Abstract
Biofilm growth of Mycobacterium smegmatis was found to be unaffected at an isoniazid concentration that inhibited growth of planktonic bacilli (i.e. at isoniazid minimum inhibitory concentration = 10 Wg ml 31 ). Significant growth (50% of drug-free control) of biofilms was observed at up to 40 Wg ml 31 and the MIC for biofilm growth showed an increase to up to 80 Wg ml 31 isoniazid. Thus, the biofilm growth modus appears to be a strategy for replicating bacilli to evade the onslaught of antibacterials. ß
Introduction
Conventionally, drug resistance is de¢ned as the ability of a microbe to grow in the presence of an elevated level of antibiotic (re£ected in an increase in the minimum inhibitory concentration (MIC)). Bio¢lms, i.e. microbes attached to a surface, are usually considered to be drug-resistant. In most bio¢lm susceptibility studies, however, survival of cells in a preformed bio¢lm (rather than the ability of the bio¢lm to grow) is recorded. Accordingly, the reported 'bio¢lm resistance' usually describes an increased resistance of cells to killing (not growth inhibition) and this is what virtually all bio¢lms are good at [1] .
Introduction
Here, we asked whether mycobacterial bio¢lm cultures are drug-resistant in the conventional sense, i.e. whether bio¢lms can grow beyond drug concentrations that inhibit growth of planktonic cells. For this study we used the non-pathogenic and fast-growing mycobacterial model organism Mycobacterium smegmatis mc 2 155 [2] . Recently it was demonstrated that M. smegmatis is capable of forming bio¢lms on polyvinyl chloride surfaces and the ¢rst genes (involved in synthesis and transport of glycopeptidolipids) essential for bio¢lm formation were identi¢ed by Kolter and co-workers [3^5]. First we optimized a polyvinyl chloride-based bio¢lm culture system to grow reproducibly exponential bio¢lms within 1 day. Then we employed this culture system to investigate the inhibitory e¡ects of isoniazid on bio¢lm growth. This frontline tuberculosis drug inhibits mycolic acid synthesis, and hence cell wall synthesis. Bio¢lms of the bacillus were found to grow signi¢cantly beyond the MIC for planktonic culture.
Materials and methods

Strain, cultivation and drugs
All experiments were conducted with M. smegmatis mc 2 155 [2] . Aliquots of seed stocks were kept at 370 ‡C. All cultures were grown in bio¢lm medium (M63 salts minimal medium supplemented with 2% glucose, 0.5% casamino acids, 1 mM MgSO 4 , and 0.7 mM CaCl 2 [5] ) at 37 ‡C. Bio¢lm cultures were grown in the 200-Wl dishes of 96-well, U-bottom, polyvinyl chloride plates (Falcon, cat. no. 353911) with lids (Falcon, cat. no. 353913) as described by Kolter and co-workers [5] with two modi¢ca-tions. (i) To produce a bio¢lm growth curve from start to stationary phase within 1 day (rather than several days) incubation was at 37 ‡C (as opposed to room temperature).
Strain, cultivation and drugs
(ii) To increase reproducibility of the growth curve inoculation was done with an exponentially growing pre-culture of de¢ned cell density (rather than tooth-picking from a colony). Brie£y, exponential phase pre-cultures (with 0.05% Tween 80, to prevent clumping) were grown overnight in tissue culture £asks (OD 600 = 0.2^0.3), washed in bio¢lm medium to remove Tween and resuspended in bio¢lm medium. The washed pre-culture was diluted to an OD 600 of 0.005 and 100-Wl aliquots (containing 10 5 cfu) were grown in dishes under gentle shaking at 100 rpm on an orbital shaker. Planktonic cultures were grown in the same way as described for bio¢lm cultures with the di¡er-ence that the 200-Wl dishes of 96-well, U-bottom, polystyrene (instead of polyvinyl chloride) plates with lids (Nunc, cat. no. 163320) were used as culture vessels.
