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. 2003 Jul;69(7):3719–3727. doi: 10.1128/AEM.69.7.3719-3727.2003

Biofilm-Specific Cross-Species Induction of Antimicrobial Compounds in Bacilli

Liming Yan 1, Kenneth G Boyd 1,†, David R Adams 2, J Grant Burgess 1,*
PMCID: PMC165162  PMID: 12839737

Abstract

An air-membrane surface (AMS) bioreactor was designed to allow bacteria to grow attached to a surface as a biofilm in contact with air. When Bacillus licheniformis strain EI-34-6, isolated from the surface of a marine alga, was grown in this reactor, cells produced antimicrobial compounds which they did not produce when they were grown in shake flask cultures. An unidentified red pigment was also produced by surface-grown cells but not by planktonically grown cells. Glycerol and ferric iron were important for the production of antimicrobial compounds and the red pigment. Release of these secondary metabolites was not due to the onset of sporulation. Cell-free spent medium recovered from beneath the reactor membrane could induce production of antimicrobial compounds and red pigment in shake flask cultures. Neither glycerol nor ferric iron was required for production of these inducer compounds. Spent medium from beneath the membrane of an AMS bioreactor culture of Bacillus subtilis strain DSM10T and Bacillus pumilus strain EI-25-8 could also induce production of antimicrobial compounds and a red pigment in B. licheniformis isolate EI-34-6 grown in shake flask cultures; however, the corresponding spent medium from shake flask cultures of DSM10T and EI-25-8 could not. These results suggest that there is a biofilm-specific cross-species signaling system which can induce planktonically grown cells to behave as if they were in a biofilm by regulating the expression of pigments and antimicrobial compounds.

Bacteria growing on the surfaces of marine algae and other organisms live in a highly competitive environment in which space and access to nutrients are limited (7, 36). Previous studies have shown that a high percentage of marine epibiotic bacteria produce antimicrobial metabolites compared with the number of planktonic isolates that produce such metabolites (5, 6, 21, 23). However, in the laboratory, most isolates stop producing antimicrobial compounds when they are continuously cultivated in shake flasks. Well-agitated suspension cultures in closed flasks represent artificial growth conditions that are very different from the natural environment. Previously, it was demonstrated that a modified roller bottle bioreactor, which mimicked the intertidal environment, could facilitate the production of antimicrobial compounds by two marine epiphytic isolates which had stopped producing them under planktonic growth conditions (43, 44). Thus, new cultivation strategies, particularly strategies mimicking the natural habitats of microorganisms with niche-mimicking bioreactors, can be used to elicit production of secondary metabolites by apparent nonproducers.

Further work showed that biofilm formation and direct exposure to the air were required for eliciting production of antimicrobial compounds by the two isolates. The dominant growth mode of bacteria in the natural environment is in biofilms attached to surfaces (22). The growth conditions in a biofilm are usually heterogeneous (42). pH gradients, for example, can develop around microcolonies within the biofilm (40), and limitations in the transport of substrates and nutrients into the biofilm can result in differential starvation of bacteria (2, 3, 20, 24). As a result, the bacterial metabolism in a biofilm is very different from that in a suspension culture (11, 12, 17, 18, 39). Despite this, there have been few studies in which the workers have investigated the effect of growth in biofilms on the production of secondary metabolites, such as antimicrobial compounds.

The bacterial densities in biofilms can often reach 1012 CFU · cm−3 (35), so it is possible that cell density-dependent signaling and gene expression systems are present in biofilms (1, 13, 14). An accepted model of quorum sensing is that bacteria produce and release chemicals as signal molecules whose concentrations increase as a function of cell density. An increase above a threshold concentration of signal molecules stimulates an alteration in gene expression and consequently leads to a change of phenotype (28, 41). Different signal molecules have been found to regulate a diverse array of physiological activities in various bacteria, including biofilm formation (4, 33, 34). All quorum-sensing-associated signal molecules that have been described were discovered in planktonic suspension cultures. However, it is almost impossible for bacteria grown in a planktonic suspension culture to reach cell densities as high as those observed in biofilms. Until now, there has been no direct evidence suggesting that there are biofilm-specific signal molecules, although the existence of such molecules may explain how certain phenotypes are expressed only within biofilms.

