Effect of Farnesol on Structure and Composition of <Emphasis Type="Italic">Staphylococcus epidermidis</Emphasis> Biofilm Matrix

Staphylococcus epidermidis is the most frequent cause of nosocomial sepsis and catheter-related infections in which biofilm formation is considered to be one of the main virulence mechanisms. Moreover, their increased resistance to conventional antibiotic therapy enhances the need to develop new therapeutical agents. Farnesol, a natural sesquiterpenoid present in many essential oils, has been described as impairing bacterial growth. The aim of this study was to evaluate the effect of farnesol on the structure and composition of biofilm matrix of S. epidermidis. Biofilms formed in the presence of farnesol (300 μM) contained less biomass, and displayed notable changes in the composition of the biofilm matrix. Changes in the spacial structure were also verified by confocal scanning laser microscopy (CSLM). The results obtained by the quantification of extracellular polymers and by wheat germ agglutinin (WGA) fluorescent detection of glycoproteins containing β(1→4)-N-acetyl-d-glucosamine support the hypothesis that farnesol causes disruption of the cytoplasmic membrane and consequently release of cellular content.     

Efficacy of novel antibacterial compounds targeting histidine kinase YycG protein

Treating staphylococcal biofilm-associated infections is challenging. Based on the findings that compound 2 targeting the HK domain of Staphylococcus epidermidis YycG has bactericidal and antibiofilm activities against staphylococci, six newly synthesized derivatives were evaluated for their antibacterial activities. The six derivatives of compound 2 inhibited autophosphorylation of recombinant YycG′ and the IC₅₀ values ranged from 24.2 to 71.2 μM. The derivatives displayed bactericidal activity against planktonic S. epidermidis or Staphylococcus aureus strains in the MIC range of 1.5–3.1 μM. All the derivatives had antibiofilm activities against the 6- and 24-h biofilms of S. epidermidis. Compared to the prototype compound 2, they had less cytotoxicity for Vero cells and less hemolytic activity for human erythrocytes. The derivatives showed antibacterial activities against clinical methicillin-resistant staphylococcal isolates. The structural modification of YycG inhibitors will assist the discovery of novel agents to eliminate biofilm infections and multidrug-resistant staphylococcal infections.     

In vitro Activity of Daptomycin,Linezolid and Rifampicin on <Emphasis Type="Italic">Staphylococcus epidermidis</Emphasis> Biofilms

Owing to their massive use, Staphylococcus epidermidis has recently developed significant resistance to several antibiotics, and became one of the leading causes of hospital-acquired infections. Current antibiotics are typically ineffective in the eradication of bacteria in biofilm-associated persistent infections. Accordingly, the paucity of effective treatment against cells in this mode of growth is a key factor that potentiates the need for new agents active in the prevention or eradication of biofilms. Daptomycin and linezolid belong to the novel antibiotic therapies that are active against gram-positive cocci. On the other hand, rifampicin has been shown to be one of the most potent, prevalent antibiotics against S. epidermidis biofilms. Therefore, the main aim of this study was to study the susceptibility of S. epidermidis biofilm cells to the two newer antimicrobial agents previously mentioned, and compare the results obtained with the antimicrobial effect of rifampicin, widely used in the prevention/treatment of indwelling medical device infections. To this end the in vitro activities of daptomycin, linezolid, and rifampicin on S. epidermidis biofilms were accessed, using these antibiotics at MIC and peak serum concentrations. The results demonstrated that at MIC concentration, rifampicin was the most effective antibiotic tested. At peak serum concentration, both strains demonstrated similar susceptibility to rifampicin and daptomycin, with colony-forming units (CFUs) reductions of approximately 3–4 log₁₀, with a slightly lower response to linezolid, which was also more strain dependent. However, considering all the parameters studied, daptomycin was considered the most effective antibiotic tested, demonstrating an excellent in vitro activity against S. epidermidis biofilm cells. In conclusion, this antibiotic can be strongly considered as an acceptable therapeutic option for S. epidermidis biofilm-associated infections and can represent a potential alternative to rifampicin in serious infections where rifampicin resistance becomes prevalent.     

