Antibacterial And Biofilm Removal Strategies Based On Micro/nanomotors In The Biomedical Field.

Bacterial infection, which can trigger varieties of diseases and tens of thousands of deaths each year, poses  serious threats to human health. Particularly, the new dilemma caused by biofilms is gradually becoming a severe and tough problem in the biomedical field. Thus, the strategies to address these problems are considered an urgent task at present. Micro/nanomotors (MNMs), also named micro/nanoscale robots, are mostly driven by chemical energy or external field, exhibiting strong diffusion and self-propulsion in the liquid media, which has the potential for antibacterial applications. In particular, when MNMs are assembled in swarms, they become robust and efficient for biofilm removal. However, there is a lack of comprehensive review discussing the progress in this aspect. Bearing it in mind and based on our own research experience in this regard, the studies on MNMs driven by different mechanisms orchestrated for antibacterial activity and biofilm removal are timely and concisely summarized and discussed in this work, aiming to show the advantages of MNMs brought to this field. In addition, an outlook was proposed, hoping to provide the fundamental guidance for future development in this area.

Coptis rhizome extract influence on Streptococcus pneumoniae through autolysin activation.

This study investigated the antibacterial properties of Coptis rhizome, a plant traditionally used for respiratory infections, against Streptoccus pneumonia (S. pneumoniae), for which there has been minimal empirical evidence of effectiveness. The study particularly examined autolysis, indirectly associated with antibacterial resistance, when using Coptis rhizome for bacterial infections. In our methodology, Coptis rhizome was processed with ethanol and distilled water to produce four different extracts: CRET30, CRET50, CRET70, and CRDW. The antibacterial activity of these extracts were tested through Minimum Inhibitory Concentration (MIC) assays, disk diffusion tests, and time-kill assays, targeting both standard (ATCC 49619) and resistant (ATCC 70067) strains. The study also evaluated the extracts' biofilm inhibition properties and monitored the expression of the lyt gene, integral to autolysis. The results prominently showed that the CRET70 extract demonstrated remarkable antibacterial strength. It achieved an MIC of 0.125 µg/mL against both tested S. pneumoniae strains. The disk diffusion assay recorded inhibition zones of 22.17 mm for ATCC 49619 and 17.20 mm for ATCC 70067. Impressively, CRET70 resulted in a 2-log decrease in bacterial numbers for both strains, showcasing its potent bactericidal capacity. The extract was also effective in inhibiting 77.40% of biofilm formation. Additionally, the significant overexpression of the lytA gene in the presence of CRET70 pointed to a potential mechanism of action for its antibacterial effects. The outcomes provided new perspectives on the use of Coptis rhizome in combating S. pneumoniae, especially significant in an era of escalating antibiotic resistance.

Environmental stressor assessment of hydrocarbonoclastic bacteria biofilms from a marine oil spill.

The environmental and economic impact of an oil spill can be significant. Biotechnologies applied during a marine oil spill involve bioaugmentation with immobilised or encapsulated indigenous hydrocarbonoclastic species selected under laboratory conditions to improve degradation rates. The environmental factors that act as stressors and impact the effectiveness of hydrocarbon removal are one of the challenges associated with these applications. Understanding how native microbes react to environmental stresses is necessary for effective bioaugmentation. Herein, Micrococcus luteus and M. yunnanensis isolated from a marine oil spill mooring system showed hydrocarbonoclastic activity on Maya crude oil in a short time by means of total petroleum hydrocarbons (TPH) at 144 h: M. luteus up to 98.79 % and M. yunnanensis 97.77 % removal. The assessment of Micrococcus biofilms at different temperature (30 °C and 50 °C), pH (5, 6, 7, 8, 9), salinity (30, 50, 60, 70, 80 g/L), and crude oil concentration (1, 5, 15, 25, 35 %) showed different response to the stressors depending on the strain. According to response surface analysis, the main effect was temperature > salinity > hydrocarbon concentration. The hydrocarbonoclastic biofilm architecture was characterised using scanning electron microscopy (SEM) and atomic force microscopy (AFM). Subtle but significant differences were observed: pili in M. luteus by SEM and the topographical differences measured by AFM Power Spectral Density (PSD) analysis, roughness was higher in M. luteus than in M. yunnanensis. In all three domains of life, the Universal Stress Protein (Usp) is crucial for stress adaptation. Herein, the uspA gene expression was analysed in Micrococcus biofilm under environmental stressors. The uspA expression increased up to 2.5-fold in M. luteus biofilms at 30 °C, and 1.3-fold at 50 °C. The highest uspA expression was recorded in M. yunnanensis biofilms at 50 °C with 2.5 and 3-fold with salinities of 50, 60, and 80 g/L at hydrocarbon concentrations of 15, 25, and 35 %. M. yunnanensis biofilms showed greater resilience than M. luteus biofilms when exposed to harsh environmental stressors. M. yunnanensis biofilms were thicker than M. luteus biofilms. Both biofilm responses to environmental stressors through uspA gene expression were consistent with the behaviours observed in the response surface analyses. The uspA gene is a suitable biomarker for assessing environmental stressors of potential microorganisms for bioremediation of marine oil spills and for biosensing the ecophysiological status of native microbiota in a marine petroleum environment.

