Keywords

Biofilm, Motilities, pH, Pseudomonas, Staphylococcus, Escherichia

Introduction

Bacterial infection is mainly related to the ability of bacteria to colonize and disseminate through their hosts by using different types of motility, by building structured biofilm and by releasing a myriad of virulence factors which lead to host cell and tissue damages [1]. Biofilms are surface-associated communities enclosed within an extracellular matrix mainly composed of polysaccharides, proteins, nucleic acids, lipids and other macromolecules and chemicals [2] which protect bacteria cells against environmental assaults and predation, including antimicrobials and host defenses. As organism area present various pH at the example of along the digestive system that present pH range from 2 to 8 [3], it’s important to understand the influence of environment pH that promote the expression of these bacterial behaviors particularly biofilm formation and motilities. Indeed, comprehension of these behaviors is important to elaborate adequate strategy in order to struggle against bacterial infection or to exploit their biotechnological potential. The present study evaluate the abilities of Escherichia coli ATCC® 25922, Staphylococcus aureus ATCC® 25923 and P. aeruginosa ATCC® 27853 strains to move and to develop biofilm under complex and minimum media and different pH condition from 5 to 8.

Materials and methods

Bacterial strains and culture conditions

Reference strains Escherichia coli ATCC® 25922, Staphylococcus aureus ATCC® 25923 and P. aeruginosa ATCC® 27853 were purchased from OXOID and grown in LB-MOPS broth.

Biofilm Formation and Quantification

The formation of biofilm was studied on 96-well micro-plates and quantified by a crystal violet staining protocol as previously described [4]. Briefly, an overnight culture of Escherichia coli ATCC® 25922, Staphylococcus aureus ATCC® 25923 or P. aeruginosa ATCC® 27853 was washed twice and diluted in fresh biofilm broth (BB) medium (Na2HPO4, 1.25 g/l; FeSO4.7H2O 0.0005 g/l; glucose, 0.05 g/l; (NH4)2SO4, 0.1 g/l; MgSO4.7H2O, 0.2 g/l and KH2PO4, 0.5 g/l) or LB-MOPS broth medium. Experiments were realized using BB or LB at four different pH (5, 6, 7 and 8) in 96-well polystyrene plates, 50 μl of the diluted culture (A600 between 0.14 and 0.16) was added to 150 μl of BB medium or LB broth. Escherichia coli ATCC® 25922, Staphylococcus aureus ATCC® 25923 and P. aeruginosa ATCC® 27853 cultures were incubated statically for 24 h at 37°C and biofilm quantification was performed by measuring the formed biofilm stained by crystal violet at A590nm with a spectrophotometer BK-UV1000 device (Biobase, China).

Motility Assays

Swimming, swarming and twitching motilities were examined by using LB agar at different pH 5, 6, 7 and 8 (0.3%, 0.6% and 1%, respectively) as described by Rasamiravaka et al. [4] and Ha et al. [5]. After sterilization and cooling (45-50°C) of LB agar, five μl of bacterial culture (A600 = 1) were inoculated at the center of each compartment of the Petri dishes and incubated at 37°C for 24 (for swimming and swarming) or 48 h (for twitching motility). Bacteria spreading from the inoculation spot were measured with sliding caliper after adequate incubation time. The inoculation spot is generally around 6mm and spreading less than 8mm was considered as non-motile mechanism.

Statistics

All experiments were performed in triplicate and repeated in 3 independent assays. The data were statistically analyzed by conducting the one-way ANOVA with Tukey’s multiple comparison tests and a P-value < 0.01 was considered as being significant

Results

Biofilm formation by Staphylococcus aureus is not influenced by variation of acidity medium. Indeed, there is no significant difference between biofilm formed in pH5, 6, 7 and 8 (Figure 1). Moreover, S. aureus form biofilm in both minimum and complex media. In regard of motilities, S. aureus was unable to spread in all BB medium agar and unable to swim and swarm in complex medium (Figure 1). However, S. aureus was able to twitch a under pH7 and 8.

Image 1

Figure 1: Effect of pH variation (from 5 to 8) and growth medium (complex and minimum media) on biofilm development and motilities by Staphylococcus aureus ATCC® 25923. (A) Biofilm formation by S. aureus grown under different pH (from 5 to 8) and medium. (B) Swimming motility of S. aureus grown under different pH (from 5 to 8) and medium. (C) Swarming motility of S. aureus grown under different pH (from 5 to 8) and medium. (D) Twitching motility of S. aureus grown under different pH (from 5 to 8) and medium. All experiments were performed in triplicate with three independent assays. Different letters (A or a and B or b) above the bars indicate data that are statistically different from each other according to the one-way ANOVA with Tukey’s multiple comparison test (p ≤ 0.01).

