Differential response of sessile and planktonic bacterial populations following exposure to antimicrobial treatment

Thesis (MSc)--University of Stellenbosch, 2004.ENGLISH ABSTRACT: The ability of biofilms to resist antimicrobial treatment, when planktonic microbes cannot, is of not only fundamental scientific interest, but also a concern in industrial and medical fields. The inability to control biofouling of water distribution networks and products, as well as recurrent infections of implanted medical devices, is not only costly, but also potentially lethal. Several mechanisms whereby biofilms are able to evade antibiotic and biocidal agents have been proposed and investigated, but no universally relevant characteristic has been identified. . Initial investigation, involving BacLightTh ! LIVEIDEAD viability probes, epifluorescence microscopy and image analysis into the ability of natural biofilm and planktonic populations, .cultured in situ in a cooling tower, to survive treatment with a commercial biocide was not conclusive. Subsequent laboratory experimentation with a bacterial isolate from the cooling tower water revealed that the ability of attached biofilms to resist antimicrobial treatment exceeded that of planktonic cells shed from the biofilm. The reduced ability of suspended cells to survive antimicrobial treatment was not statistically significant, compared to that of the biofilm (P = 0.05). This is in contrast to the wealth of literature published on the subject of biofilm antimicrobial resistance The dilution rate in the flowcells in which biofilms were cultivated was more than 100 times higher than the maximum specific growth rate of the test organism. Nevertheless, there was typically more than I x 108 cells/ml in the effluent, suggesting that a metabolically active, rapidly dividing layer of cells existed at the biofilm bulk-liquid interface, from where daughter cells continuously detached. Treatment with an antimicrobial agent resulted in a significant reduction in the viability and number of cells detached from the biofilm, suggesting that this metabolically active layer of the biofilm was more sensitive to antimicrobial treatment, possibly due to a higher specific growth rate. Antimicrobial resistance was shown to be affected by the growth rate for planktonic bacterial populations, with an increased ability to survive, correlated with a decrease in specific growth rate. This supports the contention that growth rate plays a role in the susceptibility of the active layer. The bacterial cells in the layers closest to the attachment surface of the biofilm has frequently been shown to be slow growing, due to nutrient and oxygen limitation, while the outer biofilm layer is more susceptible to unfavourable environmental conditions. It is possible that such differentiation, which results in a responsive outer biofilm layer, provides a mechanism for the protection of the cells in the deeper layers, and thus survival over time. The results presented here support several hypotheses put forth in literature to account for the increased resistance of biofilms towards antimicrobial agents. Future work will include an investigation into changes in the patterns of gene expression when a bacteria becomes attached to a surface, upon subsequent release from the biofilm, and the influence this has on the ability to resist antimicrobial treatment.AFRIKAANSE OPSOMMING: Die vermoë van aangehegte mikrobes, in teenstelling met vrydrywende mikroorganismes, om behandeling met antimikrobiese middels te oorleef, is nie net van belang vanuit 'n fundamenteel wetenskaplike oogpunt nie, maar ook betekenisvol vir die industriële en mediese velde. Die beheer van bio-bevuiling van waterverspreidingsnetwerke en produkte, sowel as herhaalde infeksies van mediese inplantings, is nie net van kostebelang nie, maar ook potensieël lewensgevaarlik. Verskeie meganismes wat biofilms in staat stelom antimikrobiese behandeling te oorleef, IS voorgestel en ondersoek, maar geen alomteenwoordige eienskap is tot dusver geïdentifiseer nie. Aanvanklike ondersoeke na die vermoë van natuurlike biofilms en planktoniese 'gemeenskappe, om biosiedbehandeling in situ in 'n lugversorgingskoeltoring se water te oorleef, was onbeslis. Die eksperimentele metodes het gebruik gemaak van BacLight™ LIVE/DEAD lewensvatbaarheidkleurstof, epifluoressensie-mikroskopie en beeldanalise. Daaropvolgende ondersoeke met 'n bakteriese isolaat vanuit die koeltoring het daarop gedui dat biofilms beter in staat is om antimikrobiese behandeling te oorleef as selle wat vrygelaat word vanuit die biofilm. Die afname in the lewensvatbaarheid van vrydrywende selle, na afloop van biosiedbehandeling, was nie statisties beduidend in vergelyking met die van die biofilm nie (P = 0.05). Die bevinding is in teenstelling met wat algemeen aanvaar word in die literatuur. Die verdunningstempo waaronder die biofilms in die vloeiselle gekweek is, was meer as 100- voudig hoër as die maksimum spesifieke groeitempo van die toetsorganisme. Ten spyte hiervan was daar tipies meer as 1 x 108 selle/ml in die uitvloeisel teenwoordig. Dit dui op 'n metabolies aktiewe, vinnig verdelende laag selle in die boonste laag van die biofilm, naaste aan die vloeistof fase, waarvandaan dogterselle voortdurend vrygestel word. Behandeling met die antimikrobiese agent het 'n beduidende afname in die lewensvatbaarheid en aantal dogterselle tot gevolg gehad, wat lei tot die gevolgtrekking dat die metabolies aktiewe laag van die biofilm meer sensitief is vir antimikrobiese behandeling, moontlik weens 'n hoër spesifieke groeitempo. Daar is verder bewys dat die vermoë om die werking van die antimikrobiese middel teen te staan, afhanklik is van die spesifieke groeitempo van planktoniese populasies. 'n Afname in groeitempo word geassosieer met 'n toename in oorlewing na antimikrobiese behandeling, wat die voorstel dat die groeitempo van die aktiewe laag 'n rol speel in die vatbaarheid daarvan, ondersteun. Dit is bekend dat die metaboliese aktiwiteit van bakteriese selle nader aan die aanhegtingsoppervlak van die biofilm verlaag is, weens 'n afname in diffusie van suurstof en nutriente in daardie deel van die biofilm. Dit is moontlik dat hierdie differensiasie, wat lei tot die vatbaarheid van die buitenste laag van die biofilm vir ongunstige omgewingstoestande, 'n oorlewingsmeganisme daarstel wat die onderliggende selle beskerm. Die resultate wat hier voorgelê word, ondersteun verskeie hipoteses wat die verhoogde weerstandbiedendheid van biofilms teen antimikrobiese middels beskryf. Toekomstige werk sluit ondersoeke in na veranderende patrone van geenuitdrukking wat plaasvind wanneer 'n bakterie in aanraking kom met 'n oppervlak, vasheg en ook weer vrygestel word, asook die invloed hiervan op die vermoë om antimikrobiese behandeling te oorleef

