Antimicrobial mechanism and the effect of atmospheric pressure N2 plasma jet on the regeneration capacity of Staphylococcus aureus biofilm

Abstract

This study systematically assessed the inactivation mechanism on Staphylococcus aureus biofilms by a N₂ atmospheric-pressure plasma jet and the effect on the biofilm regeneration capacity from the bacteria which survived, and their progenies. The total bacterial populations were 7.18?±?0.34 log10 CFU ml^?1 in biofilms and these were effectively inactivated (>5.5-log10 CFU ml^?1) within 30?min of exposure. Meanwhile, >80% of the S. aureus biofilm cells lost their metabolic capacity. In comparison, ～20% of the plasma-treated bacteria entered a viable but non-culturable state. Moreover, the percentage of membrane-intact bacteria declined to ～30%. Scanning electron microscope images demonstrated cell shrinkage and deformation post-treatment. The total amount of intracellular reactive oxygen species was observed to have significantly increased in membrane-intact bacterial cells with increasing plasma dose. Notably, the N₂ plasma treatment could effectively inhibit the biofilm regeneration ability of the bacteria which survived, leading to a long-term phenotypic response and dose-dependent inactivation effect on S. aureus biofilms, in addition to the direct rapid bactericidal effect.     

Autoinducer‐2 is produced in saliva‐fed flow conditions relevant to natural oral biofilms

Aims: We evaluated the ability of a dual‐species community of oral bacteria to produce the universal signalling molecule, autoinducer‐2 (AI‐2), in saliva‐fed biofilms. Methods and Results: Streptococcus oralis 34, S. oralis 34 luxS mutant and Actinomyces naeslundii T14V were grown as single‐ and dual‐species biofilms within sorbarods fed with 25% human saliva. AI‐2 concentration in biofilm effluents was determined by the Vibrio harveyi BB170 bioluminescence assay. After homogenizing the sorbarods to release biofilm cells, cell numbers were determined by fluorometric analysis of fluorescent antibody‐labelled cells. After 48 h, dual‐species biofilm communities of interdigitated S. oralis 34 and A. naeslundii T14V contained 3·2 × 10⁹ cells: fivefold more than single‐species biofilms. However, these 48‐h dual‐species biofilms exhibited the lowest concentration ratio of AI‐2 to cell density. Conclusions: Oral bacteria produce AI‐2 in saliva‐fed biofilms. The decrease of more than 10‐fold in concentration ratio seen between 1 and 48 h in S. oralis 34–A. naeslundii T14V biofilms suggests that peak production of AI‐2 occurs early and is followed by a very low steady‐state level. Significance and Impact of the Study: High oral bacterial biofilm densities may be achieved by inter‐species AI‐2 signalling. We propose that low concentrations of AI‐2 contribute to the establishment of oral commensal biofilm communities.     

Development and testing of a novel microcantilever technique for measuring the cohesive strength of intact biofilms

Cohesive strength is an important parameter for understanding and modeling the mechanics of biomass detachment from bacterial biofilms. It is challenging to measure the mechanical properties of biofilms, however, because biofilms may desiccate when removed from liquid medium and they are inherently fragile. Poppele and Hozalski (Poppele and Hozalski, 2003, J Microb Methods 55:607–615) presented a microcantilever method for measuring the tensile strength of detached biofilm fragments while submersed in liquid medium. Here we present a modification of the microcantilever method to quantify the strength of intact bacterial biofilms. Initial testing was performed on Pseudomonas aeruginosa biofilms and on Staphylococcus epidermidis biofilms grown in rotating disk reactors. The cohesive strength values were highly variable (i.e., coefficients of variation ranging from 71% to 143%) and ranged from 59 to 18,900 Pa for the P. aeruginosa biofilms and from 61 to 5,840 Pa for the S. epidermidis biofilms. The biofilms also appeared to be isotropic as strength did not vary with angle of testing relative to the direction of applied shear. Strength testing using both the intact and fragment methods was performed on five samples of P. aeruginosa biofilms, and the strength populations were not from the same distribution in three cases. Equivalent diameters for the fragments detached from biofilms during strength testing ranged from 5 to 500 µm, which is within the range of size of biofilm fragments observed in the effluents of lab‐scale and full‐scale bioreactors. The microcantilever is a simple yet powerful tool for measuring the cohesive strength of intact biofilms at a relevant scale. Biotechnol. Bioeng. 2010;105: 924–934. © 2009 Wiley Periodicals, Inc.     

