Important Contribution of the Novel LocuscomEBto Extracellular DNA-Dependent Staphylococcus lugdunensis Biofilm Formation

The coagulase-negative speciesStaphylococcus lugdunensisis an emerging cause of serious and potentially life-threatening infections, such as infective endocarditis. The pathogenesis of these infections is characterized by the ability ofS. lugdunensisto form biofilms on either biotic or abiotic surfaces. To elucidate the genetic basis of biofilm formation inS. lugdunensis, we performed transposon (Tn917) mutagenesis. One mutant had a significantly reduced biofilm-forming capacity and carried a Tn917insertion within the competence genecomEB. Site-directed mutagenesis and subsequent complementation with a functional copy ofcomEBverified the importance ofcomEBin biofilm formation. In several bacterial species, natural competence stimulates DNA release via lysis-dependent or -independent mechanisms. Extracellular DNA (eDNA) has been demonstrated to be an important structural component of many bacterial biofilms. Therefore, we quantified the eDNA in the biofilms and found diminished eDNA amounts in thecomEBmutant biofilm. High-resolution images and three-dimensional data obtained via confocal laser scanning microscopy (CSLM) visualized the impact of thecomEBmutation on biofilm integrity. ThecomEBmutant did not show reduced expression of autolysin genes, decreased autolytic activities, or increased cell viability, suggesting a cell lysis-independent mechanism of DNA release. Furthermore, reduced amounts of eDNA in thecomEBmutant biofilms did not result from elevated levels or activity of theS. lugdunensisthermonuclease NucI. In conclusion, we defined here, for the first time, a role for the competence genecomEBin staphylococcal biofilm formation. Our findings indicate thatcomEBstimulates biofilm formation via a lysis-independent mechanism of DNA release.

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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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Low Levels of β-Lactam Antibiotics Induce Extracellular DNA Release and Biofilm Formation in Staphylococcus aureus

mBio ◽

10.1128/mbio.00198-12 ◽

2012 ◽

Vol 3 (4) ◽

Cited By ~ 140

Author(s):

Jeffrey B. Kaplan ◽

Era A. Izano ◽

Prerna Gopal ◽

Michael T. Karwacki ◽

Sangho Kim ◽

...

Keyword(s):

Staphylococcus Aureus ◽

Biofilm Formation ◽

Bacterial Biofilm ◽

Extracellular Dna ◽

Human Pathogen ◽

Methicillin Resistant ◽

Content Type ◽

Low Level ◽

Dna Release ◽

Low Levels

ABSTRACTSubminimal inhibitory concentrations of antibiotics have been shown to induce bacterial biofilm formation. Few studies have investigated antibiotic-induced biofilm formation inStaphylococcus aureus, an important human pathogen. Our goal was to measureS. aureusbiofilm formation in the presence of low levels of β-lactam antibiotics. Fifteen phylogenetically diverse methicillin-resistantStaphylococcus aureus(MRSA) and methicillin-sensitiveS. aureus(MSSA) strains were employed. Methicillin, ampicillin, amoxicillin, and cloxacillin were added to cultures at concentrations ranging from 0× to 1× MIC. Biofilm formation was measured in 96-well microtiter plates using a crystal violet binding assay. Autoaggregation was measured using a visual test tube settling assay. Extracellular DNA was quantitated using agarose gel electrophoresis. All four antibiotics induced biofilm formation in some strains. The amount of biofilm induction was as high as 10-fold and was inversely proportional to the amount of biofilm produced by the strain in the absence of antibiotics. MRSA strains of lineages USA300, USA400, and USA500 exhibited the highest levels of methicillin-induced biofilm induction. Biofilm formation induced by low-level methicillin was inhibited by DNase. Low-level methicillin also induced DNase-sensitive autoaggregation and extracellular DNA release. The biofilm induction phenotype was absent in a strain deficient in autolysin (atl). Our findings demonstrate that subminimal inhibitory concentrations of β-lactam antibiotics significantly induce autolysin-dependent extracellular DNA release and biofilm formation in some strains ofS. aureus.IMPORTANCEThe widespread use of antibiotics as growth promoters in agriculture may expose bacteria to low levels of the drugs. The aim of this study was to investigate the effects of low levels of antibiotics on bacterial autoaggregation and biofilm formation, two processes that have been shown to foster genetic exchange and antibiotic resistance. We found that low levels of β-lactam antibiotics, a class commonly used in both clinical and agricultural settings, caused significant autoaggregation and biofilm formation by the important human pathogenStaphylococcus aureus. Both processes were dependent on cell lysis and release of DNA into the environment. The effect was most pronounced among multidrug-resistant strains known as methicillin-resistantS. aureus(MRSA). These results may shed light on the recalcitrance of some bacterial infections to antibiotic treatment in clinical settings and the evolution of antibiotic-resistant bacteria in agricultural settings.

