scholarly journals RpiRc regulates RsbU to modulate eDNA-dependent biofilm formation and in vivo virulence of Staphylococcus aureus in a mouse model of catheter infection

2019 ◽  
Author(s):  
Adrien Fischer ◽  
Myriam Girard ◽  
Floriane Laumay ◽  
Anne-Kathrin Woischnig ◽  
Nina Khanna ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Mouse Model ◽  
Biofilm Formation ◽  
Ex Vivo ◽  
Bacterial Biofilm ◽  
Extracellular Dna ◽  
Hek293 Cells ◽  
Catheter Infection ◽  
Biofilm Matrix

AbstractStaphylococcus aureus is a major human pathogen. Despite high incidence and morbidity, molecular mechanisms occurring during infection remain largely unknown. Under defined conditions, biofilm formation contributes to the severity of S. aureus related infections. Extracellular DNA (eDNA), a component of biofilm matrix released from apoptotic bacteria, is involved in biofilm structure and stability. In many bacterial biofilms, eDNA originates from cell lysis although eDNA can also be actively secreted or exported by bacterial membrane vesicles. By screening the Nebraska transposon library, we identified rpiRc as a biofilm regulator involved in eDNA regulation. RpiRc is a transcription factor from the pentose phosphate pathway (PPP) whose product is a polysaccharide intercellular adhesin (PIA) precursor. However, rpiRc mutant strain showed neither susceptibility to DispersinB® (a commercially available enzyme disrupting PIA biofilms) nor alteration of ica transcription (the operon regulating PIA production). Decreased biofilm formation was linked to Sln, an extracellular compound degrading eDNA in an autolysis independent pathway. Biofilm susceptibility to antibiotics in wt and mutant strains was tested using a similar protocol as the Calgary biofilm device. Involvement of RpiRc in S. aureus virulence was assessed ex vivo by internalization experiments into HEK293 cells and in vivo in a mouse model of subcutaneous catheter infection. While minimum inhibitory concentrations (MICs) of planktonic cells were not affected in the mutant strain, we observed increased biofilm susceptibility to almost all tested antibiotics, regardless of their mode of action. More importantly, the rpiRc mutant showed reduced virulence in both ex vivo and in vivo experiments related to decreased fnbpA-B transcription and eDNA production. RpiRc is an important regulator involved in eDNA degradation inside the matrix of mature PIA independent biofilms. These results illustrate that RpiRc contributes to increased antibiotic tolerance in mature bacterial biofilm and also to S. aureus cell adhesion and virulence during subcutaneous infection.Author summaryBiofilm formation contributes to the severity of Staphylococcus aureus related infections. Biofilm matrix is mainly composed by polysaccharide intercellular adhesion (PIA), proteins and extracellular DNA (eDNA). By screening a mutant library of S. aureus, RpiRc was identified as a new regulator of eDNA dependent biofilm formation. How RpiRc regulates biofilm and its role in S. aureus virulence was studied in four different S. aureus strains. Deletion of RpiRc resulted in a pronounced decreased eDNA dependent biofilm formation, but not PIA dependent biofilm formation. Decreased biofilm formation was not related to increased autolysis, but was linked to extracellular compounds found in the supernatant of mutant biofilms. Sln was identified as one of this compound. RpiRc deletion also decreased biofilm recalcitrance (resistance) to selected antibiotics. Involvement of RpiRc in S. aureus pathogenesis was investigated ex vivo by internalization into HEK293 cells and in vivo in a mouse model of catheter infection. RpiRc deletion resulted in decreased virulence related to decreased expression of surface proteins like the fibronectin binding proteins A and B (FnbpA-B). These results illustrate that RpiRc contributes to increased antibiotic tolerance in mature bacterial biofilm and also to S. aureus cell adhesion and virulence during subcutaneous infection.

scholarly journals Virulence of methicillin-resistant Staphylococcus aureus modulated by the YycFG two-component pathway in a rat model of osteomyelitis

