Possible involvement of the division cycle in dispersal of Escherichia coli from biofilms.

Growth rate control of adherent, sessile populations was achieved by the controlled perfusion of membrane-associated bacterial biofilms by the method of Gilbert et al. (P. Gilbert, D. G. Allison, D. J. Evans, P. S. Handley, and M. R. W. Brown, Appl. Environ. Microbiol. 55:1308-1311, 1989). Changes in cell surface hydrophobicity were evaluated with respect to growth rate for such sessile Escherichia coli cells and compared with those of suspended (planktonic) populations grown in a chemostat. Newly formed daughter cells shed at the various growth rates from the biofilm during its growth and development were also included in the study. Surface hydrophobicity decreased with growth rate similarly for both planktonic and sessile E. coli; no significant differences were noted between the two. Daughter cells dislodged from the biofilm, however, were significantly more hydrophilic than those remaining, indicating that hydrophobicity changed during the division cycle. Our data support the hypothesis that dispersal of cells from adhesive biofilms and recolonization of new surfaces reflect cell-cycle-mediated events.     

Planktonic-cell yield of a pseudomonad biofilm

Biofilm cells differ phenotypically from their free-floating counterparts. Differential growth rates in biofilms are often referred to, particularly in response to limited diffusion of oxygen and nutrients. We observed growth rates of attached Pseudomonas sp. strain CT07 cells that were notably higher than the maximum specific growth rate measured in batch culture. Despite dilution rates in continuous flow cells that exceeded the maximum planktonic specific growth rate by 58 times, sampling of the effluent revealed >10(9) cells ml(-1), suggesting that biofilms function as a source of planktonic cells through high cell yield and detachment. Further investigation demonstrated considerable planktonic cell yield from biofilms as young as 6 h, indicating that detachment is not limited to established biofilms. These biofilm-detached cells were more sensitive to a commercial biocide than associated biofilm- and chemostat-cultivated populations, implying that detached biofilm cells exhibit a character that is distinct from that of attached and planktonic cell populations.     

Planktonic-Cell Yield of a Pseudomonad Biofilm

Biofilm cells differ phenotypically from their free-floating counterparts. Differential growth rates in biofilms are often referred to, particularly in response to limited diffusion of oxygen and nutrients. We observed growth rates of attached Pseudomonas sp. strain CT07 cells that were notably higher than the maximum specific growth rate measured in batch culture. Despite dilution rates in continuous flow cells that exceeded the maximum planktonic specific growth rate by 58 times, sampling of the effluent revealed >10⁹ cells ml⁻¹, suggesting that biofilms function as a source of planktonic cells through high cell yield and detachment. Further investigation demonstrated considerable planktonic cell yield from biofilms as young as 6 h, indicating that detachment is not limited to established biofilms. These biofilm-detached cells were more sensitive to a commercial biocide than associated biofilm- and chemostat-cultivated populations, implying that detached biofilm cells exhibit a character that is distinct from that of attached and planktonic cell populations.     

Effect of growth in biofilms on chlorine susceptibility of Mycobacterium avium and Mycobacterium intracellulare

Mycobacterium avium and Mycobacterium intracellulare were grown in suspension and in biofilms, and their susceptibilities to chlorine were measured. M. avium and M. intracellulare readily adhered within 2 h, and numbers increased 10-fold in 30 days at room temperature in biofilms on both polystyrene flasks and glass beads. The chlorine resistance of M. avium and M. intracellulare cells grown and exposed to chlorine in biofilms was significantly higher than that of cells grown in suspension. Survival curves showed no evidence of a resistant, persisting population after 6 h of exposure to 1 mug chlorine/ml. The chlorine susceptibility of cells grown in biofilms and exposed in suspension (cells detached from bead surfaces) was also significantly higher than that of cells grown and exposed in suspension (planktonic cells), although it was lower than that of cells grown and exposed in biofilms. The higher resistance of the detached biofilm-grown cells was reversed upon their growth in suspension. There was a strong correlation between the chlorine susceptibility of cells of both M. avium and M. intracellulare and cell surface hydrophobicity measured by contact angle for both biofilm- and suspension-grown cells.     

