs (50%), accounting for substantial medical costs and morbidity worldwide. UPEC isolates exhibit a high degree of genetic diversity due to the possession of specialised virulence genes located on mobile genetic elements called 'Pathogenicity Islands'. Characteristic virulence traits that are present in most UPEC isolates include various adhesins (e.g., P and type I fimbriae), factors to avoid or subvert host defence systems (e.g., capsule, lipopolysaccharide), mechanisms for nutrient acquisition (e.g., siderophores) and toxins (e.g., hemolysin, cytotoxic necrotising factor 1).
There are insufficient data regarding the relationship between phylogroup and distribution of virulence factors (VFs) among UPEC strains isolated from India. Hence, the present study was undertaken to phylogroup the UPEC strains and to correlate carriage of specific virulence markers with different phylogroups and also to correlate these findings with clinical outcome of patients.
| ~ Subjects And Methods|| |
Participants and clinical isolates
The study was conducted from August 2010 to August 2013, from patients of the tertiary care hospitals in South India after obtaining permission from the Institutional Ethical Committee. A total of 156 non-repeat strains of UPEC were isolated from the study population. The study population included patients of all age groups whose urine samples grew E. coli with colony counts of >105/ml and excluded those subjects who had received antimicrobial drugs during the past 1 month, those who had asymptomatic UTI or patients with polymicrobial infections. All patients were followed up for 1 year to monitor the clinical outcome. Isolates from a patient with the same antibiogram and biochemical characters were considered as relapses. Samples were processed immediately using standard procedures.
Isolation and identification of the organism
Isolates were identified based on colony morphology on blood agar, MacConkey's agar, 4–5 suspected colonies from each bacterial plate were picked, cultured and then identified by the various biochemical tests. Biochemical tests were performed to confirm E. coli using Gram staining, catalase test, Indole, Methyl red, Voges-Proskauer test, nitrate reduction, urease production, simmons citrate agar and various sugar fermentation tests.E. coli ATCC 25922 was used as the quality control strains for antimicrobial susceptibility testing.
Bacteria were harvested from tryptone soy agar, suspended in 250 μl of sterile water, incubated at 100°C for 5 min to release the DNA, and centrifuged. The supernatant was used in the polymerase chain reaction (PCR) as described below.
The positive control E. coli strains used in the PCR assay were kindly provided by Lotte Jakobsen (Department of Microbiology and Infection Control, Statens Serum Institut, 5 Artillerivej, Build 46/202 DK-2300 Copenhagen, Denmark). The control strains included known chuA, yjaA, TSPE4.C2 positive isolates E. coli 139A, papC, cnf1, neuC positive isolates E. coli 69A and hlyA, fimH, iutA positive E. coli 139A.
Phylogenetic analysis was performed by triplex PCR-based methods as described by Clermont et al. Briefly, a combination of two genes (chuA and yjaA) and an anonymous DNA fragment (TSPE4.C2), allows the determination of the main phylogenetic groups of E. coli (these being A, B1, B2 and D).
Detection of virulence factor genes by multiplex polymerase chain reaction assay
Two sets of multiplex PCR were developed to detect following genes:
- Set 1: A PCR assay was performed to detect papC, cnf1 and neuC genes as per primers and conditions described earlier with minor modification 
Template DNA was amplified by multiplex PCR with the use of oligonucleotide primers obtained from Sigma-Aldrich Pvt. Ltd., India. The PCR was performed in a final reaction volume of 50 μl containing 750 mM Tris-HCl, 200 mM (NH4)2 SO4, 2.5 mM MgCl20.2 mM each dNTP, 0.4 μM of papC primers and 0.6 μM of cnf1 and neuC primers, 1 U of Taq DNA polymerase (5 U/μl Fermentas, India) and 4 μl template DNA. An Eppendorf thermocycler was used for amplification. The program for amplification included a step of initial denaturation at 95°C for 3 min, followed by 25 cycles of 94°C for 30 s, 61°C for 30 s and 68°C for 3 min and a final extension step at 72°C for 3 min The PCR products were loaded in 2% weight/vol agarose gel prepared in Tris-borate-EDTA buffer at 120 V for 1 h and detected by ethidium bromide staining after electrophoresis. The amplicons were visualised using the gel documentation system (Alpha Imager, Bengaluru, India).
