Antibacterial activity of a new broad-spectrum antibiotic covalently bound to titanium surfaces.

Biofilm-associated infections, particularly those caused by Staphylococcus aureus, are a major cause of implant failure. Covalent coupling of broad-spectrum antimicrobials to implants is a promising approach to reduce the risk of infections. In this study, we developed titanium substrates on which the recently discovered antibacterial agent SPI031, a N-alkylated 3, 6-dihalogenocarbazol 1-(sec-butylamino)-3-(3,6-dichloro-9H-carbazol-9-yl)propan-2-ol, was covalently linked (SPI031-Ti). We found that SPI031-Ti substrates prevent biofilm formation of S. aureus and Pseudomonas aeruginosa in vitro, as quantified by plate counting and fluorescence microscopy. To test the effectiveness of SPI031-Ti substrates in vivo, we used an adapted in vivo biomaterial-associated infection model in mice in which SPI031-Ti substrates were implanted subcutaneously and subsequently inoculated with S. aureus. Using this model, we found a significant reduction in biofilm formation (up to 98%) on SPI031-Ti substrates compared to control substrates. Finally, we demonstrated that the functionalization of the titanium surfaces with SPI031 did not influence the adhesion and proliferation of human cells important for osseointegration and bone repair. In conclusion, these data demonstrate the clinical potential of SPI031 to be used as an antibacterial coating for implants, thereby reducing the incidence of implant-associated infections. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 34:2191-2198, 2016.

Covalent immobilization of antimicrobial agents on titanium prevents Staphylococcus aureus and Candida albicans colonization and biofilm formation.

OBJECTIVES: Biofilm-associated implant infections represent a serious public health problem. Covalent immobilization of antimicrobial agents on titanium (Ti), thereby inhibiting biofilm formation of microbial pathogens, is a solution to this problem. METHODS: Vancomycin (VAN) and caspofungin (CAS) were covalently bound on Ti substrates using an improved processing technique adapted to large-scale coating of implants. Resistance of the VAN-coated Ti (VAN-Ti) and CAS-coated Ti (CAS-Ti) substrates against in vitro biofilm formation of the bacterium Staphylococcus aureus and the fungal pathogen Candida albicans was determined by plate counting and visualized by confocal laser scanning microscopy. The efficacy of the coated Ti substrates was also tested in vivo using an adapted biomaterial-associated murine infection model in which control-Ti, VAN-Ti or CAS-Ti substrates were implanted subcutaneously and subsequently challenged with the respective pathogens. The osseointegration potential of VAN-Ti and CAS-Ti was examined in vitro using human bone marrow-derived stromal cells, and for VAN-Ti also in a rat osseointegration model. RESULTS: In vitro biofilm formation of S. aureus and C. albicans on VAN-Ti and CAS-Ti substrates, respectively, was significantly reduced compared with biofilm formation on control-Ti. In vivo, we observed over 99.9% reduction in biofilm formation of S. aureus on VAN-Ti substrates and 89% reduction in biofilm formation of C. albicans on CAS-Ti substrates, compared with control-Ti substrates. The coated substrates supported osseointegration in vitro and in vivo. CONCLUSIONS: These data demonstrate the clinical potential of covalently bound VAN and CAS on Ti to reduce microbial biofilm formation without jeopardizing osseointegration.

A comparative study of infrared and microwave heating for microbial decontamination of paprika powder.

There is currently a need in developing new decontamination technologies for spices due to limitations of existing technologies, mainly regarding their effects on spices' sensory quality. In the search of new decontamination solutions, it is of interest to compare different technologies, to provide the industry with knowledge for taking decisions concerning appropriate decontamination technologies for spices. The present study compares infrared (IR) and microwave decontamination of naturally contaminated paprika powder after adjustment of water activity to 0.88. IR respectively microwave heating was applied to quickly heat up paprika powder to 98°C, after which the paprika sample was transferred to a conventional oven set at 98°C to keep the temperature constant during a holding time up to 20 min. In the present experimental set-up microwave treatment at 98°C for 20 min resulted in a reduction of 4.8 log units of the total number of mesophilic bacteria, while the IR treatment showed a 1 log unit lower reduction for the corresponding temperature and treatment time. Microwave and IR heating created different temperature profiles and moisture distribution within the paprika sample during the heating up part of the process, which is likely to have influenced the decontamination efficiency. The results of this study are used to discuss the difficulties in comparing two thermal technologies on equal conditions due to differences in their heating mechanisms.

Identification and characterization of an anti-pseudomonal dichlorocarbazol derivative displaying anti-biofilm activity.

