Elastase inhibitors, bacteriostatic agents with anti-biofilm activity effective against both single-and multi-species biofilms
阅读说明：本技术 弹性蛋白酶抑制剂、具有对单物种和多物种生物膜都有效的抗生物膜活性的抑菌剂 (Elastase inhibitors, bacteriostatic agents with anti-biofilm activity effective against both single-and multi-species biofilms ) 是由 钱培元 龙乐欣 李泳新 王若珺 蒋皓然 于 2020-08-21 设计创作，主要内容包括：本申请涉及使用基于弹性蛋白酶抑制剂(elasnin)的组合物清除生物膜和/或抑制生物膜的形成或抑制非生物膜形成生物体的污染的组合物和方法。在某些实施方案中,弹性蛋白酶抑制剂可以结合抗菌化合物和/或传统涂层成分。(The present application relates to compositions and methods for removing biofilms and/or inhibiting biofilm formation or inhibiting fouling by non-biofilm forming organisms using elastase inhibitor (elasnin) -based compositions. In certain embodiments, the elastase inhibitor can be combined with an antimicrobial compound and/or a conventional coating ingredient.)
1. A method for inhibiting biofilm formation and/or dispersing an existing biofilm, the method comprising contacting an elastase inhibitor with the biofilm or applying an elastase inhibitor to a surface.
2. The method of claim 1, wherein the method further comprises adding to the surface one or more components selected from the group consisting of antimicrobial compounds and traditional coating compositions.
3. The method of claim 1, wherein the performance or lifetime of the surface is improved.
4. The method of claim 1, wherein the surface is a medical instrument, a boat, an anchor, a dock, a buoy, a net, a heat exchanger, or a water pipe.
5. The method of claim 1, wherein the biofilm comprises gram positive bacteria.
6. The method of claim 1, wherein the biofilm comprises a plurality of species of organisms.
7. The method of claim 1, wherein the traditional coating composition is an adhesive, pigment, sealant, solvent, pH adjuster, or buffer.
8. The method of claim 1, wherein the method further comprises inhibiting the growth of a non-biofilm forming organism.
9. An anti-biofilm composition comprising an elastase inhibitor and one or more antibacterial compounds or conventional coating ingredients.
10. The composition of claim 9, wherein the antibacterial compound is vancomycin.
11. The composition of claim 9, wherein the traditional coating component is an adhesive, pigment, sealant, solvent, pH adjuster, or buffer.
Technical Field
Provided herein are anti-biofilm compositions. In particular, the present application provides methods for inhibiting biofilm formation, disrupting mature biofilms, and inhibiting attachment of biofouling organisms. The application also relates to compositions comprising elastase inhibitors (elasnins) and/or antibacterials
Anti-biofilm compositions of compounds.
Background
Biofilms are organized aggregates of microorganisms (O' Toole, g., Kaplan, h.b., & Kolter, R. (2000.) biofilmformation as microbial reduction in Microbiology,54(1),49-79) attached to surfaces. The biofilm consists of cells and a matrix of Extracellular Polymers (EPS) comprising various biopolymers such as proteins, nucleic acids, lipids and other substances that maintain The links between cells and allow cell-cell interactions (fleming, h.c., & winggent, J. (2010), The bifilm matrix nature reviews microbiology,8(9), 623). The microorganisms within the biofilm may be a single species of microorganism or a plurality of populations. Microorganisms can colonize a variety of animate and inanimate surfaces, creating a structurally and kinetically complex multifunctional biological system, enabling microorganisms to survive and resist external threats in diverse environments (Davey, m.e., & O' tools, G.A. (2000) & Microbial biologies & from environmental to molecular genetics & microbe & mol.biol.biol.rev.64, (4),847 & 867 & Hall-stodley & l. & Costerton, j.w. & stodley & P. & Bacterial biologies & P. (2004) & the natural environmental to environmental diseases & nature reviews microbiology,2(2), (95.). Bacteria form biofilms in a similar manner, despite the existence of a variety of different habitats. The process begins with the regulated attachment of planktonic cells to the surface in response to signaling molecules, followed by EPS secretion, forming small colony aggregates. Over time, microcolonies mix and develop into mature biofilms with three-dimensional structures. Cells can be detached and dispersed from mature Biofilms, colonizing elsewhere, and initiating a new cycle (Rodri i guez-Mart i nez, J.M., & Pascal, A. (2006). Antimicrobial resistance in bacterial biology. reviews. Medical Microbiology,17(3), 65-75; Donlan, R.M. (2001). Biofilm formation: a clinical release microbial Processes Diseases,33(8), 1387. 1392; Donlan, R.M. (2002). Biofilms: biological life surface Diseases, environmental Diseases, diagnosis (8), 8819).
Biofilm formation is a critical factor in bacterial survival; the delicate structure of the biofilm provides a barrier to the cells within it and provides spatial proximity and homeostasis to the cells to promote growth and differentiation, thereby facilitating the persistence of the cells in various environments (Dang, h.,&lovell, c.r. (2016), Microbial surface degradation and biochemical degradation in mineral environments microbiol. mol. biol. rev.,80(1), 91-138). Biofilms concentrate nutrients and have water channels and pore structures that enable efficient nutrient uptake, enhance metabolite transport, promote cell-cell interactions. Thus, the different environmental signals enriched by metabolism cause changes in gene expression, resulting in diverse and specific subpopulations in a particular microenvironment. When organisms reside in biofilms, organisms are persistent in the environment and more resistant than planktonic microorganisms to antibacterial treatments, toxins, protozoa, and the host immune system (antchines, l.c.m.,&Ferreira,R.B.(2011).Biofilms and bacterial virulence.Reviews in Medical Microbiology,22(1),12-16；López,D.,Vlamakis,H.,&kolter, R. (2010), Biofilms, Cold Spring Harbor perspectives in biology,2(7), a 000398). The sensitivity of cells in biofilms to various antibacterial agents is reduced by 10-fold to 1,000-fold. Once the biofilm begins to develop, it is difficult to remove and remove completely (Hengzhuang, w., Wu, h., Ciofu, o., Song, z.,&(2011) Pharmacokinetics/Pharmacodynmics of coistin and imipenem on mucoid and nonmucoid Pseudomonas aeruginosa bioinformatics and chemitherapy, 55(9), 4469-; davies, d. (2003). using mapping of biochemical responses to antibiotic agents. nature reviews Drug discovery,2(2), 114). Several mechanisms have been proposed to explain biofilm resistance (Mah, t.f.c.,&O'Toole,G.A.(2001).Mechanisms of biofilm resistance to antimicrobial agents.Trends in microbiology,9(1),34-39；Hall,C.W.,&mah, T.F, (2017), Molecular mechanisms of biological-based antigenic resistance and tolerance in pathological bacteria, fems microbiological reviews,41(3), 276-. The first mechanism is the barrier function of EPS. The EPS prevents or dilutes the diffusion of foreign substances into cells by embedding the cells within the biofilm by various EPS. Furthermore, the altered growth rate is also one of the protection mechanisms of biofilms. The lack of nutrients slows the growth of the bacteria and may cause the bacteria to shift from an exponential growth phase to a slow growth phase or a growth arrest phase. At the same time, vigorous interactions within the biofilm promote gene transfer and differentiation, which may lead to the development of resistant phenotypes affecting the efficacy of the antimicrobial agent. In addition, resistance mechanisms have been proposed, such as antimicrobial efflux pumps and antibiotic modifying enzymes. Thus, standard antibiotic therapy can only eliminate planktonic cells, but sessile cells within the biofilm can quickly recover and continue to multiply and spread.
