Biofilm Dispersal of Neisseria subflava and Other Phylogenetically Diverse Oral Bacteria

Polystyrene petri dishes containing liquid medium were inoculated with single-cell suspensions of a fresh clinical isolate of Neisseria subflava and were incubated under conditions of low vibration. N. subflava colonies grew firmly attached to the surface of the dish, while the broth remained clear. Growing colonies released cells into the medium, resulting in the appearance of 10² to 10⁴ small satellite colonies attached to the surface of the dish in an area adjacent to each mature colony after 24 h. Satellite colonies grew in patterns of streamers shaped like jets and flares emanating from mature colonies and pointing toward the center of the dish. This dispersal pattern evidently resulted from the surface translocation of detached biofilm cells by buoyancy-driven convection currents that were generated due to slight temperature gradients in the medium. Streamers of satellite colonies ranged from 2 to >40 mm in length. Satellite colonies in very long streamers were relatively uniform in size regardless of their distance from the mature colony, suggesting that mature colonies released single cells or small clusters of cells into the medium and that the detachment, surface translocation, and subsequent surface reattachment of released cells were a transitory process. Incubation of N. subflava single cells in a perfused biofilm fermentor resulted in a large spike of the number of CFU in the perfusate after 9.5 h of growth, consistent with a rapid release of cells into the medium. Biofilm colonies of several other phylogenetically diverse oral bacteria, including Actinobacillus actinomycetemcomitans, Haemophilus aphrophilus, Streptococcus mitis, and a prevalent but previously uncultured oral Streptococcus sp., exhibited similar temperature-dependent dispersal patterns in broth culture. This in vitro spreading phenotype could be a useful tool for studying biofilm dispersal in these and other nonflagellated bacteria and may have physiological relevance to biofilm dispersal in the oral cavity.     

Biofilm growth and detachment of Actinobacillus actinomycetemcomitans

The gram-negative, oral bacterium Actinobacillus actinomycetemcomitans has been implicated as the causative agent of several forms of periodontal disease in humans. When cultured in broth, fresh clinical isolates of A. actinomycetemcomitans form tenacious biofilms on surfaces such as glass, plastic, and saliva-coated hydroxyapatite, a property that probably plays an important role in the ability of this bacterium to colonize the oral cavity and cause disease. We examined the morphology of A. actinomycetemcomitans biofilm colonies grown on glass slides and in polystyrene petri dishes by using light microscopy and scanning and transmission electron microscopy. We found that A. actinomycetemcomitans developed asymmetric, lobed biofilm colonies that displayed complex architectural features, including a layer of densely packed cells on the outside of the colony and nonaggregated cells and large, transparent cavities on the inside of the colony. Mature biofilm colonies released single cells or small clusters of cells into the medium. These released cells adhered to the surface of the culture vessel and formed new colonies, enabling the biofilm to spread. We isolated three transposon insertion mutants which produced biofilm colonies that lacked internal, nonaggregated cells and were unable to release cells into the medium. All three transposon insertions mapped to genes required for the synthesis of the O polysaccharide (O-PS) component of lipopolysaccharide. Plasmids carrying the complementary wild-type genes restored the ability of mutant strains to synthesize O-PS and release cells into the medium. Our findings suggest that A. actinomycetemcomitans biofilm growth and detachment are discrete processes and that biofilm cell detachment evidently involves the formation of nonaggregated cells inside the biofilm colony that are destined for release from the colony.     

Effect of 2-hydroxyethyl methacrylate on Streptococcus spp. biofilms

Aims: The effect of different concentrations of 2‐hydroxyethyl methacrylate (HEMA) was evaluated on biofilm formation and preformed biofilm of Streptococcus mitis, Streptococcus mutans and Streptococcus oralis, alone or combined to each other. Methods and Results: Twofold serial dilution of HEMA ranged from 12 to 0·75 mmol l^?1 was added to Streptococcal broth cultures and mature biofilms in 96‐well‐microtitre plates to evaluate bacterial biomass and cell viability. HEMA affected the Streptococcal population in a strain‐specific way producing few significant effects. A reduction on biofilm formation and a detachment of preformed biofilm was recorded in Strep. mitis ATCC 6249, whereas in mixed cultures, the monomer expressed a general aggregative effect on mature biofilms. A reduction in cell viability was also recorded in an HEMA‐concentration‐dependent way in each experimental condition studied. Conclusions: These results suggest that the HEMA prevalent effects are both the reduction of bacterial adhesion to a polystyrene surface and the increase in dead cells also characterized by an aggregative status. Significance and Impact of the Study: Understanding the potential effect of HEMA, released from resin‐based materials, on oral bacteria may furnish information for surveillance of the risk reduction in secondary caries via hindering biofilm generation.     

