Urinary tract infections (UTIs) account for about 25-40% of nosocomial infections, out of which about 90% are catheter-associated (Trautner and Darouiche, 2004; Ghanwate et al., 2012). The daily incidence of bacteriuria in catheterized patients is approximately 3–10% and among patients with bacteriuria, up to 25% will develop symptoms of local UTI, while about 3% will develop bacteremia (Saint et al., 2005). Studies have also shown that the duration of catheterization is the important  factor

of bacteriuria (Stickler, 2008; Al-Mathkhury et al., 2011).

Several strategies have been attempted to control urinary catheter biofilms including application of antimicrobial ointments and lubricants, bladder instillation or irrigation, antimicrobial agents in the collection bags, impregnation of catheter with antimicrobial agent such as silver oxide or use of systemic antibiotics, but none of these strategies proved very effective (Donlan, 2001; Trautner   and     Darouiche,    2004).    Currently,    novel strategies are being attempted by researchers, including coating of catheters with enzymes, EDTA, biosurfactants, probiotics and other non-pathogenic organisms (Darouiche et al., 2001; Trautner et al., 2007; Borchert et al., 2008; Fracchia et al., 2010; Ghanwate et al., 2012; Sambanthamoorthy et al., 2014).

Collection of samples

Sterile Foley silicone catheters were purchased from the market; urine specimens were collected aseptically from outpatients attending a local hospital and from apparently healthy individuals for the isolation of urine bacteria; and high vaginal swab (HVS) specimens were collected from an apparently healthy female for isolation of Lactobacillus spp.

Isolation and identification of organisms

In order to isolate urine bacteria, loopfuls of the urine samples were cultured on MacConkey and Blood agar and incubated for 24 h at 37°C. Resultant colonies were sub-cultured onto fresh culture plates of the same media  to  obtain  pure  cultures.  Then,  isolates were characterized and identified, using standard microbiological procedures as described by Cheesebrough et al. (2000).

For Lactobacillus spp., the vaginal swab specimens were inoculated onto De Man Rogosa Sharpe (MRS) agar (Oxoid). The plates were incubated at 37°C in an anaerobic jar, for 24 to 48 h. Thereafter, the creamy-white colonies, suspected to be Lactobacillus, were sub-cultured on MRS medium to obtain pure cultures. The isolates were further characterized and identified by biochemical analyses as described by Cheesebrough et al. (2000).

Evaluation of growth of Lactobacillus in urine

Ten milliliters of urine was filter-sterilized to make the urine free from bacteria. The sterile urine was then used as a culture medium for Lactobacillus cells using an inoculum size of 10⁶ cfu. The culture was incubated at 37°C in an anaerobic jar. Viable cell counts were taken from triplicate samples, each day, for seven days, to evaluate the survival and growth of the cells in urine.

Evaluation of anti-biofilm effect of Lactobacillus using cover-slip assay

The ability of Lactobacillus cells to inhibit biofilm formation by urine bacteria was evaluated qualitatively by cover-slip assay by a modification of the method of Ghanwate et al. (2012). Sterile Petri dishes were filled each with 10 ml of MRS broth containing 1% sucrose. Then, sterile glass cover-slips were added to each Petri dish. Thereafter, each plate was inoculated with 0.1 ml of overnight culture of L. acidophilus and incubated anaerobically at 37°C for 24 h to coat the cover-slips with Lactobacillus. The next day, the Lactobacillus-coated cover-slips were extracted and introduced into culture dishes containing 10 ml broth-culture of different urine isolates (S. aureus, E. coli, Klebsiella sp. and P. aeruginosa) to produce co-cultures of the Lactobacillus and urine organisms. The dishes were then incubated aerobically at 37°C. On daily basis, a cover-slip from each culture set-up was removed, unattached cells were rinsed off with phosphate buffered saline and attached cells or biofilms were stained with 0.1% crystal violet for 5 min (for S. aureus) and Gram staining for E. coli, Klebsiella sp. and P. areuginosa. Finally, stained biofilms were observed microscopically and photographed using a 0.2 Mega pixel Motic camera. The co-cultures were monitored over a seven-day period.

Immobilization of Lactobacillus acidophilus on catheter pieces

MRS broth containing L. acidophilus cells was incubated for 48 h at 37°C. The broth was then centrifuged at 5000 rpm for 15 min. The supernatant was discarded while the pellet of cells was added into a beaker containing exactly 2% (w/v) sodium alginate solution. The catheter pieces were then introduced into the mixture and allowed to stand for 1 h. Thereafter, the catheter pieces were extracted and immersed in a beaker containing 2% CaCl₂.2H₂O to allow the formation of a gel. The set-up was incubated for 24 h for stability of the gel.

Evaluation of anti-biofilm capability of immobilized Lactobacillus cells

The catheter pieces containing immobilized L. acidophilus cells were immersed in broth cultures of the different urine isolates in separate Bijoux bottles. The bottles were allowed to stand for 7 days at 37°C. Untreated catheter sections also immersed in broth cultures of urine isolates served as control. Biofilm formation on the catheter sections was evaluated by viable cell count procedures. On each day of counting, two catheter pieces were picked 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 phosphate buffered saline. The cells in the biofilm were dispersed using a magnetic stirrer. Serial dilutions were prepared and the cells inoculated onto MacConkey and blood agar plates for viable counts.

