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Open AccessArticle

Insights on Klebsiella pneumoniae Biofilms Assembled on Different Surfaces Using Phenotypic and Genotypic Approaches

by Maria Bandeira, Vítor Borges, João P. Gomes, Aida Duarte and Luisa Jordao

Microorganisms 2017, 5(2), 16; https://doi.org/10.3390/microorganisms5020016 - 03 Apr 2017

Cited by 22 | Viewed by 6431

Klebsiella pneumoniae is a prominent etiological agent of healthcare associated infections (HAIs). In this context, multidrug-resistant and biofilm-producing bacteria are of special public health concern due to the difficulties associated with treatment of human infections and eradication from hospital environments. Here, in order [...] Read more.

Klebsiella pneumoniae is a prominent etiological agent of healthcare associated infections (HAIs). In this context, multidrug-resistant and biofilm-producing bacteria are of special public health concern due to the difficulties associated with treatment of human infections and eradication from hospital environments. Here, in order to study the impact of medical devices-associated materials on the biofilm dynamics, we performed biofilm phenotypic analyses through a classic and a new scanning electron microscopy (SEM) technique for three multidrug-resistant K. pneumoniae isolates growing on polystyrene and silicone. We also applied whole-genome sequencing (WGS) to search for genetic clues underlying biofilm phenotypic differences. We found major differences in the extracellular polymeric substances (EPS) content among the three strains, which were further corroborated by in-depth EPS composition analysis. WGS analysis revealed a high nucleotide similarity within the core-genome, but relevant differences in the accessory genome that may account for the detected biofilm phenotypic dissimilarities, such as genes already associated with biofilm formation in other pathogenic bacteria (e.g., genes coding haemogglutinins and haemolysins). These data reinforce that the research efforts to defeat bacterial biofilms should take into account that their dynamics may be contingent on the medical devices-associated materials. Full article

(This article belongs to the Special Issue The Biofilm Matrix)

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10156 KiB

Open AccessArticle

Presence of Calcium Lowers the Expansion of Bacillus subtilis Colony Biofilms

by Eisha Mhatre, Anandaroopan Sundaram, Theresa Hölscher, Mike Mühlstädt, Jörg Bossert and Ákos T. Kovács

Microorganisms 2017, 5(1), 7; https://doi.org/10.3390/microorganisms5010007 - 16 Feb 2017

Cited by 26 | Viewed by 8747

Abstract

Robust colony formation by Bacillus subtilis is recognized as one of the sessile, multicellular lifestyles of this bacterium. Numerous pathways and genes are responsible for the architecturally complex colony structure development. Cells in the biofilm colony secrete extracellular polysaccharides (EPS) and protein components [...] Read more.

Robust colony formation by Bacillus subtilis is recognized as one of the sessile, multicellular lifestyles of this bacterium. Numerous pathways and genes are responsible for the architecturally complex colony structure development. Cells in the biofilm colony secrete extracellular polysaccharides (EPS) and protein components (TasA and the hydrophobin BslA) that hold them together and provide a protective hydrophobic shield. Cells also secrete surfactin with antimicrobial as well as surface tension reducing properties that aid cells to colonize the solid surface. Depending on the environmental conditions, these secreted components of the colony biofilm can also promote the flagellum-independent surface spreading of B. subtilis, called sliding. In this study, we emphasize the influence of Ca²⁺ in the medium on colony expansion of B. subtilis. Interestingly, the availability of Ca²⁺ has no major impact on the induction of complex colony morphology. However, in the absence of this divalent ion, peripheral cells of the colony expand radially at later stages of development, causing colony size to increase. We demonstrate that the secreted extracellular compounds, EPS, BslA, and surfactin facilitate colony expansion after biofilm maturation. We propose that Ca²⁺ hinders biofilm colony expansion by modifying the amphiphilic properties of surfactin. Full article

(This article belongs to the Special Issue The Biofilm Matrix)