Susceptibility of <Emphasis Type="Italic">Candida</Emphasis> biofilms to histatin 5 and fluconazole

Candida-associated denture stomatitis has a high rate of recurrence. Candida biofilms formed on denture acrylic are more resistant to antifungals than planktonic yeasts. Histatins, a family of basic peptides secreted by the major salivary glands in humans, especially histatin 5, possess significant antifungal properties. We examined antifungal activities of histatin 5 against planktonic or biofilm Candida albicans and Candida glabrata. Candida biofilms were developed on poly(methyl methacrylate) discs and treated with histatin 5 (0.01–100 μM) or fluconazole (1–200 μM). The metabolic activity of the biofilms was measured by the XTT reduction assay. The fungicidal activity of histatin 5 against planktonic Candida was tested by microdilution plate assay. Biofilm and planktonic C. albicans GDH18, UTR-14 and 6122/06 were highly susceptible to histatin 5, with 50% RMA (concentration of the agent causing 50% reduction in the metabolic activity; biofilm) of 4.6 ± 2.2, 6.9 ± 3.7 and 1.7 ± 1.5 μM, and IC₅₀ (planktonic cells) of 3.0 ± 0.5, 2.6 ± 0.1 and 4.8 ± 0.5, respectively. Biofilms of C. glabrata GDH1407 and 6115/06 were less susceptible to histatin 5, with 50% RMA of 31.2 ± 4.8 and 62.5 ± 0.7 μM, respectively. Planktonic C. glabrata was insensitive to histatin 5 (IC₅₀ > 100 μM). Biofilm-associated Candida was highly resistant to fluconazole in the range 1–200 μM; e.g. at 100 μM only ~20% inhibition was observed for C. albicans, and ~30% inhibition for C. glabrata. These results indicate that histatin 5 exhibits antifungal activity against biofilms of C. albicans and C. glabrata developed on denture acrylic. C. glabrata is significantly less sensitive to histatin 5 than C. albicans.     

<Emphasis Type="Italic">Staphylococcus epidermidis</Emphasis> glucose uptake in biofilm versus planktonic cells

The aim of this work was to compare the glucose uptake of biofilms formed by four different Staphylococcus epidermidis strains as well as to compare between sessile and planktonic cells of the same strain. Biofilm cells showed a lower level of glucose uptake compared to planktonic cells. Moreover, glucose uptake by cells in the sessile form was strongly influenced by biofilm composition. Therefore, this work helps to confirm the phenotypic variability of S. epidermidis strains and the different behaviour patterns between sessile and free cells.     

Discovery of quinoline small molecules with potent dispersal activity against methicillin-resistant Staphylococcus aureus and Staphylococcus epidermidis biofilms using a scaffold hopping strategy

Staphylococcus aureus and Staphylococcus epidermidis are recognized as the most frequent cause of biofilm-associated nosocomial and indwelling medical device infections. Biofilm-associated infections are known to be highly resistant to our current arsenal of clinically used antibiotics and antibacterial agents. To exacerbate this problem, no therapeutic option exists that targets biofilm-dependent machinery critical to Staphylococcal biofilm formation and maintenance. Here, we describe the discovery of a series of quinoline small molecules that demonstrate potent biofilm dispersal activity against methicillin-resistant S. aureus and S. epidermidis using a scaffold hopping strategy. This interesting class of quinolines also has select synthetic analogues that demonstrate potent antibacterial activity and biofilm inhibition against S. aureus and S. epidermidis.     

In vitro activity of the antimicrobial peptide AP7121 against the human methicillin-resistant biofilm producers Staphylococcus aureus and Staphylococcus epidermidis

Abstract

In vitro activity against methicillin-resistant Staphylococcus aureus (MRSA) and Staphylococcus epidermidis biofilm producers from blood cultures of patients with prosthetic hip infections was evaluated. The Minimum Inhibitory Concentration (MIC) for AP7121 was determined and the bactericidal activity of AP7121 (MICx1, MICx4) against planktonic cells was studied at 4, 8 and 24?h. The biofilms formed were incubated with AP7121 (MICx1, MICx4) for 1 and 24?h. The anti-adhesion effect of an AP7121-treated inert surface over the highest MIC isolate was studied with scanning electron microscopy (SEM). The bactericidal activity of AP7121 against all the planktonic staphylococcal cells was observed at 4?h at both peptide concentrations. Dose-dependent anti-biofilm activity was detected. AP7121 (MICx4) showed bactericidal activity at 24?h in all isolates. SEM confirmed prevention of biofilm formation. This research showed the in vitro anti-biofilm activity of AP7121 against MRSA and S. epidermidis and the prevention of biofilm formation by them on an abiotic surface.     