Charge adaptive phytochemical-based nanoparticles for eradication of methicillin-resistant staphylococcus aureus biofilms.

The intrinsic resistance of MRSA coupled with biofilm antibiotic tolerance challenges the antibiotic treatment of MRSA biofilm infections. Phytochemical-based nanoplatform is a promising emerging approach for treatment of biofilm infection. However, their therapeutic efficacy was restricted by the low drug loading capacity and lack of selectivity. Herein, we constructed a surface charge adaptive phytochemical-based nanoparticle with high isoliquiritigenin (ISL) loading content for effective treatment of MRSA biofilm. A dimeric ISL prodrug (ISL-G2) bearing a lipase responsive ester bond was synthesized, and then encapsulated into the amphiphilic quaternized oligochitosan. The obtained ISL-G2 loaded NPs possessed positively charged surface, which allowed cis-aconityl-d-tyrosine (CA-Tyr) binding via electrostatic interaction to obtain ISL-G2@TMDCOS-Tyr NPs. The NPs maintained their negatively charged surface, thus prolonging the blood circulation time. In response to low pH in the biofilms, the fast removal of CA-Tyr led to a shift in their surface charge from negative to positive, which enhanced the accumulation and penetration of NPs in the biofilms. Sequentially, the pH-triggered release of d-tyrosine dispersed the biofilm and lipase-triggered released of ISL effectively kill biofilm MRSA. An in vivo study was performed on a MRSA biofilm infected wound model. This phytochemical-based system led to ∼2 log CFU (>99 %) reduction of biofilm MRSA as compared to untreated wound (P < 0.001) with negligible biotoxicity in mice. This phytochemical dimer nanoplatform shows great potential for long-term treatment of resistant bacterial infections.

Understanding the flow behavior around marine biofilms.

In vitro platforms capable of mimicking the hydrodynamic conditions prevailing in natural aquatic environments have been previously validated and used to predict the fouling behavior on different surfaces. Computational Fluid Dynamics (CFD) has been used to predict the shear forces occurring in these platforms. In general, these predictions are made for the initial stages of biofilm formation, where the amount of biofilm does not affect the flow behavior, enabling the estimation of the shear forces that initial adhering organisms have to withstand. In this work, we go a step further in understanding the flow behavior when a mature biofilm is present in such platforms to better understand the shear rate distribution affecting marine biofilms. Using 3D images obtained by Optical Coherence Tomography, a mesh was produced and used in CFD simulations. Biofilms of two different marine cyanobacteria were developed in agitated microtiter plates incubated at two different shaking frequencies for 7 weeks. The biofilm-flow interactions were characterized in terms of the velocity field and shear rate distribution. Results show that global hydrodynamics imposed by the different shaking frequencies affect biofilm architecture and also that this architecture affects local hydrodynamics, causing a large heterogeneity in the shear rate field. Biofilm cells located in the streamers of the biofilm are subjected to much higher shear values than those located on the bottom of the streamers and this dispersion in shear rate values increases at lower bulk fluid velocities. This heterogeneity in the shear force field may be a contributing factor for the heterogeneous behavior in metabolic activity, growth status, gene expression pattern, and antibiotic resistance often associated with nutrient availability within the biofilm.

