Exploring coaggregation mechanisms involved in biofilm formation in drinking water through a proteomic-based approach.

AIM: Coaggregation, a highly specific cell-cell interaction mechanism, plays a pivotal role in multispecies biofilm formation. While it has been mostly studied in oral environments, its occurrence in aquatic systems is also acknowledged. Considering biofilm formation's economic and health-related implications in engineered water systems, it is crucial to understand its mechanisms. Here, we hypothesized that traceable differences at the proteome level might determine coaggregation ability. METHODS AND RESULTS: Two strains of Delftia acidovorans, isolated from drinking water were studied. First, in vitro motility assays indicated more swarming and twitching motility for the coaggregating strain (C+) than non-coaggregating strain (C-). By transmission electronic microscopy, we confirmed the presence of flagella for both strains. By proteomics, we detected a significantly higher expression of type IV pilus twitching motility proteins in C+, in line with the motility assays. Moreover, flagellum ring proteins were more abundant in C+, while those involved in the formation of the flagellar hook (FlE and FilG) were only detected in C-. All the results combined suggested structural and conformational differences between stains in their cell appendages. CONCLUSION: This study presents an alternative approach for identifying protein biomarkers to detect coaggregation abilities in uncharacterized strains.

Hydrocinnamic acid and perillyl alcohol are effective against Escherichia coli biofilms when used alone and combined with antibiotics.

AIMS: The use of phytochemicals to improve the effectiveness of antibiotics is a promising strategy for the development of novel antimicrobials. In this study, the antibiofilm activity of perillyl alcohol and hydrocinnamic acid, both phytochemicals present in several plants, and two antibiotics from different classes (amoxicillin and chloramphenicol) was tested, alone and in combination, against Escherichia coli. METHODS AND RESULTS: Each molecule was tested at the minimum inhibitory concentration (MIC), 5 × MIC, and 10 × MIC, and characterized concerning biomass removal, metabolic inactivation, and cellular culturability. The highest percentages of metabolic inactivation (88.5% for 10 × MIC) and biomass reduction (61.7% for 10 × MIC) were obtained with amoxicillin. Interestingly, for 5 × MIC and 10 × MIC, phytochemicals provided a total reduction of colony-forming units (CFUs). Dual and triple combinations of phytochemicals and antibiotics (at MIC and 5 × MIC) demonstrated high efficacy in metabolic inactivation, moderate efficacy in terms of biomass reduction, and total reduction of cellular culturability for 5 × MIC. CONCLUSIONS: The results demonstrated the antibiofilm potential of phytochemicals, highlighting the advantage of phytochemical/antibiotic combinations for biofilm control.

Contribution to Understanding the Mechanisms Involved in Biofilm Formation, Tolerance and Control.

Biofilms constitute a protected mode of growth that allows the colonizing microbial cells to survive in hostile environments, even when an antimicrobial agent is present. The scientific community has come to understand many things about the growth dynamics and behavior of microbial biofilms. It is now accepted that biofilm formation is a multifactorial process that starts with the adhesion of individual cells and (auto-)coaggregates of cells to a surface. Then, attached cells grow, reproduce and secrete insoluble extracellular polymeric substances. As the biofilm matures, biofilm detachment and growth processes come into balance, such that the total amount of biomass on the surface remains approximately constant in time. The detached cells retain the phenotype of the biofilm cells, which facilitates the colonization of neighboring surfaces. The most common practice to eliminate unwanted biofilms is the application of antimicrobial agents. However, conventional antimicrobial agents often show inefficacy in the control of biofilms. Much remains to be understood in the biofilm formation process and in the development of effective strategies for biofilm prevention and control. The articles contained in this Special Issue deal with biofilms of some important bacteria (including pathogens such as Escherichia coli, Pseudomonas aeruginosa and Staphylococcus aureus) and fungi (Candida tropicalis), providing novel insights into their formation mechanisms and implications, together with novel methods (e.g., use of chemical conjugates and combinations of molecules) that can be used to disrupt the biofilm structure and kill the colonizing cells.

Effect of quorum sensing and quenching molecules on inter-kingdom biofilm formation by Penicillium expansum and bacteria.

