scholarly journals Comparative biofilm assays using Enterococcus faecalis OG1RF identify new determinants of biofilm formation

2021 ◽  
Author(s):  
Julia L. E. Willett ◽  
Jennifer L. Dale ◽  
Lucy M. Kwiatkowski ◽  
Jennifer L. Powers ◽  
Michelle L. Korir ◽  
...  
Keyword(s):  
Enterococcus Faecalis ◽  
Growth Medium ◽  
Biofilm Formation ◽  
Time Course ◽  
Opportunistic Pathogen ◽  
Genetic Determinants ◽  
Experimental Conditions ◽  
Biofilm Reactors ◽  
Biofilm Architecture ◽  
Biofilm Development

AbstractEnterococcus faecalis is a common commensal organism and a prolific nosocomial pathogen that causes biofilm-associated infections. Numerous E. faecalis OG1RF genes required for biofilm formation have been identified, but few studies have compared genetic determinants of biofilm formation and biofilm morphology across multiple conditions. Here, we cultured transposon (Tn) libraries in CDC biofilm reactors in two different media and used Tn sequencing (TnSeq) to identify core and accessory biofilm determinants, including many genes that are poorly characterized or annotated as hypothetical. Multiple secondary assays (96-well plates, submerged Aclar, and MultiRep biofilm reactors) were used to validate phenotypes of new biofilm determinants. We quantified biofilm cells and used fluorescence microscopy to visualize biofilms formed by 6 Tn mutants identified using TnSeq and found that disrupting these genes (OG1RF_10350, prsA, tig, OG1RF_10576, OG1RF_11288, and OG1RF_11456) leads to significant time- and medium-dependent changes in biofilm architecture. Structural predictions revealed potential roles in cell wall homeostasis for OG1RF_10350 and OG1RF_11288 and signaling for OG1RF_11456. Additionally, we identified growth medium-specific hallmarks of OG1RF biofilm morphology. This study demonstrates how E. faecalis biofilm architecture is modulated by growth medium and experimental conditions, and identifies multiple new genetic determinants of biofilm formation.ImportanceE. faecalis is an opportunistic pathogen and a leading cause of hospital-acquired infections, in part due to its ability to form biofilms. A complete understanding of the genes required for E. faecalis biofilm formation as well as specific features of biofilm morphology related to nutrient availability and growth conditions is crucial for understanding how E. faecalis biofilm-associated infections develop and resist treatment in patients. We employed a comprehensive approach to analysis of biofilm determinants by combining TnSeq primary screens with secondary phenotypic validation using diverse biofilm assays. This enabled identification of numerous core (important under many conditions) and accessory (important under specific conditions) biofilm determinants in E. faecalis OG1RF. We found multiple genes whose disruption results in drastic changes to OG1RF biofilm morphology. These results expand our understanding of the genetic requirements for biofilm formation in E. faecalis that affect the time course of biofilm development as well as the response to specific nutritional conditions.

scholarly journals Role ofepaQ, a Previously UncharacterizedEnterococcus faecalisGene, in Biofilm Development and Antimicrobial Resistance

2019 ◽  
Vol 201 (18) ◽  
Author(s):  
Michelle L. Korir ◽  
Jennifer L. Dale ◽  
Gary M. Dunny
Keyword(s):  
Antibiotic Resistance ◽  
Biofilm Formation ◽  
Cell Surface Hydrophobicity ◽  
Opportunistic Pathogen ◽  
Surface Hydrophobicity ◽  
Biofilm Growth ◽  
Content Type ◽  
Biofilm Architecture ◽  
High Level ◽  
Biofilm Development

