Posters

Biofilms provide physical, mechanical and chemical protection for microbes from their external environment, necessitating the use of harsh chemicals (such as sanitisers and antimicrobials) and abrasive cleaning (brushing or pigging) for their control. Biofilms have a broad impact upon the manufacturing of a wide range of fast-moving consumer goods, and biofilm contamination during their manufacture can lead to production interruption and significant economic costs to industry for cleaning and sanitisation. Biofilms formed by Pseudomonas aeruginosa (Ps. a.), a major contaminant of industrial processes, have yet to be studied in-depth with respect to the changes that occur in response to high-flow shear conditions from a combined physical and biological perspective.

The central aims of this work were to understand and elucidate the biological response of Ps. a. biofilms when grown under different, industrially-relevant fluid flow conditions; to characterise the mechanisms through which Ps. a. produces phenotypic responses, and how these in turn affect biofilm architecture. Two strains of Ps. a., PA01 and PA14, were used, known to differentially produce two exopolysaccharides (Psl and Pel respectively), integral components in the biofilm’s protective extracellular matrix (Colvin et al. 2012).

To investigate the effect of shear stress on biofilm formation, the CDC Bioreactor (CBR) was used to grow biofilms on polyethylene coupons under high or low shear conditions for 96 hours. At 24, 48, 72 and 96-hour timepoints, coupons containing biofilms were removed from the CBR and analysed by confocal laser scanning microscopy (CSLM) and biochemical assays.

Exopolysaccharide (EPS) production was quantified by CSLM image analysis in Fiji. In order to determine how EPS organisation effects wider biofilm architecture and individual structures within a maturing biofilm, Psl and Pel localisation and distribution throughout biofilms was assessed. Under low and high shear conditions, the architecture of PA01 and PA14 biofilms was compared to further identify similarities and differences in their phenotypic responses to shear stress.

We will present data that shows that Psl and Pel have distinct localisation patterns throughout PA01 and PA14 biofilms over our selected time course. PA01 and PA14 were shown to produce varying amounts of the exopolysaccharides Psl and Pel in mature biofilm structures (i.e. mushroom colonies) and throughout the entire biofilm population. Early adhesion, colony morphology and overall biofilm architecture was shown to be considerably affected by shear. Hydrodynamic conditions impose shear stress on Ps. a. biofilms, in turn affecting the structural components that make up their mature architecture.

Reference: Colvin, K.M., Irie, Y., and Tart, C.S. et al. (2012). The Pel and Psl polysaccharides provide Pseudomonas aeruginosa structural redundancy within the biofilm matrix. Environmental Microbiology, 14(8):1913-28.

How to cite: Allan, W., Webber, M., Wright, K., and Overton, T.: “Adhere today, here tomorrow” – how is exopolysaccharide production by Pseudomonas aeruginosa affected by high-flow shear conditions?, biofilms 9 conference, 29 Sep–1 Oct 2020, biofilms9-69, https://doi.org/10.5194/biofilms9-69, 2020.

Feast-famine moving bed biofilm reactors (MBBRs) were found to be removing a number of organic micropollutants effectively from wastewater in previous studies. It was hypothesized that micropollutant-degrading organisms in the biofilm communities were possibly enriched by feast-famine selective pressure. We established a MBBR operated in feast-famine regimes (alternating influent/effluent wastewater) to test the hypothesis. The development of degradation kinetics of 36 micropollutants and the microbial communities in the biofilm were assessed simultaneously for 19 time points during the 70-day adaptation.

During this adaptation, 16S rRNA gene amplicon sequencing showed that the microbial communities shifted greatly from the initial biofilm composition in the first 8 days toward a more steady development afterwards. Ammonia oxidizing bacteria (Nitrosomonas) and nitrite oxidizing bacteria (Nitrospira) were strongly enriched (both > 18 % relative abundance at day 43), which led to high nitrification capability. Notably, the biofilm absorbed and nitrified ammonia during the feast regime, while releasing stored nitrate during the famine regime. Twenty-four out of studied 36 micropollutants showed enhanced reaction rate constants k (especially for propranolol up to 6600 %) during the adaptation. Maximum k values were observed between day 22 and 67 during the adaptation. DNA concentration in the biofilm was used as a proxy for biomass, and normalized reaction rate constants relative to the DNA concentration as k_DNA were used for understanding the degradation reaction rates of MPs per DNA concentration unit. During the adaptation, the DNA concentration continuously increased suggesting growth and accumulation of microorganisms. However, k_DNA of 21 micropollutants showed a decreased removal after day 11, which suggests the relative abundance of the respective degraders decreased while their absolute abundance increased. It suggests that the colonization rates of the MP degraders were slower than the non-degraders under the selective pressure of the feast-famine regime. By mining correlations between the microbial community and k_DNA of micropollutants, 88 operational taxonomic units (OTUs) belonging to different taxonomic groups were found to correlate significantly with removal rates of micropollutants (Pearson correlation coefficients, r > 0.5, p < 0.05). Thus, these identified OTUs are potential candidates as the degraders of the respective micropollutants. In summary, the feast-famine strategy was successful for enhancing the degradation of some compounds, but the feast-famine regime in this study was not successful in selecting microorganisms in biofilm with high removal capability for many micropollutants. Nevertheless, this study contributed to a better understanding of what occurred during the adaptation process of biofilms with potential for micropollutant degradation.

How to cite: Liang, C., de Jonge, N., Carvalho, P. N., Lund Nielsen, J., and Bester, K.: Adaptation of biofilm communities in a feast-famine regime: implications for degradation of organic micropollutants, biofilms 9 conference, 29 Sep–1 Oct 2020, biofilms9-114, https://doi.org/10.5194/biofilms9-114, 2020.

The ability of bacterial pathogens to form biofilm is an important virulence mechanism in relation to its pathogenesis and transmission. Biofilms play a crucial role in survival in unfavourable environmental conditions, act as reservoirs of microbial contamination and antibiotic resistance. For intestinal pathogen Campylobacter jejuni, biofilms are considered to be a contributing factor in transmission through the food chain and currently, there are no known methods for intervention. Here we present an unconventional approach to reducing biofilm formation by C. jejuni by the application of D-amino acids (DAs), and L-amino acids (LAs). We found that DAs not LAs, except L-alanine, reduced biofilm formation by up to 70%. The treatment of C. jejuni cells with DAs changed the biofilm architecture and reduced the appearance of amyloid-like fibrils.  In addition, a mixture of DAs enhanced antimicrobial efficacy of D-Cycloserine (DCS) up to 32% as compared with DCS treatment alone. Unexpectedly, D-alanine was able to reverse the inhibitory effect of other DAs as well as DCS. Furthermore, L-alanine and D-tryptophan decreased transcript levels of alanine racemase (alr) and D-alanine-D-alanine ligase (ddlA). Our findings suggest that a combination of DAs could reduce biofilm formation, viability and persistence of C. jejuni.

How to cite: Elgamoudi, B., taha, T., and Korolik, V.: Alanine racemase as target to inhibit the Campylobacter jejuni biofilm formation by L and D-amino acids, biofilms 9 conference, 29 Sep–1 Oct 2020, biofilms9-137, https://doi.org/10.5194/biofilms9-137, 2020.

Effective biofilm disinfection is difficult to be implemented in healthcare settings and industry. In particular, surface disinfection is crucial to prevent microbial contaminations. However, disinfectants misuse has led to an increased concern on the existence of resistance and cross-resistance phenomena due to inadequate disinfection practices. The purpose of this study was the development of a formulation to be used for surface disinfection with wipes. The idea was to produce a formulation based on the combination between the quaternary ammonium compound - cetyltrimethylammonium bromide (CTAB) and a natural product - cinnamaldehyde. In addition, a new disc methodology to assess wiping efficiency was developed based on the Wiperator test (E2967-15) and on the quantitative test method for the evaluation of bactericidal and yeasticidal activity on non-porous surfaces with mechanical action employing wipes in the medical area, 4- field test (EN 16615:2015). The combination of CTAB and cinnamaldehyde was synergic in terms of antimicrobial action against Escherichia coli and Staphylococcus aureus. After stablishing the final formulation, wiping efficacy was assessed with the new methodology. In this case, a contaminated surface (6.20 ± 0.21 log₁₀ CFU of E. coli and 7.10 ± 0.06 log₁₀ CFU of S. aureus) was wiped using two different wipes in terms of composition, thickness and porosity (A and B). After wiping the contaminated surface with wipe A, without the formulation, 3.42 ± 0.46 log₁₀ CFU (E. coli) and 5.38 ± 0.20 log₁₀ CFU (S. aureus) remained on the surface while in the presence of the formulation the bacteria present were under the limit of detection for E. coli and 2.76 ± 0.22 log₁₀ CFU for S. aureus. The formulation was also able to prevent the transfer of bacteria to clean surfaces after wiping the contaminated surface. In the case of wipe A, after wiping the contaminated surface and the subsequent 2 clean surfaces, a total reduction of 4.35 ± 0.22 log₁₀ CFU and 4.27 ± 0.22 log₁₀ CFU was achieved when the wipe was impregnated with the formulation in comparison with 2.45 ± 0.41 log₁₀ CFU and 1.50 ± 0.35 log₁₀ CFU of removal just by mechanical action for E. coli and S. aureus, respectively. For wipe B a general lower reduction was observed but the same behaviour was detected with the use of the formulation when comparison to just mechanical action. This work highlights the enormous potential of combinatorial approach to increase the efficacy of already used biocides diminishing their in-use concentration and consequently their environmental and public health burden.

Acknowledgements

This work was financed by: UIDB/00511/2020 of the Laboratory for Process Engineering, Environment, Biotechnology and Energy – LEPABE - funded by national funds through the FCT/MCTES (PIDDAC); POCI-01-0145-FEDER-030219, POCI-01-0247-FEDER-035234; POCI-01-0145-FEDER-028397; POCI-01-0247-FEDER-033298; POCI-01-0145-FEDER-006939, funded by FEDER through COMPETE2020 – Programa Operacional Competitividade e Internacionalização (POCI) and by national funds (PIDDAC) through FCT/MCTES. Grant attributed by Portuguese Foundation for Science and Technology (FCT) to Joana Malheiro (SFRH/BD/103843/2014) and Manuel Simões (SFRH/BSAB/150379/2019).

How to cite: Malheiro, J. F., Borges, F., Maillard, J.-Y., and Simões, M.: A new surface wiping test to study surface disinfection by a novel chemical combination, biofilms 9 conference, 29 Sep–1 Oct 2020, biofilms9-155, https://doi.org/10.5194/biofilms9-155, 2020.

Introduction

Biofilm consists of a complex self-produced matrix of polysaccharides, DNA and proteins that 

protects bacteria from the environment including the host immune system and constitutes the main

cause of bacterial resistance against antibiotics. Research is then focused on finding alternative 

antimicrobial substances able to either hamper biofilm formation or to prevent bacterial growth. 

Recently, we showed that the antimicrobial peptide Temporin-L impairs E.coli growth by inhibiting 

cell division (Di Somma et al.; 2020; BBA). Here we investigate the effect of Temporin-L (TL) on 

biofilm formation in Pseudomonas fluorescens (P. fluorescens) both in static and dynamic conditions, 

showing that TL displays antibiofilm properties. 

Materials and methods

Biofilm formation in static conditions was performed on coverslips and analyzed by the Crystal Violet 

assay. Biofilm morphology was assessed using imaging techniques. Investigation of biofilms in 

dynamic conditions was performed in a flow chamber using a microfluidic system and images were 

recorded by confocal microscopy.

Results

The P. fluorescens cells were either grown in the presence of TL or incubated with the antimicrobial 

peptide after biofilm formation both in static and dynamic conditions using different concentrations 

of the peptide. When TL was added during cell growth, the peptide affected biofilm formation at 25 

µM. Confocal microscopy demonstrated that at this concentration P. fluorescens cells were still alive

but a clear disruption of the biofilm architecture was observed. These results had to be ascribed to a 

specific antibiofilm effect of TL. At 100 µM TL antibiofilm activity biofilm thickness was nearly 

negligible. 

