Posters

Among the Enterococci, Enterococcus faecalis is most frequently associated with human infections ranging from the urinary tract and wound infection to endocarditis and bacteraemia. These infections are often multidrug-resistant and, hence, life-threatening. Moreover, E. faecalis are often co-isolated with other pathogenic bacteria from polymicrobial biofilm-associated infections contributing to disease progression and poorer patient outcomes. Genetic tools to dissect complex interactions in biofilms and mixed microbial communities are largely limited to transposon mutagenesis and traditional allelic exchange methods requiring time- and labour-intensive two-step integration and excision screening that can take a week or more to make a single mutant. We built upon the well-characterized CRISPR interference system using streptococcal dCas9 to develop an easily-modifiable, inducible system for E. faecalis that can efficiently silence single and multiple genes in a matter of hours. We show that this system can silence genes involved in biofilm formation, antibiotic resistance, and can be used to interrogate gene essentiality. Uniquely, this tool is optimized to study genes important for biofilm initiation, maturation, and maintenance, and can be used to perturb pre-formed biofilms. This inducible CRISPRi system will be valuable to rapidly and efficiently investigate a wide range of aspects of complex enterococcal regulation networks within the biofilms, including polymicrobial biofilms. 

How to cite: Afonina, I., Lu, T., and Kline, K.: CRISPRi enables studies of enterococcal biofilm initiation, maturation and maintenance. , biofilms 9 conference, 29 Sep–1 Oct 2020, biofilms9-40, https://doi.org/10.5194/biofilms9-40, 2020.

Acinetobacter baumannii is one of the most problematic opportunist pathogen responsible for many infections worldwide (1). Besides its high capacities to acquire antibiotic resistance mechanisms, it also presents high adhesion abilities on any types of abiotic or living surfaces leading to biofilm development, a mode of growth conferring an additional protection against various treatments and allowing the infection relapse (2). A. baumannii has been recently ranked on the global priority pathogens list established by the World Health Organization for which there is an urgent need for new treatments. One interesting way to identify new therapeutic targets to eradicate this pathogen is the characterization of its post-translational modifications (PTMs) (3). The functions and extents of PTMs remain largely unknown in prokaryotic cells compared to eukaryotic cells. Lysine acetylation is an attractive and prevalent PTM in bacteria. An increasing number of investigations have been dedicated to identify acetylated proteins by proteomics. Some studies have shown that acetylation can play a pivotal role in bacterial virulence, resistance, or biofilm (4). Enzymes involved in acetylation addition (lysine acetyltranferase KAT) or removal (lysine deacetylase KDAC) would provide a better mechanistic understanding of bacterial physiology and therefore could be considered as potential therapeutic targets. So far, little information is available on these enzymes in A. baumannii (5). Recently, in a global dynamic proteome study of A. baumannii ATCC 17978 strain grown in sessile mode, we highlighted the highest protein fold change for a protein belonging to the Sir2-like family which may possess a KDAC activity (6). The aim of the current study was to evaluate the involvement of this protein in A. baumannii physiology. For this purpose, a gene deletion approach was carried out to perform different phenotype tests (drugs and oxidative stress resistance, virulence assays, motility and biofilm formation) on wild-type and mutant strains. We compared, in biofilm mode of growth, acetylomes of the WT and the mutant. Our results demonstrated more than twice acetylated proteins in mutant in comparison to the WT. Of interest, biofilm formation in mutant was sensibly decreased. These different results suggest a potential involvement of this protein in A. baumannii biofilm formation.

(1) Antunes et al. Acinetobacter baumannii: evolution of a global pathogen. Pathog. Dis. 71(2014), 292-301.

(2) Espinal et al. Effect of biofilm formation on the survival of Acinetobacter baumannii on dry surfaces. J. Hosp. Infect. 80(2012), 56–60.

(3) Richters. Targeting protein arginine methyltransferase 5 in disease. Future Med. Chem. 9(2017), 2081-2098.

(4) VanDrisse and Escalante-Semerena. Protein acetylation in bacteria. Annu. Rev. Microbiol. 73(2019), 111-132.