Detachment of Actinobacillus actinomycetemcomitans biofilm cells by an endogenous beta-hexosaminidase activity

When cultured in broth, fresh clinical isolates of the gram-negative periodontal pathogen Actinobacillus actinomycetemcomitans form tenaciously adherent biofilm colonies on surfaces such as plastic and glass. These biofilm colonies release adherent cells into the medium, and the released cells can attach to the surface of the culture vessel and form new colonies, enabling the biofilm to spread. We mutagenized A. actinomycetemcomitans clinical strain CU1000 with transposon IS903phikan and isolated a transposon insertion mutant that formed biofilm colonies which were tightly adherent to surfaces but which lacked the ability to release cells into the medium and disperse. The transposon insertion in the mutant strain mapped to a gene, designated dspB, that was predicted to encode a secreted protein homologous to the catalytic domain of the family 20 glycosyl hydrolases. A plasmid carrying a wild-type dspB gene restored the ability of biofilm colonies of the mutant strain to disperse. We expressed A. actinomycetemcomitans DspB protein engineered to contain a hexahistidine metal-binding site at its C terminus in Escherichia coli and purified the protein by using Ni affinity chromatography. Substrate specificity studies performed with monosaccharides labeled with 4-nitrophenyl groups showed that DspB hydrolyzed the 1-->4 glycosidic bond of beta-substituted N-acetylglucosamine, which is consistent with the known functions of other family 20 glycosyl hydrolases. When added to culture medium, purified DspB protein, but not heat-inactivated DspB, restored the ability of the mutant strain to release cells and disperse. DspB protein also caused the detachment of cells from preformed biofilm colonies of strain CU1000 grown attached to plastic and the disaggregation of highly autoaggregated clumps of CU1000 cells in solution. We concluded that dspB encodes a soluble beta-N-acetylglucosaminidase that causes detachment and dispersion of A. actinomycetemcomitans biofilm cells.     

Phenotypic diversification and adaptation of Serratia marcescens MG1 biofilm-derived morphotypes

We report here the characterization of dispersal variants from microcolony-type biofilms of Serratia marcescens MG1. Biofilm formation proceeds through a reproducible process of attachment, aggregation, microcolony development, hollow colony formation, and dispersal. From the time when hollow colonies were observed in flow cell biofilms after 3 to 4 days, at least six different morphological colony variants were consistently isolated from the biofilm effluent. The timing and pattern of variant formation were found to follow a predictable sequence, where some variants, such as a smooth variant with a sticky colony texture (SSV), could be consistently isolated at the time when mature hollow colonies were observed, whereas a variant that produced copious amounts of capsular polysaccharide (SUMV) was always isolated at late stages of biofilm development and coincided with cell death and biofilm dispersal or sloughing. The morphological variants differed extensively from the wild type in attachment, biofilm formation, and cell ultrastructure properties. For example, SSV formed two- to threefold more biofilm biomass than the wild type in batch biofilm assays, despite having a similar growth rate and attachment capacity. Interestingly, the SUMV, and no other variants, was readily isolated from an established SSV biofilm, indicating that the SUMV is a second-generation genetic variant derived from SSV. Planktonic cultures showed significantly lower frequencies of variant formation than the biofilms (5.05 x 10(-8) versus 4.83 x 10(-6), respectively), suggesting that there is strong, diversifying selection occurring within biofilms and that biofilm dispersal involves phenotypic radiation with divergent phenotypes.     

Mononuclear phagocyte colony formation of human spleen cells in liquid culture

We found that mononuclear phagocytes formed a distinct number of clusters and colonies on the bottom of a culture dish 7 days later but granulocytes did not, when a large number of human spleen cells were cultured in liquid medium. In all gastric cancer bearers and patients with portal hypertension operated on, however, colony formation was restricted to spleen cells from patients with advanced gastric cancer and from a group of patients with portal hypertension. These spleen cells formed mononuclear phagocyte colonies without the help of exogeneous colony stimulating factor (CSF). We further demonstrated that the colony-forming cells were glass non-adherent and nylon wool adherent, and that spontaneous colony formation required cooperation between the colony-forming cells and colony-stimulating cells adherent to a plastic surface.     