- Set 2: Another PCR assay was performed to detect hlyA, fimH and iutA genes as per primers and conditions described earlier with minor modification 
Template DNA was amplified by multiplex PCR with the use of oligonucleotide primers obtained from Sigma-Aldrich Pvt. Ltd., India. The PCR was performed in a final reaction volume of 50 μl containing 750 mM Tris-HCl, 200 mM (NH4)2 SO4, 2.5 mM MgCl20.2 mM each dNTP, 0.6 μM of hlyA primers and 0.3 μM of iutA and fimH primers, 1 U of Taq DNA polymerase (5 U/μl Fermentas, India) and 4 μl template DNA. An Eppendorf thermocycler was used for amplification. The program for amplification included a step of initial denaturation at 95°C for 3 min, followed by 25 cycles of 94°C for 30 s, 61°C for 30 s and 68°C for 3 min and a final extension step at 72°C for 3 min. The PCR products were loaded in 2% weight/vol agarose gel prepared in Tris-borate-EDTA buffer at 120 V for 1 h and detected by ethidium bromide staining after electrophoresis. The amplicons were visualised using the gel documentation system (Alpha Imager, Bengaluru, India).
Antimicrobial susceptibility testing
Antibiotic susceptibility testing was done by the modified Kirby–Bauer disk diffusion method in accordance with Clinical and Laboratory Standards Institute guidelines. The antibiotic disks (HiMedia, Mumbai, India) used were ampicillin (10 μg), piperacillin (10 μg), piperacillin/tazobactam (100/10 μg), ceftriaxone (30 μg), cefotaxime (30 μg), ciprofloxacin (5 μg), norfloxacin (10 μg), amikacin (30 μg), gentamicin (10 μg), cotrimoxazole (1.25/23.75 μg), cefoperazone + sulbactam (75/30 μg), imipenem (10 μg), meropenem (MRP; 10 μg) and ertapenem (ETP; 10 μg). Isolates were further tested for extended-spectrum beta-lactamase (ESBL) and AmpC activities by phenotypic methods, as described previously.,
Chi-square test was used to find an association between the phylogroups, VF genes and patient's clinical outcome. The analysis was performed using statistical package SPSS version 17.0 (SPSS version 17.0 IBM, USA).
| ~ Results|| |
A total of 156 patients infected by UPEC with significant bacterial counts were included in this study. Of the 156 patients, 66 (42%) were males and 90 (58%) were females with the age group of <1 = 3 (2%), 1–18 = 3 (2%), 19–44 = 43 (27.5%), 45–59 = 42 (27%) and >60 = 65 (42%). The majority of isolates 139 (89%), were community acquired infections and 17 (11%) were hospital acquired infections. The most common predisposing factors were diabetes (45.5%) followed by carcinoma (7%).
Phylogenetic grouping of the isolates was done using the results of PCR amplification of the chuA and yjaA genes and DNA fragment TSPE4.C2 [Figure 1]. Twenty-seven (17%) isolates were found to belong to phylogroup A, and 16 (10%) strains to Group B1, both phylogroups which are known to be commensal groups. Among the virulent groups (phylogroups B2 and D), 59 (38%) were from Group B2 and 54 (35%) were from Group D.
|Figure 1: Phylogenetic grouping of uropathogenic Escherichia coli : Phylogenetic Group A ([chu A−, yjaA−, TspE4.C2−] and [yjaA+, chu A−, TspE4.C2−]); Group B1 (chu A−, yjaA−, TspE4.C2+); Group B2 ([chuA+, yjaA+, TspE4.C2−] and [chuA+, yjaA+, TspE4.C2+]); and Group D ([chuA+, yjaA−, TspE4.C2−] and [chuA+, yjaA−, TspE4.C2+]). Lane 1–7: Test organism, Lane 8: Positive control: Escherichia coli 139A.|
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As might be expected, it was the isolates belonging to the virulent phylogroups, namely B2 (38%) and D (35%) that were causing maximum number of infections as compared to Group A and B1 (statistically significant, P< 0.05).