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Role of Exopolysaccharides in Pseudomonas aeruginosa Biofilm Formation and Architecture

Applied and Environmental Microbiology ◽

10.1128/aem.00637-11 ◽

2011 ◽

Vol 77 (15) ◽

pp. 5238-5246 ◽

Cited By ~ 207

Author(s):

Aamir Ghafoor ◽

Iain D. Hay ◽

Bernd H. A. Rehm

Keyword(s):

Pseudomonas Aeruginosa ◽

Biofilm Formation ◽

Laser Scanning ◽

Bacterial Biofilm ◽

Laser Scanning Microscopy ◽

Extracellular Dna ◽

Confocal Laser ◽

Content Type ◽

Biofilm Architecture

ABSTRACTPseudomonas aeruginosais an opportunistic human pathogen and has been established as a model organism to study bacterial biofilm formation. At least three exopolysaccharides (alginate, Psl, and Pel) contribute to the formation of biofilms in this organism. Here mutants deficient in the production of one or more of these polysaccharides were generated to investigate how these polymers interactively contribute to biofilm formation. Confocal laser scanning microscopy of biofilms formed in flow chambers showed that mutants deficient in alginate biosynthesis developed biofilms with a decreased proportion of viable cells than alginate-producing strains, indicating a role of alginate in viability of cells in biofilms. Alginate-deficient mutants showed enhanced extracellular DNA (eDNA)-containing surface structures impacting the biofilm architecture. PAO1 ΔpslAΔalg8overproduced Pel, and eDNA showing meshwork-like structures presumably based on an interaction between both polymers were observed. The formation of characteristic mushroom-like structures required both Psl and alginate, whereas Pel appeared to play a role in biofilm cell density and/or the compactness of the biofilm. Mutants producing only alginate, i.e., mutants deficient in both Psl and Pel production, lost their ability to form biofilms. A lack of Psl enhanced the production of Pel, and the absence of Pel enhanced the production of alginate. The function of Psl in attachment was independent of alginate and Pel. A 30% decrease in Psl promoter activity in the alginate-overproducing MucA-negative mutant PDO300 suggested inverse regulation of both biosynthesis operons. Overall, this study demonstrated that the various exopolysaccharides and eDNA interactively contribute to the biofilm architecture ofP. aeruginosa.

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Exploring the Diversity of Listeria monocytogenes Biofilm Architecture by High-Throughput Confocal Laser Scanning Microscopy and the Predominance of the Honeycomb-Like Morphotype

Applied and Environmental Microbiology ◽

10.1128/aem.03173-14 ◽

2014 ◽

Vol 81 (5) ◽

pp. 1813-1819 ◽

Cited By ~ 68

Author(s):

Morgan Guilbaud ◽

Pascal Piveteau ◽

Mickaël Desvaux ◽

Sylvain Brisse ◽

Romain Briandet

Keyword(s):

