Highlighting the limitations of static microplate biofilm assays for industrial biocide effectiveness compared to dynamic flow conditions.

The minimal inhibitory concentration of an antimicrobial required to inhibit the growth of planktonic populations (minimum inhibitory concentration [MIC]) remains the 'gold standard' even though biofilms are acknowledged to be recalcitrant to concentrations that greatly exceed the MIC. As a result, most studies focus on biofilm tolerance to high antimicrobial concentrations, whereas the effect of environmentally relevant sub-MIC on biofilms is neglected. The effect of the MIC and sub-MIC of an isothiazolinone biocide on a microbial community isolated from an industrial cooling system was assessed under static and flow conditions. The differential response of planktonic and sessile populations to these biocide concentrations was discerned by modifying the broth microdilution assay. However, the end-point analysis of biofilms cultivated in static microplates obscured the effect of sub-MIC and MIC on biofilms. A transition from batch to the continuous flow system revealed a more nuanced response of biofilms to these biocide concentrations, where biofilm-derived planktonic cell production was maintained despite an increase in the frequency and extent of biofilm sloughing. A holistic, 'best of both worlds' approach that combines the use of static and continuous flow systems is useful to investigate the potential for the development of persistent biofilms under conditions where exposure to sub-MIC and MIC may occur.

Listeria monocytogenes Biofilms Are Planktonic Cell Factories despite Peracetic Acid Exposure under Continuous Flow Conditions.

Listeria monocytogenes biofilms are ubiquitous in the food-processing environment, where they frequently show resistance against treatment with disinfectants such as peracetic acid (PAA) due to sub-lethal damage resulting in biofilm persistence or the formation of secondary biofilms. L. monocytogenes serovar ½a EGD-e biofilms were cultivated under continuous flow conditions at 10 °C, 22 °C, and 37 °C and exposed to industrially relevant PAA concentrations. The effect of PAA on biofilm metabolic activity and biomass was monitored in real-time using the CEMS-BioSpec system, in addition to daily measurement of biofilm-derived planktonic cell production. Biofilm-derived planktonic cell yields proved to be consistent with high yields during biofilm establishment (≥106 CFU.mL-1). The exposure of biofilms to the minimum inhibitory PAA concentration (0.16%) resulted in only a brief disruption in whole-biofilm metabolic activity and biofilm biomass accumulation. The recovered biofilm accumulated more biomass and greater activity, but cell yields remained similar. Increasing concentrations of PAA (0.50%, 1.5%, and 4.0%) had a longer-lasting inhibitory effect. Only the maximum dose resulted in a lasting inhibition of biofilm activity and biomass-a factor that needs due consideration in view of dilution in industrial settings. Better disinfection monitoring tools and protocols are required to adequately address the problem of Listeria biofilms in the food-processing environment, and more emphasis should be placed on biofilms serving as a "factory" for cell proliferation rather than only a survival mechanism.

Biofilm dynamics: linking in situ biofilm biomass and metabolic activity measurements in real-time under continuous flow conditions.

The tools used to study biofilms generally involve either destructive, end-point analyses or periodic measurements. The advent of the internet of things (IoT) era allows circumvention of these limitations. Here we introduce and detail the development of the BioSpec; a modular, nondestructive, real-time monitoring system, which accurately and reliably track changes in biofilm biomass over time. The performance of the system was validated using a commercial spectrophotometer and produced comparable results for variations in planktonic and sessile biomass. BioSpec was combined with the previously developed carbon dioxide evolution measurement system (CEMS) to allow simultaneous measurement of biofilm biomass and metabolic activity and revealed a differential response of these interrelated parameters to changing environmental conditions. The application of this system can facilitate a greater understanding of biofilm mass-function relationships and aid in the development of biofilm control strategies.

Survival of Planktonic and Sessile Cells of Lactobacillus rhamnosus and Lactobacillus reuteri upon Exposure to Simulated Fasting-State Gastrointestinal Conditions.

In this study, we report on the formation and resilience of Lactobacillus reuteri HFI-LD5 and Lactobacillus rhamnosus HFI-K2 biofilms cultivated in a CO2 evolution measurement system (CEMS) and exposed to biologically relevant, fasting-state gastrointestinal fluids under continuous flow conditions. For comparative purposes, planktonic and sessile populations of L. reuteri HFI-LD5 and L. rhamnosus HFI-K2 were each exposed to fasting-state gastric fluid (FSGF, pH 2.0) for 2 h, fasting-state intestinal fluid (FSIF, pH 7.5) for 6 h, and simulated colonic fluid (SCoF, pH 7.0) for 24 h. Planktonic cell numbers of L. reuteri HFI-LD5 declined from 6.6 log10 CFU/mL to 3.2 log10 CFU/mL and L. rhamnosus HFI-K2 from 6.6 log10 CFU/mL to undetectable levels after exposure to FSGF. Limited loss in viability was observed when free-floating cells were exposed to FSIF and SCoF. Sessile populations of both strains survived and recovered from the sequential exposure to all three gastric fluids despite observed detachment of biofilm biomass and a temporary decrease in metabolic activity to below detection limits, as recorded by changes in whole-biofilm CO2 production rates. The planktonic cell-focused gut microbiome-related research has most likely caused an underestimation in the overall survival ability of microorganisms in the gastrointestinal tract. Sessile cells of L. reuteri HFI-LD5 were metabolically inactive when exposed to gastric (FSGF) and intestinal (FSIF) fluids, suggesting that biofilms are formed in the small intestinal tract as survival mechanism. In the case of L. rhamnosus HFI-K2, cells were released from biofilms when suddenly exposed to pH 2.0.

