-
Methods in Molecular Biology (Clifton,... 2024The oral cavity is a habitat for different microorganisms, of which bacteria are best described. Studying different bacterial taxa and their proteins is crucial to...
The oral cavity is a habitat for different microorganisms, of which bacteria are best described. Studying different bacterial taxa and their proteins is crucial to understanding their interactions with the host and other microbes. Also, for bacteria with virulence potential, identifying novel antigenic proteins is essential to finding candidates for the development of vaccines.Here, a workflow for gel-free and label-free protein analysis of oral bacterial species grown in vitro as a biofilm and a planktonic culture is described. Details on cultivation, protein extraction and digestion, peptide cleanup, LC-MS/MS run parameters, and subsequent bioinformatics analysis are included. Challenging steps in the workflow, such as growing different types of bacteria and selecting a suitable protein database, are also discussed. This protocol provides a valuable guide for metaproteomic experiments using multi-species models of oral bacteria.
Topics: Proteomics; Mouth; Tandem Mass Spectrometry; Chromatography, Liquid; Bacterial Proteins; Humans; Bacteria; Microbiota; Biofilms; Computational Biology; Proteome; Workflow
PubMed: 38941022
DOI: 10.1007/978-1-0716-3910-8_14 -
Methods in Molecular Biology (Clifton,... 2024Wastewater treatment plants (WWTPs) are the main barrier to cope with the increased pressure of municipal and industrial wastewater on natural water resources in terms...
Wastewater treatment plants (WWTPs) are the main barrier to cope with the increased pressure of municipal and industrial wastewater on natural water resources in terms of both polluting load and produced volumes. For this reason, WWTP's efficiency should be the highest; thus, their monitoring becomes critical. In conventional WWTPs, biodegradation of pollutants mainly occurs in the biological reactors, and an increasing interest in a deeper characterization of the biomasses involved in these processes (made of biofilms, granules, and suspended activated sludge) rose up in recent years. In this sense, the meta-omics approaches were recently developed to investigate the entire set of biomolecules of a given class in a microbial community with the same general objective: the identification of the biomolecules through the sequence similarity of high degree in the already available databases. Particularly, metaproteomics concerns the identification of all proteins in a microbial community in a given moment or condition. In this chapter, a protocol for the extraction and separation of proteins from activate sludge sampled at WWTPs is proposed.
Topics: Sewage; Wastewater; Proteomics; Proteins; Waste Disposal, Fluid
PubMed: 38941010
DOI: 10.1007/978-1-0716-3910-8_2 -
Membrane protein Bcsdr2 mediates biofilm integrity, hyphal growth and virulence of Botrytis cinerea.Applied Microbiology and Biotechnology Jun 2024Grey mould caused by Botrytis cinerea is a devastating disease responsible for large losses to agricultural production, and B. cinerea is a necrotrophic model fungal...
Grey mould caused by Botrytis cinerea is a devastating disease responsible for large losses to agricultural production, and B. cinerea is a necrotrophic model fungal plant pathogen. Membrane proteins are important targets of fungicides and hotspots in the research and development of fungicide products. Wuyiencin affects the permeability and pathogenicity of B. cinerea, parallel reaction monitoring revealed the association of membrane protein Bcsdr2, and the bacteriostatic mechanism of wuyiencin was elucidated. In the present work, we generated and characterised ΔBcsdr2 deletion and complemented mutant B. cinerea strains. The ΔBcsdr2 deletion mutants exhibited biofilm loss and dissolution, and their functional activity was illustrated by reduced necrotic colonisation on strawberry and grape fruits. Targeted deletion of Bcsdr2 also blocked several phenotypic defects in aspects of mycelial growth, conidiation and virulence. All phenotypic defects were restored by targeted gene complementation. The roles of Bcsdr2 in biofilms and pathogenicity were also supported by quantitative real-time RT-PCR results showing that phosphatidylserine decarboxylase synthesis gene Bcpsd and chitin synthase gene BcCHSV II were downregulated in the early stages of infection for the ΔBcsdr2 strain. The results suggest that Bcsdr2 plays important roles in regulating various cellular processes in B. cinerea. KEY POINTS: • The mechanism of wuyiencin inhibits B. cinerea is closely associated with membrane proteins. • Wuyiencin can downregulate the expression of the membrane protein Bcsdr2 in B. cinerea. • Bcsdr2 is involved in regulating B. cinerea virulence, growth and development.
