Biofilm-specific cross-species induction of antimicrobial compounds in bacilli

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 DSM10(T) 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 DSM10(T) 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.     

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

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 DSM10^T 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 DSM10^T 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.     

Improved fermentative production of Monascus pigments in roller bottle culture

The quantities and qualities of Monascus pigments produced by the filamentous fungus Monascus anka in batch submerged, agar surface, and roller bottle cultures were compared. In roller bottles, the fungus became attached to the wall of the culture vessels and produced a larger quantity of both intracellular (1508 absorbance units g⁻¹ cell mass) and extracellular (27 absorbance units g⁻¹ cell mass) Monascus red pigments, a yield that was about 10-fold greater than that of batch submerged and agar surface cultures. The optimum time required for maximum pigment production was reduced from 7 days in batch submerged or agar surface cultures to 4 days in roller bottle culture. In the roller bottle culture, the ratio of red to yellow pigments was also greatly increased. The advantage of the rotating vessel might be due to a combination of factors, including better gas exchange, higher medium pH, efficient pigment secretion, solid support for mycelium, and retarded conidiation.     

The symbiotic role of marine microbes on living surfaces

Every surface immersed in the sea rapidly becomes covered with a biofilm. On inanimate surfaces, this is often followed by colonisation by larger organisms, and general macrofouling. On the other hand, the majority of marine organisms remain relatively free from macrofouling, although some may be covered in a thin film of epibiotic bacteria. The role of these bacteria in maintaining the health of the host has received little attention. Here we describe an ecological role for epibiotic bacteria from seaweed surfaces. These epibionts may play a protective role, releasing compounds into the surrounding seawater that help prevent extensive fouling of the surface. These compounds may also have industrial and medical applications. The relative ease of culturing these microbes, compared to other bacteria that produce active compounds suggests seaweed-associated bacteria may be useful in bioprocess applications, such as the production of antimicrobial or antifouling compounds.     

A comparative study of biofilm formation by Shiga toxigenic Escherichia coli using epifluorescence microscopy on stainless steel and a microtitre plate method

Attachment of Shiga toxigenic Escherichia coli (STEC) to surfaces and the formation of biofilms may enhance persistence in a food processing environment and present a risk of contaminating products. Seven strains of STEC and three non-STEC strains were selected to compare two biofilm quantification methods; epifluorescence microscopy on stainless steel (SS) and a microtitre plate assay. The influence of prior growth in planktonic (nutrient broth) and sessile (nutrient agar) culture on biofilm production, as well as expression of surface structures and the possession of antigen 43 (encoded by agn43) on biofilm formation were also investigated. Biofilms were produced in diluted nutrient broth at 25 degrees C for 24 and 48 h. Curli expression was determined using congo red indicator agar, while the presence of agn43 was determined using polymerase chain reaction. No correlation was found between counts for epifluorescence microscopy on SS and the absorbance values obtained with the microtitre plate method for planktonic and sessile grown cultures. Different abilities of individual STEC strains to attach to SS and microtitre plates were found with some strains attaching better to each surface following growth in either planktonic or sessile culture. All O157 STEC strains had low biofilm counts on SS for planktonic and sessile grown cultures; however, one STEC O157:H- strain (EC516) had significantly greater (p<0.05) biofilm production on microtitre plates compared to the other O157 STEC strains. EC516 and other STEC (O174:H21 and O91:H21) strains expressing curli fimbriae were found to produce significantly greater (p<0.05) biofilms on microtitre plates compared to the non-curli expressing strains. No relationship was found between the production of type-I fimbriae, motility, agn43 and bacterial physicochemical properties (previously determined) and biofilm formation on SS or microtitre plates. Variations between the two biofilm determination methods may suggest that the biofilm production on microtitre plates may not be appropriate to represent other surfaces such as SS and that caution should be taken when selecting a method to quantify biofilm production on a surface.     