Greater than 65% of nosocomial infections are associated with biofilm formation, and The mortality rate for these infections is up to 70%, including both device-related and chronic non-device related infections, contributing to additional treatment costs of more than one billion dollars per year (Del Pozo, j.l., & Patel, R. (2007). The challenge of treating biological-associated bacterial infections, clinical Pharmacology & Therapeutics,82(2), 204-. The chronic pulmonary infection Cystic Fibrosis (CF) caused by Pseudomonas aeruginosa (Pseudomonas aeruginosa) and the infection caused by Staphylococcus (Staphylococcus spp.) left on medical devices are two important examples of infections involving biofilms. Cystic Fibrosis (CF) patients are susceptible to pseudomonas aeruginosa infection and EPS produced by biofilm cells can cause severe inflammatory reactions in the lungs and disrupt lung function, which can lead to death. Staphylococci, particularly staphylococcus aureus (s.aureus) and staphylococcus epidermidis (s.epidermidis), are the most common microorganisms in human biofilm-associated infections. Are often distributed on the human skin and these bacteria can colonize the medical indwelling device, thereby facilitating transmission to other sites, causing systemic infections. Staphylococci are extremely resistant to antibiotic treatment and the immune system. This is not only because of its abundant resistant phenotype, but also because of the ability of bacteria to utilize inflammatory reaction products to induce biofilm formation. Thus, advanced devices such as intravenous catheters, prosthetic heart valves, and endotracheal tubes can cause fatal infections in humans. In addition, biofilm formation creates significant problems in various industrial activities, such as aquaculture, heat exchangers, oil and gas industry, marine transportation, and desalination of sea water. Biofilms mediate the attachment of large fouling organisms and accelerate bio-corrosion, which results in a 35% to 50% increase in fuel consumption, a 5% to 20% increase in cleaning operations costs, and a 20% to 30% increase in corrosion-related costs (16, 17). With the increasing severity of biofilm-related problems, currently available biofilm control methods are limited. These methods include physical removal, continuous antimicrobial treatment and surface coatings (Koo, h., alan, r.n., Howlin, r.p., stodley, p., & Hall-stodley, L. (2017). Targeting microbial biologics: current and reactive thermal protocols, nature Reviews Microbiology,15(12), 740). Some non-toxic "green" coatings have been developed from silicones, fluorine and fluoro-silicon to resist biofilm formation with antimicrobials in industrial and clinical settings. However, in most cases, existing methods are expensive, and most of the antibiotics currently developed target planktonic cells, thus requiring high doses for treating biofilms and possibly increasing selectivity for antibiotic resistance phenotypes leading to an increase in resistance (de Carvalho, C.C (2018). Marine biolofils: a basic microbial strain with environmental importance. Frontiers in Marine Science,5,126; Ribeiro, S.M., Felicio, M.R., as Bo, E.V., Goncales, S.Costa, F.F., Samy, R.P., & Franco, O.L. (2016). N.P.. Fr. (144). Therefore, there is a need to develop efficient, safe, environmentally friendly and low cost biofilm-targeting anti-biofilm agents.
Disclosure of Invention
Provided herein are anti-biofilm compositions. In particular, the present application provides methods for inhibiting biofilm formation, disrupting mature biofilms, and inhibiting attachment of biofouling organisms. The present application also relates to anti-biofilm compositions comprising elastase inhibitors and/or antibacterial compounds. In certain embodiments, the elastase inhibitor may be produced by Streptomyces mobaraensis (Streptomyces mobaraensis) DSM 40847.
In certain embodiments, an anti-biofilm composition comprising an elastase inhibitor and one or more antimicrobial compounds is provided. In certain embodiments, the antimicrobial component comprises, for example, vancomycin.
In certain embodiments, an anti-biofilm composition comprising an elastase inhibitor and one or more conventional surface coating components is provided. In certain embodiments, the surface coating ingredients include, for example, binders, solvents, pigments, pH adjusters, buffers, or any other ingredients that constitute, for example, a pigment, a primer, a paint, or a sealant.
In certain embodiments, the present application utilizes bacterial strains and/or by-products of their growth. The present application provides, for example, a microorganism-based preparation comprising cultured streptomyces mobaraensis DSM40847 and/or a metabolite of the microorganism.
In a preferred embodiment, there is provided a method of inhibiting biofilm formation and/or removing an existing biofilm, the method comprising applying an elastase inhibitor to a surface and/or biofilm. In certain embodiments, the addition of an elastase inhibitor-based composition to a surface enhances the performance and/or prolongs the life of the surface.
Advantageously, the present application provides environmentally friendly anti-biofilm compositions and methods of use. Elastase inhibitors can remain closely associated with the site of their application and therefore do not allow the surrounding environment, including marine and freshwater environments, to take up significant amounts of elastase inhibitors. The ability of the elastase inhibitor to remain upon application can protect the presence of uncontaminated biofilm.
Drawings
FIG. 1A shows the mass spectrum (ESI) and structure of elastase inhibitors.
FIG. 1B shows the growth of Streptomyces mobaraensis DSM40847 on GyM agar plates. Gym agar is prepared from glucose (4.0g), yeast extract (4.0g), malt extract (10.0g), CaCO3(2.0g), agar (12.0g) and distilled water (1000.0ml) (pH was adjusted to 7.2 before adding agar).
FIG. 1C shows HPLC analysis of a crude extract of Streptococcus mobaraensis DSM 40847.
FIG. 1D shows the time course of elastase inhibitor production in AM4 medium at 30 ℃.
Fig. 2A shows the minimum concentration required to inhibit 90% biofilm formation.
Fig. 2B shows the minimum concentration required to clear 50% of mature biofilm.
Fig. 2C shows cell viability after 24 hours of treatment with various antimicrobial agents.
FIG. 2D is a summary of MIC, MBC, MBIC and MBEC. The points below 0% are not shown in the figure.
Figure 3A shows MRSA biofilms after 24 hours of growth (control). Series 1 is a photograph of the biofilm observed directly. Series 2 and 3 are by FilmtracerTM 1-43Green Biofilm Cell Stain of two-dimensional and three-dimensional confocal images of Biofilm cells. Series 4 and 5 are by FilmtracerTM 2D and 3D images of Ruby Biofilm Matrix Stain on Biofilm Matrix. Confocal images were acquired under the same conditions and were quantitatively analyzed using Leica Application Suite X based on the relative fluorescence of the 3D confocal images.
FIG. 3B shows MRSA biofilms after 24 hours of growth treated with elastase inhibitors at a concentration of 4 μ g/mL (treatment). Series 1 is a photograph of the biofilm observed directly. Series 2 and 3 are by FilmtracerTM 1-43Green Biofilm Cell Stain of two-dimensional and three-dimensional confocal images of Biofilm cells. Series 4 and 5 are by FilmtracerTM 2D and 3D images of Ruby Biofilm Matrix Stain on Biofilm Matrix. Confocal images were acquired under the same conditions and were quantitatively analyzed using Leica Application Suite X based on the relative fluorescence of the 3D confocal images.
Figure 3C shows the appearance of pre-formed MRSA biofilms after 18 hours of further incubation (control). Series 1 is a photograph of the biofilm observed directly. Series 2 and 3 are by FilmtracerTM 1-43Green Biofilm Cell Stain of two-dimensional and three-dimensional confocal images of Biofilm cells. Series 4 and 5 are by FilmtracerTM 2D staining of Biofilm Matrix by Ruby Biofilm Matrix Stain anda 3D image. Confocal images were acquired under the same conditions and were quantitatively analyzed using Leica Application Suite X based on the relative fluorescence of the 3D confocal images.
FIG. 3D shows the appearance of pre-formed MRSA biofilms after 18 hours of treatment with elastase inhibitors at a concentration of 4 μ g/mL (treatment). Series 1 is a photograph of the biofilm observed directly. Series 2 and 3 are by FilmtracerTM 1-43Green Biofilm Cell Stain of two-dimensional and three-dimensional confocal images of Biofilm cells. Series 4 and 5 are by FilmtracerTM 2D and 3D images of Ruby Biofilm Matrix Stain on Biofilm Matrix. Confocal images were acquired under the same conditions and were quantitatively analyzed using Leica Application Suite X based on the relative fluorescence of the 3D confocal images.
