A Role for Secreted Immune Effectors in Microbial Biofilm Formation Revealed by Simple In Vitro Assays

This study aimed to isolate Staphylococcus aureus in refrigerated raw cow milk, and identify the presence of enterotoxin-expression genes, enterotoxin production and adherence ability, and antimicrobial resistance potential of the isolated strains. Fifty raw milk samples obtained in different dairy farms were analyzed for S. aureus and evaluated in the isolates the presence of genes associated with the production of major staphylococcal enterotoxins and biofilm formation. In vitro assays were also performed to evaluate the production of enterotoxins and adherence ability, and the antimicrobial resistance. One half (25/50) of raw milk samples presented coagulase-positive staphylococci and 95.2% of the isolates were confirmed to be S. aureus. Among them, 42.4% were carrying genes for enterotoxins production; however, only one isolate was able to produce enterotoxins. All S. aureus isolates were carrying at least two genes associated with biofilm formation and 95.2% isolates was able to adhere upon the in vitro assay. All isolates demonstrated antimicrobial resistance potential to one or more of the tested antibiotics.

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In vitro methods for the study of microbial biofilms

Biointerface Research in Applied Chemistry ◽

10.33263/briac11.031040 ◽

2011 ◽

Vol 1 (1) ◽

pp. 031-040 ◽

Cited By ~ 25

Keyword(s):

Ferrite Nanoparticles ◽

In Vitro Assays ◽

Confocal Laser ◽

Microbial Biofilm ◽

In Vivo Models ◽

Microbial Biofilms ◽

Human Pathology ◽

Biofilm Development

The emergence of microbial biofilm related infections (bacterial and fungal) has a significant impact for the human pathology in the entire world. The understanding of microbial infections related to the biofilm development on tissues or indwelling devices was possible by using different qualitative and quantitative in vitro assays, in continuous and discontinuous systems, as well as in vivo models. A necessary step for obtaining more standardized, reliable and comparable results among different laboratories is the simplification of the available techniques used for investigating the biofilm formation and properties, including the biofilms susceptibility to antibiotics. The aim of the present study was to exemplify a series of available methods for the investigation of in vitro microbial biofilms developed on inert substrata, as well as coated with ferrite nanoparticles, using as experimental model a Sacharomyces cerevisiae strain. Microbial biofilm architecture was directly examined by two microscopy techniques (inverted microscopy and confocal laser microscopy scanning). The in vitro study of the influence of suspended ferrite nanoparticles on planktonic cells growth, adherence and consecutive biofilm development on inert substrata was performed by using a simple microtiter method.

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oxyR, a LysR-Type Regulator Involved in Klebsiella pneumoniae Mucosal and Abiotic Colonization

Infection and Immunity ◽

10.1128/iai.00837-09 ◽

2009 ◽

Vol 77 (12) ◽

pp. 5449-5457 ◽

Cited By ~ 57

Author(s):

Claire Hennequin ◽

Christiane Forestier

Keyword(s):

Gastrointestinal Tract ◽

Klebsiella Pneumoniae ◽

Biofilm Formation ◽

Defense Mechanisms ◽

In Vitro Assays ◽

Intestinal Epithelial ◽

Bacterial Transcription ◽

Host Interaction

ABSTRACT Colonization of the gastrointestinal tract is the first event in Klebsiella pneumoniae nosocomial infections, followed by colonization of the bladder or respiratory tract or entry into the bloodstream. To survive in the host, bacteria must harbor specific traits and overcome multiple stresses. OxyR is a conserved bacterial transcription factor with a key role both in the upregulation of defense mechanisms against oxidative stress and in pathogenesis by enhancing biofilm formation, fimbrial expression, and mucosal colonization. A homolog of oxyR was detected in silico in the K. pneumoniae sequenced genome and amplified from the LM21 wild-type strain. To determine the role of oxyR in K. pneumoniae host-interaction processes, an oxyR isogenic mutant was constructed, and its behavior was assessed. At concentrations lower than 107 ml−1, oxyR-deficient organisms were easily killed by micromolar concentrations of H2O2 and exhibited typical aerobic phenotypes. The oxyR mutant was impaired in biofilm formation and types 1 and 3 fimbrial gene expression. In addition, the oxyR mutant was unable to colonize the murine gastrointestinal tract, and in vitro assays showed that it was defective in adhesion to Int-407 and HT-29 intestinal epithelial cells. The behavior of the oxyR mutant was also determined under hostile conditions, reproducing stresses encountered in the gastrointestinal environment: deletion of oxyR resulted in higher sensitivity to bile and acid stresses but not to osmotic stress. These results show the pleiotropic role of oxyR in K. pneumoniae gastrointestinal colonization.