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2591 KiB

Open AccessArticle

Fluorescence Lectin Bar-Coding of Glycoconjugates in the Extracellular Matrix of Biofilm and Bioaggregate Forming Microorganisms

by Thomas R. Neu and Ute Kuhlicke

Microorganisms 2017, 5(1), 5; https://doi.org/10.3390/microorganisms5010005 - 10 Feb 2017

Cited by 41 | Viewed by 8298

Abstract

Microbial biofilm systems are defined as interface-associated microorganisms embedded into a self-produced matrix. The extracellular matrix represents a continuous challenge in terms of characterization and analysis. The tools applied in more detailed studies comprise extraction/chemical analysis, molecular characterization, and visualisation using various techniques. [...] Read more.

Microbial biofilm systems are defined as interface-associated microorganisms embedded into a self-produced matrix. The extracellular matrix represents a continuous challenge in terms of characterization and analysis. The tools applied in more detailed studies comprise extraction/chemical analysis, molecular characterization, and visualisation using various techniques. Imaging by laser microscopy became a standard tool for biofilm analysis, and, in combination with fluorescently labelled lectins, the glycoconjugates of the matrix can be assessed. By employing this approach a wide range of pure culture biofilms from different habitats were examined using the commercially available lectins. From the results, a binary barcode pattern of lectin binding can be generated. Furthermore, the results can be fine-tuned and transferred into a heat map according to signal intensity. The lectin barcode approach is suggested as a useful tool for investigating the biofilm matrix characteristics and dynamics at various levels, e.g. bacterial cell surfaces, adhesive footprints, individual microcolonies, and the gross biofilm or bio-aggregate. Hence fluorescence lectin bar-coding (FLBC) serves as a basis for a subsequent tailor-made fluorescence lectin-binding analysis (FLBA) of a particular biofilm. So far, the lectin approach represents the only tool for in situ characterization of the glycoconjugate makeup in biofilm systems. Furthermore, lectin staining lends itself to other fluorescence techniques in order to correlate it with cellular biofilm constituents in general and glycoconjugate producers in particular. Full article

(This article belongs to the Special Issue The Biofilm Matrix)

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Open AccessArticle

Streptokinase Treatment Reverses Biofilm-Associated Antibiotic Resistance in Staphylococcus aureus

by Nis Pedersen Jørgensen, Natalia Zobek, Cindy Dreier, Jakob Haaber, Hanne Ingmer, Ole Halfdan Larsen and Rikke L. Meyer

Microorganisms 2016, 4(3), 36; https://doi.org/10.3390/microorganisms4030036 - 20 Sep 2016

Cited by 15 | Viewed by 5973

Abstract

Biofilms formed by Staphylococcus aureus is a serious complication to the use of medical implants. A central part of the pathogenesis relies on S. aureus’ ability to adhere to host extracellular matrix proteins, which adsorb to medical implants and stimulate biofilm formation. Being [...] Read more.

Biofilms formed by Staphylococcus aureus is a serious complication to the use of medical implants. A central part of the pathogenesis relies on S. aureus’ ability to adhere to host extracellular matrix proteins, which adsorb to medical implants and stimulate biofilm formation. Being coagulase positive, S. aureus furthermore induces formation of fibrin fibers from fibrinogen in the blood. Consequently, we hypothesized that fibrin is a key component of the extracellular matrix of S. aureus biofilms under in vivo conditions, and that the recalcitrance of biofilm infections can be overcome by combining antibiotic treatment with a fibrinolytic drug. We quantified S. aureus USA300 biofilms grown on peg-lids in brain heart infusion (BHI) broth with 0%–50% human plasma. Young (2 h) and mature (24 h) biofilms were then treated with streptokinase to determine if this lead to dispersal. Then, the minimal biofilm eradication concentration (MBEC) of 24 h old biofilms was measured for vancomycin and daptomycin alone or in combination with 10 µg/mL rifampicin in the presence or absence of streptokinase in the antibiotic treatment step. Finally, biofilms were visualized by confocal laser scanning microscopy. Addition of human plasma stimulated biofilm formation in BHI in a dose-dependent manner, and biofilms could be partially dispersed by streptokinase. The biofilms could be eradicated with physiologically relevant concentrations of streptokinase in combination with rifampicin and vancomycin or daptomycin, which are commonly used antibiotics for treatment of S. aureus infections. Fibronolytic drugs have been used to treat thromboembolic events for decades, and our findings suggest that their use against biofilm infections has the potential to improve the efficacy of antibiotics in treatment of S. aureus biofilm infections. Full article

