Patent Application
20180125066
The instant application contains a Sequence Listing which has been submitted in ASCII format via EFS-Web and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Jan. 15, 2018, is named PRN-06-CIP_Sequence_Listing_ST25.txt and is 2,746 bytes in size.
In a process referred to as quorum sensing, microorganisms, such as bacteria, communicate using chemical signaling molecules called autoinducers. Quorum sensing involves the production, release, and population-wide detection of extracellular signal molecules called autoinducers1-8. By monitoring increases and decreases in autoinducer concentration, quorum-sensing bacteria track changes in cell-population density and synchronously switch into and out of collective group behaviors. Quorum sensing allows bacteria to collectively carry out tasks that would be unsuccessful if carried out by an individual bacterium acting alone.
Both Gram-positive and Gram-negative infectious bacteria, which include human, animal, plant, and marine pathogens, use quorum sensing to control virulence. Quorum sensing also controls biofilm and in some cases streamer formation. Biofilms are communities of bacterial cells adhered to surfaces and encased in a self-produced matrix of extracellular polymeric substances. In most environments, bacteria are found predominantly in biofilms. These biofilms are also widespread in industrial systems and are associated with increased risk of infection when found in clinical environments and in indwelling medical devices. These bacterial biofilm communities can cause chronic infections in humans by colonizing, for example, in medical implants, heart valves, or lungs.
In settings involving fluid flow across the biofilm, as in the environment for example in rivers or in industrial and medical systems that are subject to flow, filamentous biofilms, called streamers, can be formed. These streamers can have a dramatic effect on the biofilm environment. In rivers, for example, the biofilm streamers can increase transient storage and cycling of nutrients and can enhance the retention of suspended particles. In industrial and medical settings, the biofilm streamers have been associated with increased issues associated with clogging and pressure drops.
Bacterial infections are treated with bactericidal or bacteriostatic molecules that impede at least five major processes: cell wall formation, DNA replication, transcription, translation or tetrahydrofolic acid synthesis. Existing methods for treating bacterial infection unfortunately exacerbate the growing antibiotic resistance problem because they inherently select for growth of bacteria that can resist the drug.
For example, Staphylococcus aureus is a human pathogen notorious for causing hospital-acquired infections as well as fatal infections that occur outside of health care settings. S. aureus is responsible for multiple fatal diseases including bacteremia, toxic shock syndrome, and medical device-related infections. Many strains of S. aureus are multi-drug resistant (i.e. Methicillin-resistant Staphylococcus aureus (MRSA))24,25. In this context, the quorum-sensing system plays a central regulatory role in S. aureus pathogenicity and biofilm dynamics1,8.
S. aureus infections that are associated with abiotic materials, such as intravenous catheters and implants, are of primary concern as S. aureus readily colonizes such medical devices, forming biofilms, biofilm streamers and initiates virulence factor production under these conditions.
In fact, methicillin-resistant S. aureus (MRSA) is a major concern due to its potent virulence coupled with resistance to many antibiotics. MRSA is the most widespread cause of hospital-associated infections in the United States and Europe with a high mortality rate. S. aureus and MRSA cause a variety of infections ranging from minor skin infections to serious illnesses such as infections of indwelling medical devices, osteomyelitis, endocarditis, sepsis, and toxic shock syndrome. S. aureus is just one example of a microorganism that uses quorum-sensing-mediated communication to control virulence factor production and to regulate biofilm formation.
Administration of compounds in general can be difficult, due to lack of or inefficient compound transport to the site of infection, compound insolubility, compound instability, complex topography (i.e. grooves/crevices at the site of infection), and resistance to chemical perturbations18-21. These issues complicate therapies, often necessitating delivery of large and frequent doses of the compounds, which in turn, can cause complications such as cytoxicity or off target effects.
Thus, what is needed are methods of modulating quorum sensing to combat bacterial infections which can be effectively delivered without causing adverse side effects. Preferably, these methods will demonstrate improved efficacy and safety over conventional methods.
