Study design

First: A cross-sectional study to determine the bacteriological profile of CAUTIs

Second: An experimental study to evaluate the role of LAB in preventing urinary catheters’ colonization and BF

Sample size

Based on a previous study, 8.2% of hospitalized patients had a CAUTI [10], using a margin of error of 5%, alpha error = 0.05, and the minimum required sample size is 116 subjects. The sample size was calculated using Epi Info 7 software.

The study was conducted over 7 months from January to July 2019. It involved 120 urine samples randomly collected from adults with indwelling Foley urinary catheters, who were admitted to the Urology Department at the Alexandria Main University Hospital (AMUH), Egypt.

Sample collection

The catheter tubing distal to the puncture site was clamped for 15–30 min to allow urine to fill the tubing. Then, the catheter port was disinfected using 70.0% ethanol. Urine samples were obtained by inserting sterile syringes aseptically into the catheter port and then transferred into labeled screw-capped sterile containers [11]. All the collected samples were transported within 2 h in an ice box to the Microbiology Laboratory of the High Institute of Public Health (HIPH) for processing.

Sample processingIsolation and antimicrobial susceptibility testing of uropathogens

A standard loop was inserted into the well-mixed urine and then spread over the surface of each of the blood, MacConkey and Sabouraud’s dextrose agar (SDA) plates. Blood and MacConkey agar plates were incubated at 37 °C for 24 h, while SDA plates were incubated at 25 °C for 48 h and up to 10 days [12]. The Quebec Colony Counter was used for counting the colonies. Plates yielded ≥ 105 CFU/mL of one or maximally two organisms were considered positive for a UTI [13].

After incubation, the identification of isolated colonies was performed according to standard microbiological methods [12]. All bacterial isolates were subjected to antibiotic susceptibility testing by the disk diffusion method, [14] according to the Clinical and Laboratory Standards Institute recommendations [15].

Microtiter plate method for the detection of BFA

The biofilm-forming ability (BFA) of the identified uropathogens was tested by the MtP method according to Stepanović et al [16]. The experiments were performed in triplicate, and the results were averaged. Three colonies of each uropathogen (isolated from an overnight culture on Mueller-Hinton agar plates) were inoculated in 3 mL of tryptic soy broth with 1% glucose (TSBglu) and incubated for 24 h at 37 °C. The cultures were adjusted to a turbidity of 0.5-McFarland standards using phosphate-buffered saline (PBS) and diluted 1 in 100 with fresh TSBglu medium. Individual wells of sterile, 96-well MtP, were filled with 200 μL of the diluted cultures; negative control wells contained uninoculated sterile broth only. After incubation (24 h at 37 °C), the contents of each well were removed by gentle tapping, and the wells were washed three times with 300 μL of sterile PBS. The plates were drained in an inverted position. The biofilms formed by bacteria adherent to the wells were heat fixed by exposure to hot air at 60 °C for 60 min and then stained with 150-μL crystal violet (2% w/v) for 15 min. Excess staining was removed by using running tap water, and the plates were kept for drying.

The optical density (OD) of each well was obtained using a microplate enzyme-linked immunosorbent assay reader (Dialab ELX800G, Vienna, Austria) at wavelength 630 nm. The experiment was performed in triplicate, and the results were averaged. The cutoff OD (ODc) was calculated as three standard deviations above the mean OD of the negative control. The isolates were classified as follows:

OD ≤ ODc — non-biofilm former

ODc ˂ OD ≤ 2 × ODc — weak biofilm former

2 × ODc ˂ OD ≤ 4 × ODc — moderate biofilm maker

4 × ODc ˂ OD — strong biofilm former

Immobilization of probiotic strains on catheter pieces [17]

Five probiotic strains (L. acidophilus, L. plantarum, L. paracasei, L. pentosus, and B. bifidum) which showed the best probiotic properties (auto-aggregation, co-aggregation, safety, and hemolytic activities) were selected from a previously conducted PhD study at the HIPH.

Five 10 mL De Man Rogosa Sharpe (MRS) broth tubes containing probiotic cells were used. Each tube was inoculated with 106 CFU of different probiotic strains (L. acidophilus, L. plantarum, L. paraquisi, L. pentosus, and B. bifidum). All tubes were incubated aerobically for 48 h at 37 °C. The broth tubes were centrifuged at 5000 rpm for 15 min. From each tube, the supernatant was discarded, while the pellet of cells was added to a beaker containing 2% (w/v) sodium alginate solution. Twelve sterile Foley catheters were purchased from the market, and each catheter was divided into 10 equal pieces. The catheter pieces were then introduced into the mixture for 1 h (24 catheter pieces in each beaker). Each of the 24 catheter pieces was extracted and immersed in a separate beaker containing 2% calcium chloride solution and incubated aerobically for 24 h to allow the formation of a gel.

From each beaker containing immobilized probiotic cells, three catheter pieces were immersed in broth cultures of each of the selected eight urine isolates (two Klebsiella pneumoniae (K. pneumoniae), two Escherichia coli (E. coli), one Proteus mirabilis (P. mirabilis), one Pseudomonas aeruginosa (P. aeruginosa), one Enterococcus spp., and one Candida spp.) in separate sterile bottles and were allowed to stand for 6 days at 37 °C. Biofilm formation on the catheter sections was evaluated by a viable cell count procedure after 2, 4, and 6 days.

On each day of counting, a catheter piece was picked up from the various cultures and rinsed with sterile distilled water to remove unattached cells. Then, the attached cells were gently scraped off from both the outer and luminal surfaces of the catheters using a wire loop and introduced into sterile beakers containing 10 ml of PBS. The cells in the biofilm were dispersed using a magnetic stirrer, and a loopful of the broth was spread on blood, MacConkey, and MRS agar plates for viable counts. Sabouraud dextrose agar was used instead of a MacConkey plate with Candida spp. Blood and MacConkey agar plates were incubated at 37 °C for 24 h, while MRS agar and SDA were incubated for 48 h (at 35 °C for MRS and 25 °C for SDA).

Colonies were counted using a Quebec Counter and expressed as follows:

No colonies/mL

Less than 50 CFU/mL

50: 100 CFU/mL

More than 100 CFU/mL

Statistical analysis

Data were analyzed using the Statistical Package for the Social Sciences software version 20. Data were presented as numbers and percentages for categorical variables and as means and standard deviations (SD) for continuous variables. All results were interpreted at a 5% level of significance.