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 10(2) to 10(4) 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.     

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.     

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.     

Phenotypic Switching in Biofilm-Forming Marine Bacterium Paenibacillus lautus NE3B01

Biofilm-forming marine bacterium Paenibacillus lautus NE3B01 was isolated from a mangrove ecosystem, Odisha, India. This isolate formed a swarming type of colony pattern on the solid culture medium with 0.5–2 % agar. Phase contrast microscopy study of a growing colony of P. lautus on solid media and swarming pattern revealed the existence of two phenotypically distinct cells (i.e. cocci and rods) across the colonies. However, in actively growing planktonic culture, only rod-shaped cells were observed. Biofilm growth studies (crystal violet assay) with the isolate showed significant biofilm formation by 6 h, and the detachment phase was observed after 18 h. Biofilm parameters (such as total biomass, roughness coefficient, biofilm thickness, etc.) of 24-h-old P. lautus biofilm were studied by confocal scanning laser microscopy (CSLM). The CSLM study showed that P. lautus formed a biofilm with an average thickness of 14.8 ± 2.6 μm, a high roughness coefficient (0.379 ± 0.103) and surface to bio-volume ratio (4.59 ± 1.12 μm²/μm³), indicating a highly uneven topography of the biofilm. This also indicates that the 24-h-old biofilm is in dispersal phase. Scanning electron microphotographs of P. lautus also supported the existence of two distinct phenotypes of P. lautus. The current findings suggest that P. lautus has two vegetative phenotypes and to decongest the overcrowded biofilm the bacterium can switch over to motile rods from nonmotile cocci and vice versa.     

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.     

Forward mutation assay of V79 cells to 6-thioguanine resistance in a soft agar technique that eliminates effects of metabolic co-operation

A technique involving culture in soft agar was used for the assay of forward mutation of V79 cells to 6-thioguanine (6TG) resistance. The main reason for the use of soft agar was to prevent reduction in recovery of mutants depending on the cell density plated for mutation selection, which is the chief problem in the liquid method, and which results mainly from metabolic co-operation due to cell-to-cell contact.V79 cells grew well in fortified soft agar medium (DMEM + 20% FBS) showing cloning efficiencies (>80%) as high as in liquid culture. Therefore, V79/HGPRT mutagenesis could be assayed quantitatively in soft agar culture.The frequency of 6TG-resistant colonies in agar selective medium increased linearly with increase in concentration of EMS. Toxicity and mutagenic responses were greater in soft agar than in liquid culture.In cultures of untreated and EMS-treated cells, more than 95% of the 6TG-resistant colonies isolated were aminopterin-sensitive.Use of soft agar for selection prevented the reduction in the number of mutants with increase in the size of incula on plating up to 1?2 × 10⁶ cells per 9-cm dish: in liquid culture, even with a lower plating number (2 × 10⁵ cells per 9-cm dish), a notable reduction in numbers of mutants was observed. This character was re-examined in a reconstruction experiment. The results show that, when up to 2 × 10⁶ cells were plated per 9-cm dish, 6TG-resistant cells were almost completely recovered from the soft agar medium, whereas only 10% were recovered from liquid culture.     

Surface proteins and the formation of biofilms by Staphylococcus aureus

Staphylococcus aureus biofilms pose a serious clinical threat as reservoirs for persistent infections. Despite this clinical significance, the composition and mechanism of formation of S. aureus biofilms are unknown. To address these problems, we used solid-state NMR to examine S. aureus (SA113), a strong biofilm-forming strain. We labeled whole cells and cell walls of planktonic cells, young biofilms formed for 12–24 h after stationary phase, and more mature biofilms formed for up to 60 h after stationary phase. All samples were labeled either by (i) [¹⁵N]glycine and l-[1-¹³C]threonine, or in separate experiments, by (ii) l-[2-¹³C,¹⁵N]leucine. We then measured ¹³C-¹⁵N direct bonds by C{N} rotational-echo double resonance (REDOR). The increase in peptidoglycan stems that have bridges connected to a surface protein was determined directly by a cell-wall double difference (biofilm REDOR difference minus planktonic REDOR difference). This procedure eliminates errors arising from differences in ¹⁵N isotopic enrichments and from the routing of ¹³C label from threonine degradation to glycine. For both planktonic cells and the mature biofilm, 20% of pentaglycyl bridges are not cross-linked and are potential surface-protein attachment sites. None of these sites has a surface protein attached in the planktonic cells, but one-fourth have a surface protein attached in the mature biofilm. Moreover, the leucine-label shows that the concentration of β-strands in leucine-rich regions doubles in the mature biofilm. Thus, a primary event in establishing a S. aureus biofilm is extensive decoration of the cell surface with surface proteins that are linked covalently to the cell wall and promote cell-cell adhesion.     

