Antimicrobial activities of YycG histidine kinase inhibitors against Staphylococcus epidermidis biofilms

Staphylococcus epidermidis has become a significant pathogen causing infections due to biofilm formation on surfaces of indwelling medical devices. Biofilm-associated bacteria exhibit enhanced resistance to many conventional antibiotics. It is therefore, important to design novel antimicrobial reagents targeting S. epidermidis biofilms. In a static chamber system, the bactericidal effect of two leading compounds active as YycG inhibitors was assessed on biofilm cells by confocal laser scanning microscopy combined with viability staining. In young biofilms (6-h-old), the two compounds killed the majority of the embedded cells at concentrations of 100 microM and 25 microM, respectively. In mature biofilms (24-h-old), one compound was still effectively killing biofilm cells, whereas the other compound mainly killed cells located at the bottom of the biofilm. In contrast, vancomycin was found to stimulate biofilm development at the MBC (8 microg mL(-1)). Even at a high concentration (128 microg mL(-1)), vancomycin exhibited poor killing on cells embedded in biofilms. The two compounds exhibited faster and more effective killing of S. epidermidis planktonic cells than vancomycin at the early stage of exposure (6 h). The data suggest that the new inhibitors can serve as potential agents against S. epidermidis biofilms when added alone or in concert with other antimicrobial agents.     

The <Emphasis Type="Italic">in vitro</Emphasis> and <Emphasis Type="Italic">in vivo</Emphasis> efficacy of fluconazole in combination with farnesol against <Emphasis Type="Italic">Candida albicans</Emphasis> isolates using a murine vulvovaginitis model

Farnesol is a quorum-sensing molecule that inhibits biofilm formation in Candida albicans. Previous in vitro data suggest that, in combination with certain antifungals, farnesol may have an adjuvant anti-biofilm agent. However, the in vivo efficacy of farnesol is very questionable. Therefore, the in vitro and in vivo activity of fluconazole combined with farnesol was evaluated against C. albicans biofilms using fractional inhibitory concentration index (FICI) determination, time-kill experiments and a murine vulvovaginitis model. The median biofilm MICs of fluconazole-sensitive C. albicans isolates ranged between 4 -> 512 mg/L and 150–300 μM for fluconazole and farnesol, respectively. These values were 512 -> 512 mg/L and > 300 μM for fluconazole-resistant clinical isolates. Farnesol decreased the median MICs of fluconazole by 2-64-fold for biofilms. Based on FICI, synergistic interaction was observed only in the case of the sessile SC5314 reference strain (FICIs: 0.16–0.27). In time-kill studies, only the 512 mg/L fluconazole and 512 mg/L fluconazole + 75 μM farnesol reduced biofilm mass significantly at each time point in the case of all isolates. The combination reduced the metabolic activity of biofilms for all isolates in a concentration- and time-dependent manner. Our findings revealed that farnesol alone was not protective in a murine vulvovaginitis model. Farnesol was not beneficial in combination with fluconazole for fluconazole-susceptible isolates, but partially increased fluconazole activity against one fluconazole-resistant isolate, but not the other one.     

Modulation of poly-N-acetylglucosamine accumulation within mature Staphylococcus epidermidis biofilms grown in excess glucose

PNAG is a major component of Staphylococcus epidermidis biofilms involved in intercellular adhesion as well as in the interaction of the biofilm with components of the host immune response. Synthesis of PNAG has been found to be regulated by several environmental factors. In the present study, the effect of glucose metabolism-dependent culture medium acidification in PNAG accumulation was evaluated. Established S. epidermidis biofilms were allowed to grow in excess glucose with or without maintained pH conditions. PNAG accumulation in these biofilms was determined by flow cytometry and fluorescence microscopy using wheat germ agglutinin as a fluorescent probe. Biofilms grown in maintained pH conditions presented significantly higher amounts of this polymer as well as higher icaA expression than biofilms grown in acidic pH conditions. Moreover, PNAG accumulation in biofilms grown in non-maintained pH conditions occurred in association with cell death. Overall, we show that glucose metabolism by decreasing the culture pH affects biofilm physiology in respect to PNAG production and cell death. The reported in vitro modulation of PNAG accumulation within S. epidermidis biofilms further highlights the role of environment on determining the biofilm physiological state.     

