Surface kinetic test method for determining rate of kill by an antimicrobial solid.

An antimicrobial-surface kinetic test which maximizes probability of cell-to-surface contact has been developed. The measurement of rate of kill by a nonleaching antimicrobial surface is based on the number of surviving bacterial cells at specific times of exposure to various amounts of total treated surface area of test substrate. This method gives information for direct comparison of rate of kill for a variety of antimicrobial surfaces in terms of rate of kill per square centimeter of surface area. Data obtained by this method can also give valuable dose response application information as an indication of the exponential efficiency of concentration in terms of treated surface area.     

Anti-adhesive and antibacterial properties of a proprietary denture cleanser

The antimicrobial efficacy and anti-adhesive attributes of a proprietary denture cleanser were evaluated. To determine cleanser antimicrobial efficacy, Streptococcus mutans was grown on denture acrylic strips which were then exposed to the cleanser. To evaluate anti-adhesive efficacy, the strips were treated with the cleanser and then placed in the Strep. mutans suspension. Following incubation, adhered bacteria were removed and enumerated by viable counting. Treated denture acrylic plates were also placed in a parallel-plate flow chamber and then exposed to Strep. oralis. Images of adhered bacteria were analysed to determine biofilm coverage. Biofilm removal force was quantified by increasing the flow rate between the acrylic plates. The cleanser exhibited a 100% kill against Strep. mutans adhered to the acrylic surface, and inhibited attachment of cells by 66%. The flow chamber study found that cleanser-treated denture acrylic allowed the formation of a multilayer biofilm which was easily removed by a slight increase in flow rate.     

In vitro method to assess the antimicrobial activity and potential efficacy of novel types of wound dressings

AIMS: To develop a simple, reproducible in vitro static diffusion method using cellulose disks and defined species to test antimicrobial efficacy of wound dressings. METHODS AND RESULTS: Cellulose disks were inoculated by immersion in cell suspensions of target species Staphylococcus epidermidis, Candida albicans and Fusobacterium nucleatum. Test and control wound dressings were cut into equal sized squares (25 x 25 mm) and applied to the surface of 10-mm thick tryptone yeast extract agar on test beds. Following a 2-h equilibration period, inoculated cellulose disks were inserted (one per dressing) at the interface between dressing and agar surface and a small weight applied over each square. At various sampling times, disks were removed and surviving cells enumerated by viable counts. Disk to disk variation for microbial loading was assessed using S. epidermidis for both initial (n = 16) and standard treatment (n = 16) conditions. The coefficient of variation was low (<5%) indicating good reproducibility for cell loading and treatment position on the test bed. Replicate assays (n = 6) using S. epidermidis and oxyzyme gels produced similar kill rates with low scatter (R2 > 0.9) indicating good reproducibility between assays. Significant differences (P < 0.05) in kill rates were observed for different target species, types of dressing and test bed conditions (+/-blood and nutrients). CONCLUSIONS: The method is reproducible and useful in tracking the death kinetics of test species, enabling the comparison of different types of dressing. SIGNIFICANCE AND IMPACT OF THE STUDY: The reported method has significant advantages over established test procedures; it can be applied equally across a wide range of target species (including anaerobes and yeasts), a wide range of conditions, and different types of surface dressings, including those relying upon oxygen diffusion.     

Antimicrobial testing for surface‐immobilized agents with a surface‐separated live–dead staining method

