Biofilm Structure Research Articles

Inhibitory Effect of DNase–Chitosan–Nisin Nanoparticles on Cell Viability, Motility, and Spatial Structures of Listeria monocytogenes Biofilms

Listeria monocytogenes biofilm contamination on food contact surfaces is a major concern for the food industry. Nanoparticle encapsulation appears as a novel strategy for food surface disinfection to prevent biofilm formation. Chitosan nanoparticles loaded with nisin and DNase I (DNase-CS-N) have been constructed to exhibit antimicrobial activity against L. monocytogenes. This study aimed to investigate their ability to inhibit L. monocytogenes biofilm formation and eliminate preformed biofilms on food contact surfaces (polystyrene, polyurethane, and stainless steel). DNase-CS-N could decrease 99% and 99.5% biofilm cell numbers at 1/2 MIC and MIC, respectively. At sub-MICs, DNase-CS-N could reduce cell motility (swimming and swarming) and slime production of L. monocytogenes. In terms of effect on biofilm elimination, DNase-CS-N at the concentration of 4 MIC led to 3–4 log reduction in biofilm cells in preformed biofilms, performing higher efficiency compared with other treatments (CSNPs, CS-N). Furthermore, the three-dimensional structure of L. monocytogenes biofilms was severely disrupted after DNase-CS-N treatment, with bacterial cells scattered on the surface. The morphology of biofilm cells was also greatly damaged with wrinkled surfaces, disrupted cell membranes, and leakage of intracellular nucleic acids and proteins. These results indicate the potential applicability of DNase-CS-N for inhibiting and eliminating L. monocytogenes biofilms on food contact surfaces.

Quantifying the fractal complexity of nutrient transport channels in Escherichia coli biofilms under varying cell shape and growth environment.

Recent mesoscopic characterization of nutrient-transporting channels in Escherichia coli has allowed the identification and measurement of individual channels in whole mature colony biofilms. However, their complexity under different physiological and environmental conditions remains unknown. Analysis of confocal micrographs of colony biofilms formed by cell shape mutants of E. coli shows that channels have high fractal complexity, regardless of cell phenotype or growth medium. In particular, colony biofilms formed by the mutant strain ΔompR, which has a wide-cell phenotype, have a higher fractal dimension when grown on rich medium than when grown on minimal medium, with channel complexity affected by glucose and agar concentrations in the medium. Osmotic stress leads to a dramatic reduction in the ΔompR cell size but has a limited effect on channel morphology. This work shows that fractal image analysis is a powerful tool to quantify the effect of phenotypic mutations and growth environment on the morphological complexity of internal E. coli biofilm structures. If applied to a wider range of mutant strains, this approach could help elucidate the genetic determinants of channel formation in E. coli colony biofilms.

Evaluation of a Burkholderia ambifaria strain from plants as a novel promising probiotic in dental caries management

ABSTRACT Background Probiotics serve as a novel preventive or therapeutic approach for dental caries owing to their ability to reverse dysbiosis and restore a healthy microbiota. Here, we identified Burkholderia ambifaria AFS098024 as a probiotic candidate isolated from plants. Methods The safety of B. ambifaria was evaluated by hemolytic activity, D-lactic acid production and antibiotic susceptibility. In vitro biofilm model derived from the saliva of caries-free and caries-active donors and in vivo rat caries model were used to assess the efficacy of B. ambifaria in caries prevention and treatment. Results B. ambifaria was safe as a probiotic candidate and it could integrate with in vitro biofilm model. It significantly reduced the biomass and lactate production of biofilms from caries-active donors and disrupted biofilm structures. B. ambifaria effectively reduced the severity of carious lesions in rat molars, regardless of the inoculation sequence. Molars pretreated or treated with B. ambifaria demonstrated notably higher enamel volumes. Additionally, colonization of rat molars by B. ambifaria persisted for 6 weeks. Conclusion The B. ambifaria strain used in this study holds promise as a probiotic for inhibiting dental caries, both in vitro and in vivo.

Laser NIR Irradiation Enhances Antimicrobial Photodynamic Inactivation of Biofilms of Staphylococcus aureus.