Isolation of bacteria from urine and HVS

Four bacteria were isolated from the urine specimens. These were: S. aureus, E. coli, Klebsiella sp. and P. aeruginosa. All four organisms were used in the biofilm studies. Two Lactobacillus species were identified from HVS, namely: L. acidophilus and Lactobacillus plantarum. The L. acidophilus was used for all the anti-biofilm studies.

Growth of Lactobacillus in urine

L. acidophilus cells were able to survive and multiply in urine, increasing in number from 6.5 x 10⁷ to 1.5 x 10⁸ cfu/ml by the fifth day of culture, after which there was a decrease in number (Figure 1).

Inhibition of urine bacteria by Lactobacillus cells on coverslips

Evaluation of the inhibitory activity of Lactobacillus cells against bacterial biofilms on coverslips showed that pre-coating of coverslip surfaces with Lactobacillus cells prior to exposure of the surfaces to urine bacteria reduced the number of urine bacterial cells attaching to the coverslips. For E. coli, cell numbers reduced relative to number of Lactobacillus cells from the third day of culture, while there was complete inhibition by the sixth day (Figure 2).

Similarly, numbers of attached cells of Klebsiella and S. aureus reduced from the third day and complete inhibition was  achieved  by  the seventh day (Figures 3 and 4). For P. aeruginosa, on the other hand, reduction in number was observed only from the fifth day and attached cells were still observed by the end of the experiment on the seventh day (Figure 5).

Inhibition of catheter biofilms by immobilized Lactobacillus cells

Evaluation of attachment and growth of urine organisms on catheter pieces containing immobilized Lactobacillus cells was compared with growth on uncoated catheter pieces. The results showed significant (p<0.05)  reduction in number of S. aureus and Klebsiella cells on the coated catheter pieces when compared with the uncoated catheters. E. coli and P. aeruginosa, on the other hand, showed no reduction. Representative graphs of the growth of S. aureus and P. aeruginosa are shown in Figures 6 and 7, respectively.

Inhibitory activity of crude bacteriocin of L. acidophilus against test isolates

The crude bacteriocin preparation from the Lactobacillus isolate  was   tested   for   its   activity   against  the  urine aisolates. The results showed that the highest activity was against S. aureus, followed by Klebsiella before E. coli (Figure 8).

The human normal bacterial flora is increasingly recog-nized as an important defense to infection. Lactobacilli in particular, are regarded to be generally safe and recognized as potent interfering bacteria. Lactobacilli and their derived substances have been used widely in both clinical and experimental trials to  inhibit  growth  of  other bacteria on various natural and artificial surfaces, including urinary catheters (Reid and Tieszer, 1994; Reid and Burton, 2002; Reid and Bruce, 2006; Maldonando et al., 2007; Ruiz et al., 2009; Barrons and Tassone, 2008; Fracchia et al., 2010; Ray, 2011; Al-Mathkhury et al., 2011; Sambanthamoorthy et al., 2014; Abd-Alkareem, 2014).

In this study, L. acidophilus was isolated from HVS of a healthy female. The Lactobacillus cells showed appreciable growth in urine for up to 5 days in culture, after which there was a decline in growth. The decline in growth  after  five  days  could   be   attributed   to  culture conditions, in which the cells had to survive in what could be described as stagnant urine.

Evaluation    of    the    anti-biofilm    potential    of    the Lactobacillus cells by cover-slip assay showed that pre-coating of the surfaces with Lactobacillus reduced the attachment  and  growth  of urine bacteria. The effect was more pronounced against E. coli, Klebsiella and Staphylococcus, with near complete inhibition being achieved by the fifth day of co-culture. For P. aeruginosa, on the other hand, clearing required longer than seven days. The inhibitory effect observed in the cover-slip assay was confirmed quantitatively using viable cell count procedures following immobilization of Lactobacillus cells on catheter surfaces and exposure to urine bacteria. In the   quantitative   assay,  however,  more  inhibition  was recorded with S. aureus and Klebsiella than with E. coli and Pseudomonas.

The results in this study agree with those of Maldonado et al. (2007), in which whole cells and acid supernatant of Lactobacillus fermentum inhibited biofilm formation and growth of Klebsiella. Westbroek et al. (2010) reported that Streptococcus pyogenes was not inhibited by Lactobacillus crispatus and Lactobacillus jensenii. In Westbroek’s    (2010)    study,    the    Lactobacillus    and Steptococcus cells were mixed before plating. It appears therefore that the inhibitory action requires pre-application of the Lactobacillus or its products to the surface before exposure   to   the   biofilm-forming   organism,    as   was demonstrated in the study by Fracchia et al. (2010). Similar results were also reported by Hall et al. (2000) and Darouiche et al. (2001), working with benign E. coli strains. In their studies, instillation of the benign E. coli strains into the bladders of patients with recurrent UTI and coating of urinary catheters with benign E. coli, respectively, significantly reduced the occurrence of UTI in the patients.