Biofilm-derived Planktonic Cell Yield: A Mechanism for Bacterial Proliferation

The development of biofilms at solid-liquid interfaces has been investigated extensively, whereas the yield of planktonic cells from biofilms has received comparatively little attention. The detachment of single cells from biofilms has been attributed mainly to the erosive action of flowing liquid or the dispersal of cells from within biofilm microcolonies. The result has been an underestimation of the active role that biofilms can play in microbial proliferation through the production and release of planktonic cells to the environment. In this study, the cultivation of Pseudomonas sp. strain CT07 biofilms in conventional flowcells, glass tubes and a novel CO2 evolution measurement system was utilized to show that biofilm-derived planktonic cell yield was initiated within 6 hours of initial surface colonization and increased in conjunction with biofilm development. The magnitude of the yield was influenced by the metabolic activity of the biofilm, which was in turn dependent on environmental conditions, such as carbon availability. The physiologically active region of the biofilm was responsible for the yield of significant numbers of planktonic cells (~107 CFU.cm-2.h-1), whereas a less active biofilm zone was optimized for survival during unfavourable conditions and shown to be responsible for the subsequent re-establishment of biofilm structure, activity and cell yield. Despite the yield of numerically considerable numbers of planktonic cells (~1010 CFU), a carbon balance revealed that the carbon investment required to maintain this yield was insignificant (~1%) compared to the amount of carbon channelled into CO2 production (~54%). Together, these results indicate that biofilm-derived planktonic cell yield represents an efficient proliferation mechanism and support the view that the biofilm lifestyle affords microbes a dual survival-proliferation strategy, where the dominant strategy depends on the prevailing environmental conditions. An alternative model of biofilm development is presented to account for planktonic cell yield during all stages of biofilm development.Ph

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

<i>Listeria monocytogenes</i> 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

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

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·cell−1·h−1 to 22.6 ± 7.58 fmol of CO2·cell−1·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

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

CITATION: Klopper, K. B., et al. Biofilm dynamics : linking in situ biofilm biomass and metabolic activity measurements in real-time under continuous flow conditions. npj Biofilms and Microbiomes, 6:42, doi:10.1038/s41522-020-00153-9.The original publication is available at https://www.nature.comPublication of this article was funded by the Stellenbosch University Open Access FundThe 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.https://www.nature.com/articles/s41522-020-00153-9Publisher's versio