When P. fluorescens cells were treated with TL following biofilm formation, confocal images 

demonstrated that the peptide exerted a strong antibiofilm effect leading to cell detachment and 

disruption the biofilm architecture. 

Discussion and Conclusions 

Investigation of TL effect on P. fluorescens showed that when added during bacterial growth this 

peptide exerted antibiofilm activity at low concentration impairing biofilm formation both in static 

and dynamic conditions, leaving most of bacterial cells still alive. However, confocal microscopy 

measurements could not detect the long necklace-like structures observed in E.coli indicating a 

different mechanism of action of TL on P. fluorescens. Furthermore, when TL was added to a 

preformed P. fluorescens biofilm, the peptide showed a strong antibiofilm activity both in static and 

dynamic conditions, suggesting that TL might penetrate biofilm architecture with a still unknown 

mechanism leading to disruption of P. fluorescens biofilm.

How to cite: Cirillo, A., Di Somma, A., Romano, A., Recupido, F., Caserta, S., Guido, S., Romanelli, A., and Duilio, A.: Antibiofilm effect of Temporin-L on Pseudomonas fluorescens, in static and dynamic conditions. , biofilms 9 conference, 29 Sep–1 Oct 2020, biofilms9-148, https://doi.org/10.5194/biofilms9-148, 2020.

Biofilms are well-organized sessile communities which exhibit an increased tolerance against antimicrobial and antibiotic treatments in comparison with their planktonic counterparts. Biofilms are ubiquitous and due to their high resilience, the problem with contamination of medical implants leads to serious health problems [1]. Within the last decades, novel therapies to prevent the formation of biofilms have been developed and, among others, antimicrobials based on metal nanoparticles (NPs) have been intensively studied [2], due to their ability to reduce biofilm formation. Silver nanoparticles (AgNPs) are known to be effective antimicrobial agents, as silver(I) has the ability to penetrate the cell and produce oxidative stress via the generation of reactive oxygen species (ROS) [3]. To understand the release mechanism of silver(I) ions, scanning electrochemical probe microscopy such as scanning electrochemical microscopy (SECM) is highly suitable.

In this contribution, biocompatible AgNPs-fluoropolymer (Ag-CF_x) composite films, prepared by ion beam sputtering (IBS) deposition [4], are investigated in respect to silver(I) release associated to the swelling of the antimicrobial film. The mechanism of the silver(I) release is studied real-time by scanning electrochemical microscopy (SECM) in combination with square-wave stripping voltammetry and the relation between controlled silver(I) release and the swelling of Ag-CF_x films will be presented, combining electrochemical techniques and atomic force microscopy (AFM).

References

[1] G.C. Anderson, et al. Innate and Induced Resistance Mechanisms of Bacterial Biofilms. In Bacterial Biofilms; Romeo, T., Ed.; Springer Berlin Heidelberg: Berlin, Heidelberg, 2008; pp 85–105.

[2] M.C. Sportelli, et al. Nano-Antimicrobials Based on Metals. In Novel Antimicrobial Agents and Strategies; John Wiley & Sons, Ltd, 2014; pp 181–218.

[3] N. Durán, et al. Nanomedicine, 2016, 12(3), 789-799.

[4] M.C. Sportelli, et al. Sci. Rep. 2017, 7 (1), 11870.

Acknowledgements

Financial support is acknowledged from European Union’s 2020 research and innovation program under the Marie Sklodowska-Curie Grant Agreement No. 813439.

How to cite: Caniglia, G., Heinzmann, A., Sportelli, M. C., Valentini, A., Cioffi, N., and Kranz, C.: Antimicriobial Activity of Silver-Nanoparticles studied by Scanning Probe Microscopy, biofilms 9 conference, 29 Sep–1 Oct 2020, biofilms9-151, https://doi.org/10.5194/biofilms9-151, 2020.

Among nosocomial infections, materials associated infections are the most frequent and severe due to biofilm formation. To prevent bacterial colonization, understanding the underlying interaction between bacteria and surface is fundamental. Herein we focused on studying how material viscoelasticity and physicochemistry can influence bacterial adhesion, using polydimethylsiloxane (PDMS) as a model material. To delineate the impact caused by bulk material from interfacial physicochemical properties, a 2 nm PDMS-like polymer layer was coated onto PDMS surfaces of different stiffness to confer comparable surface chemical properties, while retaining similar viscoelasticity for coated and uncoated PDMS species. Although the uncoated samples displayed increasing interfacial adhesion force with the decreasing Young's modulus, the nanolayer coating ensured comparable forces independent of material stiffness. The Gram negative strains Escherichia coli and Pseudomonas aeruginosa and the Gram positive strain Staphylococcus epidermidis were found to adhere respectively in similar numbers on the coated surfaces of different PDMS species, whereas the amount on the uncoated surfaces increased several fold with the decreasing modulus. The similar adhesion behaviour was noticed for abiotic polystyrene beads of similar size to bacteria, demonstrating that the interfacial chemistry of the PDMS rather than the material viscoelasticity plays a crucial role in bacterial adhesion. 

How to cite: Pan, F., Altenried, S., Liu, M., Hegemann, D., Bülbül, E., Moeller, J., Schmahl, W. W., Maniura-Weber, K., and Ren, Q.: Bacteria Adhesion on Polydimethylsiloxane Surfaces Impacted by Material Viscoelasticity or Surface Chemistry?, biofilms 9 conference, 29 Sep–1 Oct 2020, biofilms9-131, https://doi.org/10.5194/biofilms9-131, 2020.

Microbially influenced corrosion (MIC), is acknowledged to be the direct cause of catastrophic corrosion failures, with damages ranging to many billions of US$. In spite of extensive research and numerous publications, fundamental questions still remain unanswered. In 1993, J.F.D. Stott published a review paper in Corrosion Science, entitled “What progress in the understanding of microbially influenced corrosion has been made in the last 25 years?“ He concluded, “The most commonly asked question about MIC is: what will be the expected corrosion rate of material x in an environment where aggressive microorganisms proliferate?... For many materials we can no more answer this question now than we could 25 years ago.” Now, over 50 years later, that question is still open. Current MIC research does not provide data related to detection and verification in the field, diagnosing, modelling or prediction. Laboratory experiments seldom attempt to recreate relevant natural or industrial electrolytes. A sober, solution-oriented contemplation of the state-of-art and acknowledgement of the substantial deficiencies in our understanding may help shift MIC research into a direction which could actually produce useful answers.

How to cite: Flemming, H.-C., Little, B., Blackwood, D., Hinks, J., Lauro, F., Marsili, E., Okamoto, A., Rice, S., and Wade, S.: Biocorrosion research: Are we barking up the right trees?, biofilms 9 conference, 29 Sep–1 Oct 2020, biofilms9-9, https://doi.org/10.5194/biofilms9-9, 2020.

Biofilm development on medical devices is of particular concern and finding new strategies for preventing surface colonization and infection development are urgent. Antimicrobial biosurfactants such as rhamnolipids (RLs), emerge as one possible solution due their lack of resistance development. Using nanoparticles as delivery systems for these compounds may be a promising alternative in the context of biofilm-infections control. As such, the aim of this study was to encapsulate RLs into chitosan nanoparticles (RLs-NPs), test their antimicrobial activity and their biocompatibility profile.

Blank nanoparticles (b-NPs) and RLs-NPs were prepared by ionic gelation. For particles characterization, zeta potential, size distribution and encapsulation efficiency were performed. Minimal inhibitory concentration and biofilm inhibition ability were evaluated towards Staphylococcus aureus (ATCC 25923). To access NPs cytocompatibility the in vitro tetrazolium dye assay (MTT) and morphology observation were performed with a mouse fibroblastic cell line (L929).

RLs-NPs presented an encapsulation efficiency of 74.2±1.3%, a size ranging from 300 to 400 nm and a zeta potential of  37±1 mV. The minimum inhibitory concentration of RLs-NPs was 130 mg/mL and a 99% biofilm inhibition was achieved with these NPs meaning that their antimicrobial activity is also effective towards sessile bacteria. When compared to control, cell cultures grown in the presence of RLs-NPs presented no significant differences regarding the MTT reduction values and morphology analysis, suggesting that NPs up to 500 mg/mL did not significantly interfere with viability and proliferation.

The results revealed that the RLs-NPs were able to inhibit bacterial growth showing adequate cytocompatibility and might become, after additional studies, a possible approach to fight S. aureus biofilm associated infections.

Acknowledgments: Support for this work was provided by FCT through Portuguese government, PTDC/BTM-SAL/29335/2017 and Pest-UID/DTP/04138/2019

How to cite: Tomé, C., Anjos, I., Martin, V., Santos, C., Gonçalves, L., Fernades, M. H., Bettencourt, A. F., Gomes, P., and Ribeiro, I. A. C.: Can rhamnose-based glycolipids nanoparticles be an alternative to fight biofilms on medical devices?, biofilms 9 conference, 29 Sep–1 Oct 2020, biofilms9-94, https://doi.org/10.5194/biofilms9-94, 2020.

Biofilms are thought to play a serious role in the food processing environment. Direct contact with or detachment of microorganisms of biofilms could lead to product contamination resulting in diminished shelf life of the product. Packaging and filling are essential key steps for product safety, as any contamination within this step will directly impact the shelf life and safety of the product. For bottled and canned beverages filling lines are used for filling huge amounts of product in a standardised manner. Most parts of these lines are cleaned and disinfected automatically during cleaning in place (CIP) procedures. The design of these filling lines is of great importance, as accessibility and materials are crucial regarding the success of automatic cleaning and disinfection programs. Some companies implemented additional manual cleaning strategies for the complete removal of potential problem-causing sites. In the brewery setting the term biofilm is manifested since the 90s. However, until now all studies focus only on the presence of microorganisms, neglecting the presence of matrix components, which constitutes an essential component of a biofilm.  

Within this study a filling line for cans, capable of filling 60000 cans per hour (volume 0.5 l), was investigated regarding critical sites for biofilm formation. The filling line is used primarily for beer, but mixed beer drinks are also filled. We sampled 23 sites using a scraper-flocked-swab method at two time points. The first sampling was done during operation and the second sampling was conducted one month later after the automated cleaning and disinfection procedure. The samples were characterised regarding their microbial load (qPCR for 16S rRNA and 18S rRNA genes) and the presence of biofilm matrix (phenol-sulfuric assay for carbohydrates, precipitation and SDS-PAGE with subsequent silver staining for proteins and precipitation and spectrophotometric quantitative measurements for extracellular DNA).

During operation, we could identify three sites harbouring a biofilm by applying the definition of presence of microorganisms and at least two matrix components. Furthermore, there were seven sites harbouring microorganisms and one matrix component. After cleaning and disinfection no biofilm could be detected. At one site, microorganisms and one matrix component could be detected. The drastic reduction of biofilm positive sites indicates the successful removal of biofilms by the cleaning and disinfection process.

The design of future filling plants should emphasise on the principles of hygienic design, as this can help to prevent biofilm formation and targeted removal of biofilms during cleaning and disinfection. The here identified biofilm hotspots indicate potential problem-causing sites and weak-points in the design of the filling line.

How to cite: Wagner, E. M., Thalguter, S., Rychli, K., and Wagner, M.: Characterisation of biofilm hotspots at a can filling line for beer, biofilms 9 conference, 29 Sep–1 Oct 2020, biofilms9-41, https://doi.org/10.5194/biofilms9-41, 2020.

