Unique Features of Mycobacterium abscessus Biofilms Formed in Synthetic Cystic Fibrosis Medium

Characterizing Mycobacterium abscessus complex (MABSC) biofilms under host-relevant conditions is essential to the design of informed therapeutic strategies targeted to this persistent, drug-tolerant, population of extracellular bacilli. Using synthetic cystic fibrosis medium (SCFM) which we previously reported to closely mimic the conditions encountered by MABSC in actual cystic fibrosis (CF) sputum and a new model of biofilm formation, we show that MABSC biofilms formed under these conditions are substantially different from previously reported biofilms grown in standard laboratory media in terms of their composition, gene expression profile and stress response. Extracellular DNA (eDNA), mannose-and glucose-containing glycans and phospholipids, rather than proteins and mycolic acids, were revealed as key extracellular matrix (ECM) constituents holding clusters of bacilli together. None of the environmental cues previously reported to impact biofilm development had any significant effect on SCFM-grown biofilms, most likely reflecting the fact that SCFM is a nutrient-rich environment in which MABSC finds a variety of ways of coping with stresses. Finally, molecular determinants were identified that may represent attractive new targets for the development of adjunct therapeutics targeting MABSC biofilms in persons with CF.

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Effects of pH on the Properties of Membrane Vesicles Including Glucosyltransferase in Streptococcus mutans

Microorganisms ◽

10.3390/microorganisms9112308 ◽

2021 ◽

Vol 9 (11) ◽

pp. 2308

Author(s):

Yusuke Iwabuchi ◽

Tomoyo Nakamura ◽

Yasuka Kusumoto ◽

Ryoma Nakao ◽

Tsutomu Iwamoto ◽

...

Keyword(s):

Streptococcus Mutans ◽

Biofilm Formation ◽

Membrane Vesicles ◽

Extracellular Dna ◽

Tooth Surface ◽

Initial Ph ◽

Effects Of Ph ◽

Low Levels ◽

Quantitative Changes ◽

Biofilm Development

Streptococcus mutans releases membrane vesicles (MVs) and induces MV-dependent biofilm formation. Glucosyltransferases (Gtfs) are bound to MVs and contribute to the adhesion and glucans-dependent biofilm formation of early adherent bacteria on the tooth surface. The biofilm formation of S. mutans may be controlled depending on whether the initial pH tends to be acidic or alkaline. In this study, the characteristics and effects of MVs extracted from various conditions {(initial pH 6.0 and 8.0 media prepared with lactic acid (LA) and acetic acid (AA), and with NaOH (NO), respectively)} on the biofilm formation of S. mutans and early adherent bacteria were investigated. The quantitative changes in glucans between primary pH 6.0 and 8.0 conditions were observed, associated with different activities affecting MV-dependent biofilm formation. The decreased amount of Gtfs on MVs under the initial pH 6.0 conditions strongly guided low levels of MV-dependent biofilm formation. However, in the initial pH 6.0 and 8.0 solutions prepared with AA and NO, the MVs in the biofilm appeared to be formed by the expression of glucans and/or extracellular DNA. These results suggest that the environmental pH conditions established by acid and alkaline factors determine the differences in the local pathogenic activities of biofilm development in the oral cavity.

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Environment in the lung of cystic fibrosis patients stimulates the expression of biofilm phenotype in Mycobacterium abscessus

Journal of Medical Microbiology ◽

10.1099/jmm.0.001467 ◽

2022 ◽

Vol 71 (1) ◽

Author(s):

Bailey F. Keefe ◽

Luiz E. Bermudez

Keyword(s):