Listeria Monocytogenes ◽

High Throughput ◽

Biofilm Formation ◽

Spatial Organization ◽

Confocal Imaging ◽

Laser Scanning Microscopy ◽

Extracellular Dna ◽

Confocal Laser ◽

Published Data ◽

Content Type

ABSTRACTListeria monocytogenesis involved in food-borne illness with a high mortality rate. The persistence of the pathogen along the food chain can be associated with its ability to form biofilms on inert surfaces. While most of the phenotypes associated with biofilms are related to their spatial organization, most published data comparing biofilm formation byL. monocytogenesisolates are based on the quantitative crystal violet assay, which does not give access to structural information. Using a high-throughput confocal-imaging approach, the aim of this work was to decipher the structural diversity of biofilms formed by 96L. monocytogenesstrains isolated from various environments. Prior to large-scale analysis, an experimental design was created to improveL. monocytogenesbiofilm formation in microscopic-grade microplates, with special emphasis on the growth medium composition. Microscopic analysis of biofilms formed under the selected conditions by the 96 isolates revealed only weak correlation between the genetic lineages of the isolates and the structural properties of the biofilms. However, a gradient in their geometric descriptors (biovolume, mean thickness, and roughness), ranging from flat multilayers to complex honeycomb-like structures, was shown. The dominant honeycomb-like morphotype was characterized by hollow voids hosting free-swimming cells and localized pockets containing mixtures of dead cells and extracellular DNA (eDNA).

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Impact of Extracellular Nuclease Production on the Biofilm Phenotype of Staphylococcus aureus underIn VitroandIn VivoConditions

Infection and Immunity ◽

10.1128/iai.06134-11 ◽

2012 ◽

Vol 80 (5) ◽

pp. 1634-1638 ◽

Cited By ~ 57

Author(s):

Karen E. Beenken ◽

Horace Spencer ◽

Linda M. Griffin ◽

Mark S. Smeltzer

Keyword(s):

Staphylococcus Aureus ◽

Murine Model ◽

Biofilm Formation ◽

Extracellular Dna ◽

Content Type ◽

Extracellular Nuclease ◽

The Impact ◽

Increased Susceptibility

ABSTRACTRecent studies suggest that extracellular DNA promotes biofilm formation inStaphylococcus aureusand, conversely, that extracellular nucleases limit the ability to form a biofilm.S. aureusproduces at least two extracellular nucleases, and in the study described in this report, we examined the impact of each of these nucleases on biofilm formation under bothin vitroandin vivoconditions. Our results demonstrate that both nucleases impact biofilm formation in the clinical isolate UAMS-1. Under certainin vitroconditions, this impact is negative, with mutation of either or both of the nuclease genes (nuc1andnuc2) resulting in an enhanced capacity to form a biofilm. However, this effect was not apparentin vivoin a murine model of catheter-associated biofilm formation. Rather, mutation of either or both nuclease genes appeared to limit biofilm formation to a degree that could be correlated with increased susceptibility to daptomycin.

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AtlA Mediates Extracellular DNA Release, Which Contributes to Streptococcus mutans Biofilm Formation in an Experimental Rat Model of Infective Endocarditis

Infection and Immunity ◽

10.1128/iai.00252-17 ◽

2017 ◽

Vol 85 (9) ◽

Cited By ~ 13

Author(s):

Chiau-Jing Jung ◽

Ron-Bin Hsu ◽

Chia-Tung Shun ◽

Chih-Chieh Hsu ◽

Jean-San Chia

Keyword(s):