Subminimum Inhibitory Concentrations Tetracycline Antibiotics Induce Biofilm Formation in Minocycline-Resistant Klebsiella pneumonia by Affecting Bacterial Physical and Chemical Properties and Associated Genes Expression.

Biofilm formation of Klebsiella pneumoniae can protect bacteria from antibiotics and is difficult to eradicate. Thus, the influence of subinhibitory concentrations of antibiotics on bacteria is becoming increasingly important. Our study showed that subminimum inhibitory concentrations (sub-MICs) of tetracycline antibiotics can increase biofilm formation in minocycline-resistant Klebsiella pneumoniae clinical strains. However, in the bacterial adhesion and invasion experiments, the adhesion and invasion ability decreased and the survival rate of Galleria mellonella increased. Under sub-MICs of tetracycline antibiotics treatment, abnormal stretching of bacteria was observed by scanning electron microscopy. Treatment with sub-MICs of tetracyclines leads to increased surface hydrophobicity and eDNA content and decreased outer membrane permeability. The expression levels of the fimA, luxS, qseB, and qseC genes decreased, the expression level of mrkA increased, and the expression level of acrA was inconsistent under different tetracycline antibiotics treatments. Together, our results suggested that the increase in Klebsiella pneumoniae biofilm formation caused by sub-MICs of tetracycline antibiotics may occur by affecting bacterial physical and chemical properties and associated genes expression.

How well do antimicrobial mouth rinses prevent dysbiosis in an in vitro periodontitis biofilm model?

BACKGROUND: Periodontal diseases are associated with dysbiosis in the oral microbial communities. Managing oral biofilms is therefore key for preventing these diseases. Management protocols often include over-the-counter antimicrobial mouth rinses, which lack data on their effects on the oral microbiome's ecology, bacterial composition, metabolic activity, and dysbiosis resilience. This study examined the efficacy of antimicrobial mouth rinses to halt dysbiosis in in vitro oral biofilms under periodontitis-simulating conditions. METHODS: Multispecies oral biofilms were grown on hydroxyapatite discs (HADs) and rinsed daily with one of six mouth rinses. Positive and negative controls were included. After three rinses, biofilms were analyzed with viability quantitative polymerase chain reaction and visualized using scanning electron microscopy. Supernatants of rinsed biofilms were used for metabolic activity analysis. In addition, human oral keratinocytes were exposed to rinsed biofilms to assess their inflammatory response. All outputs were analyzed for correlation using Spearman coefficient. RESULTS: Product-related changes were observed in the rinsed biofilms. Three of the six tested mouth rinses could significantly prevent dysbiosis with ≥30% reduction in pathobiont abundance relative to the control. These biofilms had lower metabolic activity, and the exposed human oral keratinocyte produced less interleukin-8. Interleukin-8 production correlated to both pathobiont quantity and the metabolic activity of the biofilms. CONCLUSION: Some mouth rinses could support biofilm resilience and stop dysbiosis evolution in the biofilm model, with a clear product-related effect. Such mouth rinses can be considered for patients under maintenance/supportive periodontal therapy to prevent/delay disease recurrence. Others are more useful for different periodontal therapy stages.

Systematic analysis of the Candida albicans kinome reveals environmentally contingent protein kinase-mediated regulation of filamentation and biofilm formation in vitro and in vivo.