2019 ◽  
Vol 14 (1) ◽  
Author(s):  
Shizhou Wu ◽  
Yunjie Liu ◽  
Lei Lei ◽  
Hui Zhang
Keyword(s):  
Staphylococcus Aureus ◽  
Western Blot ◽  
Rat Model ◽  
Biofilm Formation ◽  
Bacterial Biofilm ◽  
Invasive Ability ◽  
Methicillin Resistant ◽  
Two Component ◽  
Mrsa Strains

Abstract Objectives Methicillin-resistant Staphylococcus aureus (MRSA) strains present an urgent medical problem in osteomyelitis cases. Our previous study indicated that the YycFG two-component regulatory pathway is associated with the bacterial biofilm organization of MRSA strains. The aim of this study was to investigate the regulatory roles of ASyycG in the bacterial biofilm formation and the pathogenicity of MRSA strains using an antisense RNA strategy. Methods An ASyycG-overexpressing MRSA clinical isolate was constructed. The bacterial growth was monitored, and the biofilm biomass on bone specimens was examined using scanning electron microscopy and confocal laser scanning microscopy. Furthermore, quantitative RT-PCR (QRT-PCR) analysis was used to measure the expression of yycF/G/H and icaA/D in the MRSA and ASyycG strains. The expression of the YycG protein was quantified by Western blot assays. We validated the role of ASyycG in the invasive ability and pathogenicity of the strains in vivo using histology and peptide nucleic acid fluorescent in situ hybridization. Results The results showed that overexpression of ASyycG lead to a reduction in biofilm formation and exopolysaccharide (EPS) synthesis compared to the control MRSA strains. The ASyycG strains exhibited decreased expression of the yycF/G/H and icaA/D genes. Furthermore, Western blot data showed that the production of the YycG protein was inhibited in the ASyycG strains. In addition, we demonstrated that ASyycG suppressed the invasive ability and pathogenicity of the strain in vivo using an SPF (specific pathogen free) rat model. Conclusion In summary, the overexpression of ASyycG leads to a reduction in biofilm formation and bacterial pathogenicity in vivo, which provides a potential target for the management of MRSA-induced osteomyelitis.

scholarly journals Tissue Plasminogen Activator Coating on Implant Surfaces Reduces Staphylococcus aureus Biofilm Formation

2015 ◽  
Vol 82 (1) ◽  
pp. 394-401 ◽  
Author(s):  
Jakub Kwiecinski ◽  
Manli Na ◽  
Anders Jarneborn ◽  
Gunnar Jacobsson ◽  
Marijke Peetermans ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Plasminogen Activator ◽  
Tissue Plasminogen Activator ◽  
Medical Devices ◽  
Biofilm Formation ◽  
Bacterial Biofilm ◽  
Content Type ◽  
Tissue Plasminogen

ABSTRACTStaphylococcus aureusbiofilm infections of indwelling medical devices are a major medical challenge because of their high prevalence and antibiotic resistance. As fibrin plays an important role inS. aureusbiofilm formation, we hypothesize that coating of the implant surface with fibrinolytic agents can be used as a new method of antibiofilm prophylaxis. The effect of tissue plasminogen activator (tPA) coating onS. aureusbiofilm formation was tested within vitromicroplate biofilm assays and anin vivomouse model of biofilm infection. tPA coating efficiently inhibited biofilm formation by variousS. aureusstrains. The effect was dependent on plasminogen activation by tPA, leading to subsequent local fibrin cleavage. A tPA coating on implant surfaces prevented both early adhesion and later biomass accumulation. Furthermore, tPA coating increased the susceptibility of biofilm infections to antibiotics.In vivo, significantly fewer bacteria were detected on the surfaces of implants coated with tPA than on control implants from mice treated with cloxacillin. Fibrinolytic coatings (e.g., with tPA) reduceS. aureusbiofilm formation bothin vitroandin vivo, suggesting a novel way to prevent bacterial biofilm infections of indwelling medical devices.

scholarly journals Predilection for developing a hematogenous orthopaedic implant-associated infection in older versus younger mice

2021 ◽  
Vol 16 (1) ◽  
Author(s):  
John M. Thompson ◽  
Alyssa G. Ashbaugh ◽  
Yu Wang ◽  
Robert J. Miller ◽  
Julie E. Pickett ◽  
...  
Keyword(s):  
At Risk ◽  
Staphylococcus Aureus ◽  
Mouse Model ◽  
Ex Vivo ◽  
Kirschner Wire ◽  
Age Groups ◽  
Orthopaedic Implant ◽  
X Ray ◽  
Risk Patients