Photodynamic inactivation (PDI) is a powerful technique for eradicating microorganisms, and our group previously demonstrated its effectiveness against planktonic cultures of Staphylococcus aureus bacteria using 5,10,15,20-tetrakis[4-(3-N,N-dimethylaminopropoxy)phenyl]porphyrin (TAPP) and visible light irradiation. However, biofilms exhibit a lower sensitivity to PDI, mainly due to limited penetration of the photosensitizer (PS). In the context of emerging antibacterial strategies, near-infrared treatments (NIRTs) have shown promise, especially for combating resistant strains. NIRT can act either through photon absorption by water, causing a thermal effect on bacteria, or by specific chromophores without a significant temperature increase. Our objective was to enhance biofilm sensitivity to TAPP-PDI by pretreatment with NIRT. This combined approach aims to disrupt biofilms and increase the efficacy of TAPP-PDI against bacterial biofilms. In vitro biofilm models of S. aureus RN6390 were utilized. NIRTs involved a 980 nm laser (continuous mode, 7.5 W/cm2, 30 s, totaling 225 J/cm2) post-TAPP exposure to enhance photosensitizer accumulation. Subsequent visible light irradiation at 180 J/cm2 was employed to perform PDI. Colony-forming unit counts evaluated the synergistic effect on bacterial viability. Scanning electron microscopy visualized the architectural changes in the biofilm structure. TAPP was extracted from bacteria to estimate the impact of NIRT on biofilm penetration. Using in vitro biofilm models, NIRT application following biofilm exposure to TAPP increased PS accumulation per bacteria. Under these conditions, NIRT induced a transient increase in the temperature of PBS to 46.0 ± 2.6°C (ΔT = 21.5°C). Following exposure to visible light, a synergistic effect emerged, yielding a substantial 4.4 ± 0.1-log CFU reduction. In contrast, the PDI and NIRT treatments individually caused a decrease in viability of 0.9 ± 0.1 and 0.8 ± 0.2-log respectively. Interestingly, preheating TAPP-PBS to 46°C had no significant impact on TAPP-PDI efficacy, suggesting the involvement of thermal and nonthermal effects of NIR action. In addition to the enhanced TAPP penetration, NIRT dispersed the biofilms and induced clefts in the biofilm matrix. Our findings suggest that NIR irradiation serves as a complementary treatment to PDI. This combined strategy reduces bacterial numbers at lower PS concentrations than standalone PDI treatment, highlighting its potential as an effective and resource-efficient antibacterial approach.

Fe (II)/Fe (III) regulated adaptive biofilm responses and microbial metabolic mechanisms for enhanced cycloalkane biodegradation

Oxidation of organic contaminants by iron-cycling microorganisms is an effective remediation strategy for contaminated environments. However, studies on the removal of cycloalkane using this method have been limited, and little is known about how microbes adapt to iron concentrations and how the latter stimulates contaminant degradation. Here, we investigated the biodegradation of cyclohexane (CyH) in three sequencing batch bioreactors (SBRs) with different iron concentrations (SBRs are named: Low 2.5 μM, Suit 7.5 μM, High 75 μM). The Suit reactor showed a more efficient CyH removal performance (average 98.61 ± 0.62 %) than the Low reactor (average 55.75 ± 3.38 %), which was associated with more extracellular polymeric substances (EPS) release and improved biofilm structure. The High reactor exhibited a gradual adaptation process due to the apparent accumulation of reactive oxygen species (ROS) under iron stress, with an average removal efficiency of (86.83 ± 18.81 %). However, the High reactor had a higher microbial activity (1.08-fold increase in ATPase activity) and electron transport system activity (1.18-fold increase) to facilitate direct electron transfer in the presence of insoluble iron compared to the Suit one, largely due to the dominant genus Novosphingobium. Metagenomics and metatranscriptomics analyses revealed lactone (a key intermediate) formation as a potential degradation pathway across three SBRs in our study under iron regulation. Notably, there were differences in the key functional genes expressed under different iron concentrations: genes related to anti-oxidative stress and iron-driven pathways, biofilm regulation-related pathways, and encoding protein complexes III and IV were found in the High, Suit, and Low reactors, respectively. This study enhances understanding of microbiota adaptation mechanisms to iron-disturbed conditions and provides a new insight into iron-cycling mediated biofilm formation and inhibition.

