Antagonistic effect of bacteriocin against urinary catheter associated Pseudomonas aeruginosa biofilm.

CONTEXT: Pseudomonas aeruginosa is a gram negative opportunistic bacteria causes several infections commonly colonize these devices and developing biofilms. Bacteria in biofilm can be up to 1,000 times more resistant to antibiotics than the same bacteria circulating in a planktonic state. CASE REPORT: A total of 10 isolates of Pseudomonas aeruginosa were isolated from catheter associated urinary tract infections. While carbenicillin was the most effective antibiotic, all isolates developed multidrug resistant. Both crude and purified bacteriocin showed marked inhibition activity against planktonic and biofilm of the highly resistant isolate P. aeruginosa P7.
CONCLUSION: Bacteriocin extracted from a locally isolated L. acidophilus has an anti P. aeruginosa biofilm activity also it can be used as a therapeutic agent after adequate in vivo experimentation.

Introduction

A biofilm is a thin layer of micro-organisms that adhere to the surface of an organic or inorganic structure, together with their secreted polymers. Biofilms are the predominant phenotype of nearly all bacteria in their natural habitat, whether pathogenic or environmental [1]. Indeed, bacteria in a biofilm environment can be up to 1,000 times more resistant to antibiotics than the same bacteria circulating in a planktonic state [2].

Urinary catheters are tubular latex or silicone devices, which when inserted may readily acquire biofilms on the inner or outer surfaces. The longer the urinary catheter remains in place, the greater the tendency of these organisms to develop biofilms and result in urinary tract infections [3]. Pseudomonas aeruginosa is a gram negative opportunistic bacteria causes several infections commonly colonize these devices and developing biofilms [4]. This study attempts to evaluate the impact influence of bacteriocin extracted from Lactobacillus acidophilus on biofilm of P. aeruginosa formed on Foley catheter.

Materials and Methods

Isolation and Identification

Ten isolates of P. aeruginosa were isolated from catheterized patients presented with urinary tract infections. After withdrawal of catheter, it was cut into pieces of 1 cm in length, rinsed in phosphate buffered saline then cultivated in 5 ml of Brain Heart Infusion broth (BHIB) at 37°C for 24 hr. Identification was done according to protocol illustrated by Holt et al [5].

In order to isolate L. acidophilus, one ml sample was taken from yoghurt and cultured by spreading on MRS agar (Hi-Media, India. pH 5.5). The plates were incubated at 37°C in anaerobic jar for 24-48 hour. Thereafter, smooth convex whitish to creamy colonies were isolated, sub-cultured on MRS agar medium and incubated for 24-48 hour [6]. The lactobacilli were initially identified by their ability to grow on the selective MRSA, gram-positive staining, rod shape, and catalase-negative phenotype. Biochemical analyses, including sugar fermentation profile and gas production in MRS broth [ Hi-Media, India] , were conducted as described in the second edition of Bergey's manual [7].

Antibiotic susceptibility

Antibiotic susceptibility test towards piperacillin, imipenem, Cefotaxime, Carbenicillin, Amikacin, Ceftriaxon, Ciprofloxacin, Gentamicin, Tobramycin and Trimoxazole was done according to the method of Bauer et al [8]. The isolate which resist the highest number of antibiotics was elected for further experiments.

Preparation of crude bacteriocin

An overnight culture of L. acidophilus was adjusted with MRS broth in accordance to McFarland turbidity standard tube no. 0.5 as measured by absorbance (0.08-0.1 at 625nm) using Cary 100 spectrophotometer (Varian PTY ltd., Australia) corresponding to approximately 1.5-2 × 108 cfu/ml. Afterword, the culture was propagated in the same broth at 37°C for 24 hr under anaerobic conditions. Cells were separated by centrifugation at 6000 rpm/min for 10 min at 4°C. The resulting supernatants were filtered through a 0.2-μm membrane filter. All supernatants were cultured on MRS agar in order to confirm the absence of lactobacilli cells. Thereafter, they were stored at 4°C until the assay. In parallel, Aliquots of supernatants neutralized with 1N NaOH were prepared as well [9].

Purification of bacteriocin

The bacteriocin present in the supernatant fraction was concentrated by ammonium sulphate precipitation (700 g/l). After the mixture had been stirred overnight at 4°C, the precipitate was pelleted by centrifugation at 10,000×g for 30 min. The collected precipitate was then dissolved in 0.05M sodium acetate buffer pH 5.0 and dialyzed using a 1 kDa cut-off membrane against the same buffer at 4°C overnight [10]. Inhibitory activity was assayed before and after purification. Protein concentration after each purification step was determined [11].

Inhibitory activity of crude and purified bacteriocin on planktonic P. aeruginosa P7

Well diffusion method described by Ikeagwu et al [12] was followed to detect inhibitory effect of L. acidophilus supernatants as well as purified bacteriocin on planktonic P. aeruginosa P7.

The titers of crude and purified bacteriocin were quantified by two fold serial dilutions of bacteriocin in saline solution and aliquots of 50 μl from each dilution were placed in wells in plates previously seeded with the P. aeruginosa P7. These plates were incubated aerobically at 37°C for 24 h and examined for the presence of 2 mm or larger clear zones of inhibition around the wells. The antimicrobial activity of the bacteriocin was defined as the reciprocal of the highest dilution showing inhibition of P. aeruginosa P7 and was expressed in activity units per ml (AU/ ml) [13].

