scholarly journals Critical Assessment of Methods to Quantify Biofilm Growth and Evaluate Antibiofilm Activity of Host Defence Peptides

Biomolecules ◽  
2018 ◽  
Vol 8 (2) ◽  
pp. 29 ◽  
Author(s):  
Evan Haney ◽  
Michael Trimble ◽  
John Cheng ◽  
Quentin Vallé ◽  
Robert Hancock
Keyword(s):  
Bacterial Species ◽  
Growth Conditions ◽  
Host Defence ◽  
Detection Methods ◽  
Antibiofilm Activity ◽  
Bacterial Cells ◽  
Media Composition ◽  
Microscopy Imaging ◽  
Biofilm Growth ◽  
Tetrazolium Chloride

Biofilms are multicellular communities of bacteria that can adhere to virtually any surface. Bacterial biofilms are clinically relevant, as they are responsible for up to two-thirds of hospital acquired infections and contribute to chronic infections. Troublingly, the bacteria within a biofilm are adaptively resistant to antibiotic treatment and it can take up to 1000 times more antibiotic to kill cells within a biofilm when compared to planktonic bacterial cells. Identifying and optimizing compounds that specifically target bacteria growing in biofilms is required to address this growing concern and the reported antibiofilm activity of natural and synthetic host defence peptides has garnered significant interest. However, a standardized assay to assess the activity of antibiofilm agents has not been established. In the present work, we describe two simple assays that can assess the inhibitory and eradication capacities of peptides towards biofilms that are formed by both Gram-positive and negative bacteria. These assays are suitable for high-throughput workflows in 96-well microplates and they use crystal violet staining to quantify adhered biofilm biomass as well as tetrazolium chloride dye to evaluate the metabolic activity of the biofilms. The effect of media composition on the readouts of these biofilm detection methods was assessed against two strains of Pseudomonas aeruginosa (PAO1 and PA14), as well as a methicillin resistant strain of Staphylococcus aureus. Our results demonstrate that media composition dramatically alters the staining patterns that were obtained with these dye-based methods, highlighting the importance of establishing appropriate biofilm growth conditions for each bacterial species to be evaluated. Confocal microscopy imaging of P. aeruginosa biofilms grown in flow cells revealed that this is likely due to altered biofilm architecture under specific growth conditions. The antibiofilm activity of several antibiotics and synthetic peptides were then evaluated under both inhibition and eradication conditions to illustrate the type of data that can be obtained using this experimental setup.

scholarly journals Mitigating Milk-Associated Bacteria through Inducing Zinc Ions Antibiofilm Activity

Foods ◽  
2020 ◽  
Vol 9 (8) ◽  
pp. 1094
Author(s):  
Carmel Hutchings ◽  
Satish Kumar Rajasekharan ◽  
Ram Reifen ◽  
Moshe Shemesh
Keyword(s):  
Heat Treatment ◽  
Bacterial Species ◽  
Dairy Industry ◽  
Antimicrobial Effect ◽  
Pcr Analysis ◽  
Antibiofilm Activity ◽  
Bacterial Cells ◽  
Production Environment ◽  
Bacterial Physiology ◽  
Microscopic Evaluation

Dairy products are a sector heavily impacted by food loss, often due to bacterial contaminations. A major source of contamination is associated with the formation of biofilms by bacterial species adopted to proliferate in milk production environment and onto the surfaces of milk processing equipment. Bacterial cells within the biofilm are characterized by increased resistance to unfavorable environmental conditions and antimicrobial agents. Members of the Bacillus genus are the most commonly found spoilage microorganisms in the dairy environment. It appears that physiological behavior of these species is somehow depended on the availability of bivalent cations in the environment. One of the important cations that may affect the bacterial physiology as well as survivability are Zn2+ ions. Thus, the aim of this study was to examine the antimicrobial effect of Zn2+ ions, intending to elucidate the potential of a zinc-based antibacterial treatment suitable for the dairy industry. The antimicrobial effect of different doses of ZnCl2 was assessed microscopically. In addition, expression of biofilm related genes was evaluated using RT-PCR. Analysis of survival rates following heat treatment was conducted in order to exemplify a possible applicative use of Zn2+ ions. Addition of zinc efficiently inhibited biofilm formation by B. subtilis and further disrupted the biofilm bundles. Expression of matrix related genes was found to be notably downregulated. Microscopic evaluation showed that cell elongation was withheld when cells were grown in the presence of zinc. Finally, B. cereus and B. subtilis cells were more susceptible to heat treatment after being exposed to Zn2+ ions. It is believed that an anti-biofilm activity, expressed in downregulation of genes involved in construction of the extracellular matrix, would account for the higher sensitivity of bacteria during heat pasteurization. Consequently, we suggest that Zn2+ ions can be of used as an effective antimicrobial treatment in various applications in the dairy industry, targeting both biofilms and vegetative bacterial cells.