The ecology of a biofilm is a complex function of different factors, including the presence of microbial metabolites excreted by the inhabitants of the biofilm. This study aimed to assess the effect of patulin, and N-(3-oxododecanoyl)-L-homoserine lactone (3-oxo-C12-HSL) on inter-kingdom biofilm formation between a filamentous fungus and bacteria isolated from drinking water. The filamentous fungus Penicillium expansum and the bacteria Acinetobacter calcoaceticus and Methylobacterium oryzae were used as model species. M. oryzae biofilm formation and development was more susceptible to the presence of the quenching molecules than A. calcoaceticus biofilms. Patulin reduced M. oryzae biofilm growth while 3-oxo-C12-HSL caused an increase after 48 h. The presence of P. expansum had a detrimental effect on M. oryzae cell numbers, while an advantageous effect was observed with A. calcoaceticus. The overall results reveal that quorum sensing and quenching molecules have a significant effect on inter-kingdom biofilm formation, especially on bacterial numbers.

In vitro assessment of inter-kingdom biofilm formation by bacteria and filamentous fungi isolated from a drinking water distribution system.

The main focus so far in the study of biofilm formation in drinking water has been bacteria. Studies on biofilm formation involving filamentous fungi are, therefore, scarce. This study aimed to assess and characterize the ability of these microorganisms to interact with bacteria whilst forming inter-kingdom biofilms. Biofilms were analysed in terms of total biomass, metabolic activity, bacterial colony forming units and morphology by epifluorescence microscopy. The quantitative methods revealed that biofilm mass increased over time for both single and inter-kingdom biofilms, while specific metabolic activity decreased, in general, along the time points evaluated. Microscopic data visually confirmed the biofilm mass increase over time. This study shows that fungal stage development is important in the first 24 h of biofilm formation. Inter-kingdom biofilm formation is microorganism dependent and inter-kingdom biofilms may provide an advantage to the opportunistic bacterium Acinetobacter calcoaceticus to replicate and proliferate when compared with Methylobacterium oryzae.

Standardized reactors for the study of medical biofilms: a review of the principles and latest modifications.

Biofilms can cause severe problems to human health due to the high tolerance to antimicrobials; consequently, biofilm science and technology constitutes an important research field. Growing a relevant biofilm in the laboratory provides insights into the basic understanding of the biofilm life cycle including responses to antibiotic therapies. Therefore, the selection of an appropriate biofilm reactor is a critical decision, necessary to obtain reproducible and reliable in vitro results. A reactor should be chosen based upon the study goals and a balance between the pros and cons associated with its use and operational conditions that are as similar as possible to the clinical setting. However, standardization in biofilm studies is rare. This review will focus on the four reactors (Calgary biofilm device, Center for Disease Control biofilm reactor, drip flow biofilm reactor, and rotating disk reactor) approved by a standard setting organization (ASTM International) for biofilm experiments and how researchers have modified these standardized reactors and associated protocols to improve the study and understanding of medical biofilms.

Combinatorial approaches with selected phytochemicals to increase antibiotic efficacy against Staphylococcus aureus biofilms.

Combinations of selected phytochemicals (reserpine, pyrrolidine, quinine, morin and quercetin) with antibiotics (ciprofloxacin, tetracycline and erythromycin) were tested on the prevention and control of Staphylococcus aureus biofilms. The phytochemicals were also studied for their ability to avoid antibiotic adaptation and to inhibit antibiotic efflux pumps. Morin, pyrrolidine and quercetin at subinhibitory concentrations had significant effects in biofilm prevention and/or control when applied alone and combined with antibiotics. Synergism between antibiotics and phytochemicals was found especially against biofilms of NorA overexpressing strain S. aureus SA1199B. This strain when growing with subinhibitory concentrations of ciprofloxacin developed increased tolerance to this antibiotic. However, this was successfully reversed by quinine and morin. In addition, reserpine and quercetin showed significant efflux pump inhibition. The overall results demonstrate the role of phytochemicals in co-therapies to promote more efficient treatments and decrease antimicrobial resistance to antibiotics, with substantial effects against S. aureus in both planktonic and biofilm states.

Kinetics of biofilm formation by drinking water isolated Penicillium expansum.

