Distribution of virulence genes and phylogenetics of uropathogenic Escherichia coli among urinary tract infection patients in Addis Ababa, Ethiopia

Background Urinary tract infection (UTI) is a common cause of morbidity worldwide. Uropathogenic Escherichia coli (UPEC) bacteria are the major cause of urinary tract infections. UPEC strains derive from different phylogenetic groups and possess an arsenal of virulence factors that contribute to their ability to overcome different defense mechanisms and cause disease. The objective of this study was to identify phylogroup and virulence genes of UPEC among urinary tract infection patients. Methods A cross sectional study was conducted from January 1, 2017 to October 9, 2017. E. coli bacteria were isolated from UTI patients using culture and conventional biochemical tests. Identification of phylogroup and genes that encodes for virulence factors was done using multiplex polymerase chain reaction (PCR). Data was processed and analyzed with SPSS version16.0 and Epi-info version 3.4.1 software. Results The most common urologic clinical manifestation combinations in this study were dysuria, urine urgency and urgency incontinence. The frequent UPEC virulence gene identified was fimH 164 (82%), followed by aer 109 (54.5%), hly 103 (51.5%), pap 59 (29.5%), cnf 58 (29%), sfa 50 (25%) and afa 24 (12%).There was significant association between pap gene and urine urgency (p-0.016); sfa and dysuria and urine urgency (p-0.019 and p-0.043 respectively); hly and suprapubic pain (p-0.002); aer and suprapubic pain, flank pain and fever (p-0.017, p-0.040, p-0.029 respectively). Majority of E. coli isolates were phylogroup B2 60(30%) followed by D 55(27.5%), B1 48(24%) and A 37(18.5%). There was significant association between E. coli phylogroup B2 and three virulence genes namely afa, pap, and sfa (p-0.014, p-0.002, p-0.004 respectively). Conclusion In this study the most frequent E. coli virulence gene was fimH, followed by aer, hly, pap, cnf, sfa and afa respectively. There was significant association between E. coli virulence genes and clinical symptoms of UTI. The phylogenetic analysis indicates majority of uropathogenic E. coli isolates were phylogroup B2 followed by phylogroup D. Phylogroup B2 carries more virulence genes. Hence, targeting major UPEC phylogroup and virulence genes for potential vaccine candidates is essential for better management of UTI and further research has to be conducted in this area.


Background
Urinary tract infection (UTI) remains the most common bacterial infection in human population and is also one of the most frequently occurring nosocomial infections [1]. Its annual global incidence is of almost 250 million [2][3][4]. Escherichia coli is the major etiologic agent in causing UTI, which accounts for up to 90% of cases [3]. Escherichia coli strains isolated from the urinary tract are known as uropathogenic Escherichia coli [5].
Uropathogenic E. coli strains possess an arsenal of virulence factors that contribute to their ability to overcome different defense mechanisms cause disease. These virulence factors that are located in virulence genes include fimbriae (which help bacterial adherence and invasion), iron-acquisition systems (which allow bacterial survival in the iron-limited environment of the urinary tract), flagella and toxins (which promote bacterial dissemination).Virulence genes are located on transmissible genetic elements (plasmid) and/or on the chromosome [6] so that non-pathogenic strains acquire new virulence factors from accessory DNA [7].
Escherichia coli strains derive from different phylogenetic groups; phylogenetic typing in four groups: A, B1, B2 and D. The majority of strains responsible for extraintestinal infections, including urinary tract infections, belong to group B2 or, to a lesser degree, to group D, whereas commensal isolates belong to groups A and B1 [8,9]. To the best of our knowledge, there is no information on phylogenetics and genes that encode virulence factors of uropathogenic E. coli in Ethiopia. So, knowing the phylogroup and virulence factors of E. coli responsible for UTI is important for proper management, prevention and control of urinary tract infection.