Chemical and antimicrobial treatments change the viscoelastic properties of bacterial biofilms

Changes in the viscoelastic material properties of bacterial biofilms resulting from chemical and antimicrobial treatments were measured by rheometry. Colony biofilms of Staphylococcus epidermidis or a mucoid Pseudomonas aeruginosa were subjected to a classical creep test performed using a parallel plate rheometer. Data were fit to the 4-parameter Burger model to quantify the material properties. Biofilms were exposed to the chloride salts of several common mono-, di-, and tri- valent cations, and to urea, industrial biocides, and antibiotics. Many of these treatments resulted in statistically significant alterations in the material properties of the biofilm. Multivalent cations stiffened the P. aeruginosa biofilm, while ciprofloxacin and glutaraldehyde weakened it. Urea, rifampin, and a quaternary ammonium biocide weakened the S. epidermidis biofilm. In general, there was no correspondence between the responses of the two different types of biofilms to a particular treatment. These results underscore the distinction between the killing power of an antimicrobial agent and its ability to alter biofilm mechanical properties and thereby influence biofilm removal. Understanding biofilm rheology and how it is affected by chemical treatment could lead to improvements in biofilm control.     

Effect of Farnesol on Planktonic and Biofilm Cells of <Emphasis Type="Italic">Staphylococcus epidermidis</Emphasis>

Staphylococcus epidermidis is now amongst the most important pathogenic agents responsible for bloodstream nosocomial infections and for biofilm formation on indwelling medical devices. Its increasing resistance to common antibiotics is a challenge for the development of new antimicrobial agents. Accordingly, the goal of this study was to evaluate the effect of farnesol, a natural sesquiterpenoid, on Staphylococcus epidermidis planktonic and biofilm cells. Farnesol displayed a significant inhibitory effect on planktonic cells. Small concentrations (100 μM) were sufficient to exhibit antibacterial effect on these cells. In biofilm cells the effect of farnesol was not so pronounced and it seems to be strongly dependent on the cells metabolic activity and amount of matrix. Interestingly, the effect of farnesol at 200 μM was similar to the effect of vancomycin at peak serum concentration either in planktonic or biofilm cells. Overall, the results indicate a potential antibacterial effect of farnesol against S. epidermidis, and therefore the possible action of this molecule on the prevention of S. epidermidis related infections.     

Antimicrobial activity of diterpenoids from hairy roots of Salvia sclarea L.: Salvipisone as a potential anti-biofilm agent active against antibiotic resistant Staphylococci

The antimicrobial activities of crude dichloromethane fractions from acetone extracts of Agrobacterium rhizogenes transformed roots and roots of field-grown plants of Salvia sclarea as well as four pure abietane diterpenoids isolated from the hairy root cultures were determined. The growth of Gram-positive bacteria (Staphylococcus aureus, S. epidermidis, Enterococcus faecalis) but not Gram-negative ones (Escherichia coli, Pseudomonas aeruginosa) or pathogenic fungi (Candida albicans) was inhibited by fractions tested at concentrations of 37.5-75.0 microgml(-1). Abietane diterpenoids: salvipisone, aethiopinone, 1-oxoaethiopinone and ferruginol were shown to be bacteriostatic as well as bacteriocidal for the cultures of S. aureus and S. epidermidis strains, regardless of their antibiotic susceptibility profile. This was demonstrated by using simultaneously the optical density measuring method and 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide-reduction assay. The highest activity was shown by salvipisone which demonstrated also a very interesting activity when its effect on 24-h-old staphylococcal biofilm cells viability was examined. It limited the survival of biofilms formed by S. aureus as well as by S. epidermidis, putting this compound to the list of potential anti-biofilm agents, better than most of known antibiotics.     

Increased tolerance of Staphylococcus aureus to vancomycin in viscous media

The increased viscosity observed in biofilms, adherent communities of bacterial cells embedded in a polymeric matrix, was hypothesized to induce increased tolerance of bacteria to antibiotics. To test this concept, planktonic Staphylococcus aureus cells were grown and exposed to vancomycin in media brought to specific viscosities in order to mimic the biofilm extracellular polymeric matrix. A viscous environment was observed to decrease the vancomycin susceptibility of planktonic S. aureus to levels seen for biofilms. Both planktonic S. aureus at a viscosity of 100 mPa s and staphylococcal biofilms were able to survive at >500 times the levels of the antibiotic effective against planktonic populations in standard medium. Time-dependent and dose-dependent viability curves revealed that more than one mechanism was involved in high S. aureus tolerance to vancomycin in viscous media. Increased viscosity affects antibiotic susceptibility by reducing diffusion and the mass transfer rate; this mechanism alone, however, cannot explain the increased tolerance demonstrated by S. aureus in viscous media, suggesting that viscosity may also alter the phenotype of the planktonic bacteria to one more resistant to antimicrobials, as seen in biofilms. However, these latter changes are not yet understood and will require further study.     