Cystic Fibrosis ◽

Host Cell ◽

Biofilm Formation ◽

Type Species ◽

Divalent Cations ◽

Mycobacterium Abscessus ◽

Content Type ◽

Link Type ◽

Populations At Risk

Introduction. Pulmonary infections caused by organisms of the Mycobacterium abscessus complex are increasingly prevalent in populations at risk, such as patients with cystic fibrosis, bronchiectasis and emphysema. Hypothesis. M. abscessus infection of the lung is not observed in immunocompetent individuals, which raises the possibility that the compromised lung environment is a suitable niche for the pathogen to thrive in due to the overproduction of mucus and high amounts of host cell lysis. Aim. Evaluate the ability of M. abscessus to form biofilm and grow utilizing in vitro conditions as seen in immunocompromised lungs of patients. Methodology. We compared biofilm formation and protein composition in the presence and absence of synthetic cystic fibrosis medium (SCFM) and evaluated the bacterial growth when exposed to human DNA. Results. M. abscessus is capable of forming biofilm in SCFM. By eliminating single components found in the medium, it became clear that magnesium works as a signal for the biofilm formation, and chelation of the divalent cations resulted in the suppression of biofilm formation. Investigation of the specific proteins expressed in the presence of SCFM and in the presence of SCFM lacking magnesium revealed many different proteins between the conditions. M. abscessus also exhibited growth in SCFM and in the presence of host cell DNA, although the mechanism of DNA utilization remains unclear. Conclusions. In vitro conditions mimicking the airways of patients with cystic fibrosis appear to facilitate M. abscessus establishment of infection, and elimination of magnesium from the environment may affect the ability of the pathogen to establish infection.

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Biofilm Formation by Streptococcus pneumoniae: Role of Choline, Extracellular DNA, and Capsular Polysaccharide in Microbial Accretion

Journal of Bacteriology ◽

10.1128/jb.00673-06 ◽

2006 ◽

Vol 188 (22) ◽

pp. 7785-7795 ◽

Cited By ~ 213

Author(s):

Miriam Moscoso ◽

Ernesto García ◽

Rubens López

Keyword(s):

Streptococcus Pneumoniae ◽

Biofilm Formation ◽

Laser Scanning Microscopy ◽

Extracellular Dna ◽

Dimensional Structure ◽

Confocal Laser ◽

Upper Respiratory Tract ◽

Teichoic Acids ◽

Abiotic Surfaces ◽

Biofilm Development

ABSTRACTStreptococcus pneumoniaecolonizes the human upper respiratory tract, and this asymptomatic colonization is known to precede pneumococcal disease. In this report, chemically defined and semisynthetic media were used to identify the initial steps of biofilm formation by pneumococcus during growth on abiotic surfaces such as polystyrene or glass. Unencapsulated pneumococci adhered to abiotic surfaces and formed a three-dimensional structure about 25 μm deep, as observed by confocal laser scanning microscopy and low-temperature scanning electron microscopy. Choline residues of cell wall teichoic acids were found to play a fundamental role in pneumococcal biofilm development. The role in biofilm formation of choline-binding proteins, which anchor to the teichoic acids of the cell envelope, was determined using unambiguously characterized mutants. The results showed that LytA amidase, LytC lysozyme, LytB glucosaminidase, CbpA adhesin, PcpA putative adhesin, and PspA (pneumococcal surface protein A) mutants had a decreased capacity to form biofilms, whereas no such reduction was observed in Pce phosphocholinesterase or CbpD putative amidase mutants. Moreover, encapsulated, clinical pneumococcal isolates were impaired in their capacity to form biofilms. In addition, a role for extracellular DNA and proteins in the establishment ofS. pneumoniaebiofilms was demonstrated. Taken together, these observations provide information on conditions that favor the sessile mode of growth byS. pneumoniae. The experimental approach described here should facilitate the study of bacterial genes that are required for biofilm formation. Those results, in turn, may provide insight into strategies to prevent pneumococcal colonization of its human host.

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Extracellular DNA and Type IV Pilus Expression Regulate the Structure and Kinetics of Biofilm Formation by Nontypeable Haemophilus influenzae

mBio ◽

10.1128/mbio.01466-17 ◽

2017 ◽

Vol 8 (6) ◽

Cited By ~ 16

Author(s):

Jayajit Das ◽

Elaine Mokrzan ◽

Vinal Lakhani ◽

Lucia Rosas ◽

Joseph A. Jurcisek ◽

...