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Biomimetic Approaches for the Development of New Antifouling Solutions: Study of Incorporation of Macroalgae and Sponge Extracts for the Development of New Environmentally-Friendly Coatings

International Journal of Molecular Sciences ◽

10.3390/ijms20194863 ◽

2019 ◽

Vol 20 (19) ◽

pp. 4863 ◽

Cited By ~ 5

Author(s):

Ilse Sánchez-Lozano ◽

Claudia Judith Hernández-Guerrero ◽

Mauricio Muñoz-Ochoa ◽

Claire Hellio

Keyword(s):

Biofilm Formation ◽

Gulf Of California ◽

Environmentally Friendly ◽

In Vitro Assays ◽

Economic Losses ◽

Maritime Industry ◽

Marine Biotechnology ◽

La Paz ◽

Selection Of

Biofouling causes major economic losses in the maritime industry. In our site study, the Bay of La Paz (Gulf of California), biofouling on immersed structures is a major problem and is treated mostly with copper-based antifouling paints. Due to the known environmental effect of such treatments, the search for environmentally friendly alternatives in this zone of high biodiversity is a priority to ensure the conservation and protection of species. The aim of this work was to link chemical ecology to marine biotechnology: indeed, the natural defense of macroalgae and sponge was evaluated against biofoulers (biofilm and macrofoulers) from the same geographical zone, and some coatings formulation was done for field assays. Our approach combines in vitro and field bioassays to ensure the selection of the best AF agent prospects. The 1st step consisted of the selection of macroalgae (5 species) and sponges (2 species) with surfaces harboring a low level of colonizers; then extracts were prepared and assayed for toxicity against Artemia, activity towards key marine bacteria involved in biofilm formation in the Bay of La Paz, and the potency to inhibit adhesion of macroorganisms (phenoloxidase assays). The most active and non-toxic extracts were further studied for biofouling activity in the adhesion of the bacteria involved in biofilm formation and through incorporation in marine coatings which were immersed in La Paz Bay during 40 days. In vitro assays demonstrated that extracts of Laurencia gardneri, Sargassum horridum (macroalgae), Haliclona caerulea and Ircinia sp. (sponges) were the most promising. The field test results were of high interest as the best formulation were composed of extracts of H. caerulea and S. horridum and led to a reduction of 32% of biofouling compared with the control.

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A Rare Mutation in SPLUNC1 Affects Bacterial Adherence and Invasion in Meningococcal Disease

Clinical Infectious Diseases ◽

10.1093/cid/ciz600 ◽

2019 ◽

Vol 70 (10) ◽

pp. 2045-2053

Author(s):

Bayarchimeg Mashbat ◽

Evangelos Bellos ◽

Stephanie Hodeib ◽

Fadil Bidmos ◽

Ryan S Thwaites ◽

...