Antimicrobial activity of surface attached marine bacteria in biofilms

Relatively little is known about the microbial ecology of biofilm communities or the diversity of antimicrobial molecules that they produce to regulate these communities. This study tested whether the production of antimicrobial activity in biofilm cultures is enhanced towards competing bacteria found in those biofilms. First, the production of antimicrobial activity of marine bacteria grown in biofilms was tested. Fourteen of the 105 marine isolates tested were found to produce antimicrobial factors when grown in biofilms. The antimicrobial activity produced by these isolates in biofilms was more potent and inhibited a broader range of target bacteria grown in biofilms compared to shaken liquid cultures. In a separate experiment, we found that cultivation in biofilms containing produced metabolites from an ‘inducer’ bacterium stimulated the production of antimicrobial molecules by ‘producer’ bacteria that were active against the ‘inducer’ bacterium. Overall, the study suggests that surface attached marine bacteria can target their antimicrobial activity towards competing bacteria in biofilms.     

Cross-species induction of antibacterial activity produced by epibiotic bacteria isolated from Indian marine sponge Pseudoceratina purpurea

Four antibiotic producing bacteria were isolated from the surface of the marine sponge Pseudoceratina purpurea and exposed to living cells of two human pathogenic bacteria as well as some marine fouling bacteria to induce the production of antimicrobial activity. Experimental results showed that these four marine epibiotic bacteria enhanced their antibacterial production, when exposed to these test strains. The highest induction was exhibited by the sponge isolate PS79 against fouling bacterium FB-9 (from 3 mm to 7 mm inhibition zone). All the four strains were induced and showed increased activity specifically against the challenged pathogenic or fouling bacteria tested. Specific induction by these species suggests that the induction might be attributed to the response to the chemical signals received from potential challenger strains.     

Microbial antagonism: a neglected avenue of natural products research

Competition amongst microbes for space and nutrients in the marine environment is a powerful selective force which has led to the evolution of a variety of effective strategies for colonising and growing on surfaces. We are particularly interested in the chemical ecology of marine epibiotic bacteria which live on the surfaces of marine algae or invertebrates. Over 400 strains of surface-associated bacteria from various species of seaweed and invertebrate from Scottish coastal waters were isolated and 35% of them shown to produce antimicrobial compounds. This is a much higher proportion than free living marine isolates or soil bacteria. In addition, many strains which did not normally produce antibiotics could be induced to do so by exposing them to small amounts of live cells, supernatants from other bacterial cultures or other chemicals. Thus the number of strains able to produce antibiotics appears to be much higher than previously thought. Induction of antibiotic production was elicited by other marine epibionts and also by terrestrial human pathogens such as Bacillus subtilis, Escherichia coli and Pseudomonas aeruginosa. An understanding of this type of chemical induction and the factors regulating non-constitutive secretion of antimicrobial compounds will allow more effective strategies for searching for new chemotherapeutic antibiotics to be designed.     

Biofilm formation and interactions of bacterial strains found in wastewater treatment systems

Biofilm formation and adherence properties of 13 bacterial strains commonly found in wastewater treatment systems were studied in pure and mixed cultures using a crystal violet microtiter plate assay. Four different culture media were used, wastewater, acetate medium, glucose medium and diluted nutrient broth. The medium composition strongly affected biofilm formation. All strains were able to form pure culture biofilms within 24 h in at least one of the tested culture media and three strains were able to form biofilm in all four culture media, namely Acinetobacter calcoaceticus ATCC 23055, Comamonas denitrificans 123 and Pseudomonas aeruginosa MBL 0199. The adherence properties assessed were initial adherence, cell surface hydrophobicity, and production of amyloid fibers and extracellular polymeric substances. The growth of dual-strain biofilms showed that five organisms formed biofilm with all 13 strains while seven formed no or only weak biofilm when cocultured. In dual-strain cultures, strains with different properties were able to complement each other, giving synergistic effects. Strongest biofilm formation was observed when a mixture of all 13 bacteria were grown together. These results on attachment and biofilm formation can serve as a tool for the design of tailored systems for the degradation of municipal and industrial wastewater.     

