Growing Mycobacterial Biofilm as a Model to Study Antimicrobial Resistance

Abstract

Many bacteria thrive in intricate natural communities, exhibiting key attributes of multicellularity such as communication, cooperation, and competition. The most prevalent manifestation of bacterial multicellular behavior is the formation of biofilms, often linked to pathogenicity. Biofilms offer a haven against antimicrobial agents, fostering the emergence of antimicrobial resistance. The conventional practice of cultivating bacteria in shake flask liquid cultures fails to represent their proper physiological growth in nature, consequently limiting our comprehension of their intricate dynamics. Notably, the metabolic and transcriptional profiles of bacteria residing in biofilms closely resemble those of naturally growing cells. This parallelism underscores the significance of biofilms as an ideal model for foundational and translational research. This article focuses on utilizing Mycobacterium smegmatis as a model organism to illustrate a technique for cultivating pellicle biofilms. The approach is adaptable to various culture volumes, facilitating its implementation for diverse experimental objectives such as antimicrobial studies. Moreover, the method's design enables the qualitative or quantitative evaluation of the biofilm-forming capabilities of different mycobacterial species with minor adjustments.

Introduction

Bacteria are able to survive as single-celled entities; however, in most physiologically relevant conditions, they organize into community mimetics. Biofilm is a widely recognized community organization of bacteria formed by aggregated cells encased in a self-produced matrix¹. Such assembly possesses signatures of early multicellularity and provides higher stress resilience to bacterial systems. Biofilms are often tolerant to antimicrobials and are estimated to be responsible for almost 80% of microbial infections^2,3.

Shake flask and plate-based cultures have traditionally been the usual practices for bacterial culturing. Their enormous acceptability and success can be attributed to their ease of handling, reproducibility, and scalability. However, the lack of physiological context limits the translational potential of the knowledge generated using such systems⁴. Therefore, biofilms are becoming an attractive model system to study bacterial pathophysiology. Biofilms provide a dynamic model system, closely mirroring natural conditions, allowing researchers to replicate physiological aspects such as nutrient gradients and spatial heterogeneity^5,6.

The biofilm lifestyle is particularly pertinent in mycobacterial studies, as mycobacteria, including the notorious Mycobacterium tuberculosis, are adept biofilm formers⁷. Their ability to thrive within biofilms contributes to their persistence in host tissues during infections. It poses a formidable challenge in treating mycobacterial diseases, given the inherent antibiotic resistance associated with biofilm lifestyles⁸. Biofilms also provide an ideal model system to study mycobacterial metabolism, as they allow for the investigation of the unique metabolic adaptations and nutrient utilization strategies employed by mycobacteria within complex microbial communities⁹.

While biofilm is increasingly being accepted as a better model system for mycobacterial studies¹⁰, there is a need for consistent and reproducible standard operating procedures, especially for drawing parallels among studies conducted in different laboratories. The method outlined here describes biofilm formation procedures for a mycobacterial species, M. smegmatis. M. smegmatis is a more accessible model for studying mycobacterial biofilms, given its non-pathogenicity and faster biofilm formation kinetics. The method can be modified to suit applications like antimycobacterial screening, metabolite extraction, and omics studies.

Protocol

The details of all the reagents and equipment used for the study are listed in the Table of Materials.