Abstract Background The pathogenesis of hematogenous orthopaedic implant-associated infections (HOIAI) remains largely unknown, with little understanding of the influence of the physis on bacterial seeding. Since the growth velocity in the physis of long bones decreases during aging, we sought to evaluate the role of the physis on influencing the development of Staphylococcus aureus HOIAI in a mouse model comparing younger versus older mice. Methods In a mouse model of HOIAI, a sterile Kirschner wire was inserted retrograde into the distal femur of younger (5–8-week-old) and older (14–21-week-old) mice. After a 3-week convalescent period, a bioluminescent Staphylococcus aureus strain was inoculated intravenously. Bacterial dissemination to operative and non-operative legs was monitored longitudinally in vivo for 4 weeks, followed by ex vivo bacterial enumeration and X-ray analysis. Results In vivo bioluminescence imaging and ex vivo CFU enumeration of the bone/joint tissue demonstrated that older mice had a strong predilection for developing a hematogenous infection in the operative legs but not the non-operative legs. In contrast, this predilection was less apparent in younger mice as the infection occurred at a similar rate in both the operative and non-operative legs. X-ray imaging revealed that the operative legs of younger mice had decreased femoral length, likely due to the surgical and/or infectious insult to the more active physis, which was not observed in older mice. Both age groups demonstrated substantial reactive bone changes in the operative leg due to infection. Conclusions The presence of an implant was an important determinant for developing a hematogenous orthopaedic infection in older but not younger mice, whereas younger mice had a similar predilection for developing periarticular infection whether or not an implant was present. On a clinical scale, diagnosing HOIAI may be difficult particularly in at-risk patients with limited examination or other data points. Understanding the influence of age on developing HOIAI may guide clinical surveillance and decision-making in at-risk patients.

scholarly journals An Electrostatic Net Model for the Role of Extracellular DNA in Biofilm Formation by Staphylococcus aureus

2015 ◽  
Vol 197 (24) ◽  
pp. 3779-3787 ◽  
Author(s):  
Vanina Dengler ◽  
Lucy Foulston ◽  
Alicia S. DeFrancesco ◽  
Richard Losick
Keyword(s):  
Staphylococcus Aureus ◽  
Biofilm Formation ◽  
Extracellular Dna ◽  
Matrix Proteins ◽  
Exogenous Dna ◽  
Content Type ◽  
Cytoplasmic Proteins ◽  
The Matrix ◽  
Biofilm Matrix

ABSTRACTStaphylococcus aureusis an important human pathogen that can form biofilms on various surfaces. These cell communities are protected from the environment by a self-produced extracellular matrix composed of proteins, DNA, and polysaccharide. The exact compositions and roles of the different components are not fully understood. In this study, we investigated the role of extracellular DNA (eDNA) and its interaction with the recently identified cytoplasmic proteins that have a moonlighting role in the biofilm matrix. These matrix proteins associate with the cell surface upon the drop in pH that naturally occurs during biofilm formation, and we found here that this association is independent of eDNA. Conversely, the association of eDNA with the matrix was dependent on matrix proteins. Both proteinase and DNase treatments severely reduced clumping of resuspended biofilms; highlighting the importance of both proteins and eDNA in connecting cells together. By adding an excess of exogenous DNA to DNase-treated biofilm, clumping was partially restored, confirming the crucial role of eDNA in the interconnection of cells. On the basis of our results, we propose that eDNA acts as an electrostatic net, interconnecting cells surrounded by positively charged matrix proteins at a low pH.IMPORTANCEExtracellular DNA (eDNA) is an important component of the biofilm matrix of diverse bacteria, but its role in biofilm formation is not well understood. Here we report that inStaphylococcus aureus, eDNA associates with cells in a manner that depends on matrix proteins and that eDNA is required to link cells together in the biofilm. These results confirm previous studies that showed that eDNA is an important component of theS. aureusbiofilm matrix and also suggest that eDNA acts as an electrostatic net that tethers cells together via the proteinaceous layer of the biofilm matrix.