Current knowledge on drinking water (DW) biofilms has been obtained mainly from studies on bacterial biofilms. Very few reports on filamentous fungi (ff) biofilms are available, although they can contribute to the reduction in DW quality. This study aimed to assess the dynamics of biofilm formation by Penicillium expansum using microtiter plates under static conditions, mimicking water flow behaviour in stagnant regions of drinking water distribution systems. Biofilms were analysed in terms of biomass (crystal violet staining), metabolic activity (resazurin, fluorescein diacetate and 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide [MTT]) and morphology (epifluorescence [calcofluor white M2R, FUN-1, FDA and acridine orange] and bright-field microscopies). Biofilm development over time showed the typical sigmoidal curve with noticeable different phases in biofilm formation (induction, exponential, stationary, and sloughing off). The methods used to assess metabolic activity provided similar results. The microscope analysis allowed identification of the involvement of conidia in initial adhesion (4 h), germlings (8 h), initial monolayers (12 h), a monolayer of intertwined hyphae (24 h), mycelial development, hyphal layering and bundling, and development of the mature biofilms (≥48 h). P. expansum grows as a complex, multicellular biofilm in 48 h. The metabolic activity and biomass of the fungal biofilms were shown to increase over time and a correlation between metabolism, biofilm mass and hyphal development was found.

What should be considered in the treatment of bacterial infections by multi-drug therapies: a mathematical perspective?

Bacterial infections are a global health concern with high levels of mortality and morbidity associated. The resistance of pathogens to drugs is one leading cause of this problem, being common the administration of multiple drugs to improve the therapeutic effects. This review critically explores diverse aspects involved in the treatment of bacterial infections through multi-drug therapies, from a mathematical and within-host perspectives. Five recent models were selected and are reviewed. These models fall into the following question: which drugs to select, the respective dose, the administration period to effectively eradicate the infection in the shortest period of time and with reduced side effects? In this analysis, three groups of variables were considered: pharmacokinetics, pharmacodynamics and disturbance variables. To date, there is no model that fully answers to this issue for a living organism and it is questionable whether this would be possible for any case of infection.

Antibacterial activity and mode of action of selected glucosinolate hydrolysis products against bacterial pathogens.

Plants contain numerous components that are important sources of new bioactive molecules with antimicrobial properties. Isothiocyanates (ITCs) are plant secondary metabolites found in cruciferous vegetables that are arising as promising antimicrobial agents in food industry. The aim of this study was to assess the antibacterial activity of two isothiocyanates (ITCs), allylisothiocyanate (AITC) and 2-phenylethylisothiocyanate (PEITC) against Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus and Listeria monocytogenes. The antibacterial mode of action was also characterized by the assessment of different physiological indices: membrane integrity, intracellular potassium release, physicochemical surface properties and surface charge. The minimum inhibitory concentration (MIC) of AITC and PEITC was 100 µg/mL for all bacteria. The minimum bactericidal concentration (MBC) of the ITCs was at least 10 times higher than the MIC. Both AITC and PEITC changed the membrane properties of the bacteria decreasing their surface charge and compromising the integrity of the cytoplasmatic membrane with consequent potassium leakage and propidium iodide uptake. The surface hydrophobicity was also non-specifically altered (E. coli and L. monocytogenes become less hydrophilic; P. aeruginosa and S. aureus become more hydrophilic). This study shows that AITC and PEITC have strong antimicrobial potential against the bacteria tested, through the disruption of the bacterial cell membranes. Moreover, phytochemicals are highlighted as a valuable sustainable source of new bioactive products.

A review of research advances on disinfection strategies for biofilm control in drinking water distribution systems.