Characterization of Switch Phenotypes in <Emphasis Type="Italic">Candida albicans</Emphasis> Biofilms

The aim of this study was to characterize switch phenotypes in Candida albicans biofilms. Cells of Candida albicans 192887g biofilms (24 h) were resuspended and these together with their planktonic counterparts were separately inoculated on Lee’s medium agar supplemented with arginine and zinc, at 25 °C for 9 days, for colony formation. The different switch phenotypes, as reflected by varying colony morphologies, were then examined for their (i) stability under various growth conditions, (ii) carbohydrate assimilation profiles, (iii) susceptibility to the polyene antifungal, nystatin, (iv) adhering and biofilm-forming ability, (v) filamentation, and (vi) growth rate in yeast nitrogen base medium supplemented with 100 mM glucose. Our data showed that the frequency of phenotypic switching in C. albicans biofilms was approximately 1%. Compared with the planktonic yeasts, cells derived from candidal biofilms generated one of the phenotypes less frequently (Chi-square-tests: P = 0.017). The five phenotypes derived from the biofilm growth demonstrated differing profiles for carbohydrate assimilation, adhesion, biofilm formation, filamentation, and growth rate. These findings reported here, for the first time, imply that phenotypic switching in the candidal biofilms differs from that in the planktonic growth, and affects multiple biological attributes.     

Inhibitory effect of a lipopeptide biosurfactant produced by Bacillus subtilis on planktonic and sessile cells of Trichosporon spp.

The present study aimed to investigate the inhibitory effect of a bacterial biosurfactant (TIM96) on clinical strains of Trichosporon. Additionally, the effect of TIM96 on the ergosterol content, cell membrane integrity, and the hydrophobicity of planktonic cells was assessed. The inhibitory activity of TIM96 against Trichosporon biofilms was evaluated by analyzing metabolic activity, biomass and morphology. MIC values ranged from 78.125 to 312.5 μg ml^?1 for TIM96; time-kill curves revealed that the decline in the number of fungal cells started after incubation for 6 h with TIM96 at both MIC and 2×MIC. The biosurfactant reduced the cellular ergosterol content and altered the membrane permeability and the surface hydrophobicity of planktonic cells. Incubation at 10×MIC TIM96 reduced cell adhesion by up to 96.89%, thus interfering with biofilm formation. This concentration also caused up to a 99.2% reduction in the metabolic activity of mature biofilms. The results indicate potential perspectives for the development of new antifungal strategies.     

Biofilms and Planktonic Cells of Pseudomonas aeruginosa Have Similar Resistance to Killing by Antimicrobials

Biofilms are considered to be highly resistant to antimicrobial agents. Strictly speaking, this is not the case-biofilms do not grow in the presence of antimicrobials any better than do planktonic cells. Biofilms are indeed highly resistant to killing by bactericidal antimicrobials, compared to logarithmic-phase planktonic cells, and therefore exhibit tolerance. It is assumed that biofilms are also significantly more tolerant than stationary-phase planktonic cells. A detailed comparative examination of tolerance of biofilms versus stationary- and logarithmic-phase planktonic cells with four different antimicrobial agents was performed in this study. Carbenicillin appeared to be completely ineffective against both stationary-phase cells and biofilms. Killing by this beta-lactam antibiotic depends on rapid growth, and this result confirms the notion of slow-growing biofilms resembling the stationary state. Ofloxacin is a fluoroquinolone antibiotic that kills nongrowing cells, and biofilms and stationary-phase cells were comparably tolerant to this antibiotic. The majority of cells in both populations were eradicated at low levels of ofloxacin, leaving a fraction of essentially invulnerable persisters. The bulk of the population in both biofilm and stationary-phase cultures was tolerant to tobramycin. At very high tobramycin concentrations, a fraction of persister cells became apparent in stationary-phase culture. Stationary-phase cells were more tolerant to the biocide peracetic acid than were biofilms. In general, stationary-phase cells were somewhat more tolerant than biofilms in all of the cases examined. We concluded that, at least for Pseudomonas aeruginosa, one of the model organisms for biofilm studies, the notion that biofilms have greater resistance than do planktonic cells is unwarranted. We further suggest that tolerance to antibiotics in stationary-phase or biofilm cultures is largely dependent on the presence of persister cells.     

Erosion from Staphylococcus aureus biofilms grown under physiologically relevant fluid shear forces yields bacterial cells with reduced avidity to collagen