ABSTRACTEnterococcus faecalisis a commensal of the human gastrointestinal tract; it is also an opportunistic pathogen and one of the leading causes of hospital-acquired infections.E. faecalisproduces biofilms that are highly resistant to antibiotics, and it has been previously reported that certain genes of theepaoperon contribute to biofilm-associated antibiotic resistance. Despite several studies examining theepaoperon, many gene products of this operon remain annotated as hypothetical proteins. Here, we further explore theepaoperon; we identifiedepaQ, currently annotated as encoding a hypothetical membrane protein, as being important for biofilm formation in the presence of the antibiotic daptomycin. Mutants with disruptions ofepaQwere more susceptible to daptomycin relative to the wild type, suggesting its importance in biofilm-associated antibiotic resistance. Furthermore, the ΔepaQmutant exhibited an altered biofilm architectural arrangement and formed small aggregates in liquid cultures. Our cumulative data show thatepamutations result in altered polysaccharide content, increased cell surface hydrophobicity, and decreased membrane potential. Surprisingly, severalepamutations significantly increased resistance to the antibiotic ceftriaxone, indicating that the way in which theepaoperon impacts antibiotic resistance is antibiotic dependent. These results further define the key role ofepain antibiotic resistance in biofilms and in biofilm architecture.IMPORTANCEE. faecalisis a common cause of nosocomial infection, has a high level of antibiotic resistance, and forms robust biofilms. Biofilm formation is associated with increased antibiotic resistance. Therefore, a thorough understanding of biofilm-associated antibiotic resistance is important for combating resistance. Several genes from theepaoperon have previously been implicated in biofilm-associated antibiotic resistance, pathogenesis, and competitive fitness in the GI tract, but most genes in this locus remain uncharacterized. Here, we examineepaQ,which has not been characterized functionally. We show that the ΔepaQmutant exhibits reduced biofilm formation in the presence of daptomycin, altered biofilm architecture, and increased resistance to ceftriaxone, further expanding our understanding of the contribution of this operon to intrinsic enterococcal antibiotic resistance and biofilm growth.

scholarly journals Planktonic Interference and Biofilm Alliance between Aggregation Substance and Endocarditis- and Biofilm-Associated Pili inEnterococcus faecalis

2018 ◽  
Vol 200 (24) ◽  
Author(s):  
Irina Afonina ◽  
Xin Ni Lim ◽  
Rosalind Tan ◽  
Kimberly A. Kline
Keyword(s):  
Enterococcus Faecalis ◽  
Biofilm Formation ◽  
Bacterial Species ◽  
Opportunistic Pathogen ◽  
Plasmid Transfer ◽  
Conjugative Plasmid ◽  
Content Type ◽  
Regulatory Pathways ◽  
Aggregation Substance ◽  
Biofilm Development

ABSTRACTLike many bacteria,Enterococcus faecalisencodes a number of adhesins involved in colonization or infection of different niches. Two well-studiedE. faecalisadhesins, aggregation substance (AS) and endocarditis- and biofilm-associated pili (Ebp), both contribute to biofilm formation on abiotic surfaces and in endocarditis, suggesting that they may be expressed at the same time. Because different regulatory pathways have been reported for AS and Ebp, here, we examined if they are coexpressed on the same cells and what is the functional impact of coexpression on individual cells and within a population. We found that while Ebp are only expressed on a subset of cells, when Ebp and AS are expressed on the same cells, pili interfere with AS-mediated clumping and impede AS-mediated conjugative plasmid transfer during planktonic growth. However, when the population density increases, horizontal gene transfer rates normalize and are no longer affected by pilus expression. Instead, at higher cell densities during biofilm formation, Ebp and AS differentially contribute to biofilm development and structure, synergizing to promote maximal biofilm formation.IMPORTANCEMost bacteria express multiple adhesins that contribute to surface attachment and colonization. However, the network and relationships between the various adhesins of a single bacterial species are less well understood. Here, we examined two well-characterized adhesins inEnterococcus faecalis, aggregation substance and endocarditis- and biofilm-associated pili, and found that they exhibit distinct functional contributions depending on the growth stage of the bacterial community. Pili interfere with aggregation substance-mediated clumping and plasmid transfer under planktonic conditions, whereas the two adhesins structurally complement one another during biofilm development. This study advances our understanding of howE. faecalis, a ubiquitous member of the human gut microbiome and an opportunistic pathogen, uses multiple surface structures to evolve and thrive.

scholarly journals Lon Protease Has Multifaceted Biological Functions inAcinetobacter baumannii