Strain, cultivation and drugs
Isoniazid and o£oxacin were from Sigma. Stock solutions were made in water. Drug solutions of appropriate dilutions were added as 10-Wl aliquots to the culture dishes.
Monitoring of growth
Bio¢lm growth was monitored via crystal violet staining of the cell material [4, 5] . The wells were rinsed twice with water, and 120 Wl of a 1% solution of crystal violet was added. Plates were incubated at room temperature for 30 min and rinsed with water three times. Quanti¢cation of bio¢lm formation was performed by extracting the bio¢lm-associated crystal violet with ethanol. 200 Wl ethanol was added per dish for 1 h and the contents of eight dishes were pooled for measuring absorbance of crystal violet at 570 nm. To determine cfu of bio¢lm culture, the bio¢lm was washed once and then dissolved by adding bio¢lm medium containing 0.5% Tween 80, pipetting up and down 10 times, incubation for 15 min on an orbital shaker at 100 rpm, and ¢nally pipetting up and down 10 times. Cfu were determined by plating appropriate dilutions and colony count. Crystal violet staining of the dish after Tween 80 treatment con¢rmed that virtually the entire bio¢lm had been removed from the surface. Growth of planktonic culture was determined by turbidity measurement at OD 600 and colony count after plating of appropriate dilutions.
Results

Growth of M. smegmatis bio¢lm cultures on polyvinyl chloride dishes
First, we established conditions to reproducibly generate exponentially growing bio¢lm cultures within 1 day. 200-Wl dishes of polyvinyl chloride 96-well plates were used as culture vessels. Bio¢lm growth was determined by measuring photometrically the increase in cell-bound crystal violet after staining of the bio¢lm at various time points and extraction of the dye. Variation of temperature, aeration and inoculum size showed that growth at 37 ‡C, with shaking at 100 rpm and inoculation of the dishes with 100 Wl of an exponentially growing (planktonic) pre-culture diluted to OD 600 = 0.005 resulted in reproducible bio¢lm growth curves. Fig. 1A (BF-PVC) shows that bio¢lm growth under these conditions was detectable by the crystal violet assay after 3 h. Exponential bio¢lm growth (generation time of 3 h) was observed for 15 h post inoculation.
Growth of M. smegmatis bio¢lm cultures on polyvinyl chloride dishes
To con¢rm that increase in crystal violet staining indeed correlates with increase in cell number, cfu were determined. The mild detergent Tween 80 was used to 'dissolve' the bio¢lm at various time points during exponential growth and the resulting suspension was plated for colony counting. Fig. 1C (BF-PVC) shows that bio¢lm cfu increased exponentially with a generation time of 3 h, consistent with the increase in cell-bound crystal violet.
Growth of M. smegmatis bio¢lm cultures on polyvinyl chloride dishes
The polyvinyl chloride culture dish contained, in addition to the bio¢lm bacilli growing attached to the surface, a subpopulation of planktonic cells in the liquid phase. Fig. 1B (PC-PVC) shows that no increase in turbidity (and cfu, data not shown) in the liquid phase of the culture was observed. To determine whether the planktonic subpopulation in the dish is required for the growth of the bio¢lm (or whether bio¢lm growth can be driven 'internally'), the planktonic cells were removed after 3, 6, 9, and 12 h post inoculation, the dishes were washed and 100 Wl of (cell-free) fresh medium was added. The e¡ect of the removal of the planktonic subpopulation from the culture on the growth of the bio¢lm was determined by measuring the bio¢lm after 24 h post inoculation. Removal of the planktonic subpopulation had little or no e¡ect on bio¢lm growth (data not shown). This suggests that bio¢lm growth (once the surface is 'colonized' by planktonic cells) is predominantly driven internally, i.e. by replicating cells within the ¢lm and not via attachment of planktonic cells from the liquid phase to the ¢lm. The same results were obtained when the planktonic subpopulation was removed via ¢ltration and the used (rather than fresh) medium was re-introduced to the dishes (data not shown).