Integration and proliferation of Pseudomonas aeruginosa PA01 in multispecies biofilms.

Despite an increased awareness of biofilm formation by pathogens and the role of biofilms in human infections, the potential role of environmental biofilms as an intermediate stage in the host-to-host cycle is poorly described. To initiate infection, pathogens in biofilms on inanimate environmental surfaces must detach from the biofilm and be transmitted to a susceptible individual in numbers large enough to constitute an infectious dose. Additionally, while detachment has been recognized as a discrete event in the biofilm lifestyle, it has not been studied to the same extent as biofilm development or biofilm physiology. Successful integration of Pseudomonas aeruginosa strain PA01 expressing green fluorescent protein (PA01GFP), employed here as a surrogate pathogen, into multispecies biofilm communities isolated and enriched from sink drains in public washrooms and a hospital intensive care unit is described. Confocal laser scanning microscopy indicated that PA01GFP cells were most frequently located in the deeper layers of the biofilm, near the attachment surface, when introduced into continuous flow cells before or at the same time as the multispecies drain communities. A more random integration pattern was observed when PA01GFP was introduced into established multispecies biofilms. Significant numbers of single PA01GFP cells were continuously released from the biofilms to the bulk liquid environment, regardless of the order of introduction into the flow cell. Challenging the multispecies biofilms containing PA01GFP with sub-lethal concentrations of an antibiotic, chelating agent and shear forces that typically prevail at distances away from the point of treatment showed that environmental biofilms provide a suitable habitat where pathogens are maintained and protected, and from where they are continuously released.

Biofilms' role in planktonic cell proliferation.

The detachment of single cells from biofilms is an intrinsic part of this surface-associated mode of bacterial existence. Pseudomonas sp. strain CT07gfp biofilms, cultivated in microfluidic channels under continuous flow conditions, were subjected to a range of liquid shear stresses (9.42 mPa to 320 mPa). The number of detached planktonic cells was quantified from the effluent at 24-h intervals, while average biofilm thickness and biofilm surface area were determined by confocal laser scanning microscopy and image analysis. Biofilm accumulation proceeded at the highest applied shear stress, while similar rates of planktonic cell detachment was maintained for biofilms of the same age subjected to the range of average shear rates. The conventional view of liquid-mediated shear leading to the passive erosion of single cells from the biofilm surface, disregards the active contribution of attached cell metabolism and growth to the observed detachment rates. As a complement to the conventional conceptual biofilm models, the existence of a biofilm surface-associated zone of planktonic cell proliferation is proposed to highlight the need to expand the traditional perception of biofilms as promoting microbial survival, to include the potential of biofilms to contribute to microbial proliferation.

Metabolic differentiation in biofilms as indicated by carbon dioxide production rates.

The measurement of carbon dioxide production rates as an indication of metabolic activity was applied to study biofilm development and response of Pseudomonas sp. biofilms to an environmental disturbance in the form of a moving air-liquid interface (i.e., shear). A differential response in biofilm cohesiveness was observed after bubble perturbation, and the biofilm layers were operationally defined as either shear-susceptible or non-shear-susceptible. Confocal laser scanning microscopy and image analysis showed a significant reduction in biofilm thickness and biomass after the removal of the shear-susceptible biofilm layer, as well as notable changes in the roughness coefficient and surface-to-biovolume ratio. These changes were accompanied by a 72% reduction of whole-biofilm CO2 production; however, the non-shear-susceptible region of the biofilm responded rapidly after the removal of the overlying cells and extracellular polymeric substances (EPS) along with the associated changes in nutrient and O2 flux, with CO2 production rates returning to preperturbation levels within 24 h. The adaptable nature and the ability of bacteria to respond to environmental conditions were further demonstrated by the outer shear-susceptible region of the biofilm; the average CO2 production rate of cells from this region increased within 0.25 h from 9.45 +/- 5.40 fmol of CO2 x cell(-1) x h(-1) to 22.6 +/- 7.58 fmol of CO2 x cell(-1) x h(-1) when cells were removed from the biofilm and maintained in suspension without an additional nutrient supply. These results also demonstrate the need for sufficient monitoring of biofilm recovery at the solid substratum if mechanical methods are used for biofouling control.

Planktonic cell yield is linked to biofilm development.

We report on the ability of surface-associated microbes to produce and release single planktonic cells to the bulk liquid as early as 6 h after attachment, with pure culture and mixed-species biofilms yielding up to approximately 1 x 10(6) cells/cm(2) of attachment area per hour to the effluent after 24 h. Planktonic cell production typically increased as the biofilm developed and levelled off after the biofilm reached steady-state dimensions. Microscopic observations of continuous-flow cultured biofilms revealed independent cell movement within the biofilm microenvironment compared with flow-dependent movement of mostly single cells in the bulk-liquid phase. These results indicate that the prevailing concept of detachment occurring only after the biofilm has matured is incomplete. Instead, we show that biofilms yield cells to the environment soon after initial surface contact; the extent of this yield is dependent on biofilm development, which in turn is influenced by environmental parameters such as bulk-liquid flow rates and nutrient availability. The observation that biofilms yield significant numbers of cells throughout development should lead to a greater understanding of pathogen dissemination, biofouling of products or facilities, and the role that biofilms play in microbial proliferation in the environment.

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.