2018 ◽  
Vol 201 (2) ◽  
Author(s):  
Carly Ching ◽  
Brendan Yang ◽  
Chineme Onwubueke ◽  
David Lazinski ◽  
Andrew Camilli ◽  
...  
Keyword(s):  
Acinetobacter Baumannii ◽  
Biofilm Formation ◽  
Developmental Process ◽  
Cell Envelope ◽  
Opportunistic Pathogen ◽  
Lon Protease ◽  
Content Type ◽  
Hospital Acquired Infections ◽  
Biofilm Development ◽  
Hospital Acquired

ABSTRACTAcinetobacter baumanniiis a Gram-negative opportunistic pathogen that is known to survive harsh environmental conditions and is a leading cause of hospital-acquired infections. Specifically, multicellular communities (known as biofilms) ofA. baumanniican withstand desiccation and survive on hospital surfaces and equipment. Biofilms are bacteria embedded in a self-produced extracellular matrix composed of proteins, sugars, and/or DNA. Bacteria in a biofilm are protected from environmental stresses, including antibiotics, which provides the bacteria with selective advantage for survival. Although some gene products are known to play roles in this developmental process inA. baumannii, mechanisms and signaling remain mostly unknown. Here, we find that Lon protease inA. baumanniiaffects biofilm development and has other important physiological roles, including motility and the cell envelope. Lon proteases are found in all domains of life, participating in regulatory processes and maintaining cellular homeostasis. These data reveal the importance of Lon protease in influencing keyA. baumanniiprocesses to survive stress and to maintain viability.IMPORTANCEAcinetobacter baumanniiis an opportunistic pathogen and is a leading cause of hospital-acquired infections.A. baumanniiis difficult to eradicate and to manage, because this bacterium is known to robustly survive desiccation and to quickly gain antibiotic resistance. We sought to investigate biofilm formation inA. baumannii, since much remains unknown about biofilm formation in this bacterium. Biofilms, which are multicellular communities of bacteria, are surface attached and difficult to eliminate from hospital equipment and implanted devices. Our research identifies multifaceted physiological roles for the conserved bacterial protease Lon inA. baumannii. These roles include biofilm formation, motility, and viability. This work broadly affects and expands understanding of the biology ofA. baumannii, which will permit us to find effective ways to eliminate the bacterium.

scholarly journals Interactions between Candida albicans and Enterococcus faecalis in an Organotypic Oral Epithelial Model

2020 ◽  
Vol 8 (11) ◽  
pp. 1771
Author(s):  
Akshaya Lakshmi Krishnamoorthy ◽  
Alex A. Lemus ◽  
Adline Princy Solomon ◽  
Alex M. Valm ◽  
Prasanna Neelakantan
Keyword(s):  
Candida Albicans ◽  
Enterococcus Faecalis ◽  
Biofilm Formation ◽  
Opportunistic Pathogen ◽  
Surface Erosion ◽  
Host Immune System ◽  
Microbial Invasion ◽  
Mucosal Surfaces ◽  
Life Threatening ◽  
Hyphal Formation

Candida albicans as an opportunistic pathogen exploits the host immune system and causes a variety of life-threatening infections. The polymorphic nature of this fungus gives it tremendous advantage to breach mucosal barriers and cause oral and disseminated infections. Similar to C. albicans, Enterococcus faecalis is a major opportunistic pathogen, which is of critical concern in immunocompromised patients. There is increasing evidence that E. faecalis co-exists with C. albicans in the human body in disease samples. While the interactive profiles between these two organisms have been studied on abiotic substrates and mouse models, studies on their interactions on human oral mucosal surfaces are non-existent. Here, for the first time, we comprehensively characterized the interactive profiles between laboratory and clinical isolates of C. albicans (SC5314 and BF1) and E. faecalis (OG1RF and P52S) on an organotypic oral mucosal model. Our results demonstrated that the dual species biofilms resulted in profound surface erosion and significantly increased microbial invasion into mucosal compartments, compared to either species alone. Notably, several genes of C. albicans involved in tissue adhesion, hyphal formation, fungal invasion, and biofilm formation were significantly upregulated in the presence of E. faecalis. By contrast, E. faecalis genes involved in quorum sensing, biofilm formation, virulence, and mammalian cell invasion were downregulated. This study highlights the synergistic cross-kingdom interactions between E. faecalis and C. albicans in mucosal tissue invasion.