Protein kinases are critical regulatory proteins in both prokaryotes and eukaryotes. Accordingly, protein kinases represent a common drug target for a wide range of human diseases. Therefore, understanding protein kinase function in human pathogens such as the fungus Candida albicans is likely to extend our knowledge of its pathobiology and identify new potential therapies. To facilitate the study of C. albicans protein kinases, we constructed a library of 99 non-essential protein kinase homozygous deletion mutants marked with barcodes in the widely used SN genetic background. Here, we describe the construction of this library and the characterization of the competitive fitness of the protein kinase mutants under 11 different growth and stress conditions. We also screened the library for protein kinase mutants with altered filamentation and biofilm formation, two critical virulence traits of C. albicans. An extensive network of protein kinases governs these virulence traits in a manner highly dependent on the specific environmental conditions. Studies on specific protein kinases revealed that (i) the cell wall integrity MAPK pathway plays a condition-dependent role in filament initiation and elongation; (ii) the hyper-osmolar glycerol MAPK pathway is required for both filamentation and biofilm formation, particularly in the setting of in vivo catheter infection; and (iii) Sok1 is dispensable for filamentation in hypoxic environments at the basal level of a biofilm but is required for filamentation in normoxia. In addition to providing a new genetic resource for the community, these observations emphasize the environmentally contingent function of C. albicans protein kinases.IMPORTANCECandida albicans is one of the most common causes of fungal disease in humans for which new therapies are needed. Protein kinases are key regulatory proteins and are increasingly targeted by drugs for the treatment of a wide range of diseases. Understanding protein kinase function in C. albicans pathogenesis may facilitate the development of new antifungal drugs. Here, we describe a new library of 99 protein kinase deletion mutants to facilitate the study of protein kinases. Furthermore, we show that the function of protein kinases in two virulence-related processes, filamentation and biofilm formation, is dependent on the specific environmental conditions.

The uncharacterized PA3040-3042 operon is part of the cell envelope stress response and a tobramycin resistance determinant in a clinical isolate of Pseudomonas aeruginosa.

Bacteriophages (hereafter "phages") are ubiquitous predators of bacteria in the natural world, but interest is growing in their development into antibacterial therapy as complement or replacement for antibiotics. However, bacteria have evolved a huge variety of antiphage defense systems allowing them to resist phage lysis to a greater or lesser extent. In addition to dedicated phage defense systems, some aspects of the general stress response also impact phage susceptibility, but the details of this are not well known. In order to elucidate these factors in the opportunistic pathogen Pseudomonas aeruginosa, we used the laboratory-conditioned strain PAO1 as host for phage infection experiments as it is naturally poor in dedicated phage defense systems. Screening by transposon insertion sequencing indicated that the uncharacterized operon PA3040-PA3042 was potentially associated with resistance to lytic phages. However, we found that its primary role appeared to be in regulating biofilm formation, particularly in a clinical isolate of P. aeruginosa in which it also altered tobramycin resistance. Its expression was highly growth-phase dependent and responsive to phage infection and cell envelope stress. Our results suggest that this operon may be a cryptic but important locus for P. aeruginosa stress tolerance. IMPORTANCE: An important category of bacterial stress response systems is bacteriophage defense, where systems are triggered by bacteriophage infection and activate a response which may either destroy the phage genome or destroy the infected cell so that the rest of the population survives. In some bacteria, the cell envelope stress response is activated by bacteriophage infection, but it is unknown whether this contributes to the survival of the infection. We have found that a conserved uncharacterized operon (PA3040-PA3042) of the cell envelope stress regulon in Pseudomonas aeruginosa, which has very few dedicated phage defense systems, responds to phage infection and stationary phase as well as envelope stress and is important for growth and biofilm formation in a clinical isolate of P. aeruginosa, even in the absence of phages. As homologs of these genes are found in other bacteria, they may be a novel component of the general stress response.

Three-Dimensional Rotary Culture to Model Mycobacterial Biofilms in Low-Shear Detergent-Free Liquid Suspension.

In vitro biofilm models have allowed researchers to investigate the role biofilms play in the pathogenesis, virulence, and antimicrobial drug susceptibility of a wide range of bacterial pathogens. Rotary cell culture systems create three-dimensional cellular structures, primarily applied to eukaryotic organoids, that better capture characteristics of the cells in vivo. Here, we describe how to apply a low-shear, detergent-free rotary cell culture system to generate biofilms of Mycobacterium bovis BCG. The three-dimensional biofilm model forms mycobacterial cell aggregates in suspension as surface-detached biomass, without severe nutrient starvation or environmental stress, that can be harvested for downstream experiments. Mycobacterium bovis BCG derived from cell clusters display antimicrobial drug tolerance, presence of an extracellular matrix, and evidence of cell wall remodeling, all features of biofilm-associated bacteria that may be relevant to the treatment of tuberculosis.

