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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Microalgal-based removal of contaminants of emerging concern.

The presence of contaminants of emerging concern (CECs) in the environment has been recognized as a worldwide concern. In particular, water pollution by CECs is becoming a major global problem, which requires ongoing evaluation of water resources policies at all levels and the use of effective and innovative wastewaters treatment processes for their removal. Microalgae have been increasingly recognized as relevant for wastewater polishing, including CECs removal. These microorganisms are commonly cultivated in suspension. However, the use of planktonic microalgae for wastewater treatment has limitations in terms of microbiological contamination, process effectiveness and sustainability. The use of consortia of microalgae and bacteria represents a significant advance for sustainable wastewater polishing, particularly when the microorganisms are associated as biofilms. These immobilized mixed cultures can overcome the limitations of suspended-microalgae systems and improve the performance of the involved species for CECs removal. In addition, microalgae-bacteria based systems can offer a relevant combined effect for CECs removal and biomass production enhancement. This study reviews the advantages and advances on the use of microalgae for wastewater treatment, highlighting the potential on the use of microalgae-bacteria biofilms for CECs removal and the further biomass valorisation for third-generation biofuel production.

Chlorinated cyanurates and potassium salt of peroxymonosulphate as antimicrobial and antibiofilm agents for drinking water disinfection.

The understanding of microbial susceptibility to disinfectants is an important step to provide drinking water (DW) of adequate microbiological quality. In drinking water distribution systems (DWDS) the application of disinfectants is the main approach to control microorganisms. Although chlorine has been commonly used for DW treatment, the increase of microbial resistance and the production of harmful disinfection by-products promote the necessity to seek new alternatives. This study evaluated the antimicrobial activity of sodium dichloroisocyanurate (NaDCC), trichloroisocyanuric acid (TCCA), and pentapotassium bis(peroxymonosulphate) bis(sulphate) (OXONE) against two emerging pathogens isolated from DW, Acinetobacter calcoaceticus and Stenotrophomonas maltophilia. Free chlorine from calcium hypochlorite was used for comparison. The dose and time-responses against planktonic bacteria were performed as well as the assessment of the effects on membrane integrity. Moreover, the effects against 48 h-old biofilms formed on polyvinyl chloride and stainless steel were evaluated in terms of biofilm culturability and removal. Minimum bactericidal concentrations of 2.1 and 3.1 mg/L for NaDCC, 2.5 and 3.8 mg/L for TCCA, 340 and 690 mg/L for OXONE, and 0.80 and 1.0 mg/L for free chlorine alone were obtained against S. maltophilia and A. calcoaceticus, respectively. The kinetic modeling revealed that NaDCC and TCCA caused similar inactivation rates and the time for first log reduction by OXONE was less than 10 min, for both bacteria. All the disinfectants triggered significant bacterial cytoplasmic membrane destabilization, even at sub-lethal concentrations. A 30 min treatment with the disinfectants allowed a reduction in the biofilm culturability up to 5 log. OXONE was the disinfectant with the best efficiency against both bacterial biofilms. However, none of the disinfectants caused significant biofilm removal (reduction < 1 log cells/cm2). This study highlights NaDCC, TCCA, and OXONE as promising alternatives to free chlorine for DW disinfection, particularly for planktonic growth control and biofilm culturability reduction.

The Effects of Chemical and Mechanical Stresses on Bacillus cereus and Pseudomonas fluorescens Single- and Dual-Species Biofilm Removal.

Biofilm control is mainly based on chemical disinfection, without a clear understanding of the role of the biocides and process conditions on biofilm removal. This study aims to understand the effects of a biocide (benzyldimethyldodecyl ammonium chloride-BDMDAC) and mechanical treatment (an increase of shear stress -τw) on single- and dual-species biofilms formed by Bacillus cereus and Pseudomonas fluorescens on high-density polyethene (HDPE). BDMDAC effects were initially assessed on bacterial physicochemical properties and initial adhesion ability. Then, mature biofilms were formed on a rotating cylinder reactor (RCR) for 7 days to assess the effects of chemical and mechanical treatments, and the combination of both on biofilm removal. The results demonstrated that the initial adhesion does not predict the formation of mature biofilms. It was observed that the dual-species biofilms were the most susceptible to BDMDAC exposure. The exposure to increasing τw emphasised the mechanical stability of biofilms, as lower values of τw (1.66 Pa) caused high biofilm erosion and higher τw values (17.7 Pa) seem to compress the remaining biofilm. In general, the combination of BDMDAC and the mechanical treatment was synergic in increasing biofilm removal. However, these were insufficient to cause total biofilm removal (100%; an average standard deviation of 11% for the method accuracy should be considered) from HDPE.

Bacterial coaggregation in aquatic systems.

The establishment of a sessile community is believed to occur in a sequence of steps where genetically distinct bacteria can become attached to partner cells via specific molecules, in a process known as coaggregation. The presence of bacteria with the ability to autoaggregate and coaggregate has been described for diverse aquatic systems, particularly freshwater, drinking water, wastewater, and marine water. In these aquatic systems, coaggregation already demonstrated a role in the development of complex multispecies sessile communities, including biofilms. While specific molecular aspects on coaggregation in aquatic systems remain to be understood, clear evidence exist on the impact of this mechanism in multispecies biofilm resilience and homeostasis. The identification of bridging bacteria among coaggregating consortia has potential to improve the performance of wastewater treatment plants and/or to contribute for the development of strategies to control undesirable biofilms. This study provides a comprehensive analysis on the occurrence and role of bacterial coaggregation in diverse aquatic systems. The potential of this mechanism in water-related biotechnology is further described, with particular emphasis on the role of bridging bacteria.

LegionellaDB - A Database on Legionella Outbreaks.

LegionellaDB is the first database on Legionella outbreaks; it is based on a metadata analysis of peer-reviewed manuscripts from PubMed and SCOPUS. LegionellaDB is dynamic and extensible, allowing users to search for specific outbreaks, suggest additional information to be included after curation, visualize statistical representations on specific outbreaks, and download selected data. The database is maintained online.

Biofilm control by ionic liquids.

Ionic liquids (ILs) are remarkable chemical compounds with applications in many areas of modern science. They are increasingly recognized as promising compounds to fight microorganisms in both planktonic and biofilm states, contributing to reinvent the antimicrobial pipeline. Biofilm-related infections are particularly challenging given that the scientific community has not yet identified a reliable control strategy. Understanding of the action of ILs in biofilm control is is still in a very early stage. However, given the highly tunable nature and exceptional properties of ILs, they are excellent candidates for biofilm control. Here, we review the major advances in, and challenges tothe use of ILs for effective biofilm control.

Occurrence of filamentous fungi in drinking water: their role on fungal-bacterial biofilm formation.

Water is indispensable to life and safe and accessible supply must be available to all. The presence of microorganisms is a threat to this commitment. Biofilms are the main reservoir of microorganisms inside water distribution systems and they are extremely ecologically diverse. Filamentous fungi and bacteria can coexist inside these systems forming inter-kingdom biofilms. This review has the goal of summarizing the most relevant and recent reports on the occurrence of filamentous fungi in water distribution systems along with the current knowledge and gaps about filamentous fungal biofilm formation. Special focus is given on fungal-bacterial interactions in water biofilms.