Bacterial communities forming yellow biofilms in different cave types share a common core

The walls of different types of caves under diverse geological settings (limestone, gypsum and volcanic) are colonized by biofilms of different colors: white, yellow, pink, grey, green to dark brown, but only a few colored biofilms such as the white, yellow and grey ones have been extensively studied. However, an assessment among the microbial communities originating these biofilms in different lithologies is lacking. Here we compare the yellow biofilms from two caves, Covadura and C3, in the Gypsum Karst of Sorbas in Spain, with those from two Spanish limestone caves (Pindal and Santian), and four volcanic caves in Spain and Italy (Viento, Honda del Bejenado, Grotta del Santo, Grotta di Monte Corruccio). The structure of yellow biofilms in gypsum caves closely resembles that found in other Spanish and European limestone caves. However, volcanic cave biofilms exhibit greater variability in their microbial community structure and morphologies. Biofilms from gypsum, limestone and volcanic caves were characterized by the abundance of the genera Crossiella and the gammaproteobacterial wb1-P19. The uncultured Euzebyaceae were abundant in gypsum and Spanish volcanic caves, while in the limestone and Italian volcanic caves, they were rare or absent. Nitrospira was also abundant in limestone and volcanic caves, but not in gypsum caves. Due to the abundances of Crossiella, gammaproteobacterial wb1-P19, and uncultured Euzebyaceae, in many different ecosystems, not only in caves, as recently reported, understanding the functional diversity in which these lineages are involved seems critical. Although we have studied a limited number of yellow biofilms from caves in Spain and Italy, data from other caves in USA and Russia also point out the existence of a similarity among the most abundant members composing the structure of yellow biofilms, suggesting that they share a common core.

Significance of in-vivo biofilm in non-tuberculous mycobacterial diseases

There’s been a rise of pulmonary non-tuberculous mycobacterial (NTM) disease in developed countries. Treatment for this is limited and existing treatments have an extensive duration. The disease has a long latent period and often experiences recurrence after treatment. In their work, Senior Research Scientist Dr Kentaro Yamamoto and his team based at the Leprosy Research Centre within the Department of Mycobacteriology, National Institute of Infectious Diseases (NIID) are building on the recent discovery that NTMs form biofilms in infected tissues. There is potential that biofilms formed in infected tissues could shield the bacilli from drug exposure, protect them against the host’s immune system attacks, or offer a refuge for the bacilli within the human body. Therefore the researchers believe that the removal of biofilms formed by NTMs or M. tuberculosis in the body could represent a novel treatment approach. By developing a device that can easily detect mycobacterial biofilms within the body, it would be possible to non-invasively assess a patient’s condition, including the stage of infection. Much remains to be discovered about the structure and function of mycobacterial biofilms in vivo and so the researchers want to clarify the relationship between biofilm formation in the lungs and key factors such as pathogenicity, bacterial growth, drug resistance, host immune response and the phenomena of latency and recurrence. If the team can make these clarifications, the research will benefit patients who are allergic to antimicrobials and those whose quality of life is reduced by the long-term treatment of NTM disease.

Effect of Acetic Acid on Biofilm Formation in Paracidovorax citrulli, Causal Agent of Bacterial Fruit Blotch.

The unique tissue structure of pathogenic bacteria biofilm plays an important role in its pathogenicity and bactericide resistance. Inhibition or destruction of biofilm formation of pathogenic bacteria is of great significance for the control of plant bacterial diseases. In this study, Paracidovorax citrulli was inoculated into KB medium containing acetic acid, and after shaking at 28°C and 55 r/min for 48 h, it was found that the content of extracellular polysaccharide, extracellular protein and extracellular DNA (eDNA) decreased with the increase of acetic acid concentration, which resulted in the decrease of biofilm formation, it is not even possible to form biofilms on plastic slides. When the final concentration of acetic acid in the culture medium was greater than or equal to 0.5 mg/mL, there was no biofilm on the plastic slides. Therefore, the use of acetic acid as an inhibitor of P. citrulli has a good potential for control of bacterial fruit blotch.

Role of the SaeRS Two-Component Regulatory System in Group B Streptococcus Biofilm Formation on Human Fibrinogen.