Direct analysis of bacterial viability in endotracheal tube biofilm from a pig model of methicillin-resistant Staphylococcus aureus pneumonia following antimicrobial therapy

Confocal laser scanning microscopy (CLSM) helps to observe the biofilms formed in the endotracheal tube (ETT) of ventilated subjects and to determine its structure and bacterial viability using specific dyes. We compared the effect of three different treatments (placebo, linezolid, and vancomycin) on the bacterial biofilm viability captured by CLSM. Eight pigs with pneumonia induced by methicillin-resistant Staphylococcus aureus (MRSA) were ventilated up to 96?h and treated with linezolid, vancomycin, or placebo (controls). ETT images were microscopically examined after staining with the live/dead(?) BacLight(?) Kit (Invitrogen, Barcelona, Spain) with a confocal laser scanning microscope. We analyzed 127 images obtained by CLSM. The median ratio of live/dead bacteria was 0.51, 0.74, and 1 for the linezolid, vancomycin, and control groups, respectively (P?=?0.002 for the three groups); this ratio was significantly lower for the linezolid group, compared with the control group (P?=?0.001). Images showed bacterial biofilm attached and non-attached to the ETT surface but growing within secretions accumulated inside ETT. Systemic treatment with linezolid is associated with a higher proportion of dead bacteria in the ETT biofilm of animals with MRSA pneumonia. Biofilm clusters not necessarily attach to the ETT surface.     

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.     

Inhibition of Candida albicans biofilm formation by farnesol,a quorum-sensing molecule

Farnesol is a quorum-sensing molecule that inhibits filamentation in Candida albicans. Both filamentation and quorum sensing are deemed to be important factors in C. albicans biofilm development. Here we examined the effect of farnesol on C. albicans biofilm formation. C. albicans adherent cell populations (after 0, 1, 2, and 4 h of adherence) and preformed biofilms (24 h) were treated with various concentrations of farnesol (0, 3, 30, and 300 micro M) and incubated at 37 degrees C for 24 h. The extent and characteristics of biofilm formation were then assessed microscopically and with a semiquantitative colorimetric technique based on the use of 2,3-bis(2-methoxy-4-nitro-5-sulfo-phenyl)-2H-tetrazolium-5-carboxanilide. The results indicated that the effect of farnesol was dependent on the concentration of this compound and the initial adherence time, and preincubation with 300 micro M farnesol completely inhibited biofilm formation. Supernatant media recovered from mature biofilms inhibited the ability of planktonic C. albicans to form filaments, indicating that a morphogenetic autoregulatory compound is produced in situ in biofilms. Northern blot analysis of RNA extracted from cells in biofilms indicated that the levels of expression of HWP1, encoding a hypha-specific wall protein, were decreased in farnesol-treated biofilms compared to the levels in controls. Our results indicate that farnesol acts as a naturally occurring quorum-sensing molecule which inhibits biofilm formation, and we discuss its potential for further development and use as a novel therapeutic agent.     

Biofilm penetration and disinfection efficacy of alkaline hypochlorite and chlorosulfamates

AIMS: The purpose of this study was to compare the efficacy, in terms of bacterial biofilm penetration and killing, of alkaline hypochlorite (pH 11) and chlorosulfamate (pH 5.5) formulations. METHODS AND RESULTS: Two species biofilms of Pseudomonas aeruginosa and Klebsiella pneumoniae were grown by flowing a dilute medium over inclined stainless steel slides for 6 d. Microelectrode technology was used to measure concentration profiles of active chlorine species within the biofilms in response to treatment at a concentration of 1000 mg total chlorine l(-1). Chlorosulfamate formulations penetrated biofilms faster than did hypochlorite. The mean penetration time into approximately 1 mm-thick biofilms for chlorosulfamate (6 min) was only one-eighth as long as for the same concentration of hypochlorite (48 min). Chloride ion penetrated biofilms rapidly (5 min) with an effective diffusion coefficient in the biofilm that was close to the value for chloride in water. Biofilm bacteria were highly resistant to killing by both antimicrobial agents. Biofilms challenged with 1000 mg l(-1) alkaline hypochlorite or chlorosulfamate for 1 h experienced 0.85 and 1.3 log reductions in viable cell numbers, respectively. Similar treatment reduced viable numbers of planktonic bacteria to non-detectable levels (log reduction greater than 6) within 60 s. Aged planktonic and resuspended laboratory biofilm bacteria were just as susceptible to hypochlorite as fresh planktonic cells. CONCLUSION: Chlorosulfamate transport into biofilm was not retarded whereas hypochlorite transport clearly was retarded. Superior penetration by chlorosulfamate was hypothesized to be due to its lower capacity for reaction with constituents of the biofilm. Poor biofilm killing despite direct measurement of effective physical penetration of the antimicrobial agent into the biofilm demonstrates that bacteria in the biofilm are protected by some mechanism other than simple physical shielding by the biofilm matrix. SIGNIFICANCE AND IMPACT OF THE STUDY: This study lends support to the theory that the penetration of antimicrobial agents into microbial biofilms is controlled by the reactivity of the antimicrobial agent with biofilm components. The finding that chlorine-based biocides can penetrate, but fail to kill, bacteria in biofilms should motivate the search for other mechanisms of protection from killing by antimicrobial agents in biofilms.     