scholarly journals Enterococcus faecalis Polymicrobial Interactions Facilitate Biofilm Formation, Antibiotic Recalcitrance, and Persistent Colonization of the Catheterized Urinary Tract

Pathogens ◽  
2020 ◽  
Vol 9 (10) ◽  
pp. 835
Author(s):  
Jordan R. Gaston ◽  
Marissa J. Andersen ◽  
Alexandra O. Johnson ◽  
Kirsten L. Bair ◽  
Christopher M. Sullivan ◽  
...  
Keyword(s):  
Urinary Tract ◽  
Enterococcus Faecalis ◽  
Biofilm Formation ◽  
Pioneer Species ◽  
Urinary Catheters ◽  
Health Care Settings ◽  
Biofilm Architecture ◽  
Prospective Assessment ◽  
Catheter Urine

Indwelling urinary catheters are common in health care settings and can lead to catheter-associated urinary tract infection (CAUTI). Long-term catheterization causes polymicrobial colonization of the catheter and urine, for which the clinical significance is poorly understood. Through prospective assessment of catheter urine colonization, we identified Enterococcus faecalis and Proteus mirabilis as the most prevalent and persistent co-colonizers. Clinical isolates of both species successfully co-colonized in a murine model of CAUTI, and they were observed to co-localize on catheter biofilms during infection. We further demonstrate that P. mirabilis preferentially adheres to E. faecalis during biofilm formation, and that contact-dependent interactions between E. faecalis and P. mirabilis facilitate establishment of a robust biofilm architecture that enhances antimicrobial resistance for both species. E. faecalis may therefore act as a pioneer species on urinary catheters, establishing an ideal surface for persistent colonization by more traditional pathogens such as P. mirabilis.

scholarly journals The Discrepancy between Clove and Non-Clove Cigarette Smoke-Promoted Candida albicans Biofilm Formation with Precoating RNA-aptamer

F1000Research ◽  
2021 ◽  
Vol 10 ◽  
pp. 372
Author(s):  
Boy Muchlis Bachtiar ◽  
Basri A. Gani ◽  
Astri Deviana ◽  
Nastiti Rilo Utami ◽  
Anissa Dien Andriyani ◽  
...  
Keyword(s):  
Cigarette Smoke ◽  
Growth Medium ◽  
Biofilm Formation ◽  
Morphological Changes ◽  
Inhibitory Effect ◽  
Yeast Cells ◽  
Hypha Formation ◽  
Microscopy Analysis ◽  
Light Microscopy Analysis ◽  
Biofilm Development

This study explores the influence of precoating aptamer (Ca-apt1) on C. albicans viability while the fungus was growing in the presence of exposing condensed cigarette smoke (CSC), prepared from clove (CCSC) and non-clove (NCSC) cigarettes, for 48 h. Using qPCR, we found that mRNA expression of adhesion-associated genes (ALS3 and HWP1) was impaired by precoating C. albicans yeast cells with the aptamer. Conversely, the gene transcription was upregulated when aptamer-uncoated yeast was pre-treated with either CSC. In addition, by analysing the result of MTT ([3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide] assay, we found that the presence of added CCSC or NCSC in growth medium for 48 h was significantly enhanced C. albicans biofilm development. However, the presence of precoated aptamer was significantly impaired biofilm development accelerated by the NCSC. The inhibitory effect of the Ca-apt1 was not dependent on the precoated aptamer (1 and 10%). Interestingly, we noted that the enhancer effect of treated CCSC was no longer effective when the yeast had been precoated with 10% aptamer tested. Additionally, light microscopy analysis revealed that precoating aptamer alleviates morphological changes of C. albicans (from yeast to hypha formation) that are enhanced by adding CCSC or NCSC in the growth medium. In conclusion, these results suggest that administration on Ca-ap1 exhibits a significant protective effect on CSC-induced biofilm formation by C. albicans.

scholarly journals Function of BriC Peptide in the Pneumococcal Competence and Virulence Portfolio