Fig. 3E shows a quantitative analysis of confocal images acquired in the biofilm inhibition assay.
Fig. 3F shows a quantitative analysis of confocal images acquired in the biofilm removal assay.
Fig. 4A shows anti-biofilm activity of elastase inhibitors to reduce single species biofilm formation by 90%.
Fig. 4B shows the anti-biofilm activity of elastase inhibitors to clear 50% of mature single-species biofilms.
FIG. 4C shows the MBIC/MBEC for 6 bacterial strains used in the anti-biofilm assay and elastase inhibitors against these strains.
Figure 5A provides a graph showing the anti-biofilm (weeks 2 and 3) and anti-fouling (week 4) performance of surfaces coated with PCL-based polyurethane and mixed with different concentrations of crude extract (CR, secondary metabolite of DSM 40748 extracted with 1-hexane).
Fig. 5B shows the quantification of the biomass of the biofilm observed by CLSM. Biomass was calculated using Comstat 2.1 based on CLSM images and the values significantly different between the elastase inhibitor based anti-biofilm coating and the control group are indicated by asterisks as follows: p < 0.05 and P < 0.01.
Fig. 5C shows the release rate of elastase inhibitors in artificial seawater.
Fig. 5D shows the composition of the elastase inhibitor coating used to determine the anti-biofilm performance of the elastase inhibitor.
FIG. 6A provides a comparison of the beta-diversity (Bray-Curtis) of the microbial composition between biofilms on control slides (C-1,2,3) and biofilms on a 10% wt. elastase inhibitor coating (E10-1,2,3) at the gate level.
Figure 6B shows the alpha-diversity of biofilm microbial composition at the portal level. The difference between the two types of biofilms was calculated by student t-test and is indicated by asterisks as follows: p < 0.05.
Figure 7 provides NMR analysis of elastase inhibitors.
FIG. 8 provides HPLC profiles of crude extracts of high elastase inhibitor content and crude extract/elastase inhibitor yields using different extraction solvents.
FIG. 9 shows the biological activity of 20 fragments isolated from a crude extract of secondary metabolites produced by Streptococcus mobaraensis DSM 400847 (cultured in AM4 medium and extracted with 1-butanol). Fragment 17 is an analogue of fragment 16 (an elastase inhibitor).
FIG. 10 shows the microbial composition of biofilms on control slides (C1, C2 and C3) and on genus level on slides (E10-1, E10-2 and E10-3) with a 10% by weight content of elastase inhibitor coating.
Description of sequence listing
SEQ ID NOS: 1 to 2 provide primer sequences for amplifying the hypervariable V3-V4 region of 16S rRNA in bacteria.
Detailed Description
Compositions and methods for inhibiting biofilm formation and/or removing biofilm are provided. In particular, the present application provides compositions and methods for using elastase inhibitors to inhibit and/or clear biofilms. In certain embodiments, the anti-biofilm composition may comprise an antimicrobial compound and/or a conventional coating composition.
Definition of selection
As used herein, the singular forms "a", "an" and "the" are intended to include the plural references unless the context clearly indicates otherwise. Furthermore, to the extent that the term "includes," including, "" includes, "" having, "" has, "" with, "or variants thereof are used in either the detailed description and/or the claims, such term is intended to be inclusive in a manner similar to the term" comprising. Variant terms/phrases (and any grammatical variants thereof) "comprising", "having", "including" includes the phrases "consisting essentially of … (consistency addressing of)", "consisting essentially of … (consistency addressing of)", "consisting of … (consistency)" and "consisting of … (consistency)".
The phrases "consisting essentially of …" or "consisting essentially of …" mean that the claims include embodiments that include the specified materials or steps as well as those embodiments that do not materially affect the basic and novel characteristics of the claims.
The term "about" means within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is determined or determined, i.e., the limitations of the assay system. Where particular values are described in the application and claims, unless otherwise stated, it should be assumed that the term "about" means within an acceptable error range for the particular value.
In this disclosure, ranges are described in a shorthand manner, avoiding the necessity of setting forth and describing in detail individual values and all values within the range. Any suitable value within the range can be selected as the upper, lower, or end value of the range, where appropriate. For example, a range of 1 to 10 represents the endpoints of 1 and 10, and the median of 2,3, 4, 5, 6, 7, 8, 9, and includes all intermediate ranges within 1 to 10, such as 2 to 5, 2 to 8, and 7 to 10. Moreover, when ranges are used herein, it is intended to expressly include combinations and sub-combinations of ranges (e.g., sub-ranges within the disclosed ranges) and specific embodiments therein.
As used herein, a "biofilm" is a complex aggregate of microorganisms, such as bacteria, in which cells are attached to each other and/or to a surface using a exopolysaccharide matrix. The cells in a biofilm are physiologically different from planktonic cells of the same organism, a planktonic cell being a single cell that can float or float in a liquid medium.
As used herein, "obtaining" refers to removing some or all of the microorganism-based composition from the growth vessel.
According to the present application, the harmful accumulation of materials, including living organisms or inactive substances, leads to a "fouling" process. "fouling" can lead to plugging, fouling or other unwanted buildup. "contamination" can affect the efficiency, reliability, or functionality of a target object.
Elastase inhibitor compositions
The present disclosure provides methods of inhibiting and/or removing biofilms using compositions comprising elastase inhibitors.
In preferred embodiments, the compositions and methods according to the present application utilize elastase inhibitors and/or bacterial culture extracts. The elastase inhibitors can be in purified form or a mixture of bacterial growth products, including crude extracts. The concentration of the elastase inhibitor added may be 0.01% to 90% by weight (wt%), preferably 0.1 wt% to 50 wt%, and more preferably 0.1 wt% to 20 wt%. In other embodiments, the purified elastase inhibitor may be combined with an acceptable carrier, wherein the elastase inhibitor may be present at a concentration of 0.001% to 50% by volume, preferably 0.01% to 20% by volume, more preferably 0.02% to 10% by volume.
The microorganism used according to the present application may be a natural or genetically modified microorganism. For example, a microorganism can be transformed with a specific gene to exhibit a specific characteristic. The microorganism may also be a mutant of a desired strain. As used herein, "mutant" refers to a strain, genetic variation, or subtype of a reference microorganism, wherein the mutant has one or more genetic variations (e.g., a point mutation, a missense mutation, a nonsense mutation, a deletion, a duplication, a frameshift mutation, or a repeat amplification) as compared to the reference microorganism. Methods for making mutants are well known in the art of microbiology. For example, UV mutagenesis and nitrosoguanidine are widely used for this purpose.
In certain embodiments, the microorganism is any bacterium that produces an elastase inhibitor. Elastase inhibitors and/or related bacterial culture extracts can be produced by bacteria including streptomyces mobaraensis. In a preferred embodiment, the elastase inhibitor is produced by streptomyces mobaraensis DSM 40847.
In one embodiment, the method for culturing microorganisms is performed at about 5 ℃ to about 100 ℃, preferably 15 ℃ to 60 ℃, more preferably 25 ℃ to 50 ℃. In another embodiment, the culture may be performed continuously at a constant temperature. In yet another embodiment, the culture may be subjected to a temperature change.
In one embodiment, the apparatus used in the method and culture process is sterile. The culture device, such as a reactor/vessel, may be separate from, but connected to, a sterilization unit, such as an autoclave. The culture device may also have a sterilization unit that sterilizes in situ before starting the inoculation. The air may be sterilized by methods known in the art. For example, ambient air may be passed through at least one filter prior to being introduced into the container. In other embodiments, the medium may be pasteurized, or optionally, not heated at all, wherein the use of low water activity and low pH may be utilized to control bacterial growth.
