MATERIALS AND METHODS

Bacterial isolates and growth condition: A total of 29 pathogenic Leptospira isolates were obtained from Microbiology Laboratory, Department of Molecular Biology, Faculty of Resource Science and Technology, Universiti Malaysia Sarawak. The details of the isolates are tabulated in Table 1. Leptospira noguchii and L. interrogans were used as the positive controls. All the cultures were maintained at room temperature in modified semisolid Ellinghausen-McCullough-Johnson-Harris (EMJH) broth with 100 μg mL^–1 5-fluorouracil.

Preparation of starting bacterial suspension: Mid-exponential culture was prepared by incubating the stock culture in modified semisolid EMJH broth for 1 week. It was then standardized to optical density at 420 nm of approximately 0.3-0.4 using spectrophotometer (Metertech Inc.) which corresponded to about 10⁸ bacteria mL^–1. A starting bacterial suspension inoculum of 10⁶ bacteria mL^–1 was then prepared from this mid-exponential culture¹⁵.

Microtitre plate biofilm assay: Biofilm assay was conducted in triplicate in 24-well microtitre plates (tissue culture treated, flat-bottom wells; TPP). The EMJH broth was used as the negative control. The procedure to conduct microtitre plate biofilm assay was adapted from Ristow et al.⁹ and Lee et al.¹⁶.

Briefly, liquid culture was removed every day for 10 days with time interval of 24 h. The wells were rinsed gently with distilled water to remove non-adherent planktonic cell. They were then allowed to air dry for 15 min before fixation using 2% sodium acetate. The sodium acetate solution was removed and allowed to air dry again. Cells were stained using 900 μL of 1% crystal violet solution for 20 min after which the crystal violet solution was removed. Cells were then rinsed three times with distilled water. Finally the crystal violet remained in the wells was dissolved in 1 mL of ethanol/acetone (80/20 v/v solution) prior to optical density measurement at 600 nm (OD₆₀₀). To correct the background staining of crystal violet, the mean optical density at 600 nm of the negative control was subtracted from the mean optical density at 600 nm of the biofilm formation by pathogenic Leptospira. The results were presented in mean±standard deviation.

Classification of biofilm forming ability by isolates: The ability of Leptospira to form biofilm was determined as described by Stepanovic et al.¹⁷, Marinho et al.⁷ and Saxena et al.¹⁸. The biofilm forming abilities of the 29 pathogenic Leptospira isolates and positive controls on each day were classified into four categories based on the optical density obtained. The optical density cut-off value (OD_c) is defined as three standard deviations above the mean optical density (OD) of the negative control.

Table 1:	Sample ID and sources for 29 pathogenic Leptospira isolates examined in this study

These four categories are non-adherent when OD<OD_c, weakly adherent when OD_c<OD<2×OD_c, moderately adherent when 2×OD_c<OD<4×OD_c and strongly adherent when 4×OD_c<OD.

Statistical analysis: Statistical analysis was performed using SPSS version 20.0 software (IBM Corporation, NY, USA). Differences between means for the variables were evaluated using repeated measures ANOVA. Post hoc Bonferroni test was used to compare the biofilm OD₆₀₀ mean values. The level of significance was set at p<0.05.

RESULTS

In this study, biofilm formation using microtitre plate biofilm assay for all the tested Leptospira isolates over 10 incubation days are illustrated in Fig. 1, 2 and 3. To ensure reliability and reproducibility of data the assays were conducted in triplicates. Statistical analysis indicated that there was a significant difference in the effect of time on the formation of biofilm by these bacteria (p<0.05). These bacteria were also found to be statistically significant different among themselves in producing biofilm (p<0.05).

Based on Fig. 1, 2 and 3, it was observed that all the tested Leptospira isolates showed similar pattern on biofilm growth. On 1st and 2nd day, the amount of biofilm formed as shown by OD₆₀₀, was still low. The amount starts to increase from 3rd day which indicated the increase in the number of adherent bacteria. In average, highest biofilm OD₆₀₀ mean values was obtained either on 7th or 8th day, varies among the bacteria. After achieving the highest point, there was a decrease in the biofilm OD₆₀₀ mean values. The number of tested bacteria with different biofilm forming abilities on each day is represented in Table 2. Among the 29 pathogenic Leptospira isolates and 2 positive controls examined, 32.26% each was classified either as non-adherent or weakly adherent biofilm producers on 1st day. Majority of them produced moderately and strongly adherent biofilms from 2nd to 5th day. All of them achieved strongly adherent capabilities starting on the 6th day and remained to adhere strongly until the 10th day.

