Biofilm Detachment Research Articles

Analysis of the mechanical stability and surface detachment of mature Streptococcus mutans biofilms by applying a range of external shear forces

Well-established biofilms formed by Streptococcus mutans via exopolysaccharide matrix synthesis are firmly attached to tooth surfaces. Enhanced understanding of the physical properties of mature biofilms may lead to improved approaches to detaching or disassembling these highly organized and adhesive structures. Here, the mechanical stability of S. mutans biofilms was investigated by determining their ability to withstand measured applications of shear stress using a custom-built device. The data show that the initial biofilm bulk (~ 50% biomass) was removed after exposure to 0.184 and 0.449 N m−2 for 67 and 115 h old biofilms. However, removal of the remaining biofilm close to the surface was significantly reduced (vs initial bulk removal) even when shear forces were increased 10-fold. Treatment of biofilms with exopolysaccharide-digesting dextranase substantially compromised their mechanical stability and rigidity, resulting in bulk removal at a shear stress as low as 0.027 N m−2 and > a two-fold reduction in the storage modulus (G′). The data reveal how incremental increases in shear stress cause distinctive patterns of biofilm detachment, while demonstrating that the exopolysaccharide matrix modulates the resistance of biofilms to mechanical clearance.

Biofilm-based implant infections in orthopaedics.

The demand for joint replacement surgery is continuously increasing with rising costs for hospitals and healthcare systems. Staphylococci are the most prevalent etiological agents of orthopedic infections. After an initial adhesin-mediated implant colonization, Staphylococcus aureus and Staphylococcus epidermidis produce biofilm. Biofilm formation proceeds as a four-step process: (1) initial attachment of bacterial cells; (2) cell aggregation and accumulation in multiple cell layers; (3) biofilm maturation and (4) detachment of cells from the biofilm into a planktonic state to initiate a new cycle of biofilm formation elsewhere. The encasing of bacteria in biofilms gives rise to insuperable difficulties not only in the treatment of the infection, but also in assessing the state and the nature of the infection using traditional cultural methods. Therefore, DNA-based molecular methods have been developed to provide rapid identification of all microbial pathogens. To combat biofilm-centered implant infections, new strategies are being developed, among which anti-infective or infective-resistant materials are at the forefront. Infection-resistant materials can be based on different approaches: (i) modifying the biomaterial surface to give anti-adhesive properties, (ii) doping the material with antimicrobial substances, (iii) combining anti-adhesive and antimicrobial effects in the same coating, (iv) designing materials able to oppose biofilm formation and support bone repair.

Colonization of Microbial Biofilms in Pipeline of Water Reuse

Aim of this study was to investigate biofilms attached in pipeline of water reuse from the MBR system treating sewage without chlorination in correlation to the outflow water quality. Two general pipe materials: polyvinyl chloride (PVC) and polyethylene (PE) were employed in the experiment. The peak growths were found at week 4 in both pipes. The maximum biofilms in PE pipe was 33 mgVSS/cm 2 with the growth rate of 4.75 mgVSS/cm 2 -d which was significant higher than that of PVC pipe. Biofilms examined by PCR-DGGE technique revealed five bacterial species in PE biofilms namely Sinorhizobium medicae WSM419, Sinorhizobium fredii NGR234, Geobacter sp. M18, Parachlamydia acanthamoebae UV-7, and Mycobacterium chubuense NBB4. The VSS concentrations in outflow had directly correlated to the biofilm attachment and detachment. High COD concentrations of outflow appeared during biofilm detaching phase. In summary, water quality of reuse water corresponded to the biofilms attachment and detachment in the pipeline.

In-situ biofilm characterization in membrane systems using Optical Coherence Tomography: Formation, structure, detachment and impact of flux change