A Microbiological Paradox: Viable but Nonculturable Bacteria with Special Reference to Vibrio cholerae

1996 ◽  
Vol 59 (1) ◽  
pp. 96-101 ◽  
Author(s):  
ANWARUL HUQ ◽  
RITA R. COLWELL
Keyword(s):  
Vibrio Cholerae ◽  
Culture Media ◽  
Direct Detection ◽  
Bacterial Species ◽  
Detection Methods ◽  
Bacterial Cells ◽  
Culture Methods ◽  
Viable But Nonculturable ◽  
Bacteriological Culture ◽  
Nonculturable State

The observation that directly-detectable bacterial cells are unable to grow on bacteriological culture media under certain conditions raises questions regarding the viability of these cells. Various terminologies have been used to describe substrate-responsive and metabolically-active bacterial cells that cannot be cultured. The currently-accepted term is “viable but nonculturable.” During the past 15 years, the viable but nonculturable phenomenon has been actively investigated. Bacterial pathogens in the viable but nonculturable state can maintain virulence and produce disease. These organisms may escape detection if bacteriological culture methods are solely used. Thus, methods for direct detection of specific pathogens in food, water and environmental samples are preferable. Viable but nonculturable Vibrio cholerae have been extensively studied, and several sensitive and reliable direct-detection kits have been developed. Viable but nonculturable forms of bacteria are now recognized as a common phenomenon, observable in many bacterial species, which suggests that standard bacteriological laboratory protocols for assessing microbiological safety of food and drinking water are less reliable than direct detection methods.

scholarly journals THE INITIATION OF GROWTH OF CERTAIN FACULTATIVE ANAEROBES AS RELATED TO OXIDATION-REDUCTION PROCESSES IN THE MEDIUM

1929 ◽  
Vol 49 (4) ◽  
pp. 559-573 ◽  
Author(s):  
René Dubos
Keyword(s):  
Reduction Potential ◽  
Bacterial Species ◽  
Growth Conditions ◽  
Critical Value ◽  
Oxidation Potential ◽  
Bacterial Cells ◽  
Anaerobic Conditions ◽  
Reduction Processes ◽  
Oxidation Reduction ◽  
Reducing Properties

The growth of many pathogenic organisms in plain meat infusion broth is possible only when a large inoculum is used. This requirement is much less strict when the broth cultures are incubated (a) under anaerobic conditions, (b) in fresh media very recently boiled or autoclaved, (c) in fresh media reduced by means of hydrogen, or to which small amounts of cysteine or of blood have been added. It is suggested that these findings can be accounted for by assuming that the bacterial species under consideration can multiply only in media the oxidation potential of which is below a critical value. The favorable growth conditions obtained by the procedures enumerated above may be attributed to the establishment of a proper reduction potential in the medium; the same result is obtained by using a large inoculum, owing to the reducing properties of bacterial cells.

scholarly journals Graphene-Based Sensor for Detection of Bacterial Pathogens

Sensors ◽  
2021 ◽  
Vol 21 (23) ◽  
pp. 8085
Author(s):  
Santosh Pandit ◽  
Mengyue Li ◽  
Yanyan Chen ◽  
Shadi Rahimi ◽  
Vrss Mokkapati ◽  
...  
Keyword(s):  
Antimicrobial Agents ◽  
Bacterial Colonization ◽  
Bacterial Species ◽  
Complex Structure ◽  
Growth Dynamics ◽  
Microbial Colonization ◽  
Bacterial Cells ◽  
Resistance Change ◽  
Biofilm Growth ◽  
Adherence Pattern