Identification of synergistic interactions among microorganisms in biofilms by digital image analysis.

Digital image analysis showed that reductions in biofilm plating efficiency were due to the loss of protection provided by two benzoate-degrading strains of Pseudomonas fluorescens. This loss in protection was due to the spatial separation of the protective organisms from benzoate-sensitive organisms during the dilution process. Communities were cultivated in flow cells irrigated with trypticase soy broth. When the effluent from these flow cells was plated on 0.15% benzoic acid, satellite colonies formed only in the vicinity of primary colonies. A digital image analysis procedure was developed to measure the size and spatial distribution of these satellites as a function of distance from the primary colony. The size of satellites served as a measure of growth, and the number per unit area served as a measure of survival. At the three dilutions tested, the size and concentration of satellite colonies varied inversely with distance from the primary colonies. When these measurements were plotted, the slopes were used to quantify the effect of bacterial association on the growth and survivability of the satellites. In the absence of the primary colonies, satellites grew in axenic culture only at low benzoate concentrations. Thus benzoate-degrading organisms are capable of creating a protective microenvironment for other members of biofilm communities.     

Multiparameter assessments to determine the effects of sugars and antimicrobials on a polymicrobial oral biofilm

Clinical studies indicate relationships between dental plaque, a naturally formed biofilm, and oral diseases. The crucial role of nonmicrobial biofilm constituents in maintaining biofilm structure and biofilm-specific attributes, such as resistance to shear and viscoelasticity, is increasingly recognized. Concurrent analyses of the diverse nonmicrobial biofilm components for multiparameter assessments formed the focus of this investigation. Comparable numbers of Actinomyces viscosus, Streptococcus sanguinis, Streptococcus mutans, Neisseria subflava, and Actinobacillus actinomycetemcomitans cells were seeded into multiple wells of 96-well polystyrene plates for biofilm formation. Quantitative fluorescence and confocal laser scanning microscopy (CLSM) examined the influences of dietary sugars, incubation conditions, ingredients in oral hygiene formulations, and antibiotics on biofilm components. Biofilm extracellular polymeric substances (EPS) were examined with an optimized mixture of fluorescent lectins, with biofilm proteins, lipids, and nucleic acids detected with specific fluorescent stains. Anaerobic incubation of biofilms resulted in significantly more biofilm EPS and extractable carbohydrates than those formed under aerobic conditions (P < 0.05). Sucrose significantly enhanced biofilm EPS in comparison to fructose, galactose, glucose, and lactose (P < 0.05). CLSM demonstrated thicker biofilms under sucrose-replete conditions, along with significant increases in biofilm EPS, proteins, lipids, and nucleic acids, than under conditions of sucrose deficiency (P < 0.05). Agents in oral hygiene formulations (chlorhexidine, ethanol, and sodium lauryl sulfate), a mucolytic agent (N-acetyl-L-cysteine), and antibiotics with different modes of action (amoxicillin, doxycycline, erythromycin, metronidazole, and vancomycin) inhibited biofilm components (P < 0.05). Multiparameter analysis indicated a dose-dependent inhibition of biofilm EPS and protein by chlorhexidine and sodium lauryl sulfate, along with distinctive inhibitory patterns for subinhibitory concentrations of antibiotics. Collectively, these results highlight multiparameter assessments as a broad platform for simultaneous assessment of diverse biofilm components.     

Functional differences between cells from MLR colonies grown in soft agar cultures

This report describes a method of growing soft agar colonies of human T lymphocytes activated in the MLR. Two types of colonies were demonstrated: lower colonies grew within the agar layer, and upper colonies grew on the surface of the agar layer. Three days of priming the lymphocytes in the MLR and the use of supernatants of day-3 MLR cultures to provide T cell colony growth factor were necessary for optimal colony formation. Lymphocytes obtained from colonies were grown in long-term (2 to 4 weeks) cultures to generate sufficient numbers of cells to be tested in different functional assays. Cells from both types of colonies exhibited PLT activity. Upper colony cells showed considerably higher CML activity than lower colony cells (mean percent cytotoxicity 37 +/- 5 vs 6 +/- 3). Cells from both types of colonies contained radiosensitive suppressor cell activity that inhibited the primary MLR. The suppressor cell effect of lower colony cells was specific for the original stimulator, but upper colony cells displayed nonspecific suppressive effects. For both types of colony cells, it appeared that suppressive effects were unrelated to the CML activity of these cells. These data suggest that the soft agar colony assay offers a promising approach to separate subpopulations of lymphocytes activated in the MLR.     