Biofouling causes performance loss in spiral wound nanofiltration (NF) and reverse osmosis (RO) membrane operation for process and drinking water production. The development of biofilm formation, structure and detachment was studied in-situ, non-destructively with Optical Coherence Tomography (OCT) in direct relation with the hydraulic biofilm resistance and membrane performance parameters: transmembrane pressure drop (TMP) and feed-channel pressure drop (FCP). The objective was to evaluate the suitability of OCT for biofouling studies, applying a membrane biofouling test cell operated at constant crossflow velocity (0.1 m s−1) and permeate flux (20 L m−2h−1).In time, the biofilm thickness on the membrane increased continuously causing a decline in membrane performance. Local biofilm detachment was observed at the biofilm–membrane interface.A mature biofilm was subjected to permeate flux variation (20 to 60 to 20 L m−2h−1). An increase in permeate flux caused a decrease in biofilm thickness and an increase in biofilm resistance, indicating biofilm compaction. Restoring the original permeate flux did not completely restore the original biofilm parameters: After elevated flux operation the biofilm thickness was reduced to 75% and the hydraulic resistance increased to 116% of the original values. Therefore, after a temporarily permeate flux increase the impact of the biofilm on membrane performance was stronger. OCT imaging of the biofilm with increased permeate flux revealed that the biofilm became compacted, lost internal voids, and became more dense. Therefore, membrane performance losses were not only related to biofilm thickness but also to the internal biofilm structure, e.g. caused by changes in pressure.Optical Coherence Tomography proved to be a suitable tool for quantitative in-situ biofilm thickness and morphology studies which can be carried out non-destructively and in real-time in transparent membrane biofouling monitors.

The Effects of Flushing on Water Quality and Biofilm Biomass in Different Daily Flow Patterns of Distribution System

We studied the effects of flushing on water quality and biofilm biomass in different daily flow patterns of distribution system. After building up steady biofilm layers in two flow velocity (0.25 m/s and 0.65 m/s) distribution systems consisting of PE pipe, then flushed by stepwise increased flow velocity. An increase flow velocity caused an immediate increase in turbidity and heterotrophic bacteria of water as a result of detachment of biofilms in the pipes, especially in low flow patterns distribution system. Three days later after flushing, water quality of two systems were generally lower and more stable than before flushing. Flushing strategies only limited biofilm growth to a certain extent and did not completely eliminate microorganisms from the systems.The low flow velocity distribution system was easily being disturbed by flushing.

The combined effects of shear stress and mass transfer on the balance between biofilm and suspended cell dynamics

This work investigates the effect of shear stress and mass transfer on the development of biofilms in a flow cell that mimics industrial piping. The shear stress and maximum flow velocity were estimated by computational fluid dynamics and the external mass transfer coefficient was calculated using empirical correlations for Reynolds numbers ranging from 100 to 10,000. The effect of two flow rates on the development of Escherichia coli biofilms under turbulent flow conditions was assessed and it was observed that biofilm formation was favored at the lowest flow rate. Additionally, estimations of the shear stress and external mass transfer coefficient indicate that both parameters increase with increasing flow rates. Thus, it seems that biofilm formation was being controlled by the shear stress that promoted biofilm erosion/sloughing and not by mass transfer which would potentiate biofilm growth. Our results indicate that not only efficient pre-treatment units are required on water recirculation loops in order to reduce the effective concentration of bacteria and nutrients, but also that high flow rates are preferred at all times to reduce the buildup of bacterial biofilms. For instance, high flow rates should be used during cleaning and disinfection cycles because the increase in shear stress will promote biofilm detachment and also potentiate the effect of biocides and other cleaning agents due to the increased mass transfer from the bulk solution to the surface of the biofilm.

Chemoorganotrophic Bioleaching of Olivine for Nickel Recovery

Bioleaching of olivine, a natural nickel-containing magnesium-iron-silicate, was conducted by applying chemoorganotrophic bacteria and fungi. The tested fungus, Aspergillus niger, leached substantially more nickel from olivine than the tested bacterium, Paenibacillus mucilaginosus. Aspergillus niger also outperformed two other fungal species: Humicola grisae and Penicillium chrysogenum. Contrary to traditional acid leaching, the microorganisms leached nickel preferentially over magnesium and iron. An average selectivity factor of 2.2 was achieved for nickel compared to iron. The impact of ultrasonic conditioning on bioleaching was also tested, and it was found to substantially increase nickel extraction by A. niger. This is credited to an enhancement in the fungal growth rate, to the promotion of particle degradation, and to the detachment of the stagnant biofilm around the particles. Furthermore, ultrasonic conditioning enhanced the selectivity of A. niger for nickel over iron to a value of 3.5. Pre-carbonating the olivine mineral, to enhance mineral liberation and change metal speciation, was also attempted, but did not result in improvement as a consequence of the mild pH of chemoorganotrophic bioleaching.

Glutathione-gated potassium efflux as a mechanism of active biofilm detachment.