An estimated 65% of infective diseases are associated with the presence of bacterial biofilms. Biofilm-issued planktonic cells promote blood-borne, secondary sites of infection by the inoculation of the infected sites with bacteria from the intravascular space. To investigate the potential role of early detachment events in initiating secondary infections, we studied the phenotypic attributes of Staphylococcus aureus planktonic cells eroding from biofilms with respect to expression of the collagen adhesin, CNA. The collagen-binding abilities of S. aureus have been correlated to the development of osteomyelitis and septic arthritis. In this study, we focused on the impact of CNA expression on S. aureus adhesion to immobilized collagen in vitro under physiologically relevant shear forces. In contrast to the growth phase-dependent adhesion properties characteristic of S. aureus cells grown in suspension, eroding planktonic cells expressed invariant and lower effective adhesion rates regardless of the age of the biofilm from which they originated. These results correlated directly with the surface expression level of CNA. However, subsequent analysis revealed no qualitative differences between biofilms initiated with suspension cells and secondary biofilms initiated with biofilm-shed planktonic cells. Taken together, our findings suggest that, despite their low levels of CNA expression, S. aureus planktonic cells shed from biofilms retain the capacity for metastatic spread and the initiation of secondary infection. These findings demonstrate the need for a better understanding of the phenotypic properties of eroding planktonic cells, which could lead to new therapeutic strategies to target secondary infections.     

Susceptibility of Staphylococcus epidermidis planktonic cells and biofilms to the lytic action of staphylococcus bacteriophage K

AIMS: To evaluate differences in biofilm or planktonic bacteria susceptibility to be killed by the polyvalent antistaphylococcus bacteriophage K. METHODS AND RESULTS: In this study, the ability of phage K to infect and kill several clinical isolates of Staphylococcus epidermidis was tested. Strains were grown in suspension or as biofilms to compare the susceptibility of both phenotypes to the phage lytic action. Most strains (10/11) were susceptible to phage K, and phage K was also effective in reducing biofilm biomass after 24 h of challenging. Biofilm cells were killed at a lower rate than the log-phase planktonic bacteria but at similar rate as stationary phase planktonic bacteria. CONCLUSIONS: Staphylococcus epidermidis biofilms and stationary growth phase planktonic bacteria are more resistant to phage K lysis than the exponential phase planktonic bacteria. SIGNIFICANCE OF STUDY: This study shows the differences in Staph. epidermidis susceptibility to be killed by bacteriophage K, when grown in biofilm or planktonic phenotypes.     

Effect of Growth in Biofilms on Chlorine Susceptibility of Mycobacterium avium and Mycobacterium intracellulare

Mycobacterium avium and Mycobacterium intracellulare were grown in suspension and in biofilms, and their susceptibilities to chlorine were measured. M. avium and M. intracellulare readily adhered within 2 h, and numbers increased 10-fold in 30 days at room temperature in biofilms on both polystyrene flasks and glass beads. The chlorine resistance of M. avium and M. intracellulare cells grown and exposed to chlorine in biofilms was significantly higher than that of cells grown in suspension. Survival curves showed no evidence of a resistant, persisting population after 6 h of exposure to 1 μg chlorine/ml. The chlorine susceptibility of cells grown in biofilms and exposed in suspension (cells detached from bead surfaces) was also significantly higher than that of cells grown and exposed in suspension (planktonic cells), although it was lower than that of cells grown and exposed in biofilms. The higher resistance of the detached biofilm-grown cells was reversed upon their growth in suspension. There was a strong correlation between the chlorine susceptibility of cells of both M. avium and M. intracellulare and cell surface hydrophobicity measured by contact angle for both biofilm- and suspension-grown cells.     

Erosion from Staphylococcus aureus Biofilms Grown under Physiologically Relevant Fluid Shear Forces Yields Bacterial Cells with Reduced Avidity to Collagen

An estimated 65% of infective diseases are associated with the presence of bacterial biofilms. Biofilm-issued planktonic cells promote blood-borne, secondary sites of infection by the inoculation of the infected sites with bacteria from the intravascular space. To investigate the potential role of early detachment events in initiating secondary infections, we studied the phenotypic attributes of Staphylococcus aureus planktonic cells eroding from biofilms with respect to expression of the collagen adhesin, CNA. The collagen-binding abilities of S. aureus have been correlated to the development of osteomyelitis and septic arthritis. In this study, we focused on the impact of CNA expression on S. aureus adhesion to immobilized collagen in vitro under physiologically relevant shear forces. In contrast to the growth phase-dependent adhesion properties characteristic of S. aureus cells grown in suspension, eroding planktonic cells expressed invariant and lower effective adhesion rates regardless of the age of the biofilm from which they originated. These results correlated directly with the surface expression level of CNA. However, subsequent analysis revealed no qualitative differences between biofilms initiated with suspension cells and secondary biofilms initiated with biofilm-shed planktonic cells. Taken together, our findings suggest that, despite their low levels of CNA expression, S. aureus planktonic cells shed from biofilms retain the capacity for metastatic spread and the initiation of secondary infection. These findings demonstrate the need for a better understanding of the phenotypic properties of eroding planktonic cells, which could lead to new therapeutic strategies to target secondary infections.     