Modification of a traditional live–dead staining technique based on fluorescence microscopy has yielded an improved method capable of differentiating surface‐immobilized antimicrobial agents from those agents acting via solution diffusion processes. By utilizing an inoculation chamber comprised of 50 µm polystyrene spheres as spacers between test substrate and coverslip control surfaces, three distinct bacterial cell populations can be probed by fluorescence microscopy for antimicrobial activity: (1) cells adhered to the coverslip, (2) cells adhered to the substrate, and (3) mobile cells in solution. Truly immobilized antimicrobial agents were found efficacious only at the substrate surface, while elutable agents were effective against all three populations. Glass surfaces derivatized with either quaternized poly dimethylaminoethylmethacrylate (pDMAEMA) or 3‐(trimethoxysilyl) propyldimethyloctadecyl ammonium chloride (Si‐QAC) were compared with bare glass control surfaces after contact and 4 h incubation with Staphylococcus aureus. pDMAEMA surfaces were both antimicrobial and immobilized, whereas the Si‐QAC surfaces were only observed to be antimicrobial via active diffusion. In contrast to conventional thinking, Si‐QAC surfaces showed no kill after removing all Si‐QAC elutables via rinsing procedures. The semi‐quantitative surface‐separated live–dead staining (SSLDS) technique provides mechanistic insight and represents a significant improvement relative to current microbiological test methods for evaluating immobilized, antimicrobial agents. Biotechnol. Bioeng. 2011; 108:231–236. © 2010 Wiley Periodicals, Inc.     

Surface-active antifungal polyquaternary amine

There is a distinct need for antimicrobial compounds that can act at surfaces without leaching into the environment. Such materials should be easy to synthesize, be easy to apply to surfaces, and display reasonable levels of antimicrobial and antifungal activity. Here we describe such a surface-active compound and demonstrate its ability to inhibit the growth of the filamentous fungus Aspergillus niger. We have synthesized a series of polyquaternary ammonium compounds by atom transfer radical polymerization. Two members of this series were tested for their ability to inhibit the growth of A. niger. The compounds were dried onto surfaces, and the treated surfaces were then used as growth chambers for A. niger. A water soluble polyquaternary amine compound was shown to effectively kill A. niger in solution in a dose-dependent manner. Conversely, a water insoluble polyquaternary amine compound was shown to kill only the fungi in direct contact with the material on the surface. These results have important implications for the development of effective, environmentally benign, surface-active anti-fungal compounds.     

Use of membrane filters for measurement of mycobactericidal activity of alkaline glutaraldehyde solution

Bactericidal assays of 2% alkaline glutaraldehyde solution were carried out using Millipore membranes, and the rate of kill was compared with that of mycobacteria in suspension and on Penicylinder surfaces when using the methods recommended for the official tuberculocidal test. The rate of inactivation observed on the membrane filter surface was similar to that achieved using Penicylinders. The absence of visible colonies on the treated membranes provided a direct demonstration of sterility. The use of filter membranes in tuberculocidal tests provides a simple quantitative assay.     

Use of membrane filters for measurement of mycobactericidal activity of alkaline glutaraldehyde solution.

Bactericidal assays of 2% alkaline glutaraldehyde solution were carried out using Millipore membranes, and the rate of kill was compared with that of mycobacteria in suspension and on Penicylinder surfaces when using the methods recommended for the official tuberculocidal test. The rate of inactivation observed on the membrane filter surface was similar to that achieved using Penicylinders. The absence of visible colonies on the treated membranes provided a direct demonstration of sterility. The use of filter membranes in tuberculocidal tests provides a simple quantitative assay.     

Structural determinants of antimicrobial and antiplasmodial activity and selectivity in histidine-rich amphipathic cationic peptides

Designed histidine-rich amphipathic cationic peptides, such as LAH4, have enhanced membrane disruption and antibiotic properties when the peptide adopts an alignment parallel to the membrane surface. Although this was previously achieved by lowering the pH, here we have designed a new generation of histidine-rich peptides that adopt a surface alignment at neutral pH. In vitro, this new generation of peptides are powerful antibiotics in terms of the concentrations required for antibiotic activity; the spectrum of target bacteria, fungi, and parasites; and the speed with which they kill. Further modifications to the peptides, including the addition of more hydrophobic residues at the N terminus, the inclusion of a helix-breaking proline residue or using D-amino acids as building blocks, modulated the biophysical properties of the peptides and led to substantial changes in toxicity to human and parasite cells but had only a minimal effect on the antibacterial and antifungal activity. Using a range of biophysical methods, in particular solid-state NMR, we show that the peptides are highly efficient at disrupting the anionic lipid component of model membranes. However, we also show that effective pore formation in such model membranes may be related to, but is not essential for, high antimicrobial activity by cationic amphipathic helical peptides. The information in this study comprises a new layer of detail in the understanding of the action of cationic helical antimicrobial peptides and shows that rational design is capable of producing potentially therapeutic membrane active peptides with properties tailored to their function.     