Inoculum effects on community composition and nitritation performance of autotrophic nitrifying biofilm reactors with counter‐diffusion geometry

The link between nitritation success in a membrane‐aerated biofilm reactor (MABR) and the composition of the initial ammonia‐ and nitrite‐oxidizing bacterial (AOB and NOB) population was investigated. Four identically operated flat‐sheet type MABRs were initiated with two different inocula: from an autotrophic nitrifying bioreactor (Inoculum A) or from a municipal wastewater treatment plant (Inoculum B). Higher nitritation efficiencies (NO₂^‐‐N/NH₄⁺‐N) were obtained in the Inoculum B‐ (55.2–56.4%) versus the Inoculum A‐ (20.2–22.1%) initiated reactors. The biofilms had similar oxygen penetration depths (100–150 µm), but the AOB profiles [based on 16S rRNA gene targeted real‐time quantitative PCR (qPCR)] revealed different peak densities at or distant from the membrane surface in the Inoculum B‐ versus A‐initiated reactors, respectively. Quantitative fluorescence in situ hybridization (FISH) revealed that the predominant AOB in the Inoculum A‐ and B‐initiated reactors were Nitrosospira spp. (48.9–61.2%) versus halophilic and halotolerant Nitrosomonas spp. (54.8–63.7%), respectively. The latter biofilm displayed a higher specific AOB activity than the former biofilm (1.65 fmol cell^?1 h^?1 versus 0.79 fmol cell^?1 h^?1). These observations suggest that the AOB and NOB population compositions of the inoculum may determine dominant AOB in the MABR biofilm, which in turn affects the degree of attainable nitritation in an MABR.     

In situ effective diffusion coefficient profiles in live biofilms using pulsed‐field gradient nuclear magnetic resonance

Diffusive mass transfer in biofilms is characterized by the effective diffusion coefficient. It is well documented that the effective diffusion coefficient can vary by location in a biofilm. The current literature is dominated by effective diffusion coefficient measurements for distinct cell clusters and stratified biofilms showing this spatial variation. Regardless of whether distinct cell clusters or surface‐averaging methods are used, position‐dependent measurements of the effective diffusion coefficient are currently: (1) invasive to the biofilm, (2) performed under unnatural conditions, (3) lethal to cells, and/or (4) spatially restricted to only certain regions of the biofilm. Invasive measurements can lead to inaccurate results and prohibit further (time‐dependent) measurements which are important for the mathematical modeling of biofilms. In this study our goals were to: (1) measure the effective diffusion coefficient for water in live biofilms, (2) monitor how the effective diffusion coefficient changes over time under growth conditions, and (3) correlate the effective diffusion coefficient with depth in the biofilm. We measured in situ two‐dimensional effective diffusion coefficient maps within Shewanella oneidensis MR‐1 biofilms using pulsed‐field gradient nuclear magnetic resonance methods, and used them to calculate surface‐averaged relative effective diffusion coefficient (D_rs) profiles. We found that (1) D_rs decreased from the top of the biofilm to the bottom, (2) D_rs profiles differed for biofilms of different ages, (3) D_rs profiles changed over time and generally decreased with time, (4) all the biofilms showed very similar D_rs profiles near the top of the biofilm, and (5) the D_rs profile near the bottom of the biofilm was different for each biofilm. Practically, our results demonstrate that advanced biofilm models should use a variable effective diffusivity which changes with time and location in the biofilm. Biotechnol. Bioeng. 2010;106: 928–937. © 2010 Wiley Periodicals, Inc.     

Homogenization of Pseudomonas aeruginosa PAO1 biofilms visualized by freeze‐substitution electron microscopy

A knowledge of the mechanical properties of bacterial biofilms is required to more fully understand the processes of biofilm formation such as initial adhesion or detachment. The main contribution of this article is to demonstrate the use of homogenization techniques to compute mechanical parameters of Pseudomonas aeruginosa PAO1 biofilms. For this purpose, homogenization techniques are used to analyze freeze substitution electron micrographs of the biofilm cross‐sections. The concept of a representative volume element and the study about his representativeness allows us to determine the optimal size in order to analyze these biofilm images. Results demonstrate significant heterogeneities with respect to stiffness and these can be explained by varying cell density distribution throughout the bacterial biofilms. These stiffness variations lead to different mechanical properties along the height of the biofilm. Moreover, a numerical shear stress test shows the impact of these heterogeneities on the detachment process. Several modes of detachment are highlighted according to the local strain energy in the different parts of the biofilm. Knowing where, and how, a biofilm may detach will allow better prediction of accumulation and biomass detachment. Biotechnol. Bioeng. 2013; 110: 1405–1418. © 2012 Wiley Periodicals, Inc.     

