scholarly journals Advanced understanding of prokaryotic biofilm formation using a cost-effective and versatile multi-panel adhesion (mPAD) mount

2022 ◽  
Author(s):  
Stefan Schulze ◽  
Heather Schiller ◽  
Jordan Solomonic ◽  
Orkan Telhan ◽  
Kyle Costa ◽  
...  
Keyword(s):  
Biofilm Formation ◽  
Cost Effective ◽  
Flow Chamber ◽  
Culture Conditions ◽  
Multiple Time ◽  
Wild Type ◽  
Surface Attachment ◽  
Flowing Liquid ◽  
Multiple Time Points ◽  
Static Conditions

Most microorganisms exist in biofilms, which comprise aggregates of cells surrounded by an extracellular matrix that provides protection from external stresses. Based on the conditions under which they form, biofilm structures vary in significant ways. For instance, biofilms that develop when microbes are incubated under static conditions differ from those formed when microbes encounter the shear forces of a flowing liquid. Moreover, biofilms develop dynamically over time. Here, we describe a cost-effective, 3D-printed coverslip holder that facilitates surface adhesion assays under a broad range of standing and shaking culture conditions. This multi-panel adhesion (mPAD) mount further allows cultures to be sampled at multiple time points, ensuring consistency and comparability between samples and enabling analyses of the dynamics of biofilm formation. As a proof of principle, using the mPAD mount for shaking, oxic cultures, we confirm previous flow chamber experiments showing that Pseudomonas aeruginosa wild type and a phenazine deletion mutant (Δ phz ) form biofilms with similar structure but reduced density in the mutant strain. Extending this analysis to anoxic conditions, we reveal that microcolony and biofilm formation can only be observed under shaking conditions and are decreased in the Δ phz mutant compared to wild-type cultures, indicating that phenazines are crucial for the formation of biofilms if oxygen as an electron acceptor is unavailable. Furthermore, while the model archaeon Haloferax volcanii does not require archaella for surface attachment under static conditions, we demonstrate that H. volcanii mutants that lack archaella are impaired in early stages of biofilm formation under shaking conditions. Importance: Due to the versatility of the mPAD mount, we anticipate that it will aid the analysis of biofilm formation in a broad range of bacteria and archaea. Thereby, it contributes to answering critical biological questions about the regulatory and structural components of biofilm formation and understanding this process in a wide array of environmental, biotechnological, and medical contexts.

scholarly journals Advanced understanding of prokaryotic biofilm formation using a cost-effective and versatile multi-panel adhesion (mPAD) mount

2021 ◽  
Author(s):  
Stefan Schulze ◽  
Heather Schiller ◽  
Jordan Solomonic ◽  
Orkan Telhan ◽  
Kyle Costa ◽  
...  
Keyword(s):  
Biofilm Formation ◽  
Cost Effective ◽  
Flow Chamber ◽  
Culture Conditions ◽  
Multiple Time ◽  
Wild Type ◽  
Flowing Liquid ◽  
External Stresses ◽  
Multiple Time Points ◽  
Static Conditions

AbstractMost microorganisms exist in biofilms, which comprise aggregates of cells surrounded by an extracellular matrix that provides protection from external stresses. Based on the conditions under which they form, biofilm structures vary in significant ways. For instance, biofilms that develop when microbes are incubated under static conditions differ from those formed when microbes encounter the shear forces of a flowing liquid. Moreover, biofilms develop dynamically over time. Here, we describe a cost-effective, 3D-printed coverslip holder that facilitates surface adhesion assays under a broad range of standing and shaking culture conditions. This multi-panel adhesion (mPAD) mount further allows cultures to be sampled at multiple time points, ensuring consistency and comparability between samples and enabling analyses of the dynamics of biofilm formation. As a proof of principle, using the mPAD mount for shaking, oxic cultures, we confirm previous flow chamber experiments showing that Pseudomonas aeruginosa wild type and a phenazine deletion mutant (Δphz) form similar biofilms. Extending this analysis to anoxic conditions, we reveal that microcolony and biofilm formation can only be observed under shaking conditions and are decreased in the Δphz mutant compared to wild-type cultures, indicating that phenazines are crucial for the formation of biofilms if oxygen as an electron acceptor is not available. Furthermore, while the model archaeon Haloferax volcanii does not require archaella for attachment to surfaces under static conditions, we demonstrate that H. volcanii mutants that lack archaella are negatively affected in their early stages of biofilm formation under shaking conditions.ImportanceDue to the versatility of the mPAD mount, we anticipate that it will aid the analysis of biofilm formation in a broad range of bacteria and archaea. Thereby, it contributes to answering critical biological questions about the regulatory and structural components of biofilm formation and understanding this process in a wide array of environmental, biotechnological, and medical contexts.