Chronic skin wounds are disruptions of the skin that remain in the inflammatory state for more than one month. They affect approximately 3% of the people with more than 60 years and are mostly associated with diabetic foot, pressure ulcers, blood vessels or trauma¹. Besides that, it is expected that 578 million people worldwide will suffer from the disease by 2030, that will subsequently increase the risk of developing infection and potentially result in amputation or osteomyelitis². One of the main difficulties in the wound healing process is the fact that bacteria persist due to biofilm formation. Biofilms delay not only healing, but they also make bacteria more resistant to the antimicrobial therapy. Among the bacteria present at the wound site, Staphylococcus aureus was found to be one of the most prevalent biofilm producers³. In this sense, other alternatives to fight biofilm infections should be considered due to the resistance to the current antibiotics. One possible strategy consists of using antibiotic adjuvants to enhance the activity of current drugs and to minimize or even block resistance. For that, plants are used as a resource of such adjuvant compounds. The use of natural compounds from plants have been applied in skin wound care for millennials, generating a lot of interest from the scientific community. Indeed, not only are phytochemicals great antibiotic potentiators but they also possess numerous therapeutic properties⁴. Therefore, in this study, the phenolic compound juglone (5-hydroxy-1,4-naphtoquinone) was investigated, alone and combined with fusidic acid antibiotic, for its potential to eradicate pre-formed S. aureus biofilms. Although no biomass removal was observed, there was a total loss of cell culturability (about 6-log CFU/cm² reduction) and a considerable metabolic activity reduction. Juglone reduced metabolic activity by 83% both alone and in combination with fusidic acid, which is an improvement over the 70% reduction obtained by the antibiotic. Therefore, an additive interaction of juglone when combined with fusidic acid was attained in the control of S. aureus biofilms. In addition, according with Lipinsksi’s rule of five, it was assessed that juglone possesses important molecular properties with respect to pharmacokinetics in the human body. Overall, this study reveals the great potential for the topical application of juglone as an adjuvant to the widely used fusidic acid to combat multi-drug resistant wound infections.  

References:

(1)         Manu, C., et al. J. Wound Care 27.3 (2018): 186–192.

(2)         International Diabetes Federation. https://www.diabetesatlas.org (accessed May 27, 2020).

(3)         Kadam, S., et al. Biomedicines 7.2 (2019): 35.

(4)         Borges, A.; et al. Molecules 21.7 (2016): 877.

Acknowledgements: This work was financially supported by: Base Funding - UIDB/00511/2020 of the Laboratory for Process Engineering, Environment, Biotechnology and Energy – LEPABE - funded by national funds through the FCT/MCTES (PIDDAC); - PTDC/BII-BTI/30219/2017 - POCI-01-0145-FEDER-030219; POCI-01-0247-FEDER-035234; POCI-01-145-FEDER-006939, funded by FEDER funds through COMPETE2020 – Programa Operacional Competitividade e Internacionalização (POCI) and by national funds (PIDDAC) through CEECIND/01261/2017 (Anabela Borges), SFRH/BD/138217/2018 (Diana Oliveira) and SFRH/BSAB/150379/2019 (Manuel Simões).

How to cite: Oliveira, D., Couto, F., Borges, A., Borges, F., and Simões, M.: Combinatorial effects of juglone and fusidic acid in controlling Staphylococcus aureus biofilms , biofilms 9 conference, 29 Sep–1 Oct 2020, biofilms9-154, https://doi.org/10.5194/biofilms9-154, 2020.

The presence of biofilms on stainless steel surfaces in the dairy industry greatly limits the efficiency of the cleaning procedures. The matrix of extracellular polymeric substances produced by the embedded bacteria is largely responsible for this irreversible binding. Therefore, to detach the biofilm in its entirety from the surface for microbiological identification and physico-chemical characterization is limited with the classical methods commonly used for surface sampling such as swabbing. The objective of this study is to optimize an extraction technique of biofilm formed using a dynamic CDC bioreactor system by a strain of Pseudomonas fluorescens isolated from the dairy industry during a biofilm issue. Three methods: swabbing, scraping and sonic brushing were tested in order to determine which one of these techniques allows a better recovery of the biofilm. They were also compared to sonication which is the standard method established by ASTM International. The results demonstrated that the total viable counts obtained by scraping (8.65 ± 0.07 CFU/cm2) were not significantly different from those achieved by sonication (8.74 ± 0.06 CFU/cm2) in contrast to the other two approaches, while scanning electron microscopy showed an effective removal of biofilms from surfaces by sonic brushing. In conclusion, other combinations including brushing, sonication and/or scraping must be investigated for representative sampling of biofilm on the surfaces of dairy plants.

How to cite: Niboucha, N., Goetz, C., and Jean, J.: Comparative assessment of biofilm sampling methods on stainless steel surfaces in a CDC biofilm reactor, biofilms 9 conference, 29 Sep–1 Oct 2020, biofilms9-136, https://doi.org/10.5194/biofilms9-136, 2020.

Biofilms within drinking water distribution systems can pose risks to consumers, especially when mobilised, as high concentrations of microorganisms and associated material can be released leading to degradation of water quality. Access and sampling of biofilms within drinking water pipelines can be difficult without disrupting supply in these extensive and buried systems. A novel biofilm monitoring device was developed to determine if biofilm formation rates can be used to assess microbiological water quality, track fouling rates and ultimately indicate distribution system performance. The device comprises a sample-line pipe with multiple, independent removable sections (allowing for biofilm sampling) that can be easily connected to sampling points in the distribution system. Biofilm is removed from the device and flow cytometry used to determine total and intact cell concentrations. The biomonitoring device was tested in a series of laboratory trials, to establish the impact of different flow rates and orientations on biofilm formation and to determine the optimum configuration that achieves accurate and repeatable results. Subsequently, these devices were installed in two operational systems, with different water qualities, and biofilms were sampled for two months to obtain biofilm growth rates. The results provide the first direct evidence of different biofilm formation rates in distribution systems with different water qualities. This evidence is now being used to investigate fouling rates via risk analysis and modelling. The use of the device has potential to improve understanding of biofilm behaviour and help inform biofilm and asset management to safeguard the quality of delivered drinking water.

How to cite: Pick, F., Fish, K., Husband, S., and Boxall, J.: Drinking Water Biofilm Management and Monitoring, biofilms 9 conference, 29 Sep–1 Oct 2020, biofilms9-19, https://doi.org/10.5194/biofilms9-19, 2020.

Research on the discovery of new drugs to treat bacterial biofilm infections has been a priority for biomedical and scientific communities. Actually, the sessile mode of growth accounts for more than 80% of human bacterial infections and displays increased resistance to antibiotics. To date, there are no drugs with total efficacy against biofilms and the treatment failure is a recurrent clinical situation. This lack of alternatives boosted up again the exploration of the pharmacological properties of health-promoting agents from natural origin. In this connection, the potential of phytotherapeutic agents for the treatment/prevention of complex infectious diseases, such as those that involve biofilm formation has been intensified[1]. In view of that, the main aim of the present study was to evaluate the activity of four phytochemicals bellowing to terpenoids class [cis-6-nonen-1-ol (CIS), citronellic acid (CITACID), citronellol (CITRO) and 3-7-dimethyl-1-octanol (3,7DOC)] against Staphylococcus aureus, both in planktonic and sessile state. Firstly, the minimum inhibitory and bactericidal concentrations (MIC and MBC) were determined by the broth microdilution method and culturability on plate count agar, respectively. Then, the potential of each terpenoid as resistance modifying agent was assessed by the disc diffusion method, using antibiotics from different classes. Besides, its potential to eradicate pre-formed S. aureus biofilms (24-h old) was performed using a microtiter plate assay and characterized in terms of biofilm mass removal (crystal violet staining), metabolic activity reduction (alamar blue staining) and culturability (colony forming units - CFU - counts). Considering that the selected terpenoids are chemical structurally related, i.e. present a similar backbone and differ only on the functional groups location, a structure activity relationship (SAR) analysis was also established. Both CITO and 3,7DOC presented the lowest MIC value (200 μg/mL) followed by CIS (400 μg/mL) and CITACID (1000 μg/mL). The MBC was found to be 1000 µg/mL with CIS, 2000 µg/mL with CITACID and > 2000 µg/mL (the maximum concentration tested) with CITO/3,7DOC. Apparently, the hydrophobicity of the molecules appear to affect positively its inhibitory proprieties – molecules with higher hydrophobicity presented lower MIC values. Moreover, it seems that the hydroxyl and methyl functional groups play the major influence on the antimicrobial properties. Indeed, CITO and 3,7DOC presented the higher hydrophobicity values and both had hydroxyl and methyl functional groups, possessing the lowest MIC value. Independently of the terpenoid tested, all combinations (terpenoid-antibiotic) resulted in a potentiation effect. Regarding biofilm eradication, although no biomass removal was observed, metabolic activity reductions from 25% (CIS at 5×MIC) up to 44% (CITACID at 10×MIC) and total loss of culturability (CITACID at 10×MIC; 6-log CFU/cm² reduction) was found. These effects were found to be dose dependent. Overall, the results obtained suggest that all the tested terpenoids might be interesting antibiotic adjuvants and emphasize the use of CITACID for biofilm cells inactivation. The results obtained are promising since the terpenoids studied are natural occurring flavoring ingredients, generally recognized as safe by the FDA, which are usually applied as food additives for human consumption.

References: [1] A.Borges, A.Abreu, C.Dias, M.Saavedra, F.Borges, M.Simões, Molecules 21(2016)877.

How to cite: Borges, A., Alves, A. C., and Simões, M.: Effect of selected terpenoids on antibiotic potentiation and eradication of Staphylococcus aureus biofilms – a structure activity relationship study, biofilms 9 conference, 29 Sep–1 Oct 2020, biofilms9-124, https://doi.org/10.5194/biofilms9-124, 2020.

¹Dipartimento di Chimica, Università degli Studi di Bari “Aldo Moro”, Bari, Italy; ²CNR, Istituto di Fotonica e Nanotecnologie UOS, Bari, Italy;       ³CSGI (Center for Colloid and Surface Science) c/o Dept. Chemistry, via Orabona 4, 70125 Bari, Italy.

Copper nanoparticles (CuNPs) are considered as potential antimicrobial agents due to their improved stability and safety, and longer active period than that of organic nanomaterials, with multi-targeted mechanism of action [1]. Nevertheless, metal NPs can suffer from agglomeration, reducing their antibacterial activity [2]. Cu incorporation in inorganic substrates such as metal oxides or montmorillonite (MMT) plays an important role due to the possibilities of creating an antibacterial nanomaterial with slow release of Cu species in order to obtain a prolonged antibacterial activity. Therefore, CuNPs were synthesized via a rapid electrochemical method using the inorganic micro-powders as carrier. Characterization studies on the nanocomposite were done by Scanning electron microscopy (SEM), Transmission electron microscopy (TEM), and X-ray photoelectron spectroscopy (XPS). The as-prepared Cu-based nanocomposites could be employed for inhibiting the growth of biofilms.

References

Acknowledgements

"Financial support is acknowledged from European Union’s 2020 research 
 and innovation program under the Marie Sklodowska-Curie Grant 
 Agreement No. 813439."

How to cite: Hossain, S. I., Sportelli, M. C., Picca, R. A., Ditaranto, N., and Cioffi, N.: Electrosynthetized copper based nanoantimicrobials for the inhibition of biofilms, biofilms 9 conference, 29 Sep–1 Oct 2020, biofilms9-149, https://doi.org/10.5194/biofilms9-149, 2020.

Biofilms are considered a major cause of serious health issues in human medicine and food industry, due to their resistance against harsh conditions and pharmacological treatment [1]. Biofilms are defined as three-dimensional structures encasing bacterial communities rooted in extracellular polymeric substances (EPS). These complex systems are strongly influenced by a variety of parameters including biofilm age, external conditions, nutrient deficiency, attack of exogenous agents [2]. Moreover, bacterial colonies may activate survival strategies when subjected to stress such as the presence of antimicrobial agents. Even cannibalistic behavior may occur [3], which involves the secretion of cannibalism toxins inducing the generation of lysed cells providing nutrients.

Several methodologies were developed for or adapted to biofilm formation studies enabling a more comprehensive understanding of biofilm physiology, structure, and composition. This information should facilitate the development of more effective eradication strategies. Infrared spectroscopy in attenuated total reflectance (IR-ATR) mode provides in-situ and close to real time monitoring of biofilm lifecycles providing molecular information on the various stages of biofilm formation. Given the antibiotic resistance of biofilms [4], it is of increasing importance to develop innovative methodologies for the treatment of biofilm-related infections. While our research team has shown the generic utility of antimicrobial nanoparticles (NPs) such as ZnONPs, AgNPs, CuNPs, etc. in the past [5], the current study focuses on AgNPs embedded within fluoropolymer matrices with tunable loading of the NPs. Next to morphological studies by TEM and AFM, detailed XPS investigations revealed the surface chemical composition. In addition, the kinetics of antimicrobial ion release enabled correlating the behavior of the nanocomposite to its swelling properties and 3D modification after immersion in liquids. Biofilm growth and inhibition was studied via AFM, optical microscopy and IR-ATR. The IR analysis of the biofilm allowed collecting molecular information on the biofilm behavior during long-term contact with antimicrobial surfaces. It was demonstrated that bacterial cells may re-colonize on top of dead biomass once the latter is thick enough to prevent direct interaction with the antimicrobial surface. In summary, this study represents an excellent foundation for developing an in depth understanding on the behavior of bacterial colonies and nascent biofilms in contact with surfaces decorated with nanoantimicrobials over extended periods of time. It is anticipated that an improved understanding on the stages of biofilm formation provides insight into the processes governing antimicrobial resistance phenomena. Finally, present antimicrobial material may be a useful strategy against Corona viruses. An outlook to this urging topic will be also presented.