Biofilm form and function: carbon availability affects biofilm architecture, metabolic activity and planktonic cell yield

Aims: To investigate carbon transformation by biofilms and changes in biofilm architecture, metabolic activity and planktonic cell yield in response to fluctuating carbon availability. Methods and Results: Pseudomonas sp. biofilms were cultured under alternating carbon‐replete and carbon‐limited conditions. A shift to medium without added carbon led to a 90% decrease in biofilm respiration rate and a 40% reduction in planktonic cell yield within 1 h. Attached cell division and progeny release were shown to contribute to planktonic cell numbers during carbon limitation. Development of a significantly enlarged biofilm surface area during carbon limitation facilitated a rapid increase in whole‐biofilm metabolic activity, cell yield and biomass upon the re‐introduction of carbon after 8 days of limitation. The cumulative number of planktonic cells (>10¹⁰ CFU) released from the biofilm during the cultivation period contained only 1·0% of the total carbon available to the biofilm, with 6·5% of the carbon retained in the biofilm and 54% mineralized to CO₂. Conclusions: Biofilm‐derived planktonic cell yield is a proliferation mechanism. The rapid response of biofilms to environmental perturbations facilitates the optimal utilization of resources to promote both proliferation and survival. Biofilms function as efficient catalysts for environmental carbon transformation and mineralization. Significance and Impact of the study: A greater understanding of the relationship between biofilm form and function can inform strategies intended to control and/or promote biofilm formation.     

Bacterial challenge and eicosanoids act in plasmatocyte spreading

We report on experiments designed to more thoroughly document the roles of eicosanoids as crucial elements in cell spreading and on experiments designed to test the hypothesis that in vivo bacterial infections influence cell spreading on glass surfaces. We used hemocytes prepared from tobacco hornworms, Manduca sexta (L.) (Lepidoptera: Sphingidae) and four species of bacteria (Serratia marcescens, Escherichia coli, Bacillus subtilis, and Micrococcus luteus) in each experiment. Our protocols yielded several important points: (i) hemocytes prepared from hornworms at 15 and 60 min following infection with, separately, each of the four bacterial species were fundamentally altered in size (all less than the 15‐µm counting cut‐off) and none of the hemocytes exhibited cell‐spreading behavior; (ii) the influence of bacterial challenge on cell spreading declined with incubation time post‐challenge; (iii) conditioned medium (CM) prepared by exposing hemocytes to bacterial cells in vitro exerted a strong dose‐dependent influence on cell spreading. Specifically, plasmatocytes increased in length from about 38 µm with 2.5% CM to a maximum of about 54 µm at 100% CM; and (iv) the retarding influence of dexamethasone (an eicosanoid biosynthesis inhibitor) on cell spreading was reversed by arachidonic acid, prostaglandin H₂, and CM. Taken together, these findings indicate that both bacterial infection and eicosanoids influence hemocyte‐spreading processes.     

Real-time in-situ electrochemical monitoring of Pseudomonas aeruginosa biofilms grown on air–liquid interface and its antibiotic susceptibility using a novel dual-chamber microfluidic device

Biofilms are communities of bacterial cells encased in a self-produced polymeric matrix that exhibit high tolerance toward environmental stress. Despite the plethora of research on biofilms, most P. aeruginosa biofilm models are cultured on a solid–liquid interface, and the longitudinal growth characteristics of P. aeruginosa biofilm are unclear. This study demonstrates the real-time and noninvasive monitoring of biofilm growth using a novel dual-chamber microfluidic device integrated with electrochemical detection capabilities to monitor pyocyanin (PYO). The growth of P. aeruginosa biofilms on the air–liquid interface (ALI) was monitored over 48 h, and its antibiotic susceptibility to 6 h exposure of 50, 400, and 1600 µg/ml of ciprofloxacin solutions was analyzed. The biofilm was treated directly on its surface and indirectly from the substratum by delivering the CIP solution to the top or bottom chamber of the microfluidic device. Results showed that P. aeruginosa biofilm developed on ALI produces PYO continuously, with the PYO production rate varying longitudinally and peak production observed between 24 and 30 h. In addition, this current study shows that the amount of PYO produced by the ALI biofilm is proportional to its viable cell numbers, which has not been previously demonstrated. Biofilm treated with ciprofloxacin solution above 400 µg/ml showed significant PYO reduction, with biofilms being killed more effectively when treatment was applied to their surfaces. The electrochemical measurement results have been verified with colony-forming unit count results, and the strong correlation between the PYO electrical signal and the viable cell number highlights the usefulness of this approach for fast and low-cost ALI biofilm study and antimicrobial tests.     