This summary is provided to introduce a selection of concepts in a simplified form that are further described below in the Detailed Description. This summary is not intended to identify key features or essential features of the claimed subject matter, nor is it intended to be used as an aid in determining the scope of the claimed subject matter.
The present invention targets quorum sensing (“QS”) systems utilized by microorganisms, which are crucial in microorganism infection and pathogenicity. This technology involves conjugation of QS modulating molecules (either QS antagonists or agonists14-17) to a surface to influence and/or control QS-controlled phenotypes, wherein the attached molecule retains the ability to influence bacterial behaviors.
More specifically, presented herein are methods of attaching compounds to a surface to interact with receptors involved in QS, and the ability of the modified surface to resist degradation, e.g., from long-term storage, repeated infections, host plasma components and/or flow-generated stresses. The techniques presented herein can be used to make colonization-resistant materials against S. aureus and other pathogens and promote beneficial behaviors of bacteria on surfaces. By tethering quorum modulating molecules to surfaces, site-specific targeting (e.g., at the site of the medical device) can be achieved for a wide range of applications to combat bacterial infections.
Preferred examples of altered QS phenotypes (also referred to as traits) include, but are not limited to, significant reductions in biofilm formation, biofilm streamer formation, and virulence factor production. This technology can be immediately applied to many current and urgent issues in healthcare settings, such as the accidental introduction of pathogens into patients during medical procedures and the entry of bacteria at wound sites. This technology can be used to modify existing (e.g., medical, food processing, agricultural, etc.) devices, create new devices, and can also be applied as a direct treatment of bacterial infections for patients. Beyond medicine, this technology can also be applied to other fields including, but not limited to, industrial and engineering processes, food processing, cooling towers, and mining. Additional applications include prevention of biofilm formation to reduce biofouling in industry, to halt recurrent infections in medicine, and to suppress virulence factor production to limit pathogen infectivity9-11.
Additionally, the techniques presented herein may be used to enhance QS-controlled behaviors in beneficial bacteria. For example, biofilm formation from beneficial bacterial is used in wastewater treatment and food processing, and, in the context of the microbiome, such beneficial/commensal bacterial are thought to fend off invading pathogens by relying on QS-controlled traits12,13.
Thus, the present invention relates to a method of modulating QS, biofilm production, biofilm streamer production, and/or virulence factor production by a microorganism using: (1) an antagonist to decrease QS, or (2) an agonist to increase QS, wherein the QS modulator is attached to a surface through a linker. A microorganism that is exposed to the surface will exhibit altered biofilm production, biofilm streamer production, and/or virulence factor production. In some embodiments, inhibiting QS will lead to a decrease in biofilm production, biofilm streamer production, and/or virulence factor production. In other embodiments, agonizing QS will lead to a decrease in biofilm production, biofilm streamer production, and/or virulence factor production.
For example, a QS modulator molecule attached to a surface can be used to promote or inhibit the pathogenic behaviors of the microorganism on a surface, including, but not limited to, a medical device and/or at any wound site in patients. By conjugating a QS modulating molecule to a surface, the surface will then promote or inhibit QS, in turn, leading to an alteration in biofilm formation, biofilm streamer formation, and/or virulence factor production.
Alternatively, a QS modulator molecule attached to a surface can be used to promote beneficial behaviors of the microorganism on a surface, including, but not limited to, in food processing, engineering or industrial settings. By conjugating a QS modulating molecule to a surface, the surface will then control QS regulated beneficial phenotypes, including, but not limited to, enzyme or metabolite production, such as enzymes that can degrade plastics and petroleum products, enzymes that help digestion in humans, and metabolites that can be consumed by animals or humans.
In specific microorganisms, a QS agonist can actually repress biofilm formation and/or virulence factor expression. These microorganisms are virulent at low cell density and in response to QS autoinducers, can escape the host cell defenses. For example, Vibrio cholerae dissociates from the host's epithelial cells at high cell densities to become extremely contagious. In this situation, a QS agonist attached to a surface, rather than a QS antagonist, could be used to inhibit biofilm formation and thus repress virulence. Examples of such microorganisms include, but are not limited to S. aureus, Vibrio cholerae, Vibrio parahaemolyticus, Vibrio vulnificus, and Vibrio harveyi.