Keyword(s):

Middle Ear ◽

In Silico ◽

Biofilm Formation ◽

Rational Design ◽

Bacterial Biofilm ◽

Confocal Imaging ◽

Extracellular Dna ◽

Fractal Structures ◽

Biofilm Development

ABSTRACT Biofilms formed in the middle ear by nontypeable Haemophilus influenzae (NTHI) are central to the chronicity, recurrence, and refractive nature of otitis media (OM). However, mechanisms that underlie the emergence of specific NTHI biofilm structures are unclear. We combined computational analysis tools and in silico modeling rooted in statistical physics with confocal imaging of NTHI biofilms formed in vitro during static culture in order to identify mechanisms that give rise to distinguishing morphological features. Our analysis of confocal images of biofilms formed by NTHI strain 86-028NP using pair correlations of local bacterial densities within sequential planes parallel to the substrate showed the presence of fractal structures of short length scales (≤10 μm). The in silico modeling revealed that extracellular DNA (eDNA) and type IV pilus (Tfp) expression played important roles in giving rise to the fractal structures and allowed us to predict a substantial reduction of these structures for an isogenic mutant (ΔcomE) that was significantly compromised in its ability to release eDNA into the biofilm matrix and had impaired Tfp function. This prediction was confirmed by analysis of confocal images of in vitro ΔcomE strain biofilms. The fractal structures potentially generate niches for NTHI survival in the hostile middle ear microenvironment by dramatically increasing the contact area of the biofilm with the surrounding environment, facilitating nutrient exchange, and by generating spatial positive feedback to quorum signaling. IMPORTANCE NTHI is a major bacterial pathogen for OM, which is a common ear infection in children worldwide. Chronic OM is associated with bacterial biofilm formation in the middle ear; therefore, knowledge of the mechanisms that underlie NTHI biofilm formation is important for the development of therapeutic strategies for NTHI-associated OM. Our combined approach using confocal imaging of NTHI biofilms formed in vitro and mathematical tools for analysis of pairwise density correlations and agent-based modeling revealed that eDNA and Tfp expression were important factors in the development of fractal structures in NTHI biofilms. These structures may help NTHI survive in hostile environments, such as the middle ear. Our in silico model can be used in combination with laboratory or animal modeling studies to further define the mechanisms that underlie NTHI biofilm development during OM and thereby guide the rational design of, and optimize time and cost for, benchwork and preclinical studies. IMPORTANCE NTHI is a major bacterial pathogen for OM, which is a common ear infection in children worldwide. Chronic OM is associated with bacterial biofilm formation in the middle ear; therefore, knowledge of the mechanisms that underlie NTHI biofilm formation is important for the development of therapeutic strategies for NTHI-associated OM. Our combined approach using confocal imaging of NTHI biofilms formed in vitro and mathematical tools for analysis of pairwise density correlations and agent-based modeling revealed that eDNA and Tfp expression were important factors in the development of fractal structures in NTHI biofilms. These structures may help NTHI survive in hostile environments, such as the middle ear. Our in silico model can be used in combination with laboratory or animal modeling studies to further define the mechanisms that underlie NTHI biofilm development during OM and thereby guide the rational design of, and optimize time and cost for, benchwork and preclinical studies.

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The exopolysaccharide–eDNA interaction modulates 3D architecture of Bacillus subtilis biofilm

10.21203/rs.2.18238/v3 ◽

2020 ◽

Author(s):

Na Peng ◽

Peng Cai ◽

Monika Mortimer ◽

Yichao Wu ◽

Chunhui Gao ◽

...

Keyword(s):

Biofilm Formation ◽

Laser Scanning ◽

Laser Scanning Microscopy ◽

Extracellular Dna ◽

Titration Calorimetry ◽

Confocal Laser ◽

Physical Interaction ◽

Matrix Components ◽

Biofilm Development ◽

Early Phases

Abstract Background Bacterial biofilms are a surface-adherent microbial community in which individual cells are surrounded by a self-produced extracellular matrix of polysaccharide, extracellular DNA (eDNA) and proteins. Interactions among matrix components within the biofilms are responsible for creating an adaptable structure during biofilm development. However, it is unclear how the interaction among matrix components contributes to the construction of the three-dimensional (3D) biofilm architecture. Results DNase I treatment could significantly inhibit B. subtilis biofilm formation in early phases. Confocal laser scanning microscopy (CLSM) and image analysis revealed that eDNA was cooperative with exopolysaccharide (EPS) in early stages of B. subtilis biofilm development, while EPS played a major structural role in the later stages. In addition, deletion of EPS production gene epsG in B. subtilis SBE1 resulted in loss of the interaction between EPS and eDNA, and reduction of biofilm biomass in pellicles at air-liquid interface. The physical interaction between these two essential biofilm matrix components was confirmed by isothermal titration calorimetry (ITC). Conclusions The biofilm 3D structures become interconnected through surrounding eDNA and EPS. eDNA interacts with EPS in the early phases of biofilm development, while EPS mainly participates in the maturation of biofilm. The findings of this study provide better understanding of the role of interaction between eDNA and EPS in shaping the biofilm 3D matrix structure and biofilm formation.