Preparation of alginate-quaternary ammonium complex beads and evaluation of their antimicrobial activity

Alginate-quaternary ammonium complex beads with antimicrobial activity were prepared by the reaction of sodium alginate (SA) with 3-(trimethoxysilyl)propyl-octadecyldimethylammonium chloride (TSA) in acid solution, followed by crosslinking with CaCl(2). FTIR spectroscopy analysis showed that the resulting complex was formed mainly through covalent bonds between the hydroxyl groups of SA and the methoxysilyl groups of TSA. The complex beads exhibited a maximum swelling of 20% in water at 37 degrees C and were not hydrolyzed in water during experiments lasting for 30 days. The in vitro antimicrobial activity of the complex beads was evaluated against four species of bacteria and fungi. The test microorganisms were completely eliminated within 20 min when treated with 5% (v/v) complex beads, which showed a wide spectrum of excellent antimicrobial activity. The antimicrobial activity of the complex beads was retained after 10 cycles of washing and drying. The present results indicate that these SA-TSA complex beads are a new type of insoluble cationic polymer that can kill or remove microorganisms in water by mere contact without releasing the reactive agent.     

Kinetic evaluation of claimed synergistic paraben combinations using a factorial design.

The antimicrobial effects of methyl and propyl parabens are investigated, with Escherichia coli as test organism, with a view to determining whether the parabens act synergistically. At appropriate concentrations, the parabens killed E. coli cells according to first order kinetics and the bactericidal effects were quantified by the first order kill rate constants. Combinations of methyl or propyl parabens, at concentrations which slow down or inhibit bacterial growth when used singly, produced definite kill. In this sense, the parabens are therefore synergistic since in combination they produce an effect which is not observed when they are used singly. This effect is not true synergism as shown by the results of our experiments with a factorial design. Analysis of variance indicated no significant interaction between the two parabens.     

Antimicrobial properties of delmopinol against oral bacteria

Antimicrobial properties of the morpholine derivative delmopinol hydrochloride were investigated in vitro . Minimum inhibitory concentrations were measured for a range of oral bacteria by broth and agar dilution techniques and minimum bactericidal concentrations were measured in broth. The rate of kill was investigated by determination and analysis of kill curves. The results indicated that delmopinol is a relatively weak antimicrobial in comparison with chlorhexidine. However, it may selectively affect certain species of oral bacteria which are of importance in plaque formation.     

A more accurate method for measurement of tuberculocidal activity of disinfectants

The current Association of Official Analytical Chemists method for testing tuberculocidal activity of disinfectants has been shown to be inaccurate and to have a high degree of variability. An alternate test method is proposed which is more accurate, more precise, and quantitative. A suspension of Mycobacterium bovis BCG was exposed to a variety of disinfectant chemicals and a kill curve was constructed from quantitative data. Data are presented that show the discrepancy between current claims, determined by the Association of Official Analytical Chemists method, of selected commercially available products and claims generated by the proposed method. The effects of different recovery media were examined. The data indicated that Mycobacteria 7H11 and Middlebrook 7H10 agars were equal in recovery of the different chemically treated cells, with Lowenstein-Jensen agar having approximately the same recovery rate but requiring incubation for up to 3 weeks longer for countability. The kill curves generated for several different chemicals were reproducible, as indicated by the standard deviations of the slopes and intercepts of the linear regression curves.     

A more accurate method for measurement of tuberculocidal activity of disinfectants.

The current Association of Official Analytical Chemists method for testing tuberculocidal activity of disinfectants has been shown to be inaccurate and to have a high degree of variability. An alternate test method is proposed which is more accurate, more precise, and quantitative. A suspension of Mycobacterium bovis BCG was exposed to a variety of disinfectant chemicals and a kill curve was constructed from quantitative data. Data are presented that show the discrepancy between current claims, determined by the Association of Official Analytical Chemists method, of selected commercially available products and claims generated by the proposed method. The effects of different recovery media were examined. The data indicated that Mycobacteria 7H11 and Middlebrook 7H10 agars were equal in recovery of the different chemically treated cells, with Lowenstein-Jensen agar having approximately the same recovery rate but requiring incubation for up to 3 weeks longer for countability. The kill curves generated for several different chemicals were reproducible, as indicated by the standard deviations of the slopes and intercepts of the linear regression curves.     

