Secondary Biofilm Research Articles

Metainterface Heterostructure Enhances Sonodynamic Therapy for Disrupting Secondary Biofilms.

Implant-related secondary infections are a challenging clinical problem. Sonodynamic therapy (SDT) strategies are promising for secondary biofilm infections by nonsurgical therapy. However, the inefficiency of SDT in existing acoustic sensitization systems limits its application. Therefore, we take inspiration from popular metamaterials and propose the design idea of a metainterface heterostructure to improve SDT efficiency. The metainterfacial heterostructure is defined as a periodic arrangement of heterointerface monoclonal cells that amplify the intrinsic properties of the heterointerface. Herein, we develop a TiO2/Ti2O3/vertical graphene metainterface heterostructure film on titanium implants. This metainterface heterostructure exhibits extraordinary sonodynamic and acoustic-to-thermal conversion effects under low-intensity ultrasound. The modulation mechanisms of the metainterface for electron accumulation and separation are revealed. The synergistic sonodynamic/mild sonothermal therapy disrupts biofilm infections (antibacterial rates: 99.99% for Staphylococcus aureus, 99.54% for Escherichia coli), and the osseointegration ability of implants is significantly improved in in vivo tests. Such a metainterface heterostructure film lays the foundation for the metainterface of manipulating electron transport to enhance the catalytic performance and holding promise for addressing secondary biofilm infections.

A Preliminary Evaluation on the Antifungal Efficacy of VT-1161 against Persister Candida albicans Cells in Vulvovaginal Candidiasis.

Persister cells are a small fraction of the microbial population that survive lethal concentrations of antimicrobial agents. Candida albicans causes vaginal candidiasis, including recurrent vulvovaginal candidiasis, and may survive common antifungal treatments. The triazole VT-1161 is an antifungal agent that specifically targets fungal CYP51, as opposed to the human CYP enzyme. This work illustrates a new role of VT-1161 in eradicating the biofilm created from the persister cells of a primary biofilm of a clinical vaginal isolate of C. albicans. Antifungal activity was determined by the minimum inhibitory concentration (MIC), and the primary biofilm was treated with amphotericin B to obtain persister cells that were able to form a new biofilm. Results obtained using the new azole VT-1161 showed that VT-1161 not only eradicated a secondary biofilm formed from the persister-derived biofilm and counteracted the adhesion of C. albicans in vitro to human cells but also ameliorated C. albicans-induced infection in vivo in Galleria mellonella larvae, suggesting that it could be proposed as an alternative therapeutic strategy for the treatment of recurrent candidiasis.

Fungal symbionts impact cyanobacterial biofilm durability and photosynthetic efficiency

Cyanobacteria contribute to over 25% of the world's net primary photosynthetic production and are pivotal in mitigating greenhouse gas emissions.1 This study unveils a previously unobserved symbiotic relationship between benthic cyanobacteria and fungi that have also adapted to life as a plant endophyte. The interaction suggests an initial phase of lichenization. We isolated Leptolyngbya frigida from the Naracauli stream, which emanates from abandoned Zn industrial waste in Sardinia. Seasonally, L.frigida participates in a biomineralization processes, mitigating the Zn transfer to rivers and, subsequently, the sea.2,3,4L.frigida is a benthic cyanobacterium that establishes a biofilm on the stream bed. Notably, the area predominantly features Juncus acutus. From these roots, endophytic fungi were predominantly isolated as Clonostachys rosea, a fungus recognized for its biocontrol capabilities against plant pathogens. An intriguing observation was made when L.frigida was cultured with C.rosea on a low-carbohydrate agar medium: the fungal mycelium transformed into wall-less forms, a phenomenon not documented previously. In liquid environments, the resulting biofilm first settled at the container's bottom. Even upon rising to the surface, this biofilm remained pigment rich. Concurrently, a secondary biofilm began its formation at the bottom. These fungal-integrated biofilms displayed enhanced resilience and superior photosynthetic performance compared to those without fungal presence. Moreover, the symbiotic relationship significantly amplified O2 emission and CO2 sequestration by the biofilm.

Small Molecule Inhibition of an Exopolysaccharide Modification Enzyme is a Viable Strategy To Block Pseudomonas aeruginosa Pel Biofilm Formation.