1. Sauton's media preparation

1. Prepare 50 mL of 2.5% potassium dihydrogen phosphate solution by carefully weighing 1.25 g of potassium dihydrogen phosphate and dissolving it in 50 mL of deionized water.
2. Prepare 50 mL of 2.5% magnesium sulfate solution by weighing 2.56 g of magnesium sulfate and dissolving it in 50 mL of deionized water.
3. Prepare 10 mL of 5% ferric ammonium citrate solution by weighing 0.5 g of ferric ammonium citrate and dissolving it in 10 mL of deionized water. Store it in an amber tube.
4. Prepare 100 mL of 20% glucose solution by weighing 20 g of glucose and dissolving it in 100 mL of deionized water. Filter-sterilize using a 50 mL sterile syringe and a 0.2 µM PVDF syringe filter in a tube.
5. Prepare 10 mL of sterile 1% zinc sulfate solution by carefully weighing 0.1 g of zinc sulfate and dissolving it in 10 mL of deionized water. Filter-sterilize using a 10 mL sterile syringe and a 0.2 µM PVDF syringe filter in the laminar hood.
6. Mix the prepared components in 750 mL of deionized water as described in Table 1. Measure the pH of the solution and adjust it to 7. Make up the volume to 900 mL with deionized water.
7. Autoclave the media at 15 PSI and 121 °C for 15-20 min. Let it cool down. Then, add 100 µL of filter-sterilized 1% zinc sulfate. Add 100 mL of filter-sterilized 20% glucose and dissolve it well.

2. Preparation of filter-sterilized 5% Tween-80

1. Dissolve 0.5 mL of Tween-80 in 9.5 mL of deionized water.
2. Filter-sterilize the solution using a 10 mL sterile syringe and a 0.2 µM PVDF syringe filter in the laminar hood.

3. Preparing the primary culture

1. In the laminar hood, use a serological pipette to dispense 2 mL of autoclaved LB media into two sterile 14 mL test tubes.
2. Add 20 µL of filter-sterilized 5% Tween-80 to the test tubes using a micropipette.
3. Inoculate M. smegmatis into one of the tubes by adding its cryo-stock using an inoculation loop. The other test tube serves as a media control.
4. Incubate the tubes for 48 h in a shaker incubator at 300 rpm and 37 °C.
   NOTE: The media control tube should not show any growth.

4. Preparing the secondary culture

1. In the laminar hood, use a serological pipette to dispense 2 mL of autoclaved LB media into two sterile 14 mL test tubes.
2. Add 20 µL of filter-sterilized 5% Tween-80 to the test tubes using a micropipette.
3. Add 20 µL of the primary culture to one of the tubes using a micropipette. Keep the other tube as a media control.
4. Incubate the cultures in a shaker incubator at 300 rpm and 37 °C until they reach an OD₆₀₀ of 0.6 to 0.8.
   NOTE: It generally takes 12 to 14 hours to reach an OD₆₀₀ of 0.6 to 0.8. Other cell densities may also be used, but a defined OD₆₀₀ value helps with comparative studies.

5. Setting up the biofilm

1. In the laminar hood, dispense 6 mL of Sauton's media supplemented with 2% glucose into each well of a 6-well plate using a serological pipette.
2. Inoculate the respective wells with 120 µL (i.e., 2%) of the secondary inoculum using a micropipette. Keep one uninoculated well as a media control.
3. Mix the media and culture by pipetting up and down with a 1 mL micropipette. Cover the lid and carefully seal the plate with paraffin film.
4. Incubate the plate undisturbed in a static incubator at 37 °C for 7 days.

6. Setting up biofilms for stage-specific observations

NOTE: The overall biofilm setup is the same as described in step 5. To harvest or image biofilm at different points in time, it is suggested that the same biofilm be set up in multiple sets on separate plates.

1. To observe biofilms between day 3 and day 6, prepare four plates with identical conditions and use one plate per day for imaging.
   NOTE: The biofilm layer starts appearing on the air-medium interface beginning on the third day of plate setup.

7. Estimating the biofilm development

1. Visual estimate
   1. Image the plates using a gel documentation system with epi-white light illumination (Figure 1).
      NOTE: Imaging gives a gross qualitative and quantitative estimate of the biofilm formed. Any good quality camera can be used as an alternative.
2. Dry weight estimate
   1. Perform dry-weight measurement for more precise quantification.
   2. To measure dry weight, begin by weighing a blotting paper.
      NOTE: Filter paper can also be used instead of blotting paper.
   3. Extract the biofilm from the 6-well plate and transfer it onto the paper using a spatula.
   4. Carefully collect most of the biofilm from the culture, being cautious not to transfer excessive amounts of the media onto the paper.
   5. Place the biofilm on the blotting paper.
   6. Place it in an incubator at 50 °C for 0.5-1 h until the biofilm dries completely.
   7. Measure the combined weight of the paper and biofilm.
      NOTE: Dry Weight of biofilm=(Weight of biofilm +paper)-(Weight of paper)