Biofilm detachment often has detrimental effects such as pipe obstruction and infection, yet the detachment mechanisms underlying dispersal remain largely unknown. In this study, a stress response mechanism known as glutathione-gated potassium efflux (GGKE) was evaluated as an active detachment mechanism in the dispersal of Pseudomonas aeruginosa biofilms. N-ethylmaleimide (NEM) was used to activate potassium efflux proteins (Kef) associated with the GGKE pathway. This stress response mechanism was hypothesized to lead to altered cation concentration, which can potentially affect polymer bridging in biofilms, and ultimately cause biofilm detachment. Results showed the activation of GGKE by NEM exposure caused biofilm detachment without inducing a measurable change in viability, and detached biomass concentration and composition were dependent on NEM concentration. More detached biomass was observed with higher concentrations of NEM, with a trend of increasing polymer detachment. The detachment was likely resulting from a weakened biofilm structural integrity induced by bridge denaturing from GGKE activation. This study is important in understanding biofilm detachment from engineered systems such as membrane aerated bioreactors.

Anaerobic treatment of tequila vinasses under seasonal operating conditions: Start-up, normal operation and restart-up after a long stop and starvation period

This study examines the performance of an anaerobic fixed-film bioreactor under seasonal operating conditions prevailing in medium and small size Tequila factories: start-up, normal operation and particularly, during the restart-up after a long stop and starvation period. The proposed start-up procedure attained a stable biofilm in a rather short period (28days) despite unbalanced COD/N/P ratio and the use of non-acclimated inoculum. The bioreactor was restarted-up after being shut down for 6months during which the inoculum starved. Even when biofilm detachment and bioreactor clogging were detected at the very beginning of restart-up, results show that the bioreactor performed better as higher COD removal and methane yield were attained. CE-SSCP and Q-PCR analyses, conducted on the biofilm prokaryotic communities for each operating condition, confirmed that the high COD removal results after the bioreactor clogging and the severe starvation period were mainly due to the stable archaeal and resilient bacterial populations.

Soft Robotic Concepts in Catheter Design: an On‐Demand Fouling‐Release Urinary Catheter

Infectious biofilms are problematic in many healthcare-related devices and are especially challenging and ubiquitous in urinary catheters. This report presents an on-demand fouling-release methodology to mechanically disrupt and remove biofilms, and proposes this method for the active removal of infectious biofilms from the previously inaccessible main drainage lumen of urinary catheters. Mature Proteus mirabilis crystalline biofilms detach from silicone elastomer substrates upon application of strain to the substrate, and increasing the strain rate increases biofilm detachment. The study presents a quantitative relationship between applied strain rate and biofilm debonding through an analysis of biofilm segment length and the driving force for debonding. Based on this mechanism, hydraulic and pneumatic elastomer actuation is used to achieve surface strain selectively within the lumen of prototypes of sections of a fouling-release urinary catheter. Proof-of-concept prototypes of sections of active, fouling-release catheters are constructed using techniques typical to soft robotics including 3D printing and replica molding, and those prototypes demonstrate release of mature P. mirabilis crystalline biofilms (e.g., ≈90%) from strained surfaces. These results provide a basis for the development of a new urinary catheter technology in which infectious biofilms are effectively managed through new methods that are entirely complementary to existing approaches.

Selective Reactivity of Monochloramine with Extracellular Matrix Components Affects the Disinfection of Biofilm and Detached Clusters

The efficiency of monochloramine disinfection was dependent on the quantity and composition of extracellular polymeric substances (EPS) in biofilms, as monochloramine has a selective reactivity with proteins over polysaccharides. Biofilms with protein-based (Pseudomonas putida) and polysaccharide based EPS (Pseudomonas aeruginosa), as well as biofilms with varied amount of polysaccharide EPS (wild-type and mutant P. aeruginosa), were compared. The different reactivity of EPS components with monochloramine influenced disinfectant penetration, biofilm inactivation, as well as the viability of detached clusters. Monochloramine transport profiling measured by a chloramine-sensitive microelectrode revealed a broader diffusion boundary layer between bulk and biofilm surface in the P. putida biofilm compared to those of P. aeruginosa biofilms. The reaction with proteins in P. putida EPS multiplied both the time and the monochloramine mass required to achieve a full biofilm penetration. Cell viability in biofilms was also spatially influenced by monochloramine diffusion and reaction within biofilms, showing a lower survival in the surface section and a higher persistence in the middle section of the P. putida biofilm compared to the P. aeruginosa biofilms. While polysaccharide EPS promoted biofilm cell viability by obstructing monochloramine reactive sites on bacterial cells, protein EPS hindered monochloramine penetration by reacting with monochloramine and reduced its concentration within biofilms. Furthermore, the persistence of bacterial cells detached from biofilm (over 70% for P. putida and ∼40% for polysaccharide producing P. aeruginosa) suggested that currently recommended monochloramine residual levels may underestimate the risk of water quality deterioration caused by biofilm detachment.