Streptococcus agalactiae, also known as Group B Streptococcus or GBS, is a commensal colonizer of human vaginal and gastrointestinal tracts that can also be a deadly pathogen for newborns, pregnant women, and the elderly. The SaeRS two-component regulatory system (TCS) positively regulates the expression of two GBS adhesins genes, but its role in the formation of biofilm, an important step in pathogenesis, has not been investigated. In the present study, we set up a novel model of GBS biofilm formation using surfaces coated with human fibrinogen (hFg). Biofilm mass and structure were analyzed by crystal violet staining and three-dimensional fluorescence microscopy, respectively. GBS growth on hFg resulted in the formation of a mature and abundant biofilm composed of bacterial cells and an extracellular matrix containing polysaccharides, proteins, and extracellular DNA (eDNA). Enzymatic and genetic analysis showed that GBS biofilm formation on hFg is dependent on proteins and eDNA in the extracellular matrix and on the presence of covalently linked cell wall proteins on the bacterial surface but not on the type-specific capsular polysaccharide. In the absence of the SaeR regulator of the SaeRS TCS, there was a significant reduction in biomass formation, with reduced numbers of bacterial cells, reduced eDNA content, and disruption of the biofilm architecture. Overall, our data suggest that GBS binding to hFg contributes to biofilm formation and that the SaeRS TCS plays an important role in this process.

Soft X-ray spectromicroscopic proof of a reversible oxidation/reduction of microbial biofilm structures using a novel microfluidic in situ electrochemical device

In situ electrochemistry on micron and submicron-sized individual particles and thin layers is a valuable, emerging tool for process understanding and optimization in a variety of scientific and technological fields such as material science, process technology, analytical chemistry, and environmental sciences. Electrochemical characterization and manipulation coupled with soft X-ray spectromicroscopy helps identify, quantify, and optimize processes in complex systems such as those with high heterogeneity in the spatial and/or temporal domain. Here we present a novel platform optimized for in situ electrochemistry with variable liquid electrolyte flow in soft X-ray scanning transmission X-ray microscopes (STXM). With four channels for fluid control and a modular design, it is suited for a wealth of experimental conditions. We demonstrate its capabilities by proving the reversible oxidation and reduction of individual microbial biofilm structures formed by microaerophilic Fe(II)-oxidizing bacteria, also known as twisted stalks. We show spectromicroscopically the heterogeneity of the redox activity on the submicron scale. Examples are also provided of electrochemical modification of liquid electrolyte species (Fe(II) and Fe(III) cyanides), and in situ studies of electrodeposited copper nanoparticles as CO2 reduction electrocatalysts under reaction conditions.

Deciphering stratified structure and microbiota assembly of biofilms from a pyrite-based biofilter driven by mixotrophic denitrification

The precise structure and assembly process of pyrite-based biofilms remain poorly understood. The polysaccharides (PN), proteins (PS), and extracellular DNA were enriched in the soluble extracellular polymeric substance (EPS), loosely bound EPS, and tightly bound EPS, respectively, indicating a significant stratified structure of biofilms. The tryptophan facilitated mixotrophic metabolic processes. Both dominant (>1%) and rare species (<0.01 %) harbored core bacteria, including sulfur autotrophic bacteria, sulfate-reducing bacteria, and heterotrophic bacteria. Furthermore, partial least-squares path modeling quantified the contributions of total phosphorus (TP) (λ = 0.32), dissolved organic matter (DOC) (λ = 0.29), and NH4+–N (λ = 0.26) to variations in the microbial community. Nonmetric multidimensional scaling analysis revealed three distinct stages in biofilm development: colonization (0–36 d), succession (36–149 d), and maturation/old (149–215 d). Furthermore, neutral community model indicated that stochastic processes drove the colonization and maturation/old stages, while deterministic processes dominated the succession stage. This study offered valuable insights into the regulation of pyrite-based engineered ecosystems.

Metabolic interactions shape emergent biofilm structures in a conceptual model of gut mucosal bacterial communities

The gut microbiome plays a major role in human health; however, little is known about the structural arrangement of microbes and factors governing their distribution. In this work, we present an in silico agent-based model (ABM) to conceptually simulate the dynamics of gut mucosal bacterial communities. We explored how various types of metabolic interactions, including competition, neutralism, commensalism, and mutualism, affect community structure, through nutrient consumption and metabolite exchange. Results showed that, across scenarios with different initial species abundances, cross-feeding promotes species coexistence. Morphologically, competition and neutralism resulted in segregation, while mutualism and commensalism fostered high intermixing. In addition, cooperative relations resulted in community properties with little sensitivity to the selective uptake of metabolites produced by the host. Moreover, metabolic interactions strongly influenced colonization success following the invasion of newcomer species. These results provide important insights into the utility of ABM in deciphering complex microbiome patterns.