The biomass content of the culture medium of the bacterial growth broth may be, for example, from 5g/l to 180g/l or more. In one embodiment, the broth medium has a solids content of from 10g/l to 150 g/l.
In one embodiment, the anti-biofilm composition comprises a bacterial culture produced according to the methods of the present application.
Microbial growth byproducts produced by the target microorganism may be retained in the microorganism or secreted into the liquid culture medium. In another embodiment, the method for producing microbial growth byproducts may further include the step of concentrating and purifying the microbial growth byproducts of interest. In yet another embodiment, the liquid culture medium may comprise a compound that stabilizes the activity of the microbial growth by-product.
Preparation of an anti-biofilm article
One elastase inhibitor-based product of the present application is a bacterial growth broth containing only bacteria and/or elastase inhibitors produced by bacteria and/or any residual nutrients. The bacterial growth product can be used directly without extraction or purification. Extraction and purification can be readily accomplished using standard extraction and/or purification methods or techniques, if desired.
In a preferred embodiment, the elastase inhibitor can be extracted from the bacteria by n-hexane to obtain a crude extract. The crude extract can be further processed to form a coating. The coating layer of the crude extract (CR coating) can be prepared by a mixed solution of the crude extract and the polyester. Typically, the crude extract can be added at about 1% to about 50%, about 5% to about 25%, or about 10% by weight content, and polyesters such as poly (epsilon-caprolactone) diol, including PCL-PU80, can be added at about 50% to about 99%, about 5% to about 75%, or about 90% by weight content, and the composition can be dissolved by vigorous stirring in xylene and THF (v: v ═ 1:2) at about 25 ℃. After thorough mixing, the solution may coat the surface. The surface may be dried at about 5 ℃ to about 50 ℃, about 10 ℃ to about 37 ℃, or about 15 ℃ to about 25 ℃ for a longer period of at least 1 hour, 2 hours, 4 hours, 8 hours, 24 hours, 72 hours, 1 week, 2 weeks to remove the solvent. Coatings with different concentrations of crude extract and/or polyester can be prepared following the same procedure.
In harvesting the anti-biofilm composition from the growth vessel, other components may be added when the resulting product is placed in a vessel and/or pipeline (or otherwise transported for use). Additives can be, for example, dyes, pigments, pH adjusters, buffers, salts, adhesion promoting compounds, solvents (e.g., isopropyl alcohol, ethyl alcohol), antimicrobial agents, other microorganisms, and other ingredients specific to the intended use.
In certain embodiments, the anti-biofilm composition may be added to existing compositions conventionally used to coat surfaces. Furthermore, the anti-biofilm composition may be applied to the surface before, simultaneously with or after the application of a composition conventionally used for coating surfaces.
In certain embodiments, the anti-biofilm compositions of the present application include a binder that is primarily responsible for the attachment of the anti-biofilm composition to a target object and/or surface. The binder compound may be selected from, for example, acrylic, alkyd, acrylic, acrylamide, phenolic-alkyd, polyacrylamide, polyurethane, silicone-alkyd, polyester, epoxy, vinyl acetate-ethylene, vinyl-alkyd, inorganic binders (sodium, potassium ethyl silicate, lithium, etc.), organic binders (carbon based), and the like,(Daubert Chemical Company, Inc., Chicago, IL), aliphatic-urethane resins, and oil-modified urethane resins.
In certain embodiments, the anti-biofilm compositions of the present application include pigments or dyes that can provide color to the primer. The pigments or dyes may be natural, synthetic, inorganic or organic. The pigment or dye may be selected from, for example, titanium dioxide, zinc oxide, zinc yellow, yellow dyes, benzidine yellow, chromium oxide green, phthalocyanine blue, ultramarine blue, vermilion, pigment brown 6, red 170, dioxazine violet, carbon black, iron (II) oxide, quartz sand(SiO2) Talc, barite (BaSO)4) Kaolin and limestone (CaCO)3)。
In certain embodiments, one of the solvents for the composition is selected from mineral or organic solvents including, for example, ethanol, butanol, propanol, aliphatic hydrocarbons, cycloaliphatic hydrocarbons, xylene, toluene, ketones, and/or isopropanol.
In certain embodiments, the composition further comprises water as a solvent. The water may be filtered through granular activated carbon, deionized, distilled or treated by reverse osmosis. In addition, pH adjusting agents may be used to increase or decrease pH, thereby (preferably) facilitating dissolution of various components in the anti-biofilm composition. The water-based anti-biofilm composition may be acrylic-based or latex-based. The latex may be derived from natural sources such as, for example, flowering plants (angiosperms), or preferably, the latex is synthetically derived from, for example, polymerized styrene. The acrylic binder for the biofilm-resistant composition may be produced from acrylic resins, which are synthetic thermoplastics.
In certain embodiments, the anti-biofilm composition may be oil-based. Synthetic resins or natural resins may be used in combination with any of the above solvents to produce an oil-based resin. Alkyd resins may be used, for example, in the compositions of the present application. Alkyd resins may be produced using natural oils such as, for example, linseed oil, safflower oil, soybean oil, sunflower oil, tung oil or castor oil.
In one embodiment, the elastase inhibitor-based product may also comprise a buffering agent comprising an organic acid and an amino acid or salts thereof. Suitable buffering agents include, for example, citrate, gluconate, tartrate, malate, acetate, lactate, oxalate, aspartate, malonate, glucoheptonate, pyruvate, galactarate, glucarate, tartronate, glutamate, glycine, lysine, glutamine, methionine, cysteine, arginine, and mixtures thereof. Phosphoric acid and phosphorous acid or their salts may also be used. The use of synthetic buffers is suitable, but it is preferred to use natural buffers, such as the organic and amino acids listed above or their salts.
In another embodiment, a pH adjusting agent may be added to the composition, the pH adjusting agent comprising potassium hydroxide, ammonium hydroxide, potassium carbonate or bicarbonate, hydrochloric acid, nitric acid, sulfuric acid, or a mixture.
The anti-biofilm article may be applied with a composition that facilitates the attachment of the anti-biofilm article to the surface to be treated. The adhesion-promoting substance may be a component of the anti-biofilm article, or the adhesion-promoting substance may be applied simultaneously or sequentially with the anti-biofilm article. Examples of useful adhesion promoters include maleic acid, crotonic acid, fumaric acid, polyesters, polyamides, polyethers, polyacrylates, and polyurethanes.
Other additives that may be used in the anti-biofilm composition include water softeners, chelating agents, preservatives and antioxidants, which are added in amounts effective to achieve their intended function. The identification and use of such additives and the amounts of such additives are well within the skill of the art. Suitable water softeners include linear phosphate esters, styrene-maleic acid copolymers and polyacrylates. Suitable chelating agents include 1, 3-dimethyl-2-imidazolidinone; 1-phenyl-3-isoheptyl-1, 3-propanedione; and 2-hydroxy-5-nonyl acetophenone oxime. Examples of corrosion inhibitors include 2-aminomethylpropanol, diethylethanolamine benzotriazole, and methylbenzotriazole. Antioxidants suitable for use herein include (BHT)2, 6-di-tert-butyl-p-cresol, (BHA)2, 6-di-tert-butyl-p-anisole, Eastman inhibitor oabbm-oxalylbis (benzylidene hydrazide), and Eastman DTBMA 2, 5-di-tert-butylhydroquinone.
Other suitable additives that may be included in formulations according to the present application include materials commonly used in such formulations. The additive may be, for example, a carrier, other microorganism-based composition produced in the same or different equipment, viscosity modifier, preservative, tracer, biocide, desiccant, flow control agent, defoamer, UV stabilizer, antiskinning agent, thickener, emulsifier, lubricant, solubility control agent, chelating agent, and/or stabilizer.