Methods
A cross sectional study was conducted from January 1, 2017 to October 9, 2017 in selected health facilities of Addis Ababa, Ethiopia; Namely Tikur Anbessa Specialized Hospital, Yekatit 12 Hospital and Zewditu memorial Hospital. These governmental hospitals were selected because they have microbiology laboratories that perform culture and antimicrobial sensitivity testing. They are also referral hospitals so most patients from Addis Ababa visit these hospitals. Clinical data were collected using a well-designed questionnaire.
The proposal of this study was ethically approved by the Institutional Review Board (IRB) of Addis Ababa University, College of Health Sciences. Permission was obtained from Medical directors of Tikur anbessa specialized Hospital, Yekatit 12 Hospital and Zewditu Hospital. Written informed consent was obtained from each patient participated in the study. Research participants were those patients coming to Tikur Anbessa Specialized Hospital, Zewditu Memorial Hospital and Yekatit 12 Hospital that were diagnosed with urinary tract infections and gave urine sample for microbiological investigation. The study participants' age was > 1 year old (Those children age < 1 year were excluded from the study because it is difficult to obtain urine from these patients). Socio-demographic and clinical data were collected from the patient directly and from patient record respectively by data collectors using well designed questionnaire. Urine sample processing and microbiological investigations were conducted without delay in the Microbiology laboratory. Mid-stream urine sample was collected using sterile container from patients diagnosed with urinary tract infections. Escherichia coli isolates were presumptively identified by colonial morphology on MacConkey agar (Oxoid, UK), and further identified and confirmed by conventional biochemical tests. A sample was considered as positive for UTI if a single organism was cultured at a concentration of > 10 5 CFU (colony forming unit) per milliliter of urine [10]. Patients having at least two of the following complaints: dysuria, urine urgency, frequency, incontinence, suprapubic pain, flank pain or costo-vertebral angle tenderness, fever (> 38°C) and chills was considered as urinary tract infection.
Bacterial DNA extraction DNA extraction was performed using an alkaline heat lysis method. Escherichia coli strains were grown on LB medium at 37°C overnight. Bacteria colonies were inoculated and suspended in 1.5 ml centrifuge tubes containing 200 μl of 1xPBS solution, and then 800 μl of 0.05 M NaOH added and mixed by vortexing. The sample/mixture was incubated at 60°C for 45 min. After 45 min 240 μl 1 M Tris-Cl was added to neutralize NaOH and centrifuged at 13,000 rpm for 3 min. One thousand microliters of the supernatant were stored at -20°C as a template DNA stock [12,13].

