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 > 105 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.

In-vitro antimicrobial susceptibility testing of the bacterial isolates was performed by Kirby-Bauer disc diffusion method. The following antimicrobial agents were used with their respective concentration: trimethoprim-sulfamethoxazole (SXT) (1.25/23.75 μg), ampicillin (AMP) (10 μg), nalidixic acid (NA) (30 μg), amoxicillin-clavulanate (AMC) (20/10 μg), ceftazidime (CAZ) (30 μg), tetracycline (TE) (30 μg), cefotaxime (CTX) (30 μg), ceftriaxone (CRO) (30 μg), gentamicin (CN) (10 μg), ciprofloxacin (CIP) (5 μg), amikacin (AK) (30 μg), norfloxacin (NOR) (10 μg), nitrofurantoin (F) (300 μg), meropenem (MEM) (10 μg), imipenem (IM) (10 μg) and chloramphenicol (C) (30 μg) (Oxoid, UK). The antibiotic disks were firmly placed on sterile Mueller-Hinton Agar (Oxoid, UK) plates previously seeded with a 24 h old culture of the isolate (106 CFU/ml of 0.5 McFarland Standard). The plates were incubated at 37 °C for 24 h and diameter of zones of inhibitions was measured using caliper and compared with the standard set by CLSI [11]. E. coli ATCC 25922 was used as reference strain. Molecular characterization of E. coli isolates was conducted in college of Human Medicine, Michigan State University, USA.

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 fimbriae [fimH], pili associated with pyelonephritis [pap], S and F1C fimbriae [sfa and foc], afimbrial adhesins [afa], hemolysin [hly], cytotoxic necrotizing factor [cnf], and aerobactin [aer] [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.

Table 1 Primers for uropathogenic Escherichia coli virulence genes PCR assay [12, 16]
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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 SuperMix (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 Cl1212strains 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 2 Primers used for phylogenetic of uropathogenic Escherichia coli [23]
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Table 3 Interpretation of amplified PCR products for phylogrouping E. coli [22]
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Data analysis

SPSS version 16.0 and Epi-info version 3.4.1 softwares 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).

Table 4 Common combinations of clinical manifestations
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The antimicrobial susceptibility patterns of 200 E. coli isolates which were subjected to similar testing procedure showed that E. coli isolates had highest resistance (86.5%) to ampicillin followed by ceftazidime (84%), ceftriaxone (80.5%), tetracycline (80%), trimethoprim-sulfamethoxazole (68.5%) and cefotaxime (66%) (see Table 5).

Table 5 Antimicrobial susceptibility patterns of E. coli isolates
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Virulence genes were amplified and detected successfully in 198 (99%) E. coli isolates. The most frequent E. coli virulence gene 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%) (see Fig. 1).

Fig. 1
figure 1

Virulence genes of uropathogenic Escherichia coli. (Proportion of uropathogenic E. coli virulence genes. The most frequent virulence gene was fim H, followed by aer, hly, pap, cnf, sfa and afa)

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The virulence genes fimH (456 bp), afa (672 bp), pap (328 bp), sfa (410 bp), aer (269 bp), cnf (693 bp) and hly (556 bp) were successfully amplified. One kilobase plus (1 kb plus) DNA ladder was used to determine the base pair size (see Fig. 2).

Fig. 2
figure 2

Representative 1.2% agarose gel electrophoresis of uropathogenic Escherichia coli virulence genes isolated from urinary tract infection patients. (Lane M, 1-kb plus ladder; lane 1–15, amplified E. coli PCR products (sample 81–95) with the following virulence genes, pap (328 bp), fimH (456 bp) and afa (672 bp); lane 16, strain J96 positive control; lane 17, strain K10 positive control and lane 20, distilled water as negative control)

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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) (see Table 6).

Table 6 Association between virulence genes of E. coli and clinical data of urinary tract infections
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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).

Fig. 3
figure 3

Representative 1.2% agarose gel electrophoresis of uropathogenic Escherichia coli genes used to classify Escherichia coli into different phylogroup. (Lane M, 1-kb plus ladder; lane 1–15, amplified PCR products (sample 111–125) with the following Escherichia coli phylogrouping genes; arp A (400 bp), chu A (288 bp), yja A (211 bp) and TspE4C2 (152 bp); lane 16, strain CFT073 positive control; lane 17, strain J96 positive control and lane 20, strain K10 positive control)

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Phylogenetic analysis indicates majority of uropathogenic Escherichia coli isolates were group B2 60(30%) followed by group D 55(27.5%), group B1 48(24%) and group A 37(18.5%) (see Fig. 4).