Topics: Botrytis; Biofilms; Virulence; Hyphae; Plant Diseases; Fragaria; Fungal Proteins; Membrane Proteins; Vitis; Spores, Fungal; Gene Deletion
PubMed: 38940906
DOI: 10.1007/s00253-024-13238-8 -
Microbial Ecology Jun 2024The primary purpose of the study, as part of the planned conservation work, was to uncover all aspects of autochthonous biofilm pertaining to the formation of numerous...
The primary purpose of the study, as part of the planned conservation work, was to uncover all aspects of autochthonous biofilm pertaining to the formation of numerous deterioration symptoms occurring on the limestone Rožanec Mithraeum monument in Slovenia. Using state-of-the-art sequencing technologies combining mycobiome data with observations made via numerous light and spectroscopic (FTIR and Raman) microscopy analyses pointed out to epilithic lichen Gyalecta jenensis and its photobiont, carotenoid-rich Trentepohlia aurea, as the origin of salmon-hued pigmented alterations of limestone surface. Furthermore, the development of the main deterioration symptom on the monument, i.e., biopitting, was instigated by the formation of typical endolithic thalli and ascomata of representative Verrucariaceae family (Verrucaria sp.) in conjunction with the oxalic acid-mediated dissolution of limestone. The domination of lichenized fungi, as the main deterioration agents, both on the relief and surrounding limestone, was additionally supported by the high relative abundance of lichenized and symbiotroph groups in FUNGuild analysis. Obtained results not only upgraded knowledge of this frequently occurring but often overlooked group of extremophilic stone heritage deteriogens but also provided a necessary groundwork for the development of efficient biocontrol formulation applicable in situ for the preservation of similarly affected limestone monuments.
Topics: Biofilms; Lichens; Calcium Carbonate; Slovenia; Ascomycota; Mycobiome
PubMed: 38940862
DOI: 10.1007/s00248-024-02404-0 -
Current Microbiology Jun 2024Garlic (Allium sativum L.), particularly its volatile essential oil, is widely recognized for medicinal properties. We have evaluated the efficacy of Indian Garlic...
Garlic (Allium sativum L.), particularly its volatile essential oil, is widely recognized for medicinal properties. We have evaluated the efficacy of Indian Garlic Essential Oil (GEO) for antimicrobial and antibiofilm activity and its bioactive constituents. Allyl sulfur-rich compounds were identified as predominant phytochemicals in GEO, constituting 96.51% of total volatile oils, with 38% Diallyl trisulphide (DTS) as most abundant. GEO exhibited significant antibacterial activity against eleven bacteria, including three drug-resistant strains with minimum inhibitory concentrations (MICs) ranging from 78 to 1250 µg/mL. In bacterial growth kinetic assay GEO effectively inhibited growth of all tested strains at its ½ MIC. Antibiofilm activity was evident against two important human pathogens, S. aureus and P. aeruginosa. Mechanistic studies demonstrated that GEO disrupts bacterial cell membranes, leading to the release of nucleic acids, proteins, and reactive oxygen species. Additionally, GEO demonstrated potent antioxidant activity at IC 31.18 mg/mL, while its isolated constituents, Diallyl disulphide (DDS) and Diallyl trisulphide (DTS), showed effective antibacterial activity ranging from 125 to 500 µg/mL and 250-1000 µg/mL respectively. Overall, GEO displayed promising antimicrobial and antibiofilm activity against enteric bacteria, suggesting its potential application in the food industry.
Topics: Garlic; Oils, Volatile; Antioxidants; Anti-Bacterial Agents; Biofilms; Microbial Sensitivity Tests; Staphylococcus aureus; Allyl Compounds; Phytochemicals; Sulfides; Bacteria; Pseudomonas aeruginosa; Disulfides; India; Plant Oils; Humans; Plant Extracts
PubMed: 38940852
DOI: 10.1007/s00284-024-03753-2 -
International Journal of Systematic and... Jun 2024A Gram-negative, strictly aerobic bacterial strain was isolated from asymptomatic leaf tissue of a wild yam plant. Optimal growth was observed at 28 °C and pH 7, and...