2018 ◽  
Author(s):  
Surya D. Aggarwal ◽  
Rory Eutsey ◽  
Jacob West-Roberts ◽  
Arnau Domenech ◽  
Wenjie Xu ◽  
...  
Keyword(s):  
Murine Model ◽  
Biofilm Formation ◽  
Opportunistic Pathogen ◽  
Nasopharyngeal Colonization ◽  
Point Of Entry ◽  
Abiotic Surfaces ◽  
Biofilm Development

AbstractStreptococcus pneumoniae (pneumococcus) is an opportunistic pathogen that causes otitis media, sinusitis, pneumonia, meningitis and sepsis. The progression to this pathogenic lifestyle is preceded by asymptomatic colonization of the nasopharynx. This colonization is associated with biofilm formation; the competence pathway influences the structure and stability of biofilms. However, the molecules that link the competence pathway to biofilm formation are unknown. Here, we describe a new competence-induced gene, called briC, and demonstrate that its product promotes biofilm development and stimulates colonization in a murine model. We show that expression of briC is induced by the master regulator of competence, ComE. Whereas briC does not substantially influence early biofilm development on abiotic surfaces, it significantly impacts later stages of biofilm development. Specifically, briC expression leads to increases in biofilm biomass and thickness at 72h. Consistent with the role of biofilms in colonization, briC promotes nasopharyngeal colonization in the murine model. The function of BriC appears to be conserved across pneumococci, as comparative genomics reveal that briC is widespread across isolates. Surprisingly, many isolates, including strains from clinically important PMEN1 and PMEN14 lineages, which are widely associated with colonization, encode a long briC promoter. This long form captures an instance of genomic plasticity and functions as a competence-independent expression enhancer that may serve as a precocious point of entry into this otherwise competence-regulated pathway. Moreover, overexpression of briC by the long promoter fully rescues the comE-deletion induced biofilm defect in vitro, and partially in vivo. These findings indicate that BriC may bypass the influence of competence in biofilm development and that such a pathway may be active in a subset of pneumococcal lineages. In conclusion, BriC is a part of the complex molecular network that connects signaling of the competence pathway to biofilm development and colonization.

scholarly journals Enterococcus faecalis Polymicrobial Interactions Facilitate Biofilm Formation, Antibiotic Recalcitrance, and Persistent Colonization of the Catheterized Urinary Tract

2020 ◽  
Author(s):  
Jordan Gaston ◽  
Marissa Andersen ◽  
Alexandra Johnson ◽  
Kirsten Bair ◽  
Christopher Sullivan ◽  
...  
Keyword(s):  
Urinary Tract ◽  
Enterococcus Faecalis ◽  
Biofilm Formation ◽  
Pioneer Species ◽  
Urinary Catheters ◽  
Healthcare Settings ◽  
Biofilm Architecture ◽  
Prospective Assessment ◽  
Catheter Urine

Indwelling urinary catheters are common in healthcare settings and can lead to catheter-associated urinary tract infection (CAUTI). Long-term catheterization causes polymicrobial colonization of the catheter and urine, for which the clinical significance is poorly understood. Through prospective assessment of catheter urine colonization, we identified Enterococcus faecalis and Proteus mirabilis as the most prevalent and persistent co-colonizers. Clinical isolates of both species successfully co-colonized in a murine model of CAUTI, and they were observed to co-localize on catheter biofilms during infection. We further demonstrate that P. mirabilis preferentially adheres to E. faecalis during biofilm formation, and that contact-dependent interactions between E. faecalis and P. mirabilis facilitate establishment of a robust biofilm architecture that enhances antimicrobial resistance for both species. E. faecalis may therefore act as a pioneer species on urinary catheters, establishing an ideal surface for persistent colonization by more traditional pathogens such as P. mirabilis.

scholarly journals The SiaABC threonine phosphorylation pathway controls biofilm formation in response to carbon availability in Pseudomonas aeruginosa