Rumex japonicus Houtt. Leaves: the chemical composition and anti-fungal activity

BackgroundCandida albicans is a pathogenic commensal fungus. Trichophyton mentagrophytes and Trichophyton rubrum are the leading pathogens of dermatophysis. Rumex japonicus Houtt. has a miraculous effect on the treatment of tinea skin disease, but its mechanism has not been clarified. Purpose: This paper investigated the anti-fungal ingredients of the leaves of Rumex japonicus Houtt. (RJH-L) and the mechanism of the anti-fungal (Trichophyton mentagrophytes, Trichophyton rubrum and Candida albicans). MethodFirst, the chemical composition analysis of RJH-L was conducted by acid extraction and alcohol precipitation, high performance liquid chromatography (HPLC) and nuclear magnetic resonance spectroscopy (NMR); in vitro anti-fungal experiments were carried out, including the minimum inhibitory concentration (MIC) and the minimum fungicidal concentration (MFC) spore germination rate, germ tube production rate, nucleic acid and protein leakage rate, biofilm structure, PCR, etc., to study the mechanism of action of RJH-L anti-fungal and anti-biofilm activity. ResultSeven monomer compounds were obtained: anthraquinones (rhein, emodin and aloe-emodin); polyphenols (ferulic acid, p-coumaric acid), and flavonoids (rutin and quercetin). The results of in vitro anti-fungal experiments showed that the extracts of RJH-L had strong inhibitory effect on both fungi (MIC: 1.96 µg/mL-62.50 µg/mL), of which emodin had the strongest effect on Trichophyton mentagrophytes; and rhein had the strongest effect on Candida albicans and Trichophyton rubrum. The above active components can inhibit the germination of fungal spores and germ tube, change cell membrane permeability, prevent hyphal growth, destroy the biofilm structure, and down-regulate the expression of agglutinin-like sequencefamily1 of biofilm growth. ConclusionThis study shows that RJH-L are rich in polyphenols, flavonoids, and anthraquinones, and play a fungicidal role.

Redox potential shapes spatial heterogeneity of mixed-cultured electroactive biofilm treating wastewater

The core of bioelectrochemical systems (BESs) is electrochemically active microorganisms (EAMs), which exert spatial heterogeneity on electrode surface and influences BESs performance. Setting an optimal potential is an effective strategy for improving and optimizing BESs performance, however, how the electrode potential affects spatial structure of microbial community within anode biofilm is not known. Using a complex substrate-fed BES with a wastewater inoculum, this study investigated the community structure and composition of the stratified biofilm developed under the potential of −0.3 V, 0 V, +0.3 V and +0.6 V (vs. saturated calomel electrode) by freezing microtome method and high-throughput sequencing analysis. The spatial heterogeneity of biofilm community was found to be dependent on the electrode potential and a less-stratified community structure was observed for +0.6 V than other potentials. Within the biofilms, the inner layers selected more Geobacter and the outer layers enriched more Acinetobacter and Serratia, potentially suggested a stratification of electron transfer pathway and metabolite-based interspecies communications. The results demonstrated the response of spatial heterogeneity of anode biofilm community to the change of electrode potential, which help to understand the selective and enrichment of kinetically efficient anodic microbiome by electron potential.

Veillonella parvula acts as a pathobiont promoting the biofilm virulence and cariogenicity of Streptococcus mutans in adult severe caries.