The presence of biofilms in drinking water distribution systems (DWDS) is responsible for water quality deterioration and a possible source of public health risks. Different factors impact the biological stability of drinking water (DW) in the distribution networks, such as the presence and concentration of nutrients, water temperature, pipe material composition, hydrodynamic conditions, and levels of disinfectant residual. This review aimed to evaluate the current state of knowledge on strategies for DW biofilm disinfection through a qualitative and quantitative analysis of the literature published over the last decade. A systematic review method was performed on the 562 journal articles identified through database searching on Web of Science and Scopus, with 85 studies selected for detailed analysis. A variety of disinfectants were identified for DW biofilm control such as chlorine, chloramine, UV irradiation, hydrogen peroxide, chlorine dioxide, ozone, and others at a lower frequency, namely, electrolyzed water, bacteriophages, silver ions, and nanoparticles. The disinfectants can impact the microbial communities within biofilms, reduce the number of culturable cells and biofilm biomass, as well as interfere with the biofilm matrix components. The maintenance of an effective residual concentration in the water guarantees long-term prevention of biofilm formation and improves the inactivation of detached biofilm-associated opportunistic pathogens. Additionally, strategies based on multi-barrier processes by optimization of primary and secondary disinfection combined with other water treatment methods improve the control of opportunistic pathogens, reduce the chlorine-tolerance of biofilm-embedded cells, as well as decrease the corrosion rate in metal-based pipelines. Most of the studies used benchtop laboratory devices for biofilm research. Even though these devices mimic the conditions found in real DWDS, future investigations on strategies for DW biofilm control should include the validity of the promising strategies against biofilms formed in real DW networks.

Coaggregation dynamics in drinking water biofilms and implications for chlorine disinfection.

Biofilms in drinking water (DW) systems persistently challenge traditional disinfection methods due to intricate microbial interactions, with coaggregation playing a crucial role in forming multispecies biofilms. This study examined the implications of coaggregation on tolerance towards sodium hypochlorite (NaOCl) disinfection. Dual-species biofilms were formed for seven days on polyvinyl chloride coupons, comprising a strain of the emerging pathogen Stenotrophomonas maltophilia and the coaggregating strain Delftia acidovorans 005 P. For comparison, dual-species biofilms were also formed with a non-coaggregation strain (D. acidovorans 009 P). The minimum bactericidal concentration (MBC) for each planktonic strain varied (D. acidovorans: 1 mg/L, S. maltophilia: 1.5 mg/L) below the safe DW treatment limits. However, high NaOCl doses (10 ×MBC and 100 ×MBC,) showed low efficacy against dual-species biofilms, indicating significant biofilm tolerance to disinfection. Membrane damage occurred at sub-MBC without culturability loss, underscoring biofilm resilience. The biofilm analysis revealed a complex interplay between the composition of extracellular polymeric substances and the architecture, which was influenced by the presence of the coaggregating strain. Overall, coaggregation significantly influenced biofilm formation and resilience, impacting NaOCl disinfection. These findings underscore the challenges of microbial interactions in biofilms, emphasizing the need for improved disinfection strategies to control biofilms in drinking water systems.

Elucidating bacterial coaggregation through a physicochemical and imaging surface characterization.

Bacterial coaggregation is a highly specific type of cell-cell interaction, well-documented among oral bacteria, and involves specific characteristics of the cell surface of the coaggregating strains. However, the understanding of the mechanisms promoting coaggregation in aquatic systems remains limited. This gap is critical to address, given the broad implications of coaggregation for multispecies biofilm formation, water quality, the performance of engineered systems, and diverse biotechnological applications. Therefore, this study aims to comprehensively characterize the cell surface of the coaggregating strain Delftia acidovorans 005P, isolated from drinking water, alongside a non-coaggregating strain, D. acidovorans 009P. By analyzing two strains of the same species, we aim to identify the factors contributing to the coaggregation ability of strain 005P. To achieve this, we employed a combination of physicochemical characterization, Fourier-transform infrared spectroscopy (FTIR), and advancing imaging techniques [transmission electron microscopy and cryo-electron tomography (cryo-ET)]. The coaggregating strain (005P) exhibited higher surface hydrophobicity, negative surface charge, and cell surface and co-adhesion energies than the non-coaggregating strain (009P). The chemical characterization of bacterial surfaces through FTIR revealed subtle differences, particularly in spectral regions linked to carbohydrates and phosphodiesters/amide III of proteins (860-930 cm-1 and 1212-1240 cm-1, respectively). Cryo-ET highlighted significant differences in pili structures between the strains, such as variations in length, frequency, and arrangement. The pili in the 005P strain, identified as pili-like adhesins, serve as key mediators of coaggregation. By integrating physicochemical analyses and high-resolution imaging techniques, this study conclusively links the coaggregation ability of D. acidovorans 005P to its unique pili characteristics, emphasizing their crucial role in microbial coaggregation in aquatic environments.

Beyond Penicillin: The Potential of Filamentous Fungi for Drug Discovery in the Age of Antibiotic Resistance.

