Abstract
Pathogenic Leptospira spp. are the causative agent of leptospirosis. Biofilm formation in leptospires is a new area of study, and its role in pathogenesis is not fully explored. As in other biofilm-forming bacteria, Leptospira biofilm may play a significant role in antibiotic resistance. In this study, the antimicrobial susceptibility of Leptospira biofilm was investigated by 96-well plate assay using Alamar Blue. Leptospira biofilm showed five to sixfold increase in resistance in all the strains used. The range of minimal bactericidal concentrations for penicillin G, ampicillin, tetracycline, and doxycycline was 1,600 U/ml, 800–1,600 μg/ml, 800–1,600 μg/ml, and 800–1,600 μg/ml, respectively. In agar substrate, the biofilm showed six- to sevenfold increase in resistance to antibiotics compared to planktonic cell. The present study emphasizes the importance of biofilm formation and its antibiotic susceptibility patterns. This could pave the way for devising appropriate strategy to prevent the occurrence of potential chronic leptospirosis in endemic areas and also during an outbreak situation.
Introduction
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Leptospira was believed to be a solitary organism until it was found to form biofilm in the environment and in vitro.5,6 Studies are indicative that pathogenic strains can colonize the host and form biofilm and cell aggregations in animals.4,7 Antibiotic treatments failed to eradicate leptospires that colonize infected mice. 4
Ample information regarding the susceptibility of Leptospira against various antimicrobial agents exists in the literature. 8 However, there is no study regarding the susceptibility of different morphological forms of Leptospira, namely aggregates and biofilm. Consideration of all morphological forms of the microbes would be necessary for a complete assessment of its antibiotic susceptibility. Hence, we hypothesize that the cell aggregation and biofilm formation may be the cause of antibiotic resistance. In the present study, we subjected the biofilm-forming leptospiral strain to antibiotics and compared the minimal bactericidal concentration (MBC) with that of its planktonic form.
Materials and Methods
Bacterial strains and biofilm formation assay
Stock strains of Leptospira spp. were recovered from the Regional Medical Research Centre (ICMR), Port Blair, India, and these are listed in Table 1. The leptospiral strains were screened for their ability to adhere or form biofilm to the walls of the wells in 96-well plates. The biofilm formation assay was performed in Ellinghausen–McCullough–Johnson–Harris (EMJH) liquid medium, supplemented with enrichment medium (Bovine Serum Albumin + Tween 80), which was followed by the methodology described earlier. 6 Briefly, the biofilms were allowed to form by incubating at 30°C for 4–7 days without shaking. Every 24 hr, the growth medium was discarded and freshly added. Fresh EMJH medium was added to the wells as a control. Each well was washed three times with phosphate-buffered saline (PBS) under aseptic conditions to eliminate unbound bacteria. The experiment was repeated for 10 strains (Table 1) of Leptospira, and the ability of these strains to form biofilm was determined as per the crystal violet staining methodology, as described by O'Toole. 9
Environmental biofilm isolate. 6
OD, optical density; SD, standard deviation.
Substrate coating
Twenty-five microliters of 2% molten agar (BD Difco™) in distilled water (autoclaved) was uniformly coated in the wells of polystyrene 96-well plate (Corning) and tested as the binding substrate.
Antibiotic stock preparation
Stock solutions of antimicrobial substances, namely penicillin G, ampicillin, tetracycline, and doxycycline (Sigma-Aldrich), were prepared as per the National Committee for Clinical Laboratory Standards document M100-S24. 10
Antimicrobial susceptibility of Leptospira biofilm
Antimicrobial susceptibility of Leptospira biofilms was carried out as previously described by Pettit et al., 11 with the following modifications. The Leptospira biofilm was grown on a U-bottom 96-well (polyvinyl plate) tissue culture microtiter plate and non-tissue culture-treated polystyrene 96-well plates (coated with agar), as described above. These were washed twice with 250 μl PBS solution. Serial twofold dilutions ranging from 1,600 to 0.02 μg/ml for antibiotics were prepared in EMJH medium. A 200 μl sample of each concentration was added to corresponding wells, and plates were incubated for 24 hr at 30°C. EMJH medium without antibiotic was added to wells containing each of the test strains, which served as control. After the exposure, solution was discarded, and the wells were filled (100 μl) with PBS. The viability of Leptospira in the biofilm was checked using Alamar Blue (AB), as per the methodology described elsewhere. 11 Briefly, 10 μl AB was added to each well and incubated for 6 hr at 30°C. After incubation, the solution was aspirated to sterile flat-bottom 96-well tissue culture microtiter plate. Ten microliters of AB in sterile PBS was kept as control. The optical density (OD) of the plate was read at 570–600 nm. The MBC was determined using the OD value. Another set of experiment was performed to check the viability of leptospires using conventional modified method described by Mataraci and Dosler. 12 Briefly, after the antibiotic exposure, the wells were scraped thoroughly, especially the well edges. Well contents were transferred to 1 ml PBS solution and sonicated at 40 Hz for 30 sec by switching on and off for five times (Labsonic; Sartorius Stedim Biotech). The disintegrated bacterial suspension (0.5 ml) was inoculated into fresh EMJH medium and incubated at 30°C for 1 month. During this period, the growth was monitored regularly. The MBC was considered as the lowest concentration of antibiotic that prevented bacterial regrowth.