(5) Carabetta and Cristea. Regulation, function, and detection of protein acetylation in bacteria. J. Bacteriol. 199(2017), e00107-17.

(6) Kentache et al. Global dynamic proteome study of a pellicle-forming Acinetobacter baumanii strain. Mol. Cell. Proteomics. 16(2017), 100-112.

How to cite: Robin, B., Massier, S., Potron, A., Vuillemenot, J.-B., Sauvage, S., Jouenne, T., Perrot, V., Dé, E., and Hardouin, J.: A SIR2 family protein impacts biofilm formation by post-translational modifications in Acinetobacter baumannii, biofilms 9 conference, 29 Sep–1 Oct 2020, biofilms9-127, https://doi.org/10.5194/biofilms9-127, 2020.

Background: Human milk oligosaccharides (HMOs) are the third most abundant component of breast milk, after fat and lactose, that promote infant health. Recent studies have shown that HMOs demonstrated antimicrobial and antibiofilm activity against different strains. Cystic fibrosis (CF), it is one of the major respiratory diseases, the clinical management and definitive treatment of CF biofilm-mediated chronic bacterial lung infection remains a challenge.

Objective: In this study, we examine HMOs antibiofilm activity against pathogens isolated from CF patients.

Methods and results: In current work, we investigated the antibiofilm activity of the saccharide fraction obtained from pooled human milk of 9 donors against strains of: Acinetobacter baumannii, Pseudomonas aeruginosa, Enterobacteriaceae, Staphylococcus aureus and Burkholderia cenocepacia, an intrinsically multi-resistant pathogen associated with high mortality in CF patients. We tested the ability of HMOs to inhibit biofilm formation and to eradicate matured biofilms. Live/dead staining of the biofilms and CLSM image acquisition were used.

The pooled HMOs showed a biofilm eradicating effect on most tested pathogens. The HMOs effectively killed the bacteria at high concentration (20 mg/ml, corresponds to the concentration in human milk), but visible reduction of viable bacteria and biofilm mass was observed already at lower concentrations that varied between the species. The biofilm mass was also reduced in almost all pathogenic biofilms.

The data presented in this paper supporting the importance and potential inhibitory effect of HMOs in biofilm formation. HMOs could potentially be used as novel therapeutics to treat or prevent infectious disease in patient with CF.

How to cite: Jarzynka, S., Strom, K., Makarewicz, O., Minkiewicz-Zochniak, A., Koryszewska-Baginska, A., and Oledzka, G.: Antibiofilm activity of Human Milk Oligosaccharides against pathogens isolated from cystic fibrosis patients, biofilms 9 conference, 29 Sep–1 Oct 2020, biofilms9-147, https://doi.org/10.5194/biofilms9-147, 2020.

In our attempts to improve the biofilm formation for productive bacteria (here the gram-negative seawater bacterium Paracoccus seriniphilus), we focus on the attachment of single bacteria to a solid surface as the first step of the biofilm formation process. Beside adhesion forces and elasticity of the bacteria, we investigate the minimal detachment forces due to lateral shear forces.

In order to investigate the influence of shear forces on already adhered bacteria in the laboratory, the Lateral Force Microscopy(LFM) was used first.The tip is moved laterally towards the adherent cell with different lateral forces until the cell detaches and thus the force required to shear the cell is determined.

By applying LFM, we found a correlation between the applied force and the number of moved bacteria as well as between the number of detached bacteria and the surface energy of the substrate. Further, any structuring of the substrate hinders the detachment substantially [1]. In agreement with the vertical adhesion forces, the bacteria are harder to detach at pH 4 than at pH 7.

In order to get closer to reality, the next step is to examine the (lateral) scanning force microscopic measurements under the influence of a flowing liquid and compare them with the LFM measurements. In combination with digital holography and proteome analysis, a better understanding of biofilm formation under the influence of a flowing liquid is to be achieved.