Pseudomonas aeruginosa PAO1 Preferentially Grows as Aggregates in Liquid Batch Cultures and Disperses upon Starvation

In both natural and artificial environments, bacteria predominantly grow in biofilms, and bacteria often disperse from biofilms as freely suspended single-cells. In the present study, the formation and dispersal of planktonic cellular aggregates, or ‘suspended biofilms’, by Pseudomonas aeruginosa in liquid batch cultures were closely examined, and compared to biofilm formation on a matrix of polyester (PE) fibers as solid surface in batch cultures. Plankton samples were analyzed by laser-diffraction particle-size scanning (LDA) and microscopy of aggregates. Interestingly, LDA indicated that up to 90% of the total planktonic biomass consisted of cellular aggregates in the size range of 10–400 µm in diameter during the growth phase, as opposed to individual cells. In cultures with PE surfaces, P. aeruginosa preferred to grow in biofilms, as opposed to planktonicly. However, upon carbon, nitrogen or oxygen limitation, the planktonic aggregates and PE-attached biofilms dispersed into single cells, resulting in an increase in optical density (OD) independent of cellular growth. During growth, planktonic aggregates and PE-attached biofilms contained densely packed viable cells and extracellular DNA (eDNA), and starvation resulted in a loss of viable cells, and an increase in dead cells and eDNA. Furthermore, a release of metabolites and infective bacteriophage into the culture supernatant, and a marked decrease in intracellular concentration of the second messenger cyclic di-GMP, was observed in dispersing cultures. Thus, what traditionally has been described as planktonic, individual cell cultures of P. aeruginosa, are in fact suspended biofilms, and such aggregates have behaviors and responses (e.g. dispersal) similar to surface associated biofilms. In addition, we suggest that this planktonic biofilm model system can provide the basis for a detailed analysis of the synchronized biofilm life cycle of P. aeruginosa.     

A simple in vitro model for growth control of bacterial biofilms

The perfused biofilm fermenter was found to be unsuitable for the long-term culture and growth rate control of Staphylococcus aureus and Pseudomonas aeruginosa biofilms. In a simplified approach, biofilms of these organisms were grown within Sorbarod filter plugs which were perfused with culture medium. Pseudo-steady states were established which were stable over several days at which the growth rate of the biofilm was reproducible, measurable and significantly slower than in broth culture. Environmental scanning electron microscopy of dissected Sorbarods demonstrated an association of cells with the surfaces of individual cellulose fibres, and growth characteristic of biofilms. Relatively high cell numbers recovered from the Sorbarod model facilitated biochemical investigations of biofilm populations and cells released spontaneously from them. SDS-PAGE demonstrated significant differences between the protein profiles of biofilm and eluted populations, which include, in Staph. aureus, the repression of a 48 kDa protein and increased expression of a 21 kDa protein relative to planktonic controls cultured at equivalent growth rates. The paper demonstrates the suitability of the approach for the culture of biofilm samples which are suitable for biochemical analysis.     

A simple in vitro model for growth control of bacterial biofilms

The perfused biofilm fermenter was found to be unsuitable for the long-term culture and growth rate control of Staphylococcus aureus and Pseudomonas aeruginosab biofilms. In a simplified approach, biofilms of these organisms were grown within Sorbarod filter plugs which were perfused with culture medium. Pseudo-steady states were established which were stable over several days at which the growth rate of the biofilm was reproducible, measurable and significantly slower than in broth culture. Environmental scanning electron microscopy of dissected Sorbarods demonstrated an association of cells with the surfaces of individual cellulose fibres, and growth characteristic of biofilms. Relatively high cell numbers recovered from the Sorbarod model facilitated biochemical investigations of biofilm populations and cells released spontaneously from them.
SDS-PAGE demonstrated significant differences between the protein profiles of biofilm and eluted populations, which include, in Staph. aureus , the repression of a 48 kDa protein and increased expression of a 21 kDa protein relative to planktonic controls cultured at equivalent growth rates. The paper demonstrates the suitability of the approach for the culture of biofilm samples which are suitable for biochemical analysis.     