Regulation of human B-cell colony growth

PHA-induced B-cell enriched populations from venous blood of healthy adults developed into B-cell colonies. Analyses of individual colonies revealed that 80-85% of the cells in each colony were surface membrane immunoglobulin positive. Most colonies, 84%, contained surface IgM-bearing cells. Only a few, 16%, were found with surface IgG-bearing cells. Surface IgM- and surface IgG-bearing cells were not observed in the same colony. Thirty-nine percent of the colonies contained cells bearing surface IgD in addition to either surface IgM- or surface IgG-bearing cells. There was no evidence of cytoplasmic immunoglobulin in the colony cells. The development of B-cell colonies was T-cell dependent; it appears that at least two different T-cell subpopulations, one with low density (D = 1.05) and the other with high density (D = 1.08) are responsible for this helper effect. Monocytes were found to inhibit B-cell colony formation; the inhibition was mainly by endogenous prostaglandin E2 (PGE2) synthetized and released by monocytes. The addition of physiological concentrations of synthetic PGE2 to monocyte-depleted B-cell enriched populations inhibited B-cell colony growth, this paralleled the effect of endogenous PGE2 released by monocytes. Indomethacin (10-5 M) obviated the inhibitory effect of monocytes.     

The in vitro effect of manuka honeys on growth and adherence of oral bacteria

Honey has been used since ancient times and more recently, for the healing of wounds and against infectious diseases. The aim of our study was to investigate the effect of two manuka honeys showing different potencies of their antibacterial activity, on potentially pathogenic oral bacteria. The antimicrobial activity was examined by determining the MIC and MBC using the macro dilution broth technique. The effect on the adherence was tested on growing cells of Streptococcus mutans on a glass surface and on a multi-species biofilm grown on saliva-coated hydroxyapatite discs. As expected, the antibacterial activity of manuka 1 (with higher potency of antibacterial activity) was the most important. The two tested honeys weakly inhibited the adherence of S.mutans cells to a glass surface at sub-MIC concentration. Manuka 1 showed a total inhibition of multi-species biofilm at the concentration of 200 μg/ml manuka 2 inhibited biofilm formation weakly at the concentration of 200 μg/ml but firmly at the concentration of 500 μg/ml. Our findings suggest that manuka honeys might be able to reduce oral pathogens within dental plaque. These two honeys appear to be able to control dental biofilm deposit.     

Modeling the interactions of Lactobacillus curvatus colonies in solid medium: consequences for food quality and safety

The growth process of Lactobacillus curvatus colonies was quantified by a coupled growth and diffusion equation incorporating a volumetric rate of lactic acid production. Analytical solutions were compared to numerical ones, and both were able to predict the onset of interaction well. The derived analytical solution modeled the lactic acid concentration profile as a function of the diffusion coefficient, colony radius, and volumetric production rate. Interaction was assumed to occur when the volume-averaged specific growth rate of the cells in a colony was 90% of the initial maximum rate. Growth of L. curvatus in solid medium is dependent on the number of cells in a colony. In colonies with populations of fewer than 10(5) cells, mass transfer limitation is not significant for the growth process. When the initial inoculation density is relatively high, colonies are not able to grow to these sizes and growth approaches that of broth cultures (negligible mass transfer limitation). In foods, which resemble the model solid system and in which the initial inoculation density is high, it will be appropriate to use predictive models of broth cultures to estimate growth. For a very low initial inoculation density, large colonies can develop that will start to deviate from growth in broth cultures, but only after large outgrowth.     