Impact of the <Emphasis Type="Italic">Staphylococcus epidermidis</Emphasis> LytSR two-component regulatory system on murein hydrolase activity,pyruvate utilization and global transcriptional profile

Background  

Staphylococcus epidermidis has emerged as one of the most important nosocomial pathogens, mainly because of its ability to colonize implanted biomaterials by forming a biofilm. Extensive studies are focused on the molecular mechanisms involved in biofilm formation. The LytSR two-component regulatory system regulates autolysis and biofilm formation in Staphylococcus aureus. However, the role of LytSR played in S. epidermidis remained unknown.     

In Vitro Model of Bacterial Biofilm Formation on Polyvinyl Chloride Biomaterial

The aim of the study was to establish an in vitro model of Staphylococcus epidermidis biofilms on polyvinyl chloride (PVC) material, and to investigate bacterial biofilm formation and its structure using the combined approach of confocal laser scanning microscope (CLSM) and scanning electron microscope (SEM). Staphylococcus epidermidis bacteria (stain RP62A) were incubated with PVC pieces in Tris buffered saline to form biofilms. Biofilm formation was examined at 6, 12, 18, 24, 30, and 48 h. Thicknesses of these biofilms and the number, and percentage of viable cells in biofilms were measured. CT scan images of biofilms were obtained using CLSM and environmental SEM. The results of this study showed that Staphylococcus epidermidis biofilm is a highly organized multi-cellular structure. The biofilm is constituted of large number of viable and dead bacterial cells. Bacterial biofilm formation on the surface of PVC material was found to be a dynamic process with maximal thickness being attained at 12–18 h. These biofilms became mature by 24 h. There was significant difference in the percentage of viable cells along with interior, middle, and outer layers of biofilms (P < 0.05). Staphylococcus epidermidis biofilm is sophisticated in structure and the combination method involving CLSM and SEM was ideal for investigation of biofilms on PVC material.     

In vitro activity of vancomycin, quinupristin/dalfopristin, and linezolid against intact and disrupted biofilms of staphylococci

 

Shed cells or disrupted parts of the biofilm may enter the circulation causing serious and very hard to treat biofilm-associated infections. The activity of antimicrobial agents against the shed cells/disrupted biofilms is largely unknown.

Methods

We studied the in vitro susceptibility of intact and disrupted biofilms of thirty clinical isolates of methicillin-resistant and methicillin–susceptible Staphylococcus aureus (MRSA and MSSA) and Staphylococcus epidermidis to vancomycin, quinupristin/dalfopristin, and linezolid and compared it to that of the suspended (planktonic) cells.

Results

Bacteria in the disrupted biofilms were as resistant as those in the intact biofilms at the minimum inhibitory concentrations of the antibiotics. At higher concentrations, bacteria in the disrupted biofilms were significantly (P < 0.001) less resistant than those in the intact biofilms but more resistant than the planktonic cells. Quinupristin/dalfopristin showed the best activity against cells of the disrupted biofilms at concentrations above MICs and vancomycin, at 500 and 1,000 μg/ml, was significantly more active against the biofilms of MRSA and S. epidermidis

Conclusion

The difficulty of treating biofilm-associated infections may be attributed not only to the difficulty of eradicating the biofilm focus but also to the lack of susceptibility of cells disrupted from the biofilm to antimicrobial agents.     

苦参碱对表皮葡萄球菌生物被膜作用初探

通过中药有效成分苦参碱对表皮葡萄球菌生物被膜抑制作用的研究,为表皮葡萄球菌生物被膜引起的相关感染提供新的治疗途径。利用XTT减低法评价苦参碱对表皮葡萄球菌初始粘附及生物被膜内细菌代谢的影响,镜下观察该药对表皮葡萄球菌生物被膜的形态学影响。结果表明:苦参碱对表皮葡萄球菌生物被膜菌的SMIC50和SMIC80分别为62.5 mg/L和500 mg/L;1 000 mg/L浓度的苦参碱对表皮葡萄球菌早期粘附有抑制作用;250 mg/L浓度的苦参碱对表皮葡萄球菌生物被膜的形态有显著影响。因此可见,苦参碱对表皮葡萄球菌生物被膜的形成与粘附均有抑制作用。     

Holo and apo-transferrins interfere with adherence to abiotic surfaces and with adhesion/invasion to HeLa cells in Staphylococcus spp.