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.     

Chloraminated drinking water does not generate bacterial resistance to antibiotics in Pseudomonas aeruginosa biofilms

Aim: To determine if exposure of Pseudomonas aeruginosa biofilms to chloraminated drinking water can lead to individual bacteria with resistance to antibiotics. Methods and Results: Biofilms of P. aeruginosa PA14 were grown in drinking water in a Kadouri drip‐fed reactor; the biofilms were treated with either 0·5 mg l^‐1 or 1·0 mg l^‐1 of chloramine for 15 or 21 days; control biofilms were grown in water without chloramine. Fewer isolates with antibiotic resistance were obtained from the chloramine‐treated biofilms as compared to the control. Minimum inhibitory concentrations (MIC) for selected antibiotic‐resistant isolates were determined using ciprofloxacin, tobramycin, gentamicin, rifampicin and chloramphenicol. All of the isolates tested had increased resistance over the wildtype to ciprofloxacin, rifampicin and chloramphenicol, but were not resistant to tobramycin or gentamicin. Conclusions: Under these test conditions, there was no detectable increase in antibiotic resistance in P. aeruginosa exposed as biofilms to disinfectant residues in chloraminated drinking water. Significance and Impact of the study: Chloramine in drinking water, while unable to kill biofilm bacteria, does not increase the potential of P. aeruginosa to become resistant to antibiotics.     

激光扫描共聚焦显微镜观察细菌生物膜形成的方法学研究

目的:建立激光扫描共聚焦显微镜观察生物膜形成过程的方法,为进一步研究生物膜的形成机制奠定基础。方法:以临床分离金葡菌X428为研究对象,在盖玻片上形成生物膜,分别于接种后的4、8、12、16、24和48h取出玻片,采用免疫荧光技术标记多糖和细菌,激光扫描共聚焦显微镜(CLSM)观察生物膜形成情况。结果:取得了生物膜形成过程的不同时间点的CLSM图像,4h时细菌在盖玻片上粘附形成小菌落,8h和12h细菌聚集成簇,多糖基质产生并逐渐增多,至16h形成成熟生物膜结构;24h和48h生物膜已经播散,其结构变小。结论:应用免疫荧光技术和激光扫描共聚焦显微镜技术研究生物膜形成过程是一种简便可行的方法。     

Antimicrobial activity of tigecycline alone or in combination with rifampin against Staphylococcus epidermidis in biofilm

Staphylococcus epidermidis is a commensal inhabitant of the healthy human skin, but in the recent years, it has been recognized as a nosocomial pathogen especially in immunocompromised patients. The pathogenesis of S. epidermidis is thought to be based on its capacity to form biofilms on the surface of medical devices, where bacterial cells may persist, protected from host defence and antimicrobial agents. Rifampin has been shown to be one of the most active antimicrobial agents in the eradication of the staphylococcal biofilm. However, this antibiotic should not be used in monotherapy. Therefore, one of the objectives of our research was to study the efficacy of the tigecycline/rifampin combination against methicillin-resistant S. epidermidis embedded in biofilms. Of the 80 clinically significant S. epidermidis isolates, 75 strains possess the ability to form a biofilm. These bacteria formed the biofilm via ica-dependent mechanisms. However, other biofilm-associated genes, including aap (encoding accumulation-associated protein) and bhp (coding cell wall-associated protein), were present in 85 and 29 % of isolates, respectively. The biofilm structures of S. epidermidis strains were also analyzed in confocal laser scanning microscopy (CLSM) and the obtained image demonstrated differences in their architecture. In vitro studies showed that the MIC value for tigecycline against S. epidermidis growing in the biofilm ranged from 0.125 to 2 μg/mL. Tigecycline in combination with rifampin demonstrated higher activity against bacteria embedded in biofilms than tigecycline alone.     