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The Antibacterial and Anti-Biofilm Activity of Metal Complexes Incorporating 3,6,9-Trioxaundecanedioate and 1,10-Phenanthroline Ligands in Clinical Isolates of Pseudomonas aeruginosa from Irish Cystic Fibrosis Patients

Antibiotics ◽

10.3390/antibiotics9100674 ◽

2020 ◽

Vol 9 (10) ◽

pp. 674

Author(s):

Megan O’Shaughnessy ◽

Pauraic McCarron ◽

Livia Viganor ◽

Malachy McCann ◽

Michael Devereux ◽

...

Keyword(s):

Cystic Fibrosis ◽

Pseudomonas Aeruginosa ◽

Biofilm Formation ◽

Post Treatment ◽

Extracellular Dna ◽

Clinical Isolates ◽

Reference Laboratory ◽

Synergistic Activity ◽

Chronic Infections ◽

The Individual

Chronic infections of Pseudomonas aeruginosa in the lungs of cystic fibrosis (CF) patients are problematic in Ireland where inherited CF is prevalent. The bacteria’s capacity to form a biofilm in its pathogenesis is highly virulent and leads to decreased susceptibility to most antibiotic treatments. Herein, we present the activity profiles of the Cu(II), Mn(II) and Ag(I) tdda-phen chelate complexes {[Cu(3,6,9-tdda)(phen)2]·3H2O·EtOH}n (Cu-tdda-phen), {[Mn(3,6,9-tdda)(phen)2]·3H2O·EtOH}n (Mn-tdda-phen) and [Ag2(3,6,9-tdda)(phen)4]·EtOH (Ag-tdda-phen) (tddaH2 = 3,6,9-trioxaundecanedioic acid; phen = 1,10-phenanthroline) towards clinical isolates of P. aeruginosa derived from Irish CF patients in comparison to two reference laboratory strains (ATCC 27853 and PAO1). The effects of the metal-tdda-phen complexes and gentamicin on planktonic growth, biofilm formation (pre-treatment) and mature biofilm (post-treatment) alone and in combination were investigated. The effects of the metal-tdda-phen complexes on the individual biofilm components; exopolysaccharide, extracellular DNA (eDNA), pyocyanin and pyoverdine are also presented. All three metal-tdda-phen complexes showed comparable and often superior activity to gentamicin in the CF strains, compared to their activities in the laboratory strains, with respect to both biofilm formation and established biofilms. Combination studies presented synergistic activity between all three complexes and gentamicin, particularly for the post-treatment of established mature biofilms, and was supported by the reduction of the individual biofilm components examined.

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Subinhibitory Concentrations of Mupirocin Stimulate Staphylococcus aureus Biofilm Formation by Upregulating cidA

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01912-19 ◽

2020 ◽

Vol 64 (3) ◽

Cited By ~ 3

Author(s):

Ye Jin ◽

Yinjuan Guo ◽

Qing Zhan ◽

Yongpeng Shang ◽

Di Qu ◽

...

