Background: Urinary tract infections (UTIs) are the most prevalent bacterial infection in humans. The uropathogenic E. coli (UPEC) express a wide range of virulence factors that contribute to their pathogenicity. The emergence of Multidrug resistance(MDR)-associated UTI is increasing off late. Hence this study was undertaken to monitor the distribution of virulence factors among UPEC strains and to note the antibiogram, outcome and type of associated UTI.

Methods: A prospective cross-sectional time-bound study of 6 months was done on clinically significant urinary isolates of Escherichia coli. Detection of haemolysin production and serum resistance was done by phenotypic methods. Genotypic characterization of the virulence genes (papC, iutA, hlyA, cnf1) was done by multiplex PCR. Demographic data, clinical history, antibiogram and type of UTI were collected from clinical case records.

Results: 75 E. coli isolates from patients with suspected urinary tract infections were included. Females had a higher preponderance of UTI (66.7%).93% of the patients were adults and the remaining 7% were from the paediatric population.  24 (32%) isolates showed haemolysis by plate haemolysis method, and all 75 (100%) isolates were serum resistant. Out of 75 isolates, 65 were positive for at least one of the four targeted genes, while the remaining 10 isolates were negative for all 4 genes. Multidrug resistance was found in 40 (53.3%) isolates. 97.4% of the UTI cases had a favourable clinical outcome at discharge. Mortality due to urosepsis was 2.6%.

Conclusion: The association of hemolysin production with resistance to imipenem and norfloxacin in UPEC strains was significant. The presence of the hlyA gene is positively associated with ceftazidime resistance. Nitrofurantoin, piperacillin tazobactam and cefaperazone sulbactam maybe suitable candidates for empirical therapy of UTIs. Drugs like aminoglycosides, carbapenems and fosfomycin may be used as reserve drugs in the treatment of MDR-UTI. However, inappropriate usage can gradually increase antibiotic resistance. Hence, proper selection of antibiotics in hospitals taking into account the local antibiogram is needed to reduce the emergence of antibiotic resistance.

Methodology

Specimen processing: Clean catch midstream urine samples received from suspected cases of UTI were processed within one hour of collection. The samples were inoculated onto MacConkey’s agar by semi-quantitative method, Cystine-Lactose-electrolyte-deficient agar, and UTI chrome agar. The culture plates were incubated at 37℃ for 24 hrs. Urine samples with pure growth of E. coli, with a colony count of ≥ 10⁵ CFU/ml or ≥ 10³ CFU/ml in symptomatic patients, were considered significant. The study included 75 urinary isolates of E. coli. The isolates were identified based on colony morphology and standard biochemical tests. Antibiotic susceptibility testing was performed by the modified Kirby–Bauer disk diffusion/ Vitek2 Compact (Biomerieux, France) system, and interpretation was done as per the Clinical and Laboratory Standards Institute guidelines.

Variables and data source

Phenotypic methods for detection of haemolysin production and serum resistanc

The detection of α-haemolysin produced by E. coli was performed by plate haemolysis test. The presence of a zone of complete lysis of erythrocytes around the colony and clearing of the medium on 5% sheep blood agar, is suggestive of α-haemolysin production.

Serum resistance was studied by using fresh overnight culture of isolates as per the method described by Sharma et al. The UPEC strains were considered serum sensitive if the viable count dropped to 1% of the initial value and serum resistant if ≥ 90% of organisms survived after 180 minutes.

Genotypic characterization of virulence genes of UPEC:3,10

DNA extraction was performed by boiling method. The spectrum of virulence genes in UPEC strains was detected using two sets of multiplex PCR as shown below. Table 1 shows the PCR mastermix preparation used. The primer sequence of the mentioned genes is shown in Table 2. PCR was performed in a final reaction volume of 50 μl. 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 amplicons are visualized using the gel documentation system.

The required data were retrieved from the clinical case records and the cases of complicated, and uncomplicated UTIs were identified. Uncomplicated UTIs are those that occur in healthy individuals without any of the predisposing factors for UTI. Complicated  UTIs occur in individuals with underlying functional or structural abnormalities of the genitourinary tract.

Statistical Analysis

All the data was entered into an excel sheet and analyzed using IBM SPSS version 25. The continuous and categorical variables have been represented as mean ± standard deviation and frequency percentages respectively. The association between the variables was analyzed using the chi-square test.

Excel software