Biofilm-producing ability of methicillin-resistant Staphylococcus aureus clinically isolated in China.

BACKGROUND: Staphylococcus aureus, a commensal bacterium, colonizes the skin and mucous membranes of approximately 30% of the human population. Apart from conventional resistance mechanisms, one of the pathogenic features of S. aureus is its ability to survive in a biofilm state on both biotic and abiotic surfaces. Due to this characteristic, S. aureus is a major cause of human infections, with Methicillin-Resistant Staphylococcus aureus (MRSA) being a significant contributor to both community-acquired and hospital-acquired infections. RESULTS: Analyzing non-repetitive clinical isolates of MRSA collected from seven provinces and cities in China between 2014 and 2020, it was observed that 53.2% of the MRSA isolates exhibited varying degrees of ability to produce biofilm. The biofilm positivity rate was notably high in MRSA isolates from Guangdong, Jiangxi, and Hubei. The predominant MRSA strains collected in this study were of sequence types ST59, ST5, and ST239, with the biofilm-producing capability mainly distributed among moderate and weak biofilm producers within these ST types. Notably, certain sequence types, such as ST88, exhibited a high prevalence of strong biofilm-producing strains. The study found that SCCmec IV was the predominant type among biofilm-positive MRSA, followed by SCCmec II. Comparing strains with weak and strong biofilm production capabilities, the positive rates of the sdrD and sdrE were higher in strong biofilm producers. The genetic determinants ebp, icaA, icaB, icaC, icaD, icaR, and sdrE were associated with strong biofilm production in MRSA. Additionally, biofilm-negative MRSA isolates showed higher sensitivity rates to cefalotin (94.8%), daptomycin (94.5%), mupirocin (86.5%), teicoplanin (94.5%), fusidic acid (81.0%), and dalbavancin (94.5%) compared to biofilm-positive MRSA isolates. The biofilm positivity rate was consistently above 50% in all collected specimen types. CONCLUSIONS: MRSA strains with biofilm production capability warrant increased vigilance.

Initial therapeutic evidence of a borosilicate bioactive glass (BSG) and Fe3O4 magnetic nanoparticle scaffold on implant-associated Staphylococcal aureus bone infection.

Implant-associated Staphylococcus aureus (S. aureus) osteomyelitis is a severe challenge in orthopedics. While antibiotic-loaded bone cement is a standardized therapeutic approach for S. aureus osteomyelitis, it falls short in eradicating Staphylococcus abscess communities (SACs) and bacteria within osteocyte-lacuna canalicular network (OLCN) and repairing bone defects. To address limitations, we developed a borosilicate bioactive glass (BSG) combined with ferroferric oxide (Fe3O4) magnetic scaffold to enhance antibacterial efficacy and bone repair capabilities. We conducted comprehensive assessments of the osteoinductive, immunomodulatory, antibacterial properties, and thermal response of this scaffold, with or without an alternating magnetic field (AMF). Utilizing a well-established implant-related S. aureus tibial infection rabbit model, we evaluated its antibacterial performance in vivo. RNA transcriptome sequencing demonstrated that BSG + 5%Fe3O4 enhanced the immune response to bacteria and promoted osteogenic differentiation and mineralization of MSCs. Notably, BSG + 5%Fe3O4 upregulated gene expression of NOD-like receptor and TNF pathway in MSCs, alongside increased the expression of osteogenic factors (RUNX2, ALP and OCN) in vitro. Flow cytometry on macrophage exhibited a polarization effect towards M2, accompanied by upregulation of anti-inflammatory genes (TGF-ß1 and IL-1Ra) and downregulation of pro-inflammatory genes (IL-6 and IL-1ß) among macrophages. In vivo CT imaging revealed the absence of osteolysis and periosteal response in rabbits treated with BSG + 5%Fe3O4 + AMF at 42 days. Histological analysis indicated complete controls of SACs and bacteria within OLCN by day 42, along with new bone formation, signifying effective control of S. aureus osteomyelitis. Further investigations will focus on the in vivo biosafety and biological mechanism of this scaffold within infectious microenvironment.