Antibiotics are a staple in current medicine for the therapy of infectious diseases. However, their extensive use and misuse, combined with the high adaptability of bacteria, has dangerously increased the incidence of multi-drug-resistant (MDR) bacteria. This makes the treatment of infections challenging, especially when MDR bacteria form biofilms. The most recent antibiotics entering the market have very similar modes of action to the existing ones, so bacteria rapidly catch up to those as well. As such, it is very important to adopt effective measures to avoid the development of antibiotic resistance by pathogenic bacteria, but also to perform bioprospecting of new molecules from diverse sources to expand the arsenal of drugs that are available to fight these infectious bacteria. Filamentous fungi have a large and vastly unexplored secondary metabolome and are rich in bioactive molecules that can be potential novel antimicrobial drugs. Their production can be challenging, as the associated biosynthetic pathways may not be active under standard culture conditions. New techniques involving metabolic and genetic engineering can help boost antibiotic production. This study aims to review the bioprospection of fungi to produce new drugs to face the growing problem of MDR bacteria and biofilm-associated infections.

Interactions between Penicillium brevicompactum/Penicillium expansum and Acinetobacter calcoaceticus isolated from drinking water in biofilm development and control.

Bacteria and filamentous fungi (ff) are commonly encountered in biofilms developed in drinking water (DW) distribution systems (DWDS). Despite their intimate ecological relationships, researchers tend to study bacteria and ff separately. This work assesses the impact of bacteria-ff association in biofilm formation and tolerance to chlorination. One strain of Acinetobacter calcoaceticus isolated from DW was used as a model bacterium. Penicillium brevicompactum and P. expansum isolated from DW were the ff selected. Single species and inter-kingdom adhesion and biofilm formation occurred under two shear stress (τ) conditions (0.05 and 1.6 Pa). The sessile structures were further characterized in terms of biomass production, respiratory activity and structure. The results showed that 1.6 Pa of shear stress and A. calcoaceticus-ff association favoured biofilm production. Inter-kingdom biofilms produced more biomass than A. calcoaceticus single species and reduced A. calcoaceticus susceptibility to disinfection, particularly to high sodium hypochlorite (SHC) concentrations. In addition, P. brevicompactum formed single species biofilms highly resistant to removal and inactivation by SHC. The presence of P. brevicompactum or P. expansum in inter-kingdom biofilms significantly decreased SHC removal and inactivation effects in comparison to the bacterial biofilms alone, proposing that using bacteria to form biofilms representative of DWDS can provide inaccurate conclusions, particularly in terms of biofilm production and susceptibility to disinfection.

A procedure to harmonize the hydrodynamic force during microbial cultivation in shaking flasks.

Shake flask cultivation is a routine technique in microbiology and biotechnology laboratories where cell growth can be affected by the hydrodynamic conditions, which depend on the agitation velocity, shaking diameter, and shake flask size. Liquid agitation is implemented inherently to increase aeration, substrate transfer to the cells, and prevent sedimentation, disregarding the role of hydrodynamics in microbial growth and metabolism. Here, we present a simple approach to help standardize the hydrodynamic forces in orbital shakers to increase the experimental accuracy and reproducibility and give students a better knowledge of the significance of the agitation process in microbial growth.

Realism-based assessment of the efficacy of potassium peroxymonosulphate on Stenotrophomonas maltophilia biofilm control.

The potential of pentapotassium bis(peroxymonosulphate) bis(sulphate) (OXONE) to control biofilms in drinking water distribution systems (DWDS) was evaluated and compared to chlorine disinfection. Mature biofilms of drinking water (DW)-isolated Stenotrophomonas maltophilia were formed using a simulated DWDS with a rotating cylinder reactor (RCR). After 30 min of exposure, OXONE at 10 × minimum bactericidal concentration (MBC) caused a significant 4 log reduction of biofilm culturability in comparison to the unexposed biofilms and a decrease in the number of non-damaged cells below the detection limit (4.8 log cells/cm2). The effects of free chlorine were restricted to approximately 1 log reduction in both biofilm culturability and non-damaged cells. OXONE in synthetic tap water (STW) at 25 ºC was more stable over 40 days than free chlorine in the same conditions. OXONE solution exhibited a disinfectant decrease of about 10% of the initial concentration during the first 9 days, and after this time the values remained stable. Whereas possible reaction of chlorine with inorganic and organic substances in STW contributed to free chlorine depletion of approximately 48% of the initial concentration. Electron paramagnetic resonance (EPR) spectroscopy studies confirmed the presence of singlet oxygen and other free radicals during S. maltophilia disinfection with OXONE. Overall, OXONE constitutes a relevant alternative to conventional DW disinfection for effective biofilm control in DWDS.

