This prospective, analytic study was done for a period of six months. A total of 100 urine samples were collected from patients of all age groups and both the sexes with a urinary catheter for at least two days suffering from symptoms of UTIs. The study was approved by the Institutional Human Ethical Committee (IHEC) and informed written consent was taken from the patients before collection of samples. Samples were collected under complete aseptic conditions with a sterile syringe from the distal end of the urinary catheter and transferred to a sterile urine container and transported immediately to the laboratory without any delay.
Urine samples were inoculated on Blood Agar, MacConkey’s agar and Cystine lactose electrolyte deficient (CLED) medium with a calibrated loop to determine colony-forming units (CFU). All specimens with bacteriuria of >103
colony- forming units (cfu)/mL urine (which defines CAUTI) of one or two organisms were analysed to determine their causative uropathogens and their antibiotic susceptibilities.3
Identification of isolates was done by colony morphology, gram staining and standard biochemical tests. Bacterial susceptibility to antimicrobial agents was determined by the Kirby Bauer disk diffusion method on Muller-Hinton agar. Isolates were categorised as susceptible, moderately susceptible, and resistant, based upon interpretive criteria developed by the Clinical and Laboratory Standards Institute (CLSI).4
Antibiotic discs (Hi-Media) ampicillin (10μg), cefuroxime (30μg), cefotaxime (30μg), amikacin (30μg), gentamicin (10μg), cotrimoxazole (1.25/23.75μg), norfloxacin (10μg), netilmycin (30μg), nitrofurantoin (300μg), nalidixicacid (30μg), ciprofloxacin (5μg), erythromycin (15μg), oxacillin (1μg), linezolide (30μg), imipenem (10μg) and vancomycin (30 μg) were used for antimicrobial susceptibility tests.
Detection of biofilms was done by tube adherence method and Congo red agar method.
Tube adherence method by Christensen et al 5
Suspension of tested strains was incubated in the glass tubes containing Brain Heart Infusion Broth (broth) aerobically at the temperature of 35°C for a period of two days. Then the supernatant discarded and the glass tube was stained by 0.1% safranin solution, washed with distilled water three times and dried. A positive result is defined as the presence of a layer of stained material adhered to the inner wall of the tubes. The exclusive observation of a stained ring at the liquid-air interface should be considered negative
Congo red agar method by Freeman et al 6
Suspension of tested strains was inoculated onto a specially prepared solid medium - brain heart infusion broth (BHI) supplemented with 5% sucrose and Congo red. The medium was composed of BHI (37 gms/L), sucrose (50 gms/L), agar no.1 (10 gms/L) and Congo red stain (0.8 gms/L). Congo red was prepared as concentrated aqueous solution and autoclaved at 121°C for 15 minutes, separately from other medium constituents and was then added when the agar had cooled to 55°C. Plates were inoculated and incubated aerobically for 24 to 48 hours at 37°C.
A positive result was indicated by black colonies with a dry crystalline consistency. Weak slime producers usually remained pink; though occasional darkening at the centres of colonies was observed. A darkening of the colonies with the absence of a dry crystalline colonial morphology indicated an indeterminate result. The experiment was performed in triplicate and repeated three times.
Biofilm positive by any one these two methods were taken as positive for biofilm production.
The following international reference strains were used as controls: the biofilm producers S. epidermidis ATCC 35984 (positive control) and the non-biofilm producers S. epidermidis ATCC 12228 (negative control).
The statistical analysis was done by taking percentage and simple ratios.