A Gram-negative, strictly aerobic bacterial strain was isolated from asymptomatic leaf tissue of a wild yam plant. Optimal growth was observed at 28 °C and pH 7, and catalase and oxidase activities were detected. Polyphasic taxonomic and comparative genomics revealed that strain LMG 33091 represents a novel species of . The nearest phylogenetic neighbours of strain LMG 33091 were NBRC 14164 (with 99.79 % 16S rRNA sequence identity), KL28 (99.28 %) and (99.07 %) ATCC 23835. MALDI-TOF MS analysis yielded distinct profiles for strain LMG 33091 and the nearest phylogenetic neighbours. Average nucleotide identity analyses between the whole genome sequence of strain LMG 33091 and of the type strains of its nearest-neighbour taxa yielded values below the species delineation threshold and thus confirmed that the strain represented a novel species, for which we propose the name sp. nov., with strain LMG 33091 (=GMI12077= CFBP 9143) as the type strain.
Topics: Pseudomonas; Phylogeny; RNA, Ribosomal, 16S; DNA, Bacterial; Plant Leaves; Sequence Analysis, DNA; Bacterial Typing Techniques; Dioscorea; Whole Genome Sequencing; Base Composition; Fatty Acids; Genome, Bacterial
PubMed: 38940814
DOI: 10.1099/ijsem.0.006395 -
Journal of Bacteriology Jun 2024The cystic fibrosis (CF) lung environment is conducive to the colonization of bacteria as polymicrobial biofilms, which are associated with poor clinical outcomes for...
UNLABELLED
The cystic fibrosis (CF) lung environment is conducive to the colonization of bacteria as polymicrobial biofilms, which are associated with poor clinical outcomes for persons with CF (pwCF). spp. are highly prevalent in the CF airway, but its role in the CF lung microbiome is poorly understood. Some studies have shown spp. to be associated with better clinical outcomes for pwCF, while others show that high abundance of spp. is correlated with exacerbations. Our lab previously reported a polymicrobial culture system consisting of four CF-relevant pathogens that can be used to study microbial behavior in a more clinically relevant setting. Here, we use this model system to identify genetic pathways that are important for survival in the context of the polymicrobial community. We identified genes related to reactive oxygen species as differentially expressed in monoculture versus growth of this microbe in the mixed community. Genetic studies identified Dpr as important for survival in the community. We show that Dpr, a DNA-binding ferritin-like protein, and PerR, a peroxide-responsive transcriptional regulator of Dpr, are important for protecting from phenazine-mediated toxicity in co-culture with and when exposed to hydrogen peroxide, both of which mimic the CF lung environment. Characterizing such interactions in a clinically relevant model system contributes to our understanding of microbial behavior in the context of polymicrobial biofilm infections.
IMPORTANCE
spp. are recognized as a highly prevalent pathogen in cystic fibrosis (CF) airway infections. However, the role of this microbe in clinical outcomes for persons with CF is poorly understood. Here, we leverage a polymicrobial community system previously developed by our group to model CF airway infections as a tool to investigate a - interaction involving reactive oxygen species (ROS). We show that protection against ROS is required for survival in a clinically relevant polymicrobial system. Using this model system to study interspecies interactions contributes to our broader understanding of the complex role of spp. in the CF lung.
PubMed: 38940597
DOI: 10.1128/jb.00176-24 -
Applied and Environmental Microbiology Jun 2024This study performed microbial analysis of nutrient film technique (NFT) hydroponic systems on three indoor farms in Singapore (the "what"). To justify the necessity of...
UNLABELLED
This study performed microbial analysis of nutrient film technique (NFT) hydroponic systems on three indoor farms in Singapore (the "what"). To justify the necessity of sanitizing hydroponic systems, strong biofilm-forming bacteria were isolated from the facility and investigated for their influence on colonization on polyvinyl chloride (PVC) coupons in hydroponic nutrient solutions (the "why"). Finally, sanitization solutions were evaluated with both laboratory-scale and field-scale tests (the "how"). As a result, the microbiome composition in NFT systems was found to be highly farm specific. The strong biofilm formers C2 and C3 were found to facilitate the attachment and colonization of on PVC coupons. When forming dual-species biofilms, the presence of C2 and C3 also significantly promoted the growth of ( < 0.05). Compared with hydrogen peroxide (HO) and sodium percarbonate (SPC), sodium hypochlorite (NaOCl) exhibited superior efficacy in biofilm removal. At 50 ppm, NaOCl reduced the Typhimurium, C2, and C3 counts to <1 log CFU/cm within 12 h, whereas neither 3% HO nor 1% SPC achieved this effect. In operational hydroponic systems, the concentration of NaOCl needed to achieve biofilm elimination increased to 500 ppm, likely due to the presence of organic matter accumulated during crop cultivation and the greater persistence of naturally formed multispecies biofilms. Sanitization using 500 ppm NaOCl for 12 h did not impede subsequent plant growth, but chlorination byproduct chlorate was detected at high levels in the hydroponic solution and in plants in the sanitized systems without rinsing.