Pseudomonas aeruginosa strains resistant towards all currently available antibiotics are increasingly encountered, raising the need for new anti-pseudomonal drugs. We therefore conducted a medium-throughput screen of a small-molecule collection resulting in the identification of the N-alkylated 3,6-dihalogenocarbazol 1-(sec-butylamino)-3-(3,6-dichloro-9H-carbazol-9-yl)propan-2-ol (MIC = 18.5 µg mL⁻¹). This compound, compound 1, is bacteriostatic towards a broad spectrum of Gram-positive and Gram-negative pathogens, including P. aeruginosa. Importantly, 1 also eradicates mature biofilms of P. aeruginosa. 1 displays no cytotoxicity against various human cell types, pointing to its potential for further development as a novel antibacterial drug.

Infrared decontamination of oregano: effects on Bacillus cereus spores, water activity, color, and volatile compounds.

Infrared (IR) heating, a novel technology for decontaminating oregano, was evaluated by investigating the reduction of inoculated Bacillus cereus spores and the effect on water activity (a(w)), color, and headspace volatile compounds after exposure to IR treatment. Conditioned oregano (a(w) 0.88) was IR-treated in a closed heating unit at 90 and 100 °C for holding times of 2 and 10 min, respectively. The most successful reduction in B. cereus spore numbers (5.6 log units) was achieved after a holding time of 10 min at 90 °C, while treatment at 100 °C for the same time resulted in a lower reduction efficiency (4.7 log units). The lower reduction at 100 °C was probably due to a reduced aw (aw 0.76) during IR treatment or possibly to the alteration or loss of volatile compounds possessing antimicrobial properties. The green color of oregano was only slightly affected, while the composition of volatile compounds was clearly altered by IR heating. However, two of the key aroma compounds, carvacrol and thymol, were only slightly affected, compared to the effect on the other studied compounds, indicating that the typical oregano aroma can likely be preserved. In conclusion, IR heating shows potential for the successful decontamination of oregano without severe alteration of its color or the key aroma compounds, carvacrol and thymol.

Adhesion of Streptococcus mitis and Actinomyces oris in co-culture to machined and anodized titanium surfaces as affected by atmosphere and pH.

BACKGROUND: With the rising demand for osseointegrated titanium implants for replacing missing teeth, often in patients with a history of periodontitis, implant-related infections have become an issue of growing concern. Novel methods for treating and preventing implant-associated infections are urgently needed. The aim of this study was to investigate if different pH, atmosphere and surface properties could restrict bacterial adhesion to titanium surfaces used in dental implants. METHODS: Titanium discs with machined or anodized (TiUnite™) surface were incubated with a co-culture of Streptococcus mitis and Actinomyces oris (early colonizers of oral surfaces) at pH 5.0, 7.0 and 9.0 at aerobic or anaerobic atmosphere. The adhesion was analysed by counting colony forming (CFU) units on agar and by confocal laser scanning microscopy (CLSM). RESULTS: The CFU analysis showed that a pH of 5.0 was found to significantly decrease the adhesion of S. mitis, and an aerobic atmosphere, the adhesion of A. oris. S. mitis was found in significantly less amounts on the anodized surface than the machined surface, while A. oris was found in equal amounts on both surfaces. The CLSM analysis confirmed the results from the CFU count and provided additional information on how the two oral commensal species adhered to the surfaces: mainly in dispersed clusters oriented with the groves of the machined surface and the pores of the anodized surface. CONCLUSIONS: Bacterial adhesion by S. mitis and A. oris can be restricted by acidic pH and aerobic atmosphere. The anodized surface reduced the adhesion of S. mitis compared to the machined surface; while A. oris adhered equally well to the pores of the anodized surface and to the grooves of the machined surface. It is difficult to transfer these results directly into a clinical situation. However, it is worth further investigating these findings from an in vitro perspective, as well as clinically, to gain more knowledge of the effects acid pH and aerobic atmosphere have on initial bacterial adhesion.

Effects of carbon dioxide on neurotoxin gene expression in nonproteolytic Clostridium botulinum Type E.

Carbon dioxide is an antimicrobial gas commonly used in modified atmosphere packaging. In the present study, the effects of carbon dioxide on the growth of and neurotoxin production by nonproteolytic Clostridium botulinum type E were studied during the growth cycle. Quantitative reverse transcription-PCR and an enzyme-linked immunosorbent assay were used to quantify expression of the type E botulinum neurotoxin gene (cntE) and the formation of type E neurotoxin. The expression levels of cntE were similar in two strains, with relative expression peaking in the transition between exponential phase and stationary phase. In stationary phase, cntE mRNA expression declined rapidly. The cntE mRNA half-life was calculated to be approximately 9 minutes. Neurotoxin formation occurred in late exponential phase and stationary phase. High carbon dioxide concentrations delayed growth by increasing the lag time and decreasing the maximum growth rate. The effects of carbon dioxide concentration on relative neurotoxin gene expression and neurotoxin formation were significant. Expression of cntE mRNA and the formation of extracellular neurotoxin were twofold higher with a headspace carbon dioxide concentration of 70% (vol/vol) compared to 10% (vol/vol). This finding sheds a new, cautionary light on the potential risks of botulism associated with the use of modified atmosphere packaging.

Strategies for overcoming PCR inhibition.