In this study, we designed a novel bioreactor in which semipermeable membranes are used as a support to allow formation of a biofilm at an air-solid interface. We called this apparatus an air-membrane surface (AMS) bioreactor. Bacillus licheniformis strain EI-34-6, isolated from the surface of the marine alga Palmaria palmata, was studied by using the AMS bioreactor. Production of antimicrobial compounds by EI-34-6 was elicited when the organism was grown in the AMS bioreactor but was not when it was grown in shake flask cultures. The major antimicrobial component was identified as bacitracin. Further studies on the mechanism of the induction effect in the AMS bioreactor indicated that there are biofilm-specific inducer compounds.

MATERIALS AND METHODS

Bacterial strains and media.

Two marine epiphytic strains, EI-34-6 and EI-25-8, were isolated from the surfaces of the seaweeds P. palmata and Laminaria digitata, respectively. These strains were selected because of their ability to produce antimicrobial compounds, which are active predominantly against gram-positive bacteria. In addition, Bacillus subtilis strain DSM10T was used for cross-species induction studies. Unless otherwise stated, strains were grown in NGF medium containing 13 g of nutrient broth base (Oxoid, Basingstoke, United Kingdom) per liter of distilled water, 1% (vol/vol) glycerol, and 0.16 g of FeCl3 per liter (pH 6.2). When necessary, nutrient broth (NB) was also used. Bacteriological agar no. 1 (1.5%, wt/wt; Oxoid) was added to make the corresponding agar medium.

Identification of marine epiphytic bacteria.

EI-34-6 and EI-25-8 were identified by 16S ribosomal DNA (rDNA) sequence analysis and additional biochemical tests. The oligonucleotide primers used to sequence the target gene were designated 27F (5′-AGA GTT TGA TCC TGG CTC AG-3′) and 1525R (5′-AAG GAG GTG [AT]TC CAG CC-3′). Sequencing was performed by MWG-Biotech UK Ltd. The sequences obtained were aligned with those in GenBank by using BLAST (19). Identification was assisted by biochemical analysis. The Gram stain, Schaeffer-Fulton spore stain, oxidase, catalase, propionate utilization, starch hydrolysis, casein hydrolysis, and gelatin hydrolysis tests were carried out as described by Collins et al. (9) and Parry et al. (32). For anaerobic cultivation, EI-34-6 was grown on plates containing marine agar 2216 (Difco), nutrient agar, and Columbia base agar (Oxoid) in an anaerobic jar (Anaerocult A; Merck).

AMS bioreactor cultivation.

Bacteria were cultivated on the surface of a semipermeable membrane disc in contact with the air. Three kinds of semipermeable membranes were used: nylon (diameter, 47 mm; pore size, 0.2 μm; Whatman), flat dialysis (pore size, 12,000 to 14,000 Da; Visking), and cellophane (pore size, 12,000 to 14,000 Da; Courtaulds Films). The flat dialysis and cellophane membranes were cut into discs having a diameter of 45 mm. All membranes were autoclaved (121°C, 15 min) and then placed in small shallow dishes (diameter, 26 mm; height, 10 mm) which had been prefilled with sterile liquid medium (approximately 5 ml), so that the membranes were in contact with medium on one side and with air on the other side and were held in place by surface tension (Fig. 1). EI-34-6 (which had been grown in a liquid suspension in marine broth at 28°C for 4 days) was swabbed onto the surface of each type of membrane. Each inoculated membrane in a dish was placed in a deep petri dish (diameter, 55 mm; height, 18 mm; Bibby-Sterilin) to maintain sterility and incubated at 28°C. After cultivation for 7 days, the antimicrobial activity of the liquid beneath the membrane was assayed.