Keyword(s):

Staphylococcus Aureus ◽

Biofilm Formation ◽

Laser Scanning ◽

Multidrug Resistant ◽

Laser Scanning Microscopy ◽

Extracellular Dna ◽

Confocal Laser ◽

Content Type ◽

Subinhibitory Concentrations ◽

Biofilm Development

ABSTRACT Previous studies have shown that the administration of antibiotics at subinhibitory concentrations stimulates biofilm formation by the majority of multidrug-resistant Staphylococcus aureus (MRSA) strains. Here, we investigated the effect of subinhibitory concentrations of mupirocin on biofilm formation by the community-associated (CA) mupirocin-sensitive MRSA strain USA300 and the highly mupirocin-resistant clinical S. aureus SA01 to SA05 isolates. We found that mupirocin increased the ability of MRSA cells to attach to surfaces and form biofilms. Confocal laser scanning microscopy (CLSM) demonstrated that mupirocin treatment promoted thicker biofilm formation, which also correlated with the production of extracellular DNA (eDNA). Furthermore, quantitative real-time PCR (RT-qPCR) results revealed that this effect was largely due to the involvement of holin-like and antiholin-like proteins (encoded by the cidA gene), which are responsible for modulating cell death and lysis during biofilm development. We found that cidA expression levels significantly increased by 6.05- to 35.52-fold (P < 0.01) after mupirocin administration. We generated a cidA-deficient mutant of the USA300 S. aureus strain. Exposure of the ΔcidA mutant to mupirocin did not result in thicker biofilm formation than that in the parent strain. We therefore hypothesize that the mupirocin-induced stimulation of S. aureus biofilm formation may involve the upregulation of cidA.

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Staphylococcal Biofilm Development: Structure, Regulation, and Treatment Strategies

Microbiology and Molecular Biology Reviews ◽

10.1128/mmbr.00026-19 ◽

2020 ◽

Vol 84 (3) ◽

Cited By ~ 5

Author(s):

Katrin Schilcher ◽

Alexander R. Horswill

Keyword(s):

Extracellular Matrix ◽

Biofilm Formation ◽

Three Dimensional ◽

Treatment Strategies ◽

Extracellular Dna ◽

Content Type ◽

Structural And Functional Properties ◽

Characteristic Features ◽

Biofilm Development ◽

Development Structure

SUMMARY In many natural and clinical settings, bacteria are associated with some type of biotic or abiotic surface that enables them to form biofilms, a multicellular lifestyle with bacteria embedded in an extracellular matrix. Staphylococcus aureus and Staphylococcus epidermidis, the most frequent causes of biofilm-associated infections on indwelling medical devices, can switch between an existence as single free-floating cells and multicellular biofilms. During biofilm formation, cells first attach to a surface and then multiply to form microcolonies. They subsequently produce the extracellular matrix, a hallmark of biofilm formation, which consists of polysaccharides, proteins, and extracellular DNA. After biofilm maturation into three-dimensional structures, the biofilm community undergoes a disassembly process that leads to the dissemination of staphylococcal cells. As biofilms are dynamic and complex biological systems, staphylococci have evolved a vast network of regulatory mechanisms to modify and fine-tune biofilm development upon changes in environmental conditions. Thus, biofilm formation is used as a strategy for survival and persistence in the human host and can serve as a reservoir for spreading to new infection sites. Moreover, staphylococcal biofilms provide enhanced resilience toward antibiotics and the immune response and impose remarkable therapeutic challenges in clinics worldwide. This review provides an overview and an updated perspective on staphylococcal biofilms, describing the characteristic features of biofilm formation, the structural and functional properties of the biofilm matrix, and the most important mechanisms involved in the regulation of staphylococcal biofilm formation. Finally, we highlight promising strategies and technologies, including multitargeted or combinational therapies, to eradicate staphylococcal biofilms.

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Pseudomonas aeruginosa leucine aminopeptidase influences early biofilm composition and structure via vesicle-associated anti-biofilm activity

10.1101/784918 ◽

2019 ◽

Author(s):

Caitlin N. Esoda ◽

Meta J. Kuehn

Keyword(s):