On analysis of the VF genes of the 156 isolates, a majority of strains (140; 90%) were possessing the fimH gene and very few isolates (8; 5%) were harbouring neuC genes [Figure 2] and [Figure 3]. The distribution of other VFs genes is summarised in [Table 1]. Among the 45 hlyA positive isolates 37 (80%) were also co-harbouring cnf1 gene (statistically significant, P< 0.05).
|Figure 2: Multiplex polymerase chain reaction assays for neuC, cnf1, papC genes. Lane 1: 100 bp DNA ladder; Lane 2: Positive control (Escherichia coli 69A); Lane 3: Negative control; Lane 4 and 5: Test isolates.|
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|Figure 3: Multiplex polymerase chain reaction assays for hlyA, fimH, iutA genes. Lane 1: 100 bp DNA ladder; Lane 2: Negative control; Lane 3: Positive control (Escherichia coli 139A); Lane 5 and 6: Test isolates.|
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|Table 1: Prevalence of virulence factor genes among the isolates (n=156)|
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A study of the possession of multiple VF genes revealed that 19 isolates possessed five VF genes, 22 isolates were observed to possess 4 VF genes, 38 strains contained 3 VF genes, 45 with 2 and 29 isolates were positive with 1 VF gene. However, no virulence genes (targeted) were detected in case of 3 isolates.
On analysis of the distribution of virulence genes among the phylogroups, we found that the presence of all virulence genes was significantly higher (P< 0.05) among B2 and D isolates [Table 2].
Of our study population, maximum number of patients (69%) recovered with appropriate antibiotic treatment. Relapses were seen in 29% patients. On analysis of the distribution of the virulence genes among the outcome groups, we observed that presence of papC, cnf1 and hlyA was significantly higher (P< 0.05) among the isolates from relapse patients [Table 3].
|Table 3: Distribution of the virulence genes among different clinical outcome groups|
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Results of Kirby–Bauer disk diffusion methods indicated that, of the 151 isolates, (97%) were fully susceptible to ETP and similarly, 148 (95%), 143 (92%) and 139 (89%) isolates were susceptible to MRP, imipenem and nitrofurantoin, respectively. Resistance pattern of other antibiotics is summarised in [Figure 4]. Of the total of 156 isolates 107 (68.50%) were ESBL producers and 46 (29.50%) were AmpC producers.
|Figure 4: Resistance pattern of commonly used antibiotics to the isolates.|
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| ~ Discussion|| |
In our study population, we found that age was an important risk factor for susceptibility to infection with UPEC strain. Elderly patients (>60 years) were more susceptible to infection when compared with any other age group. Several investigators have reported the same., We also found a higher proportion of females with UTI compared to males [Figure 5]. This finding is similar to other studies done by other investigators.,, Studies by others such as that of Janifer et al. and Eshwarappa et al. also found diabetes to be the most common factor associated with complicated UTI which is similar to our study where we found around 1 in 2 patients were diabetic.
UPEC strains which routinely cause infections have been shown to belong to phylogroups B2 and D. Results of our study indicated that approximately 70% of the E. coli isolates from our patients belonged to phylogenetic Group B2 and D which is in agreement with previous findings.,, The least frequently isolated phylogenetic group in our study was Group B1 which is also in accordance with similar studies done elsewhere.,
In our study, we found a high prevalence of type-1 fimbriae producing isolates (90%). Several recent studies such as that of, Kudinha et al., Mora et al. have also demonstrated a high prevalence of fimH genes among the UPEC isolates.