FIG. 1.

FIG. 1.

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AMS bioreactor. The small chamber beneath the membrane is filled with liquid medium, and the membrane disc is held in place by surface tension. Bacteria were inoculated onto the surface of a semipermeable nylon membrane. The AMS bioreactor was placed in a sterile petri dish during growth to maintain sterility.

Antimicrobial activity assay.

The following four pathogenic test strains were used: methicillin-resistant Staphylococcus aureus strains MRSA9551 and MRSA14986 and vancomycin-resistant Enterococcus strains VRE788 and VRE1349. Liquid media (1.5 ml) from various EI-34-6 cultures were transferred into 1.5-ml Eppendorf tubes and centrifuged (5,000 × g, 10 min, room temperature). The antimicrobial activities of spent media were assessed after they were filtered twice with a Nalgene membrane filter (pore size, 0.2 μm). Sterile 6-mm-diameter paper discs (Whatman) were saturated with the supernatants and left to dry at room temperature. The process was then repeated; altogether, approximately 60 μl of supernatant was added to each disc. Antimicrobial activity assays with these pathogenic strains were performed by using diagnostic sensitive test agar (Oxoid) at 37°C. Inhibition zones were measured after 12 h.

Determination of cell densities.

Bacteria from shake flask cultures were diluted 10−6 with sterile NGF medium. Then 0.1-ml portions of each culture were plated onto NGF agar plates. The plates were incubated at 28°C for 2 days. Cell densities were estimated by counting the number of CFU on each plate. For biofilms, a sterile precalibrated needle was used to measure the thickness of the biofilm. The area of the biofilm was calculated as an ellipse. A horizontal axis (a) and a vertical axis (b) were measured, and the area (A) equation A = Πab was used; the results were expressed in square millimeters. Membranes were then aseptically moved into wells of a new sterile Nunclon tray. The biofilms were removed with sterile cell scrapers (Costar), and 5 ml of distilled water was added to resuspend the bacteria. The total cell number (CFU) on each membrane was also estimated by dilution and plate counting. The cell density within the biofilm was calculated as follows: Db = (Bb × 106)/(ATb), where Db is the cell density in the biofilm (in CFU per cubic centimeter), Bb is the total number of CFU in the biofilm, Tb is the average thickness of the biofilm (in millimeters), and A is the area of the biofilm.

Determination of the optimal concentrations of glycerol and FeCl3.

NB containing 1% (vol/vol) glycerol and different concentrations of FeCl3 (0, 6, 10, 30, 60 mg liter−1 and 0.12 and 0.2 g liter−1) were used. FeCl3 precipitates from solution in NB (pH 7.0) when it is present at concentrations higher than 0.25 g liter−1 (1.5 mM). These media were used to cultivate EI-34-6 by both the AMS bioreactor (nylon membrane) and shake flask methods. NB containing 1 mM FeCl3 and different concentrations of glycerol (0, 0.5, 1, 2, 5, and 10% [vol/vol]) were also used to cultivate EI-34-6 as described above. Antimicrobial activity assays were carried out after cultivation for 7 days.

Determination of percentage of sporulation.

The sporulation levels of EI-34-6 in AMS bioreactor and shake flask cultures were compared. Bacteria were removed from a nylon membrane AMS bioreactor culture with a loop and then diluted and dispersed in 1 ml of NB. A planktonic suspension culture (50 μl) was also diluted in the same way. Both samples were stained by the Schaeffer-Fulton method (32). Spores (which stained blue-green) and vegetative cells (which stained red) were counted with a microscope. The percentage of sporulation (ratio of the number of spores to the total cell number) was calculated by determining the average for five random fields of vision.

Effects of metabolites from AMS bioreactor-grown cells on shake flask cultures.