Advantageously, according to the present application, the anti-biofilm article may comprise a broth medium in which the microorganisms are grown. The article can be, for example, a broth culture of at least 0.01% by weight, 0.1% by weight, 1% by weight, 5% by weight, 10% by weight, 25% by weight, 50% by weight, 75% by weight, or 100% by weight. The amount of biomass in the preparation can be, for example, any value from 0% by weight to 100% by weight, including all percentages therebetween.
Optionally, the article may be stored prior to use. Preferably with a short storage time. Thus, the storage time may be less than 60 days, 45 days, 30 days, 20 days, 15 days, 10 days, 7 days, 5 days, 3 days, 2 days, 1 day, or 12 hours. In a preferred embodiment, if living cells are present in the preparation, the preparation is stored at a cooler temperature, e.g., less than 20 ℃,15 ℃, 10 ℃ or 5 ℃.
In certain embodiments, the anti-biofilm compositions of the present application may comprise an antimicrobial agent. The antimicrobial agent may be bactericidal or bacteriostatic. An exemplary antimicrobial agent is vancomycin; however, other contemplated antibacterial agents include beta-lactam antibiotics, daptomycin, fluoroquinolones (e.g., ciprofloxacin), metronidazole, nitrofurantoin, co-sulfamethoxazole, telithromycin, and aminoglycoside antibiotics.
Use of elastase inhibitors in anti-biofilm compositions
In a preferred embodiment, a method is provided for applying an anti-biofilm composition directly onto an existing biofilm or onto a surface that can be contaminated by a biofilm, wherein an elastase inhibitor and/or a bacterial culture comprising an elastase inhibitor is applied onto the biofilm or the surface. The use of the anti-biofilm composition according to the present application may provide various improvements after application to a biofilm, a surface and/or a target object. The stated elements of this application are not an exhaustive review of all applications.
In certain embodiments, the anti-biofilm composition can disrupt the structure of a biofilm, including EPS. The anti-biofilm composition can inhibit biofilm-forming organisms from building biofilm structures, mainly EPS, water channels and pore structures.
The anti-biofilm compositions of the present application can be applied to the surface of various inorganic or organic target objects, for example, metals including steel, aluminum, iron; organic matter including wood, coral, paper, cotton, silk, hair, skin, fur, rubber, or plants; plastic; a mineral comprising gypsum; glass; concrete; gypsum; clay; or stucco. The surface can be used in a variety of industries including medical equipment, aquaculture, fishing, desalination of seawater, water purification, nuclear power plants, and marine and freshwater sailing. The surface may be a pipe, pipeline, needle, pump, propeller, hull, deck, railing, buoy, barge, dock and chain, rope. The composition can be applied to a target subject in a temperature range, an aqueous environment, or other induced stress conditions. The anti-biofilm composition may be added to conventional coated articles such as paints, primers, varnishes, stains and sealants. In addition, the anti-biofilm composition may be applied to the target object before, simultaneously with, or after application of a conventional coated article.
The composition may be applied to a surface or biofilm by spraying using, for example, a spray bottle or a pressurized spray device. The composition may also be applied using a cloth or brush, wherein the composition is rubbed, spread or brushed onto a surface or biofilm. In addition, the composition may be applied to a surface or biofilm by dipping, soaking or immersing the surface into a container having the composition therein.
In certain embodiments, the elastase inhibitor-based composition can inhibit biofilm formation and/or clear mature or immature biofilms of bacteria, while having a non-lethal effect on cells. If bacteriostatic or bactericidal activity is desired, compositions based on elastase inhibitors may be combined with an antibacterial agent. Preferably, the biofilm consists of gram-positive bacteria, but may also be other bacteria including gram-negative bacteria.
The anti-biofilm composition may be added to existing antimicrobial preparations. Furthermore, the anti-biofilm composition may be applied to the target object before, simultaneously with, or after the application of the antimicrobial article.
In certain embodiments, elastase inhibitors can inhibit or clear non-biological membrane-forming organisms. These organisms may include macroscopic organisms that settle on or near a biofilm, including marine organisms such as (for example) shellfish and algae.
In a preferred embodiment, there is provided a method of inhibiting biofilm formation and/or removing an existing biofilm, the method comprising applying an elastase inhibitor to a surface. In certain embodiments, the addition of an elastase inhibitor-based composition to a surface enhances the performance and/or prolongs the life of the surface. Performance may be improved by reducing friction, such as the hull of a ship or the wings of an aircraft. The lifetime may be extended by reducing the frequency of cleaning contaminated surfaces and/or reducing bio-corrosion due to biological sources. In certain embodiments, the anti-biofilm composition comprising an elastase inhibitor extends the life of a surface and/or target object to which the anti-biofilm composition comprising an elastase inhibitor is applied by inhibiting contamination of the surface and/or target object by living organisms or inactive substances. The present application can be used to inhibit the deposition of organisms or sediments. Thus, the present application is able to delay or completely eliminate the necessity of preventative maintenance in connection with the removal of deposits and deposits, as well as the need to replace or repair device components.
Materials and methods
Strains, culture media and antibiotics
The 12 actinomycete strains were purchased from the German Collection of microorganisms and strains (DSMZ, Braunschweig, Germany). The strains to be tested MRSA ATCC 43300, Escherichia coli (E.coli) ATCC 25922 and Staphylococcus aureus ATCC 25923 are purchased from American type culture Collection; marine staphylococcus aureus B04 was a laboratory stock solution isolated from marine biofilms, and bacillus subtilis 168 was obtained from the laboratory stock solution. Soybean meal was purchased from Wugumf of china; soluble starch is available from Affymetrix, usa; magnesium sulfate hydrate was purchased from Riedel-De Haen, Germany; bacterial peptones are available from Oxoid in italy; mueller Hinton Broth (MHB) was purchased from Fluka AG, Switzerland; phosphate Buffered Saline (PBS) was purchased from Thermo Fisher Scientific Inc; LB, glucose, MTT, vancomycin, chloramphenicol, and 1-butanol were purchased from VWR in the United kingdom; and all other chemicals were supplied by Sigma Chemical ltd.
Screening of secondary metabolites and isolation of compounds
Stock cultures of 12 actinomycetes were inoculated into 50ml of AM4, AM5 and AM6 medium with glass beads (to break up globular colonies) and incubated at 22 ℃ and 30 ℃ on a rotary shaker (170 rpm). AM4 medium is prepared from soybean powder (20g/L), bactopeptone (2g/L), glucose (20g/L), soluble starch (5g/L), yeast extract (2g/L), NaCl (4g/L), K2HPO4(0.5g/L)、MgSO4·7H2O (0.5g/L) and CaCO3(2g/L) composition, pH 7.8. The AM5 medium consisted of malt extract (10g/L), yeast extract (4g/L) and glucose (4g/L), with a pH of 6 to 7. AM6 medium was composed of soluble starch (20g/L), glucose (10g/L), bacterial peptone (5g/L), yeast extract (5g/L) and CaCO3(5g/L) and pH of 7.2 to 7.5. Extracting the culture broth with 1-butanol on days 3, 5 and 7; the crude extract was dissolved in DMSO for storage and bioassays. The pure compound was separated by reverse phase high performance liquid chromatography (HPLC, Waters 2695, Milford, MA, USA) using a Semi-Prep C18 column (10X 250mm) eluting the Semi-Prep C18 column at a flow rate of 3ml/min with a 55-min gradient of 5% to 95% aqueous Acetonitrile (ACN) containing 0.05% Tetrahydrofuran (TFA).