Staphylococcus aureus and Staphylococcus epidermidis are the major cause of infections associated with implanted medical devices. Colonization on abiotic and biotic surfaces is often sustained by biofilm forming strains. Human natural defenses can interfere with this virulence factor. We investigated the effect of human apo-transferrin (apo-Tf, the iron-free form of transferrin, Tf) and holo-transferrin (holo-Tf, the iron-saturated form) on biofilm formation by CA-MRSA S. aureus USA300 type (ST8-IV) and S. epidermidis (a clinical isolate and ATCC 35984 strain). Furthermore S. aureus adhesion and invasion assays were performed in a eukaryotic cell line. A strong reduction in biofilm formation with both Tfs was obtained albeit at very different concentrations. In particular, the reduction in biofilm formation was higher with apo-Tf rather than obtained with holo-Tf. Furthermore, while S. aureus adhesion to eukaryotic cells was not appreciably affected, their invasion was highly inhibited in the presence of holo-Tf, and partially inhibited by the apo form. Our results suggest that Tfs could be used as antibacterial adjuvant therapy in infection sustained by staphylococci to strongly reduce their virulence related to adhesion and cellular invasion.     

Amoxicillin and specific bacteriophage can be used together for eradication of biofilm of <Emphasis Type="Italic">Klebsiella pneumoniae</Emphasis> B5055

Despite the efficacy of antibiotics as well as bacteriophages in treatment of bacterial infections, their role in treatment of biofilm associated infections is still under consideration especially in case of older biofilms. Here, efficacy of bacteriophage alone or in combination with amoxicillin, for eradication of biofilm of Klebsiella pneumoniae B5055 has been assessed. Planktonic cells as well as biofilm of K. pneumoniae B5055 grown in 96-well microtiter plates were exposed to bacteriophage and amoxicillin at various Multiplicity of Infections (MoIs) as well as at three different antibiotic concentrations (512, 256 and 128 μg/ml), respectively. After exposure to 256 μg/ml (MIC) of amoxicillin, bacterial load of planktonic culture as well as 1-day-old biofilm was reduced by a log factor of 4.1 ± 0.31 (P = 0.008) and 1.24 ± 0.27 (P < 0.05), respectively but reduction in the bacterial load of mature biofilm (8-day-old) was insignificant (P = 0.23). When 8-day-old biofilm was exposed to higher antibiotic concentration (512 μg/ml) or phage alone (MoI = 0.01) a log reduction of 2.97 ± 0.11 (P = 0.182) and 3.51 ± 0.19 (P = 0.073), respectively was observed. While on exposing to a combination of both the amoxicillin and phage, a significant reduction (P < 0.01) in bacterial load of the biofilm was seen. Hence, when antibiotic was used in combination with specific bacteriophage a greater destruction of the biofilm structure suggested that the phages could be used successfully along with antibiotic therapy. An added advantage of the combination therapy would be its ability to check formation of resistant mutants that otherwise develop easily upon using phage or antibiotic alone.     

Human cathelicidin peptide LL37 inhibits both attachment capability and biofilm formation of Staphylococcus epidermidis

Aims: The aim of this work was to investigate the possible effect of human cathelicidin antimicrobial peptide LL37 on biofilm formation of Staphylococcus epidermidis, a major causative agent of indwelling device‐related infections. Methods and Results: We performed initial attachment assay and biofilm formation solid surface assay in microtitre plates, as well as growth experiment in liquid medium using laboratory strain Staph. epidermidis ATCC35984. We found that already a low concentration of the peptide LL37 (1 mg l^?1) significantly decreased both the attachment of bacteria to the surface and also the biofilm mass. No growth inhibition was observed even at 16 mg l^?1 concentration of LL37, indicating a direct effect of the peptide on biofilm production. Conclusions: As biofilm protects bacteria during infections in humans and allows their survival in a hostile environment, inhibition of biofilm formation by LL37 may have a key role to prevent bacterial colonization on indwelling devices. Significance and Impact of the Study: Our findings suggest that this host defence factor can be a potential candidate in prevention and treatment strategies of Staph. epidermidis infections in humans.     