scholarly journals Motility and Chemotaxis in Agrobacterium tumefaciens Surface Attachment and Biofilm Formation

2007 ◽  
Vol 189 (22) ◽  
pp. 8005-8014 ◽  
Author(s):  
Peter M. Merritt ◽  
Thomas Danhorn ◽  
Clay Fuqua
Keyword(s):  
Agrobacterium Tumefaciens ◽  
Biofilm Formation ◽  
Bacterial Species ◽  
Wild Type ◽  
Swarming Motility ◽  
Surface Attachment ◽  
Directed Motility ◽  
Suppressor Mutants ◽  
Static Conditions ◽  
Specific Deletion

ABSTRACT Bacterial motility mechanisms, including swimming, swarming, and twitching, are known to have important roles in biofilm formation, including colonization and the subsequent expansion into mature structured surface communities. Directed motility requires chemotaxis functions that are conserved among many bacterial species. The biofilm-forming plant pathogen Agrobacterium tumefaciens drives swimming motility by utilizing a small group of flagella localized to a single pole or the subpolar region of the cell. There is no evidence for twitching or swarming motility in A. tumefaciens. Site-specific deletion mutations that resulted in either aflagellate, flagellated but nonmotile, or flagellated but nonchemotactic A. tumefaciens derivatives were examined for biofilm formation under static and flowing conditions. Nonmotile mutants were significantly deficient in biofilm formation under static conditions. Under flowing conditions, however, the aflagellate mutant rapidly formed aberrantly dense, tall biofilms. In contrast, a nonmotile mutant with unpowered flagella was clearly debilitated for biofilm formation relative to the wild type. A nontumbling chemotaxis mutant was only weakly affected with regard to biofilm formation under nonflowing conditions but was notably compromised in flow, generating less adherent biomass than the wild type, with a more dispersed cellular arrangement. Extragenic suppressor mutants of the chemotaxis-impaired, straight-swimming phenotype were readily isolated from motility agar plates. These mutants regained tumbling at a frequency similar to that of the wild type. Despite this phenotype, biofilm formation by the suppressor mutants in static cultures was significantly deficient. Under flowing conditions, a representative suppressor mutant manifested a phenotype similar to yet distinct from that of its nonchemotactic parent.

Bacterial Adhesion to Nanomodified Surfaces: Dynamic Flow Effects onS. aureusandP. aeruginosa

2013 ◽  
Vol 1498 ◽  
pp. 79-84
Author(s):  
Mary C. Machado ◽  
Keiko M. Tarquinio ◽  
Thomas J. Webster
Keyword(s):  
Bacterial Adhesion ◽  
Biofilm Formation ◽  
Cost Effective ◽  
Clinical Problem ◽  
Flow Chamber ◽  
High Morbidity ◽  
Pressure Gradients ◽  
Dynamic Flow ◽  
Endotracheal Tubes ◽  
Flow Effects

AbstractVentilator associated pneumonia (VAP) is a serious and costly clinical problem. Specifically, receiving mechanical ventilation over 24 hours increases the risk of VAP and is associated with high morbidity, mortality and medical costs. Cost effective endotracheal tubes (ETTs) that are resistant to bacterial infection would help to prevent this problem. The objective of this study was to determine differences in bacterial growth on nanomodified and unmodified ETTs under dynamic airway conditions. A bench top model based upon the general design of Hartmann et al. (1999) was constructed to test of the effectiveness of nanomodified ETTs under the airflow conditions present in the airway. Twenty-four hour studies performed in a dynamic flow chamber showed a marked difference in the biofilm formation on different areas of unmodified tubes. Areas where tubes were curved, such as at the entrance to the mouth and the connection between the oropharynx and the larynx, seemed to collect the largest amount of biofilm.The biofilm formation on ETTs in the airflow system after 24 hours showed a large difference depending upon where tubes were oriented within the apparatus. This illustrates the importance of dynamic flow on biofilm formation in pediatric ETTs. It is of particular interest that increased biofilm density on both unmodified and nanomodified tubes appeared to occur at curves in the tube where changes in flow pattern occurred. This emphasizes the need for more accurate models of airflow within pediatric ETTs, suggesting that not only does flow affect pressure gradients along the tube, but in fact, determines the composition of the film itself. More testing is needed to determine the effects of biofilm formation on the efficiency of ETT under airflow, however this study provides significant evidence for nanomodification alone (without the use of antibiotics) to decrease bacteria function.