Adult severe caries (ASC) brings severe oral dysfunction and treatment difficulties to patients, and yet no clear pathogenic mechanism for it has been found. This study is focused on the composition of dental plaque microbiome profiles in order to identify disease-relevant species and to investigate into their interactions with the S. mutans. Samples of dental plaque were collected for metagenomic analysis. The acidification, aciduricity, oxidative stress tolerance, and gtf (glucosyltransferase) gene expression of S. mutans cocultured with V. parvula which was identified as ASC-related dominant bacterium. The biofilm formation and extracellular exopolysaccharide (EPS) synthesis of dual-strain were analyzed with scanning electron microscopy (SEM), crystal violet (CV) staining, live/dead bacterial staining, and confocal laser scanning microscopy (CLSM). Furthermore, rodent model experiments were performed to validate the in vivo cariogenicity of the dual-species biofilm. The most significantly abundant taxon found associated with ASC was V. parvula. In vitro experiments found that V. parvula can effectively promote S. mutans mature biofilm formation with enhanced acid resistance, hydrogen peroxide detoxicity, and biofilm virulence. Rodent model experiments revealed that V. parvula was incapable of causing disease on its own, but it significantly heightened the biofilm virulence of S. mutans when being co-infected and augmented the progression, quantity, and severity of dental caries. Our findings demonstrated that V. parvula may act as a synergistic pathobiont to modulate the metabolic activity, spatial structure, and pathogenicity of biofilms of S. mutans in the context of ASC.IMPORTANCEAdult severe caries (ASC), as a special type of acute caries, is rarely reported and its worthiness of further study is still in dispute. Yet studies on the etiology of severe caries in adults have not found a clear pathogenic mechanism for it. Knowledge of the oral microbiota is important for the treatment of dental caries. We discovered that the interaction between V. parvula and S. mutans augments the severity of dental caries in vivo, suggesting V. parvula may act as a synergistic pathobiont exacerbating biofilm virulence of S. mutans in ASC. Our findings may improve the understanding of ASC pathogenesis and are likely to provide a basis for planning appropriate therapeutic strategies.

Piezoelectric-Enhanced Nanocatalysts Trigger Neutrophil N1 Polarization against Bacterial Biofilm by Disrupting Redox Homeostasis.

Strategies of manipulating redox signaling molecules to inhibit or activate immune signals have revolutionized therapeutics involving reactive oxygen species (ROS). However, certain diseases with dual resistance barriers to the attacks by both ROS and immune cells, such as bacterial biofilm infections of medical implants, are difficult to eradicate by a single exogenous oxidative stimulus due to the diversity and complexity of the redox species involved. Here, this work demonstrates that metal-organic framework (MOF) nanoparticles capable of disrupting the bacterial ROS-defense system can dismantle bacterial redox resistance and induce potent antimicrobial immune responses in a mouse model of surgical implant infection by simultaneously modulating redox homeostasis and initiating neutrophil N1 polarization in the infection microenvironment. Mechanistically, the piezoelectrically enhanced MOF triggers ROS production by tilting the band structure and acts synergistically with the aurintricarboxylic acid loaded within the MOF, which inhibits the activity of the cystathionine γ-cleaving enzyme. This leads to biofilm structure disruption and antigen exposure through homeostatic imbalance and synergistic activation of neutrophil N1 polarization signals. Thus, this study provides an alternative but promising strategy for the treatment of antibiotic-resistant biofilm infections.

Effect of &lt;i&gt;Staphylococcus aureus&lt;/i&gt; Cell-Free Culture Liquid on the Structure and Biochemical Composition of &lt;i&gt;Klebsiella pneumoniae&lt;/i&gt; and &lt;i&gt;Pseudomonas aeruginosa&lt;/i&gt; Biofilms

Recently acquired data suggest that many infections are associated with formation of multispecies biofilms, in which both antibiotic sensitivity and the permeability of the extracellular matrix differ from those of monocultures. In this work, we show that addition of cell-free culture liquid of Staphylococcus aureus to the biofilms of Klebsiella pneumoniae and Pseudomonas aeruginosa increased the content of α- and β-polysaccharides in the matrix up to twofold, which in turn probably affected the biofilm structure. Increased content of the polysaccharide component was also confirmed by a significantly increased expression of the K. pneumoniae pgaA gene and of the P. aeruginosa pelA and pslA genes in the presence of S. aureus culture liquid.