Susceptibility testing of antimicrobial agents
The Minimum Inhibitory Concentration (MIC) and the Minimum Bactericidal Concentration (MBC) were determined using MRSA ATCC 43300 and escherichia coli ATCC 25922 according to the clinical and laboratory standards institute guidelines CLSI M100 (2018). Will be about 105An overnight culture of CFU/ml bacterial strain was inoculated into MHB and treated with a range of concentrations of elastase inhibitor (or crude extract and antibiotics). After 24 hours of incubation, the lowest concentration showing no visible bacterial growth was recorded as MIC. After MIC determination, 1ml of the bacterial suspension from each well was diluted appropriately with broth mediumAnd coated on Mueller Hinton Agar (MHA) plates to determine MBC. MBC was defined as the lowest concentration at which the antimicrobial caused > 99.9% cell reduction. Each assay was performed multiple times and repeated 3 times.
Anti-biofilm assay
The Minimum Biofilm Inhibitory Concentration (MBIC) and minimum biofilm clearance concentration (MBEC) were determined as described previously.
MBIC assay
The overnight culture of the test strain was diluted to about 10 with LB and 0.5% glucose7And treated with various concentrations of elastase inhibitors in 96-well cell culture plates. The cells were then incubated at 37 ℃ for 24 hours and washed twice with 1 XPBS to remove non-adherent planktonic cells. After washing, an MTT staining assay was performed to assay living cells in biofilms, as MTT can react with activated succinate dehydrogenase in living cell mitochondria to form blue-violet formazan, which can be read at 570nm after being dissolved in DMSO.
MBEC assay
The ability of elastase inhibitors to clear preformed mature biofilms was tested by MBEC assay. An overnight culture of the test strain was cultured in a 96-well cell culture plate for 24 hours, as in the MBIC assay, but without the addition of elastase inhibitors to form a biofilm. The formed biofilm was then washed twice with 1 × PBS and treated with a range of concentrations of elastase inhibitor (diluted with LB and 0.5% glucose) and incubated at 37 ℃ for a further 24 hours. After incubation, each well was washed twice with 1X PBS and subjected to MTT assay to determine viable cells in the remaining biofilm.
Concentration response Curve study
MRSA ATCC 43300 was used for concentration response curve studies. Will be 4X 105CFU/ml of exponentially growing MRSA was inoculated into MHB with different concentrations of elastase inhibitor and vancomycin in 15ml Falcon tubes. The tube was incubated at 37 ℃ for 24 hours on a rotary shaker; 1ml of culture broth in each tube was diluted with MHB, and 1ml of the diluted bacteria was plated on MHA plates for CFU counting. Each concentration was applied to two platesOn plate, and repeat 3 experiments.
Yield monitoring and comparison of extraction efficiency
Stock cultures of Streptococcus mobaraensis DSM40847 were incubated in AM4 medium as described in Secondary metabolites screening and compound isolation. 1ml of the culture broth was removed every 12 hours, and the elastase inhibitors were extracted with 1ml of 1-butanol, Ethyl Acetate (EA) and hexane, respectively. The solvent was then removed by evaporation. The crude extract was dissolved in methanol and quantified by HPLC analysis using a Phenomenex Luna C18 column. Three experiments were performed. The peak of the elastase inhibitor was identified by retention time and its concentration was calculated based on the established standard curve. By using dimethyl sulfoxide-d on a Bruker AV500 spectrometer (500MHz)6Nuclear Magnetic Resonance (NMR) analysis of the NMR spectra of the labelled 1H, 1H-1H-COSY, 1H-13C-HSQC and 1H-13C-HMBC (1H-NMR DMSO-d)6:δH＝2.50ppm；DMSO-d6δ C ═ 39.50ppm) elucidates the structure of elastase inhibitors.
Coating formulations
4L of culture broth of Streptococcus mobaraensis DSM40847 was extracted by n-hexane (incubated as described for secondary metabolite screening and compound isolation) to obtain a crude extract with high elastase inhibitor content. The crude extract coating (CR coating) was prepared by mixing the solutions. Typically, for a 10% by weight CR coating, PCL-PU80(0.90g, 90% by weight) and the crude extract (0.10g, 10% by weight) were dissolved in xylene and THF (v: v ═ 1:2) by vigorous stirring at 25 ℃. After mixing well, the solution was coated onto a glass slide and dried at room temperature for one week to remove the solvent. Coatings with different concentrations of crude extract were prepared following the same procedure.
Marine natural biofilm analysis and release rate determination
As previously described, in 2019, 4 months, at Yung Shue O Fish Farm in hong kong, CR-coated slides were exposed to moist water (22 ° 20'16.7 "N, 114 ° 16' 08.0" E) for 2 weeks, 3 weeks, and 4 weeks. Transporting the slides back to true in a cooler with in situ seawaterLaboratory and washed twice with autoclaved and 0.22 μm filtered seawater to remove loose particles and cells; then, pass through FilmtracerTMLIVE/DEADTMThe Biofilm visualization Kit stained the Biofilm and sent for Confocal Laser Scanning Microscopy (CLSM) observation. Meanwhile, the release rate of the elastase inhibitor was determined by measuring the concentration using High Performance Liquid Chromatography (HPLC) under static conditions. The plates coated with the crude extract and polymer were dipped into an assay vessel containing 100ml of sterile artificial seawater. After 24 hours of immersion, 10ml of seawater was taken and extracted with the same volume of dichloromethane, and then dichloromethane was removed under nitrogen. After drying, the extract was resuspended in 100ml of methanol and analyzed by HPLC as described above. The release rate was measured weekly for 4 weeks and multiple measurements were made for each concentration.
CLSM Observation Using biofilm cell and matrix staining
Biofilms were grown on glass coverslips as described for the MBIC and MBEC assays. The treated biofilm was then washed twice with 1 XPBS and with Filmtracer at room temperatureTM 1-43Green Biofilm Cell Stain and FilmtracerTM Ruby Biofilm Matrix Stain was stained in the dark for 30 minutes. Cells and matrix in the biofilm were observed at 488nm using a Leica Sp8 confocal microscope.
DNA extraction, 16S rRNA gene sequencing and analysis
Biofilm samples on the surface of the coated slides were collected with autoclaved cotton and stored in DNA storage buffer (10mM Tris-HCl; 0.5mM EDTA, pH 8.0) at-80 ℃. Prior to extraction, the sample was vortexed several times to release the microbial cells into the DNA storage buffer. All samples were then centrifuged at 10,000rpm for 1 minute and the supernatant discarded. After sequential treatment with 10mg/mL lysozyme and 20mg/mL proteinase K, DNA was extracted from the microbial cells to be treated using a microbial genomic DNA extraction kit (Tiangen Biotech, Beijing, China) according to the manufacturer's protocol.
The quality of the DNA samples was controlled using NanoDrop (test for DNA purity, OD260/OD280) and agarose gel electrophoresis (test for DNA degradation and potential contamination). The hypervariable V3-V4 region of the prokaryotic 16S rRNA gene (forward primer: 5'-CCTAYGGGRBGCASCAG-3' (SEQ ID NO: 1); reverse primer: 5'-GGACTACNNGGGTATCTAAT-3' (SEQ ID NO:2)) was used to amplify DNA from biological membranes by Polymerase Chain Reaction (PCR). The PCR products were purified prior to library construction and sequenced in NovaSeq 6000System from Novogene (Beijing, China). The reads were 250bp in length and each pair of reads had a 50bp overlap. Quality control of paired end readings was performed using NGS QC Toolkit (version 2.0). The 16S rRNA gene amplification data were analyzed using the software package QIIME2 and then pooled using Q2_ manifest _ maker in QIIME 2. Low quality reads and chimeras were removed using dada2 command in QIIME 2. To normalize for uneven sequencing depth, 500,000 filtered reads of each sample were picked. The Operational Taxonomic Units (OTUs) were reclassified from the pooled reads with 97% sequence similarity using a classifier trained by the naive bayes method. The canonical sequence was then recovered using the feature classifier classification-sklern script in QIIME 2. Alpha-diversity analysis (observation of OTU and Shannon diversity) was performed using the script "QIIME diversity alpha" in QIIME 2. The beta-diversity analysis based on the Bray-Curtis distance was performed by clustering analysis in the software PAST (version 3.0). Furthermore, the classification structure was plotted in Excel (Office 365MSO 64 bits) based on relative abundance.