Physiology of biofilms of thermophilic bacilli—potential consequences for cleaning

Thermophilic Bacillus species readily attached and grew on stainless steel surfaces, forming mature biofilms of >10^6.0 cells/cm² in 6 h on a surface inoculated with the bacteria. Clean stainless steel exposed only to pasteurized skim milk at 55 °C developed a mature biofilm of >10^6.0 cells/cm² within 18 h. When bacilli were inoculated onto the steel coupons, 18-h biofilms were 30 m thick. Biofilm growth followed a repeatable pattern, with a reduction in the numbers of bacteria on the surface occurring after 30 h, followed by a recovery. This reduction in numbers was associated with the production of a substance that inhibited the growth of the bacteria. Variations in the environment, including pH and molarity, affected the viability of the cells. Chemicals that attack the polysaccharide matrix of the biofilm were particularly effective in killing and removing cells from the biofilm, demonstrating the importance of polysaccharides in the persistence of these biofilms. Treatment of either the biofilm or a clean stainless steel surface with lysozyme killed biofilm cells and prevented the attachment of any bacteria exposed to the surface. This suggests that lysozyme may have potential as an alternative control method for biofilms of these bacteria.     

Requirement for Cell Dispersion Prior to Selection of Induced Azaguanine-Resistant Colonies of Chinese Hamster Cells

With V79 Chinese hamster cell cultures treated with a mutagen, the maximum frequency of colonies resistant to 8-azaguanine (AZG) was attained when the cells were dispersed after a suitable expression time before adding the selection medium. V79–4 cells were exposed to 500 µM MMS, 7 µM AFAA, or 10 µM MNNG and allowed to multiply before being reseeded at 4 x 10⁴ cells/60 mm dish and selected with 10 µg/ml AZG. Maximum frequencies of 4 x 10^-5, 4 x 10^-4, and 2.4 x 10^-3 were obtained about 100, 130, and 200 hrs after exposure to MMS, AFAA, and MNNG, respectively. The maximum frequencies following MMS or MNNG treatments were about 10-fold greater than those obtained when induction and selection of AZG-resistant colonies were performed in the same culture dish. The reseeding of treated cells eliminated the possibility of metabolic cooperation within mosaic colonies of wild-type and mutant cells and achieved expression of the induced changes before intercolony crossfeeding reduced the frequency of resistant colonies.—AZG-resistant colonies were selected in medium containing dialyzed fetal bovine serum, and the selection medium was replaced at least twice. Both serum dialysis and selection medium replacement were necessary for consistent achievement of background frequencies of resistant colonies near 10^-6. Reconstruction experiments with AZG-resistant V79 lines showed that the efficiency of recovery of resistant cells in the selection medium was constant over a range of 0–20 colonies observed/dish. A mixed population of V79 and AZG-resistant cells was also correctly analyzed by the procedure used in mutagenesis studies.     