[1] K. Huttenlochner, N. Davoudi, C. Schlegel, M. Bohley, C. Müller-Renno, J. C. Aurich, R. Ulber, and C. Ziegle "Paracoccus seriniphilus adhered on surfaces: Resistance of a seawater bacterium against shear forces under the influence of roughness, surface energy, and zeta potential of the surfaces." Biointerphases 13.5 (2018)

How to cite: Müller-Renno, C., Seehase, J., Huttenlochner, K., Chodorski, J., Ulber, R., and Ziegler, C.: Bacteria and biofilms under the influence of shear, biofilms 9 conference, 29 Sep–1 Oct 2020, biofilms9-22, https://doi.org/10.5194/biofilms9-22, 2020.

Bacteria living in biofilms tolerate much higher antibiotic concentrations compared to planktonic bacteria and can cause chronic infections. Among the most difficult pathogens to treat, Pseudomonas aeruginosa is responsible for many biofilm-related infections and for much of the mortality associated with airway infections in cystic fibrosis. We speculated that there are specific genes responsible for increased antibiotic resistance in biofilms and aimed to identify them in P. aeruginosa. By doing so, a better understanding of biofilm-mediated resistance can be achieved and new bacterial targets can be identified. A P. aeruginosa transposon mutant library was screened to assess the impact on biofilm formation and the biofilm resistance toward antibiotics. Briefly, the biofilm resistance was estimated by following the re-growth of biofilm cells exposed to different concentrations of antibiotics. A few candidates, e. g. the response regulator CbrB, involved in nutrient uptake, have been identified as crucial for biofilm formation and resistance towards antibiotics. Further characterization of these interesting genes has been carried out to explore the underlying mechanism of resistance. Such knowledge can lead to the identification of susceptibility of P. aeruginosa biofilm and help to develop tools to treat persistent infections.

How to cite: Valentin, J., Varadarajan, A. R., Ahrens, C. H., van der Mei, H., and Ren, Q.: Better treatment options through a better understanding of Pseudomonas aeruginosa biofilm formation and biofilm-mediated resistance, biofilms 9 conference, 29 Sep–1 Oct 2020, biofilms9-138, https://doi.org/10.5194/biofilms9-138, 2020.

Staphylococcus aureus biofilms play important roles during infection. The main components of these biofilms are well studied; however, we lack the full understanding of factors and genes involved in regulation of biofilm formation. To screen for essential and non-essential biofilm regulatory genes in S. aureus, we have created a pooled inducible CRISPR interference library. The pooled library is designed to allow knockdown of every transcriptional unit in the S. aureus genome, thus targeting both essential and non-essential genes. We used our library in S. aureus Newman, a strain which forms structured macrocolonies on agar plates. We performed an unbiased screen of 1500 macrocolonies and found 10 macrocolonies with stably altered structures. The genotypes of these macrocolonies were determined by sequencing the single guide RNAs of the CRISPR interference system. As a proof of the validity of the approach, we identified several genes previously reported to be implicated in biofilm and macrocolony formation, including ica-genes, and metabolic genes of the TCA-cycle and gluconeogenesis. In addition, three new genes (two encoding putative enzymes and one hypothetical genes) whose depletion resulted in completely altered macrocolonies were also identified. The molecular mechanisms explaining the roles of these proteins in biofilm formation are currently under investigation.

How to cite: Kjos, M., Morales Angeles, D., Mårli, M. T., Heggenhougen, M. V., de Bakker, V., Liu, X., and Veening, J.-W.: CRISPR interference knockdown screen identifies novel proteins involved in formation of structured macrocolonies in Staphylococcus aureus., biofilms 9 conference, 29 Sep–1 Oct 2020, biofilms9-105, https://doi.org/10.5194/biofilms9-105, 2020.