In vitro efficacy of bismuth thiols against biofilms formed by bacteria isolated from human chronic wounds

Aims: The purpose of this study was to evaluate the antimicrobial efficacy of thirteen bismuth thiol preparations for bactericidal activity against established biofilms formed by two bacteria isolated from human chronic wounds. Methods: Single species biofilms of a Pseudomonas aeruginosa or a methicillin‐resistant Staphylococcus aureus were grown in either colony biofilm or drip‐flow reactors systems. Biofilms were challenged with bismuth thiols, antibiotics or silver sulfadiazine, and log reductions were determined by plating for colony formation. Conclusions: Antibiotics were ineffective or inconsistent against biofilms of both bacterial species tested. None of the antibiotics tested were able to achieve >2 log reductions in both biofilm models. The 13 different bismuth thiols tested in this investigation achieved widely varying degrees of killing, even against the same micro‐organism in the same biofilm model. For each micro‐organism, the best bismuth thiol easily outperformed the best conventional antibiotic. Against P. aeruginosa biofilms, bismuth‐2,3‐dimercaptopropanol (BisBAL) at 40–80 μg ml^?1 achieved >7·7 mean log reduction for the two biofilm models. Against MRSA biofilms, bismuth‐1,3‐propanedithiol/bismuth‐2‐mercaptopyridine N‐oxide (BisBDT/PYR) achieved a 4·9 log reduction. Significance and Impact of the Study: Bismuth thiols are effective antimicrobial agents against biofilms formed by wound bacteria and merit further development as topical antiseptics for the suppression of biofilms in chronic wounds.     

Inhibitory effect of the human liver-derived antimicrobial peptide hepcidin 20 on biofilms of polysaccharide intercellular adhesin (PIA)-positive and PIA-negative strains of Staphylococcus epidermidis

Staphylococcus epidermidis plays a major role in biofilm-related medical device infections. Herein the anti-biofilm activity of the human liver-derived antimicrobial peptide hepcidin 20 (hep20) was evaluated against polysaccharide intercellular adhesin (PIA)-positive and PIA-negative clinical isolates of S. epidermidis. Hep20 markedly inhibited biofilm formation and bacterial cell metabolism of PIA-positive and PIA-negative strains, but the decrease in biofilm biomass only partially correlated with a decrease in viable bacteria. Confocal microscope images revealed that, in the presence of hep20, both PIA-positive and PIA-negative strains formed biofilms with altered architectures and reduced amounts of extracellular matrix. Co-incubation of hep20 with vancomycin produced no synergistic effect, evaluated as number of viable cells, both in preventing biofilm formation and in treating preformed biofilms. In contrast, biofilms obtained in the presence of hep20, and then exposed to vancomycin, displayed an increased susceptibility to vancomycin. These results suggest that hep20 may inhibit the production/accumulation of biofilm extracellular matrix.     

Pseudomonas aeruginosa and Saccharomyces cerevisiae Biofilm in Flow Cells

Many microbial cells have the ability to form sessile microbial communities defined as biofilms that have altered physiological and pathological properties compared to free living microorganisms. Biofilms in nature are often difficult to investigate and reside under poorly defined conditions¹. Using a transparent substratum it is possible to device a system where simple biofilms can be examined in a non-destructive way in real-time: here we demonstrate the assembly and operation of a flow cell model system, for in vitro 3D studies of microbial biofilms generating high reproducibility under well-defined conditions^2,3.The system consists of a flow cell that serves as growth chamber for the biofilm. The flow cell is supplied with nutrients and oxygen from a medium flask via a peristaltic pump and spent medium is collected in a waste container. This construction of the flow system allows a continuous supply of nutrients and administration of e.g. antibiotics with minimal disturbance of the cells grown in the flow chamber. Moreover, the flow conditions within the flow cell allow studies of biofilm exposed to shear stress. A bubble trapping device confines air bubbles from the tubing which otherwise could disrupt the biofilm structure in the flow cell.The flow cell system is compatible with Confocal Laser Scanning Microscopy (CLSM) and can thereby provide highly detailed 3D information about developing microbial biofilms. Cells in the biofilm can be labeled with fluorescent probes or proteins compatible with CLSM analysis. This enables online visualization and allows investigation of niches in the developing biofilm. Microbial interrelationship, investigation of antimicrobial agents or the expression of specific genes, are of the many experimental setups that can be investigated in the flow cell system.