Genetic diversity, biofilm formation, and Vancomycin resistance of clinical Clostridium innocuum isolates

BackgroundClostridium innocuum, previously considered a commensal microbe, is a spore-forming anaerobic bacterium. C. innocuum displays inherent resistance to vancomycin and is associated with extra-intestinal infections, antibiotic-associated diarrhea, and inflammatory bowel disease. This study seeks to establish a multilocus sequence typing (MLST) scheme to explore the correlation between C. innocuum genotyping and its potential pathogenic phenotypes.MethodsFifty-two C. innocuum isolates from Linkou Chang Gung Memorial Hospital (CGMH) in Taiwan and 60 sequence-available C. innocuum isolates from the National Center for Biotechnolgy Information Genome Database were included. The concentrated sequence of housekeeping genes in C. innocuum was determined by amplicon sequencing and used for MLST and phylogenetic analyses. The biofilm production activity of the C. innocuum isolates was determined by crystal violet staining.ResultsOf the 112 C. innocuum isolates, 58 sequence types were identified. Maximum likelihood estimation categorized 52 CGMH isolates into two phylogenetic clades. These isolates were found to be biofilm producers, with isolates in clade I exhibiting significantly higher biofilm production than isolates in clade II. The sub-inhibitory concentration of vancomycin seemed to minimally influence biofilm production by C. innocuum isolates. Nevertheless, C. innocuum embedded in the biofilm structure demonstrated resistance to vancomycin treatments at a concentration greater than 256 µg/mL.ConclusionsThis study suggests that a specific genetic clade of C. innocuum produces a substantial amount of biofilm. Furthermore, this phenotype assists C. innocuum in resisting high concentrations of vancomycin, which may potentially play undefined roles in C. innocuum pathogenesis.

Response of bacterial and fungal communities in natural biofilms to bioavailable heavy metals in a mining-affected river

Biofilms, known as “microbial skin” in rivers, respond to rapid and sensitive environmental changes. However, the ecological response mechanisms of bacterial and fungal communities in river biofilms toward heavy metal pollution (HMP) remains poorly understood. This study focused on the key driving factors of bacterial and fungal community diversity and composition and their ecological response mechanisms within periphytic biofilms of Asia's largest Pb–Zn mining area. The diversity, dominant bacterial taxa, and bacteria structure in biofilms were influenced by biologically available heavy metal (HM) fractions, with Ni-F3 (17.96 %) and Pb-F4 (16.27 %) as the main factors affecting the bacterial community structure. Fungal community structure and α-diversity were more susceptible to physicochemical parameters (pH and nutrient elements). Partial least squares path modeling revealed that environmental factors influencing bacterial and fungal communities in biofilms were ranked as water quality > metal fractions > total metals. Dispersal limitation was the most critical ecological process in bacterial (56.9 %) and fungal (73.4 %) assembly. The proportion of heterogeneous selection by bacteria (39.5 %) was higher than that of fungus (26.2 %), which increased with increasing HMP. Bacterial communities had a higher migration rate (0.48) and ecological drift proportion (3.6 %), making them more prone to escape environmental stress. Fungal communities exhibited more keystone species, larger niche width (23.24 ± 13.04 vs. 9.72 ± 5.48), higher organization level, and a more stable co-occurrence network than bacterial communities, which enabled them to better adapt to high environmental pollution levels. These findings expanded the understanding of the spatiotemporal dynamics of microbial communities within biofilms in HM-polluted watersheds and provided new insights into the ecological responses of microbial communities to HMP.

Antimicrobial effects and metabolomics analysis of the cell-free supernatant of Limosilactobacillus fermentum

In this study, Listeria monocytogenes and Escherichia coli were selected as reference bacteria, and the inhibitory effects and mechanisms of cell-free supernatants (CFS) of Limosilactobacillus fermentum 733 on its free-living bacteria and biofilm were investigated in depth. The findings demonstrated that L. fermentum 733 CFS displayed notable suppressive effects on L. monocytogenes and E. coli, which were caused by the loss of the cell membranes' structural integrity. This disturbance resulted in the release of intracellular adenosine triphosphate (ATP), alkaline phosphatase (ALP), and reactive oxygen species (ROS). Scanning electron microscopy and laser scanning confocal microscopy showed that L. fermentum 733 CFS disrupted L. monocytogenes and E. coli biofilm structure. L. fermentum 733 CFS inhibited partial population sensing and biofilm-associated gene expression of L. monocytogenes and E. coli, shown by fluorescence quantitative PCR (qPCR). Metabolomics analysis showed that L. fermentum 733 CFS strongly inhibited L. monocytogenes and E. coli by the disruption of both cellular structure and intracellular. These results offer novel perspectives and a theoretical foundation for the prevention of lactic acid bacteria (LAB) infections caused by L. monocytogenes and E. coli.