Sessile Legionella pneumophila is able to grow on surfaces and generate structured monospecies biofilms

Currently, models for studying Legionella pneumophila biofilm formation rely on multi-species biofilms with low reproducibility or on growth in rich medium, where planktonic growth is unavoidable. The present study describes a new medium adapted to the growth of L. pneumophila monospecies biofilms in vitro. A microplate model was used to test several media. After incubation for 6 days in a specific biofilm broth not supporting planktonic growth, biofilms consisted of 5.36 ± 0.40 log (cfu cm^?2) or 5.34 ± 0.33 log (gu cm^?2). The adhered population remained stable for up to 3 weeks after initial inoculation. In situ confocal microscope observations revealed a typical biofilm structure, comprising cell clusters ranging up to ～300 μm in height. This model is adapted to growing monospecies L. pneumophila biofilms that are structurally different from biofilms formed in a rich medium. High reproducibility and the absence of other microbial species make this model useful for studying genes involved in biofilm formation.     

pH, redox potential and local biofilm potential microenvironments within Geobacter sulfurreducens biofilms and their roles in electron transfer

The limitation of pH inside electrode‐respiring biofilms is a well‐known concept. However, little is known about how pH and redox potential are affected by increasing current inside biofilms respiring on electrodes. Quantifying the variations in pH and redox potential with increasing current is needed to determine how electron transfer is tied to proton transfer within the biofilm. In this research, we quantified pH and redox potential variations in electrode‐respiring Geobacter sulfurreducens biofilms as a function of respiration rates, measured as current. We also characterized pH and redox potential at the counter electrode. We concluded that (1) pH continued to decrease in the biofilm through different growth phases, showing that the pH is not always a limiting factor in a biofilm and (2) decreasing pH and increasing redox potential at the biofilm electrode were associated only with the biofilm, demonstrating that G. sulfurreducens biofilms respire in a unique internal environment. Redox potential inside the biofilm was also compared to the local biofilm potential measured by a graphite microelectrode, where the tip of the microelectrode was allowed to acclimatize inside the biofilm. Biotechnol. Bioeng. 2012; 109: 2651–2662. © 2012 Wiley Periodicals, Inc.     

Biofilm model calibration and microbial diversity study using Monte Carlo simulations

Mathematical models are useful tools for studying and exploring biological conversion processes as well as microbial competition in biological treatment processes. A single‐species biofilm model was used to describe biofilm reactor operation at three different hydraulic retention times (HRT). The single‐species biofilm model was calibrated with sparse experimental data using the Monte Carlo filtering method. This calibrated single‐species biofilm model was then extended to a multi‐species model considering 10 different heterotrophic bacteria. The aim was to study microbial diversity in bulk phase biomass and biofilm, as well as the competition between suspended and attached biomass. At steady state and independently of the HRT, Monte Carlo simulations resulted only in one unique dominating bacterial species for suspended and attached biomass. The dominating bacterial species was determined by the highest specific substrate affinity (ratio of µ/K_S). At a short HRT of 20 min, the structure of the microbial community in the bulk liquid was determined by biomass detached from the biofilm. At a long HRT of 8 h, both biofilm detachment and microbial growth in the bulk liquid influenced the microbial community distribution. Biotechnol. Bioeng. 2013; 110: 1323–1332. © 2012 Wiley Periodicals, Inc.     