EFFECT OF TOPOGRAPHICALLY PATTERNED POLY(DIMETHYLSILOXANE) SURFACES ON Pseudomonas aeruginosa ADHESION AND BIOFILM FORMATION

Nano LIFE ◽  
2012 ◽  
Vol 02 (04) ◽  
pp. 1242004 ◽  
Author(s):  
JOHN F. LING ◽  
MARY V. GRAHAM ◽  
NATHANIEL C. CADY
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Biofilm Formation ◽  
Antimicrobial Agents ◽  
Lower Surface ◽  
Bacterial Attachment ◽  
Initial Surface ◽  
Direct Role ◽  
Surface Attachment ◽  
Bacterial Surface ◽  
Static Conditions

Bacterial pathogens, such as Pseudomonas aeruginosa, readily form biofilms on surfaces, limiting the efficacy of antimicrobial and antibiotic treatments. To mitigate biofilm formation, surfaces are often treated with antimicrobial agents, which have limited lifetime and efficacy. Recent studies have shown that well-ordered topographic patterns can limit bacterial attachment to surfaces and limit biofilm formation. In this study, nano and microscale patterned poly(dimethylsiloxane) surfaces were evaluated for their ability to affect adhesion and biofilm formation by Pseudomonas aeruginosa. Feature size and spacing were varied from 500 nm to 2 μm and included repeating arrays of square pillars, holes, lines and biomimetc Sharklet™ patterns. Bacterial surface adhesion and biofilm formation was assessed in microfluidic flow devices and under static conditions. Attachment profiles under static and fluid flow varied within topography types, sizes and spacing. Pillar structures of all sizes yielded lower surface attachment than line-based patterns and arrays of holes. This trend was also observed for biomimetic Sharklet™ patterns, with reduced bacterial attachment to "raised" features as compared to "recessed" features. Notably, none of the topographically patterned surfaces outperformed smooth surfaces (without topography) for resisting cell adhesion. Initial surface attachment patterns were indicative of subsequent biofilm formation and coverage, suggesting a direct role of surface topography in biofilm-based biofouling.

Alternative Sigma Factor σB Is Not Essential for Listeria monocytogenes Surface Attachment

2005 ◽  
Vol 68 (2) ◽  
pp. 311-317 ◽  
Author(s):  
UTE SCHWAB ◽  
YUEWEI HU ◽  
MARTIN WIEDMANN ◽  
KATHRYN J. BOOR
Keyword(s):  
Stainless Steel ◽  
Listeria Monocytogenes ◽  
Single Cells ◽  
Foodborne Pathogen ◽  
Plate Count ◽  
Initial Surface ◽  
Wild Type ◽  
Surface Attachment ◽  
Steel Analysis ◽  
Static Conditions

Listeria monocytogenes is a foodborne pathogen frequently isolated from the food processing environment. Multiple lines of evidence suggested a possible role for the L. monocytogenes alternative transcription factor sigma B (σB) in surface attachment and biofilm formation. Therefore, through plate count and microscopic techniques, the L. monocytogenes 10403S strain and an otherwise isogenic ΔsigB strain were tested for attachment to stainless steel. Analysis of microscopic images revealed that after 72 h at 24°C under static conditions the tested L. monocytogenes strains attached uniformly to surfaces as single cells. Both strains were capable of rapid attachment (i.e., numbers of attached cells were essentially the same after either 5 min or 24 h of incubation). Numbers of attached ΔsigB cells were significantly lower than those of the wild-type strain after 48 and 72 h of incubation at 24°C (P = 0.001). Similar numbers of the ΔsigB strain attached to stainless steel regardless of temperature (24 or 37°C); however, ΔsigB cells attached at higher relative numbers in the presence of 6% NaCl after 48 and 72 h. Furthermore, in the presence of Pseudomonas fluorescens, similarly high numbers of wild-type and ΔsigB cells attached to the surfaces, forming mixed biofilms. Our data suggest that σB is not required for initial surface attachment of L. monocytogenes.