Microbial colonization to biomedical surfaces and biofilm formation is one of the key challenges in the medical field. Recalcitrant biofilms on such surfaces cause serious infections which are difficult to treat using antimicrobial agents, due to their complex structure. Early detection of microbial colonization and monitoring of biofilm growth could turn the tide by providing timely guidance for treatment or replacement of biomedical devices. Hence, there is a need for sensors, which could generate rapid signals upon bacterial colonization. In this study, we developed a simple prototype sensor based on pristine, non-functionalized graphene. The detection principle is a change in electrical resistance of graphene upon exposure to bacterial cells. Without functionalization with specific receptors, such sensors cannot be expected to be selective to certain bacteria. However, we demonstrated that two different bacterial species can be detected and differentiated by our sensor due to their different growth dynamics, adherence pattern, density of adhered bacteria and microcolonies formation. These distinct behaviors of tested bacteria depicted distinguishable pattern of resistance change, resistance versus gate voltage plot and hysteresis effect. This sensor is simple to fabricate, can easily be miniaturized, and can be effective in cases when precise identification of species is not needed.

scholarly journals The Core Proteome of Biofilm-Grown Clinical Pseudomonas aeruginosa Isolates

Cells ◽  
2019 ◽  
Vol 8 (10) ◽  
pp. 1129 ◽  
Author(s):  
Jelena Erdmann ◽  
Janne G. Thöming ◽  
Sarah Pohl ◽  
Andreas Pich ◽  
Christof Lenz ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Bacterial Species ◽  
Protein Profile ◽  
Opportunistic Pathogen ◽  
Growth Conditions ◽  
Protein Quantification ◽  
Clinical Isolates ◽  
Biofilm Growth ◽  
Regulatory Pathways ◽  
Data Independent Acquisition

Comparative genomics has greatly facilitated the identification of shared as well as unique features among individual cells or tissues, and thus offers the potential to find disease markers. While proteomics is recognized for its potential to generate quantitative maps of protein expression, comparative proteomics in bacteria has been largely restricted to the comparison of single cell lines or mutant strains. In this study, we used a data independent acquisition (DIA) technique, which enables global protein quantification of large sample cohorts, to record the proteome profiles of overall 27 whole genome sequenced and transcriptionally profiled clinical isolates of the opportunistic pathogen Pseudomonas aeruginosa. Analysis of the proteome profiles across the 27 clinical isolates grown under planktonic and biofilm growth conditions led to the identification of a core biofilm-associated protein profile. Furthermore, we found that protein-to-mRNA ratios between different P. aeruginosa strains are well correlated, indicating conserved patterns of post-transcriptional regulation. Uncovering core regulatory pathways, which drive biofilm formation and associated antibiotic tolerance in bacterial pathogens, promise to give clues to interactions between bacterial species and their environment and could provide useful targets for new clinical interventions to combat biofilm-associated infections.

scholarly journals Evaluating the Effects of Disinfectants on Bacterial Biofilms Using a Microfluidics Flow Cell and Time-Lapse Fluorescence Microscopy

2020 ◽  
Vol 8 (11) ◽  
pp. 1837
Author(s):  
Milos Legner ◽  
James Jonkman ◽  
Dean Swift
Keyword(s):  
Fluorescence Microscopy ◽  
Bacterial Species ◽  
Bacterial Biomass ◽  
Time Lapse ◽  
Flow Cell ◽  
Bacterial Cells ◽  
Bacterial Strains ◽  
Microscopy Imaging ◽  
Microscopy Method ◽  
Time Lapse Imaging

A commercially available microfluidics flow cell was utilized together with widefield fluorescence microscopy to evaluate the effects of disinfectants on bacterial strains. The flow cell’s inner surface supports the formation of biofilms of numerous bacterial species. The modular setup of the flow cell accessories allows connection to syringes, pumps and collection vials, facilitating aseptic experiments in a controlled fluidics environment which can be documented with precisely timed microscopy imaging. The flow cell is inoculated with a suspension of bacteria in a nutrient medium and incubated for several days allowing bacterial cells to form a biofilm. Shortly before performing an assay, the biofilm is labelled with a dual-fluorescent DNA probe which distinguishes unharmed and damaged bacteria. Then a disinfectant sample (or control) is gently injected and time-lapse imaging is used for quantifying the course of bacterial biomass response. We use a simplified widefield microscopy method that allows intensive recording and quantification of time series of two-dimensional frames for tracking the course of disinfectant action on a variety of microbial strains. This procedure has potential for the rapid evaluation of novel products.