8. Antibiotic tolerance assay in biofilms

1. Estimate the MIC90 of rifampicin in M. smegmatis planktonic culture following the protocol described by Irith Wiegand et al. using Sauton's media¹¹. Add the antibiotic when the cultures reach an OD₆₀₀ of 0.2.
2. Set up the plates for biofilm growth as described in step 5 of the protocol.
3. On the fourth day of incubation, add rifampicin to cultures at the MIC90 concentration (64 µg/mL) from the sides of the well using a micropipette in the laminar hood.
   NOTE: Do this carefully without disturbing the biofilm too much. Leave one of the wells with biofilm untreated.
4. Slowly swirl the plates to mix the antibiotics.
5. Cover the sides of the plates with paraffin film and keep them in the incubator for further growth.
6. After 24 h, remove the plates from the laminar hood and add Tween-80 to a final concentration of 0.2% in all the wells containing biofilm.
7. Once again, cover the plates with paraffin film and keep them at 4 °C and 100 rpm for 12 h.
8. Take the plates out in a laminar hood and homogenize the constituents with a 1 mL micropipette.
9. Wash the constituents with Sauton's media using the protocol described in Anand et al.¹².
10. Finally, resuspend the cells in 6 mL Sauton's media and dilute to final concentrations of 10^-4, 10^-5, 10^-6, and 10^-7.
11. Spread 100 µL of each dilution on LB-agar plates using autoclaved glass beads.
12. Seal all the plates using paraffin film and keep them in an incubator at 37 °C.
13. After 3 days of incubation, count colonies on each plate. Only consider the plates with a colony count between 30 and 300.
14. Calculate CFU/mL and the relative decrease in CFU/mL using the provided formulas¹³.
    NOTE: CFU/mL = (No. of colonies × Dilution factor) / (Volume plated (in mL))
    Relative decrease in CFU/mL = ((CFU/mL in Untreated - CFU/mL in Treated)) / ((CFU/mL in Untreated)) × 100

Representative Results

Biofilm pellicles become visible to the naked eye from the third day onwards. Although biofilm grows on Sauton's media without 2% glucose, an improvement was observed in the reticulation when it was added. We obtained 10.48 mg ± 3.13 mg (n = 4) of biofilm dry weight from each well of a 24-well plate with 1.5 mL of Sauton's media (supplemented with 2% glucose) grown for four days. In Figure 2, biofilm development was visible from day 3 to day 6. It starts forming a film with slight reticulation on day 3, and by day 5, it is fully matured. From day 6 onwards, the biofilm starts to disintegrate. The MIC90 of rifampicin (64 µg/mL) reduced the biofilm's CFU by 25% ± 12% (n = 3) only.

Figure 1: Biofilms of M. smegmatis after 4 days of incubation in Sauton's media. (1) Without glucose and inoculated with 2% inoculum. (2) Without glucose and inoculated with 4% inoculum. (3) With 2% glucose and inoculated with 2% inoculum. (4) With 2% glucose and inoculated with 4% inoculum. (5) and (6) are media controls. Here, the samples are in standard 6-well plates. The photographs are captured without any magnification. Please click here to view a larger version of this figure.

Figure 2: M. smegmatis biofilm morphology from the 3^rd day post inoculation to the 6^th day. Here, the samples are in standard 24-well plates. The photographs are captured without any magnification. Please click here to view a larger version of this figure.

S. No.	Component			Working concentration (w/v)		Stock solution concentration		Volume/ Weight of stock solution
1	Potassium dihydrogen phosphate			0.05%		2.50%		20 mL
2	Magnesium sulfate			0.05%		2.50%		20 mL
3	Ferric ammonium citrate			0.01%		5%		1 mL
4	Glycerol			6%		100%		60 mL
5	L- Asparagine			0.40%		NA		4 g
6	Citric acid			0.20%		NA		2 g

Table 1: Components for Sauton's media preparation.