Detection of virulence genes of uropathogenic Escherichia coli
The genetic determinants that are studied includes those coding for type 1 [12,[14][15][16].Specific primers were used to amplify sequences of the fim, pap, sfa/foc, afa, hly, cnf, and aer operons. Details of primer sequences and predicted sizes of the amplified products are given in Table 1.
Detection of fim, pap and afa, and sfa/foc and aer sequences were done by multiplex PCR while hly and cnf detection were done by single-plex PCR [12,16,17]. PCR amplification of bacterial DNA extracts was done in a total volume of 25 μl containing 20 μl of Platinum® PCR Super-Mix (The mixture contains Mg ++ , dNTPs and recombinant Taq DNA polymerase at concentrations sufficient to allow amplification during PCR), 1.5 μl template DNA and1.5-2 μl (30 pmol of each) of the primers [12,16].
The amplification was carried out in a multiplex PCR [T100™ Thermal cycler (BIO RAD) & PTC-200 Peltier Thermal cycler (MJ Research)]. Conditions consisted of an initial denaturation at 94°C for 10 min, followed by 30 cycles of denaturation at 94°C for 2 min, annealing at a specific temperature for 30 s (Multiplex PCR for fimH, afa and pap annealing temperature used was 60°C; Multiplex PCR for sfa and aer annealing temperature used was 55°C; annealing temperature of Single-plex PCR for cnf and hly was 45°C and 50°C respectively) and 72°C for 1 min, and final extension at 72°C for 10 min. A 4.5 μl aliquot of the PCR product was mixed with 6x blue loading dye on parafilm and loaded on 1.2% agarose gel electrophoresis stained with 10 μL 10,000x GelRed. Electrophoresis was carried out for 120 min at 110 V on TAE buffer system and the gel was imaged under UV light (E-gel Imager; life technologies, USA). Amplified DNA fragments of specific sizes were detected by UV-induced fluorescence and the size of the amplicons were estimated by comparing them with the 1 kb plus DNA ladder (Invitrogen™) included on the same gel [12,16].
Strain J96 was used as positive control for pap, sfa/foc, hly, cnf, and fimH sequences and the strain K10 was used as positive control for afa. The positive control for aer was J96 and Cl 1212 strains and distilled water is used as negative control [18][19][20].
Phylogenetics grouping of uropathogenic Escherichia coli E. coli strains responsible for extra-intestinal infection are far more likely to be members of phylogroups B2 or D than A or B1 [7,8,21]. This study used PCR assay to detect the genes chuA and yjaA, and an anonymous DNA fragment TspE4.C2 found in E. coli isolates to classify E. coli isolates into phylogroups A, B1, B2 or D [22] (See Table 2). All PCR reactions were carried out in a 25 μl volume containing 20 μl of 10X buffer (supplied with Taq polymerase), 2 mM each dNTP, 2 U of Taqpolymerase (Invitrogen™ Super mix); the amounts of primer used are 20 pmol (2 μl of each primers). PCR reactions (T100™ Thermal cycler, BIO RAD) were performed under the following conditions: denaturation 4 min at 94°C, 30 cycles of 5 s at 94°C and 20 s at 59°C, and a final extension step of 5 min at 72°C [23,24]. Interpretation of amplified PCR products for phylogrouping E. coli was done according to Clermont et al. [22] (See Table 3). Table 1 Primers for uropathogenic Escherichia coli virulence genes PCR assay [12,16] Virulence factor Target gene(s) Primer Name Primer Sequence (5′-3′) Size of amplicon (bp) were used for data analysis. Regression and Chi-square test was performed to asses' relationship between variables. P value < 0.05 was considered as significant.

Results
Urine samples of 780 study participants who had complaints of urologic symptoms of urinary tract infections were cultured and 200 (25.6%) Escherichia coli isolates were identified by biochemical tests. Among study participants, 265 (34%) were males and 515 (66%) were females. The most common urologic clinical manifestation combinations in this study were dysuria; urine urgency and urgency incontinence followed by dysuria and urgency incontinence (see Table 4).

Phylogenetics of uropathogenic Escherichia coli
The distribution of phylogenetic groups amongst Escherichia coli isolates was determined by the following genes; arp A (400 bp), chu A (288 bp), yja A (211 bp) and an anonymous DNA fragment that is found in E. coli worldwide; TspE4C2 (152 bp). One kilobase plus (1 kb plus) DNA ladder was used to determine the base pair size (see Fig. 3).