Modelling epilithic biofilms combining hydrodynamics, invertebrate grazing and algal traits

Summary This model of stream epilithic biofilm biomass dynamics is based on the system of equations from Uehlinger et al. () and the term for autogenic detachment of biofilm from Boulêtreau et al. (). Its new features are (i) a mathematical term based on estimated feeding activity of biofilm‐dwelling invertebrates, (ii) local hydrodynamics considered as the principal factor governing algal traits and biofilm structure and (iii) a variable degree of parameterisation that was adjusted to biofilm biomass conditions. Biofilm biomass was monitored over a one‐year period in the Garonne river in France (September 2008–2009). An allometric approach was used to estimate the feeding activity of biofilm‐dwelling invertebrates based on their energetic requirements. Diatom functional diversity was also monitored to find how it varied with overall biofilm growth patterns. The one‐year monitoring period was divided into six biofilm biomass cycles, with each cycle consisting of a phase of biofilm growth as the main process, followed by detachment. This model reproduced the observed data as a complex of biofilm growth/detachment cycles using different sets of empirical parameters which allowed (i) the dominant processes involved in each biofilm cycle to be evaluated and (ii) the six cycles of biofilm growth/detachment to be reproduced. This accounted for the observed patterns more effectively than a parameterisation using a single set of empirical parameters. High flow had a severe effect on biofilm dynamics through chronic and catastrophic detachment. Presumably as a result, assemblages of diatoms shifted towards species that were firmly attached and protected by mucilage. During low flow (and when temperature was high), biofilm dynamics was mainly affected by autogenic detachment and grazer activity. The grazing pressure of the dominant biofilm‐dwelling invertebrates (Nematoda and larvae of Chironomidae and Trichoptera) was fairly low (a maximum of 6% of biofilm biomass ingested daily); nevertheless, their presence in the biofilm seemed to favour biofilm autogenic detachment.

Investigating electrochemical removal of bacterial biofilms from stainless steel substrates

Electrochemical removal of biofilms deserves attention because of its ease of use and environmentally friendly nature. We investigated the influence of electrode potential and treatment time on the removal of a 10-day old Pseudomonas aeruginosa biofilm formed on stainless steel 316L substrates. At electrode potentials more positive than −1.5V vs. Ag/AgCl, lower removal rates were observed and only partial removal of the biofilm was achieved during a 1-min time interval. Electrostatic repulsion between the film and electrode surface is believed to drive biofilm detachment under these conditions. However, when the biofilm-coated substrates were treated at potentials negative of −1.5V vs. Ag/AgCl, complete removal of a biofilm was achieved within seconds. Under these conditions, vigorous evolution of hydrogen gas is believed to be responsible for the film removal, mechanically detaching the bacteria and extracellular polymeric matrix from the substrate. Stainless steel substrates were also subjected to repeated cycles of biofilm formation and electrochemical removal. High removal efficiencies were maintained throughout this process suggesting the potential of the proposed technology for application on conductive surfaces in various industrial settings.

Phenol-Soluble Modulins: Novel Virulence-Associated Peptides of Staphylococci

Phenol-soluble modulins (PSMs), a novel class of small peptides with an amphipathic α-helical structure and strong surfactant-like properties, are produced by most staphylococci, especially pathogenic Staphylococcus aureus and Staphylococcus epidermidis. PSMs can: induce the production of proinflammatory cytokines; recruit, activate and lyse neutrophils to help staphylococci evade immune damage; lyse erythrocytes and are associated with the hemolysis of staphylococcal disease; facilitate the structuring and detachment of staphylococcal biofilms and disseminate biofilm-associated infection; and kill competing microbes and act as weapons in interbacterial warfare. Therefore, PSMs are considered to be critical virulence-associated factors and to play important roles in the pathogenesis of staphylococci. This review summarizes the classification, structure, expression regulation and biological functions of PSMs. The possible means to prevent PSM-associated diseases are also outlined in order to emphasize the need to investigate PSMs as potential targets for drug and vaccine design against staphylococcal infections.