Surpassing nature: rational design of sterile-surface materials

The rise of multidrug-resistant pathogens and recalcitrance of biofilm infections present a formidable challenge to combating infectious diseases. There are numerous disinfectants and antiseptics for treating materials in hospitals and community settings, and devices such as catheters impregnated with anti-infectives have been introduced into practice. However, there are many limitations of materials impregnated with a leaching antibacterial agent. Recently, non-leaching, permanent, sterile-surface materials have been developed in which one end of a long-chained hydrophobic polycation containing antimicrobial monomers is attached covalently to the surface of a material, for example, cotton or plastic. The polymeric chain allows the antimicrobial moieties to permeate into, and kill, the cells of the pathogen. These sterile-surface materials kill both air- and waterborne pathogens and are not susceptible to existing resistance mechanisms.     

耐甲氧西林葡萄球菌的分布及耐药性分析

目的：了解我院临床分离的耐甲氧西林葡萄球菌（MRS）的分布及耐药性。方法：用Kirby-Bauer法对我院临床分离的394株MRS进行药敏试验并对其标本来源和病区分布进行分析。结果：471株葡萄球菌中MRS394株,占83.7%。各类标本中,检出MRS最多的是血液（40.36%),检出MRS最多的科室是ICU（23.35%）。MRS除对万古霉素和替考拉宁耐药率为0外,对其余8种抗生素的耐药率高达27.7%-84.7%。结论：本地区MRS的检出率高,血液标本和ICU病房检出MRS最多,MRS耐药形势严峻,值得高度重视。     

Kinetic evaluation of claimed synergistic paraben combinations using a factorial design

D. GILLILAND, A. LI WAN PO AND E. SCOTT. 1992. The antimicrobial effects of methyl and propyl parabens are investigated, with Escherichia coli as test organism, with a view to determining whether the parabens act synergistically. At appropriate concentrations, the parabens killed E. coli cells according to first order kinetics and the bactericidal effects were quantified by the first order kill rate constants. Combinations of methyl or propyl parabens, at concentrations which slow down or inhibit bacterial growth when used singly, produced definite kill. In this sense, the parabens are therefore synergistic since in combination they produce an effect which is not observed when they are used singly. This effect is not true synergism as shown by the results of our experiments with a factorial design. Analysis of variance indicated no significant interaction between the two parabens.     

Polypropylene with embedded copper metal or copper oxide nanoparticles as a novel plastic antimicrobial agent

Aims: To develop novel polypropylene composite materials with antimicrobial activity by adding different types of copper nanoparticles. Methods and Results: Copper metal (CuP) and copper oxide nanoparticles (CuOP) were embedded in a polypropylene (PP) matrix. These composites present strong antimicrobial behaviour against E. coli that depends on the contact time between the sample and the bacteria. After just 4 h of contact, these samples are able to kill more than 95% of the bacteria. CuOP fillers are much more effective eliminating bacteria than CuP fillers, showing that the antimicrobial property further depends on the type of copper particle. Cu²⁺ released from the bulk of the composite is responsible for this behaviour. Moreover, PP/CuOP composites present a higher release rate than PP/CuP composites in a short time, explaining the antimicrobial tendency. Conclusions: Polypropylene composites based on copper nanoparticles can kill E. coli bacteria depending on the release rate of Cu²⁺ from the bulk of the material. CuOP are more effective as antimicrobial filler than CuP. Significance and Impact of the Study: Our findings open up novel applications of these ion‐copper‐delivery plastic materials based on PP with embedded copper nanoparticles with great potential as antimicrobial agents.     