EI-34-6 cultures were grown in shake flasks (30 ml of NGF medium, shaken for 4 days) and used as reporter cultures. Spent media (2 ml) from various EI-34-6 cultures were then added to these shake flask cultures to measure their abilities to elicit antimicrobial compound production. The following samples were added in separate experiments: spent NGF medium from an AMS bioreactor culture (AMS-NGF); interstitial fluid from an EI-34-6 AMS bioreactor biofilm culture; and spent NB from an AMS bioreactor culture (AMS-NB) without glycerol or FeCl3. The following three controls were used: no spent medium; fresh NGF medium; and spent media from corresponding shake flask cultures. All spent media were filtered twice with a 0.2-μm-pore-size syringe filter and further checked for sterility prior to addition. A well-mixed aliquot of the supernatant (1.5 ml) from each of four flasks was removed and tested for antimicrobial activity just after addition of the spent medium. The flasks were then shaken at 28°C for a further 4 days, and the antimicrobial activity of the supernatant was tested again. Any pH changes in the shake flask cultures and the AMS bioreactor cultures were determined. Experiments were carried out in quadruplicate.

Spent media from the AMS bioreactor cultures of EI-34-6 were also treated by autoclaving and trypsin (Sigma-Aldrich, Dorset, United Kingdom) and DNase (Sigma-Aldrich) digestion, and their abilities to elicit production of antimicrobial compounds and red pigment were determined.

B. subtilis strain DSM10T was grown in AMS bioreactor and shake flask cultures by using NGF medium and NB, respectively. Bacillus pumilus strain EI-25-8 was also grown in AMS bioreactor and in shake flask cultures with NB. Cell-free spent media (2 ml) from the DSM10T and EI-25-8 cultures were added to reporter cultures to determine whether compounds from these species could elicit synthesis of antimicrobial agents and/or pigment by B. licheniformis strain EI-34-6.

Identification of the major antimicrobial compound.

Butanol was used to extract the antimicrobial compounds from 6-liter portions of EI-34-6 cultures in scaled-up AMS bioreactors (43). The evaporated extract was purified by antimicrobial activity assay-guided fractionation by using a combination of C18 reverse-phase column chromatography (Sep-Pak) and reverse-phase high-performance liquid chromatography (Waters 501 pump, Waters 484 tunable UV [220-nm] absorbance detector, Pharmacia Fine Chemicals chart recorder, and Phenomenex KROMASIL 5 C18 preparative column, Waters Associates μBondapak C18 P/N analytical column; 30% [vol/vol] CH3CN at a flow rate of 3 ml · min−1). The purified antimicrobial compound was identified by UV spectroscopy, 1H and 13C nuclear magnetic resonance spectroscopy, and mass spectrometry and was compared with pure bacitracin (Sigma-Aldrich) as a reference.

RESULTS

Identification of marine epiphytic bacteria.

EI-34-6 is a gram-, catalase-, and oxidase-positive motile rod. The almost complete 16S rDNA sequence was obtained (GenBank accession number AJ293011) and exhibited the highest level of similarity (1,475 of 1,476 positions; 99% similarity) to the B. licheniformis sequence. Strain EI-34-6 grew well under anaerobic conditions on Columbia agar; its colonies adhered strongly to the agar surface, and the propionate reaction was positive, which distinguished this strain from B. subtilis (32). EI-34-6 also had the following properties in common with the B. licheniformis type strain: positive for citrate utilization, nitrate reduction, starch hydrolysis, casein hydrolysis, and gelatin hydrolysis. EI-34-6 was therefore identified as a marine isolate of B. licheniformis. The 16S rDNA sequence of EI-25-8 was also determined (GenBank accession number AJ494728), and this strain was tentatively identified as B. pumilus (43).

Identification of the major antimicrobial compound.

Following fractionation by reverse-phase high-performance liquid chromatography, only one peak exhibited activity against all target strains. Approximately 50 mg of the antimicrobial compound was purified, and on the basis of its chemical structure it was determined to be bacitracin (43). During the initial Sep-Pak fractionation, the eluted red pigment was not active.