In other specific microorganisms, a QS antagonist can repress biofilm formation and/or virulence factor expression. These microorganisms are virulent at high cell density, and in response to QS autoinducers, can damage the host cells. In this situation, a QS antagonist attached to a surface, rather than a QS agonist, could be used to inhibit biofilm formation and/or repress virulence. Examples of such microorganisms include, but are not limited to Pseudomonas aeruginosa and Enterococcus faecalis.
As used herein, the QS autoinducers, a biofilm, a biofilm streamer, and/or a virulence factor are produced or formed by a microorganism(s). In preferred embodiments, the microorganism is selected from the following groups: bacteria, archaea, protozoa, fungi, and/or algae. In further embodiments, the bacteria, archaea, protozoa, fungi, and/or algae are pathogenic to humans, animals and/or plants. Alternatively, the bacteria, archaea, protozoa, fungi, and/or algae are beneficial to humans, animals and/or plants. In further embodiments the bacteria, archaea, protozoa, fungi, or algae are common to industrial settings, including, but not limited to, industrial fluid handling processes, medical processes, agricultural processes, and/or machinery. In further embodiments, the bacteria, archaea, protozoa, fungi, or algae are common to an apparatus and/or process that involves fluid flow.
In still further embodiments, the bacteria are selected from the following genera: Abiotrophia, Achromobacter, Acidaminococcus, Acidovorax, Acinetobacter, Actinobacillus, Actinobaculum, Actinomadura, Actinomyces, Aerococcus, Aeromonas, Afipia, Agrobacterium, Alcaligenes, Alloiococcus, Alteromonas, Amycolata, Amycolatopsis, Anabaena, Anabaenopsis, Anaerobospirillum, Anaerorhabdus, Aphanizomenon, Arachnia, Arcanobacterium, Arcobacter, Arthrobacter, Atopobium, Aureobacterium, Bacillus, Bacteroides, Balneatrix, Bartonella, Bergeyella, Bifidobacterium, Bilophila, Bordetella, Borrelia, Brachyspira, Branhamella, Brevibacillus, Brevibacterium, Brevundimonas, Brucella, Burkholderia, Buttiauxella, Butyrivibrio, Calymmatobacterium, Camesiphon, Campylobacter, Capnocytophaga, Capnylophaga, Cardiobacterium, Catonella, Cedecea, Cellulomonas, Centipeda, Chlamydia, Chlamydophila, Chromobacterium, Chryseomonas, Chyseobacterium, Citrobacter, Clostridium, Collinsella, Comamonas, Corynebacterium, Coxiella, Cryptobacterium, Cyanobacteria, Cylindrospermopsis, Delftia, Dermabacter, Dermatophilus, Desulfomonas, Desulfovibrio, Dialister, Dichelobacter, Dolosicoccus, Dolosigranulum, Edwardsiella, Eggerthella, Ehrlichia, Eikenella, Empedobacter, Enterobacter, Enterococcus, Erwinia, Erysipelothrix, Escherichia, Eubacterium, Ewingella, Exiguobacterium, Facklamia, Filifactor, Flavimonas, Flavobacterium, Francisella, Fusobacterium, Gardnerella, Gemella, Globicatella, Gloeobacter, Gordona, Haemophilus, Hafnia, Hapalosiphon, Helicobacter, Helococcus, Hemophilus, Holdemania, Ignavigranum, Johnsonella, Kingella, Klebsiella, Kocuria, Koserella, Kurthia, Kytococcus, Lactobacillus, Lactococcus, Lautropia, Leclercia, Legionella, Leminorella, Leptospira, Leptospirae, Leptotrichia, Leuconostoc, Listeria, Listonella, Lyngbya, Megasphaera, Methylobacterium, Microbacterium, Micrococcus, Microcystis, Mitsuokella, Mobiluncus, Moellerella, Moraxella, Morganella