(This article belongs to the Special Issue The Biofilm Matrix)

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Open AccessArticle

Achromobacter Species Isolated from Cystic Fibrosis Patients Reveal Distinctly Different Biofilm Morphotypes

by Signe M. Nielsen, Niels Nørskov-Lauritsen, Thomas Bjarnsholt and Rikke L. Meyer

Microorganisms 2016, 4(3), 33; https://doi.org/10.3390/microorganisms4030033 - 14 Sep 2016

Cited by 34 | Viewed by 7406

Abstract

Achromobacter species have attracted attention as emerging pathogens in cystic fibrosis. The clinical significance of Achromobacter infection is not yet fully elucidated; however, their intrinsic resistance to antimicrobials and ability to form biofilms renders them capable of establishing long-term chronic infections. Still, many [...] Read more.

Achromobacter species have attracted attention as emerging pathogens in cystic fibrosis. The clinical significance of Achromobacter infection is not yet fully elucidated; however, their intrinsic resistance to antimicrobials and ability to form biofilms renders them capable of establishing long-term chronic infections. Still, many aspects of Achromobacter biofilm formation remain uncharacterized. In this study, we characterized biofilm formation in clinical isolates of Achromobacter and investigated the effect of challenging the biofilm with antimicrobials and/or enzymes targeting the extracellular matrix. In vitro biofilm growth and subsequent visualization by confocal microscopy revealed distinctly different biofilm morphotypes: a surface-attached biofilm morphotype of small aggregates and an unattached biofilm morphotype of large suspended aggregates. Aggregates consistent with our in vitro findings were visualized in sputum samples from cystic fibrosis patients using an Achromobacter specific peptide nucleic acid fluorescence in situ hybridization (PNA-FISH) probe, confirming the presence of Achromobacter biofilms in the CF lung. High antibiotic tolerance was associated with the biofilm phenotype, and biocidal antibiotic concentrations were up to 1000 fold higher than for planktonic cultures. Treatment with DNase or subtilisin partially dispersed the biofilm and reduced the tolerance to specific antimicrobials, paving the way for further research into using dispersal mechanisms to improve treatment strategies. Full article

(This article belongs to the Special Issue The Biofilm Matrix)

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3374 KiB

Open AccessArticle

Biofilms from Klebsiella pneumoniae: Matrix Polysaccharide Structure and Interactions with Antimicrobial Peptides

by Monica Benincasa, Cristina Lagatolla, Lucilla Dolzani, Annalisa Milan, Sabrina Pacor, Gianfranco Liut, Alessandro Tossi, Paola Cescutti and Roberto Rizzo

Microorganisms 2016, 4(3), 26; https://doi.org/10.3390/microorganisms4030026 - 10 Aug 2016

Cited by 16 | Viewed by 6018

Abstract

Biofilm matrices of two Klebsiella pneumoniae clinical isolates, KpTs101 and KpTs113, were investigated for their polysaccharide composition and protective effects against antimicrobial peptides. Both strains were good biofilm producers, with KpTs113 forming flocs with very low adhesive properties to supports. Matrix exopolysaccharides were [...] Read more.