INTRODUCTIONThe use of conventional and real-time PCR is to some extent restricted by the presence of PCR inhibitors. This is particularly so when the techniques are applied directly to complex biological samples such as clinical, environmental, or food samples for the detection of microorganisms. PCR inhibitors can originate from the sample itself, or as a result of the method used to collect or otherwise prepare the sample. Either way, inhibitors can dramatically reduce the sensitivity and amplification efficiency of PCR. This article discusses methods of reducing inhibition and designing reliable and sensitive conventional and real-time PCR experiments.

Quantitative interaction effects of carbon dioxide, sodium chloride, and sodium nitrite on neurotoxin gene expression in nonproteolytic Clostridium botulinum type B.

The effects of carbon dioxide, sodium chloride, and sodium nitrite on type B botulinum neurotoxin (BoNT/B) gene (cntB) expression in nonproteolytic Clostridium botulinum were investigated in a tryptone-peptone-yeast extract (TPY) medium. Various concentrations of these selected food preservatives were studied by using a complete factorial design in order to quantitatively study interaction effects, as well as main effects, on the following responses: lag phase duration (LPD), growth rate, relative cntB expression, and extracellular BoNT/B production. Multiple linear regression was used to set up six statistical models to quantify and predict these responses. All combinations of NaCl and NaNO(2) in the growth medium resulted in a prolonged lag phase duration and in a reduction in the specific growth rate. In contrast, the relative BoNT/B gene expression was unchanged, as determined by the cntB-specific quantitative reverse transcription-PCR method. This was confirmed when we measured the extracellular BoNT/B concentration by an enzyme-linked immunosorbent assay. CO(2) was found to have a major effect on gene expression when the cntB mRNA levels were monitored in the mid-exponential, late exponential, and late stationary growth phases. The expression of cntB relative to the expression of the 16S rRNA gene was stimulated by an elevated CO(2) concentration; the cntB mRNA level was fivefold greater in a 70% CO(2) atmosphere than in a 10% CO(2) atmosphere. These findings were also confirmed when we analyzed the extracellular BoNT/B concentration; we found that the concentrations were 27 ng x ml(-1). unit of optical density(-1) in the 10% CO(2) atmosphere and 126 ng x ml(-1). unit of optical density(-1) in the 70% CO(2) atmosphere.

Relative neurotoxin gene expression in clostridium botulinum type B, determined using quantitative reverse transcription-PCR.

A quantitative reverse transcription-PCR (qRT-PCR) method was developed to monitor the relative expression of the type B botulinum neurotoxin (BoNT/B) gene (cntB) in Clostridium botulinum. The levels of cntB mRNA in five type B strains were accurately monitored by using primers specific for cntB and for the reference gene encoding the 16S rRNA. The patterns and relative expression of cntB were different in the different strains. Except for one of the strains investigated, an increase in cntB expression was observed when the bacteria entered the early stationary growth phase. In the proteolytic strain C. botulinum ATCC 7949, the level of cntB mRNA was four- to fivefold higher than the corresponding levels in the other strains. This was confirmed when we quantified the production of extracellular BoNT/B by an enzyme-linked immunosorbent assay and measured the toxicity of BoNT/B by a mouse bioassay. When the effect of exposure to air on cntB expression was investigated, no decline in the relative expression was observed in spite of an 83% reduction in the viable count based on the initial cell number. Instead, the level of cntB mRNA remained the same. When there was an increase in the sodium nitrite concentration, the bacteria needed a longer adjustment time in the medium before exponential growth occurred. In addition, there was a reduction in the expression of cntB compared to the expression of the 16S rRNA gene at higher sodium nitrite concentrations. This was most obvious in the late exponential growth phase, but at the highest sodium nitrite concentration investigated, 45 ppm, a one- to threefold decline in the cntB mRNA level was observed in all growth phases.

Pre-PCR processing: strategies to generate PCR-compatible samples.

Polymerase chain reaction (PCR) is recognized as a rapid, sensitive, and specific molecular diagnostic tool for the analysis of nucleic acids. However, the sensitivity and kinetics of diagnostic PCR may be dramatically reduced when applied directly to biological samples, such as blood and feces, owing to PCR-inhibitory components. As a result, pre-PCR processing procedures have been developed to remove or reduce the effects of PCR inhibitors. Pre-PCR processing comprises all steps prior to the detection of PCR products, that is, sampling, sample preparation, and deoxyribonucleic acid (DNA) amplification. The aim of pre-PCR processing is to convert a complex biological sample with its target nucleic acids/cells into PCR-amplifiable samples by combining sample preparation and amplification conditions. Several different pre-PCR processing strategies are used: (1) optimization of the DNA amplification conditions by the use of alternative DNA polymerases and/or amplification facilitators, (2) optimization of the sample preparation method, (3) optimization of the sampling method, and (4) combinations of the different strategies. This review describes different pre-PCR processing strategies to circumvent PCR inhibition to allow accurate and precise DNA amplification.