Biosynthesis of the Pel exopolysaccharide in Pseudomonas aeruginosa requires all seven genes of the pelABCDEFG operon. The periplasmic modification enzyme PelA contains a C-terminal deacetylase domain that is necessary for Pel-dependent biofilm formation. Herein, we show that extracellular Pel is not produced by a P. aeruginosa PelA deacetylase mutant. This positions PelA deacetylase activity as an attractive target to prevent Pel-dependent biofilm formation. Using a high-throughput screen (n = 69,360), we identified 56 compounds that potentially inhibit PelA esterase activity, the first enzymatic step in the deacetylase reaction. A secondary biofilm inhibition assay identified methyl 2-(2-pyridinylmethylene) hydrazinecarbodithioate (SK-017154-O) as a specific Pel-dependent biofilm inhibitor. Structure-activity relationship studies identified the thiocarbazate as a necessary functional group and that the pyridyl ring could be replaced with a phenyl substituent (compound 1). Both SK-017154-O and compound 1 inhibit Pel-dependent biofilm formation in Bacillus cereus ATCC 10987, which has a predicted extracellular PelA deacetylase in its pel operon. Michaelis-Menten kinetics determined SK-017154-O to be a noncompetitive inhibitor of PelA, while compound 1 did not directly inhibit PelA esterase activity. Cytotoxicity assays using human lung fibroblast cells showed that compound 1 is less cytotoxic than SK-017154-O. This work provides proof of concept that biofilm exopolysaccharide modification enzymes are important for biofilm formation and can serve as useful antibiofilm targets. IMPORTANCE Present in more than 500 diverse Gram-negative and 900 Gram-positive organisms, the Pel polysaccharide is one of the most phylogenetically widespread biofilm matrix determinants found to date. Partial de-N-acetylation of this α-1,4 linked N-acetylgalactosamine polymer by the carbohydrate modification enzyme PelA is required for Pel-dependent biofilm formation in Pseudomonas aeruginosa and Bacillus cereus. Given this and our observation that extracellular Pel is not produced by a P. aeruginosa PelA deactylase mutant, we developed an enzyme-based high-throughput screen and identified methyl 2-(2-pyridinylmethylene) hydrazinecarbodithioate (SK-017154-O) and its phenyl derivative as specific Pel-dependent biofilm inhibitors. Michaelis-Menten kinetics revealed SK-017154-O is a noncompetitive inhibitor and that its noncytotoxic, phenyl derivative does not directly inhibit P. aeruginosa PelA esterase activity. We provide proof of concept that exopolysaccharide modification enzymes can be targeted with small molecule inhibitors to block Pel-dependent biofilm development in both Gram-negative and Gram-positive bacteria.

Concepts of species and strain in relation to microorganisms under formation of biofilms as superorganismic systems

Nowadays, many biological terms receive new interpretations, especially the concept of species. The species is the main structural unit of living things. It emerges, develops, and, when living conditions change either disappear or transforms into other species. There is no clear and unified definition of species. The achievements of contemporary molecular genetic studies indicate that the majority of microorganisms exist mainly not in a free-floating condition, but in formed associations or consortia. Microbial interactions occur by transferring molecular and genetic information, and various mechanisms such as secondary metabolites, siderophores, quorum-sensing system, biofilm formation, and cell transduction signals can be involved in this exchange. Therefore, for a deeper understanding of the concept of "species" in biology, it is necessary to take into account not only morphological and physiological criteria, but also to consider species from the viewpoint of systems biology, and to bear in mind factors of horizontal gene transfer. Therefore, the concept of "species" can be considered in a broader context, in particular within ecosystems with all assimilation relations.

The taxonomic complex Centaurea stoebe s. l. (Asteraceae) in the flora of Ukraine

Nowadays, many biological terms receive new interpretations, especially the concept of species. The species is the main structural unit of living things. It emerges, develops, and, when living conditions change either disappear or transforms into other species. There is no clear and unified definition of species. The achievements of contemporary molecular genetic studies indicate that the majority of microorganisms exist mainly not in a free-floating condition, but in formed associations or consortia. Microbial interactions occur by transferring molecular and genetic information, and various mechanisms such as secondary metabolites, siderophores, quorum-sensing system, biofilm formation, and cell transduction signals can be involved in this exchange. Therefore, for a deeper understanding of the concept of "species" in biology, it is necessary to take into account not only morphological and physiological criteria, but also to consider species from the viewpoint of systems biology, and to bear in mind factors of horizontal gene transfer. Therefore, the concept of "species" can be considered in a broader context, in particular within ecosystems with all assimilation relations.