The intracellular second messenger cyclic diguanylate (c-di-GMP) is broadly conserved in bacteria, where it influences processes such as virulence, stress resistance and biofilm development. In the plant-beneficial bacterium Pseudomonas putida KT2440, the response regulator with diguanylate cyclase activity CfcR is the main contributor to c-di-GMP levels in the stationary phase of growth. When overexpressed, CfcR increases c-di-GMP levels and gives rise to a pleiotropic phenotype that includes enhanced biofilm formation and crinkly colony morphology. Our group has previously reported that insertion mutants in argG and argH, the genes that encode the last two enzymes in the arginine biosynthesis pathway, do not display the crinkly colony morphology phenotype and show decreased c-di-GMP levels even in the presence of cfcR in multicopy (Ramos-González, M.I. et al. 2016. Front. Microbiol. 7, 1093). Here we present results indicating that L-arginine acts both as an environmental and as a metabolic signal that influences the lifestyles of P. putida through the modulation of c-di-GMP levels and changes in the expression of structural elements of biofilms. Exogenous L-arginine partially restores c-di-GMP levels in arginine biosynthesis mutants, a response that is transduced through CfcR and possibly (an)other diguanylate cyclase(s). At least three periplasmic binding proteins, each forming part of an amino acid transport system, contribute in different ways to the response to external L-arginine. We propose that the turnover of the second messenger c-di-GMP is modulated by the state of global arginine pools in the cell resulting both from anabolism and from uptake.

How to cite: Barrientos-Moreno, L., Molina-Henares, M. A., Ramos-González, M. I., and Espinosa-Urgel, M.: Cellular L-arginine pools modulate c-di-GMP turnover and biofilm formation in Pseudomonas putida, biofilms 9 conference, 29 Sep–1 Oct 2020, biofilms9-130, https://doi.org/10.5194/biofilms9-130, 2020.

The ability to colonize different environmental niches by bacteria is most often determined by the ability to form biofilms - complex, multicellular communities. This, in turn, depends on both cellular and extracellular factors such as genetic background of the strain, type of surface (biotic or abiotic) to which bacteria attach, availability of nutrients, temperature, etc. Pseudomonas donghuensis P482 strain is a little-known isolate from tomato rhizosphere, exhibiting antimicrobial activity towards bacterial and fungal plant pathogens. Studies have shown that it efficiently colonizes plant rhizosphere and forms biofilm on artificial surfaces. Which genetic or environmental factors underlie the mechanism of biofilm formation were yet to be elucidated. The presented research aimed at identifying those factors. Basing on the analysis of genome, knock-out mutants of the P482 strain were constructed in the genes potentially involved in biofilm formation and further analyzed for motility, colony morphology, attachment to artificial surfaces in different culture conditions, and colonization of maize and tomato rhizosphere.

How to cite: Rajewska, M., Matuszewska, M., and Jafra, S.: Cellular and environmental factors influencing biofilm formation and colonization of plant tissue by a beneficial strain of bacteria, Pseudomonas donghuensis P482., biofilms 9 conference, 29 Sep–1 Oct 2020, biofilms9-108, https://doi.org/10.5194/biofilms9-108, 2020.

Introduction

Since antibiotics were discovered, bacteria have demonstrated the ability to develop resistance by many different mechanisms. According to WHO reports from 2014, there has been an alarming increase in the antibiotic resistant bacterial strains in most parts of the world¹. Our previous results showed that a nanoantibiotic (NAB) design created in our laboratory², composed of a cerium oxide core, mesoporous silica shell loaded with capsaicin, and a chitosan coating, are effective against planktonic E. coli. However, most of the pathogenic bacteria form biofilms during infections. That is why the next stage of studying NAB is to determine whether they are effective against biofilms of different species. Moreover, the results of NAB efficiency against planktonic E. coli did not clearly show the contribution of the antibiotic drug component of NAB – capsaicin. Hence, the first step of the current study is to determine whether and to what degree, mesoporous silica nanoparticles (MSN) – serving as NAB model in this case - penetrate biofilms as a function of particle shape and surface coating; as well as finding the efficient concentration of capsaicin against E. coli and S. aureus  to optimize the NAB dosing against biofilms.

Aim

To check in vitro penetration of MSN on S. aureus biofilm and antibacterial activity of NAB and pure capsaicin on E. coli and S. aureus biofilms.

