The present study included 245 urine samples. 90 samples were collected from children with cancer attending the Pediatric Department of Minia National Oncology Institute (case group) and 155 samples were collected from children without cancer attending the Pediatric outpatient clinics of Minia University hospital (control group) during the period from October 2020 till October 2021. Children aged 2–18 years with clinical symptoms and signs of urinary tract infection (fever, dysuria, frequency, neutropenia or with bacteriuria >105 organisms/ml) were included. Phenotypic and genotypic characterization was carried out in the Microbiology and Immunology Department, Faculty of Medicine, Minia University.

Bacterial isolation

E. coli isolates were identified and isolated according to the standard methods using Gram staining, colony morphology (on MacConkey and EMB agar) and standard biochemical tests (IMVC) [4]. Confirmed E. coli isolates were stored in trypticase soy broth with sterilized 15% glycerol at −20 °C.

Antibiotic susceptibility testing

All E. coli isolates were examined for antibiotic susceptibility by the micro-dilution method following the Clinical and Laboratory Standards Institute (CLSI) recommendations [5].The panel of antibiotics was as follows: cefazolin, ampicillin-sulbactam, cefoxitin, levofloxacin, meropenem, tetracycline, ceftazidime and nitrofurantoin.

Detection of biofilm formation

The E. coli isolates were tested phenotypically for their ability to form biofilm by the Microtiter Plate Method (MTP), and genotypically by detection of biofilm encoding genes by PCR.

Microtiter Plate method (MTP)

This quantitative test is considered the gold-standard method for biofilm detection. Briefly, 3–5 of isolated pure colonies were suspended in Muller Hinton broth (MHP) with 2% sucrose and then incubated for 18 h at 37 °C in a stationary condition. After adjusting the turbidity of bacterial broth to 0.5 McFarland standard, 200 μl of bacterial broth was inoculated into a sterile MTP wells except the wells of the last column that was used as a negative control containing sterile MHP. The inoculated plates were incubated for 48 h. The contents of wells were decanted and the remaining bacteria in the wells were washed with saline. The bacteria in the wells were stained using 150 μl of crystal violet (0.1%) for 15 min at room temperature. The stain was aspirated by pipette and washed with water. One hundred fifty μl of 95% ethanol was pipetted into each well gently and the wells were covered to minimize evaporation covered to minimize evaporation. The results were interpreted by an ELISA reader at 620 nm. The interpretation and grading (weak- moderate and strong) of biofilm production was done according to the criteria of Stepanovic et al. [6].

DNA extraction

DNA extraction used overnight broth culture of all E.coli isolates. 1.5 ml of bacterial broth was pipetted into sterile microtubes. The microtubes were centrifuged at 5000 × g for 3 min. The supernatant was discarded, and the pellet was suspended in 200 μl molecular biology-grade water and mixed by pipetting. The tubes were boiled at 100 °C in a water bath for 14 min; cooled quickly on ice for 20 min, followed by centrifugation for 3 min at 5000 × g. Two hundred μl of the supernatant containing DNA was put into a newly labeled tube for PCR analysis.

Detection of ST131 E. coli isolates

ST131 E. coli isolates were identified by the presence of pabB or trpA genes using multiplex PCR. ST131-O16 was identified by being positive for the trpA gene and ST131-O25b were identified by those positive for the pabB gene.

Detection of carbapenem resistance genes

All phenotypic carbapenem-resistant E. coli (CRE) isolates were molecularly tested for the presence of the following carbapenem -resistant genes: blaVIM, blaNDM, blaKPC, and blaIMP genes using multiplex PCR.

Multiplex PCR was carried out in 50 µl reactions containing 25 µl hot start 2x Taq DNA Polymerase Master Mix, 6 µl of the extracted DNA and 10 pmol of each of the forward and reverse primers. The reaction was completed by nuclease-free water. The PCR amplification was performed with an initial denaturation at 94 °C for 10 min, followed by 30 cycles of denaturation for 5 s at 94 °C and annealing for 20 s followed by a final extension for 5 min at 72 °C.

Detection of biofilm encoding genes

All E. coli were molecularly tested for the presence of lasR, pelA, and lecA biofilm genes by conventional PCR. PCR reaction was performed in 25 μl of reaction mixture containing 3 μl of extracted DNA, 12.5 μl Taq PCR Master Mix, and 100 pmol (1 μl) of each primer [7,8,9]. PCR conditions for the amplification step were: denaturation at 94 °C for 1 min, annealing was different according to the tested gene, and extension at 72 °C for 1 min. Cycling was followed by a final extension at 72 °C for 10 min. Primer sequences, annealing temperatures and amplicon sizes of all study genes were presented in Supplementary Table S1.

Statistical analysis

SPSS Version 20.0 statistic software was utilized for carrying out the statistical analysis (SPSS Inc., Chicago, IL, USA). Chi-squared tests were performed for categorical data. A two-tailed P-value of <0.05 was considered statistically significant.