Discussion
In this study higher proportion of urinary tract infections in females (66%) than in males (34%) were observed. UTI is more common in females than in males because structurally the female urethra is less effective in preventing the bacterial entry for colonization i.e. the urethra is shorter and wider. Escherichia coli is common because it is a normal flora in large intestine and can easily be acquired via faecal contamination with urinary tract especially in female it causes ascending UTI [25]. The highest incidence of urinary tract infections was observed in the age groups 26-45. This could be due to the fact that this age group is sexually active. Sexual intercourse may access entry of bacteria in to bladder. Identification of virulence factors that are encoded by uropathogenic E. coli are important for pathogenesis, severity of urinary tract infection, targets for vaccine and drug development [26]. In our study fimH adhesion gene was the most common and present in 164 (82%) uropathogenic E. coli isolates which is in agreement with studies conducted in Romania, 86% [16]; Mongolia, 89.9% [27], Iran, 86.17% [28], 79.67% [29] and China, 87.4% [30]. Targeting fimH as vaccine candidate is important for prevention of UTI and currently vaccine targeting fimH as potential vaccine candidate is under investigation. Antibodies against fimH prevent colonization of urinary tract by UPEC isolates [26].In this study, we found no significant association between fimH gene and clinical symptoms of UTI (p > 0.05), but this does not mean fimH is not involved in pathogenesis of UTI.
Iron is generally required for bacterial growth during infection. Thus UPEC stains uses iron acquisition genes like aerobactin, aer [46]. In this study we found 109 (54.5%) uropathogenic E. coli isolates carries aerobactin (aer) genes which is similar to studies conducted in Romania, 54% [16]; Tunisia, 52% [12]; Egypt, 51% [34] and Poland, 52.6% [42]; but lower than studies conducted in South Korea, 81% [39] and Iran, 73.1% [45]. Currently, Siderophore proteins are under investigation for potential vaccine candidate against UTI [26].There was significant association between aer gene and suprapubic pain, flank pain and fever (p-0.017, p-0.040, p-0.029 respectively). Thus, the high prevalence of aer gene in our study may be due to UPEC utilizes aerobactin virulence gene as a means of acquisition of iron and associated with clinical features of suprapubic pain, flank pain and fever which were observed in most UTI patients.
From clinical point of view UTI is classified as upper (proximal) urinary tract infection and lower (distal) urinary tract infection. Upper urinary tract infection includes pyelonephritis while lower urinary tract infection includes cystitis and urethritis. The common clinical symptom of upper urinary includes flank pain, fever, chills and costovertebral angle tenderness. The common clinical symptom of lower urinary includes dysuria and frequency. The above mentioned clinical symptoms of UTI that is correlated with virulence genes should be interpreted in this aspect.
The difference in virulence genes prevalence between our study and different studies abroad may be due to sample size difference and methodology difference. Several virulence determinants are the product of different genes, which can be detected by PCR method [16,28,42,45]. However, when there is mutation at the level of the corresponding gene, this will lead to negative PCR. Thus negative PCR doesn't mean absence of specific virulence gene [12].
Phylogenetic analyses have shown that virulent uropathogenic E. coli strains belonged typically to group B2 and less often to group D [38,39]. Our finding is in agreement with studies conducted in Denmark [38], Pakistan [35], South Korea [39], Poland [42] and Mexico [31] where it was found that the majority of isolates of E. coli predominantly belong to phylogenetic group B2. These findings are indicative of virulent strains of UPEC are common in study area among UTI patients and measures needs to be taken to combat these virulence strains through designing and implementing appropriate prevention and control strategies.
In our study the phylogenetic analysis indicated majority of uropathogenic E. coli isolates were group B2 60(30%) followed by group D 55(27.5%), group B1 48(24%) and group A 37(18.5%) which is in agreement with study conducted by Munkhdelger et al [27], where B2 (33.8%) was dominant strains followed by D (28.4%) strains, A (19.6%) strains and B1 (18.2%) strains. Similar study conducted by Kot et al [42], showed that 38.1% E. coli strains belonged to phylogenetic group B2, 35.3% to group D, 18.5% to group A, and 8.1% to group B1. Phylogenetic group A, represented 18% of isolates, which was higher than in studies conducted in South Korea 3.44% [39] and Iran 0.7% [47], but some studies found phylogroup A was the dominant phylogroup [9,15,48] suggesting that the colon may be the main reservoir for strains that cause urinary tract infections [9]. In some studies, phylogroup D was the dominant strain [25,49]. These different prevalence of the phylogenetic groups may be due to health status of the host, and geographic conditions, or variations in methodology and sample size [50].
In our study there was significant association between E. coli phylogroup B2 and three virulence genes namely afa, pap, and sfa (p-0.014, p-0.002, p-0.004 respectively). This finding is explained by the fact that E. coli strains belonging to phylogroup B2 contained a greater number of virulence genes than E. coli than other phylogroup as reported by other studies on UPEC isolates [38,51]. There was also significant association between E. coli phylogroup D and two virulence genes namely fimH and pap (p-0.043, p-0.019 respectively) which is in agreement with a study conducted in Thailand [25].
In this study high prevalence of drug resistance to ceftazidime (84%), ceftriaxone (80.5%), and cefotaxime (66%) was observed [52]. Resistance to ceftriaxone was observed in 80.5% of UPEC isolates, which is comparable to studies conducted in Nigeria 86% [13], India 81.8% [53] and China 84.8% [30], but higher than studies conducted in Nigeria 23.3% [54] and Mexico 10.2% [21]. Resistance to cefotaxime was observed in 66% of UPEC isolates, which is in agreement with studies conducted in Nigeria 68% [13], India 66.66% [55] and Iraq 78% [37], but higher than studies conducted in Ethiopia 18.7% [56], Nigeria 4.4% [9] and South Korea 7.8% [43]. Ceftriaxone is recommended for treatment of severe pyelonephritis in Ethiopia by national standard treatment guidelines [57]. Resistance to ceftriaxone could be due to transmission of resistant strains/isolates among hospitalized patients and noncompliance with medication. To reduce the incidence of resistance, empirical antibiotic selection in treatment of UTI must be based on the knowledge of local prevalence of causative uropathogens and their respective antimicrobial sensitivities rather than on universal guidelines [58].