Extracellular protease in <Emphasis Type="Italic">Actinomycetes</Emphasis> culture supernatants inhibits and detaches <Emphasis Type="Italic">Staphylococcus</Emphasis><Emphasis Type="Italic">aureus</Emphasis> biofilm formation

Bacterial biofilms are associated with chronic infections due to their resistance to antimicrobial agents. Staphylococcus aureus is a versatile human pathogen and can form biofilms on human tissues and diverse medical devices. To identify novel biofilm inhibitors of S. aureus, the supernatants from a library of 458 Actinomycetes strains were screened. The culture supernatants (1% v/v) of more than 10 Actinomycetes strains inhibited S. aureus biofilm formation by more than 80% without affecting the growth. The culture supernatants of these biofilm-reducing Actinomycetes strains contained a protease (equivalent to 0.1 μg proteinase K ml⁻¹), which both inhibited S. aureus biofilm formation and detached pre-existing S. aureus biofilms. This study suggests that protease treatment could be a feasible tool to reduce and eradicate S. aureus biofilms.     

Species-specific probes and real-time PCR as a tool for fast detection and differentiation of 15 bacteria relevant in intensive care medicine

Detection of bacterial DNA from laboratory-prepared specimens such as water, urine, and blood has the potential to improve diagnostic tools in microbiology. A novel real-time PCR-based assay was developed and its performance and robustness were evaluated for a panel of spiked suspensions of 15 clinically relevant bacteria. As low as ten colony forming units (CFU)/100 μl were detectable. No cross-reactivity was observed, except for Staphylococcus aureus and Staphylococcus epidermidis. Nevertheless, S. aureus and S. epidermidis were reliably differentiated by melting curve analysis. The protocol was also validated with three groups containing a mixture of five spiked bacteria each, with the result of reliable differentiation. This novel assay allows an exact identification of 15 microbes relevant in intensive care medicine, including mixed infections, in a one run experiment in less than 4 h.     

Characterization of clinical strains of MSSA,MRSA and MRSE isolated from skin and soft tissue infections and the antibacterial activity of ZnO nanoparticles

Staphylococcus aureus, particularly methicillin-resistant S. aureus (MRSA), is an important cause of pyogenic skin and soft tissue infections (SSTIs). MRSA is an important pathogen in the healthcare sector that has neither been eliminated from the hospital nor community environment. In humans, S. aureus causes superficial lesions in the skin and localized abscesses, pyogenic meningitis/encephalitis, osteomyelitis, septic arthritis, invasive endocarditis, pneumonia, urinary tract infections and septicemia. Investigations focused in the search of other alternatives for the treatment of MRSA infections are in progress. Among the range of compounds whose bactericidal activity is being investigated, ZnO nanoparticles (ZnO–NPs) appears most promising new unconventional antibacterial agent that could be helpful to confront this and other drug-resistant bacteria. The aim of present study is to investigate the antibacterial potential of ZnO–NPs against Staphylococcus species isolated from the pus and wounds swab from the patients with skin and soft tissue infections in a tertiary care hospital of north India. ZnO–NPs (≈19.82 nm) synthesized by sol–gel process were characterized using scanning electron microscopy, X-ray diffraction , and Atomic force microscopy. The antibacterial potential was assessed using time-dependent growth inhibition assay, well diffusion test, MIC and MBC test and colony forming units methods. ZnO–NPs inhibited bacterial growth of methicillin-sensitive S. aureus (MSSA), MRSA and methicillin-resistant S. epidermidis (MRSE) strains and were effective bactericidal agents that were not affected by drug-resistant mechanisms of MRSA and MRSE.     