Growth of planktonic cultures in polystyrene dishes
To carry out a comparative analysis of the inhibitory e¡ects of drugs on the growth of bio¢lm and planktonic culture we also established a 96-well plate-based culture system to grow exponential planktonic cultures in 200-Wl dishes. To do this we made use of the ¢nding that M. smegmatis does not form bio¢lms on polystyrene surfaces. We characterized growth of planktonic culture in dishes of polystyrene 96-well plates and demonstrated the absence of bio¢lm formation on this surface. Polystyrene dishes were inoculated with exponentially growing planktonic pre-culture as described above for the seeding of polyvinyl chloride dishes and growth of the planktonic culture was monitored by turbidity measurement and cfu determination. Fig. 1B (PC-PS) shows that the planktonic culture grew exponentially with a generation time of 3 h up to 24 h post inoculation (see Fig. 1C (PC-PS) for cfu). Taken together, we have established a culture system based on polyvinyl chloride and polystyrene dishes for the growth of bio¢lm and planktonic culture, respectively. Both bio¢lm and planktonic cultures showed a similar generation time of about 3 h (Fig. 1C) .
Growth-inhibitory e¡ect of isoniazid on planktonic and bio¢lm cultures
Exponentially growing planktonic and bio¢lm cultures (grown in polystyrene and polyvinyl chloride dishes, respectively) were exposed to increasing concentrations of isoniazid. The drug was added to the dishes at the beginning (0 h, Fig. 2A) , and during early (6 h, Fig. 2B ) and late (12 h, Fig. 2C ) exponential phase. The e¡ect of isoniazid on growth was monitored by measuring turbidity of the planktonic cultures and crystal violet staining of the bio¢lm cultures after 24 h of drug exposure. The isoniazid dose^response curves for planktonic cultures curves shown in Fig. 2A^C (PC-PS) demonstrate as expected the strong inhibitory e¡ect of isoniazid on suspension cultures, independent of the age of the exponentially growing culture. The MIC was about 10 Wg ml 31 . In contrast, little or no inhibitory e¡ect on bio¢lm growth was observed when isoniazid was added at 10 Wg ml 31 to exponentially grow- Fig. 1 . Growth of M. smegmatis in polyvinyl chloride and polystyrene dishes. A: Growth on the surface of polyvinyl chloride (BF-PVC) and polystyrene dishes (BF-PS) ('bio¢lm growth') as determined by measurement of cell-bound crystal violet at OD 570 . B: Growth in the liquid phase in polyvinyl chloride (PC-PVC) and polystyrene dishes (PC-PS) ('planktonic growth') as determined by turbidity measurement at OD 600 . C: Growth of bio¢lm in polyvinyl chloride dishes (BF-PVC) and planktonic culture in polystyrene dishes (PC-PS) during exponential growth phase as determined by plating and colony count. The experiments were carried out three times and a representative set of curves is shown. Fig. 2 . Growth-inhibitory e¡ect of isoniazid on planktonic culture grown in polystyrene dishes (PC-PS) and bio¢lm cultures grown in polyvinyl chloride dishes (BF-PVC). Dose^response curves are shown. The drug was added at the beginning (A) and 6 h (B) and 12 h (C) post inoculation. After 24 h exposure time the e¡ect of the drugs on growth was determined by crystal violet staining of bio¢lm culture in polyvinyl chloride dishes and by turbidity measurement of planktonic culture grown in polystyrene dishes. The growth of drug-free cultures during the 24 h after drug exposure time was taken as 100% growth. The MIC for bio¢lm growth in C was about 80 Wg ml 31 . The experiments were carried out three times and a representative set of curves is shown. It is interesting to note that the slight increase of MIC for the planktonic culture from A to C (PC-PS) was not seen in cultures containing Tween 80. This might indicate that the observed MIC increase is due to some clumping of bacilli. The same drug exposure experiments were carried out with the following modi¢cations. (i) The e¡ect on growth was determined after 24 post inoculation (rather that 24 h exposure time). (ii) The e¡ect on bio¢lm growth was determined after 24 h of drug exposure as described above but after the removal of the planktonic subpopulation and re-introduction of fresh medium with the appropriate drug concentrations. Both experiments, change of exposure time and removal of planktonic bacilli, yielded the same conclusions. ing bio¢lms at 6 and 12 h post inoculation (Fig. 2B,C (BF-PVC) ). Furthermore, the bio¢lm cultures showed marked growth (50% of drug-free control) for isoniazid concentrations as high as four times the MIC of planktonic culture (Fig. 2C (BF-PVC) ). The MIC for bio¢lm growth was found to be as high as 80 Wg ml 31 , i.e. eight times the MIC for planktonic growth (Fig. 2C) . Taken together, the comparative analyses of the growth-inhibitory e¡ects of isoniazid on planktonic and bio¢lm culture revealed a markedly reduced susceptibility of the bio¢lm culture to the drug.