Cystic Fibrosis ◽

Pseudomonas Aeruginosa ◽

Outer Membrane ◽

Biofilm Formation ◽

Leucine Aminopeptidase ◽

Membrane Vesicles ◽

Outer Membrane Vesicles ◽

Bacterial Biofilm ◽

Biofilm Architecture ◽

Biofilm Development

AbstractPseudomonas aeruginosa, known as one of the leading causes of disease in cystic fibrosis (CF) patients, secretes a variety of proteases. These enzymes contribute significantly to P. aeruginosa pathogenesis and biofilm formation in the chronic colonization of CF patient lungs, as well as playing a role in infections of the cornea, burn wounds and chronic wounds. We previously characterized a secreted P. aeruginosa peptidase, PaAP, that is highly expressed in chronic CF isolates. This leucine aminopeptidase is highly expressed during infection and in biofilms, and it associates with bacterial outer membrane vesicles (OMVs), structures known to contribute to virulence mechanisms in a variety of Gram-negative species and one of the major components of the biofilm matrix. We hypothesized that PaAP may play a role in P. aeruginosa biofilm formation. Using a lung epithelial cell/bacterial biofilm coculture model, we show that PaAP deletion in a clinical P. aeruginosa background alters biofilm microcolony composition to increase cellular density, while decreasing matrix polysaccharide content, and that OMVs from PaAP expressing strains but not PaAP alone or in combination with PaAP deletion strain-derived OMVs could complement this phenotype. We additionally found that OMVs from PaAP expressing strains could cause protease-mediated biofilm detachment, leading to changes in matrix and colony composition. Finally, we showed that the OMVs could also mediate the detachment of biofilms formed by both non-self P. aeruginosa strains and Klebsiella pneumoniae, another respiratory pathogen. Our findings represent novel roles for OMVs and the aminopeptidase in the modulation of P. aeruginosa biofilm architecture.ImportanceBiofilm formation by the bacterial pathogen P. aeruginosa is known to contribute to drug- resistance in nosocomial infections and chronic lung infections of cystic fibrosis patients. In order to treat these infections more successfully, the mechanisms of bacterial biofilm development must be elucidated. While both bacterially-secreted aminopeptidase and outer membrane vesicles have been shown to be abundant in P. aeruginosa biofilm matrices, the contributions of each of these factors to the steps in biofilm generation have not been well studied. This work provides new insight as to how these bacterial components mediate the formation of a robust, drug-resistant extracellular matrix and implicates outer membrane vesicles as active components of biofilm architecture, expanding our overall understanding of P. aeruginosa biofilm biology.

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Biofilm formation in the life cycle of the cellulolytic actinomycete Thermobifida fusca

Biofilms ◽

10.1017/s1479050508002238 ◽

2008 ◽

pp. 1-11 ◽

Cited By ~ 4

Author(s):

A. N. Alonso ◽

P. J. Pomposiello ◽

S. B. Leschine

Keyword(s):

Life Cycle ◽

Biofilm Formation ◽

Cellulose Degradation ◽

Extracellular Dna ◽

Thermobifida Fusca ◽

General Strategy ◽

Dialysis Tubing ◽

Biofilm Production ◽

Mycelial Pellets ◽

Biofilm Development

ABSTRACTActinomycetes have been used with enormous success in industrial processes; however, little is known about biofilm formation by these filamentous microbes, or community development on insoluble substrates such as cellulose. We hypothesized that biofilm formation is a general strategy used by actinomycetes in the degradation of cellulose, and that it may serve as a means for these microbes to secure nutrients and persist in their environments. The objective of this study was to examine biofilm production byThermobifida fusca, an actinomycete that rapidly degrades cellulose by means of a well-characterized extracellular cellulase system.Thermobifida fuscacells grew as biofilms attached to both nutritive (e.g. dialysis tubing membrane) and non-nutritive surfaces. Dialysis tubing was colonized byT. fuscaaleuriospores but not by mycelial pellets, except when mycelial pellets were disrupted by sonication. Microscopic examination of surface-attached growth revealed structures characteristic of biofilms, with cells embedded in fibrous material suggestive of an extracellular polymeric matrix. Concanavalin A bound to the extracellular polymeric substance of biofilms and mycelial pellets, indicating alpha-linkedd-mannosyl and/or alpha-linkedd-glucosyl residues. The carbohydrate content of both biofilms and mycelial pellets increased during growth. Also, DNase I inhibited biofilm production, suggesting a role for extracellular DNA inT. fuscabiofilm development. Cellulose degradation and expression ofcelE(encoding endoglucanase E5) was similar forT. fuscabiofilms and mycelial pellets. Results of this study indicate that, in the life cycle of this actinomycete, cellulose is specifically colonized by aleuriospores, which germinate, grow and degrade cellulose, ultimately developing into biofilms encased in a carbohydrate-containing exopolymeric matrix, a hallmark of biofilm production.

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