Apparent antimutagenic effect of ultraviolet irradiation

Strain SL3367 is a S. typhimurium LT2 hisG46 stock which spontaneously reverts to His⁺ at a high frequency. Plates of defined medium with 1% (v/v) nutrient broth inoculated with ca. 10⁸ washed SL3367 cells were incubated, untreated or after UV irradiation. After 2 days at 37°C, an average of 165 His⁺ colonies were obtained per control plate but significantly fewer, 105 His⁺ colonies, on plates irradiated at a fluence of 7 J/m². The dry weight of bacteria in washings from plates incubated 14 h (by which time growth of His^? cells had ceased) was the same for irradiated and non-irradiated plates but the yield of colony-forming units from irradiated plates was less than from control plates, by about the same factor as the reduction in yield of His⁺ colonies caused by the same fluence. Washings from incubated irradiated plates, but not those from control plates, contained long filaments as well as bacteria of normal size; on transfer to nutrient-agar slide cultures cells normal size grew into microcolonies but filaments did not grow. The reduced plateau yield of viable His^? cells caused by consumption of much of the growth-limiting supply of histidine by irradiated cells growing into non-viable filaments reduces the number of auxotrophic bacteria at risk for spontaneous reversion and so accounts for the apparent antimutagenic effect of UV irradiation. This effect was partly reversed by the presence of d,l-pantoyl lactone in the selection medium, and was also observed for yield of Trp⁺ colonies from trpE8 cultures with a high spontaneous reversion rate. Treatments not inducing cell filamentation did not result in the depression of spontaneous revertants and were detected as being mutagenic. The apparent antimutagenic effect may be expected for reversion of any auxotroph, unless masked by induced revertants and is particularly apparent in an auxotroph which reverts spontaneously at high frequency.     

Aggregatibacter actinomycetemcomitans biofilm killing by a targeted ciprofloxacin prodrug

A pH-sensitive ciprofloxacin prodrug was synthesized and targeted against biofilms of the periodontal pathogen Aggregatibacter actinomycetemcomitans (Aa). The dose required to reduce the viability of a mature biofilm of Aa by ～80% was in the range of ng?cm^?2 of colonized area (mean biofilm density 2.33?×?10⁹?cells?cm^?2). A mathematical model was formulated that predicts the temporal change in the concentration of ciprofloxacin in the Aa biofilm as the drug is released and diffuses into the bulk medium. The predictions of the model were consistent with the extent of killing obtained. The results demonstrate the feasibility of the strategy to induce mortality, and together with the mathematical model, provide the basis for design of targeted antimicrobial prodrugs for the topical treatment of oral infections such as periodontitis. The targeted prodrug approach offers the possibility of optimizing the dose of available antimicrobials in order to kill a chosen pathogen while leaving the commensal microbiota relatively undisturbed.     

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 10⁵ 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.     

A Widely Used In Vitro Biofilm Assay Has Questionable Clinical Significance for Enterococcal Endocarditis

Biofilm formation may play an important role in the pathogenesis of infections caused by Enterococcus faecalis, including endocarditis. Most biofilm studies use a polystyrene dish assay to quantify biofilm biomass. However, recent studies of E. faecalis strains in tissue and animal models suggest that polystyrene dish results need to be interpreted with caution. We evaluated 158 clinical E. faecalis isolates using a polystyrene dish assay and found variation in biofilm formation, with many isolates forming little biofilm even when different types of media were used. However, all tested clinical isolates were able to form biofilms on porcine heart valve explants. Dextrose-enhanced biofilm formation in the polystyrene dish assay was found in 6/12 (50%) of clinical isolates tested and may explain some, but not all of the differences between the polystyrene dish assay and the heart valve assay. These findings suggest that in studies assessing the clinical relevance of enterococcal biofilm-forming ability, ex vivo biofilm formation on a relevant tissue surface may be warranted to validate results of in vitro assays.     