Biofilm formation by Escherichia coli is similar to S. aureus; indeed, Escherichia coli form biofilm at all tested pH when grown in complex and minimum media (Figure 2). However, they develop more consistent biofilm when grown in minimum media. In regard of motilities, Escherichia coli was unable to spread in all BB medium agar and unable to swim in complex medium (Figure 2). However, a slight swarming (10mm) which is not influenced by pH variation is observed in complex medium and interestingly, Escherichia coli twitch more under acidic pH than neutral and alkaline pH condition.

Image 2

Figure 2: Effect of pH variation (from 5 to 8) and growth medium (complex and minimum media) on biofilm development and motilities by Escherichia coli ATCC® 25922. (A) Biofilm formation by E. coli grown under different pH (from 5 to 8) and medium. (B) Swimming motility of E. coli grown under different pH (from 5 to 8) and medium. (C) Swarming motility of E. coli grown under different pH (from 5 to 8) and medium. (D) Twitching motility of E. coli grown under different pH (from 5 to 8) and medium. All experiments were performed in triplicate with three independent assays. Different letters (A or a and B or b) above the bars indicate data that are statistically different from each other according to the one-way ANOVA with Tukey’s multiple comparison test (p ≤ 0.01).

Biofilm formation by P. aeruginosa is influence by acidity of medium. Indeed, pH7 represent the optimum acidity for biofilm formation as in acidic environment (pH5 and 6) P. aeruginosa fail to form biofilm under both complex and minimum media. At pH8 P. aeruginosa form biofilm but significantly reduced compared to that of biofilm biomass at pH7 (Figure 3). Interestingly, swimming, swarming and twitching motilities are not affected by pH variation in complex medium suggesting that regulation of flagellar movement and pili retraction are not influenced by pH variation, whereas P. aeruginosa was not able to spread in BB medium agar (Figure 3).

Image 3

Figure 3: Effect of pH variation (from 5 to 8) and growth medium (complex and minimum media) on biofilm development and motilities by Pseudomonas aeruginosa ATCC® 27853. (A) Biofilm formation by P. aeruginosa grown under different pH (from 5 to 8) and medium. (B) Swimming motility of P. aeruginosa grown under different pH (from 5 to 8) and medium. (C) Swarming motility of P. aeruginosa grown under different pH (from 5 to 8) and medium. (D) Twitching motility of P. aeruginosa grown under different pH (from 5 to 8) and medium. All experiments were performed in triplicate with three independent assays. Different letters (A or a and B or b) above the bars indicate data that are statistically different from each other according to the one-way ANOVA with Tukey’s multiple comparison test (p ≤ 0.01).

Discussion

This study indicates that the pH value will affect biofilm and motility behaviors differently, depending upon the tested organism.

S. aureus and E. coli biofilm formation seems to be not influenced by pH variation suggesting that the mechanism regulating biofilm formation in both strains is not pH dependent. As a non-motile bacterium, S. aureus was expected to not able to swim and swarm. However, the presence of twitching motilities is intriguing. The twitching motilities observed in pH 7 and 8 may be not linked to a twitching mechanism but rather sliding motilities. Indeed, S. aureus are able to slide which is sliding is a kind of surface translocation produced by the expansive forces in a growing culture in combination with special surface properties [6]. The sliding is a passive motility and the spreading depends on bacterial growth and QS-regulated surfactant phenol soluble modulin (PSM) so it’s suggested that S. aureus grown and produce well the surfactant in pH 7 and 8 condition. E. coli twitch well particularly under acidic condition suggesting the full activation of pili at this condition. As E. coli do not swim and slightly swarm the flagella in not activate under LB medium.

For P. aeruginosa Biofilm formation is influenced by pH variation, further studies should be addressed which of the 5 steps for biofilm formation is really pH dependent. Moreover, influence of pH variation should be also investigated for mechanisms considered to regulate biofilm formation such as quorum sensing, two component system GacS/GacA and the c-di-GMP pool concentration. One point that is already clear is that swarming and twitching motilities, which are important for biofilm formation [7] seems not to be affected by pH variation. Finally, BB medium are not probably adequate for motilities testing of P. aeruginosa, E. coli and S. aureus strains as well as LB medium is not appropriate for testing swimming an swarming of E. coli and S. aureus strains. Thus, use of other medium should be investigated to evaluate which components are really necessary for biofilm formation.

Conclusion

This preliminary study demonstrated that pH may influence bacterial behavior formation particularly for Pseudomonas aeruginosa. Further investigation is necessary to shed light the mechanism of this phenomenon.