Growth-inhibitory e¡ect of isoniazid on planktonic and bio¢lm cultures
Is the observed phenotypic drug resistance of exponentially growing bio¢lms against isoniazid speci¢c to this drug or does it indicate development of general drug resistance? To answer this question the e¡ect of the £uoro-quinolone o£oxacin (a replication inhibitor) on exponentially growing planktonic and bio¢lm cultures was determined in the same way described for isoniazid (i.e. at 0, 6 and 12 h post inoculation). The shape of the doser esponse curves and the MIC for o£oxacin (4 Wg ml 31 ) were found to be independent of the mode of growth and independent of the age of exponentially growing cultures (data not shown). This demonstrates that the observed resistance of M. smegmatis bio¢lm culture for isoniazid is speci¢c for this drug and does not re£ect the development of general drug resistance in bio¢lm bacilli.
Discussion
We demonstrate here for the ¢rst time that bio¢lm cultures of a Mycobacterium are capable of growing at higher drug concentrations (i.e. have higher MICs) than suspension cultures : the MIC of isoniazid that inhibited exponentially growing bio¢lm was found to be up to eight times higher than the MIC for planktonic culture. Why do the bacilli, when growing as a surface-attached culture, show resistance when compared to bacilli growing in suspension? The bio¢lm literature o¡ers essentially three possible explanations for bio¢lm-associated drug resistance: (i) slower bio¢lm growth, (ii) decreased drug penetration into the bio¢lm due to architecture and/or extracellular matrix of the bio¢lm, (iii) resistance mechanisms expressed speci¢cally in bio¢lm bacilli but not in planktonic cells (reviewed in [1] ). The generation times of bio¢lm and planktonic M. smegmatis cultures were found to be similar. Hence, slower growth of the bio¢lm culture as a mechanism for the increase in MIC appears not to play a role. This leaves drug penetration problems and expression of (unknown) bio¢lm-speci¢c resistance mechanisms as possible causes for the observed resistance against isoniazid. We used the genetically tractable and fast-growing M. smegmatis for this study. Hence, our work provides the basis for a genetic dissection of the bio¢lm-associated drug resistance phenomenon uncovered in this work. Isolation of mutant strains that have lost the ability to form a bio¢lm at elevated isoniazid levels (but retain this ability under drug-free conditions) is currently under way.
Discussion
Our present work demonstrates that M. smegmatis has a second phenotypic (as opposed to genetic) drug resistance strategy to evade the onslaught of antibacterials. Previously, we reported that the bacillus is capable of undergoing a hypoxia-induced dormancy response in which the organism exits from the cell cycle and di¡erentiates into a de¢ned quiescent form that is resistant to killing by drugs [6] . The work reported here shows that the bacterium is furthermore capable of 'di¡erentiating' into a multicellular, surface-attached form that is resistant to growth inhibition by antimycobacterials.

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