Biofilm assay

The method of Jones and Versalovic [14] was followed with some modifications in order to produce biofilm on urinary catheter. Briefly: Foley catheter was cut into 1 cm pieces posted in 20 ml of BHIB containing P. aeruginosa P7 cultured in a final concentration of 1.5 × 108 cfu/ml. containers were incubated aerobically at 37°C for 24 hrs. Media and planktonic cells were removed by decantation and two washes with distilled water (DW) were done. Two hundred microliters of BHIB supplemented with bacteriocin (1/32 v/v) were added and incubated for another 24 hrs. All pieces were washed twice with DW while the adherent cells were stained with crystal violet (1% w/v) for 10 minutes. Thereafter, the catheter pieces were washed with DW. Finally, the crystal violet was redissolved with ethanol and the OD480 was determined spectrophotometrically. Hence absorbency will represent the biofilm thickness. Foley catheter pieces placed in sterile BHIB were designated as a blank. Control was prepared by cultivating Foley catheter pieces with bacterial culture free of bacteriocin. Simultaneously, viable count following the procedure described by Harley and Prescott [15] was carried out to determine the viability of bacterial cells within the biofilm. All assays were done in triplicates.

Statistical analysis

Data is presented as mean ± standard deviation. ANOVA and LSD0.05 were employed for data analysis using Microsoft office Excel 2007 application.

Discussion

A total of 10 isolates of Pseudomonas aeruginosa were isolated from catheter associated urinary tract infections. Morphologically all of these isolates were gram negative, non-sporing, capsulated, and motile bacilli, produced typical grapes like odor. They were also positive for oxidase, pyocyanin production as well as growth test at 4 and 42°C. All isolates were non glucose fermenter and succeeded in grown on cetrimide agar.

Data presented in Figure 1 showed that most isolates (9 isolates) were resistant to ceftriaxon; while carbenicillin was the most effective antibiotic. All isolates developed multidrug resistant. However, isolate P7 developed resistance against the highest number of antibiotics under investigation; therefore, it was elected for further experiments.

Fig. 1

Antibiotic resistance of Pseudomonas aeruginosa isolated from urinary catheter associated urinary tract infections.

Purification steps of bacteriocin are depicted in Table 1. The bacteriocin of L. acidophilus was recovered with an increase in specific activity from 10 to 26.6 AU/mg after precipitation by 70% saturation of the culture broth with ammonium sulphate. These results agreed with findings of Mojgani et al [13], Invanova et al [16] and Ogunbanwo et al [17]. Mojgani et al [13] reported that the increase in activity could be due to release of active monomers from bacteriocin complexes. During salt precipitation various amount of the protein was fractionated as a surface pellicle, this might be due to the association of bacteriocin molecules with the hydrophobic globular micelle like structure in the supernatant fluid.

Table 1

Purification of bacteriocin produced by Lactobacillus acidophilus

Purified bacteriocin showed significant activity against the multi drug resistant P. aeruginosa isolate over the crude bacteriocin (P< 0.05) as it shown in Table 2.

Table 2

Inhibition of multi drug resistant Pseudomonas aeruginosa P7 by Lactobacillus acidophilus bacteriocin.

Treating the biofilm formed on the Foley catheter by P. aeruginosa P7 with crude and purified bacteriocin caused a significant reduction (P < 0.05) in absorbancy (i.e. biofilm thickness) reached 0.536 ± 0.04 and 0.299 ± 0.07, respectively, in comparison to control (0.733 ± 0.13) as it diagrammed in Figure 2.

Fig. 2

Inhibitroy effect of L. acidophilus bacteriocin on P. aeruginosa P7 biofilm. P= 0.004. LSD= 0.15

On the other hand, bacteriocin of L. acidophilus significantly reduced the viable cell count in biofilm from 5.33 × 1011 to 303 and 605.33 cfu/ml after treating biofilm formed on Foley catheter with crude and purified bacteriocin, respectively.

Although the sensitivity of gram negative bacteria to bacteriocins produced by lactic acid bacteria is not common. L. acidophilus has already been reported to produce bacteriocins effective against pathogenic bacteria [18-21].

The mechanism by which bacteriocins affect bacterial growth can be explained by affection on cellular membranes instability and permeability via formation of complex or ionic canals through binding itself receiving particles such as lipids or proteins, lead to the dispersion and lose ability to form protons propelling force [22]. Westbroek et al. [23] mentioned that abundant of researches involved the remarkable ability of Lactobacilli in inhibiting pathogens growth through its bactericidal activity (such as production of bacteriocins, hydrogen peroxide and lactic acid as a byproduct of metabolism) and allow the body's immune system to overcome the infection without the use of antimicrobials. Ramos et al [23] stated that lactobacilli supernatants diminished the quorum signals (acyl-homoserine-lactones) produced by P. aeruginosa. Given that these signals are necessary for biofilm formation, hence, such diminishing of signals may cause an interruption in biofilm formation.

The present work can conclude that although P. aeruginosa biolim is hardly to be eradicated by pseudomandal antibiotics, the bacteriocin extracted from a locally isolated L. acidophilus has an anti P. aeruginosa biolim activity also it can be used as a therapeutic agent after adequate in vivo experimentation.