Cell growth optimization in microcarrier culture

Three monkey kidney cell lines and primary chicken embryo cells were grown in microcarrier culture. The carrier support was DEAE-Sephandex gel beads at low anion exchange capacity prepared according to a protocol developed at the Massachusetts Institute of Technology. The growth rate of the cells and the final cell density in microcarrier culture was dependent on the concentration of the beads in culture and on the size of the initial cell inoculum. A bead concentration of 1.0 to 2.0 mg of beads/ml of tissue culture medium and a cell inoculum of 20,000 cells/cm2 of bead surface appeared to be optimal. The efficiency of the microcarrier culture system was compared to that of stationary and roller bottle cultures. Stationary flasks gave cell densities about twofold higher than maximal densities in roller bottles and about threefold and twofold higher than cell densities in microcarrier culture at a bead concentration of 2.5 and 1.0 mg/ml, respectively. In terms of cell yield per milliliter of tissue culture medium, the microcarrier culture was superior to roller bottle and stationary cultures. An advantage of the microcarrier culture system is its suitability for scale up into large volume production units.     

Cross-Species Induction of Antimicrobial Compounds, Biosurfactants and Quorum-Sensing Inhibitors in Tropical Marine Epibiotic Bacteria by Pathogens and Biofouling Microorganisms

Enhancement or induction of antimicrobial, biosurfactant, and quorum-sensing inhibition property in marine bacteria due to cross-species and cross-genera interactions was investigated. Four marine epibiotic bacteria (Bacillus sp. S3, B. pumilus S8, B. licheniformis D1, and Serratia marcescens V1) displaying antimicrobial activity against pathogenic or biofouling fungi (Candida albicans CA and Yarrowia lipolytica YL), and bacteria (Pseudomonas aeruginosa PA and Bacillus pumilus BP) were chosen for this study. The marine epibiotic bacteria when co-cultivated with the aforementioned fungi or bacteria showed induction or enhancement in antimicrobial activity, biosurfactant production, and quorum-sensing inhibition. Antifungal activity against Y. lipolytica YL was induced by co-cultivation of the pathogens or biofouling strains with the marine Bacillus sp. S3, B. pumilus S8, or B. licheniformis D1. Antibacterial activity against Ps. aeruginosa PA or B. pumilus BP was enhanced in most of the marine isolates after co-cultivation. Biosurfactant activity was significantly increased when cells of B. pumilus BP were co-cultivated with S. marcescens V1, B. pumilus S8, or B. licheniformis D1. Pigment reduction in the quorum-sensing inhibition indicator strain Chromobacterium violaceum 12472 was evident when the marine strain of Bacillus sp. S3 was grown in the presence of the inducer strain Ps. aeruginosa PA, suggesting quorum-sensing inhibition. The study has important ecological and biotechnological implications in terms of microbial competition in natural environments and enhancement of secondary metabolite production.     

Cell growth optimization in microcarrier culture

Summary Three monkey kidney cell lines and primary chicken embryo cells were grown in microcarrier culture. The carrier support was DEAE-Sephadex gel beads at low anion exchange capacity prepared according to a protocol developed at the Massachusetts Institute of Technology. The growth rate of the cells and the final cell density in microcarrier culture was dependent on the concentration of the beads in culture and on the size of the initial cell inoculum. A bead concentration of 1.0 to 2.0 mg of beads/ml of tissue culture medium and a cell inoculum of 20,000 cells/cm² of bead surface appeared to be optimal. The efficiency of the microcarrier culture system was compared to that of stationary and roller bottle cultures. Stationary flasks gave cell densities about twofold higher than maximal densities in roller bottles and about threefold and twofold higher than cell densities in microcarrier culture at a bead concentration of 2.5 and 1.0 mg/ml, respectively. In terms of cell yield per millitier of tissue culture medium, the microcarrier culture was superior to roller bottle and stationary cultures. An advantage of the microcarrier culture system is its suitability for a scale up into large volume production units.     

Artificial cell-immobilization: a model simulating immobilization in natural environments?