一株病原拮抗野生菌株的筛选、鉴定及其发酵工艺优化

为获得广谱抗菌功能野生菌株并提高其发酵产物中抗菌物质的含量。采用管碟法和菌丝生长速率法筛选功能菌株,ITS序列分析鉴定功能菌株,通过响应面法和正交设计优化发酵生产抗菌物质的工艺。筛选到一株强效、广谱抗菌功能菌株,鉴定为Cerrena sp.,其发酵产物对金黄色葡萄球菌、大肠杆菌、白色念珠菌、枯草芽孢杆菌和水稻纹枯病菌有显著拮抗作用。该菌株的摇甁发酵配方及培养条件为：马铃薯13.99 g/L,蔗糖 41.58 g/L,VB1 0.027 g/L,麸皮7 g/L,KH₂PO₄ 2 g/L,MgSO₄·7H₂O 2 g/L;摇床温度28 ℃、发酵周期10 d、种龄4 d、接种量8%、初始pH为5.0、装液量110 ml/250 ml。该菌株有明显抑菌活性,发酵工艺优化后抗菌活性提高了30.37%,为该菌株今后的应用、抗菌剂的分离提纯和产业化提供了实验依据。     

Postsecretory processing generates multiple cathelicidins for enhanced topical antimicrobial defense

The production of antimicrobial peptides and proteins is essential for defense against infection. Many of the known human antimicrobial peptides are multifunctional, with stimulatory activities such as chemotaxis while simultaneously acting as natural antibiotics. In humans, eccrine appendages express DCD and CAMP, genes encoding proteins processed into the antimicrobial peptides dermcidin and LL-37. In this study we show that after secretion onto the skin surface, the CAMP gene product is processed by a serine protease-dependent mechanism into multiple novel antimicrobial peptides distinct from the cathelicidin LL-37. These peptides show enhanced antimicrobial action, acquiring the ability to kill skin pathogens such as Staphylococcus aureus and Candida albicans. Furthermore, although LL-37 may influence the host inflammatory response by stimulating IL-8 release from keratinocytes, this activity is lost in subsequently processed peptides. Thus, a single gene product encoding an important defense molecule alters structure and function in the topical environment to shift the balance of activity toward direct inhibition of microbial colonization.     

Biomolecular Surfaces for the Capture and Reprogramming of Circulating Tumor Cells

Circulating Tumor Cells(CTC)have the potential to be used clinically as a diagnostic tool and a treatment tool in the fieldof oncology.As a diagnostic tool,CTC may be used to indicate the presence of a tumor before it is large enough to cause noticeablesymptoms.As a treatment tool,CTC isolated from patients may be used to test the efficacy of chemotherapy options topersonalize patient treatment.One way for tumors to spread is through metastasis via the circulatory system.CTC are able toexploit the natural leukocyte recruitment process that is initially mediated by rolling on transient selectin bonds.Our capturedevices take advantage of this naturally occurring recruitment step to isolate CTC from whole blood by flowing samples throughselectin and antibody-coated microtubes.Whole blood was spiked with a known concentration of labeled cancer cells and thenperfused through pre-coated microtubes.Microtubes were then rinsed to remove unbound cells and the number of labeled cellscaptured on the lumen was assessed.CTC were successfully captured from whole blood at a clinically relevant level on the orderof 10 cells per mL.Combination tubes with selectin and antibody coated surface exhibited higher capture rate than tubes coatedwith selectin alone or antibody alone.Additionally,CTC capture was demonstrated with the KG 1 a hematopoietic cell line andthe DU 145 epithelial cell line.Thus,the in vivo process of selectin-mediated CTC recruitment to distant vessel walls can be usedin vitro to target CTC to a tube lumen.The biomolecular coatings can also be used to capture CTC of hematopoietic andepithelial tumor origin and is demonstrated to sensitivities down to the order of 10 CTC per mL.In a related study aimed at reducing the blood borne metastatic cancer load,we have shown that cells captured to a surfacecan be neutralized by a receptor-mediated biochemical signal.In the proposed method we have shown that using a combinedselectin and TRAIL(TNF Related Apoptosis Inducing Ligand or Apo 2L)functionalized surface we are abl