PLoS ONE ◽  
2020 ◽  
Vol 15 (11) ◽  
pp. e0241019
Author(s):  
Wee-Han Poh ◽  
Jianqing Lin ◽  
Brendan Colley ◽  
Nicolai Müller ◽  
Boon Chong Goh ◽  
...  
Keyword(s):  
Biofilm Formation ◽  
Critical Role ◽  
Dynamic Modelling ◽  
Opportunistic Pathogen ◽  
Crystallographic Analysis ◽  
Carbon Substrate ◽  
Carbon Availability ◽  
Cellular Aggregation ◽  
Biofilm Development

The critical role of bacterial biofilms in chronic human infections calls for novel anti-biofilm strategies targeting the regulation of biofilm development. However, the regulation of biofilm development is very complex and can include multiple, highly interconnected signal transduction/response pathways, which are incompletely understood. We demonstrated previously that in the opportunistic, human pathogen P. aeruginosa, the PP2C-like protein phosphatase SiaA and the di-guanylate cyclase SiaD control the formation of macroscopic cellular aggregates, a type of suspended biofilms, in response to surfactant stress. In this study, we demonstrate that the SiaABC proteins represent a signal response pathway that functions through a partner switch mechanism to control biofilm formation. We also demonstrate that SiaABCD functionality is dependent on carbon substrate availability for a variety of substrates, and that upon carbon starvation, SiaB mutants show impaired dispersal, in particular with the primary fermentation product ethanol. This suggests that carbon availability is at least one of the key environmental cues integrated by the SiaABCD system. Further, our biochemical, physiological and crystallographic data reveals that the phosphatase SiaA and its kinase counterpart SiaB balance the phosphorylation status of their target protein SiaC at threonine 68 (T68). Crystallographic analysis of the SiaA-PP2C domain shows that SiaA is present as a dimer. Dynamic modelling of SiaA with SiaC suggested that SiaA interacts strongly with phosphorylated SiaC and dissociates rapidly upon dephosphorylation of SiaC. Further, we show that the known phosphatase inhibitor fumonisin inhibits SiaA mediated phosphatase activity in vitro. In conclusion, the present work improves our understanding of how P. aeuruginosa integrates specific environmental conditions, such as carbon availability and surfactant stress, to regulate cellular aggregation and biofilm formation. With the biochemical and structural characterization of SiaA, initial data on the catalytic inhibition of SiaA, and the interaction between SiaA and SiaC, our study identifies promising targets for the development of biofilm-interference drugs to combat infections of this aggressive opportunistic pathogen.

scholarly journals Effects ofBacillusSerine Proteases on the Bacterial Biofilms

2017 ◽  
Vol 2017 ◽  
pp. 1-10 ◽  
Author(s):  
Olga Mitrofanova ◽  
Ayslu Mardanova ◽  
Vladimir Evtugyn ◽  
Lydia Bogomolnaya ◽  
Margarita Sharipova
Keyword(s):  
Biofilm Formation ◽  
Quantitative Methods ◽  
Time Course ◽  
Extracellular Polymeric Substances ◽  
Proteolytic Enzymes ◽  
Opportunistic Pathogen ◽  
Glutamyl Endopeptidase ◽  
Bacterial Cells ◽  
Biofilm Growth ◽  
Tract Infections

Serratia marcescensis an emerging opportunistic pathogen responsible for many hospital-acquired infections including catheter-associated bacteremia and urinary tract and respiratory tract infections. Biofilm formation is one of the mechanisms employed byS. marcescensto increase its virulence and pathogenicity. Here, we have investigated the main steps of the biofilm formation byS. marcescensSR 41-8000. It was found that the biofilm growth is stimulated by the nutrient-rich environment. The time-course experiments showed thatS. marcescenscells adhere to the surface of the catheter and start to produce extracellular polymeric substances (EPS) within the first 2 days of growth. After 7 days,S. marcescensbiofilms maturate and consist of bacterial cells embedded in a self-produced matrix of hydrated EPS. In this study, the effect ofBacillus pumilus3-19 proteolytic enzymes on the structure of 7-day-oldS. marcescensbiofilms was examined. Using quantitative methods and scanning electron microscopy for the detection of biofilm, we demonstrated a high efficacy of subtilisin-like protease and glutamyl endopeptidase in biofilm removal. Enzymatic treatment resulted in the degradation of the EPS components and significant eradication of the biofilms.

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