Comparative effects of adenine analogs upon metabolic cooperation between Chinese hamster cells with different levels of adenine phosphoribosyltransferase activity

Colony formation by variant Chinese hamster cells highly resistant to adenine analogs and deficient in adenine phosphoribosyltransferase (APRT) activity was measured after co-cultivation with APRT⁺, CHO-K1 cells in medium containing one of three different adenine analogs. Depending upon the density of APRT⁺ cells and the specific adenine analog, large differences in the recovery of APRT^? colonies were observed. The particular adenine analog and APRT⁺ cell density were more significant factors in the recovery of APRT^? colonies than the concentration of the analog or the level of APRT activity. The number of wild-type cells (CHO-K1) required to inhibit formation of APRT^? colonies by 50% (mean lethal density; MLD₅₀) with 65 μg/ml 8-aza-adenine (AzA) as the selective drug was 8.0 × 10⁵ cells/100 mm dish (1.5 × 10⁴/cm²). With 100 μg/ml 2,6-diaminopurine (DAP) the MLD₅₀ for CHO-K1 was 4.0 × 10⁵ cells/100 mm dish (7.3 × 10³/cm²). The MLD₅₀ for CHO-K1 when the DAP concentration was decreased to 50 μg/ml was only slightly higher, 5 × 10⁵ cells/100 mm dish (9.1 × 10³/cm²). The most toxic effect was observed with 2-fluoroadenine (FA). The MLD₅₀ for CHO-K1 in 2 μg/ml FA was 4.5 × 10⁴ cells/100 mm dish (8.2 × 10²/cm²), a cell density which permits minimal direct contact between APRT⁺ and APRT^? cells. The toxic effects of FA on individually resistant, APRT^? cells were found to be mediated by metabolites released into the medium by dying APRT⁺ cells. This metabolite toxicity to APRT^? cells was also demonstrated in mixtures with cells having only 8% of wild-type APRT activity. The MLD₅₀ for these APRT⁺ (8%) cells in 2 μg/ml FA was 7.5 × 10⁴ cells/100 dish (1.4 × 10³/cm²), a small difference from the MLD₅₀ for cells with wild-type levels of APRT activity. The differences in the recovery of APRT^? colonies from mixtures with APRT⁺ cells in these three adenine analogs are critical to the design of procedures for the selection of APRT^? cells from populations of APRT⁺ cells and emphasize the importance of establishing the parameters of metabolic cooperation, not only in terms of cell density but also with regard to the particular selective agent, in any experiment designed to determine precise mutation rates or to test putative mutagens upon mammalian cells in culture.     

Ecological Advantages of Autolysis during the Development and Dispersal of Pseudoalteromonas tunicata Biofilms

In the ubiquitous marine bacterium Pseudoalteromonas tunicata, subpopulations of cells are killed by the production of an autocidal protein, AlpP, during biofilm development. Our data demonstrate an involvement of this process in two parameters, dispersal and phenotypic diversification, which are of importance for the ecology of this organism and for its survival within the environment. Cell death in P. tunicata wild-type biofilms led to a major reproducible dispersal event after 192 h of biofilm development. The dispersal was not observed with a ΔAlpP mutant strain. Using flow cytometry and the fluorescent dye DiBAC₄(3), we also show that P. tunicata wild-type cells that disperse from biofilms have enhanced metabolic activity compared to those cells that disperse from ΔAlpP mutant biofilms, possibly due to nutrients released from dead cells. Furthermore, we report that there was considerable phenotypic variation among cells dispersing from wild-type biofilms but not from the ΔAlpP mutant. Wild-type cells that dispersed from biofilms showed significantly increased variations in growth, motility, and biofilm formation, which may be important for successful colonization of new surfaces. These findings suggest for the first time that the autocidal events mediated by an antibacterial protein can confer ecological advantages to the species by generating a metabolically active and phenotypically diverse subpopulation of dispersal cells.     

High Diversity among Environmental Escherichia coli Isolates from a Bovine Feedlot