, Mycobacterium, Mycoplasma, Myroides, Neisseria, Nocardia, Nocardiopsis, Nodularia, Nostoc, Ochrobactrum, Oeskovia, Oligella, Orientia, Paenibacillus, Pantoea, Parachlamydia, Pasteurella, Pediococcus, Peptococcus, Peptostreptococcus, Phormidium, Photobacterium, Photorhabdus, Phyllobacterium, Phytoplasma, Planktothrix, Plesiomonas, Porphyromonas, Prevotella, Propionibacterium, Proteus, Providencia, Pseudoanabaena, Pseudomonas, Pseudonocardia, Pseudoramibacter, Psychrobacter, Rahnella, Ralstonia, Rhodococcus, Rickettsia, Rochalimaea, Roseomonas, Rothia, Ruminococcus, Salmonella, Schizothrix, Selenomonas, Serpulina, Serratia, Shewenella, Shigella, Simkania, Slackia, Sphaerotilus, Sphingobacterium, Sphingomonas, Spirillum, Spiroplasma, Spirulina, Staphylococcus, Stenotrophomonas, Stomatococcus, Streptobacillus, Streptococcus, Streptomyces, Succinivibrio, Sutterella, Suttonella, Tatumella, Tissierella, Trabulsiella, Treponema, Trichodesmium, Tropheryma, Tsakamurella, Turicella, Umezakia, Ureaplasma, Vagococcus, Veillonella, Vibrio, Weeksella, Wolinella, Xanthomonas, Xenorhabdus, Yersinia, and Yokenella.
In still further embodiments the bacteria are selected from the following species: Acinetobacter baumannii, Actinobacillus actinomycetemcomitans, Actinobacillus pleuropneumoniae, Actinomyces bovis, Actinomyces Israelii, Bacillus anthracis, Bacillus ceretus, Bacillus coagulans, Bacillus liquefaciens, Bacillus popillae, Bacillus subtilis, Bacillus thuringiensis, Bacteroides distasonis, Bacteroides fragilis, Bacteroides thetaiotaomicron, Bacteroides vulgatus, Bartonella bacilliformis, Bartonella Quintana, Beneckea parahaemolytica, Bordetella bronchiseptica, Bordetella parapertussis, Bordetella pertussis, Borelia burgdorferi, Brevibacterium lactofermentum, Brucella abortus, Brucella canis, Brucella melitensis, Brucella suis, Burkholderia cepacia, Burkholderia mallei, Burkholderia pseudomallei, Campylobacter fetus, Campylobacter jejuni, Campylobacter pylori, Cardiobacterium hominis, Chlamydia pneumoniae, Chlamydia psittaci, Chlamydia trachomatis, Chlamydophila abortus, Chlamydophila caviae, Chlamydophila felis, Chlamydophila pneumonia, Chlamydophila psittaci, Chryseobacterium eningosepticum, Clostridium botulinum, Clostridium butyricum, Clostridium coccoides, Clostridium dijficile, Clostridium leptum, Clostridium tetani, Corynebacterium xerosis, Cowdria ruminantium, Coxiella burnetii, Edwardsiella tarda, Ehrlichia sennetsu, Eikenella corrodens, Elizabethkingia meningoseptica, Enterobacter aerogenes, Enterobacter cloacae, Enterococcus faecalis, Escherichia coli, Escherichia hirae, Flavobacterium meningosepticum, Fluoribacter bozemanae, Francisella tularensis, Francisella tularensis biovar Tularensis, Francisella tularensis subsp. Holarctica, Francisella tularensis subsp. nearctica, Francisella tularensis subsp. Tularensis, Francisella tularensis var. palaearctica, Fudobascterium nucleatum, Fusobacterium necrophorum, Haemophilus ducreyi, Haemophilus influenzae, Helicobacter pylori, Kingella kingae, Klebsiella mobilis, Klebsiella oxytoca, Klebsiella pneumoniae, Lactobacillus acidophilus, Lactobacillus casei, Lactobacillus hilgardii, Lactobacillus pentosus, Lactobacillus plantarum, Lactobacillus rhamnosus, Lactococcus lactis, Legionella bozemanae corrig., Legionella pneumophila, Leptospira