Complete photodynamic inactivation of Pseudomonas aeruginosa biofilm with use of potassium iodide and its comparison with enzymatic pretreatment.

Pseudomonas aeruginosa, a gram-negative bacterium, accounts for 7% of all hospital-acquired infections. Despite advances in medicine and antibiotic therapy, P. aeruginosa infection still results in high mortality rates of up to 62% in certain patient groups. This bacteria is also known to form biofilms, that are 10 to 1000 times more resistant to antibiotics compared to their free-floating counterparts. Photodynamic Inactivation (PDI) has been proved to be an effective antimicrobial technique for microbial control. This method involves the incubation of the pathogen with a photosensitizer (PS), then, a light at appropriated wavelength is applied, leading to the production of reactive oxygen species that are toxic to the microbial cells. Studies have focused on strategies to enhance the PDI efficacy, such as a pre-treatment with enzymes to degrade the biofilm matrix and/or an addition of inorganic salts to the PS. The aim of the present study is to evaluate the effectiveness of PDI against P. aeruginosa biofilm in association with the application of the enzymes prior to PDI (enzymatic pre-treatment) or the addition of potassium iodide (KI) to the photosensitizer solution, to increase the inactivation effectiveness of the treatment. First, a range of enzymes and PSs were tested, and the best protocols for combined treatments were selected. The results showed that the use of enzymes as a pre-treatment was effective to reduce the total biomass, however, when associated with PDI, mild bacterial reductions were obtained. Then, the use of KI in association with the PS was evaluated and the results showed that, PDI mediated by methylene blue (MB) in the presence of KI was able to completely eradicate the biofilm. However, when the PDI was performed with curcumin and KI, no additive reduction was observed. In conclusion, out of all strategies evaluated in the present study, the most promising strategy to improve PDI against P. aeruginosa biofilm was the use of KI in association with MB, resulting in eradication with 108 log bacterial inactivation.

The plastisphere can protect Salmonella Typhimurium from UV stress under simulated environmental conditions.

Plastic waste is found with increasing frequency in the environment, in low- and middle-income countries. Plastic pollution has increased concurrently with both economic development and rapid urbanisation, amplifying the effects of inadequate waste management. Distinct microbial communities can quickly colonise plastic surfaces in what is collectively known as the 'plastisphere'. The plastisphere can act as a reservoir for human pathogenic bacteria, including Salmonella enterica sp. (such as S. Typhimurium), which can persist for long periods, retain pathogenicity, and pose an increased public health risk. Through employing a novel mesocosm setup, we have shown here that the plastisphere provides enhanced protection against environmental pressures such as ultraviolet (UV) radiation and allows S. Typhimurium to persist at concentrations (>1x103 CFU/ml) capable of causing human infection, for up to 28 days. Additionally, using a Galleria Mellonella model of infection, S. Typhimurium exhibits greater pathogenicity following recovery from the UV-exposed plastisphere, suggesting that the plastisphere may select for more virulent variants. This study demonstrates the protection afforded by the plastisphere and provides further evidence of environmental plastic waste acting as a reservoir for dangerous clinical pathogens. Quantifying the role of plastic pollution in facilitating the survival, persistence, and dissemination of human pathogens is critical for a more holistic understanding of the potential public health risks associated with plastic waste.

High-throughput combination assay for studying biofilm formation of uropathogenic Escherichia coli.