We also observed that around 63% urine isolates were positive with iutA genes. Several studies on UPEC also found the higher prevalence of iutA gene.,,
In the present study, we also observed that approximately 1 in 2 isolates were positive for the papC genes, a finding which is supported by other investigator's findings where they found about half of their study isolates carried the papC gene.,
In our study, around one in three isolates were found to possess the hlyA gene. Several studies have shown that the haemolysin plays a significant role in the virulence of urinary isolates.,,
We also observed that approximately one in three isolates was carrying the cnf1 g ene. Several groups of investigators have also reported the same prevalence rate regarding the possession of cnf1 gene.,,
An interesting finding of our study was the co-carriage of both hlyA and cnf1 genes among the isolates, Several epidemiological studies have consistently shown that UPEC strains that make cnf1 also produce hly.,
However, in our study, the prevalence of neuC gene among the isolates was significantly low in compare to other targeted genes.
Several investigators reported that strains belong to UPEC were also harbouring multiple VF genes,,, In our study also multiple VF genes were observed in several isolates. It was detected that of the six VF traits that were targeted, around one in eight isolates were positive with at least five VFs, and at least one VF gene was observed in one in six isolates. However, around 3% of isolates were negative for all the targeted virulence genes, on phylogenetic analysis of those isolates it was revealed that they belonged to phylogroup A, which indicated that they were normal commensals.
In correlation between the outcome of infection with possession of virulence genes we found that papC, cnf1 and hlyA play an important role in recurrent infections. This finding is similar to the study done by Ejrnás et al wherein they also reported that E. coli causing persistence/relapse had a higher number of VF genes. However, in the present study, no correlation was observed regarding improvement and mortality with the possession of virulence genes in the UPEC stains. This may be due to patient-related factors such as age, diabetes, malignancy and other underlying conditions.
The rapid increase in the rate of antibiotic resistance of UPEC isolates is a major cause of concern. In our study isolates, we observed a high degree of resistance pattern to commonly used antibiotics such as ampicillin, piperacillin, ciprofloxacin, norfloxacin and ceftazidime. We also observed that 20% isolates were resistant to piperacillin/tazobactam and around 24% of the isolates were resistant to cefoperazone/sulbactam which is quite alarming. Higher sensitivity was observed in nitrofurantoin (89%), ETP (97%) and other carbapenem group of drugs. A study by Sharma et al. in Mangalore also reported high prevalence of antibiotic resistance among the E. coli isolates.
By phenotypic methods around 69% of the isolates were ESBL producers. Other studies from India have also reported 50%–65% prevalence of ESBL producers among E. coli isolates.,
In our study population, we found that around 30% of isolates were AmpC producers by phenotypic methods. A study from India has reported a 30%–50% prevalence rate of AmpC production among E. coli.,
| ~ Conclusion|| |
Our findings indicate that virulent as well as commensal strains are equally capable of causing UTI and also the virulence genes may help such isolates to persist even with appropriate chemotherapy and be responsible for recurrent infections.
Our study had certain limitations, first, because this study is a retrospective analysis, limited patient information has been collected. Second, clinical outcome can be influenced by host factors, time of presentation and antibiotic choice in addition to the phenotypical and genotypical characters of the infecting E. coli strains.
We are grateful to Manipal University, Manipal, India and Association of Physicians, Karnataka, for providing infrastructure and financial support respectively, to conduct the study. We would like to thanks, Lotte Jakobsen MSc. (Biology), PhD. Department of Microbiology and Infection Control, Statens Serum Institut, 5 Artillerivej, Build 46/202 DK-2300 Copenhagen, Denmark for providing us the positive control isolates for the study.
Financial support and sponsorship
API, Karnataka, India.
Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5]
[Table 1], [Table 2], [Table 3]