Methods

To investigate NAB efficiency on biofilms MBEC-high-throughput assay³ was performed. Equal biofilms formed on peg-lids were incubated with different concentrations of NAB and capsaicin. After different time point biofilms were sonicated and plated on agar plated to perform CFU counting. To determine the efficient concentration of capsaicin, biofilms were formed in 12 well plates and then incubated with different concentrations of capsaicin. To visualize inhibitory effect, plating for CFU counting and Resazurin assay were applied. To evaluate the penetration of particles, labeled and non-labeled particles were added to fully grown St. aureus biofilms, incubated and visualized with confocal microscopy and structured illumination microscopy.

Results

Conclusion

References

How to cite: Slita, A., Govardhanam, P., Opstad, I., Sen Karaman, D., and Rosenholm, J.: Evaluation of mesoporous silica nanoparticles-based nanoantibiotics and capsaicin on E. coli and S. aureus biofilms, biofilms 9 conference, 29 Sep–1 Oct 2020, biofilms9-133, https://doi.org/10.5194/biofilms9-133, 2020.

Background:

The adhesion of planktonic bacteria to a surface (biotic or abiotic), and their subsequent ability to aggregate into multicellular communities called biofilms, is a major driving force of failing antibiotic therapy and persistence in chronic infections caused by a variety of pathogens (e.g., Pseudomonas aeruginosa, Escherichia coli, and Staphylococcus aureus) and plaguing healthcare systems worldwide. Biofilms are estimated to be involved in over 80% of all microbial infections in humans, and commonly exhibit extreme resistance to conventional antimicrobial treatments. Consequently, there is an urgent need for novel antimicrobial agents, which target biofilm residing cells. Here, we present the development and evaluation of a new generation of dual-acting nitroxide functionalised antibiotics with potent biofilm eradication activity.

Methodology:

Synthetic organic chemistry was utilised to produce a new generation of nitroxide functionalised antibiotics with targeted biofilm eradication capabilities. These compounds were tested for biofilm eradication and/or dispersal of several bacterial species using the MBEC^TM device, a reproducible high-throughput static biofilm formation system. Mature biofilms were treated with serial dilutions of the specific test agent(s) and recovered bacterial numbers were quantified by absorbance spectroscopy at 600 nm or plating for viable cell counts. Treated biofilms were also stained with Live/Dead (SYTO-9/PI) bacterial viability kit and analysed by fluorescence and confocal laser scanning microscopy.

Results:

Nitroxide functionalised antibiotics exhibit potent biofilm-eradication activity against a variety of medically important pathogens, including P. aeruginosa, uropathogenic E. coli, and S. aureus. In Minimal Biofilm Eradication Concentration (MBEC) assays nitroxide functionalised antibiotics were 64-fold more potent against S. aureus biofilms, and at least 2-fold more potent against uropathogenic E. coli biofilms than the parent antibiotic ciprofloxacin.

Conclusions:

Currently, antibiotics are often entirely ineffective against biofilm infections. Nitroxide functionalised antibiotics represent a promising new strategy, which could circumvent the resistance of Gram-positive and Gram-negative biofilms to conventional treatments.

How to cite: Verderosa, A., Fairfull-Smith, K., and Totsika, M.: Functionalising antibiotics with nitroxides as an effective broad-spectrum biofilm eradication strategy. , biofilms 9 conference, 29 Sep–1 Oct 2020, biofilms9-12, https://doi.org/10.5194/biofilms9-12, 2020.

Antibiotic treatment regularly fails to cure patients suffering from infections caused by adaptively resistant microbial communities, referred to as biofilms. Even though at least two thirds of all clinical infections are associated with biofilms, there are no biofilm-specific therapies on the market or in clinical trials. Pseudomonas aeruginosa is a remarkably antibiotic resistant, nosocomial pathogen and biofilm-former that causes morbidity and mortality especially in cystic fibrosis and immunocompromised patients. This project aims to identify regulatory genes associated with drug resistance in P. aeruginosa biofilms to provide novel biofilm-specific targets for the design of potent drugs. A genome-wide screen of P. aeruginosa burn wound isolate UCBPP-PA14 identified 362 genes involved in biofilm formation, including dozens of regulatory and hypothetical genes. I will discuss regulatory as well as metabolic genes corresponding to the known resistome of antimicrobials.