A study of the marine diatom Haslea ostrearia was performed in controlled culture conditions. Biofilm formation by cells grown in liquid medium was studied through macroscopic events and extracellular polysaccharides (EPS) production. Alcian blue staining and image analysis methods were used to quantify EPS production in regard to the usual pattern (lag, exponential and stationary phases) of microalgal growth. Simultaneously, an artificial immobilization of H. ostrearia cells was carried out using an agar layer. Samples from liquid medium and artificially immobilized cultures were studied in scanning electron microscopy. They revealed the structure of the entrapping material and progressive changes of the natural biofilm along culture ageing. The use of artificially immobilized cultures for solute diffusivity investigations is discussed. This revised version was published online in July 2006 with corrections to the Cover Date.     

Production of antibacterial compounds and biofilm formation by Roseobacter species are influenced by culture conditions

Bacterial communities associated with marine algae are often dominated by members of the Roseobacter clade, and in the present study, we describe Roseobacter phenotypes that may provide this group of bacteria with selective advantages when colonizing this niche. Nine of 14 members of the Roseobacter clade, of which half were isolated from cultures of the dinoflagellate Pfiesteria piscicida, produced antibacterial compounds. Many non-Roseobacter marine bacteria were inhibited by sterile filtered supernatants of Silicibacter sp. TM1040 and Phaeobacter (formerly Roseobacter) strain 27-4, which had the highest production of antibacterial compound. In contrast, Roseobacter strains were susceptible only when exposed to concentrated compound. The production of antibacterial compound was influenced by the growth conditions, as production was most pronounced when bacteria were grown in liquid medium under static conditions. Under these conditions, Silicibacter sp. TM1040 cells attached to one another, forming rosettes, as has previously been reported for Phaeobacter 27-4. A spontaneous Phaeobacter 27-4 mutant unable to form rosettes was also defective in biofilm formation and the production of antibacterial compound, indicating a possible link between these phenotypes. Rosette formation was observed in 8 of 14 Roseobacter clade strains examined and was very pronounced under static growth in 5 of these strains. Attachment to surfaces and biofilm formation at the air-liquid interface by these five strains was greatly facilitated by growth conditions that favored rosette formation, and rosette-forming strains were 13 to 30 times more efficient in attaching to glass compared to strains under conditions where rosette formation was not pronounced. We hypothesize that the ability to produce antibacterial compounds that principally inhibit non-Roseobacter species, combined with an enhancement in biofilm formation, may give members of the Roseobacter clade a selective advantage and help to explain the dominance of members of this clade in association with marine algal microbiota.     

Multisurface Glass Roller Bottle for Growth of Animal Cells in Culture

A multisurface glass roller bottle has been constructed for the growth of animal cells in culture. This bottle contains five concentrically placed glass cylinders that provide additional surfaces for the growth of animal cells. The bottle occupies the same space as a standard roller bottle, but it contains nine times the surface area of a standard bottle. L and HeLa cells can be grown in the bottle with cell yields 5- to 10-fold greater than in a standard bottle. L cells can be induced to produce interferon in the multisurface bottle.     

Lecithinase activity of Proteus vulgaris

Lecithinase production is described as a new biochemical property of P. vulgaris strains grown in a selective agar medium containing brilliant green, crystal violet and lecithin (BCL agar), the authors' own modification of egg-yolk culture medium. By using this BCL agar as a medium inhibiting the swarming growth of P. vulgaris cultures the authors succeeded in identifying 12 lecithinase-positive strains among the P. vulgaris isolates obtained from patients with Crohn's disease. Of 50 P. mirabilis strains tested in parallel none gave the positive test for lecithinase production in this medium.     