Attachment to and biofilm formation on abiotic surfaces by Acinetobacter baumannii: involvement of a novel chaperone-usher pili assembly system

Microbiology ◽  
2003 ◽  
Vol 149 (12) ◽  
pp. 3473-3484 ◽  
Author(s):  
Andrew P. Tomaras ◽  
Caleb W. Dorsey ◽  
Richard E. Edelmann ◽  
Luis A. Actis
Keyword(s):  
Acinetobacter Baumannii ◽  
Biofilm Formation ◽  
Prototype Strain ◽  
Culture Conditions ◽  
Sequencing Analysis ◽  
Cell Aggregates ◽  
Surface Attachment ◽  
Abiotic Surfaces ◽  
Air Interface ◽  
Dna Sequencing Analysis

Acinetobacter baumannii causes severe infections in compromised patients, survives on abiotic surfaces in hospital environments and colonizes different medical devices. In this study the analysis of the processes involved in surface attachment and biofilm formation by the prototype strain 19606 was initiated. This strain attaches to and forms biofilm structures on plastic and glass surfaces, particularly at the liquid–air interface of cultures incubated stagnantly. The cell aggregates, which contain cell stacks separated by water channels, formed under different culture conditions and were significantly enhanced under iron limitation. Electron and fluorescence microscopy showed that pili and exopolysaccharides are part of the cell aggregates formed by this strain. Electron microscopy of two insertion derivatives deficient in attachment and biofilm formation revealed the disappearance of pili-like structures and DNA sequencing analysis showed that the transposon insertions interrupted genes with the highest similarity to hypothetical genes found in Pseudomonas aeruginosa, Pseudomonas putida and Vibrio parahaemolyticus. Although the products of these genes, which have been named csuC and csuE, have no known functions, they are located within a polycistronic operon that includes four other genes, two of which encode proteins related to chaperones and ushers involved in pili assembly in other bacteria. Introduction of a copy of the csuE parental gene restored the adherence phenotype and the presence of pili on the cell surface of the csuE mutant, but not that of the csuC derivative. These results demonstrate that the expression of a chaperone-usher secretion system, some of whose components appear to be acquired from unrelated sources, is required for pili formation and the concomitant attachment to plastic surfaces and the ensuing formation of biofilms by A. baumannii cells.

Reduced Resistance to Air Flow from Nanomodified Endotracheal Tubes

2011 ◽  
Vol 1316 ◽  
Author(s):  
Mary C. Machado ◽  
Keiko M. Tarquinio ◽  
Thomas J. Webster
Keyword(s):  
Biofilm Formation ◽  
Cost Effective ◽  
Clinical Problem ◽  
Flow Chamber ◽  
High Morbidity ◽  
Ventilator Associated Pneumonia ◽  
Pressure Gradients ◽  
Dynamic Flow ◽  
Endotracheal Tubes ◽  
Significant Evidence

AbstractVentilator associated pneumonia (VAP) is a serious and costly clinical problem. Specifically, receiving mechanical ventilation over 24 hours increases the risk of VAP and is associated with high morbidity, mortality and medical costs. Cost effective endotracheal tubes (ETTs) that are resistant to bacterial infection would help to prevent this problem. The objective of this study was to determine differences in bacterial growth on nanomodified and unmodified ETTs under dynamic airway conditions, a bench top model based upon the general design of Hartmann et al. (1999) was constructed to test of the effectiveness of nanomodified ETTs under the airflow conditions present in the airway. Twenty-four hour studies performed in a dynamic flow chamber showed a marked difference in the biofilm formation on different areas of unmodified tubes. Areas where tubes were curved, such as at the entrance to the mouth and the connection between the oropharynx and the larynx, seemed to collect the largest amount of biofilm. On the nanomodified tubes biofilm formation was markedly different occurring on smaller pieces.The biofilm formation on ETTs in the airflow system after 24 hours showed a large difference depending upon where tubes were oriented within the apparatus. This illustrates the importance of dynamic flow on biofilm formation in pediatric ETTs. It is of particular interest that increased biofilm density on both unmodified and nanomodified tubes appeared to occur at curves in the tube where changes in flow pattern occured. This emphasizes the need for more accurate models of airflow within pediatric ETTs, suggesting that not only does flow affect pressure gradients along the tube, but in fact, determines the composition of the film itself. More testing is needed to determine the effects of biofilm formation on the efficiency of ETT under airflow, however this study provides significant evidence for nanomodification alone (without the use of antibiotics) to decrease bacteria function.

scholarly journals Glycoproteomics of Haloferax volcanii reveals an extensive glycoproteome and concurrence of different N-glycosylation pathways