In Vitro Antibacterial Activity of 4-Phenyl-1-(2-phenyl-allyl)pyridinium bromide: A Novel Class of Pyridinium Based Antibacterial Compounds

The continuing increase in the incidence of multi drug resistant pathogenic bacteria and shortage of new antimicrobial agents are the prime driver in efforts to identify the novel antimicrobial classes. In vitro antibacterial activity of 4-phenyl-1-(2-phenylallyl) pyridinium bromide was tested against Gram positive Staphylococcus aureus, Streptococcus species, Bacillus subtilis, and Gram negative Klebsiella aerogenes and Escherichia coli using disk diffusion method. Among them S. aureus showed strong antibacterial activity (21.99 ± 0.03 mm) while E. coli showed very little activity (8.97 ± 0.06 mm) towards the compound. The MIC of 4-phenyl-1-(2-phenyl-allyl)-pyridinium bromide for 90% S. aureus was ≤20 μg/ml and was compared with phenoxymethylpenicillin, cloxacillin, erythromycin and vancomycin. When 4-phenyl-1-(2-phenyl-allyl)pyridinium bromide showed MIC at ≤20 μg/ml, all others showed MIC at ≤100 μg/ml. Strong antibacterial activity of 4-phenyl-1-(2-phenyl-allyl)pyridinium bromide against S. aureus indicates that there is a possibility to use it as an effective antibacterial agent.     

Susceptibility of staphylococcal biofilms to enzymatic treatments depends on their chemical composition

Bacterial infections are serious complications after orthopaedic implant surgery. Staphylococci, with Staphylococcus epidermidis as a leading species, are the prevalent and most important species involved in orthopaedic implant-related infections. The biofilm mode of growth of these bacteria on an implant surface protects the organisms from the host’s immune system and from antibiotic therapy. Therapeutic agents that disintegrate the biofilm matrix would release planktonic cells into the environment and therefore allow antibiotics to eliminate the bacteria. An addition of a biofilm-degrading agent to a solution used for washing–draining procedures of infected orthopaedic implants would greatly improve the efficiency of the procedure and thus help to avoid the removal of the implant. We have previously shown that the extracellular staphylococcal matrix consists of a poly-N-acetylglucosamine (PNAG), extracellular teichoic acids (TAs) and protein components. In this study, we accessed the sensitivity of pre-formed biofilms of five clinical staphylococcal strains associated with orthopaedic prosthesis infections and with known compositions of the biofilm matrix to periodate, Pectinex Ultra SP, proteinase K, trypsin, pancreatin and dispersin B, an enzyme with a PNAG-hydrolysing activity. We also tested the effect of these agents on the purified carbohydrate components of staphylococcal biofilms, PNAG and TA. We found that the enzymatic detachment of staphylococcal biofilms depends on the nature of their constituents and varies between the clinical isolates. We suggest that a treatment with dispersin B followed by a protease (proteinase K or trypsin) could be capable to eradicate biofilms of a variety of staphylococcal strains on inert surfaces.     

sarA negatively regulates Staphylococcus epidermidis biofilm formation by modulating expression of 1 MDa extracellular matrix binding protein and autolysis‐dependent release of eDNA

Biofilm formation is essential for Staphylococcus epidermidis pathogenicity in implant‐associated infections. Nonetheless, large proportions of invasive Staphylococcus epidermidis isolates fail to form a biofilm in vitro. We here tested the hypothesis that this apparent paradox is related to the existence of superimposed regulatory systems suppressing a multicellular biofilm life style in vitro. Transposon mutagenesis of clinical significant but biofilm‐negative S. epidermidis 1585 was used to isolate a biofilm positive mutant carrying a Tn917 insertion in sarA, chief regulator of staphylococcal virulence. Genetic analysis revealed that inactivation of sarA induced biofilm formation via overexpression of the giant 1 MDa extracellular matrix binding protein (Embp), serving as an intercellular adhesin. In addition to Embp, increased extracellular DNA (eDNA) release significantly contributed to biofilm formation in mutant 1585ΔsarA. Increased eDNA amounts indirectly resulted from upregulation of metalloprotease SepA, leading to boosted processing of autolysin AtlE, in turn inducing augmented autolysis and release of eDNA. Hence, this study identifies sarA as a negative regulator of Embp‐ and eDNA‐dependent biofilm formation. Given the importance of SarA as a positive regulator of polysaccharide mediated cell aggregation, the regulator enables S. epidermidis to switch between mechanisms of biofilm formation, ensuring S. epidermidis adaptation to hostile environments.