Uropathogenic Escherichia coli, the most common cause for urinary tract infections, forms biofilm enhancing its antibiotic resistance. To assess the effects of compounds on biofilm formation of uropathogenic Escherichia coli UMN026 strain, a high-throughput combination assay using resazurin followed by crystal violet staining was optimized for 384-well microplate. Optimized assay parameters included, for example, resazurin and crystal violet concentrations, and incubation time for readouts. For the assay validation, quality parameters Z' factor, coefficient of variation, signal-to-noise, and signal-to-background were calculated. Microplate uniformity, signal variability, edge well effects, and fold shift were also assessed. Finally, a screening with known antibacterial compounds was conducted to evaluate the assay performance. The best conditions found were achieved by using 12 µg/mL resazurin for 150 min and 0.023% crystal violet. This assay was able to detect compounds displaying antibiofilm activity against UMN026 strain at sub-inhibitory concentrations, in terms of metabolic activity and/or biomass.

Simultaneous Biogas Upgrading and Valuable Chemical Production Using Homoacetogens in a Membrane Biofilm Reactor.

Biogas produced from anaerobic digestion usually contains impurities, particularly with a high content of CO2 (15-60%), thus decreasing its caloric value and limiting its application as an energy source. H2-driven biogas upgrading using homoacetogens is a promising approach for upgrading biogas to biomethane and converting CO2 to acetate simultaneously. Herein, we developed a novel membrane biofilm reactor (MBfR) with H2 and biogas separately supplied via bubbleless hollow fiber membranes. The gas-permeable hollow fibers of the MBfR enabled high H2 and CO2 utilization efficiencies (∼98% and ∼97%, respectively) and achieved concurrent biomethane (∼94%) and acetate (∼450 mg/L/d) production. High-throughput 16S rRNA gene amplicon sequencing suggested that enriched microbial communities were dominated by Acetobacterium (38-48% relative abundance). In addition, reverse transcription quantitative PCR of the functional marker gene formyltetrahydrofolate synthetase showed that its expression level increased with increasing H2 and CO2 utilization efficiencies. These results indicate that Acetobacterium plays a key role in CO2 to acetate conversion. These findings are expected to facilitate energy-positive wastewater treatment and contribute to the development of a new solution to biogas upgrading.

Mechanisms of microbial co-aggregation in mixed anaerobic cultures.

Co-aggregation of anaerobic microorganisms into suspended microbial biofilms (aggregates) serves ecological and biotechnological functions. Tightly packed aggregates of metabolically interdependent bacteria and archaea play key roles in cycling of carbon and nitrogen. Additionally, in biotechnological applications, such as wastewater treatment, microbial aggregates provide a complete metabolic network to convert complex organic material. Currently, experimental data explaining the mechanisms behind microbial co-aggregation in anoxic environments is scarce and scattered across the literature. To what extent does this process resemble co-aggregation in aerobic environments? Does the limited availability of terminal electron acceptors drive mutualistic microbial relationships, contrary to the commensal relationships observed in oxygen-rich environments? And do co-aggregating bacteria and archaea, which depend on each other to harvest the bare minimum Gibbs energy from energy-poor substrates, use similar cellular mechanisms as those used by pathogenic bacteria that form biofilms? Here, we provide an overview of the current understanding of why and how mixed anaerobic microbial communities co-aggregate and discuss potential future scientific advancements that could improve the study of anaerobic suspended aggregates. KEY POINTS: • Metabolic dependency promotes aggregation of anaerobic bacteria and archaea • Flagella, pili, and adhesins play a role in the formation of anaerobic aggregates • Cyclic di-GMP/AMP signaling may trigger the polysaccharides production in anaerobes.

Investigating the Effect of Melittin Peptide in Preventing Biofilm Formation, Adhesion and Expression of Virulence Genes in Listeria monocytogenes.