Modelling clogging and biofilm detachment in sponge carrier media

Sponge carrier media provide a large surface area for biofilm support; however, little information is known about how to model their dual nature as a moving bed and as porous media. To investigate the interaction of mass transfer and detachment with bio-clogging, a novel biofilm model framework was built based on individual-based modelling, and hydrodynamics were modelled using the lattice Boltzmann method. The combined model structure enabled the simulation of oxygen and biomass distribution inside the porous network as well as inside the biofilm. In order to apply the model to moving bed biofilm reactors (MBBR), biofilm detachment due to abrasion (carrier collisions) was modelled to be dependent on intracarrier distance. In the initial growth stage, biofilm grew homogeneously on the internal skeleton after which a more discontinuous growth developed which significantly increased permeability. Low detachment rates caused clogging in the outer pores which limited growth of biofilm to the surface region of the sponge. High detachment rates on the surface enabled deeper oxygen penetration with higher internal biomass activity. The degree of clogging was also sensitive to the presence of extracellular polymeric substances because of its large spatial occupancy.

Reverse Osmosis Biofilm Dispersal by Osmotic Back-Flushing: Cleaning via Substratum Perforation

We have demonstrated the application of osmotic back-flushing (OBF) for the removal of biofilms from reverse osmosis (RO) membranes and proposed a new biofilm dispersal mechanism. OBF was conducted in a laboratory-scale RO test cell by introducing a sequence of hypersaline solution (1.5 M NaCl) flushes into the feedwater, while still maintaining the applied hydraulic pressure (13.8 bar). OBF resulted in significant biofilm detachment, leaving a thin, perforated bacterial film (24 μm thickness) with vertical cavities ranging from 15 to 50 μm in diameter. Application of OBF led to significant reductionin the biovolume (70–79%) and substantial removal of total organic carbon and proteins (78 and 66%, respectively), resulting in 63% permeate water flux recovery. Our findings demonstrate the potential of this chemical-free RO membrane cleaning method while highlighting the possible challenges of the technique.

On the influence of the culture conditions in bacterial antifouling bioassays and biofilm properties: Shewanella algae, a case study.

BackgroundA variety of conditions (culture media, inocula, incubation temperatures) are employed in antifouling tests with marine bacteria. Shewanella algae was selected as model organism to evaluate the effect of these parameters on: bacterial growth, biofilm formation, the activity of model antifoulants, and the development and nanomechanical properties of the biofilms.The main objectives were:1) To highlight and quantify the effect of these conditions on relevant parameters for antifouling studies: biofilm morphology, thickness, roughness, surface coverage, elasticity and adhesion forces.2) To establish and characterise in detail a biofilm model with a relevant marine strain.ResultsBoth the medium and the temperature significantly influenced the total cell densities and biofilm biomasses in 24-hour cultures. Likewise, the IC50 of three antifouling standards (TBTO, tralopyril and zinc pyrithione) was significantly affected by the medium and the initial cell density. Four media (Marine Broth, MB; 2% NaCl Mueller-Hinton Broth, MH2; Luria Marine Broth, LMB; and Supplemented Artificial Seawater, SASW) were selected to explore their effect on the morphological and nanomechanical properties of 24-h biofilms. Two biofilm growth patterns were observed: a clear trend to vertical development, with varying thickness and surface coverage in MB, LMB and SASW, and a horizontal, relatively thin film in MH2. The Atomic Force Microscopy analysis showed the lowest Young modulii for MB (0.16 ± 0.10 MPa), followed by SASW (0.19 ± 0.09 MPa), LMB (0.22 ± 0.13 MPa) and MH2 (0.34 ± 0.16 MPa). Adhesion forces followed an inverted trend, being higher in MB (1.33 ± 0.38 nN) and lower in MH2 (0.73 ± 0.29 nN).ConclusionsAll the parameters significantly affected the ability of S. algae to grow and form biofilms, as well as the activity of antifouling molecules. A detailed study has been carried out in order to establish a biofilm model for further assays. The morphology and nanomechanics of S. algae biofilms were markedly influenced by the nutritional environments in which they were developed. As strategies for biofilm formation inhibition and biofilm detachment are of particular interest in antifouling research, the present findings also highlight the need for a careful selection of the assay conditions.