2021 ◽  
Author(s):  
Stefan Schulze ◽  
Friedhelm Pfeiffer ◽  
Benjamin A. Garcia ◽  
Mechthild Pohlschroder
Keyword(s):  
Biofilm Formation ◽  
Cell Biology ◽  
Culture Conditions ◽  
Haloferax Volcanii ◽  
Spectrometric Analysis ◽  
Wild Type ◽  
Cellular Processes ◽  
Phenotypic Characterisation ◽  
Mutant Strains ◽  
Glycosylation Pathway

AbstractGlycosylation is one of the most complex post-translational protein modifications. Its importance has been established not only for eukaryotes but also for a variety of prokaryotic cellular processes, such as biofilm formation, motility and mating. However, comprehensive glycoproteomic analyses are largely missing in prokaryotes. Here we extend the phenotypic characterisation of N-glycosylation pathway mutants in Haloferax volcanii and provide a detailed glycoproteome for this model archaeon through the mass spectrometric analysis of intact glycopeptides. Using in-depth glycoproteomic datasets generated for the wild-type and mutant strains as well as a reanalysis of datasets within the Archaeal Proteome Project, we identify the largest archaeal glycoproteome described so far. We further show that different N-glycosylation pathways can modify the same glycosites under the same culture conditions. The extent and complexity of the Hfx. volcanii N-glycoproteome revealed here provides new insights into the roles of N-glycosylation in archaeal cell biology.

scholarly journals Culture Conditions of Roseobacter Strain 27-4 Affect Its Attachment and Biofilm Formation as Quantified by Real-Time PCR

2006 ◽  
Vol 72 (4) ◽  
pp. 3011-3015 ◽  
Author(s):  
Jesper Bartholin Bruhn ◽  
Janus Anders Juul Haagensen ◽  
Dorthe Bagge-Ravn ◽  
Lone Gram
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Biofilm Formation ◽  
Three Dimensional ◽  
Growth Conditions ◽  
Culture Conditions ◽  
Probiotic Bacterium ◽  
Antibacterial Compound ◽  
Pcr Method ◽  
Static Conditions

ABSTRACT The fish probiotic bacterium Roseobacter strain 27-4 grows only as rosettes and produces its antibacterial compound under static growth conditions. It forms three-dimensional biofilms when precultured under static conditions. We quantified attachment of Roseobacter strain 27-4 using a direct real-time PCR method and demonstrated that the bacteria attached more efficiently to surfaces during static growth than under aerated conditions.

scholarly journals Csp1, A Cold-Shock Protein Homolog in Xylella fastidiosa Influences Pili Formation, Stress Response, and Gene Expression

2021 ◽  
Author(s):  
Wei Wei ◽  
Lindsey Price Burbank ◽  
Teresa Sawyer
Keyword(s):  
Gene Expression ◽  
Nucleic Acid ◽  
Biofilm Formation ◽  
Cold Shock ◽  
Xylella Fastidiosa ◽  
Cold Shock Protein ◽  
Nucleic Acid Binding ◽  
Wild Type ◽  
Surface Attachment

Bacterial cold shock-domain proteins (CSPs) are conserved nucleic acid binding chaperones that play important roles in stress adaptation and pathogenesis. Csp1 is a temperature-independent cold shock protein homolog in Xylella fastidiosa, a bacterial plant pathogen of grapevine and other economically important crops. Csp1 contributes to stress tolerance and virulence in X. fastidiosa. However, besides general single stranded nucleic acid binding activity, little is known about the specific function(s) of this protein. To further investigate the role(s) of Csp1, we compared phenotypic differences between wild type and a csp1 deletion mutant (Δcsp1). We observed decreases in cellular aggregation and surface attachment with the Δcsp1 strain compared to the wild type. Transmission electron microscopy imaging revealed that Δcsp1 had reduced pili compared to the wild type and complemented strains. The Δcsp1 strain also showed reduced survival after long term growth, in vitro. Since Csp1 binds DNA and RNA, its influence on gene expression was also investigated. Long-read Nanopore RNA-Seq analysis of wild type and Δcsp1 revealed changes in expression of several genes important for attachment and biofilm formation in Δcsp1. One gene of intertest,pilA1, encodes a type IV pili subunit protein and was up regulated in Δcsp1. Deleting pilA1 increased surface attachment in vitro and reduced virulence in grapevines.X. fastidiosa virulence depends on bacterial attachment to host tissue and movement within and between xylem vessels. Our results show Csp1 may play a role in both virulence and stress tolerance by influencing expression of genes important for biofilm formation.

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