How to cite: Dostert, M., Belanger, C. R., Blimkie, T. M., Falsafi, R., Dhillon, B. K., Lee, A. H., and Hancock, R. E.: Identifying biofilm regulators as novel targets for antimicrobial drug design, biofilms 9 conference, 29 Sep–1 Oct 2020, biofilms9-119, https://doi.org/10.5194/biofilms9-119, 2020.

Women with Cystic Fibrosis (CF) have a significantly lower life expectancy compared to men, which is indicated by an earlier impairment of lung function due to chronic colonization of pathogenic bacteria like Pseudomonas aeruginosa (PA). Reasons for this “CF gender gap” until now have not yet been fully clarified and are assumed to be multifactorial.

This study aims to shed light on the contribution of sex hormones to the CF-Gender gap considering microbial endocrinology. Therefor the study investigates whether the sex hormone estradiol, whose blood serum concentrations are significantly fluctuating within the female cycle and during pregnancy, has a regulatory influence on the development of PA biofilms in the context of CF.

For that purpose, biofilms of PA isolates from CF-patients are treated in vitro with various estradiol concentrations and are examined in a comparative study quantitatively regarding the total biomass, e.g. via crystal violet staining, and qualitatively, e.g. via scanning electron microscopy, to characterize the ultrastructure of the biofilm.

We observed that estradiol induces biofilm-forming capacity of CF-PA-isolates with respect to the total biomass and modulates the biofilm structure especially concerning the distribution and clustering of bacteria.

The observed in vitro correlation between estradiol concentration and extent of biofilm growth provides a possible microbiological explanation for gender differences in CF disease progression.

These insights and further research on possible underlying mechanisms might be relevant in the long-term for new approaches in personalized treatment for female CF patients.

Acknowledgement

This work is supported by a financial grant from Mukoviszidose Institut gGmbH, Bonn, the research and development arm of the German Cystic Fibrosis Association Mukoviszidose e.V., the Christiane Herzog foundation. We further thank the Equal Opportunities Office of the University Siegen as well as the DAAD PPP Frankreich (Project-ID 55976814) for financial support.

How to cite: Afzal, F., Le Gall, T., Montier, T., and Müller, M.: Impact of estradiol on biofilm formation of Pseudomonas aeruginosa clinical isolates from cystic fibrosis patients, biofilms 9 conference, 29 Sep–1 Oct 2020, biofilms9-107, https://doi.org/10.5194/biofilms9-107, 2020.

Pseudomonas putida rapidly forms a biofilm, after which its biomass usually disperses to half its initial amount. We have observed different biofilm dynamics of P. putida in a complex medium LB and a minimal medium M9+glc+CAA and inquired about the importance of extracellular factors for the formation of P. putida biofilm.

The proteinaceous component of LB increases the biomass of P. putida biofilm. Supplementation of M9 with tryptone but not CAA increased the biofilm biomass. Proteinase K treatment of LB medium reduced the biomass of P. putida biofilm. At the same time, growth rate or maximum OD of planktic bacteria in used media did not correlate with biofilm biomass of the same media. Thus, peptides appeared to have a positive effect on the biofilm as an extracellular factor and not as a source of C and N.

We replaced tryptone in M9 medium with positively charged poly-L-lysine (MW. 1000-5000 Da), negatively charged poly-L-glutaminic acid (MW. 1500-5500 Da) or neutral poly-LD-alanine (MW. 3000-7000). Poly-lysine and poly-glutamic acid had a slight positive effect on the biomass of P. putida wild type strain PSm biofilm and poly-alanine did not affect the biofilm.

We have previously shown that overexpression of fis in P. putida strain F15 increases biofilm biomass by increasing the lapA expression, the main adhesin gene of biofilm. Using media similar to that used for the wild-type strain for strain F15, we ascertained that only poly-lysine out of these three polypeptides restored the positive effect of fis-overexpression on the biofilm biomass. At the same time, the positive impact of fis-overexpression was absent in lapA deletion mutant strain, but not in lapF deletion mutant strain.