Biofilm production assessed using microtitre plate biofilm assay revealed 100% strong biofilm producers in this study as shown by the result from 6th day. Among the rat isolates, strongest biofilm producer is P38 with OD₆₀₀ at 16.700±0.265 on 8th day. Among the 14 soil samples examined, P18 is the strongest biofilm producer with OD₆₀₀ at 21.760±0.332 on 7th day.

Fig. 1:	Biofilm quantification for 9 Leptospira noguchii isolates which were all isolated from soil samples and positive control L. noguchii. Error bars represented the standard deviation derived from triplicate measurements

Fig. 2:

Biofilm quantification for 6 Leptospira noguchii isolates (P38, P39, P40, P20, P22 and P26), 4 Leptospira borgpetersenii isolates (P3, P34, P35 and P7) and negative control. The P38, P39 and P40 were isolated from rats, P20, P22, P26 and P7 were isolated from water samples, P3, P34 and P35 were isolated from soil samples. Error bars represented the standard deviation derived from triplicate measurements

Fig. 3:

Biofilm quantification for positive control Leptospira interrogans, 7 L. interrogans isolates (P4, P5, P9, P11, P28, P30 and P31), 2 Leptospira weilii isolates (P12 and P24) and 1 Leptospira santarosai (P29). The P4 and P5 were isolated from rats, P9, P11, P28, P30, P31 and P12 were isolated from water samples, P24 and P29 were isolated from soil samples. Error bars represented the standard deviation derived from triplicate measurements

Table 2:	Number of tested bacteria with different biofilm forming abilities on different day

For water samples, P22 is known to be the strongest biofilm producer with OD₆₀₀ at 19.793±0.144 on the 7th day. Post hoc Bonferroni test implied that these three isolates (P18, P22 and P38) were statistically different from each other and positive controls in biofilm formation when pair wise comparisons were made (p<0.05).

DISCUSSION

Biofilm formation by bacteria is usually characterized by distinct stages of attachment (reversible and irreversible), growth and detachment¹. In this study, pathogenic Leptospira isolates were found to develop biofilm through different stages of process mimics to other bacteria. Result indicated that biomass was formed at the bottom of 24-well microtitre plates for all the 29 pathogenic Leptospira isolates and positive controls. The process started when Leptospira cells reaching and binding to the surface of 24-well microtitre plates. Following the cell adhesion, they developed surface-sensing responses and adhered to the conditioning film that trapped the surrounding nutrients and fluids^16,1.

From 1-6 days, the attached cells formed microcolonies and they were held together by the presence of exopolysaccharide matrix. This matrix contained extracellular DNA, proteins and dead cell debris which protected the cells from stressful environmental conditions¹⁹. These microcolonies expanded and multiplied until they grew into mature state as macrocolonies with three dimensional structures on the 7th and 8th day.

After maturation, bacteria inside the macrocolonies dispersed and either survived as planktonic cultures or start to die. As a result, the process of biofilm formation by pathogenic Leptospira in this study could be considered as a dynamic and genetically regulated process. This was in accordance with previous study by Hu et al.²⁰ who observed that different bacteria species produce biofilm through similar process and hence suggested that biofilm formation is a genetically regulated process. A similar observation was noted by Sun et al.²¹ that biofilm formation of Staphylococcus aureus is a dynamic process.

Determination of biofilm formation has been assessed using different methods but no standardized protocol has been established to determine the biofilm forming ability of different bacteria species. For example, bacteria film lining a culture tube is often stained with a cationic dye and visually scaled in tube test method. This method is qualitative and therefore, subjective in result interpretation¹⁷. In this study, microtiter plate assay was chosen, in which the optical density of the stained Leptospira biofilm was determined spectrophotometrically.

Although microtiter plate assay suffers from major drawback of biofilm detection on the bottom of well only, this assay is considered most frequently used method in biofilm quantification^22,18. The optical density measurement obtained from microtiter plate assay eliminates the subjectivity of tube test in interpreting the obtained results and predicts clinical relevance more reliably than tube test¹⁷. Besides, microtiter plate assay is easy to conduct when only application of different dyes such as crystal violet used in this study, resazurin or dimethyl methylene blue enables the quantitative biofilm measurement²³.