How to cite: Sportelli, M. C., Caniglia, G., Quarto, R., Picca, R. A., Valentini, A., Bart, H., Mizaikoff, B., Kranz, C., and Cioffi, N.: Evidence of cannibalism during long-term biofilm-antimicrobials interaction, biofilms 9 conference, 29 Sep–1 Oct 2020, biofilms9-144, https://doi.org/10.5194/biofilms9-144, 2020.

Biofilms are suggested to be a source of contamination in the food producing environment leading to food spoilage or the transmission of food-borne pathogens. However, to date, research has mainly focused on the presence of (biofilm-forming) bacteria within food processing environments, without analysing the associated biofilm matrix components.

The aim of this study was to identify biofilm hotspots in a meat processing environment by analysing the presence of microorganisms (by cultivation and targeted quantitative real-time PCR based on 16S rRNA) and the major matrix components carbohydrates, extracellular DNA and proteins. Sampling included 47 distinct food contact surfaces and 61 distinct non-food contact surfaces from eleven rooms within an Austrian meat processing plant, either during operation or after cleaning and disinfection. Additionally, we isolated and characterized bacteria found in biofilms. The biofilm forming capacity of eleven isolates, was tested, using a static biofilm model. Additionally, two different multi-species settings were tested combining three strains, each. Biofilms were grown on stainless-steel slides for seven days at 10 °C, to mimic conditions found in the food producing environment.

Overall, we identified ten biofilm positive sites, among them seven of which were sampled during operation and three after cleaning and disinfection. Five biofilms were detected on food contact surfaces (cutters and associated equipment and a screw conveyor) and five on non-food contact surfaces (drains and water hoses) resulting in 9.3 % of the sites being classified as biofilm positive. From these sites we cultivated bacteria of 29 different genera. The most prevalent bacteria belonged to the genera Brochothrix, Pseudomonas and Psychrobacter. From each biofilm we isolated bacteria from four to 12 different genera, indicating the presence of multi-species biofilms.

Culturing of eleven isolates of different species (all detected in the mentioned biofilms, representing typical residential and spoilage bacteria in the meat processing environment) showed that there are differences of individual strains to produce matrix components and biomass on stainless steel slides.  Brochothrix, Carnobacterium and Kocuria produced only detectable amounts of carbohydrates but neither eDNA nor proteins. The Acinetobacter and the Flavobacterium isolates were able to produce two of the measured components and six strains were capable of producing all types of analysed matrix components, among them a Pseudomonas fragi isolate. The minimal mean bacterial load detected was 5.4 log CFU/cm² formed by the Psychrobacter strain.

Different isolates showed differences in matrix formation ability, possible contributing in different amounts to the matrix production in multi-species biofilms, indicating that multi-species biofilms are a key survival mechanism for microorganisms within the food processing environment.

Currently, we are testing two different multi-species biofilms in our model. Hereby we cultivate three species detected in the cutter-associated biofilms and other three species detected in the water hose-associated biofilms together to mimic these biofilms. This work ultimately showed the presence of multi-species biofilms within the meat processing environment, thereby identifying various sources of potential contamination. Data on the presence, formation and composition of biofilms (i.e. chemical and microbiological) will help to prevent and reduce biofilm formation within food processing environments.

How to cite: Wagner, E. M., Pracser, N., Thalguter, S., Fischel, K., Rammer, N., Beer, C., Palmetzhofer, A. L., Pinior, B., Roch, F.-F., Pospíšilová, L., Alispahic, M., Rychli, K., and Wagner, M.: Identification of multi-species biofilms in the meat processing environment and characterisation of involved bacteria in a mono- and multi-species biofilm model, biofilms 9 conference, 29 Sep–1 Oct 2020, biofilms9-44, https://doi.org/10.5194/biofilms9-44, 2020.

The selection of materials for plumbing application has potential implications on the chemical and microbiological quality of the delivered water. This work aims to evaluate the action of materials with different copper content (0, 57, 96 and 100%) on biofilm formation and control by chlorination and mechanical stress. A strain of Stenotrophomonas maltophilia isolated from drinking water was used as model microorganism and biofilms were developed in a rotating cylinder reactor (RCR) using realism-based shear stress conditions. Biofilms were characterized phenotypically and exposed to three control strategies: 10 mg/l of free chlorine for 10 min; an increased shear stress (equivalent to 1.5 m/s of fluid velocity); and the combination of both treatments. Biofilms formed on the copper materials had lower wet mass and produced significantly lower amounts of extracellular proteins than those formed on stainless steel (0% of copper content). Although, the effects of copper materials on biofilm cell density was not significant, these materials had important impact on the efficacy of chemical and/or mechanical treatments. Biofilms formed on 96 or 100% copper materials had lower content of culturable bacteria than that observed on stainless steel after exposure to chlorine or shear stress. The mechanical treatment used had no relevant effects in biofilm control. The combination of chemical and mechanical treatments only caused higher culturability reduction than chlorine in biofilms formed on 57% copper alloy. The number of viable cells present in bulk water after biofilm treatment with chlorine was lower when biofilms were formed on any of the copper surface. The overall results are of potential importance on the selection of materials for drinking water distribution systems, particularly for house and hospital plumbing systems to overcome the effects from chlorine decay. Copper alloys may have a positive public health impact by reducing the number of viable cells in the delivered water after chlorine exposure and improving the disinfection of DW systems. Moreover, the results demonstrate that residual chlorine and mechanical stress, two strategies conventionally used for disinfection of drinking water distribution systems, failed in S. maltophilia biofilm control.

Acknowledgements:

This work was the result of the projects: UIDB/00511/2020 of the Laboratory for Process Engineering, Environment, Biotechnology and Energy – LEPABE - funded by national funds through the FCT/MCTES (PIDDAC); PTDC/BII-BTI/30219/2017 - POCI-01-0145-FEDER-030219; POCI-01-0145-FEDER-006939, funded by FEDER funds through COMPETE2020 – Programa Operacional Competitividade e Internacionalização (POCI) and by national funds (PIDDAC) through FCT/MCTES; NORTE-01-0145-FEDER-000005 – LEPABE-2-ECO-INNOVATION, supported by Norte Portugal Regional Operational Programme (NORTE 2020), under PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund (ERDF).

How to cite: Gomes, I. B., Simões, L., and Simões, M.: Influence of different copper materials on biofilm control using chlorine and mechanical stress , biofilms 9 conference, 29 Sep–1 Oct 2020, biofilms9-123, https://doi.org/10.5194/biofilms9-123, 2020.

Burkholderia cepacia (B. cepacia) is one of nine species the Burkholderia cepacia complex, a group of gram-negative, motile, non-spore-forming and rod-shaped bacteria. Contamination by B. cepacia is found in different industrial issues. B. cepacia affect manufacturing process chains by contaminating the working fluids with planktonic cells and biofilms. Because of the opportunistic pathogenicity to plants, animals, humans and and the multi-drug resistance, B. cepacia is difficult to treat. An alternative treatment method could be the use of herbal raw materials, such as essential oils and their active ingredients. This study aims: (i) to identify the antimicrobial potential of essential oils on the growth of four B. cepacia isolates, (ii) to analyse the influence of active ingredients, on planktonic growth and biofilm formation, (iii) to better understand the impact of commercial and naturally biocides to cell agglomeration as a precursor to mature biofilms. Starting with agar dilution method to evaluate the antimicrobial potential of twenty-three essential oils against B. cepacia (Burk_09, Burk_23, Burk_52 and Burk_309) isolated from cathodic dip coating systems and the wild type (DSM_7288), it was all ready possible to identify eight essential oils that inhibit the growth of B. cepacia. Serial microdilution was used to determine the minimal inhibitory concentration (MIC) of the essential oils for growth and biofilm formation inhibition of B. cepacia. The MIC of Melaleuca alternifolia and Citrus aurantium dulcis essential oils were tested equally for all strains. Essential oils contain active ingredients against the growth of multi-drug resistant and pathogenic bacteria. From twelve active substances among others, Terpinen-4-ol and Geraniol were identified that inhibited growth and biofilm formation. It is concluded that essential oils and active ingredients have a good antimicrobial potential, demonstrating a possible more environmental-friendly alternative to commercial biocides applying in industrial fluids.

How to cite: Huth-Herms, K., Kintzel, A., Brehmer, A., Hein, C., and Uhlmann, P. Dr. h. c. Dr.-Ing. E.: Influence of essential oils on the biofilm formation and cell agglomeration of Burkholderia cepacia from industrial environment, biofilms 9 conference, 29 Sep–1 Oct 2020, biofilms9-50, https://doi.org/10.5194/biofilms9-50, 2020.

Bacterial resistance to conventional antibiotics combined with the increasing awareness of the essential role of biofilms in nosocomial infections caused by medical devices has led to a growing interest in new antimicrobial strategies. Since the formation of bacterial resistances represents a permanent risk in the drug treatment of biofilms, the optimisation of surface properties to avoid microbial attachment is gaining further attention. Besides the haematological field, especially in the respiratory and oral sectors, biofilm-forming microorganisms cause major problems. Due to microbial attachment being mainly determined by the surface properties of the respective substrate material, the medically established polymer PVDF was provided with different microstructures in the size s of 1 µm ≤ s ≤ 200 µm in order to influence the wettability. These structures were applied to injection moulding tools by high and ultra precision milling, electrical discharge machining as well as laser machining. The injection moulded, microstructured PVDF samples showed pyramidal, cup-shaped, channel-shaped and random structures in the micrometer range and led to contact angles Ɵ in the range of 50° ≤ Ɵ ≤ 110°. These samples were then tested for their influence on bacterial attachment by typical representatives of haematologic as well as respiratory and oral biofilm formers Pseudomonas stutzeri and Streptococcus salivarius. The microbial growth and the formed biofilms were analysed after 24 h and 72 h via crystal violet staining and fluorescence microscopy. In comparison to unstructured surfaces, a significant reduction of bacterial attachment was found, which correlated with the respective contact angle and surface roughness, the microgeometry of the structures and the cell morphology of the tested microorganisms. Especially the laser structured, channel-shaped surface showed a highly reduced biofilm formation for both strains. The results offer great potential for the reduction of biofilm formation on medical devices. This technology can also be used in the water treatment sector, such as pipe linings, filter surfaces and sensor housings. The economic large-scale implementation of these microstructures requires further research.

How to cite: Uhlmann, P. Dr. h. c. Dr.-Ing. E., Hein, C., Brehmer, A., and Huth-Herms, K.: Inhibitory effect of microstructured PVDF surfaces on the microbial attachment of respiratory and oral biofilm forming microorganisms, biofilms 9 conference, 29 Sep–1 Oct 2020, biofilms9-57, https://doi.org/10.5194/biofilms9-57, 2020.

In the reclaimed wastewater reuse using drip irrigation, one of the main issues is the bio-clogging of drippers and decrease of water distribution efficiency in field level. However, the relation between the complex flow created along the dripper (in general formed by a milli-channel with labyrinth geometry) and the biofouling development are rarely studied.

In order to improve the knowledge of these mechanisms, the objective was to combine the numerical flow simulations to three-dimensional measurements of biofilm along a milli-fluidic system (nominal flow rate 1L/h) fed by treated wastewater. At first, using the Optical Coherence Tomography (OCT) method and based to Qian et al, 2018 studies, the bio-clogging structure was measured at different levels of fouling (up to 77% of channel volume). Secondly, the new fouled dripper geometries were integrated to 3D CFD models (using comsol multiphysics software) to analyse the effect of biofilm on flow topology and the dripper hydraulic parameters (pressure drop, shear stress, turbulence kinetic energy in particular).