Combined treatment of Pseudomonas aeruginosa biofilms with bacteriophages and chlorine

Bacterial biofilms are a growing concern in a broad range of areas. In this study, a mixture of RNA bacteriophages isolated from municipal wastewater was used to control and remove biofilms. At the concentrations of 400 and 4 × 10⁷ PFU/mL, the phages inhibited Pseudomonas aeruginosa biofilm formation by 45 ± 15% and 73 ± 8%, respectively. At the concentrations of 6,000 and 6 × 10⁷ PFU/mL, the phages removed 45 ± 9% and 75 ± 5% of pre‐existing P. aeruginosa biofilms, respectively. Chlorine reduced biofilm growth by 86 ± 3% at the concentration of 210 mg/L, but it did not remove pre‐existing biofilms. However, a combination of phages (3 × 10⁷ PFU/mL) and chlorine at this concentration reduced biofilm growth by 94 ± 2% and removed 88 ± 6% of existing biofilms. In a continuous flow system with continued biofilm growth, a combination of phages (a one‐time treatment at the concentration of 1.9 × 10⁸ PFU/mL for 1 h first) with chlorine removed 97 ± 1% of biofilms after Day 5 while phage and chlorine treatment alone removed 89 ± 1% and 40 ± 5%, respectively. For existing biofilms, a combined use of a lower phage concentration (3.8 × 10⁵ PFU/mL) and chlorination with a shorter time duration (12 h) followed by continuous water flushing removed 96 ± 1% of biofilms in less than 2 days. Laser scanning confocal microscopy supplemented with electron microscopy indicated that the combination treatment resulted in biofilms with lowest cell density and viability. These results suggest that the combination treatment of phages and chlorine is a promising method to control and remove bacterial biofilms from various surfaces. Biotechnol. Bioeng. 2013; 110: 286–295. © 2012 Wiley Periodicals, Inc.     

In vitro and ex vivo biofilms of dermatophytes: a new panorama for the study of antifungal drugs

Abstract

This study describes an ex vivo model that creates an environment for dermatophyte biofilm growth, with features that resemble those of in vivo conditions, designing a new panorama for the study of antifungal susceptibility. Regarding planktonic susceptibility, MIC ranges were 0.125-1?µg ml^?1 for griseofulvin and 0.000097-0.25?µg ml^?1 for itraconazole and terbinafine. sMIC50 ranges were 2->512?µg ml^?1 for griseofulvin and 0.25->64?µg ml^?1 for itraconazole and terbinafine. CLSM images demonstrated a reduction in the amount of cells within the biofilm, but hyphae and conidia were still observed and biofilm biomass was maintained. SEM analysis demonstrated a retraction in the biofilm matrix, but fungal structures and water channels were preserved. These results show that ex vivo biofilms are more tolerant to antifungal drugs than in vitro biofilms, suggesting that environmental and nutritional conditions created by this ex vivo model favor biofilm growth and robustness, and hence drug tolerance.     

Biofilm formation by haloarchaea

A fluorescence‐based live‐cell adhesion assay was used to examine biofilm formation by 20 different haloarchaea, including species of Halobacterium, Haloferax and Halorubrum, as well as novel natural isolates from an Antarctic salt lake. Thirteen of the 20 tested strains significantly adhered (P‐value < 0.05) to a plastic surface. Examination of adherent cell layers on glass surfaces by differential interference contrast, fluorescence and confocal microscopy showed two types of biofilm structures. Carpet‐like, multi‐layered biofilms containing micro‐ and macrocolonies (up to 50 μm in height) were formed by strains of Halobacterium salinarum and the Antarctic isolate t‐ADL strain DL24. The second type of biofilm, characterized by large aggregates of cells adhering to surfaces, was formed by Haloferax volcanii DSM 3757^T and Halorubrum lacusprofundi DL28. Staining of the biofilms formed by the strongly adhesive haloarchaeal strains revealed the presence of extracellular polymers, such as eDNA and glycoconjugates, substances previously shown to stabilize bacterial biofilms. For Hbt. salinarum DSM 3754^T and Hfx. volcanii DSM 3757^T, cells adhered within 1 day of culture and remained viable for at least 2 months in mature biofilms. Adherent cells of Hbt. salinarum DSM 3754^T showed several types of cellular appendages that could be involved in the initial attachment. Our results show that biofilm formation occurs in a surprisingly wide variety of haloarchaeal species.     