Infective Endocarditis ◽

Streptococcus Mutans ◽

Mutant Strain ◽

Rat Model ◽

Calcium Ions ◽

Biofilm Formation ◽

Laser Scanning Microscopy ◽

Extracellular Dna ◽

Wild Type ◽

Content Type

ABSTRACT Host factors, such as platelets, have been shown to enhance biofilm formation by oral commensal streptococci, inducing infective endocarditis (IE), but how bacterial components contribute to biofilm formation in vivo is still not clear. We demonstrated previously that an isogenic mutant strain of Streptococcus mutans deficient in autolysin AtlA (ΔatlA) showed a reduced ability to cause vegetation in a rat model of bacterial endocarditis. However, the role of AtlA in bacterial biofilm formation is unclear. In this study, confocal laser scanning microscopy analysis showed that extracellular DNA (eDNA) was embedded in S. mutans GS5 floes during biofilm formation on damaged heart valves, but an ΔatlA strain could not form bacterial aggregates. Semiquantification of eDNA by PCR with bacterial 16S rRNA primers demonstrated that the ΔatlA mutant strain produced dramatically less eDNA than the wild type. Similar results were observed with in vitro biofilm models. The addition of polyanethol sulfonate, a chemical lysis inhibitor, revealed that eDNA release mediated by bacterial cell lysis is required for biofilm initiation and maturation in the wild-type strain. Supplementation of cultures with calcium ions reduced wild-type growth but increased eDNA release and biofilm mass. The effect of calcium ions on biofilm formation was abolished in ΔatlA cultures and by the addition of polyanethol sulfonate. The VicK sensor, but not CiaH, was found to be required for the induction of eDNA release or the stimulation of biofilm formation by calcium ions. These data suggest that calcium ion-regulated AtlA maturation mediates the release of eDNA by S. mutans, which contributes to biofilm formation in infective endocarditis.

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Functional Specificity of Extracellular Nucleases of Shewanella oneidensis MR-1

Applied and Environmental Microbiology ◽

10.1128/aem.07895-11 ◽

2012 ◽

Vol 78 (12) ◽

pp. 4400-4411 ◽

Cited By ~ 14

Author(s):

Magnus Heun ◽

Lucas Binnenkade ◽

Maximilian Kreienbaum ◽

Kai M. Thormann

Keyword(s):

Biofilm Formation ◽

Bacterial Species ◽

Shewanella Oneidensis ◽

Large Cell ◽

Sole Source ◽

Extracellular Dna ◽

Divalent Metal Ions ◽

Content Type ◽

Highly Active ◽

Extracellular Nuclease

ABSTRACTBacterial species such asShewanella oneidensisMR-1 require extracellular nucleolytic activity for the utilization of extracellular DNA (eDNA) as a source of nutrients and for the turnover of eDNA as a structural matrix component during biofilm formation. We have previously characterized two extracellular nucleases ofS. oneidensisMR-1, ExeM and ExeS. Although both are involved in biofilm formation, they are not specifically required for the utilization of eDNA as a nutrient. Here we identified and characterized EndA, a third extracellular nuclease ofShewanella. The heterologously overproduced and purified protein was highly active and rapidly degraded linear and supercoiled DNAs of various origins. Divalent metal ions (Mg2+or Mn2+) were required for function.endAis cotranscribed withphoA, an extracellular phosphatase, and is not upregulated upon phosphostarvation. Deletion ofendAabolished both extracellular degradation of DNA byS. oneidensisMR-1 and the ability to use eDNA as a sole source of phosphorus. PhoA is not strictly required for the exploitation of eDNA as a nutrient. The activity of EndA prevents the formation of large cell aggregates during planktonic growth. However, in contrast to the findings for ExeM,endAdeletion had only minor effects on biofilm formation. The findings strongly suggest that the extracellular nucleases ofS. oneidensisexert specific functions required under different conditions.