Listeria monocytogenes is a notable food-borne pathogen that has the ability to create biofilms on different food processing surfaces, making it more resilient to disinfectants and posing a greater risk to human health. This study assessed melittin peptide's anti-biofilm and anti-pathogenicity effects on L. monocytogenes ATCC 19115. Melittin showed minimum inhibitory concenteration (MIC) of 100 µg/mL against this strain and scanning electron microscopy images confirmed its antimicrobial efficacy. The OD measurement demonstrated that melittin exhibited a strong proficiency in inhibiting biofilms and disrupting pre-formed biofilms at concentrations ranging from 1/8MIC to 2MIC and this amount was 92.59 ± 1.01% to 7.17 ± 0.31% and 100% to 11.50 ± 0.53%, respectively. Peptide also reduced hydrophobicity and self-aggregation of L. monocytogenes by 35.25% and 14.38% at MIC. Melittin also significantly reduced adhesion to HT-29 and Caco-2 cells by 61.33% and 59%, and inhibited invasion of HT-29 and Caco-2 cells by 49.33% and 40.66% for L. monocytogenes at the MIC value. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) revealed melittin's impact on gene expression, notably decreasing inlB (44%) and agrA (45%) gene expression in L. monocytogenes. flaA and hly genes also exhibited reduced expression. Also, significant changes were observed in sigB and prfA gene expression. These results underscore melittin's potential in combating bacterial infections and biofilm-related challenges in the food industry.

Effects of two fillers and process conditions on the water treatment efficiency of a continuous packed bed biofilm reactor.

This study evaluated the treatment efficiency of two selected fillers and their combination for improving the water quality of aquaculture wastewater using a packed bed biofilm reactor (PBBR) under various process conditions. The fillers used were nanosheet (NS), activated carbon (AC), and a combination of both. The results indicated that the use of combined fillers and the hydraulic retention time (HRT) of 4 h significantly enhanced water quality in the PBBR. The removal rates of chemical oxygen demand, NO2-─N, total suspended solids（TSS）, and chlorophyll a were 63.55%, 74.25%, 62.75%, and 92.85%, respectively. The microbiota analysis revealed that the presence of NS increased the abundance of microbial phyla associated with nitrogen removal, such as Nitrospirae and Proteobacteria. The difference between the M1 and M2 communities was minimal. Additionally, the microbiota in different PBBR samples displayed similar preferences for carbon sources, and carbohydrates and amino acids were the most commonly utilized carbon sources by microbiota. These results indicated that the combination of NS and AC fillers in a PBBR effectively enhanced the treatment efficiency of aquaculture wastewater when operated at an HRT of 4 h. The findings provide valuable insights into optimizing the design of aquaculture wastewater treatment systems.

Optimizing chitosan derived from Metapenaeus affinis: a novel anti-biofilm agent against Pseudomonas aeruginosa.

Pseudomonas aeruginosa is a commonly found Gram-negative bacterium in healthcare facilities and is renowned for its ability to form biofilms and its virulence factors that are controlled by quorum sensing (QS) systems. The increasing prevalence of multidrug-resistant strains of this bacterium poses a significant challenge in the field of medicine. Consequently, the exploration of novel antimicrobial agents has become a top priority. This research aims to optimize chitosan derived from white shrimp (Metapenaeus affinis) using the Response Surface Methodology (RSM) computational approach. The objective is to investigate chitosan's potential as a solution for inhibiting QS activity and biofilm formation in P. aeruginosa ATCC 10,145. Under optimized conditions, chitin was treated with NaOH (1.41 M) for 15.75 h, HCl (7.49% vol) for 2.01 h, and at a deacetylation temperature of 81.15 °C. The resulting chitosan exhibited a degree of deacetylation (DD%) exceeding 93.98%, as confirmed by Fourier-transform infrared (FTIR) spectral analysis, indicating its high purity. The extracted chitosan demonstrated a significant synergistic antibiotic effect against P. aeruginosa when combined with ceftazidime, enhancing its bactericidal activity by up to 15-fold. In addition, sub-MIC (minimum inhibitory concentration) concentrations of extracted chitosan (10 and 100 µg/mL) successfully reduced the production of pyocyanin and rhamnolipid, as well as the swimming motility, protease activity and biofilm formation ability in comparison to the control group (P < 0.05). Moreover, chitosan treatment downregulated the RhlR and LasR genes in P. aeruginosa when compared to the control group (P < 0.05). The optimized chitosan extract shows significant potential as a coating agent for surgical equipment, effectively preventing nosocomial infections caused by P. aeruginosa pathogens.