A Biomimetic Gellan-Based Hydrogel as a Physicochemical Biofilm Model

Biofilm-forming microorganisms are ubiquitous, but continuous cultivation of these microorganisms with predictable biofilm growth and structural properties remains challenging. The development of a reliable simulated biofilm has been limited by a lack of information about the microorganism subpopulations and fluid-structure interactions involved in biofilm formation and detachment due to mechanical stress. This paper presents a gellan-based hydrogel as an alternative material for a simulated physicochemical biofilm. The mechanical properties of the hydrogel in terms of the storage (G') and loss (G'') moduli can be tuned and adapted to imitate biofilms of different strengths by changing the concentration of gellan and mono(Na+) or divalent (Mg2+) ions. The storage modulus of the hydrogel ranges from 2 to 20 kPa, and the loss modulus ranges from 0.1 to 2.0 kPa. The material constants of the hydrogels and biofilms of Pseudomonas putida KT2440 were experimentally determined by rheometric analysis. A simplified biofilm imitate based on highly hydrolyzed gellan hydrogels was established by using experimental design techniques that permitted independent analyses regardless of growth. This model system design was compared to real biofilms and was adapted to mimic the mechanical properties of biofilms by changing the hydrogel composition, resulting in biofilm-like viscoelastic behavior. The use of a gellan-based hydrogel enables the imitation of biofilm behavior in the absence of growth effects, thus simplifying the system. Biofilm characterization tools can be tested and verified before their application to the measurement of slow-growing, highly variable biofilms to estimate system errors, which are often smaller than the biological variations. In general, this method permits faster and more reliable testing of biofilm mechanical properties.

A Modeling and Simulation Study of the Role of Suspended Microbial Populations in Nitrification in a Biofilm Reactor

Many biological wastewater treatment processes are based on bacterial biofilms, i.e. layered aggregates of microbial populations deposited on surfaces. Detachment and (re-)attachment leads to an exchange of biomass between the biofilm and the surrounding aqueous phase. Traditionally, mathematical models of biofilm processes do not take the contribution of the suspended, non-attached bacteria into account, implicitly assuming that these are negligible due to the relatively small amount of suspended biomass compared to biofilm biomass. In this paper, we present a model for a nitrifying biofilm reactor that explicitly includes both types of biomass. The model is derived by coupling a reactor mass balance for suspended populations and substrates with a full one-dimensional Wanner-Gujer type biofilm model. The complexity of this model, both with respect to mathematical structure and number of parameters, prevents a rigorous analysis of its dynamics, wherefore we study the model numerically.Our investigations show that suspended biomass needs to be considered explicitly in the model if the interests of the study are the details of the nitrification process and its intermediate steps and compounds. However, suspended biomass may be neglected if the primary interests are the overall reactor performance criteria, such as removal rates. Furthermore, it can be expected that changes in the biofilm area, attachment, detachment, and dilution rates are more likely to affect the variables primarily associated with the second step of nitrification, while the variables associated with the first step tend to be more robust.

Image-Based 384-Well High-Throughput Screening Method for the Discovery of Skyllamycins A to C as Biofilm Inhibitors and Inducers of Biofilm Detachment in Pseudomonas aeruginosa

To date, most antibiotics have primarily been developed to target bacteria in the planktonic state. However, biofilm formation allows bacteria to develop tolerance to antibiotics and provides a mechanism to evade innate immune systems. Therefore, there is a significant need to identify small molecules to prevent biofilm formation and, more importantly, to disperse or eradicate preattached biofilms, which are a major source of bacterial persistence in nosocomial infections. We now present a modular high-throughput 384-well image-based screening platform to identify Pseudomonas aeruginosa biofilm inhibitors and dispersal agents. Biofilm coverage measurements were accomplished using non-z-stack epifluorescence microscopy to image a constitutively expressing green fluorescent protein (GFP)-tagged strain of P. aeruginosa and quantified using an automated image analysis script. Using the redox-sensitive dye XTT, bacterial cellular metabolic activity was measured in conjunction with biofilm coverage to differentiate between classical antibiotics and nonantibiotic biofilm inhibitors/dispersers. By measuring biofilm coverage and cellular activity, this screen identifies compounds that eradicate biofilms through mechanisms that are disparate from traditional antibiotic-mediated biofilm clearance. Screening of 312 natural-product prefractions identified the cyclic depsipeptide natural products skyllamycins B and C as nonantibiotic biofilm inhibitors with 50% effective concentrations (EC50s) of 30 and 60 μM, respectively. Codosing experiments of skyllamycin B and azithromycin, an antibiotic unable to clear preattached biofilms, demonstrated that, in combination, these compounds were able to eliminate surface-associated biofilms and depress cellular metabolic activity. The skyllamycins represent the first known class of cyclic depsipeptide biofilm inhibitors/dispersers.