Bacteria and microalgae associations in periphyton-mechanisms and biotechnological opportunities.

Phototrophic and heterotrophic microorganisms coexist in complex and dynamic structures called periphyton. These structures shape the biogeochemistry and biodiversity of aquatic ecosystems. In particular, microalgae-bacteria interactions are a prominent focus of study by microbial ecologists and can provide biotechnological opportunities for numerous applications (i.e. microalgal bloom control, aquaculture, biorefinery, and wastewater bioremediation). In this review, we analyze the species dynamics (i.e. periphyton formation and factors determining the prevalence of one species over another), coexisting communities, exchange of resources, and communication mechanisms of periphytic microalgae and bacteria. We extend periphyton mathematical modelling as a tool to comprehend complex interactions. This review is expected to boost the applicability of microalgae-bacteria consortia, by drawing out knowledge from natural periphyton.

Drinking-water isolated Delftia acidovorans selectively coaggregates with partner bacteria and facilitates multispecies biofilm development.

Coaggregation plays an important role in the development of multispecies biofilms in different environments, often serving as an active bridge between biofilm members and other organisms that, in their absence, would not integrate the sessile structure. The ability of bacteria to coaggregate has been reported for a limited number of species and strains. In this study, 38 bacterial strains isolated from drinking water (DW) were investigated for their ability to coaggregate, in a total of 115 pairs of combinations. Among these isolates, only Delftia acidovorans (strain 005P) showed coaggregating ability. Coaggregation inhibition studies have shown that the interactions mediating D. acidovorans 005P coaggregation were both polysaccharide-protein and protein-protein, depending on the interacting partner bacteria. Dual-species biofilms of D. acidovorans 005P and other DW bacteria were developed to understand the role of coaggregation on biofilm formation. Biofilm formation by Citrobacter freundii and Pseudomonas putida strains highly benefited from the presence of D. acidovorans 005P, apparently due to the production of extracellular molecules/public goods favouring microbial cooperation. This was the first time that the coaggregation capacity of D. acidovorans was demonstrated, highlighting its role in providing a metabolic opportunity for partner bacteria.

Hydrocinnamic Acid and Perillyl Alcohol Potentiate the Action of Antibiotics against Escherichia coli.

The treatment of bacterial infections has been troubled by the increased resistance to antibiotics, instigating the search for new antimicrobial therapies. Phytochemicals have demonstrated broad-spectrum and effective antibacterial effects as well as antibiotic resistance-modifying activity. In this study, perillyl alcohol and hydrocinnamic acid were characterized for their antimicrobial action against Escherichia coli. Furthermore, dual and triple combinations of these molecules with the antibiotics chloramphenicol and amoxicillin were investigated for the first time. Perillyl alcohol had a minimum inhibitory concentration (MIC) of 256 µg/mL and a minimum bactericidal concentration (MBC) of 512 µg/mL. Hydrocinnamic acid had a MIC of 2048 µg/mL and an MBC > 2048 µg/mL. Checkerboard and time-kill assays demonstrated synergism or additive effects for the dual combinations chloramphenicol/perillyl alcohol, chloramphenicol/hydrocinnamic acid, and amoxicillin/hydrocinnamic acid at low concentrations of both molecules. Combenefit analysis showed synergism for various concentrations of amoxicillin with each phytochemical. Combinations of chloramphenicol with perillyl alcohol and hydrocinnamic acid revealed synergism mainly at low concentrations of antibiotics (up to 2 µg/mL of chloramphenicol with perillyl alcohol; 0.5 µg/mL of chloramphenicol with hydrocinnamic acid). The results highlight the potential of combinatorial therapies for microbial growth control, where phytochemicals can play an important role as potentiators or resistance-modifying agents.