Modulation of Gut Microbiota through Dietary Phytochemicals as a Novel Anti-infective Strategy

2020 ◽  
Vol 17 (4) ◽  
pp. 498-506 ◽  
Author(s):  
Pavan K. Mujawdiya ◽  
Suman Kapur
Keyword(s):  
Microbial Community ◽  
Quorum Sensing ◽  
Population Density ◽  
Gut Microbiota ◽  
Biofilm Formation ◽  
Chemical Interaction ◽  
Bacterial Species ◽  
Critical Role ◽  
Bacterial Cells ◽  
Gut Microbes

: Quorum Sensing (QS) is a phenomenon in which bacterial cells communicate with each other with the help of several low molecular weight compounds. QS is largely dependent on population density, and it triggers when the concentration of quorum sensing molecules accumulate in the environment and crosses a particular threshold. Once a certain population density is achieved and the concentration of molecules crosses a threshold, the bacterial cells show a collective behavior in response to various chemical stimuli referred to as “auto-inducers”. The QS signaling is crucial for several phenotypic characteristics responsible for bacterial survival such as motility, virulence, and biofilm formation. Biofilm formation is also responsible for making bacterial cells resistant to antibiotics. : The human gut is home to trillions of bacterial cells collectively called “gut microbiota” or “gut microbes”. Gut microbes are a consortium of more than 15,000 bacterial species and play a very crucial role in several body functions such as metabolism, development and maturation of the immune system, and the synthesis of several essential vitamins. Due to its critical role in shaping human survival and its modulating impact on body metabolisms, the gut microbial community has been referred to as “the forgotten organ” by O`Hara et al. (2006) [1]. Several studies have demonstrated that chemical interaction between the members of bacterial cells in the gut is responsible for shaping the overall microbial community. : Recent advances in phytochemical research have generated a lot of interest in finding new, effective, and safer alternatives to modern chemical-based medicines. In the context of antimicrobial research various plant extracts have been identified with Quorum Sensing Inhibitory (QSI) activities among bacterial cells. This review focuses on the mechanism of quorum sensing and quorum sensing inhibitors isolated from natural sources.

scholarly journals Cell Surface Expression of Nrg1 Protein in Candida auris

2021 ◽  
Vol 7 (4) ◽  
pp. 262
Author(s):  
Anuja Paudyal ◽  
Govindsamy Vediyappan
Keyword(s):  
Cell Surface ◽  
Growth Conditions ◽  
Zinc Ion ◽  
Surface Proteins ◽  
Cell Surface Expression ◽  
Unexpected Finding ◽  
Candida Auris ◽  
Cell Surface Proteins ◽  
Biofilm Growth

Candida auris is an emerging antifungal resistant human fungal pathogen increasingly reported in healthcare facilities. It persists in hospital environments, and on skin surfaces, and can form biofilms readily. Here, we investigated the cell surface proteins from C. auris biofilms grown in a synthetic sweat medium mimicking human skin conditions. Cell surface proteins from both biofilm and planktonic control cells were extracted with a buffer containing β-mercaptoethanol and resolved by 2-D gel electrophoresis. Some of the differentially expressed proteins were excised and identified by mass spectrometry. C. albicans orthologs Spe3p, Tdh3p, Sod2p, Ywp1p, and Mdh1p were overexpressed in biofilm cells when compared to the planktonic cells of C. auris. Interestingly, several proteins with zinc ion binding activity were detected. Nrg1p is a zinc-binding transcription factor that negatively regulates hyphal growth in C. albicans. C. auris does not produce true hypha under standard in vitro growth conditions, and the role of Nrg1p in C. auris is currently unknown. Western blot analyses of cell surface and cytosolic proteins of C. auris against anti-CalNrg1 antibody revealed the Nrg1p in both locations. Cell surface localization of Nrg1p in C. auris, an unexpected finding, was further confirmed by immunofluorescence microscopy. Nrg1p expression is uniform across all four clades of C. auris and is dependent on growth conditions. Taken together, the data indicate that C. auris produces several unique proteins during its biofilm growth, which may assist in the skin-colonizing lifestyle of the fungus during its pathogenesis.