Keyword(s):

Meningococcal Disease ◽

Biofilm Formation ◽

Immune Defense ◽

Dominant Negative ◽

In Vitro Assays ◽

Dominant Negative Mutant ◽

Antibiofilm Activity ◽

Invasive Infections ◽

Disease Study

Abstract Background Neisseria meningitidis (Nm) is a nasopharyngeal commensal carried by healthy individuals. However, invasive infections occurs in a minority of individuals, with devastating consequences. There is evidence that common polymorphisms are associated with invasive meningococcal disease (IMD), but the contributions of rare variants other than those in the complement system have not been determined. Methods We identified familial cases of IMD in the UK meningococcal disease study and the European Union Life-Threatening Infectious Disease Study. Candidate genetic variants were identified by whole-exome sequencing of 2 patients with familial IMD. Candidate variants were further validated by in vitro assays. Results Exomes of 2 siblings with IMD identified a novel heterozygous missense mutation in BPIFA1/SPLUNC1. Sequencing of 186 other nonfamilial cases identified another unrelated IMD patient with the same mutation. SPLUNC1 is an innate immune defense protein expressed in the nasopharyngeal epithelia; however, its role in invasive infections is unknown. In vitro assays demonstrated that recombinant SPLUNC1 protein inhibits biofilm formation by Nm, and impedes Nm adhesion and invasion of human airway cells. The dominant negative mutant recombinant SPLUNC1 (p.G22E) showed reduced antibiofilm activity, increased meningococcal adhesion, and increased invasion of cells, compared with wild-type SPLUNC1. Conclusions A mutation in SPLUNC1 affecting mucosal attachment, biofilm formation, and invasion of mucosal epithelial cells is a new genetic cause of meningococcal disease.

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In vitro and in vivo model systems to study microbial biofilm formation

Journal of Microbiological Methods ◽

10.1016/j.mimet.2010.08.018 ◽

2010 ◽

Vol 83 (2) ◽

pp. 89-105 ◽

Cited By ~ 242

Author(s):

Tom Coenye ◽

Hans J. Nelis

Keyword(s):

Biofilm Formation ◽

Model Systems ◽

In Vivo Model ◽

Microbial Biofilm

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Induction of Biofilm Formation in Klebsiella pneumoniae ATCC 13884 by Several Drugs: The Possible Role of Quorum Sensing Modulation

Antibiotics ◽

10.3390/antibiotics7040103 ◽

2018 ◽

Vol 7 (4) ◽

pp. 103 ◽

Cited By ~ 5

Author(s):

Elizabeth Cadavid ◽

Sara Robledo ◽

Wiston Quiñones ◽

Fernando Echeverri

Keyword(s):

Quorum Sensing ◽

Klebsiella Pneumoniae ◽

Biofilm Formation ◽

Bacterial Resistance ◽

Homoserine Lactone ◽

In Vitro Assays ◽

Bacterial Populations ◽

Other Substances ◽

Microplate Reader

Bacterial resistance is caused by several biochemical factors, the formation of biofilm being one of the main causes. This process is triggered by Quorum Sensing (QS), through the production of endogenous molecules, although other substances such as natural products can also do this. In this work, we aimed to determine whether some drugs are involved in the induction of biofilm formation in Klebsiella pneumoniae ATCC 13884, and thus, increase bacterial resistance. For this, the effect of 22 drugs on K. pneumoniae ATCC 13884 growth was determined at sub-plasmatic concentrations; the production of autoinducer lactones was established by HPLC and with a biosensor. The induction of biofilm formation was determined through crystal violet assay at 585 nm in a microplate reader and using urethral catheters. According to the in vitro assays, some drugs were found to induce biofilm formation in K. pneumoniae ATCC 13884. The effect of acetaminophen, hydrochlorothiazide, and progesterone stood out. The first drug caused several changes in the biochemistry of K. pneumoniae ATCC 13884 related to QS: high synthesis of N-hexanoyl-homoserine lactone, increasing bacterial populations by 27% and biofilm formation by 49%, and a more gentamicin resistant biofilm. Furthermore, it increased the colonization area of urethral catheters. Hydrochlorothiazide showed the biggest increase in the induction of biofilm formation of 51%, and progesterone displayed the greatest ability to provoke bacterial mass adherence but had no effects on K. pneumoniae ATCC 13884 bacterial population growth.

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Attenuation of Pseudomonas aeruginosa Quorum Sensing by Natural Products: Virtual Screening, Evaluation and Biomolecular Interactions

International Journal of Molecular Sciences ◽

10.3390/ijms21062190 ◽

2020 ◽

Vol 21 (6) ◽

pp. 2190 ◽

Cited By ~ 3

Author(s):

Lin Zhong ◽

Vinothkannan Ravichandran ◽

Na Zhang ◽

Hailong Wang ◽

Xiaoying Bian ◽

...