Results and Discussion
The intensity of biofilm formation varies among strains and number of passages. 5 When the biofilm formation of 10 leptospiral strains on polyvinyl chloride was scored, four strains ranked high and three strains scored medium, while the other three strains did not show any visible biofilm formation until the twenty-first day (Table 1), as reported earlier. 6 However, the biofilm formation in the presence of 2% agar was uniform, which provided viscous environment for aggregation of leptospires. 13 As described by Pettit et al., 11 to make the assay as efficient as possible, the shortest possible AB reduction endpoint was determined for leptospires (data not shown). The antimicrobial susceptibility of planktonic Leptospira was determined by MBC estimation. MBCs are defined as the lowest drug concentration, which results in 50% reduction of AB with a purplish well, 90 min after the addition of AB. The MBCs of planktonic cells were 25–100 U/ml, 12.5–50 μg/ml, 50–100 μg/ml, and 50–100 μg/ml for penicillin G, ampicillin, tetracycline, and doxycycline, respectively. For biofilm, five- to sixfold increase in resistance was observed, and the MBCs were 1,600 U/ml, 800–1,600 μg/ml, 800–1,600 μg/ml, and 800–1,600 μg/ml for penicillin G, ampicillin, tetracycline, and doxycycline, respectively. When agar was used as a substrate for biofilm formation, the resistance to antibiotics had similar MBC values. This showed an increase of one to twofold, with >1,600 forming biofilm without substrate, for all the antibiotics and the strains used. However, the resistance was six- to sevenfold higher than planktonic cells (Table 2). LVW agar was recently reported as a new medium for testing the antibiotic susceptibility of leptospires. 14 However, no significant difference in biofilm formation or antibiotic susceptibility was observed when agar/agarose/LVW agar medium was used as a substratum (data not shown). These findings can be correlated with other studies on Pseudomonas aeruginosa, 15 Staphylococcus spp.,11,16 and so forth, which may be due to multifactorial trait of microbial biofilm, by which they become resistant to antibiotics, that is, physical barrier of extrapolymeric substance, altered chemical microenvironment within the biofilm, metabolically inactive subpopulation in biofilm, and the presence of persister cells, which may serve to repopulate the biofilm. 15
Broth microdilution MBCs (run 1/run 2 for each) are given in micrograms per milliliter (units per milliliter for penicillin G).
MBC, minimal bactericidal concentration.
Biofilm bacteria are a major concern for clinicians in the treatment of infectious disease because of their resistance to a wide range of antibiotics. 17 Recently, the Leptospira research has focused on exploring the possibilities of formation of biofilm within the host as well as the environment. It has been established that Leptospira, like other biofilm-forming microbes, are capable of adhering to inert substances in vitro.5,6 The present study is the first attempt to estimate the susceptibility patterns in Leptospira biofilm.
Due to the lack of standard method for estimating the antibiotic susceptibility of Leptospira biofilm, AB method was adopted in this study, as described for Staphylococcus spp. biofilm.11,16 AB has been successfully used earlier for antimicrobial susceptibility testing for planktonic leptospires. 18 This method was found to be efficient in the studies of antileptospiral activities of plant extracts. 19 As described earlier, the results were visually appreciable, except for the colored compounds, namely doxycycline and tetracycline, where MBC could be determined spectrophotometrically. 19
The colonization of proximal renal tubules of mammalian host by pathogenic leptospires is believed to proceed via the formation of cell aggregates and biofilm, leading to maintenance of chronic carriage. In C57BL/6 mice, live imaging of bioluminescent leptospires demonstrates that renal colonization of L. interrogans provides stealth escape from the blood defense mechanism and antibiotics. 4 In this study, antibiotic treatment with penicillin, ciprofloxacin, and doxycycline failed to eliminate all the leptospires from the tubules in chronic stage. Similarly, the present study has demonstrated resistance toward antibiotics through the formation of biofilm in vitro. These results are in line with the data from the literature, which report that penicillin or other antibiotics proved efficient only in the early stage of infection. 4
In conclusion, the present study emphasizes the importance of biofilm formation in leptospires and its antibiotic susceptibility pattern. Thus, this study could facilitate in devising appropriate strategies to prevent potential chronic leptospirosis in endemic areas and also during an outbreak situation.
Footnotes
Acknowledgment
The authors are thankful to the Indian Council of Medical Research, New Delhi, for providing intramural financial grants for the study. This work is a part of the PhD thesis work of Mr. K. Vinod Kumar, Regional Medical Research Centre (ICMR), Port Blair, affiliated to the Pondicherry University, Puducherry, India.
Disclosure Statement
No competing financial interests exist.