scholarly journals Revealing the Metabolic Alterations during Biofilm Development of Burkholderia cenocepacia Based on Genome-Scale Metabolic Modeling

Metabolites ◽  
2021 ◽  
Vol 11 (4) ◽  
pp. 221
Author(s):  
Ozlem Altay ◽  
Cheng Zhang ◽  
Hasan Turkez ◽  
Jens Nielsen ◽  
Mathias Uhlén ◽  
...  
Keyword(s):  
Systems Biology ◽  
Alternative Pathway ◽  
Growth Conditions ◽  
Pentose Phosphate ◽  
Burkholderia Cenocepacia ◽  
Bacterial Cells ◽  
Metabolic Remodeling ◽  
Transcriptomics Data ◽  
Biofilm Development ◽  
Genome Scale

Burkholderia cenocepacia is among the important pathogens isolated from cystic fibrosis (CF) patients. It has attracted considerable attention because of its capacity to evade host immune defenses during chronic infection. Advances in systems biology methodologies have led to the emergence of methods that integrate experimental transcriptomics data and genome-scale metabolic models (GEMs). Here, we integrated transcriptomics data of bacterial cells grown on exponential and biofilm conditions into a manually curated GEM of B. cenocepacia. We observed substantial differences in pathway response to different growth conditions and alternative pathway susceptibility to extracellular nutrient availability. For instance, we found that blockage of the reactions was vital through the lipid biosynthesis pathways in the exponential phase and the absence of microenvironmental lysine and tryptophan are essential for survival. During biofilm development, bacteria mostly had conserved lipid metabolism but altered pathway activities associated with several amino acids and pentose phosphate pathways. Furthermore, conversion of serine to pyruvate and 2,5-dioxopentanoate synthesis are also identified as potential targets for metabolic remodeling during biofilm development. Altogether, our integrative systems biology analysis revealed the interactions between the bacteria and its microenvironment and enabled the discovery of antimicrobial targets for biofilm-related diseases.

scholarly journals Elucidation of Gram-Positive Bacterial Iron(III) Reduction for Kaolinite Clay Refinement

Molecules ◽  
2021 ◽  
Vol 26 (11) ◽  
pp. 3084
Author(s):  
Hao Jing ◽  
Zhao Liu ◽  
Seng How Kuan ◽  
Sylvia Chieng ◽  
Chun Loong Ho
Keyword(s):  
Iron Reduction ◽  
Growth Conditions ◽  
Cellular Interaction ◽  
Kaolin Clay ◽  
Media Composition ◽  
Gram Positive ◽  
Kaolinite Clay ◽  
Gram Positive Bacteria ◽  
Rich Media ◽  
The Media

Recently, microbial-based iron reduction has been considered as a viable alternative to typical chemical-based treatments. The iron reduction is an important process in kaolin refining, where iron-bearing impurities in kaolin clay affects the whiteness, refractory properties, and its commercial value. In recent years, Gram-negative bacteria has been in the center stage of iron reduction research, whereas little is known about the potential use of Gram-positive bacteria to refine kaolin clay. In this study, we investigated the ferric reducing capabilities of five microbes by manipulating the microbial growth conditions. Out of the five, we discovered that Bacillus cereus and Staphylococcus aureus outperformed the other microbes under nitrogen-rich media. Through the biochemical changes and the microbial behavior, we mapped the hypothetical pathway leading to the iron reduction cellular properties, and found that the iron reduction properties of these Gram-positive bacteria rely heavily on the media composition. The media composition results in increased basification of the media that is a prerequisite for the cellular reduction of ferric ions. Further, these changes impact the formation of biofilm, suggesting that the cellular interaction for the iron(III)oxide reduction is not solely reliant on the formation of biofilms. This article reveals the potential development of Gram-positive microbes in facilitating the microbial-based removal of metal contaminants from clays or ores. Further studies to elucidate the corresponding pathways would be crucial for the further development of the field.

Sign in / Sign up

Export Citation Format

Share Document