Fig. 4
figure 4

Phylogroup distribution of uropathogenic Escherichia coli isolates. (The most common phylogroup of uropathogenic Escherichia coli isolates was phylogroup B2 followed by phylogroup D, phylogroup B1 and phylogroup A)

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In this study there was significant association between Escherichia coli phylogroup B2 and three virulence genes namely afa, pap, and sfa (p-0.014, p-0.002, p-0.004 respectively). Similarly, there was significant association between Escherichia coli phylogroup D and two virulence genes namely fimH and pap (p-0.043, p-0.019 respectively). There was significant association between Escherichia coli phylogroup A and virulence genes fimH and afa (p-0.011, p-0.002 respectively). Phylogroup B1 has significant association with pap gene (p-0.001). The virulence factor that encodes pap gene has significant association with Escherichia coli phylogroup B2, D and B1 (p-0.002, p-0.019, p-0.001 respectively) (see Table 7).

Table 7 Association between phylogroup and virulence genes of uropathogenic Escherichia coli
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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.

Pyelonephritis associated pili (pap) gene was found in 59 (29.5%) uropathogenic E. coli isolates which is comparable to study conducted in Iran, 30.2% [6]; Mexico, 24.7% [31]; Romania, 36% [16] and Brazil, 32% [32]; but lower than studies conducted in Iran, 50.4% [28], 57% [33] and Egypt 54% [34]. In this study, there was significant association between presence of pap gene and urine urgency (p-0.016), which was commonly observed clinical symptom in most UTI patients. This indicates that UPEC uses pap genes as virulence factor to cause UTIs.

S and F1C fimbriae (sfa gene) was found in 50 (25%) uropathogenic E. coli isolates which is similar to studies conducted in Pakistan, 27% [35]; Romania, 23% [16]; Tunisia, 34% [12]; Iran, 32% [36] and Iraq, 22.7% [37]; but lower than studies conducted in Denmark, 46% [38]; Iran, 81% [33] and South Korea, 100% [39] and higher than studies conducted in Mongolia, 8.8% [27] and China, 8% [30]. In our study, there was significant association between presence of sfa gene, and dysuria and urine urgency (p-0.019 and p-0.043 respectively). This indicates that sfa genes are important for pathogenesis of UPEC to cause UTI and responsible for clinical symptoms of UTIs.

Afa adhesin (afa gene) was found in 24 (12%) uropathogenic E. coli isolates which is similar to studies conducted in Iran, 12% [33]; Mexico, 12.8% [31]; Brazil, 11% [32] and Romania, 14% [16]. Afimbrial adhesins (afa) may favor establishment of chronic and/or recurrent urinary tract infections [40]. In this study, we found no significant association between afa gene and clinical symptoms of UTI (p > 0.05).

Uropathogenic E. coli secretes toxins like α-haemolysin (hlyA) and cytotoxic necrotizing factor 1 (cnf1). Hly Alpha promotes bladder cell exfoliation and cell lysis, which facilitates iron and nutrient acquisition by the bacteria. Cnf1involved in bladder cell exfoliation and increased levels of bacterial internalization [26, 41].

In this study 103 (51.5%) uropathogenic E. coli isolates carries hemolysin (hly) gene which is comparable to studies conducted in Iran, 50.4% [28] and South Korea, 62% [39]; but higher than studies conducted in Zimbabwe, 12.5% [14]; Tunisia, 19% [12]; Poland, 18.5% [42]; Mexico, 15.4% [31] and China, 11.6% [30].In our study, hemolysin gene was significantly associated with suprapubic pain (p-0.002). This indicates that hemolysin may be responsible for clinical manifestation in UTI patients. Alpha-hemolysin encoded by hlyA is an extracellular cytolytic protein toxin that is produced by up to 50% of UPEC isolates. Alpha-hemolysin has been associated with clinical severity in UTI patients [43]. Currently vaccine against hlyA that protect renal damage is under investigation [44].

In our study we found 58 (29%) uropathogenic E. coli isolates carries cytotoxic necrotizing factor 1 (cnf1) which is similar to studies conducted in Iran, 36.5% [45] and Pakistan, 20% [35]; but higher than studies conducted in Tunisia, 3% [12]; Romania, 13% [16] and Poland, 12.1% [42]. In this study, we found no significant association between cnf1 gene and clinical symptoms of UTI (p > 0.05).

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 costo-vertebral 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 mis-assigned 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.