Discussion

The multicellular lifestyle of microbes was described almost a century ago; however, clinical studies remain sparse, mostly due to the lack of robust methods¹⁴. Methods described in works on biofilm biology are often difficult to adapt. Here, the detailed methodology, aided by demonstrations of critical steps, is expected to improve the reproducibility of the protocols.

The method of biofilm production described in this article is scalable, requiring a proportional increase in medium volume and inoculum size. Applications like metabolite extraction require a higher culture volume, whereas antimicrobial susceptibility assays require a smaller biomass¹². Additionally, the size of the inoculum can be adjusted to tailor the rate of biofilm formation. This flexibility allows researchers to appropriately control the duration of experiments.

Growth conditions and the microenvironment play crucial roles in biofilm physiology¹⁵. Notably, the presence of glucose is not obligatory for forming a biofilm; nonetheless, the inclusion of 2% glucose has been observed to enhance the reticulation and stability of the biofilm. Another interesting observation is the biofilm enhancement potential of placing a glass coverslip at the bottom of the culture wells. The addition of Tween-80 in both primary and secondary cultures is crucial for mitigating cell aggregation.

Biofilm development involves distinct growth phases such as attachment, maturation, and dispersal. While the biofilm setup described here is typically incubated for 7 days, pellicles become visible to the naked eye from the 3^rd day onwards. This setup allows for studying the temporal dynamics of biofilm development. While this article describes the estimation of dry weight to assess biofilm biomass, the setup is also amenable to conventional crystal violet staining or microscopy-based estimation^12,16.

The production of biofilms by mycobacteria plays a significant role in their pathogenicity and confers enhanced resistance to drugs^8,17. We observed seven to eight times higher antibiotic tolerance in biofilm cultures compared to shake flask cultures. Several groups use biofilms as a model system for studying bacterial physiology; a wider adoption of this growth mode by the mycobacterial research community can provide newer insights into the pathophysiology of various pathogenic species within this genus.

Materials

Name	Company	Catalog Number	Comments
0.2 µM PVDF syringe filter	Axiva	SFNY04 R
1 mL tips	Genetix	GXM-611000 C
10 µL tips	Genetix	GXM-6110 C
200 µL tips	Genetix	GXM-61200C
6-well polypropylene plates	Tarsons	980010
Amber tubes	Tarsons	546051
Autoclave	Hospharma
Biosafety Cabinet A II	MSET
Blotting paper	Any suitable vendor
Centrifuge	Eppendorf
Citric acid	Sigma	251275
Cuvettes	Bio-Rad	2239955
Ferric ammonium citrate	Sigma	F5879
Gel documentation system	Bio-Rad
Glass Beads	Sigma	G8772
Glucose	Sigma	49139
Glycerol	Sigma	G5516
Inoculation loops	Genaxy	HS81121C
L-Aspargine	Sigma	A0884
LB-agar	Himedia	M1151
LB-media	Himedia	M575
M. smegmatis mc2155 cryo-stock	ATCC	700084
Magnesium sulfate	Sigma	M2643
Micropipettes	Gilson
Parafilm	Tarsons
Petri Dish	Tarsons	460020
pH meter	Labman Scientific Instruments
Plate Reader	Tecan
Polypropylene test tubes	Genaxy	GEN-14100-PS
Potassium phosphate monobasic	Sigma	P5379
Rifampicin	MedchemExpress	HY-B0272
Serological pipette	SPL Life Sciences	95210
Shaker Incubator	Eppendorf
Spatula
Spectrophotometer	Thermo Scientific
Static Incubator	CARON
Sterile 10 mL syringe	Becton Dickinson	309642
Sterile 50 mL syringe	Becton Dickinson	309653
Tween-80	Sigma	P1754
Weighing balance	Sartorius
Zinc sulfate	Sigma	Z0251