STAGES OF BIOFILMS FORMATION BY THE LEADING PATHOGENS IN CHILDREN WITH PYELONEPHRITIS ON CONGENITAL HYDRONEPHROSIS BACKGROUND DEPENDING ON CHILD’S AGE

The aim: to study the stages of primary and secondary biofilms formation by the leading pathogens in children with pyelonephritis on congenital hydronephrosis background depending on child’s age.
 Materials and methods. Venflons, catheters, urine were used as material for microbiological study. Identification of microorganisms was provided with MICRO-LA-TESTÒID kits. Isolates were tested for ability to form biofilms in Petri dishes with d=40 mm. The morphological structure of the biofilms was studied by scanning electron microscopy.
 Results: The study of structural and functional features of biofilms formation by leading pathogens of in children with pyelonephritis on congenital hydronephrosis background depending on child’s age revealed a number of features and patterns. In addition to the classical stages of biofilms formation as 3D structure there was found a dissemination of planktonic cells with the release of bacteria or loss of single fragments that spread throughout the body and attach to the substrate with the formation of a new or secondary biofilm. In children under 3 years it was shown that the cocci attachment to the substrate appeared faster than in gram-negative rods and had appearance of separate structures. The longest stage of primary biofilms formation in young children was the co-aggregation. Detecting an ability to colonize with the formation of a secondary biofilm in isolates established that the longest stage was re-adsorption and the shortest was re-aggregation, which lasted 2 hours in all detected pathogens. In middle-aged children, the duration of adhesion stage was reduced by 1-2 hours compared with it in young children.
 Conclusions. Scientific data about the stages of biofilms formation by microorganisms, causative agents of pyelonephritis in children was updated. Adhesion stage of isolates from elder children with pyelonephritis on background of congenital hydronephrosis underwent faster in the formation of secondary biofilms than in primary, and it formed the possibility for chronic process and the development of recurrences. The duration of each stage in biofilms formation by causative agents of pyelonephritis in children with congenital hydronephrosis depended on the age of the child and the properties of microorganisms

‘Secondary biofilms’ could cause failure of peracetic acid high-level disinfection of endoscopes

The reduced susceptibility of biofilms to disinfectants presents a challenge to the successful reprocessing of medical equipment. This study examined the effect of residual biomass remaining after previous disinfection with peracetic acid (PAA) on the tolerance of subsequent mature Pseudomonas aeruginosa biofilms to PAA. The effect of enzymatic degradation of specific components of the extracellular polymeric substance (EPS) of P. aeruginosa biofilm on the effectiveness of PAA disinfection was also evaluated. The susceptibility of biofilm grown on the biomass of PAA-killed biofilm to PAA was compared with the PAA susceptibility of biofilm grown in wells of a 24-well plate by evaluating their viability using the plate count assay. The effect of PAA on biofilm biomass was measured using crystal violet quantification of total biofilm biomass, while its effect on the polysaccharide and protein components of biofilm EPS was quantified using the phenol-sulphuric acid assay or Bradford assay, respectively. A confocal microscope was used to visualize the distribution of living and dead cells in biofilms grown on residual biofilm biomass. The presence of residual biomass from previously disinfected biofilms significantly enhanced the tolerance of subsequent biofilms. A 96-h-old 'secondary biofilm' formed on disinfected biomass survived PAA concentrations of 4000 ppm, which exceeds the concentrations used in practice for high-level disinfection. These observations indicate that, under certain circumstances, recolonization of residual EPS can cause failure of disinfection of medical equipment such as endoscopes, and emphasizes the importance of cleaning endoscopes prior to disinfection.

Influence of probiotics on biofilm formation and diversity of bacteria colonising crop sorghum ensiled with unsalable vegetables