Susceptibility of Staphylococcus aureus biofilms to reactive discharge gases

Formation of bacterial biofilms at solid–liquid interfaces creates numerous problems in both industrial and biomedical sciences. In this study, the susceptibility of Staphylococcus aureus biofilms to discharge gas generated from plasma was tested. It was found that despite distinct chemical/physical properties, discharge gases from oxygen, nitrogen, and argon demonstrated very potent and almost the same anti-biofilm activity. The bacterial cells in S. aureus biofilms were killed (>99.9%) by discharge gas within minutes of exposure. Under optimal experimental conditions, no bacteria and biofilm re-growth from discharge gas treated biofilms was found. Further studies revealed that the anti-biofilm activity of the discharge gas occurred by two distinct mechanisms: (1) killing bacteria in biofilms by causing severe cell membrane damage, and (2) damaging the extracellular polymeric matrix in the architecture of the biofilm to release biofilm from the surface of the solid substratum. Information gathered from this study provides an insight into the anti-biofilm mechanisms of plasma and confirms the applications of discharge gas in the treatment of biofilms and biofilm related bacterial infections.     

Impact of Irgarol 1051 on the larval development and metamorphosis of Balanus amphitrite Darwin,the diatom Amphora coffeaformis and natural biofilm

The effect of Irgarol 1051 on the biofilm-forming diatom, Amphora coffeaformis, and on natural biofilm (NBF) was assessed. A reduction in the number of A. coffeaformis cells within a biofilm was observed after treatment with Irgarol 1051, confirming its role as an inhibitor of photosynthetic activity. The impact of this compound on the development of nauplii of Balanus amphitrite was evaluated through its impact on Chaetoceros calcitrans, which was provided as food for the larvae. A reduction in the number of cells of C. calcitrans was observed when treated with Irgarol 1051. When larvae of B. amphitrite were reared using C. calcitrans in the presence of Irgarol 1051, their mortality increased with an increase in the concentration of Irgarol 1051 (13% at 1 µg l^?1 to 40% at 1000 µg l^?1) compared with the control (6%). Nauplii reared in the presence of Irgarol 1051 developed more slowly (6–7 days) compared with control larvae (4–5 days). Cyprid bioassay results indicated an increase in percentage metamorphosis (76%) when NBFs were treated with the highest concentration of Irgarol 1051, compared with untreated biofilm (28%). The enhanced rate of metamorphosis appeared to be related to an increase in bacterial numbers in the biofilm, which may have been due to lysis of diatoms caused by Irgarol 1051. A. coffeaformis biofilms grown in the presence of antibiotics showed a significant reduction in cell numbers, which on further treatment with Irgarol 1051 showed an increase in cell numbers. Thus, it can be hypothesised that A. coffeaformis cells that were subjected to stress twice may have expressed resistant genes. Furthermore, if plasmids are present in the biofilms, they may enhance transfer to the surviving cells making them more resistant to hostile conditions.     

The Effect of a Cationic Porphyrin on Pseudomonas aeruginosa Biofilms

Current studies have indicated the utility of photodynamic therapy using porphyrins in the treatment of bacterial infections. Photoactivation of porphyrins results in the production of singlet oxygen (¹O₂) that damages biomolecules associated with cells and biofilms, e.g., proteins, polysaccharides, and DNA. The effect of a cationic porphryin on P. aeruginosa PAO1 biofilms was assessed by exposing static biofilms to 5,10,15,20-tetrakis(1-methyl-pyridino)-21H,23H-porphine, tetra-p-tosylate salt (TMP) followed by irradiation. Biofilms were visualized using confocal laser scanning microscopy (CLSM) and cell viability determined using the LIVE/DEAD BacLight viability assay and standard plate counts. At a concentration of 100 μM TMP, there was substantial killing of P. aeruginosa PAO1 wild-type and pqsA mutant biofilms with little disruption of the biofilm matrix or structure. Exposure to 225 μM TMP resulted in almost complete killing as well as the detachment of wild-type PAO1 biofilms. In contrast, pqsA mutant biofilms that contain less extracellular DNA remained intact. Standard plate counts of cells recovered from attached biofilms revealed a 4.1-log₁₀ and a 3.9-log₁₀ reduction in viable cells of wild-type PAO1 and pqsA mutant strains, respectively. Our results suggest that the action of photoactivated TMP on P. aeruginosa biofilms is two-fold: direct killing of individual cells within biofilms and detachment of the biofilm from the substratum. There was no evidence of porphyrin toxicity in the absence of light; however, biofilms pretreated with TMP without photoactivation were substantially more sensitive to tobramycin than untreated biofilms.