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LuxS Mediates Iron-Dependent Biofilm Formation, Competence, and Fratricide in Streptococcus pneumoniae

Infection and Immunity ◽

10.1128/iai.05644-11 ◽

2011 ◽

Vol 79 (11) ◽

pp. 4550-4558 ◽

Cited By ~ 59

Author(s):

Claudia Trappetti ◽

Adam J. Potter ◽

Adrienne W. Paton ◽

Marco R. Oggioni ◽

James C. Paton

Keyword(s):

Streptococcus Pneumoniae ◽

Biofilm Formation ◽

Extracellular Dna ◽

Wild Type ◽

Nasopharyngeal Colonization ◽

Content Type ◽

Planktonic Form ◽

Dna Release ◽

Amplification Loop

ABSTRACTDuring infection,Streptococcus pneumoniaeexists mainly in sessile biofilms rather than in planktonic form, except during sepsis. The capacity to form biofilms is believed to be important for nasopharyngeal colonization as well as disease pathogenesis, but relatively little is known about the regulation of this process. Here, we investigated the effect of exogenous iron [Fe(III)] as well as the role ofluxS(encoding S-ribosylhomocysteine lyase) on biofilm formation byS. pneumoniaeD39. Fe(III) strongly enhanced biofilm formation at concentrations of ≥50 μM, while Fe(III) chelation with deferoxamine was inhibitory. Importantly, Fe(III) also upregulated the expression ofluxSin wild-type D39. AluxS-deficient mutant (D39luxS) failed to form a biofilm, even with Fe(III) supplementation, whereas a derivative overexpressingluxS(D39luxS+) exhibited enhanced biofilm formation capacity and could form a biofilm without added Fe(III). D39luxSexhibited reduced expression of the major Fe(III) transporter PiuA, and the cellular [Fe(III)] was significantly lower than that in D39; in contrast, D39luxS+ had a significantly higher cellular [Fe(III)] than the wild type. The release of extracellular DNA, which is an important component of the biofilm matrix, also was directly related toluxSexpression. Similarly, genetic competence, as measured by transformation frequency as well as the expression of competence genescomD,comX,comW,cglA, anddltAand the murein hydrolasecbpD, which is associated with fratricide-dependent DNA release, all were directly related toluxSexpression levels and were further upregulated by Fe(III). Moreover, mutagenesis ofcbpDblocked biofilm formation. We propose that competence, fratricide, and biofilm formation are closely linked in pneumococci, and thatluxSis a central regulator of these processes. We also propose that the stimulatory effects of Fe(III) on all of these parameters are due to the upregulation ofluxSexpression, and that LuxS provides for a positive Fe(III)-dependent amplification loop by increasing iron uptake.

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Confocal and SEM imaging to demonstrate food pathogen- biofilm biocontrol by pyocyanin from Pseudomonas aeruginosa BTRY1

International Journal of Bioassays ◽

10.21746/ijbio.2017.01.007 ◽

2016 ◽

Vol 6 (01) ◽

pp. 5218

Author(s):

Laxmi Mohandas ◽

Anju T. R. ◽

Sarita G. Bhat*

Keyword(s):

Pseudomonas Aeruginosa ◽

Biofilm Formation ◽

Laser Scanning ◽

Laser Scanning Microscopy ◽

Confocal Laser ◽

Antibiofilm Activity ◽

Opportunistic Pathogens ◽

Redox Active ◽

Sem Imaging ◽

The Impact

An assortment of redox-active phenazine compounds like pyocyanin with their characteristic blue-green colour are synthesized by Pseudomonas aeruginosa, Gram-negative opportunistic pathogens, which are also considered one of the most commercially valuable microorganisms. In this study, pyocyanin from Pseudomonas aeruginosa BTRY1 from food sample was assessed for its antibiofilm activity by micro titer plate assay against strong biofilm producers belonging to the genera Bacillus, Staphylococcus, Brevibacterium and Micrococcus. Pyocyanin inhibited biofilm activity in very minute concentrations. This was also confirmed by Scanning Electron Microscopy (SEM) and Confocal Laser Scanning Microscopy (CLSM). Both SEM and CLSM helped to visualize the biocontrol of biofilm formation by eight pathogens. The imaging and quantification by CLSM also established the impact of pyocyanin on biofilm-biocontrol mainly in the food industry.

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