Approximately 280 Escherichia coli isolates were isolated from a bovine feedlot at the University of Connecticut campus via enrichment in lauryl tryptose broth and random selection from MacConkey plates. The E. coli subspecies diversity was estimated by employing whole-cell BOX-PCR genomic fingerprints. A total of 89 distinct operational taxonomic units (OTUs) were identified by employing a criterion of 85% fingerprint similarity as a surrogate for an OTU, while the Chao1 index estimated the E. coli population richness at 128 OTUs. One genotype (at a similarity level of 60%) dominated the population at 66% regardless of sampling depth or location, while no significant vertical distribution pattern was observed in terms of genotype, mobility, antibiotic resistance profile, or biofilm-forming ability. Motility, measured by a soft agar assay, had a very broad range among the E. coli population and was positively correlated with biofilm-forming ability in minimal medium (Spearman's rank correlation coefficient r = 0.619, P < 10⁻⁴) but not in Luria broth. Only an estimated 48% of the population possessed gene agn43, which encodes Ag43, a phase-variable outer membrane protein that has been implicated in biofilm formation in minimal medium. We observed significantly more biofilm formation in both minimal medium and Luria broth for agn43⁺ strains, with a larger effect in minimal medium. This study represents an exhaustive inventory of extant E. coli population diversity at a bovine feedlot and reveals significant subspecies heterogeneity in interfacial behavior.     

Effect of surface roughness, biofilm coverage and biofilm structure on the electrochemical efficiency of microbial cathodes

Biofilms of Geobacter sulfurreducens were formed under chronoamperometry at −0.5 V and −0.6 V vs. Ag/AgCl on stainless steel cathodes and tested for fumarate reduction. Increasing the surface roughness Ra from 2.0 μm to 4.0 μm increased currents by a factor of 1.6. The overall current density increased with biofilm coverage. When the current density was calculated with respect to the biofilm-coated area only, values up to 280 A/m² were derived. These values decreased with biofilm coverage and indicated that isolated cells or small colonies locally provide higher current density than dense colonies. Steel composition affected the current values because of differences in biofilm structure and electron transfer rates. Biofilms formed under polarisation revealed better electrochemical characteristics than biofilm developed at open circuit. This work opens up new guidelines for the design of microbial cathodes: a uniform carpet of isolated bacteria or small colonies should be targeted, avoiding the formation of large colonies.     

Characterization of Biofilm Formation in [Pasteurella] pneumotropica and [Actinobacillus] muris Isolates of Mouse Origin

[Pasteurella] pneumotropica biotypes Jawetz and Heyl and [Actinobacillus] muris are the most prevalent Pasteurellaceae species isolated from laboratory mouse. However, mechanisms contributing to their high prevalence such as the ability to form biofilms have not been studied yet. In the present investigation we analyze if these bacterial species can produce biofilms in vitro and investigate whether proteins, extracellular DNA and polysaccharides are involved in the biofilm formation and structure by inhibition and dispersal assays using proteinase K, DNase I and sodium periodate. Finally, the capacity of the biofilms to confer resistance to antibiotics is examined. We demonstrate that both [P.] pneumotropica biotypes but not [A.] muris are able to form robust biofilms in vitro, a phenotype which is widely spread among the field isolates. The biofilm inhibition and dispersal assays by proteinase and DNase lead to a strong inhibition in biofilm formation when added at the initiation of the biofilm formation and dispersed pre-formed [P.] pneumotropica biofilms, revealing thus that proteins and extracellular DNA are essential in biofilm formation and structure. Sodium periodate inhibited the bacterial growth when added at the beginning of the biofilm formation assay, making difficult the assessment of the role of β-1,6-linked polysaccharides in the biofilm formation, and had a biofilm stimulating effect when added on pre-established mature biofilms of [P.] pneumotropica biotype Heyl and a majority of [P.] pneumotropica biotype Jawetz strains, suggesting that the presence of β-1,6-linked polysaccharides on the bacterial surface might attenuate the biofilm production. Conversely, no effect or a decrease in the biofilm quantity was observed by biofilm dispersal using sodium periodate on further biotype Jawetz isolates, suggesting that polysaccharides might be incorporated in the biofilm structure. We additionally show that [P.] pneumotropica cells enclosed in biofilms were less sensitive to treatment with amoxicillin and enrofloxacin than planktonic bacteria. Taken together, these findings provide a first step in understanding of the biofilm mechanisms in [P.] pneumotropica, which might contribute to elucidation of colonization and pathogenesis mechanisms for these obligate inhabitants of the mouse mucosa.