Limitations of the study
The triplex PCR phylogroup assignment used in this study has limitations like many strains could be potentially misassigned as this method has low sensitivity and expected to fail in allocating recombinant variants. Exclusion of infants < 1 years and not differentiating nosocomial and community acquired infections are the limitation of this study. Whole Genome Sequencing, Pulse Field Gel Electrophoresis and MLVA were not done.

Conclusions
In our study the most frequent uropathogenic E. coli virulence gene was fimH, followed by aer, hly, pap, cnf, sfa and afa respectively. The most common urologic clinical manifestation combinations in this study were dysuria, urine urgency and urgency incontinence. There was significant association between pap gene and urine urgency; sfa and dysuria and urine urgency; hly and suprapubic pain; aer and suprapubic pain, flank pain and fever. The phylogenetic analysis indicates majority of uropathogenic E. coli isolates were phylogroup B2 followed by phylogroup D. There was significant association between E. coli phylogroup B2 and three virulence genes namely afa, pap, and sfa. Hence, targeting major uropathogenic Escherichia coli phylogroup and virulence genes for potential vaccine candidates is essential for better management of UTI and further research has to be conducted in this area.
Abbreviations aer: aerobactin; afa: afimbrial adhesins; bp: base pair; CFU: Colony forming unit; CLSI: Clinical and Laboratory Standards Institute; cnf: cytotoxic necrotizing factor; DNA: Deoxyribonucleic acid; dNTPs: deoxynucleoside triphosphates; fimH: type1 fimbriae; hly: hemolysin; IRB: Institutional Review Board; NaOH: Sodium Hydroxide; pap: pili associated with pyelonephritis; PCR: Polymerase chain reaction; sfa: S and F1C fimbriae; UPEC: uropathogenic E. coli; UTI: Urinary tract infection Funding This research was supported by Arba Minch University, Addis Ababa University and Michigan State University. The funding agencies had no involvement in the design of the study, data collection and analysis, interpretation of data and writing the manuscript.

Availability of data and materials
The datasets generated and/or analyzed during the current study are not publicly available due to ethical and confidentiality reasons but are available from the corresponding author on reasonable request under the Ethics Committee's approval.

Ethics approval and consent to participate
The proposal of this study was ethically approved by the Institutional Review Board (IRB) of Addis Ababa University, College of Health Sciences. Permission was obtained from Medical directors of Tikur anbessa specialized Hospital, Yekatit 12 Hospital and Zewditu Hospital. Written informed consent was obtained from each patient participated in the study. The study participants' age was > 1 year old. Before starting data collection, the purpose of the study was explained to all study participants and written informed consent was obtained. For age group 1-16 years old written consent was obtained from the parents or guardians.

Consent for publication
Not applicable.