Statistical analysis
Statistical analysis of all data was performed using GraphPad Prism 8.0.2 software. The biofilm composition on CR-coated slides was compared to controls using unpaired t-tests.
All patents, patent applications, provisional applications, and publications referred to or cited herein are incorporated by reference in their entirety, including all figures and tables, to the extent they are consistent with the explicit teachings of this specification.
The following is an example illustrating a process for carrying out the present application. These examples should not be construed as limiting. Unless otherwise indicated, all percentages are weight percentages and all solvent mixture proportions are volume percentages.
Example 1 metabolite production by Actinomycetes
The inevitable increase in resistance of microorganisms to antibiotics poses a serious threat to modern medicine, placing tremendous pressure on new drug development. However, drug development suffers from rising costs. The world-wide antibiotic market is still dominated by antibiotics discovered decades ago. Researchers have begun looking for ways to reuse old drugs. In our bioassay-directed drug development, four isolated antibacterial compounds were identified from various actinomycete bacteria, and the elastase inhibitor in them was the only compound active for adherent cells. Elastase inhibitors consisting of SatoshiFor the first time, elastase inhibitors were found to be novel human granulocyte elastase inhibitors produced by Streptomyces nobolithoensis (KM 2753 strain). Elastase inhibitors inhibit human granulocyte elastase, but have little activity against pancreatic elastase, chymotrypsin and trypsin. The low toxicity and specific activity make it an ideal candidate for the treatment of acute arthritis, various inflammations, emphysema and pancreatitis. However, there is no known report of the use of elastase inhibitors as anti-biofilm agents.
The biological activity of the secondary metabolites produced by the 12 actinomycete strains under different culture conditions was evaluated against the gram-positive bacteria MRSA and gram-negative escherichia coli (table 1). Four major compounds were found to have antibacterial activity against MRSA. Of these strains, only the compound produced by Streptococcus mobaraensis DSM40847 has anti-biofilm activity. The compound was subsequently purified by High Performance Liquid Chromatography (HPLC) and structurally characterized by ultra high performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) and Nuclear Magnetic Resonance (NMR) analysis (fig. 1C and 1A). The molecular weight was 392.3 as shown by UPLC-MS/MS, and the structural elucidation by NMR analysis revealed that the biologically active fragment isolated from crude extract of streptococcus mobaraensis (fig. 1A) was a known compound, an elastase inhibitor (fig. 7). The production of elastase inhibitors (fig. 1D) in strain DSM40847 was then explored. As shown in FIG. 1D, the yield of elastase inhibitor increased rapidly 12 hours after inoculation and peaked the next day, with a yield of 332 mg/L. The yield of elastase inhibitor remained constant with slight variations (289. + -.83 mg/L) within the indicated range.
Table 1 shows the results of bioassay of crude extracts from 12 actinomycete strains against methicillin-resistant Staphylococcus aureus and Escherichia coli.
Example 2 Elastase inhibitor Activity
Due to the presence of EPS and various tolerance mechanisms, biofilms are often resistant to traditional antibiotic treatment. Vancomycin has been the treatment of choice for the treatment of most MRSA infections in conventional therapy. To determine the activity of elastase inhibitors on biofilms and planktonic cells, a comparison was made with vancomycin and the Minimum Inhibitory Concentration (MIC), the Minimum Bactericidal Concentration (MBC), the Minimum Biofilm Inhibitory Concentration (MBIC) and the minimum biofilm clearance concentration (MBEC) were determined as MRSA.
The effect of elastase inhibitors on planktonic cells was determined based on the results of MIC, MBC and concentration-response assays. According to the results, planktonic cells were sensitive to both elastase inhibitors and vancomycin, with the same MIC, ranging from 0.5. mu.g/ml to 2. mu.g/ml. MBC was determined and the results indicated that elastase inhibitors showed bacteriostatic activity, with MBC greater than 100. mu.g/ml, whereas vancomycin showed strong bactericidal activity, with MBC between 20. mu.g/ml and 50. mu.g/ml (FIG. 2D). The effect of the agent on planktonic cells is further revealed by the concentration-response curve in fig. 2C. In the range of 0. mu.g/ml to 50. mu.g/ml, the activity of vancomycin is concentration-dependent, which results in a dramatic decrease in cell density with increasing concentration. In contrast, there was no significant difference in cell density as the compound concentration of the elastase inhibitor varied from 0 μ g/ml to 100 μ g/ml.
MBIC and MBEC were tested to explore the effect of elastase inhibitors on biofilm cells. An increase in resistance was observed in the adherent cells, while elastase inhibitors showed optimal activity against the adherent cells. Both elastase inhibitor and vancomycin inhibited biofilm formation with MBIC from 1.25 μ g/ml to 2.5 μ g/ml (FIG. 2A). Significant differences were observed between the two agents in the MBEC assay. Cells in the developing mature biofilm are resistant to antibiotics. Vancomycin greater than 10 μ g/ml was required to reduce cell density by 50%. Interestingly, the activity of elastase inhibitors was not affected by biofilm resistance, and elastase inhibitors at a concentration of 1.25 μ g/ml were able to clear 50% of the mature biofilm within 24 hours (fig. 2B).
Consistent with previous studies, bacteria developed resistance to vancomycin in pre-formed mature biofilms, whereas no increase in resistance was observed in elastase inhibitor treated adherent cells.
Example 3 Effect of Elastase inhibitors on biofilm architecture
To investigate the effect of elastase inhibitors on biofilm structure, biofilm cell and matrix staining was used for observation under a Confocal Laser Scanning Microscope (CLSM). In the biofilm inhibition assay, untreated biofilms (fig. 3A) showed a clear shape with high density of organized cells and matrix. However, the biofilm treated with elastase inhibitor (fig. 3B) showed a significant decrease in cell and matrix density, and both cells and matrix were randomly dispersed. In the biofilm removal assay, cells in the pre-formed biofilm became dispersed and released into the culture medium after treatment with elastase inhibitors (fig. 3D). Confocal images demonstrate that the distribution pattern of cells following elastase inhibitor treatment is significantly altered. Untreated biofilm cells (fig. 3C) were distributed as clusters of cell aggregates with rough edges; whereas the elastase inhibitor treated biofilm cells were distributed as narrow stripes with smooth edges (fig. 3D). Similarly, high density organized biofilm matrix becomes sparse and dispersed after treatment with elastase inhibitors. According to the quantitative analysis, both cells and matrix were significantly reduced after elastase inhibitor treatment. The cell and matrix densities of the biofilms treated with elastase inhibitors were reduced by about 80% and 35%, respectively, in the biofilm inhibition assay compared to untreated biofilms (fig. 3E). For the biofilm clearance assay, the reduction in cells and matrix was over 50% and 70%, respectively (fig. 3F).
Elastase inhibitors destroy the biofilm matrix but have no lethal effect on the cells. As mentioned in many studies, the resistance of biofilm matrix to biofilm cells is crucial. The non-lethal effect of elastase inhibitors and their activity in biofilm matrix disruption further confirms that elastase inhibitors are biofilm-targeting agents and that the use of elastase inhibitors poses less risk for developing antimicrobial resistance, as the emergence of antimicrobial resistance is only associated with the use of bactericidal antibiotics. Furthermore, the anti-biofilm activity of elastase inhibitors against MRSA is also noteworthy. The continued emergence of multidrug resistance in bacteria such as MRSA, VRSA and VRE puts pressure on modern medicine. The effective clearance efficiency of elastase inhibitors suggests the potential for their use in inducing the dissociation of biofilms, and elastase inhibitors have great potential in the treatment of biofilm-associated diseases of MRSA in combination with other antibiotics.