In this study, the quantity of biofilm as shown by crystal violet staining increased in a time-dependent manner to reach maximal OD₆₀₀ mean values of approximately 12 on the 7th and 8th day. This value is in contrast to the study by Ristow et al.⁹ who reported the maximal OD₆₀₀ mean values of approximately 2 following crystal violet staining after 2 days incubation for Leptospira biflexa, a saprophytic.

At highest biofilm cell saturation (7th and 8th day), the absorbance value for pathogenic Leptospira was approximately 6 times higher than that of saprophytic Leptospira⁹. Therefore, it was inferred that pathogenic Leptospira produced considerable higher biofilm formation than saprophytic Leptospira. In an earlier study by Brihuega et al.²⁴ to assess the biofilm formation of swine isolate of L. interrogans serovar Pomona and L. biflexa serovar Patoc, pathogenic strain was found to develop a thicker layer of biofilm compared to the saprophytic strain on 5th day. Besides, the coexistence of L. interrogans with Azospirillum brasilense cells formed a dense layer of biofilm, where they reached maximum cell adherence on glass slides on 2nd day²⁵.

Another reason which supported the different abilities of pathogenic and saprophytic Leptospira in biofilm formation is the discovery of some colonization factors that have been identified in pathogenic Leptospira but not in saprophytic Leptospira. This may be attributed to the different adhesins which contribute to the initial infection. It was reported that a 36 kDa fibronectin-binding protein isolated from the outer sheath of L. interrogans serovar Icterohaemorrhagiae is known to be present only in pathogenic strains^12,26.

It was noticed that OD₆₀₀ mean values for pathogenic Leptospira isolates dropped either on the 8th and 9th day. Previous study by Charyeva et al.²⁷ revealed that the biofilm produced by Staphylococcus epidermidis decreased between 3 and 7 days. In a study by Ristow et al.⁹, they stated that OD₆₀₀ mean values for saprophytic Leptospira isolates decreased on the 3rd day. Thus, this indicated that pathogenic Leptospira could sustain the ability to produce biofilm better than saprophytic Leptospira. The ability to sustain biofilm could have implication in the infection by Leptospira strains. After exposure of mucous membrane or abraded skin, pathogenic Leptospira quickly establish systemic infection by crossing tissue barriers and blood invasion. They adhere to extracellular matrix components such as Collagen type I, type IV, laminin and fibronectin²⁸. Atzingen et al.²⁹ stated that attachment to extracellular matrix correlates with virulence, since the attachment to extracellular matrix is more effective in pathogenic Leptospira than intermediate and saprophytic Leptospira.

The variability in the quantity of biofilm production is common as different isolates from pathogenic Leptospira produced biofilm at different adherent capabilities as shown in this study. However, based on classification as used by Stepanovic et al.¹⁷, the isolates are all considered to be strong biofilm producers which form strong biofilm on microtitre plates. The strong biofilm production in pathogenic Leptospira is a key factor for survival in diverse environment. This will subsequently contributed to the pathogenesis of Leptospira indirectly since leptospirosis is commonly caused by pathogenic Leptospira. This is in agreement to Hu et al.²⁰ who mentioned that biofilm formation by bacteria is important in the pathogenesis of many bacterial infections. More than 60% of all the human bacterial infections have been attributed to the persistence of biofilm formation by the respective bacteria³⁰. Bacteria pathogens may rely on the development of biofilm to aid in host establishment, population expansion and most importantly in disease proliferation³¹.

CONCLUSION

The microtitre plate biofilm assay, a simple method for biofilm quantification has been successfully used in this study to assess the biofilm forming abilities among the pathogenic Leptospira isolates. Overall, the 29 Leptospira isolates examined in this study possess the ability to produce strong biofilm, however, the amount of biofilm formed was different from isolate to isolate. The role of biofilm in the pathogenesis of human infection cannot be denied. Biofilm formation can lead to serious implications in medical field, public health, industry and environment. Future study on the identification of genes involved in biofilm formation by pathogenic Leptospira is of utmost important to plan for further prevention approaches against leptospirosis outbreak.

ACKNOWLEDGMENT

This study was funded by Ministry of Higher Education Malaysia under Fundamental Research Grant Scheme FRGS/STO3(02)/1209/2014(10).