Antimicrobial agent production by EI-34-6 when it was grown in the AMS bioreactor.

EI-34-6 did not produce detectable levels of an antimicrobial compound when it was grown by using shake flask cultivation in NB or NGF medium with or without various membrane fragments which became submerged during cultivation. Shake flask cultures also were not pigmented. However, when the organism was grown in the AMS bioreactor (also in NGF medium), liquid from beneath the membrane exhibited activity against four target strains. A thick biofilm also formed at the air-membrane interface, and the biofilm was dark red regardless of the type of membrane used (Fig. 2). No antimicrobial activity or pigmentation was observed if NB was used for AMS bioreactor cultivation. A nylon membrane was used for all further experiments.

FIG. 2.

FIG. 2.

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Production of antimicrobial compounds by B. licheniformis EI-34-6 grown by using shake flask (SF) cultivation or an AMS bioreactor. Three types of semipermeable membranes were used. In the case of shake flask cultures, fragments of nylon membrane, flat dialysis membrane, and cellophane were submerged in the liquid media. EI-34-6 produced antimicrobial compounds when it was grown in the AMS bioreactor and not when it was grown in shake flasks, regardless of the type of membrane. In addition, production of pigment was also observed only when EI-34-6 was grown in the AMS bioreactor. The data are means of quadruplicate experiments.

If the antimicrobial agent-producing dark red biofilm was washed thoroughly with fresh sterile NGF medium and then submerged in 30 ml of NGF medium for shake flask cultivation, production of an antimicrobial compound and a red pigment was not observed. In this case, the biofilm maintained its original shape and remained attached to the nylon surface during cultivation in the shake flask. When this experiment was repeated without washing, however, production of antimicrobial compounds and red pigment was detected, but to a lesser extent and with more variability (Fig. 3).

FIG. 3.

FIG. 3.

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Production of bacitracin when the biofilms from AMS cultures (producing bacitracin) were submerged in shake flask cultures. With washing, biofilm cultures did not show activity when they were submerged in liquid NGF medium. Without washing, cultures were active when they were submerged in liquid NGF medium, but the results were more variable. In both cases, biofilms were cultivated in shake flasks for 7 days. The data are means of quadruplicate experiments.

Effects of FeCl3 and glycerol on production of antimicrobial compounds.

The concentrations of FeCl3 and glycerol had a significant effect on production of the antimicrobial compound by EI-34-6 when it was grown in the AMS bioreactor. Within the limits tested, the greater the FeCl3 concentration, the greater the antimicrobial activity (Fig. 4A). The dose-response curve suggested that the optimal glycerol concentration was 0.5% to ∼1% (Fig. 4B). However, the dose-response effects of FeCl3 and glycerol were observed only when EI-34-6 was grown in the AMS bioreactor.

FIG. 4.

FIG. 4.

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Effects of FeCl3 (A) and glycerol (B) on antimicrobial activity. When the FeCl3 concentration was increased, the activity against all of the target strains increased. The activity reached a maximum at a glycerol concentration between 0.5 and 2% (vol/vol). The activity decreased if the concentration of glycerol was more than 2% (vol/vol). The data are means of quadruplicate experiments.

Relationship between antimicrobial activity and sporulation of EI-34-6.

We investigated whether antimicrobial compound production by EI-34-6 when it was grown in the AMS bioreactor was due to higher levels of sporulation. No activity against the target strains was observed in shake flask cultures even when the levels of sporulation rose. However, antimicrobial activity and production of the red pigment by the AMS bioreactor culture were apparent from the third day (Fig. 5). In terms of level of sporulation, there was little difference between shake flask and AMS bioreactor cultures.

FIG. 5.

FIG. 5.

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Relationship between the level of sporulation of EI-34-6 and the production of antimicrobial compounds. Similar levels of sporulation were observed when EI-34-6 was grown in AMS bioreactor and shake flask cultures; however, only organisms grown in the AMS bioreactor culture exhibited activity against the target strains. The data are means of quadruplicate experiments.