The results show that the main areas of biofilm growth correspond to vortices zones where fluid velocity, turbulent kinetic energy values and shear stress are lowest. When the level of clogging increases, the numerical plot of stream lines show local perturbation and reduction of vortices areas caused by their interactions with the biofilm structure. There is also a gradual increase in pressure drop along the milli-channel comparing to initial clean dripper. Finally, by characterising the flowrate in function of inlet pressure and according to Karmeli, 1977, the increase of biofilm formation induces also a modification of the global flow regime in the dripper, i.e. the transfer from a turbulent to a laminar regime.

Qian, J., Horn, H., Tarchitzky, J., Chen, Y., Katz, S., Wagner, M., (2017). Water quality and daily temperature cycle affect biofilm formation in drip irrigation devices revealed by optical coherence tomography. Biofouling 33, 211–221. 
Karmeli, D. (1977). Classification and flow regime analysis of drippers. J Agric Eng Res 22:165–173

How to cite: Ait Mouheb, N., Qian, J., Wagner, M., and Horn, H.: Interaction of flow field and biofilm formation in a dripper supplied by reclaimed wastewater, biofilms 9 conference, 29 Sep–1 Oct 2020, biofilms9-88, https://doi.org/10.5194/biofilms9-88, 2020.

In order to optimize operative parameters in wastewater treatment plants, drip irrigation systems as well as in biofilm reactors, it is necessary to understand biofilm development and proliferation under certain conditions. Additionally, the physical structure of biofilms is of great interest since it determines the interaction with its microenvironment, while knowledge about the mechanical behavior of biofilms is important for applying e.g., cleaning procedures.

In the past two years we refined a fully automated monitoring and cultivation setup that enabled replicate biofilm cultivations and investigation by means of optical coherence tomography (OCT). OCT as an imaging modality is ideal for biofilms since it allows for the monitoring of structure and deformation in real-time and noninvasively.

With this setup it was possible to analyze the effect of iron on biofilm growth and behavior with a minimum of N = 10 biofilm replicates including a statistical treatment. At least eight structural parameters of biofilms grown in flow cells could be analyzed and statistically quantified, providing insights into the structural integrity of biofilms and to their interface. Thereby, the results clearly show the positive effect of iron on Bacillus subtilis biofilms regarding biomass production and differentiation to mature biofilms. Further gravimetric and optical analyses prove the incorporation of iron as iron oxide-hydroxides and explain the positive effect on the biofilm’s matrix. Initial experiments under mechanical stress confirm the withstanding of high flow rates as well as a high compressibility of the biofilms.

How to cite: Gierl, L., Horn, H., and Wagner, M.: Iron as a biofilm control agent: manipulation of biofilm development and differentiation, biofilms 9 conference, 29 Sep–1 Oct 2020, biofilms9-75, https://doi.org/10.5194/biofilms9-75, 2020.

Aerobic granular sludge is one of the most promising technologies in wastewater treatment of the past decades. By now, around 50 full scale plant have been set up at full scale under the trademark Nereda©.

With approx. 50 % of the chemical oxygen demand (COD) particulate organic matter is a crucial fraction of municipal wastewater. Theories about its degradation in granular sludge reactors typically start with the assumption that particulate organic matter is adsorbed at the granule surface after the feeding phase (De Kreuk et al. 2010, Pronk et al. 2015). Despite of the ideal case, unattached particles after the feeding phase would be available for degradation under aerobic conditions or could be washed out with the effluent. To extent the knowledge about the degradation process, the present study aimed at visualizing the transport and fate of particulate organic matter into and through a bed of granular sludge. The main perspectives are to directly show their distribution and retention mechanism inside a granular sludge bed.

Magnetic Resonance Imaging (MRI) was successfully applied to visualize the different fractions of a granular sludge bed resolved in time and space (x, y, z). According to particle size, three particle consortia have been chosen to represent municipal wastewater:

Dextran coated super paramagnetic iron oxide nanoparticles (SPIONs, mean diameter d_mean = 20 nm) served as model particles for colloids. As a reference for toilet paper, paramagnetically tagged microcrystalline cellulose with a size fraction between 1 and 20 µm was used. The results are supplemented by the use of real wastewater particles with a size fraction between 28 and 100 µm. No paramagnetic tagging was applied in the latter case.

The retention mechanism is found to be size dependent. Colloidal particles are able to attach and penetrate the granules. Therefore, they will constantly release substrate during their degradation inside the granule. In contrast, larger particles accumulate within the void space between the granules. Moreover, the formation of particle layers indicates that most of the particles are not attached to the biomass and remain mobile after an initial feeding phase. Thus, they remain available under aerobic conditions and might be partially washed out with the effluent if no attachment is taking place in the aerobic mixing phase (Ranzinger et al. 2020).

References:

De Kreuk, M., Kishida, N., Tsuneda, S. and Van Loosdrecht, M. (2010) Behavior of polymeric substrates in an aerobic granular sludge system. Water research 44(20), 5929-5938.

Pronk, M., Abbas, B., Al-Zuhairy, S., Kraan, R., Kleerebezem, R. and Van Loosdrecht, M. (2015) Effect and behaviour of different substrates in relation to the formation of aerobic granular sludge. Applied microbiology and biotechnology 99(12), 5257-5268.

Ranzinger, F., Matern, M., Layer, M., Guthausen, G., Wagner, M., Derlon, N. and Horn, H. (2020) Transport and retention of artificial and real wastewater particles inside a bed of settled aerobic granular sludge assessed applying magnetic resonance imaging. Water Research X, 100050.

How to cite: Ranzinger, F., Matern, M., Layer, M., Guthausen, G., Wagner, M., Derlon, N., and Horn, H.: Magnetic resonance imaging (MRI) as non-invasive approach for quantifying the transport of particulate organic matter within a bed of settled aerobic granules, biofilms 9 conference, 29 Sep–1 Oct 2020, biofilms9-101, https://doi.org/10.5194/biofilms9-101, 2020.

Biofouling is responsible for more than 45% of all membrane fouling associated problems and is highly relevant for the performance of Reverse Osmosis systems (RO). Biofouling has a tremendous negative impact on the quality and quantity of permeate water and is responsible for high operational and maintenance costs associated with such systems. Current strategies targeting biofilm control on membrane systems often include the overuse of disinfectants which most of the time fail to effectively prevent biofouling build-up, can lead to the formation of dangerous disinfection by-products and represents high amounts of discharged biocides.

The present work aims to study how millimetric (1-3 mm length) alumina particles, functionalized with a well-known quaternary ammonium compound biocide (benzalkonium chloride) and immobilized into a Particle Biocide Bed Reactor can effectively contribute to mitigate biofilm formation in membrane systems. For that, the functionalized particles were chemically characterized, and their antimicrobial activity was assessed in batch and recirculation assays and quantified in terms of Culturability and Propidium Iodide (PI) uptake. Special attention has been given to biocides’s (free and immobilized) mechanism of action and potential biocide release was evaluated by High Performance Liquid Chromatography (HPLC) measurements.

The preliminary experiments indicate that the immobilized biocide (equivalent biocide concentration of 3 g/L) has an antimicrobial activity against Pseudomonas fluorescens (initial concentration 10⁸ CFU/mL) by reducing 4 logs after 30 min and 8 logs after 1 h. On the other hand, the control assays (functionalized particles in water with no bacteria), also shows a biocide release between 0.8 and 1% to the bulk water after 30 min, both in batch and in the Particle Bed Reactor with recirculation experiments. No significant biocide increase is observed in the bulk liquid studies for two weeks. Nonetheless, some changes in the functionalization approach are being made to improve the biocidal anchoring to the particle.

How to cite: Barros, A. C., Pereira, A., and Melo, L. F.: Millimetric particles functionalized with biocide to improve biofouling control in RO system , biofilms 9 conference, 29 Sep–1 Oct 2020, biofilms9-153, https://doi.org/10.5194/biofilms9-153, 2020.

Introduction

The presence of biofilms in drinking water distribution systems (DWDS) leads to a number of issues, i.e. secondary (biological) drinking water contamination, pipe damage and increased flow resistance. Among other operational factors, the selection of pipe material plays an important role in biofilm development. Up to now, the studies that have investigated this correlation provide contradictory results in terms of which material might be the most advantageous in the DWDS biofilm control strategy. Hence, to understand the influence of pipe material on biofilm formation, we focused on developing a standardized methodology that allows a multi-stage assessment of biofilm development on real pipe materials.

Results

Development of the methodology consisted of three steps: 1) material coupon sterilization, 2) biofilm cultivation and 3) biofilm analysis, using  transparent polyvinyl chloride (PVC) as a study material. For the coupon sterilization, methods utilizing immersion in different disinfectant solutions with and without pre-cleaning by rubbing the coupons in a surfactant solution. The results showed that mechanical cleaning before washing  is crucial and without it, reproducible sterilization was difficult to achieve. Biofilm formation on the PVC coupons was performed in a 6-well plate assay (24, 48 and 72 h; under agitation) using DWDS biofilm strains (Sphingomonas spp. and Pseudomonas extremorientalis) and Pseudomonas aeruginosa as a positive control. Bacterial fitness and ability to secrete EPS and form biofilms on the PVC surfaces were tested by monitoring optical density (OD600 nm), chemical oxygen demand (COD) and protein concentration. The formed biofilm and the morphology of attached bacteria were visualized using crystal violet staining (that allow qualitative (bright field microscopy) and quantitative (OD at 570 nm) evaluation), by scanning electron microscopy (SEM) and DNA staining (4′,6-diamidino-2-phenylindole; DAPI) with fluorescence microscopy. Combination of those techniques gave a complete overview of patterns involved in biofilm development by selected drinking water bacterial strains in presence of a PVC surface. The developed methodology was also applied  for the analysis of bacterial growth on real-grade pipe materials, such as PVC and polyethylene (PE), to understand their role in biofilm formation.

Conclusions

Implementation of various analytical and microscopic techniques is important in understanding mechanisms behind biofilm development in DWDS and the influence of pipe material in the process. The proposed approach allows the observation of biofilm formation in time, but also of the typical bacterial morphology of attached cells. In this study it was shown that to obtain reproducible results, it is crucial to select an appropriate sterilization technique and the influence of mechanical cleaning cannot be ignored in preparation of polymeric surfaces.

How to cite: Sójka, O., van Rijn, P., van der Mei, H., and Gagliano, M. C.: Multi-stage assessment of biofilm growth by drinking water bacteria on polymeric pipe materials , biofilms 9 conference, 29 Sep–1 Oct 2020, biofilms9-48, https://doi.org/10.5194/biofilms9-48, 2020.

A full-scale biofilm system using fluidized-carriers integrated with anaerobic-anoxic–aerobic process (treatment capacity of 3.75 × 10⁵ m³ d⁻¹) was used for municipal wastewater treatment. The results indicated relatively higher removal efficiencies of 86% total nitrogen (TN), 97% ammonium (NH₄-N) and 97% total phosphorus (TP) were achieved, with 0.32 mg L⁻¹ TP, 0.81 mg L⁻¹ NH₄-N and 8.07 mg L⁻¹ TN in the effluent, which meet the Class A of Discharge standard of pollutants for municipal wastewater treatment plant (GB18918-2002) of China. The results of microbial analysis indicated that the dominant microorganisms in the suspended sludge were Proteobacteria and Bacteroidetes at phylum level and β-Proteobacter at class level. The dominant microorganism in the biofilm was Proteobacteria at phylum level, with γ-Proteobacter (17.5%), β-Proteobacter (14%) and δ-Proteobacter (13.08%) distributed at class levels. The presence of Proteobacteria and Bacteroidetes in this system may be related with the phosphorus removal. A reddish color biofilm was formed on the surface of fluidized-carriers in the anaerobic tank and showed specific anammox ability, this may be related with the dominance of 0.0278% Planctomycetaceae at family level and 0.0278% Planctomycetales at order level. Besides the denitrification effects, the possible anammox bacteria present in the anaerobic tank might have also contributed to high nitrogen removal efficiency.