Physicochemical properties of Shiga toxigenic Escherichia coli

AIMS: To investigate the physicochemical surface properties, such as cellular surface charge, hydrophobicity and electron donor/acceptor potential of a selection of Shiga toxigenic Escherichia coli (STEC) isolates grown in broth and agar culture. METHODS AND RESULTS: Cellular surface charge was determined using zeta potential measurements. Hydrophobicity of the isolates was determined using bacterial adhesion to hydrocarbons assay, hydrophobic interaction chromatography and contact angle measurements. Microbial adhesion to solvents was used to determine the electron donor/acceptor characteristics. No differences of surface charge measurements were found between broth and agar grown cultures. Isolates belonging to serogroup O157 and serotypes O26:H11 and O111:H- were significantly (P < 0.05) less negatively charged than other STEC serotypes tested. All strains were hydrophilic with most methods and demonstrated a lower hydrophobicity in agar culture compared with broth culture. All strains demonstrated a strong microbial adhesion to chloroform indicating that STEC possess an electron donor and basic character. A relationship between serogroup O157 and other STEC serotypes was apparent using principal-component analysis (PCA). CONCLUSIONS: Combining the results for physicochemical properties using PCA differentiated between strains belonging to the O157 serogroup and other STEC/non-STEC strains. PCA found similar results for broth and agar grown cultures. SIGNIFICANCE AND IMPACT OF THE STUDY: Particular serotypes of STEC possess similar physicochemical properties which may play a role in their pathogenicity or potential attachment to various surfaces.     

Effect of Bacteria Associated with the Green Alga Ulva reticulata on Marine Micro- and Macrofouling

The green alga Ulva reticulata (Forsskal) is often free from biofouling in Hong Kong waters. An early study indicated that bioactive substances from this alga inhibit settlement of the polychaete Hydroides elegans (Haswell). It is also predicted that epibiotic bacteria protect this alga from micro- and macrofouling. In this study, bacterial strains from the surface of U. reticulata were isolated and their inhibitive activities on micro- and macrofouling assayed. The strains were identified by 16S rRNA analysis as belonging to the genera Alteromonas , Pseudoalteromonas and Vibrio . There was no significant effect of these strains or their extracts (aqueous and ethanol) on the growth of five Vibrio strains isolated from natural biofilm. Two bacterial strains ( Alteromonas sp. and Vibrio sp. 3) were non-toxic to the benthic diatom Nitzschia paleacea (Grunow) while the other five strains caused a low level of mortality. No one bacterial strain was toxic to the larvae of H. elegans . Aqueous extract of one of the isolated bacterial species, i.e. Vibrio sp. 2, significantly ( p <0.00001) inhibited the settlement and metamorphosis of H. elegans larvae. The putative antifouling compounds have a molecular weight of >100 kD. On the other hand, biofilm of Pseudoalteromonas sp. 2 and aqueous extract of Vibrio sp. 2 suppressed the settlement of larvae induced by 3-isobutyl-1-methylxanthine (IBMX). Other epibiotic bacteria and their extracts had neither inhibitive nor inductive effects on larval settlement of H. elegans . The results indicate that the antifouling mechanism of U. reticulata may be dependent not only on materials from the macroalga itself but also on the epibiotic bacteria on the algal surface.     

Application of a multiple surface tissue culture propagator for the production of cell monolayers,virus, and biochemicals

A new concept of tissue culture equipment and procedures was developed for the mass-scale growth of several types of animal tissue cells in monolayers on multiple glass surfaces. Continuous, cell lines, primary and diploid cell strains were grown in this equipment. Cells studied include primary bovine kidney, human diploid WI-38, human foreskin, and mouse CCL1 cells. Photomicrographic comparisons of cells grown by these techniques indicate they are morphologically identical to tissue culture cells grown in glass bottles or tubes. The growth of the tissue culture cells in the propagator was monitored by carbohydrate Utilization and acid production. Large-scale production of viruses and biochemicals on cells grown in the multiple-plate tissue culture propagator was accomplished. Virus titers were equal to those obtained from conventional bottle or tube cultures for several strains of influenza, parainfluenza, and respiratory syneytial viruses. High-titred mouse interferon was also produced in this system. In addition to tissue culture cell production, Eaton agent, Mycoplasma pneumoniae was grown on the multiple glass surfaces on a mass scale.