Characterization and gene cloning of a novel serine protease with nematicidal activity from Trichoderma pseudokoningii SMF2

Pseudomonas aeruginosa is a pathogenic bacterium widely investigated for its high incidence in clinical environments and its ability to form strong biofilms. During biofilm development, sessile cells acquire physiological characteristics differentiating them from planktonic cells. But after treatment with disinfectants, or to ensure survival of the species in hostile environments, biofilm cells can detach. This complicates disinfection procedures. This study aimed to physiologically characterize cells detached from a P. aeruginosa biofilm and to compare them with their sessile and planktonic counterparts. We first tested planktonic growth kinetics and capacities to form new biofilms. Then we investigated cell-surface properties. And finally, we tested in vitro susceptibility to antibiotics. The results first indicated that sessile and detached cells have similar planktonic growth kinetics and cell-surface properties, distinguishable from those of planktonic cells. Interestingly, the three populations exhibited different biofilm-forming capacities, suggesting that there is a transitional phenotype between sessile and planktonic states, at least during the first hours following cell detachment. It is important to consider this observation when developing treatments to optimize disinfection processes. Surprisingly, the three populations showed the same antibiotic susceptibility profile.     

Pseudomonas pseudoalcaligenes KF707 upon biofilm formation on a polystyrene surface acquire a strong antibiotic resistance with minor changes in their tolerance to metal cations and metalloid oxyanions

The susceptibility to various biocides was examined in planktonic cells and biofilms of the obligate aerobe, PCBs degrader, Pseudomonas pseudoalcaligenes KF707. The toxicity of two antibiotics, amikacin and rifampicin, three metalloid oxyanions (AsO(2) (-), SeO(3) (2-), TeO(3) (2-)) and three metal cations (Cd(2+), Ni(2+), Al(3+)) was tested at two stages of the biofilm-development (4 and 24 h) and compared to planktonic cells susceptibility. Mature biofilms formed in rich (LB, Luria-Bertani) medium were thicker (23 mum) than biofilms grown in minimal (SA saccarose-arginine) medium (13 mum). Early grown (4 h) SA-biofilms, which consisted of a few sparse/attached cells, were 50-100 times more resistant to antibiotics than planktonic cells. Conversely, minor changes in tolerance to metal(loid)s were seen in both SA- and LB-grown biofilms. In contrast to planktonic cells, no reduction of TeO(3) (2-) to elemental Te(0) or SeO(3) (2-) to elemental Se(0) was seen in KF707 biofilms. The data indicate that: (a) metal tolerance in KF707 biofilms, under the growth and exposure conditions described here, is different than antibiotic tolerance; (b) KF707 planktonic cells and biofilms, are almost equally susceptible to killing by metal cations and oxyanions, and (c) biofilm-tolerance to TeO(3) (2-) and SeO(3) (2-) is not linked to metalloid reduction; this means that KF707 planktonic cells and biofilms differ in their physiology and strategy to counteract metalloid toxicity.     

Biofilm formation and biocide susceptibility testing of Mycobacterium fortuitum and Mycobacterium marinum

The ability of non-tuberculous mycobacteria to form biofilms may allow for their increased resistance to currently used biocides in medical and industrial settings. This study examines the biofilm growth of Mycobacterium fortuitum and Mycobacterium marinum, using the MBEC trade mark assay system, and compares the susceptibility of planktonic and biofilm cells to commercially available biocides. With scanning electron microscopy, both M. fortuitum and M. marinum form biofilms that are morphologically distinct. Biocide susceptibility testing suggested that M. fortuitum biofilms displayed increased resistance over their planktonic state. This is contrasted with M. marinum biofilms, which were generally as or more susceptible over their planktonic state.     

The HIV aspartyl protease inhibitor ritonavir impairs planktonic growth,biofilm formation and proteolytic activity in Trichosporon spp.

This study evaluated the effect of the protease inhibitor ritonavir (RIT) on Trichosporon asahii and Trichosporon inkin. Susceptibility to RIT was assessed by the broth microdilution assay and the effect of RIT on protease activity was evaluated using azoalbumin as substrate. RIT was tested for its anti-biofilm properties and RIT-treated biofilms were assessed regarding protease activity, ultrastructure and matrix composition. In addition, antifungal susceptibility, surface hydrophobicity and biofilm formation were evaluated after pre-incubation of planktonic cells with RIT for 15 days. RIT (200 μg ml^?1) inhibited Trichosporon growth. RIT (100 μg ml^?1) also reduced protease activity of planktonic and biofilm cells, decreased cell adhesion and biofilm formation, and altered the structure of the biofilm and the protein composition of the biofilm matrix. Pre-incubation with RIT (100 μg ml^?1) increased the susceptibility to amphotericin B, and reduced surface hydrophobicity and cell adhesion. These results highlight the importance of proteases as promising therapeutic targets and reinforce the antifungal potential of protease inhibitors.