In conclusion, the formation of P. putida biofilm depends on polypeptides in the environment. The enhancing effect of positively charged polypeptides appears to be evident in the presence of LapA, a key factor for P. putida biofilm.

How to cite: Teras, R., Ainelo, H., and Puhm, M.: Peptides’ involvement in Pseudomonas putida biofilm formation, biofilms 9 conference, 29 Sep–1 Oct 2020, biofilms9-38, https://doi.org/10.5194/biofilms9-38, 2020.

The molecule c-di-GMP is a bacterial second messenger that controls various processes such as motility or biofilm formation in bacteria [1]. To synthesize and degrade c-di-GMP, enzymes called diguanylate cyclases (DGC) containing a GGDEF-domain and phosphodiesterases (PDE) containing an EAL-domain or HD-GYP-domain are important [1, 2]. Pseudomonas aeruginosa, a model organism for biofilm formation and dispersion, encodes for 18 GGDEF, 5 EAL, 16 GGDEF / EAL, and 3 HD-GYP-domain-containing proteins [3].
One of the GGDEF / EAL-containing proteins is NbdA. This protein also harbors an N-terminal membrane anchored MHYT-domain, that is predicted to be a sensor for NO, CO or O₂ [4]. In this work, recombinant and affinity purified NbdA was tested for its PDE activity. Three different methods were used to measure the PDE activity of NbdA: a bis-pNPP-assay in which the conversion of the pseudosubstrate bis-pNPP into p-nitrophenol was detected spectroscopically, an HPLC-analysis of an enzymatic assay with the native substrate c-di-GMP, and a MANT-c-di-GMP-assay in which a fluorescently labeled form of the presumed substrate c-di-GMP was utilized.
To establish these methods, the two known phosphodiesterases, PdeH from Escherichia coli [5] and RocR from P. aeruginosa [6], were also produced and tested. Subsequently, three variants of NbdA were investigated: the full-length version and two truncated versions of the protein. Activity was further assessed using functional complementation of an E. coli phosphodiesterase deficient strain with full-length and truncated NbdA variants confirming PDE activity in vivo.

[1] Hengge, R. (2009) Nature Rev. Microbiol. 7: 263-273.

[2] Römling, U., Gomelsky, M., Galperin, M.Y. (2005). Mol. Microbiol. 57: 629–639.

[3] Valentini, M., Filloux, A. (2016). J. Biol. Chem. 291: 12547–12555.

[4] Galperin, M.Y., Gaidenko, T.A., Mulkidjanian, A.Y., Nakano, M., und Price, C.W. (2001). FEMS Microbiol. Lett. 205, 17–23.

[5] Pesavento, C., Becker, G., Sommerfeldt, N., Possling, A., Tschowri, N., Mehlis, A., Hengge, R. (2008). Genes Dev. 22: 2434–2446.

[6] Chen et al. (2012) Chen, M.W., Kotaka, M., Vonrhein, C., Bricogne, G., Rao, F., Chuah, M.L.C., Svergun, D., Schneider, G., Liang, Z.-X., Lescar, J.  (2012). Signaling. J. Bacteriol. 194: 4837–4846

How to cite: Scherhag, A., Rüger, M., Gerbracht, K., Rehner, J., Zehner, S., and Frankenberg-Dinkel, N.: Phosphodiesterase activity of NbdA from Pseudomonas aeruginosa, biofilms 9 conference, 29 Sep–1 Oct 2020, biofilms9-89, https://doi.org/10.5194/biofilms9-89, 2020.

In Pseudomonas aeruginosa, the orphan two-component sensor SagS contributes to both, the transition to biofilm formation and to biofilm cells gaining their heightened tolerance to antimicrobials. However, little is known about the identity of the signals or conditions sensed by SagS to induce the switch to the sessile, drug tolerant mode of growth. Using a modified Biolog phenotype assay to screen for compounds that modulate attachment in a SagS-dependent manner, we identified glucose-6-phosphate to enhance attachment in a manner dependent on the glucose-6-phosphate concentration and SagS. The stimulatory effect was not limited to the attachment as glucose-6-phosphate likewise enhanced biofilm formation. We show that exposure to glucose-6-phosphate results in decreased swarming motility but increased cellular c-di-GMP levels in biofilms. Genetic analysis indicated that the diguanylate cyclase NicD is an activator of biofilm formation and is not only required for enhanced biofilm formation in response to glucose-6-phosphate but also interacts with SagS. Our findings indicate glucose-6-phosphate to likely mimic a signal or conditions sensed by SagS to activate its motile-sessile switch function. Additionally, our findings provide new insight into the interfaces between the ligand-mediated TCS signaling pathway and c-di-GMP levels.