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

mBio ◽  
2012 ◽  
Vol 3 (4) ◽  
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.

scholarly journals Anti-Biofilm Effects of Synthetic Antimicrobial Peptides Against Drug-Resistant Pseudomonas aeruginosa and Staphylococcus aureus Planktonic Cells and Biofilm

Molecules ◽  
2019 ◽  
Vol 24 (24) ◽  
pp. 4560 ◽  
Author(s):  
Seong-Cheol Park ◽  
Min-Young Lee ◽  
Jin-Young Kim ◽  
Hyeonseok Kim ◽  
Myunghwan Jung ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Pseudomonas Aeruginosa ◽  
Antimicrobial Peptides ◽  
Biofilm Formation ◽  
Extracellular Dna ◽  
Therapeutic Drug ◽  
Dependent Manner ◽  
Drug Resistant ◽  
And Staphylococcus Aureus

Biofilm-associated infections are difficult to manage or treat as biofilms or biofilm-embedded bacteria are difficult to eradicate. Antimicrobial peptides have gained increasing attention as a possible alternative to conventional drugs to combat drug-resistant microorganisms because they inhibit the growth of planktonic bacteria by disrupting the cytoplasmic membrane. The current study investigated the effects of synthetic peptides (PS1-2, PS1-5, and PS1-6) and conventional antibiotics on the growth, biofilm formation, and biofilm reduction of drug-resistant Pseudomonas aeruginosa and Staphylococcus aureus. The effects of PS1-2, PS1-5, and PS1-6 were also tested in vivo using a mouse model. All peptides inhibited planktonic cell growth and biofilm formation in a dose-dependent manner. They also reduced preformed biofilm masses by removing the carbohydrates, extracellular DNA, and lipids that comprised extracellular polymeric substances (EPSs) but did not affect proteins. In vivo, PS1-2 showed the greatest efficacy against preformed biofilms with no cytotoxicity. Our findings indicate that the PS1-2 peptide has potential as a next-generation therapeutic drug to overcome multidrug resistance and to regulate inflammatory response in biofilm-associated infections.

scholarly journals Micrococcal Nuclease stimulates Staphylococcus aureus Biofilm Formation in a Murine Implant Infection Model

2022 ◽  
Vol 11 ◽  
Author(s):  
Abigail M. Forson ◽  
Colin W. K. Rosman ◽  
Theo G. van Kooten ◽  
Henny C. van der Mei ◽  
Jelmer Sjollema
Keyword(s):  
Staphylococcus Aureus ◽  
Biofilm Formation ◽  
Polyvinylidene Fluoride ◽  
Histological Analysis ◽  
Ex Vivo ◽  
Extracellular Dna ◽  
Neutrophil Extracellular Trap ◽  
Micrococcal Nuclease ◽  
Infection Model ◽  
The Impact

Advancements in contemporary medicine have led to an increasing life expectancy which has broadened the application of biomaterial implants. As each implant procedure has an innate risk of infection, the number of biomaterial-associated infections keeps rising. Staphylococcus aureus causes 34% of such infections and is known as a potent biofilm producer. By secreting micrococcal nuclease S. aureus is able to escape neutrophil extracellular traps by cleaving their DNA-backbone. Also, micrococcal nuclease potentially limits biofilm growth and adhesion by cleaving extracellular DNA, an important constituent of biofilms. This study aimed to evaluate the impact of micrococcal nuclease on infection persistence and biofilm formation in a murine biomaterial-associated infection-model with polyvinylidene-fluoride mesh implants inoculated with bioluminescent S. aureus or its isogenic micrococcal nuclease deficient mutant. Supported by results based on in-vivo bioluminescence imaging, ex-vivo colony forming unit counts, and histological analysis it was found that production of micrococcal nuclease enables S. aureus bacteria to evade the immune response around an implant resulting in a persistent infection. As a novel finding, histological analysis provided clear indications that the production of micrococcal nuclease stimulates S. aureus to form biofilms, the presence of which extended neutrophil extracellular trap formation up to 13 days after mesh implantation. Since micrococcal nuclease production appeared vital for the persistence of S. aureus biomaterial-associated infection, targeting its production could be a novel strategy in preventing biomaterial-associated infection.