Keyword(s):

Pseudomonas Aeruginosa ◽

Natural Products ◽

Quorum Sensing ◽

Biofilm Formation ◽

Bacterial Species ◽

In Vitro Assays ◽

Biomolecular Interactions ◽

Multi Drug Resistance ◽

In Silico Studies

Natural products play vital roles against infectious diseases since ancient times and most drugs in use today are derived from natural sources. Worldwide, multi-drug resistance becomes a massive threat to the society with increasing mortality. Hence, it is very crucial to identify alternate strategies to control these ‘super bugs’. Pseudomonas aeruginosa is an opportunistic pathogen reported to be resistant to a large number of critically important antibiotics. Quorum sensing (QS) is a cell–cell communication mechanism, regulates the biofilm formation and virulence factors that endow pathogenesis in various bacteria including P. aeruginosa. In this study, we identified and evaluated quorum sensing inhibitors (QSIs) from plant-based natural products against P. aeruginosa. In silico studies revealed that catechin-7-xyloside (C7X), sappanol and butein were capable of interacting with LasR, a LuxR-type quorum sensing regulator of P. aeruginosa. In vitro assays suggested that these QSIs significantly reduced the biofilm formation, pyocyanin, elastase, and rhamnolipid without influencing the growth. Especially, butein reduced the biofilm formation up to 72.45% at 100 µM concentration while C7X and sappanol inhibited the biofilm up to 66% and 54.26% respectively. Microscale thermophoresis analysis revealed that C7X had potential interaction with LasR (KD = 933±369 nM) and thermal shift assay further confirmed the biomolecular interactions. These results suggested that QSIs are able to substantially obstruct the P. aeruginosa QS. Since LuxR-type transcriptional regulator homologues are present in numerous bacterial species, these QSIs may be developed as broad spectrum anti-infectives in the future.

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Evaluation of polysaccharide intercellular adhesion (PIA) and glycerol teichoic acid (Gly-TA) arisen antibodies to prevention of biofilm formation in Staphylococcus aureus and Staphylococcus epidermidis strains

BMC Research Notes ◽

10.1186/s13104-019-4736-8 ◽

2019 ◽

Vol 12 (1) ◽

Cited By ~ 1

Author(s):

Sanaz Amir Gholami ◽

Hamid Reza Goli ◽

Mohammad Reza Haghshenas ◽

Bahman Mirzaei

Keyword(s):

Staphylococcus Aureus ◽

Biofilm Formation ◽

Teichoic Acid ◽

Intercellular Adhesion ◽

Significant Rise ◽

In Vitro Assays ◽

Control Group ◽

Polysaccharide Intercellular Adhesion ◽

Native Condition

Abstract Objective Staphylococcus aureus and S. epidermidis as opportunistic pathogens, notable for their frequency and severity of infections are recognized as the most usual reasons for medical device-associated infections that strike hospitalized patients and also immunocompromised individuals. In this study, the polysaccharide intercellular adhesion (PIA) and Glycerol teichoic acid) Gly-TA) as two major macromolecules in the biofilm formation process were purified under the native condition and their structure was analyzed by using colorimetric assays and Fourier Transform Infrared spectroscopy (FTIR). Afterward, the immune response of macromolecules and the mixture of them were assessed by measuring total IgG titers. Subsequently, biofilm inhibitory effects of raising antibodies to biofilm former S. aureus and S. epidermidis were evaluated. Results Obtained data were shown a significant rise in levels of antibodies in immunized mice with mentioned antibodies in comparison with the control group. According to the obtained findings, mentioned antibodies could eliminate S. aureus and S. epidermidis biofilm formation in vitro assays. This survey confirms the proposal that immunization of mice with a mixture of Gly-TA and PIA vaccine could be secure and protected against S. epidermidis and S. aureus infection.

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