How to cite: Xiao, K.: Nitrogen and phosphorus removal using fluidized-carriers in a full-scale A2O biofilm system, biofilms 9 conference, 29 Sep–1 Oct 2020, biofilms9-27, https://doi.org/10.5194/biofilms9-27, 2020.

Chitosan (CS), a natural non-toxic polysaccharide, shows intrinsic antimicrobial activity against a wide range of pathogens. CS and CS-based biomaterials can be effective additives in food and medicine-related industries to inhibit growth of pathogens. The application of inorganic nanophases, such as metal and metal oxide nanoparticles, has received attention due to their broad and pronounced antimicrobial activity. Upon combination with CS, which can act as stabilizer, with active inorganic nanophases, robust synergistic nanoantimicrobial (NAM) systems can be produced. These hybrid NAMs offer an alternative strategy to fight antimicrobial resistance and overcome limitations of conventional antibiotics. Bioactive ZnO, Cu and Ag nanophases produced by green electrochemical approach [Nanomaterials, 10(3) (2020), 473] and laser ablation in solution [(Coll. Surf. A, 559 (2018), 148-158), (Food packaging shelf, 22 (2019), 1000422)] can be combined with antimicrobial CS to develop synergistic antimicrobial nanohybrids with amplified biological action. CS-based NAMs were preliminary characterized by electron microscopies and spectroscopic techniques. Hybrid NAMs may find application in the control and inhibition of biofilm growth.

Acknowledgements
Financial support is acknowledged from European Union’s 2020 research and innovation program under the Marie Sklodowska-Curie Grant Agreement No. 813439

How to cite: Kukushkina, E. A., Sportelli, M. C., Ditaranto, N., Picca, R. A., and Cioffi, N.: Preventing biofilms by chitosan-based nanoantimicrobials (NAMs), biofilms 9 conference, 29 Sep–1 Oct 2020, biofilms9-150, https://doi.org/10.5194/biofilms9-150, 2020.

In the oil and gas industry, internal corrosion represents one of the major threats to asset lifetime and integrity. Of the types of internal corrosion, microbiologically influenced corrosion (MIC) is the most difficult to predict and monitor due to the unpredictable nature of microbial growth and the minimal metal loss resulting in through wall failure (pitting). MIC results from biofilm communities interacting directly and indirectly with the metal. Due to the structure and nature of these pipelines, directly monitoring sessile growth is impossible. As a result, most MIC monitoring is done through planktonic cells retrieved from fluid samples as a proxy for sessile populations.

Growth curves are one of the most fundamental methods of quantitatively assessing microbial growth. In the lab, pure cultures are measured using optical densities, biomass staining, direct microscopic counting and counting colony forming units (CFU) on specialized media while more advanced techniques involve quantitative PCR (qPCR) of key genes. While PCR technologies are more easily transferred from the field to the lab, CFU counts are impossible in the field. Alternatives to the CFU are colorimetric activity assays such as “bug bottles” or biological activity reaction test (BART) bottles but aren’t sensitive and require long incubation times. More sensitive assays such as ATP measurements are also used but can be misleading as high metabolically active samples will give higher cell count equivalents than a metabolically slow community of an identical size.

To systematically evaluate a best practice, we conducted growth curves in a lab scenario using six pure cultures and techniques predominantly used in the field to determine how these techniques compare and accurately measure microbial growth. The six species used are Acetobacterium woodii, Bacillus subtilis, Desulfovibrio vulgaris, Geoalkalibacter subterraneus, Pseudomonas putida and Thauera aromatica. The techniques used are optical density at 600 nm, ATP activity measurements using a luciferase-based assay, DNA concentration and 16S rRNA copy numbers.

It was found that most lines of data follow the expected sigmoidal growth curve to varying degrees for all species. OD₆₀₀ readings follow the expected sigmoidal curves, exhibiting a lag phase, log growth phase and a stationary phase. ATP peaks during mid log phase and quickly declines, never showing a distinct stationary phase, while DNA concentrations closely follow the OD₆₀₀ readings but decline to death phase more rapidly. qPCR of the 16S rRNA genes revealed this data followed the same trends but was less susceptible to fluctuations.

Assessing microbial biofilms in the environment and on anthropogenic industrial infrastructure is extremely challenging given sampling, storage and transportation to the lab.  This work begins to establish best practices for growth of environmental communities to be followed.  Cumulatively, this work shows that each approach supports the expected growth curve. Considerations should be made if all field data is of a single type, e.g. ATP, as it measures activity and not total cell count. Collecting even two lines of evidence in the field will greatly improve the quality of assessment and strengthen any conclusions regarding assessment of microbial growth.

How to cite: Brown, D. and Turner, R.: Reconstruction of an industry related biofilm into a proxy model community – Challenges around Field and lab based microbial growth analysis, biofilms 9 conference, 29 Sep–1 Oct 2020, biofilms9-52, https://doi.org/10.5194/biofilms9-52, 2020.

Introduction:

Thermophilic sporeformers are present in raw milk at very low concentration and resist to pasteurisation applicated to destruct vegetative and pathogenic cells. Those spores can adhere to stainless steel due to their hydrophobicity and can form biofilms. Early stage biofilms are important because it can increase the matrix and the adhesion of other cells. Because of those biofilms, the three main species: Geobacillus stearothermophilus, Anoxybacillus flavithermus and Bacillus licheniformis can resists to Cleaning In Place (CIP) procedure, and contaminate a new process.

Material and Methods:

Early stage adhesion was conducted on stainless steel submerged by milk inoculated with a fresh culture of bacteria (G. stearothermophilus (N=15), A. flavithermus (N=32) and B. licheniformis (N=15)) for 6h of growth at 55°C under agitation. The ability of sporeformers to form biofilms under those conditions were measured by image analysis after a fluorescent coloration (acridine orange) and random photography. A coverage percentage was calculated by ImageJ ; and a positive threshold was set up at 5% of covering.

The efficiency of CIP procedures were obtained after a caustic soda and nitric acid treatment during different duration and temperature of treatment. Tested biofilms were formed in milk during 12h at 55°C, in stainless steel microplates (96 wells) on the same species (3 strains for each) under agitation. Surviving spores were enumerated by the microcolony method.

Results:

Early stage adhesion shows that 62.5 % (N=20) of A. flavithermus strains can form biofilm within 6h, whereas only 6.7% (N=1) of G. stearothermophilus and 0% (N=0) of B. licheniformis biofilm in 6h at 55°C on submerged stainless steel. However, the maximum covering % on A. flavithermus was 35%; while on the only biofilm forming strain of G. stearothermophilus, this percentage reach 75%. Image analysis also shows biofilm structure from 2D to 3D.

The presence and the resistance of spores to chemical cleaning was highly variable within strains. Nitric acid appears to be more effective than caustic soda against biofilms formed by vegetative cells and spores from these strains.

Significance:

Those results shows that strong biofilms are mainly composed of spore and are very resistant to CIP used in dairy industries. That is why a better understanding of control methods can lead to a finer and suitableness use of cleaning products.

How to cite: Delaunay, L., Postollec, F., Leguerinel, I., and Mathot, A.-G.: Screening of fast biofilm formation on stainless steel by thermophilic sporeformers originated from dairy powder and their resistance against CIP, biofilms 9 conference, 29 Sep–1 Oct 2020, biofilms9-86, https://doi.org/10.5194/biofilms9-86, 2020.

Background: Spread of pathogenic microbes and antibiotic-resistant bacteria in healthcare settings and public spaces is a serious public health challenge. Materials and surface-treatments that prevent solid surface colonization and biofilm formation or impede touch-transfer of viable microbes could provide means to decrease pathogen transfer from high-touch surfaces in critical applications. Both, ZnO and Ag nanoparticles have shown a great potential in antimicrobial applications. Although antimicrobial properties of such nanoparticle suspensions are well studied, less is known about nano-enabled solid surfaces.

Results: Here we demonstrate that solid surfaces coated with nano-ZnO or nano-ZnO/Ag composites possess species-selective medium-dependent antibiofilm activity against Escherichia coli MG1655, Staphylococcus aureus ATCC25923 and Candida albicans CAI4. Colonization of nano-ZnO surfaces by E. coli and S. aureus was decreased in oligotrophic (nutrient-poor, no growth) conditions with E. coli showing higher sensitivity to Ag and S. aureus to Zn, respectively. Nano-ZnO inhibited bacterial biofilm formation in a dose-dependent manner in oligotrophic conditions reaching maximum of 2.12 and 3.49 log reduction on dense nano-ZnO surface compared to uncoated surface after 72 h for E. coli and S. aureus, respectively. Minor to no effect was observed for bacterial biofilms in growth medium (nutrient-rich, supporting exponential growth). Addition of Ag to the sparse nano-ZnO surfaces had transient negative effect on E. coli biofilm formation in oligotrophic conditions with an additional 0.5-1.6 log reduction in harvested viable cells (3-48 h post-inoculation, respectively) compared with sparse nano-ZnO without added Ag. This additional reduction decreased to a non-significant 0.34 log by 72 h. Inversely, compared to uncoated surfaces, nano-ZnO surfaces enhanced biofilm formation by C. albicans in oligotrophic conditions by 1.27 log increase in viable attached cells at 48 h time point and just a minor transient negative effect was seen in nutrient-rich medium. However, enhanced C. albicans biofilm formation on nano-ZnO surfaces in oligotrophic conditions was effectively counteracted by the addition of Ag.

Conclusion: Our results not only showed that nano-ZnO and nano-ZnO/Ag coated solid surfaces have the potential to effectively decrease surface colonization by the bacteria E. coli and S. aureus but also indicated the importance of the use of application-appropriate test conditions and exposure medium in antimicrobial surface testing. Possible selective enhancement of biofilm formation by the yeast C. albicans on Zn-enabled surfaces should be taken into account in antimicrobial surface development.

This work was funded by Estonian Research Council Grants EAG20, PRG749.

How to cite: Rosenberg, M., Visnapuu, M., Vija, H., Kisand, V., Kasemets, K., Kahru, A., and Ivask, A.: Selective antibiofilm properties of nano-ZnO and nano-ZnO/Ag coated surfaces, biofilms 9 conference, 29 Sep–1 Oct 2020, biofilms9-74, https://doi.org/10.5194/biofilms9-74, 2020.

Avoiding undesired growth of biofilm is a fundamental challenge for all surfaces in long-term contact with aqueous media. Slippery liquid infused porous substrates (SLIPS) are a promising type of surface for preventing biofilm attachment. The effectiveness of SLIPS is based on the liquid/liquid interface between the medium and the surface, which prevents biofilm attachment. However, the long-term stability of these surfaces is problematic: under shear force, the oil layer is removed and the repellent effect is lost. Here, we study correlations between the porosity of the infused substrate and the ability to uphold the SLIPS oil-film under low shear and high shear force conditions. For this purpose, we manufacture substrates with different porosity and surface roughness in porous fluorinated polymer “Fluoropor”, which we have recently introduced. The porous layers were infused with fluorinated oil and their roughness was studied by white light interferometry. We find that SLIPS samples with smaller pores more effectively reduce Pseudomonas aeruginosa biofilm growth in a seven-day microfluidic flow cell experiment. With its easy production, simple adjustment of porosity and the possibility to attach the polymer to various technical substrates during polymerization, Fluoropor is a very promising material for producing stable SLIPS. When produced with small pores, Fluoropor is also transparent and enables the real-time observation of biofilm growth by optical examination. Thus, Fluoropor SLIPS provides an easy approach to reduce bacteria adhesion and bio fouling in many technical applications.

How to cite: Keller, N., Bruchmann, J., Sollich, T., Richter, C., Thelen, R., Sachsenheimer, K., Kotz, F., Schwartz, T., Helmer, D., and Rapp, B. E.: Study of Biofilm Growth on Slippery Liquid-Infused Porous Surfaces Made from Fluoropor, biofilms 9 conference, 29 Sep–1 Oct 2020, biofilms9-68, https://doi.org/10.5194/biofilms9-68, 2020.