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

2012 ◽  
Vol 80 (5) ◽  
pp. 1634-1638 ◽  
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.

scholarly journals PspC domain-containing protein (PCP) determines Streptococcus mutans biofilm formation through bacterial extracellular DNA release and platelet adhesion in experimental endocarditis

2021 ◽  
Vol 17 (2) ◽  
pp. e1009289
Author(s):  
Chiau-Jing Jung ◽  
Chih-Chieh Hsu ◽  
Jeng-Wei Chen ◽  
Hung-Wei Cheng ◽  
Chang-Tsu Yuan ◽  
...  
Keyword(s):  
Streptococcus Mutans ◽  
Biofilm Formation ◽  
Heart Valves ◽  
Platelet Adhesion ◽  
Bacterial Biofilm ◽  
Extracellular Dna ◽  
Dependent Manner ◽  
Dna Release

Bacterial extracellular DNA (eDNA) and activated platelets have been found to contribute to biofilm formation by Streptococcus mutans on injured heart valves to induce infective endocarditis (IE), yet the bacterial component directly responsible for biofilm formation or platelet adhesion remains unclear. Using in vivo survival assays coupled with microarray analysis, the present study identified a LiaR-regulated PspC domain-containing protein (PCP) in S. mutans that mediates bacterial biofilm formation in vivo. Reverse transcriptase- and chromatin immunoprecipitation-polymerase chain reaction assays confirmed the regulation of pcp by LiaR, while PCP is well-preserved among streptococcal pathogens. Deficiency of pcp reduced in vitro and in vivo biofilm formation and released the eDNA inside bacteria floe along with reduced bacterial platelet adhesion capacity in a fibrinogen-dependent manner. Therefore, LiaR-regulated PCP alone could determine release of bacterial eDNA and binding to platelets, thus contributing to biofilm formation in S. mutans-induced IE.

scholarly journals Daptomycin and Tigecycline Have Broader Effective Dose Ranges than Vancomycin as Prophylaxis against a Staphylococcus aureus Surgical Implant Infection in Mice

2012 ◽  
Vol 56 (5) ◽  
pp. 2590-2597 ◽  
Author(s):  
Jared A. Niska ◽  
Jonathan H. Shahbazian ◽  
Romela Irene Ramos ◽  
Jonathan R. Pribaz ◽  
Fabrizio Billi ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Effective Dose ◽  
Biofilm Formation ◽  
Low Dose ◽  
Ex Vivo ◽  
High Dose ◽  
Implant Infection ◽  
Content Type ◽  
Surgical Implant

ABSTRACTVancomycin is widely used for intravenous prophylaxis against surgical implant infections. However, it is unclear whether alternative antibiotics used to treat methicillin-resistantStaphylococcus aureus(MRSA) infections are effective as prophylactic agents. The aim of this study was to compare the efficacies of vancomycin, daptomycin, and tigecycline as prophylactic therapy against a methicillin-sensitiveS. aureus(MSSA) or MRSA surgical implant infection in mice. MSSA or MRSA was inoculated into the knee joints of mice in the presence of a surgically placed medical-grade metallic implant. The efficacies of low- versus high-dose vancomycin (10 versus 110 mg/kg), daptomycin (1 versus 10 mg/kg), and tigecycline (1 versus 10 mg/kg) intravenous prophylaxis were compared usingin vivobioluminescence imaging,ex vivobacterial counts, and biofilm formation. High-dose vancomycin, daptomycin, and tigecycline resulted in similar reductions in bacterial burden and biofilm formation. In contrast, low-dose daptomycin and tigecycline were more effective than low-dose vancomycin against the implant infection. In this mouse model of surgical implant MSSA or MRSA infection, daptomycin and tigecycline prophylaxis were effective over a broader dosage range than vancomycin. Future studies in humans will be required to determine whether these broader effective dose ranges for daptomycin and tigecycline in mice translate to improved efficacy in preventing surgical implant infections in clinical practice.

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