Replacement of the spent NGF medium under the nylon membrane with fresh NGF medium every 2 days delayed sporulation compared with the sporulation in a single-batch culture and kept the levels of sporulation below 2%. However, production of antimicrobial compounds was not affected by this procedure (Fig. 6). Thus, it appears that antibiotic production by AMS bioreactor cultures is not dependent on sporulation.

FIG. 6.

FIG. 6.

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Effect of reduced sporulation on production of antimicrobial compounds by AMS bioreactor cultures. Although the levels of sporulation remained low compared with those of single-batch cultures (Fig. 5), the antimicrobial activity was not affected. The data are means of quadruplicate experiments.

Effect of metabolites from AMS bioreactor-grown cells on shake flask cultures.

None of the reporter cultures of EI-34-6 showed detectable activity immediately after addition of 2 ml of spent medium from a 7-day AMS bioreactor culture or fresh NGF medium. Addition of spent medium did not significantly change the pH of the shake flask cultures. After 4 days, the cultures in flasks which were supplemented with spent medium from the AMS-NGF culture or with interstitial fluid from biofilms turned red and produced bacitracin (Fig. 7). All the control shake flask cultures remained unpigmented, and supernatants from them did not show detectable activity. Neither purified nor commercially obtained bacitracin showed this induction effect. If spent medium from an AMS bioreactor culture was added to a shake flask without EI-34-6 inoculum, no antimicrobial activity or pigment production was observed.

FIG. 7.

FIG. 7.

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Induction effects of cell-free spent media from different EI-34-6 cultures on EI-34-6 shake flask cultures. There was no detectable activity in any of the shake flask cultures immediately after 2 ml of cell-free spent medium was added. However, after continued growth for an additional 4 days, bacitracin production was observed. The test strain used was strain MRSA9551. The data are means of quadruplicate experiments.

Cell-free spent medium from an EI-34-6 AMS-NB culture was also able to induce production of both bacitracin and the red pigment by reporter cultures (Fig. 7). However, spent medium from the corresponding shake flask culture in NB was not able to induce production (Table 1). The minimum volume of spent NB required to induce antimicrobial compound production was 2 ml. Interestingly, even though EI-34-6 cells grown in an AMS-NB culture did not exhibit red pigment or antimicrobial compound production, spent medium from these cells was able to induce synthesis of these compounds in reporter cultures.

TABLE 1.

Effects of spent media on production of antimicrobial compounds by EI-34-6 NGF shake flask cultures

Growth medium Production of antimicrobial compounds with spent media froma:
Shake flask cultures
AMS bioreactor cultures
B. subtilis DSM10T B. pumilus EI-25-8 EI-34-6 Induced EI-34-6 B. subtilis DSM10T B. pumilus EI-25-8 EI-34-6
NGF − NG − + + NG +
NB − − − +/− + + +
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a

+, EI-34-6 shake flask culture exhibited antimicrobial activity and red color (For example, the spent NGF medium from the DSM10T AMS bioreactor culture induced the reporter culture; however, the spent medium from the corresponding shake flask culture did not. To determine antimicrobial activity MRSA9551 was used as the test strain); −, EI-34-6 shake flask culture did not exhibit antimicrobial activity or a red color; +/−, both induction and no induction were observed; NG, bacteria did not grow.

In addition, spent medium from the EI-34-6 reporter culture, which had been induced by the spent media from EI-34-6 AMS bioreactor cultures to produce bacitracin and the red pigment, could subsequently elicit production of these secondary metabolites in new reporter cultures. After autoclaving and trypsin and DNase treatment, spent media from the AMS bioreactor cultures was still able to elicit secondary metabolite production (Fig. 7).