Biofilm matrices of two Klebsiella pneumoniae clinical isolates, KpTs101 and KpTs113, were investigated for their polysaccharide composition and protective effects against antimicrobial peptides. Both strains were good biofilm producers, with KpTs113 forming flocs with very low adhesive properties to supports. Matrix exopolysaccharides were isolated and their monosaccharide composition and glycosidic linkage types were defined. KpTs101 polysaccharide is neutral and composed only of galactose, in both pyranose and furanose ring configurations. Conversely, KpTs113 polysaccharide is anionic due to glucuronic acid units, and also contains glucose and mannose residues. The susceptibility of the two strains to two bovine cathelicidin antimicrobial peptides, BMAP-27 and Bac7(1–35), was assessed using both planktonic cultures and biofilms. Biofilm matrices exerted a relevant protection against both antimicrobials, which act with quite different mechanisms. Similar protection was also detected when antimicrobial peptides were tested against planktonic bacteria in the presence of the polysaccharides extracted from KpTs101 and KpTs113 biofilms, suggesting sequestering adduct formation with antimicrobials. Circular dichroism experiments on BMAP-27 in the presence of increasing amounts of either polysaccharide confirmed their ability to interact with the peptide and induce an α-helical conformation. Full article

(This article belongs to the Special Issue The Biofilm Matrix)

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Review

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7648 KiB

Open AccessReview

EPS—Then and Now

by Hans-Curt Flemming

Microorganisms 2016, 4(4), 41; https://doi.org/10.3390/microorganisms4040041 - 18 Nov 2016

Cited by 212 | Viewed by 15103

Abstract

“Slime” played a brief and spectacular role in the 19th century founded by the theory of primordial slime by Ernst Haeckel. However, that substance was never found and eventually abandoned. Further scientific attention slowly began in the 1930s referring to slime as a [...] Read more.

“Slime” played a brief and spectacular role in the 19th century founded by the theory of primordial slime by Ernst Haeckel. However, that substance was never found and eventually abandoned. Further scientific attention slowly began in the 1930s referring to slime as a microbial product and then was inspired by “How bacteria stick” by Costerton et al. in 1978, and the matrix material was considered to be polysaccharides. Later, it turned out that proteins, nucleic acids and lipids were major other constituents of the extracellular polymeric substances (EPS), an acronym which was highly discussed. The role of the EPS matrix turns out to be fundamental for biofilms, in terms of keeping cells in proximity and allowing for extended interaction, resource capture, mechanical strength and other properties, which emerge from the life of biofilm organisms, including enhanced tolerance to antimicrobials and other stress. The EPS components are extremely complex and dynamic and fulfil many functional roles, turning biofilms into the most ubiquitous and successful form of life on Earth. Full article

(This article belongs to the Special Issue The Biofilm Matrix)

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Open AccessReview

A Look inside the Listeria monocytogenes Biofilms Extracellular Matrix

by Angelo Colagiorgi, Pierluigi Di Ciccio, Emanuela Zanardi, Sergio Ghidini and Adriana Ianieri

Microorganisms 2016, 4(3), 22; https://doi.org/10.3390/microorganisms4030022 - 05 Jul 2016

Cited by 65 | Viewed by 8767

Abstract

Listeria monocytogenes is a foodborne pathogen able to persist in food industry and is responsible for a severe illness called listeriosis. The ability of L. monocytogenes to persist in environments is due to its capacity to form biofilms that are a sessile community [...] Read more.

Listeria monocytogenes is a foodborne pathogen able to persist in food industry and is responsible for a severe illness called listeriosis. The ability of L. monocytogenes to persist in environments is due to its capacity to form biofilms that are a sessile community of microorganisms embedded in a self-produced matrix of extracellular polymeric substances (EPS’s). In this review, we summarized recent efforts performed in order to better characterize the polymeric substances that compose the extracellular matrix (ECM) of L. monocytogenes biofilms. EPS extraction and analysis led to the identification of polysaccharides, proteins, extracellular DNA, and other molecules within the listerial ECM. All this knowledge will be useful for increasing food protection, suggesting effective strategies for the minimization of persistence of L. monocytogenes in food industry environments. Full article

(This article belongs to the Special Issue The Biofilm Matrix)

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