The ability of microorganisms to form biofilms has become a major problem in the dairy industry in Canada, notably by affecting the quality and the safety of the by-products. Established biofilms are difficult to remove during the CIP cleaning system and may become resistant to sanitizers. Therefore, it is important to identify and characterize the microorganisms associated to biofilm in the Canadian dairy industry, allowing to develop improvement strategies of biofilm control. The purpose of this study is to evaluate the ability to form biofilms by spoilage microorganisms isolated in processing plants in Canada. For this purpose, 19 strains were isolated from problems associated with the formation of biofilms in the dairy industry and identified using a MALDI-TOF mass spectrometer. The single species biofilm production of these isolates was then measured after a crystal violet coloration using 96-well microplates. The results revealed different biofilm formation profiles depending of the isolates in culture medium. Indeed, 7/19 isolates are moderate or strong biofilm producers and 12/19 isolates are negative or weak biofilm producers. Furthermore, enzymatic treatments revealed that the composition of the biofilms was different depending of the species but also the isolates. In conclusion, the results suggest that some of the isolates collected in the dairy industry have the ability to produce moderate or strong biofilms and thus, to facilitate the persistence of other spoilage microorganisms but also potential pathogenic microorganisms such as Listeria monocytogenes. The characterization of those biofilms will be helpful to the development of an effective approach allowing a better control of the biofilms in the dairy industry.

How to cite: Goetz, C., Niboucha, N., and Jean, J.: Study of the ability to form biofilms of microorganisms isolated from the milk industry in Canada , biofilms 9 conference, 29 Sep–1 Oct 2020, biofilms9-134, https://doi.org/10.5194/biofilms9-134, 2020.

Biofilms are omnipresent in industrial cooling water circuits and frequently lead to technical and economic problems. Yet, methods to remove biofilms may be inefficient, due to the EPS layer, which provides a protective layer against penetration of disinfectants. In combination with stricter legislation and increased environmental awareness, this has opened the search for alternative strategies to control biofilms in cooling water systems.

In this study we implement a novel biofilm removal strategy, in which we target the intercellular language involved in the formation of biofilm structures. This language consists of signalling molecules (autoinducers) excreted by biofilm forming bacteria, a process referred to as quorum sensing (QS). We aim to alter QS via the activation of an antagonistic process called quorum quenching (QQ). Quorum quenching is a process that naturally occurs in bacterial communities; hence the final product is environment, - and user-friendly and thus a valuable alternative to the oxidising chemical products that are often used to clean cooling water circuits.

Here we present the first application of a QQ-product tested on pilot scale using parameters that resemble industrial evaporative cooling towers. This setting is particularly interesting as it an open system, fed with various types of make-up water, and comprised of biofilms adjusted to high operating temperatures.

The QQ product was tested using a closed, tube-like system, under continuous flow, fed with a propionic, - and acetic acid rich synthetic medium. Hydraulic retention time (HRT) was gradually shifted over a time span of 6 weeks. Heterotrophic plate counts were acquired once per week from the planktonic and biofilm phase. A pilot without the addition of QQ was ran in parallel as a control.

Our results show that the QQ product reduced and delayed the formation of biofilm compared to the control. Interestingly, this difference diminished when the HRT was modified. Metagenomic analysis of the biofilm phase, revealed that 16S rRNA sequences corresponding to the QQ were also strongly reduced during this shift, indicating a wash-out of the QQ product. The exact interaction of HRT and presence of QQ will be analysed in more depth using QQ specific qPCR primers.

How to cite: KleinJan, H., Meunier, C., Nonet, S., and Michel, M.: Talk2clean: application of probiotics to control biofilm in industrial water circuits – a innovative application , biofilms 9 conference, 29 Sep–1 Oct 2020, biofilms9-95, https://doi.org/10.5194/biofilms9-95, 2020.

Biofouling is a natural process in marine environments with associated economic and ecological problems. Thus, understanding the conditions that affect cyanobacterial biofilm development is crucial to develop new antibiofouling strategies and decrease the impact of biofilms in the marine environment. In this study, we investigated the relative importance of shear forces and surface hydrophobicity on biofilm development by two coccoid cyanobacteria with different biofilm formation capacities. The strong biofilm-forming Synechocystis salina was used along with the weaker biofilm-forming Cyanobium sp. Biofilms were developed in defined hydrodynamic conditions using glass (a model hydrophilic surface) and a polymeric epoxy coating (a hydrophobic surface) as substrates. Biofilms developed in both surfaces at lower shear conditions contained a higher number of cells and presented higher values for wet weight, thickness, and chlorophyll a content. The impact of hydrodynamics on biofilm development was generally stronger than the impact of surface hydrophobicity, but a combined effect of these two parameters strongly affected biofilm formation for the weaker biofilm-producing organism. The antibiofilm performance of the polymeric coating was confirmed at the hydrodynamic conditions prevailing in ports. Shear forces were shown to have a profound impact on biofilm development in marine settings regardless of the fouling capacity of the existing flora and the hydrophobicity of the surface.

How to cite: Faria, S. I., Teixeira-Santos, R., Romeu, M. J., Morais, J., Vasconcelos, V., and Mergulhão, F. J.: The Impact of Shear Forces and Surface Hydrophobicity on Coccoid Cyanobacterial Biofilm Development, biofilms 9 conference, 29 Sep–1 Oct 2020, biofilms9-33, https://doi.org/10.5194/biofilms9-33, 2020.

The chemical disinfectant chlorine, as chlorine gas (Cl₂) and sodium hypochlorite (NaOCl), has been commonly used for drinking water treatment.^1,2 Although the recommended residual concentration of free chlorine allows to some extent the control of microbial growth in the bulk water, the occurrence of biofilms in chlorinated drinking water distribution systems (DWDS) has been frequently reported.^3,4 Therefore, the main goal of this study was the study of alternative biocides to control biofilm development in DWDS. The effects of sodium dichloroisocyanurate (NaDCC), trichloroisocyanuric acid (TCCA), and pentapotassium bis(peroxymonosulphate) bis(sulphate) (OXONE®) were analysed against two emerging pathogens isolated from drinking water, Acinetobacter calcoaceticus and Stenotrophomonas maltophilia. The determination of the minimum bactericidal concentrations (MBC) of the selected biocides were based on the European Standard EN 1276, with MBC between 1.56 to 6.25 mg/L for NaDCC, 2.5 to 3.75 mg/L for TCCA, and 172 to 688 mg/L for OXONE®. Inactivation curves were developed and fitted to microbial survival models. The effects of biocides on cytoplasmic membrane integrity were assessed by propidium iodide uptake. The action on biofilm control was analysed against 48 h old biofilms developed on polyvinyl chloride (PVC) and stainless steel (SS) coupons using a 24-wells microtiter plate assay. The bacteria culturability and removal assessment were determined by colony forming units (CFU) enumeration on R2A agar, and by 4’,6-diamidino-2-phenylindole (DAPI) staining, respectively. This study reinforces biofilms as chronic contaminants of DWDS and highlights that the understanding of antimicrobial susceptibility of microorganisms to biocides is an important step in the design of effective biofilm control strategies in order to provide to consumers drinking water of adequate microbiological quality.

References

[1] K.V. Ellis (1991), Crit. Rev. Environ. Control. 20:5-6, 341-407.

[2] N. Pichel, M. Vivar, and M. Fuentes (2019), Chemosphere. 218, 1014-1030.

[3] S. Liu, C. Gunawan, N. Barraud, S.A. Rice, E.J. Harry, and R. Amal (2016), Environ. Sci. Technol. 50, 8954-8976.

[4] L.C. Simões and M. Simões (2013), RSC Adv. 3, 2520-2533.

Acknowledgements

This work was financially supported by: Base Funding - UIDB/00511/2020 of the Laboratory for Process Engineering, Environment, Biotechnology and Energy – LEPABE - funded by national funds through the FCT/MCTES (PIDDAC); PTDC/BII-BTI/30219/2017 - POCI-01-0145-FEDER-030219; POCI-01-0145-FEDER-006939, funded by FEDER funds through COMPETE2020 – Programa Operacional Competitividade e Internacionalização (POCI) and by national funds (PIDDAC) through FCT/MCTES; and PhD grant awarded to Isabel Maria Oliveira (SFRH/BD/138117/2018).

How to cite: Oliveira, I. M., Simões, L. C., and Simões, M.: The effects of different biocides against selected drinking water-isolated bacteria in planktonic and sessile states, biofilms 9 conference, 29 Sep–1 Oct 2020, biofilms9-125, https://doi.org/10.5194/biofilms9-125, 2020.

The development of new biocidal formulations targeting cells in biofilms is still a scientific challenge ^1,2. The current arsenal of biocides is clearly limited in controlling biofilms ³. Therefore, novel molecules to control biofilms are needed. This study assessed the antimicrobial activity of glycolic acid (GA) and glyoxal (GO) against Bacillus cereus and Pseudomonas fluorescens, two species commonly found in industrial biofilms. GA and GO are two glycolysis by-products approved as biocides for surface disinfection, whose antimicrobial action remains to be understood. Their antimicrobial activity was determined according to the European Standard EN 1276 ⁴. The mode of action was assessed according to the effects on the cell envelope (surface hydrophobicity and cell membrane damages) and cell replication. P. fluorescens was eradicated by both selected compounds, while B. cereus was only partially reduced even under high concentrations. According to the survival curves, P. fluorescens cells had the same susceptibility to both compounds. B. cereus cells were more susceptible due to cumulative damages. The dose-activity curves proposed that the selected compounds interacted chemically with cell targets - GA and GO were able to disturb cell integrity, causing changes in cell hydrophobicity and further membrane damages. In terms of cell replication, GA caused negligible changes in lag time length and in the maximum cell growth, while GO was found to act as a bacteriostatic. Thus, GA was found to be an oxidant (acid group) and membrane-active compound (alcohol group). On the other hand, GO had cell growth inhibitory (nucleophilic group) effects. These compounds were further applied against B. cereus and P. fluorescens biofilms, promoting strong inactivation and removal effects. The combination of GA and GO with traditional biocides is likely to represent a new, and much needed, generation of disinfectant formulations for industrial biofilm control.

References

1 Yuksel, F. N., Buzrul, S., Akcelik, M. & Akcelik, N. Inhibition and eradication of Salmonella Typhimurium biofilm using P22 bacteriophage, EDTA and nisin. Biofouling 34, 1046-1054, doi:10.1080/08927014.2018.1538412 (2019).

2 Araújo, P. A. et al. Combination of selected enzymes with cetyltrimethylammonium bromide in biofilm inactivation, removal and regrowth. Food Res Int 95, 101-107, doi:10.1016/j.foodres.2017.02.016 (2017).

3 Capita, R. et al. Effect of low doses of biocides on the antimicrobial resistance and the biofilms of Cronobacter sakazakii and Yersinia enterocolitica. Sci Rep 9, 15905, doi:10.1038/s41598-019-51907-1 (2019).

4 European Standard EN-1276 Chemical disinfectants and antiseptics in: Quantitative suspension test for the evaluation of bactericidal activity of chemical disinfectants and antiseptics used in food, industrial, domestic, and institutional areas - Test method and requirements (phase 2, step 1) (2009).

How to cite: Fernandes, S. and Simões, M.: The glycolysis by-products glycolic acid and glyoxal cause antimicrobial and antibiofilm effects, biofilms 9 conference, 29 Sep–1 Oct 2020, biofilms9-142, https://doi.org/10.5194/biofilms9-142, 2020.

Geobacter sulfurreducens is a gram- rod shaped microorganism and the model system for direct extracellular electron transfer (EET). Although several details of the molecular mechanisms of EET are deciphered [1, 2], there is still a significant lack of knowledge, for instance on kinetics or cell growth. It is the aim of this study to provide information about the influence of the anode potential on yield coefficients on the single cell level, i.e. the moles of substrate consumed or moles of electrons transferred to the anode per cell. This information is highly relevant for physiological as well as technical considerations and for further improvement of bioelectrochemical systems (BES). Therefore, single and double chamber bioelectrochemical reactors were operated in batch mode using either the “standard” graphite rods or a microscopy slide sputter-coated with 10 nm of Cr and 25 nm of AuPd as anode material for allowing analysis of the biofilms with CLSM, as a tool for cell number determination.
We demonstrate that the anode potential and the electrode material has a strong influence on the kinetics of initial growth as well as on the yield coefficients of early stage anode biofilms of Geobacter sulfurreducens, but does not influence other parameters like coulombic efficiency. For instance, in single chamber reactors, the observed lag time, expressed as the time when the biofilm delivered a current density of 1 µA cm^-2, was of 1.76±1.02, 6.99±2.26 days and 5.32±1.82 days at -200mV, 0 mV and +200mV (vs. Ag/AgCl sat. KCl) respectively with the sputter-coated glass and of 2.44±0.48 days, 1.38±0.46 days and 1.73±1.45 at -200 mV, 0 mV and +200 mV (vs. Ag/AgCl sat. KCl) respectively using graphite rods.