Cell-free spent media from AMS-NB cultures of B. pumilus strain EI-25-8 and AMS-NGF and AMS-NB cultures of B. subtilis DSM10T were all able to elicit production of bacitracin and red pigment by B. licheniformis EI-34-6. However, spent media from the corresponding shake flask cultures were not able to elicit such production (Table 1). The minimum volumes of spent media from both EI-25-8 and DSM10T cultures required to induce production were approximately 2 ml.

DISCUSSION

When B. licheniformis EI-34-6 cells were grown in a biofilm at an air-membrane interface (Fig. 1), they produced secondary metabolites which were not produced when the cells were grown in shake flasks. Different types of semipermeable membranes gave similar results, suggesting that the production of antimicrobial compounds was not due to the chemical composition of the membrane (Fig. 2). The physical environment of the bacteria when they were grown in the AMS bioreactor was similar to the physical environment when they were grown on an agar surface or in the natural environment on the surface of a seaweed. Production of bacitracin and red pigment was also detected in EI-34-6 colonies on NGF agar plates.

The effects of FeCl3 and glycerol on the production of bacitracin and red pigment confirmed that FeCl3 and glycerol are required for the synthesis of these two secondary metabolites. Coproduction of a pigment and other antimicrobial compounds has also been observed in Pseudoalteromonas (16). The red pigment produced by EI-34-6 has not been identified. Nevertheless, a biosynthetic pathway for a red pigment, pulcherrimin, produced by B. subtilis, has been reported and is known to be regulated by certain carbohydrates, ferric iron, and oxygen (38). The red pigment produced in this study may be a compound similar to pulcherrimin.

In order to further understand mechanisms by which growth in the AMS bioreactor affected the production of bacitracin and red pigment, the relationship between sporulation and the production of antimicrobial compounds by EI-34-6 was investigated. Production of antimicrobial compounds by bacilli often occurs at the onset of sporulation (26, 29). However, our results demonstrate that the production of bacitracin by EI-34-6 grown in the AMS bioreactor was not dependent on the level of sporulation (Fig. 5 and 6).

Another significant difference between cells grown in the AMS bioreactor and cells grown in shake flask cultures is cell density. The cell density of an EI-34-6 biofilm grown in the AMS bioreactor was 1012 CFU/cm3, while the maximum cell density of the corresponding shake flask culture was 108 CFU/ml (Table 2). The high cell density may elicit bacitracin production in the AMS bioreactor cultures. However, once the biofilm was washed and submerged in a shake flask culture, metabolite production was not detected, although the biofilm still maintained its shape. The density of the cells which were attached to the membrane but were in the shake flask culture was still high. At the same time, the cell density in the induced shake flask cultures remained 108 CFU/ml. Therefore, a high cell density is not the only factor required for production of bacitracin. When the experiment described above was carried out without prewashing of the biofilm, the biofilms submerged in shake flask cultures sometimes produced bacitracin and red pigment (Fig. 3). Clearly, compounds which were secreted from the biofilm-grown cells and which could be removed by washing played a key role in triggering the secretion of bacitracin and red pigment in shake flask cultures.

TABLE 2.

Cell densities of EI-34-6 grown in shake flask cultures and within biofilms in AMS bioreactors

Cultivation method Cell densitya
Shake flask culturesb
    Nylon membrane (1.1 ± 0.6) × 108
    Dialysis membrane (1.8 ± 0.3) × 108
    Cellophane (0.9 ± 0.2) × 108
AMS bioreactor cultures
    Nylon membrane (1.2 ± 0.7) × 1012
    Dialysis membrane (0.9 ± 0.3) × 1012
    Cellophane (1.9 ± 0.6) × 1012
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a

The cell densities in shake flask cultures are expressed in CFU per milliliter, and the cell densities in biofilm cultures on membranes are expressed in CFU per cubic centimeter.

b

Three types of semipermeable membranes were submerged in NGF liquid media when shake flask cultivation was carried out.

