The Major Autolysin of Streptococcus gordonii Is Subject to Complex Regulation and Modulates Stress Tolerance, Biofilm Formation, and Extracellular-DNA Release

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

Infection and Immunity ◽

10.1128/iai.00775-15 ◽

2015 ◽

Vol 83 (12) ◽

pp. 4682-4692 ◽

Cited By ~ 12

Author(s):

Nithya Babu Rajendran ◽

Julian Eikmeier ◽

Karsten Becker ◽

Muzaffar Hussain ◽

Georg Peters ◽

...

Keyword(s):

Biofilm Formation ◽

Bacterial Species ◽

Laser Scanning Microscopy ◽

Extracellular Dna ◽

Staphylococcus Lugdunensis ◽

Content Type ◽

Competence Gene ◽

Independent Mechanism ◽

Dna Release ◽

The Impact

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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Effect of nicotine on Staphylococcus aureus biofilm formation and virulence factors

Scientific Reports ◽

10.1038/s41598-019-56627-0 ◽

2019 ◽

Vol 9 (1) ◽

Cited By ~ 1

Author(s):

Le Shi ◽

Yang Wu ◽

Chen Yang ◽

Yue Ma ◽

Qing-zhao Zhang ◽

...

Keyword(s):

Staphylococcus Aureus ◽

Virulence Factors ◽

Biofilm Formation ◽

Alveolar Epithelial Cells ◽

Extracellular Dna ◽

Bacterial Invasion ◽

Initial Attachment ◽

Accessory Gene ◽

Nicotine Treatment ◽

Dna Release

AbstractStaphylococcus aureus is a common pathogen in chronic rhinosinusitis (CRS) patients, the pathogenesis of which involves the ability to form biofilms and produce various virulence factors. Tobacco smoke, another risk factor of CRS, facilitates S. aureus biofilm formation; however, the mechanisms involved are unclear. Here, we studied the effect of nicotine on S. aureus biofilm formation and the expression of virulence-related genes. S. aureus strains isolated from CRS patients and a USA300 strain were treated with nicotine or were untreated (control). Nicotine-treated S. aureus strains showed dose-dependent increases in biofilm formation, lower virulence, enhanced initial attachment, increased extracellular DNA release, and a higher autolysis rate, involving dysregulation of the accessory gene regulator (Agr) quorum-sensing system. Consequently, the expression of autolysis-related genes lytN and atlA, and the percentage of dead cells in biofilms was increased. However, the expression of virulence-related genes, including hla, hlb, pvl, nuc, ssp, spa, sigB, coa, and crtN was downregulated and there was reduced bacterial invasion of A549 human alveolar epithelial cells. The results of this study indicate that nicotine treatment enhances S. aureus biofilm formation by promoting initial attachment and extracellular DNA release but inhibits the virulence of this bacterium.

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Characterization of Hydrogen Peroxide-Induced DNA Release by Streptococcus sanguinis and Streptococcus gordonii

Journal of Bacteriology ◽

10.1128/jb.00906-09 ◽

2009 ◽

Vol 191 (20) ◽

pp. 6281-6291 ◽

Cited By ~ 70

Author(s):

Jens Kreth ◽

Hung Vu ◽

Yongshu Zhang ◽

Mark C. Herzberg

Keyword(s):

Hydrogen Peroxide ◽

Cell Adhesion ◽

Extracellular Dna ◽

Release Mechanism ◽

Streptococcus Gordonii ◽

Oral Biofilm ◽

Streptococcus Sanguinis ◽

Production Of Hydrogen ◽

Dna Release ◽

Biofilm Development

ABSTRACT Extracellular DNA (eDNA) is produced by several bacterial species and appears to contribute to biofilm development and cell-cell adhesion. We present data showing that the oral commensals Streptococcus sanguinis and Streptococcus gordonii release DNA in a process induced by pyruvate oxidase-dependent production of hydrogen peroxide (H2O2). Surprisingly, S. sanguinis and S. gordonii cell integrity appears unaffected by conditions that cause autolysis in other eDNA-producing bacteria. Exogenous H2O2 causes release of DNA from S. sanguinis and S. gordonii but does not result in obvious lysis of cells. Under DNA-releasing conditions, cell walls appear functionally intact and ribosomes are retained over time. During DNA release, intracellular RNA and ATP are not coreleased. Hence, the release mechanism appears to be highly specific for DNA. Release of DNA without detectable autolysis is suggested to be an adaptation to the competitive oral biofilm environment, where autolysis could create open spaces for competitors to invade. Since eDNA promotes cell-to-cell adhesion, release appears to support oral biofilm formation and facilitates exchange of genetic material among competent strains.

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