The objective of this study was to characterise in situ digestion kinetics and bacterial colonisation of crop sorghum ensiled with unsalable carrot or pumpkin at 0, 20 or 40% dry matter (DM). Silages with or without the application of a commercial probiotic were incubated in situ for 0, 3, 6, 9, 24 and 48h. Calculation of in situ digestion kinetics was conducted for DM, organic matter and neutral detergent fibre (aNDF). The V4 region of the 16S rRNA gene was sequenced to determine the composition and diversity of bacteria colonising the silage. Organic matter and DM digestion kinetics indicated that greater vegetable inclusion increased (P < 0.05) the soluble fraction and effective degradability. Bacterial richness at 48h incubation was greater (P = 0.02) in 20% carrot and 40% pumpkin treatments, compared with the control. An effect of level × probiotic was observed with increased Shannon diversity (P = 0.01) for 40% carrot and 20% pumpkin probiotic treatments, respectively. Primary colonising bacteria were members of the Prevotella genus, dominating after 3 and 6h of incubation. The abundance of Prevotella increased by 4.1% at 3h (P < 0.01) and by 4.7% at 9h incubation with probiotics, compared with the control. Secondary biofilm colonisers included members of Treponema, Saccharofermentans, Fibrobacter, Ruminobacter and Anaerosporobacter genera, dominant from 9 h incubation onward. This study demonstrated that including unsalable vegetables at 20 or 40% DM increases the soluble fraction and effective degradability of sorghum silage during in situ digestion and increases diversity of bacteria colonising ensiled vegetables within the rumen. KEY POINTS: • Ensiling unsalable vegetables is a viable strategy to reduce food waste. • Ensiled vegetables increased in situ soluble fraction and effective degradability. • Bacterial richness at 48 h incubation improved with 20% carrot or 40% pumpkin. • Diversity of colonising rumen bacteria increased with carrot or pumpkin inclusion.

High Exoelectrogenic Biofilms Formation in Microbial Fuel Cells

Background. Microbial fuel cells are devices in which electricity is generated by microorganisms called exoelectrogens. During the process of anaerobic respiration exoelectrogens emit electrons outside the cell. These electrons can be transferred to the anode of biofuel cell via several different mechanisms. Electricity generation in microbial fuel cells depends primarily on the electrochemical activity of the exoelectrogens present in the anode space. Nowadays, the usage of microorganisms, immobilized as biofilms on the anode, is constantly increasing. Natural sources for exoelectrogens selection such as activated sludge, biofilter biofilms, sediments of seas and rivers have a very diverse microbial composition. Therefore, it is important to immobilize relatively deficient in natural sources exoelectrogens on the anode during the biofilm formation process. The main research areas are the development of a technique for obtaining of electroactive biofilms enriched with exoelectrogens along with reduction of the period of biofilm formation process. Objective . We set a goal to study the process of high exoelectrogenic biofilm formation basing on the combination of different methods of exoelectrogens isolation and immobilization at the anode of a microbial fuel cell. Methods . A three-stage technique was used to obtain a highly exoelectrogenic biofilm which, due to the combination of typical isolation and immobilization techniques of exoelectrogens, allows obtaining the biofilm in which the vast majority of microorganisms are exoelectrogens. In the first stage, a biofilter biofilm was used as a source of exoelectrogens. The biofilm formed in the first stage was used as an inoculum for the second stage of biofilm formation. During the second stage an additional selective factor (applied additional potential in the electrical circuit of the microbial fuel cell) was used. The third stage of biofilm formation was the isolation of exoelectrogens capable of reducing ferum (III) compounds from secondary biofilm with subsequent application of these cells as inoculum. Results. The usage of the proposed method allows obtaining of a biofilm enriched with exoelectrogenic bacteria. The maximum current density generated by the biofilm, obtained during the first stage, reaches 140 μA/cm 2 , during the second – 400 μA/cm 2 , during the third – 615 μA/cm 2 . The duration of biofilm formation at each stage was 110 h, 40 h, and 60 h, respectively. Conclusions. It has been proven that the duration of biofilm formation is reduced almost twice as a result of a combination of typical methods of isolation and immobilization of exoelectrogens; obtained biofilm has high electrochemical activity and properties similar to biofilm, formed by pure cultures of exoelectrogens.

Draft Genome Sequence of Bacillus velezensis Lzh-a42, a Plant Growth-Promoting Rhizobacterium Isolated from Tomato Rhizosphere.

ABSTRACTThe plant growth-promoting rhizobacterium Bacillus velezensis strain Lzh-a42, which has antimicrobial activity, was isolated from tomato rhizosphere. Here, we report its genome sequence, which includes several predicted functional genes related to secondary metabolite biosynthesis, antimicrobial activity, and biofilm synthesis.