Addition of small quantities of cell-free spent medium or interstitial fluid from an AMS bioreactor culture to a shake flask culture could elicit production of bacitracin and the red pigment in the latter culture (Fig. 7). Addition of spent media did not significantly change the pH of the shake flask cultures; therefore, the induction was not due to changes in pH. If spent medium were supplying bacitracin precursors, then a dose-response effect should have been observed. However, this was not the case, since at volumes below a certain added volume (2 ml), no antimicrobial activity was detected and addition of a greater volume (10 ml) did not result in greater activity (43). Spent medium was added when bacteria reached the stationary phase in the shake flask cultures, and the cell density or total cell number after induction was not significantly different from that before addition of the spent medium.

The observed effects may have been due to the presence in the biofilm of inducer compounds that elicited the production of bacitracin and the red pigment. The spent medium beneath the nylon membrane and the cell-free interstitial fluid from the biofilm had similar inducing effects. Inducer compounds probably diffused into the medium beneath the nylon membrane, while some of the compounds remained in the biofilm. A biofilm grown at the air-membrane interface facilitated accumulation of inducer compounds. However, when the biofilm was washed before it was submerged in the shake flask, these inducer compounds were removed from the biofilm. Thus, it would be difficult for the inducer compounds to accumulate to a threshold level in the shake flask culture. Without washing, inducer compounds remaining in the biofilm could still elicit production of bacitracin and the red pigment. Ferric iron, glycerol, and growth within a biofilm formed at an air-membrane interface were all necessary for production of bacitracin and the red pigment by EI-34-6. However, spent media from EI-34-6 AMS-NB cultures (without glycerol and ferric iron) still exhibited inducing activity, although the spent media were not active. This suggests that the antimicrobial compounds themselves were not inducers. Ferric iron and glycerol are important for the production of antimicrobial compounds but are not required for production of the putative inducer compounds.

Our results suggest that initially, the physical environment provided by the AMS bioreactor is necessary for production of inducer compounds, which can subsequently elicit synthesis of bacitracin and the red pigment. However, once these inducer compounds have accumulated to a sufficient level, the physical environment is no longer necessary for induction. Thus, EI-34-6 cells grown within a biofilm can transfer expression of biofilm-specific phenotypes to planktonically grown cells by releasing certain compounds, which are resistant to heat and to DNase and protease digestion. Table 1 shows that spent media from AMS bioreactor cultures of both B. subtilis strain DSM10T and B. pumilus isolate EI-25-8 could also induce cultures of EI-34-6 to produce bacitracin and the red pigment, indicating that there was cross-species cell signaling (8, 27, 28).

The approaches used here have also been used to study cell-cell signaling molecules involved in other quorum-sensing phenomena in bacteria (15, 25, 30, 37). However, induction of bacitracin synthesis in biofilms is not entirely a cell density-dependent process, although the release of inducer compounds does appear to be density dependent. This biofilm-specific release of secondary metabolites could also result from the complex physical conditions in the AMS bioreactor biofilm, including high cell density, oxygen availability, a shortage of free water, and other heterogeneous conditions not found in shake flask cultures. Quorum-sensing studies have usually focused on bacteria growing in a homogeneous environment; however, in our studies we have examined the effects of different physical conditions on bacterial cell-cell communication in a heterogeneous environment. The ability of Bacillus species to release antimicrobial compounds once a biofilm has started to grow may also explain why Bacillus species are often found to be dominant in both medically (10) and environmentally important biofilms (31).

Our results also have important implications for the screening and discovery of antimicrobial compounds because specific molecules may be produced only when organisms are grown in biofilms. Further studies are being directed towards development of new bioreactors which can provide environmentally relevant growth conditions. The use of signal molecules to mimic certain biofilm-specific responses in liquid suspensions may also prove to be useful.

Acknowledgments

We thank the Scottish Hospital Endowments Research Trust for providing a research grant. Liming Yan thanks Heriot-Watt University for providing a partial scholarship.

We thank Sebastian Amyes of the Department of Medical Microbiology, University of Edinburgh, for the gift of pathogenic strains.

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