How to cite: Park, S., Dingemans, J., Gowett, M., and Sauer, K.: The SagS sensory protein modulates biofilm formation and c-di-GMP levels by Pseudomonas aeruginosa in response to glucose-6-phosphate., biofilms 9 conference, 29 Sep–1 Oct 2020, biofilms9-122, https://doi.org/10.5194/biofilms9-122, 2020.

Colonization of textiles and subsequent metabolic degradation of sweat and sebum components by axillary skin bacteria cause the characteristic sweat malodor and discoloring of dirty clothes. Once inside the textile, the bacteria can form biofilms that are hard to remove by conventional washing. When the biofilm persists after washing, the textiles retain the sweat odor. In addition to posing a huge industrial problem, textile biofilms constitute an interesting case study of bacterial behavior in periodically wetted and dried substrates with varying surface hydrophobicity. Here we aim to study the bacterial behavior in each of the four stages of the bacterial lifecycle in textiles: adhesion, growth, drying and washing. To accomplish this, we designed a novel in vitro model to mimic physiological sweating while wearing cotton and polyester textiles. The hydrophobic polyester adhered bacteria more strongly and absorbed more sebum, the bacteria’s primary nutrient source. Bacteria were therefore initially more active in polyester textiles than in cotton. However, polyester did not bind water as well as cotton. The increased water content of cotton allowed the bacteria to retain a higher activity after the textile had dried. However, neither of the textiles retained enough water upon drying to prevent the bacteria from irreversibly adhering to the textile fibers by capillary action. This demonstrates that bacterial colonization depends on the hydrophobic and hygroscopic properties of the colonized material while highlighting the possibility of controlling bacterial behavior by either changing the surface properties or the surrounding environment.

How to cite: Moellebjerg, A. and Meyer, R.: The bacterial lifecycle in cotton and polyester textiles, biofilms 9 conference, 29 Sep–1 Oct 2020, biofilms9-118, https://doi.org/10.5194/biofilms9-118, 2020.

Microalgae are typically found in freshwater and marine systems and they harbor a mostly a beneficial growth promoting microbiota. We have recently isolated several small proteins from the microbiomes of microalga (Scenedesmus quadricauda, Microasterias crux-melintensis, Chlorella saccherophilia) and have tested them for their role in either inhibition of biofilm formation and/or biofilm degradation. Thereby we have identified two candidate proteins that showed promising activities on biofilm inhibition and degradation. These proteins were designated Pµ84 and Pµ19 and strongly affected biofilm formation in several human- and plant-pathogenic bacteria. Recombinant and purified Pµ84 and Pµ19 were applied in biofilm assays in microtiter plates and reduced biofilms formed by Stenotrophomonas maltophilia, Pseudomonas aeruginosa and Klebsiella pneumonia. If expressed in the different hosts, biofilms were reduced by a factor of 40% and if applied as exogenous proteins, biofilms were reduced up to 20%. Pµ84 application also resulted in a delayed biofilm formation and biofilm formation was affected by a factor of 17%. The microprotein Pµ19 consist of 57 aa and Pµ84 consists of 49 aa. Ongoing work elucidates the mechanism of Pµ84 and Pµ19 on the reduction of biofilm in order to achieve the optimal activity.

How to cite: Han, Y., Streit, W. R., and Krohn, I.: The phycobiota-derived proteins Pµ84 and Pµ19 suppress bacterial biofilm formation in gram-negative pathogens , biofilms 9 conference, 29 Sep–1 Oct 2020, biofilms9-63, https://doi.org/10.5194/biofilms9-63, 2020.