Effect of organic loading rate on the removal of DMF, MC and IPA by a pilot-scale AnMBR for treating chemical synthesis-based antibiotic solvent wastewater

This study focuses on the effects of organic loading rate (OLR) on the removal of N,N-Dimethylformamide(DMF), m-Cresol (MC) and isopropyl alcohol (IPA) by a pilot-scale anaerobic membrane bioreactor (AnMBR) for treating chemical synthesis-based antibiotic solvent wastewater at period of improved influent COD concentration with decreased HRT. The whole process was divided into five stages in terms of the variation of OLR ranging from 3.9 to 12.7 kg COD/(m3·d). During 249 days of operating time, the average DMF, MC, IPA removal efficiency were 96.9%,98.2% and 96.4%, respectively. Cake layer was accumulated on the membrane surface acted as a dynamic secondary biofilm which lead to the increase of physical removal rate. In addition, mathematical statistical models was built on the linear regression techniques for exploring the inner relationship between EPS and the performance of the AnMBR.

The Influence of Biofilm Treatment Systems on Particle Size Distribution in Three Wastewater Treatment Plants

This article presents the results of a research study that analyzed the effect that secondary biofilm treatment systems had on particle size distribution (PSD) in the effluent of wastewater treatment plants. This study focused on three biofilm technologies (i.e. a submerged biofilter system, a trickling filter system, and a rotating biological contactor system) in three working urban wastewater treatment plants. For this purpose, the variation of the fit parameters was analyzed after modeling the PSD with the power law. The greatest reduction in particle number was obtained with the submerged biofilter system, followed by the trickling filter system. In contrast, the rotating biological contactor showed the smallest reduction in particle number under the conditions of our study. It was also found that the variation of the fit parameters of the PSD, caused by the wastewater treatments was related to other wastewater parameters, such as the chemical oxygen demand, suspended solids, and the mean particle size. This showed a direct relation between these parameters and the particles in wastewater.

Improvement of the anodic bioelectrocatalytic activity of mixed culture biofilms by a simple consecutive electrochemical selection procedure

In this paper we demonstrate that the anodic, bioelectrocatalytic performance of wastewater inoculum based, mixed culture microbial biofilms can be considerably improved by using a consecutive, purely electrochemical selection and biofilm acclimatization procedure. The procedure may represent an alternative to a repetitive mechanical biofilm removal, re-suspension and electrochemically facilitated biofilm formation. By using the proposed technique, the bioelectrocatalytic current density was increased from the primary to the secondary biofilm from 250 μA cm −2 to about 500 μA cm −2; and the power density of respective microbial fuel cells could be increased from 686 mW m −2 to 1487 mW m −2. The electrochemical characterization of the biofilms reveals a strong similarity to Geobacter sulfurreducens biofilms, which may indicate a dominating role of this bacterium in the biofilms.

Inactivation of Salmonella spp. from secondary and tertiary effluents by UV irradiation

The aim of this study was to verify the efficiency of UV irradiation in the inactivation of Salmonella spp. in treated wastewater with different levels of turbidity and exposed to increasing doses of UV irradiation. Experiments were carried out in a batch reactor and in a real scale reactor. Salmonellae obtained from clinical samples were seeded into autoclaved wastewater collected from a wastewater treatment plant (WWTP) comprising an association of a UASB reactor followed by three submerged aerated biofilters (BAF) and one tertiary filter. The results showed that salmonellae were not inactivated in effluents from the UASB reactor indicating that the presence of suspended solids was an important obstacle to UV penetration in bacteria. However, UV irradiation was efficient in inactivating Salmonella of effluents from aerated secondary and tertiary biofilm reactors.

Apparent surface associated lag time in growth of primary biofilm cells.

The ability of microorganisms to form biofilms has been well documented. Bacterial cells make a transition from a planktonic state to a sessile state, replicate, and subsequently populate a surface. In this study, organisms that initially colonize a ``clean'' surface are referred to as ``primary'' biofilm cells. The progeny of the first generation of sessile cells are known as ``secondary'' biofilm cells. This study examined the growth of planktonic, primary, and secondary biofilm cells of a green fluorescent protein producing (GFP+) Pseudomonas aeruginosa PA01. Biofilm experiments were performed in a parallel plate flow cell reactor with a glass substratum. Individual cells were tracked over time using a confocal scanning laser microscope (CSLM). Primary cells experience a lag in their growth that may be attributed to adapting to a sessile environment or undergoing a phenotypic change. This is referred to as a surface associated lag time. Planktonic and secondary biofilm cells both grew at a faster rate than the primary biofilm cells under the same nutrient conditions.