Antimicrobial effects of herbal extracts on Streptococcus mutans and normal oral streptococci

Streptococcus mutans is associated with dental caries. A cariogenic biofilm, in particular, has been studied extensively for its role in the formation of dental caries. Herbal extracts such as Cudrania tricuspidata, Sophora flavescens, Ginkgo biloba, and Betula Schmidtii have been used as a folk remedy for treating diseases. The purpose of this study was to evaluate and compare the antibacterial activity of herbal extracts against normal oral streptococci, planktonic and biofilm of S. mutans. Streptococcus gordonii, Streptococcus oralis, Streptococcus salivarius, Streptococcus sanguinis, and S. mutans were cultivated with brain heart infusion broth and susceptibility assay for the herbal extracts was performed according to the protocol of Clinical and Laboratory Standard Institute. Also, S. mutans biofilm was formed on a polystyrene 12-well plate and 8-well chamber glass slip using BHI broth containing 2% sucrose and 1% mannose after conditioning the plate and the glass slip with unstimulated saliva. The biofilm was treated with the herbal extracts in various concentrations and inoculated on Mitis-Salivarius bacitracin agar plate for enumeration of viable S. mutans by counting colony forming units. Planktonic S. mutans showed susceptibility to all of the extracts and S. mutans biofilm exhibited the highest level of sensitivity for the extracts of S. flavescens. The normal oral streptococci exhibited a weak susceptibility in comparison to S. mutans. S. oralis, however, was resistant to all of the extracts. In conclusion, the extract of S. flavescens may be a potential candidate for prevention and management of dental caries.     

Proteolysis and amino acid recycling during nitrogen deprivation inSchizophyllum commune

Transfer of exponentially growing colonies ofSchizophyllum commune to nitrogen-deficient media for 6–60 h resulted in increased general proteolytic activity and decreased extractable protein when compared with controls. A concomitant increase in free radiolabeled leucine was detected in nitrogen-deprived colonies. Radiolabel was found in new surface hyphae following transfer of previously labeled colonies to media containing no label. The concentration of label present in the new growth indicates that this label is likely to have originated from proteolytic release of label from proteins in the older, central portions of the colony. The release of substantial amounts of label into the growth medium suggests that an extra-mycelial pathway may account, at least in part, for translocation of amino acids released by proteolysis.     

Clockwise and counterclockwise pinwheel colony morphologies of Bacillus subtilis are correlated with the helix hand of the strain

Helical macrofiber-producing strains of Bacillus subtilis grown on fresh complex medium semisolid surfaces formed "pinwheel"-shaped colonies. Clockwise pinwheel projections arose from colonies of strains that produce right-handed helical macrofibers in fluid cultures. Most strains able to make left-handed helical macrofibers in fluid grew as disorganized wavy colonies without directed projections. A phage-resistant left-handed mutant was found that produces very tight colonies with pinwheel projections that lie counterclockwise relative to the colony. The pinwheel colony morphology is interpreted therefore in terms of the cell surface organization and helical growth.     

Hybridoma cell lines secreting monoclonal antibodies against foot-and-mouth disease virus (FMDV). II. Cloning conditions

Three methods of cloning hybridoma cells--picking colonies from the masterplate, limit dilution cloning, and cloning in semi-solid medium over macrophage (m phi) feeder layers--were compared. Cloning in semi-solid medium was found to be the most efficient and reliable, especially with our relatively slow growing anti-foot-and-mouth disease virus (FMDV) antibody secreting hybridoma cells. The optimum culture dish for this cloning was the 6-well (6W) dish (well diameter 1.5 cm), while the optimum cloning procedure was 10 to 10(2) hybridoma cells in 1% (w/v) methylcellulose in Dulbecco's minimal essential medium (DMEM) supplemented with 50% (v/v) conditioned medium, 10% (v/v) foetal or donor calf serum and 2 mM glutamine, over a 24-48 h old culture of syngeneic m phi in each well of the 6W plate. This could, however, produce problems in that colony formation was sometimes 'loose', and confident picking of individual colonies was impaired. Such a problem could be avoided by using a solid agar interface of 1% (w/v) agar Noble in DMEM or RPMI 1640 medium between the feeder cells and the hybridoma cells in semi-solid medium.     