References

[1] Annu Rev Microbiol. 2012; 66:391-409

[2] Microbial Electrochemical Technology Sustainable Platform for Fuels, Chemicals and Remediation Biomass, Biofuels and Biochemicals 2019, Pages 339-351

How to cite: Scarabotti, F., Korth, B., Rago, L., Bühler, K., and Harnisch, F.: The influence of the potential on single cell yield coefficients of early stage anode biofilms of Geobacter sulfurreducens, biofilms 9 conference, 29 Sep–1 Oct 2020, biofilms9-143, https://doi.org/10.5194/biofilms9-143, 2020.

Drinking water represents a reservoir for microorganisms. Microorganisms from water are able to attach to the inner surface of a water hose and nourish themselves by the leaking carbon molecules. Through the interaction of different species a multi-species biofilm can develop. Especially in the food processing environment biofilms in water hoses represent a risk factor. Within the food processing sector water hoses are often used to remove disinfecting agents from freshly cleaned surfaces, after the cleaning and disinfection procedure. When biofilms are located inside these water hoses, cells or cell clusters can detach, subsequently contaminating cleaned food contact surfaces.

We checked water hoses as a biofilm hotspot in a meat processing facility by using a flocked swab for biofilm sampling inside the water hose and accessory parts (i.e. nozzle). The bacterial load (culture-based and DNA-based) and the presence of matrix components (carbohydrates, proteins and extracellular DNA) were analysed.  

Herby we identified three from six tested water hoses to harbour a biofilm, by being positive for microorganisms and at least two matrix components. This clearly states the need for further understanding of biofilm formation in water hoses. Within the three other hoses, microorganisms could be detected, but no matrix components. We could isolate twelve genera of the water hose associated biofilms using one growth medium (TSA) and two different incubation temperatures (10 °C and 20 °C). There was only on genus that was present within all three water hose biofilms, which was Rhodococcus. Previously this genus was isolated from a shower head (Lee 2013), and is known to catabolise a wide range of organic compounds. This potentially enables the growth in a nutrient poor environment like the water hose providing secondary colonisers launch aid to contribute to the biofilm. The genera Flavobacterium, Microbacterium and Stenotrophomonas were shared among two of the water hose biofilms. Experiments to assess the biofilm forming ability of isolates of these genera using a mono-species static biofilm model indicate that all three species are able to produce matrix and can therefore be regarded as biofilm producers.

To date, there is limited information about biofilm development and presence in water hoses, especially in the food processing environment. This first identification of biofilms in water hoses and associated parts emphasizes the need of further research on this topic and detailed monitoring at these sites to prevent recontamination. A currently ongoing microbiome study on the water, the used water hoses, and the water-contacting food contact sites in a meat processing facility will give further details about the biofilm presence and possible transmission of microorganisms encountered there.

References:

Lee, Yoonjin. 2013. “An Evaluation of Microbial and Chemical Contamination Sources Related to the Deterioration of Tap Water Quality in the Household Water Supply System.” International Journal of Environmental Research and Public Health 10(9):4143–60.

How to cite: Wagner, E. M., Thalguter, S., Rychli, K., and Wagner, M.: The microbiome of water and water-associated biofilms in meat processing facilities, biofilms 9 conference, 29 Sep–1 Oct 2020, biofilms9-43, https://doi.org/10.5194/biofilms9-43, 2020.

Biofilms are inextricably linked to the hydrodynamics of flow through a filter and yet engineers rarely explicitly engineer this interaction. We developed a system that links computer simulation and 3-D printing to optimize filter media geometry and biofilm function. The main objective is to prototype filter media to passively induce vortices that roll down a surface, imposing oscillating flow in the channel to enhance biofilm formation. Thus, a 2-D model was developed and linked to a 3-D printer. The model was solved to determine the wall shear stress distribution with time. The experiments showed that the thickest, densest and most extended biofilms were formed for the strongest oscillations in the channel due to the presence of a filter medium. This is a speeding-up innovation in the design and implementation of small-scale biofiltration systems for rural communities.

How to cite: Tsagkari, E., Connelly, S., Liu, Z., McBride, A., and Sloan, W.: The role of filter media geometry in tap water biofilms , biofilms 9 conference, 29 Sep–1 Oct 2020, biofilms9-1, https://doi.org/10.5194/biofilms9-1, 2020.

Introduction: An increasing microbial resistance to known antibiotics raises the demand for new antimicrobials. New antibacterial agent should have a good activity against planktonic as well as biofilm bacteria. Quaternary ammonium compounds (QACs), are widely used in medicine, have proven antimicrobial properties, and are low toxic and low irritating. In this study new QACs were evaluated for their biofilm eradication efficiency as antibacterial compounds and as irrigants in combination with Er:YAG photoacoustic streaming.

Aims: To evaluate the effectiveness of new QACs against E. faecalis biofilms and to increase the effectiveness of QACs with laser treatment.

Method: The biofilm of E. faecalis were grown on titanium surface. The fraction of the dead cells and the biofilm surface coverage was determined with LIVE/DEAD^TM using confocal microscopy (CLSM) before and after QACs treatment. To enhance to effectiveness the biofilm samples were pretreated with QACs followed by short laser Er:YAG photoacoustic streaming treatment.

Results: All tested QACs were effective against the E. faecalis biofilms. The best anti-biofilm compounds were N-Alkylimidazolinium derivatives. Compared to planktonic bacteria the bacteria in the biofilm were up to 10 fold more resistant. The fraction of the dead bacteria that were treated with QACs followed by Er:YAG photoacoustic streaming increased significantly compared to the chemical treatment alone. In addition, the biofilm surface coverage decreased after laser treatment.

Conclusions: The results suggest that new QACs have a great potential as antibacterial compounds effective against biofilms of E. faecalis. The laser treatment can significantly improve the effectiveness of QACs treatment.

How to cite: Hympanova, M., Terlep, S., Markova, A., Prchal, L., Dogsa, I., Marek, J., and Stopar, D.: The susceptibility of E. faecalis biofilm against selected new quaternary ammonium compounds, biofilms 9 conference, 29 Sep–1 Oct 2020, biofilms9-30, https://doi.org/10.5194/biofilms9-30, 2020.

L- and D-Cateslytin (CTL) are antimicrobial peptides (AMP) derived from chromogranin A, a protein of the stress response system. Their antimicrobial properties have been thoroughly characterized and already exploited in biomaterials. However, effects on biofilms of yeast and bacteria have never been specifically addressed. We have investigated the impact of both L and D configurations of CTL on the growth of biofilms formed by Candida albicans, Escherichia coli or Staphylococcus aureus microorganisms.

The study was conducted in different media and two strategies of treatment were tested, consisting of administrating the peptide either just at the beginning of biofilm development i.e. on just adhering pioneer microbial cells or on a biofilm already allowed to develop for 24h. We also considered whether the peptide was modified in contact with the medium or/and microbial metabolites. Planktonic and sessile populations of microbial cells were analyzed by spectrophotometry, crystal violet staining, MTT and confocal microscopy with staining by Syto9Ò and propidium iodide. Identification of the peptides and their derived fragments was investigated by HPLC and Mass-Spectroscopy.

In general, CTL-D exhibited higher antibiofilm performances than CTL-L. In addition, concentrations necessary to inhibit biofilm formation were found to vary from ten to eighty times the MICs determined in planktonic cultures. Nevertheless, the results also demonstrate that sessile microorganisms and biofilms are sensitive to CTL (L and D conformations) differently that planktonic populations. Significant (p-value < 0.01) effects were observed on both sessile and planktonic populations and with both strategies of treatments, but they highly varied with medium, species and CTL configuration. Typically, better antibiofilm effect than common antibiotics was reached in some specific conditions, while enhancement of aggregation or biofilm formation occurred in another medium and for other doses. Nevertheless,

Finally, this confirms the quality of CTL peptides as new antimicrobial agents and reveals their anti-biofilm properties. This also specifies the conditions of use necessary to benefit of the highest performances.

How to cite: Ploux, L., Jin, M., Hellé, S., Betscha, C., Strub, J.-M., and Metz-Boutigue, M.-H.: The versatile effect of L- and D-Cateslytin on bacteria and yeast biofilms according to configuration, medium and dose, biofilms 9 conference, 29 Sep–1 Oct 2020, biofilms9-4, https://doi.org/10.5194/biofilms9-4, 2020.

Abstract

The growth of biofilms, causing biofouling on the membrane and feed spacer surface, is an unavoidable phenomenon in reverse osmosis. Biofouling can lead to unacceptable losses in product quality and quantity, and membrane lifetime. Process conditions such as crossflow velocity and nutrient concentration in the feed water strongly affect the development of biofilms. To improve system performance, understanding the relation between process conditions, biofilm development, and system performance is key. Optical coherence tomography (OCT), is increasingly applied to characterize biofilm structure in-situ and non-destructively. In OCT, near-infrared light is used to capture 2D and 3D images from within optical scattering media. In spacer filled channels with representative biodegradable nutrient conditions in the feed, biofilms often develop heterogeneously and dispersed. In such systems, commonly used structural parameters such as average thickness, average roughness, and average porosity may not be reflected in the system performance.

In this study, biofilm structural and spatial parameters are explored with the objective to link biofouling in spacer filled channels to system performance indicators. For this purpose, biofilms are grown in membrane fouling simulators at different nutrient concentrations and flow rates. Biofilm development on the feed spacer and on the membrane and system performance (pressure drop, transmembrane pressure, rejection) are monitored. Understanding the impact of (i) feed water quality and flow rate on biofilm growth and of (ii) biofilm structure and spatial distribution on system performance will lead to the development of more effective strategies for biofouling control.

Keywords

Biofouling; desalination; drinking water production; reverse osmosis; optical coherence tomography; feed spacer; biofilm structure

How to cite: Huisman, K. T., Blankert, B., Bucs, S., and Vrouwenvelder, J. S.: Understanding how operating conditions affect biofouling structure in spacer filled membrane filtration channels using optical coherence tomography, biofilms 9 conference, 29 Sep–1 Oct 2020, biofilms9-77, https://doi.org/10.5194/biofilms9-77, 2020.

In the last decade concern about the presence of biofilms in food processing plants has increased. Biofilms in the environment of food processing plants represent a threat to food quality, safety and shelf-life. These biofilms can host pathogenic bacteria such as Listeria, Salmonella and Campylobacter, as well as spoilage microorganisms. Additionally, biofilms show some degree of resistance to conventional detergents and disinfectants that hinders their removal and favors re-growth. Therefore, there is a need for sanitizing products and protocols that are highly efficient at removing biofilms and suitable for food processing plants. Enzymatic detergents have recently been introduced as an alternative to conventional products against biofilms in food processing plants. These detergents contain one or more enzymes that disrupt the EPS of the biofilms, making the microorganisms present in the biofilm more vulnerable to disinfectants. Enzymatic detergents have been proofed to be more efficient in degrading biofilms than conventional detergents reducing both, EPS content and bacterial counts. Finally, higher efficiency on biofilm removal was observed after completing the entire sanitizing procedure (cleaning + disinfection) using an enzymatic detergent than a conventional detergent. These tests confirm the great potential of enzymatic detergents to remove biofilms.

How to cite: Berga, M., Ylla, I., and Latorre, M.: Use of enzymatic detergents to remove biofilms in food industries, biofilms 9 conference, 29 Sep–1 Oct 2020, biofilms9-132, https://doi.org/10.5194/biofilms9-132, 2020.