Example 4-Elastase inhibitors illustrating the preference for biofilms of gram-positive bacteria
A total of 6 strains (fig. 4C) were selected to test the elastase inhibitor activity on biofilms, including staphylococcus aureus ATCC 25923, staphylococcus aureus B04, MRSA ATCC 43300, bacillus subtilis 168, escherichia coli ATCC 25922, and pseudomonas aeruginosa PAO 1. The same pattern was observed in gram-positive strains for both MBIC and MBEC; whereas gram-negative strains show a completely different trend. The MBIC of elastase inhibitors of all gram positive bacteria was in the range of 1.25. mu.g/ml to 2.5. mu.g/ml (1.25. mu.g/ml inhibited biofilm by at least 80%) except that the MBIC of Bacillus subtilis 168 was 2.5. mu.g/ml to 5. mu.g/ml (FIG. 4A). Changes in MBEC were observed; staphylococcus aureus B04 is the most resistant strain to elastase inhibitors, with MBEC 2.5. mu.g/ml to 5. mu.g/ml; the most sensitive strain was Staphylococcus aureus ATCC 25923, which has the lowest MBEC, ranging from 0.625 μ g/ml to 1.25 μ g/ml; and MBEC were 1.25. mu.g/ml to 2.5. mu.g/ml for MRSA and Bacillus subtilis (FIG. 4B). No activity was observed when gram negative bacteria (e.coli and pseudomonas aeruginosa) were treated with elastase inhibitors. The development of biofilm formation was not affected by elastase inhibitors, even at concentrations of 100 μ g/ml, and the viability of biofilm cells was constant with increasing elastase inhibitor concentration in both the MBIC and MBEC assays. Overall, the above results indicate that elastase inhibitors may have specific activity against gram-positive bacteria.
Example 5 inhibition of Multi-species biofilm formation and attachment of Large biofouling organisms by Elastase inhibitor-based coatings
Considering that elastase inhibitors have high anti-biofilm efficiency, an anti-biofilm coating based on elastase inhibitors was prepared and immersed in a fish farm to evaluate the efficiency to natural marine biofilms (fig. 5D). It is noted that in the present study, the crude extract of Streptomyces mobaraensis DSM40847 containing a very high concentration of elastase inhibitor (equal to 336.64mg/L in n-hexane, FIG. 8) was used instead of pure elastase inhibitor. The extract was mixed with polyurethane (polymer) based on poly-epsilon-caprolactone and coated directly on the surface of the glass slide. The concentration of the coating was calculated based on the weight percentage of the crude extract in the total coating (polymer and crude extract). Thus, other compounds in the fractionated extract may have an effect on our in situ test results. However, their effect should be negligible, since no active effect of minor compounds was detected in the crude extract (fig. 9).
The release rate of the elastase inhibitor from the coating was found to be dependent on both time and concentration (fig. 5C). Overall, the elastase inhibitor is released from the coating at a relatively low rate throughout the period; the higher the concentration, the faster the elastase inhibitor is released into the artificial seawater. For a concentration of 10 wt%, the highest release rate occurring in the second week was about 5 μ g days-1cm-2(ii) a For other concentrations, the maximum release rate in the first week was about 4 μ g days-1cm-2. The release rate decreases with time and depends on the total amount of elastase inhibitor remaining in the coating. After four weeks of immersion, the release rate was reduced to about 1 μ g day for concentrations of 10 wt% and 5 wt%-1cm-2And the release rate was reduced to 0.5. mu.g day for concentrations of 1.5 wt% and 2.5 wt%-1cm-2。
The performance of the anti-biofilm coatings was analyzed weekly from the second to the fourth week by direct observation and CLSM observation. According to quantitative analysis of CLSM images, the mean biofilm biomass on slides without elastase inhibitor was 116.44 μm in the second week3μm-2259.95 μm in the third week3μm-2(ii) a While the average biomass of the biofilm, as measured on 5 wt% and 10 wt% coated slides, was less than 0.1 μm in the second week3μm-2And less than 120 μm in the third week3μm-2. For low concentrations (1.5 wt.% and 2.5 wt.%) of coating, there was no significant difference in average biomass at the second week from the uncoated slides (61.97 μm, respectively)3μm-2And 84.73 μm3μm-2) However, at week three, the biomass was significantly lower than that of the control (259.95 μm)3μm-2) Average biomass of about 125 μm3μm-2And 145 μm3μm-2(FIG. 5B). At the fourth week, slides coated with low concentrations of elastase inhibitor (1.5 wt%, 2.5 wt%, and control) were contaminated with large marine organisms, while slides coated with high concentrations of elastase inhibitor showed anti-biofouling activity and had little larvae deposited except in a small area near the edge due to the edge effect commonly found on test plates (fig. 5A). In general, the anti-biofilm coating based on elastase inhibitors inhibited the formation of multi-species bacterial biofilms during the first two weeks. However, after four weeks of immersion, large biological contaminating organisms eventually covered the slides coated with low concentrations of elastase inhibitor, probably due to a reduced release of elastase inhibitor after three weeks.
In addition to biofilm-related infections, the formation of marine biofilms on various submerged surfaces such as ship structures and offshore infrastructure is also a serious problem. Mixed population biofilms have more complex structures within the biofilm due to increased inter-species communication than single population biofilms; thus, mixed species biofilms are generally significantly more resistant to antimicrobial treatment (30). The elastase inhibitor-based coatings of the present application significantly inhibit multi-species biofilm formation. Notably, only minute amounts of elastase inhibitors are released into the surrounding environment during processing, thereby limiting indirect effects on unintended organisms.
Example 6 modification of microbial communities of Natural Marine biofilms by Elastase inhibitors
Since few biofilms were formed at the end of the second week, but macroscopic contaminating organisms overgrown at the end of the fourth week, only three week old biofilms formed on a 10 wt% coating and those on a control slide (coated with only poly epsilon-caprolactone based polyurethane) were selected for 16S amplicon analysis to determine the change in biofilm microbial communities triggered by elastase inhibitors. A total of 3,000,000 16S rRNA gene sequences (500,000 per sample) were assigned to 31 phyla (Proteobacteria were assigned to the species level). The microbial composition of the biofilm differed between the 10 wt% coating and the control slide as confirmed by the α -diversity and β -diversity analyses (fig. 10). In the Bray-Curtis dissimilarity (. beta. -diversity) dendrogram (FIG. 6A), the control and treatment groups were divided into two groups based on the difference in microbial abundance between samples; the observed OTU and Shannon diversity of the treated biofilms was significantly lower than that of the control group (figure 6B), indicating that both species abundance and diversity were reduced in the treated biofilms.
It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims. Furthermore, any elements or limitations of any invention or embodiment thereof disclosed herein can be combined with any and/or all other elements or limitations (alone or in any combination) or any other invention or embodiment thereof disclosed herein, and all such combinations are considered to be within the scope of this application, without limitation.
Sequence listing
<110> hong Kong university of science and technology
China Ocean Mineral Resources Research and Development Association
<120> Elastase inhibitors, bacteriostatic agents with anti-biofilm activity effective against both single-and multi-species biofilms
<130> HKUS.148X
<150> 62/890786
<151> 2019-08-23
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<170> PatentIn version 3.5
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<213> Streptomyces sulfurochromogenes (Streptomyces Sulphureus)
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<213> Streptomyces sulfurochromogenes (Streptomyces Sulphureus)
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