IMPORTANCE
This study's significance lies first in its elucidation of the necessity of sanitizing hydroponic farming systems. The microbiome in hydroponic systems, although mostly nonpathogenic, might serve as a hotbed for pathogen colonization and thus pose a risk for food safety. We thus explored sanitization solutions with both laboratory-scale and field-scale tests. Of the three tested sanitizers, NaOCl was the most effective and economical option, whereas one must note the vital importance of rinsing the hydroponic systems after sanitization with NaOCl.
PubMed: 38940566
DOI: 10.1128/aem.00672-24 -
Microbiology Spectrum Jun 2024We used phage display, antibody engineering, and high-throughput assays to identify antibody-accessible targets of . We report the discovery of monoclonal antibodies...
We used phage display, antibody engineering, and high-throughput assays to identify antibody-accessible targets of . We report the discovery of monoclonal antibodies (mAbs) binding to type 3 fimbrial proteins, including MrkA. We found that anti-MrkA mAbs were cross-reactive to a diverse panel of clinical isolates, representing different O-serotypes. mAbs binding to MrkA have previously been described and have been shown to provide prophylactic protection, although only modest protection when dosed therapeutically in a murine lung infection model. Here, we used a combination of binding and opsonophagocytic killing studies using a high-content imaging platform to provide a possible explanation for the modest therapeutic efficacy reported in that model. Our work shows that expression of type 3 fimbriae in culture is not homogenous within a bacterial population. Instead, sub-populations of bacteria that do, and do not, express type 3 fimbriae exist. In a high-content opsonophagocytic killing assay, we showed that MrkA-targeting antibodies initially promote killing by macrophages; however, over time, this effect is diminished. We hypothesize the reason for this is that bacteria not expressing MrkA can evade opsonophagocytosis. Our data support the fact that MrkA is a conserved, immunodominant protein that is antibody accessible on the surface of and suggest that additional studies should evaluate the potential of using anti-MrkA antibodies in different stages of infection (different sites in the body) as well as against biofilms in the body during infection and associated with medical devices.IMPORTANCEThere is an unmet, urgent need for the development of novel antimicrobial therapies for the treatment of infections. We describe the use of phage display, antibody engineering, and high-throughput assays to identify antibody-accessible targets of . We discovered monoclonal antibodies (mAbs) binding to the type 3 fimbrial protein MrkA. The anti-MrkA mAbs were found to be highly cross-reactive, binding to all strains tested from a diverse panel of clinical isolates, and were active in an opsonophagocytic killing assay at pM concentrations. MrkA is important for biofilm formation; thus, our data support further exploration of the use of anti-MrkA antibodies for preventing and/or controlling in biofilms and during infection.
PubMed: 38940542
DOI: 10.1128/spectrum.00400-24 -
Journal of Oral Microbiology 2024The diversity and delicate balance of the oral microbiome contribute to oral health, with its disruption leading to oral and systemic diseases. Toothpaste includes...
The diversity and delicate balance of the oral microbiome contribute to oral health, with its disruption leading to oral and systemic diseases. Toothpaste includes elements like traditional additives such as sodium lauryl sulfate (SLS) as well as novel postbiotics derived from probiotics, which are commonly employed for maintaining oral hygiene and a healthy oral cavity. However, the response of the oral microbiota to these treatments remains poorly understood. In this study, we systematically investigated the impact of SLS, and toothpaste containing postbiotics (hereafter, postbiotic toothpaste) across three systems: biofilms, animal models, and clinical populations. SLS was found to kill bacteria in both preformed biofilms (mature biofilms) and developing biofilms (immature biofilms), and disturbed the microbial community structure by increasing the number of pathogenic bacteria. SLS also destroyed periodontal tissue, promoted alveolar bone resorption, and enhanced the extent of inflammatory response level. The postbiotic toothpaste favored bacterial homeostasis and the normal development of the two types of biofilms , and attenuated periodontitis and gingivitis via modulation of oral microecology. Importantly, the postbiotic toothpaste mitigated the adverse effects of SLS when used in combination, both and . Overall, the findings of this study describe the impact of toothpaste components on oral microflora and stress the necessity for obtaining a comprehensive understanding of oral microbial ecology by considering multiple aspects.
PubMed: 38939048
DOI: 10.1080/20002297.2024.2372224