专一性PCR和变性梯度胶电泳协助从焦化废水处理装置中分离优势功能菌Thauera属菌株

【目的】在专一性PCR和变性梯度胶电泳(DGGE)的协助下,从废水处理装置的微生物群落中分离较难分离的功能菌Thauera。【方法和结果】本研究首先使用Thauera特异性PCR-DGGE的方法鉴定了焦化废水处理装置反硝化池生物膜中的Thauera在6种培养基、好氧/厌氧条件下的生长情况。挑选Thauera多样性较高的培养基1/10 NB与MMQ在好氧条件下进行分离培养。然后使用Thauera特异性PCR方法确定分离得到的菌落是否呈阳性,并使用DGGE的方法检验其是否为纯菌。使用不同培养基对含有Thauera的混合菌落进行进一步纯化,DGGE检测发现MMP培养基对混合细菌菌落Q20中的Thauera有明显的富集作用。经过Thauera特异性PCR结合DGGE检测对Thauera属细菌进行追踪,将混合菌落在MMP培养基上多次划线,最终分离得到纯菌。通过这种方法,从反硝化池样品中分离获得了3株在样品中最为主要的Thauera菌株。【结论】以特异性分子标记为导向分离培养细菌,不仅提高了分离效率及细菌筛选的灵敏度,还能协助分离常规方法难以分离的细菌。     

Strategies for adaptation to antibiotics in wild type <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> and in the strains with small colony phenotype

Emergence of ciprofloxacin stress-induced mutants in the cultures of a collection strain Pseudomonas aeruginosa ATCC 27853 and of two strains with a small colony phenotype, which were isolated from a swimming pool biofilm, was studied. In biofilm cultures of the small colony phenotype strains, which were already resistant to hypochlorite, prolonged incubation (up to 16 days) with sublethal ciprofloxacin concentrations was shown to result in emergence of the cells, which are resistant to the antibiotic and form colonies on media with rifampicin (100 μg/mL) and streptomycin (50 μg/mL). Under the same conditions, the mechanisms of temporary adaptation are switched on in the cells of strain ATCC 27853, which enabled its shortterm survival at an average level in liquid media and provided for colony formation on solid medium with ciprofloxacin (0.2 μg/mL). Only 20% of these colonies remained viable when transferred to a higher antibiotic concentration (2 μg/mL).     

Confocal Raman microspectroscopy as a tool for studying the chemical heterogeneities of biofilms in situ

AIMS: To investigate the use of confocal Raman microspectroscopy (CRM) for the analysis of the structure, composition and development of fully hydrated biofilms. METHODS AND RESULTS: Pseudomonas aeruginosa PAO1 biofilms were cultured in a flow cell in minimal nutrient medium (artificial sea water) and their development was followed for up to 3 weeks. The spectroscopic signature of the biofilm cells and extracellular polymeric substances (EPS) were differentiated and their distribution in biofilm colonies and within water channels was mapped in-plane and -depth. The colonies were initially amorphous, mainly composed of cells with no detectable amount of EPS. They developed rapidly to give round colonies composed of a cellular core enclosed in a sheath of EPS. The EPS continued to increase and spread throughout the biofilm to become the dominating feature of aged colonies. Colonies with a liquid core morphology - characteristic of the seeding dispersal process - were also observed. CONCLUSIONS: This study demonstrated that CRM can be used to monitor the distribution of biofilm components in fully hydrated undisturbed biofilms over time. SIGNIFICANCE AND IMPACT OF THE STUDY: Confocal Raman microspectroscopy facilitates the analysis of hydrated, live bacterial biofilms as a function of space and time, thus making it a suitable technique for investigating the effects of various additives and environmental factors on biofilm growth.     

Role of cell surface hydrophobicity in Candida albicans biofilm

Overall cell surface hydrophobicity (CSH) is predicted to play an important role during biofilm formation in Candida albicans but is the result of many expressed proteins. This study compares the CSH status and CSH1 gene expression in C. albicans planktonic cells, sessile biofilm, and dispersal cells. Greater percentages of hydrophobic cells were found in non-adhered (1.5 h) and dispersal forms (24 or 48 h) (41.34±4.17% and 39.52±7.45%, respectively), compared with overnight planktonic cultures (21.69±3.60%). Results from quantitative real-time PCR confirmed greater up-regulation of the CSH1 gene in sessile biofilm compared with both planktonic culture and dispersal cells. Up-regulation was also greater in dispersal cells compared with planktonic culture. The markedly increased CSH found both in C. albicans biofilm, and in cells released during biofilm formation could provide an advantage to dispersing cells building new biofilm.