alexanderi, Leptospira borgpetersenii, Leptospira fainei, Leptospira inadai, Leptospira interrogans, Leptospira kirschneri, Leptospira noguchii, Leptospira santarosai, Leptospira weilii, Leuconostoc lactis, Leuconostoc oenos, Listeria ivanovii, Listeria monocytogenes, Moraxella catarrhalis, Morganella morganii, Mycobacterium africanum, Mycobacterium avium, Mycobacterium avium subspecies paratuberculosis, Mycobacterium bovis, Mycobacterium bovis strain BCG, Mycobacterium intracellulare, Mycobacterium kansasii, Mycobacterium leprae, Mycobacterium marinum, Mycobacterium tuberculosis, Mycobacterium typhimurium, Mycobacterium ulcerans, Mycoplasma hominis, Mycoplasma mycoides, Mycoplasma pneumoniae, Neisseria gonorrhoeae, Neisseria meningitidis, Neorickettsia sennetsu, Nocardia asteroides, Orientia tsutsugamushi, Pasteurella haemolytica, Pasteurella multocida, Plesiomonas shigelloides, Propionibacterium acnes, Proteus mirabilis, Proteus morganii, Proteus penneri, Proteus rettgeri, Proteus vulgaris, Providencia alcalifaciens, Providencia rettgeri, Pseudomonas aeruginosa, Pseudomonas mallei, Pseudomonas pseudomallei, Pyrococcus abyssi, Rickettsia akari, Rickettsia canadensis, Rickettsia canadensis corrig, Rickettsia conorii, Rickettsia montanensis, Rickettsia montanensis corrig, Rickettsia prowazekii, Rickettsia rickettsii, Rickettsia sennetsu, Rickettsia tsutsugamushi, Rickettsia typhi, Rochalimaea quintana, Salmonella arizonae, Salmonella choleraesuis subsp. arizonae, Salmonella enterica subsp. Arizonae, Salmonella enteritidis, Salmonella paratyphi, Salmonella typhi, Salmonella typhimurium, Selenomonas nominantium, Selenomonas ruminatium, Serratia marcescens, Shigella boydii, Shigella dysenteriae, Shigella flexneri, Shigella sonnei, Spirillum minus, Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus equi, Staphylococcus lugdunensis, Stenotrophomonas maltophila, Streptobacillus moniliformis, Streptococcus agalactiae, Streptococcus bovis, Streptococcus ferus, Streptococcus pneumoniae, Streptococcus pyogenes, Streptococcus viridans, Streptomyces ghanaenis, Streptomyces hygroscopicus, Streptomyces phaechromogenes, Treponema carateum, Treponema denticola, Treponema pallidum, Treponema pertenue, Vibrio cholerae, Vibrio parahaemolyticus, Vibrio vulnificus, Xanthomonas maltophilia, Yersinia enterocolitica, Yersinia pestis, Yersinia pseudotuberculosis, and Zymomonas mobilis.
In still further embodiments, the bacteria are from the class of bacteria known as Fusospirochetes. In further embodiments, the microorganism comprises fungi. In still further embodiments, the fungi are selected from the following genera: Candida, Saccharomyces, and Cryptococcus.
Such pathogenic bacteria can cause bacterial infections and disorders related to such infections that include, but are not limited to, the following: acne, rosacea, skin infection, pneumonia, otitis media, sinusitus, bronchitis, tonsillitis, and mastoiditis related to infection by Streptococcus pneumoniae, Haemophilus influenzae, Moraxella catarrhalis, Staphylococcus aureus, Peptostreptococcus spp. or Pseudomonas spp.; pharynigitis, rheumatic fever, and glomerulonephritis related to infection by Streptococcus pyogenes, Groups C and G streptococci, Clostridium diptheriae, or Actinobacillus haemolyticum; respiratory tract infections related to infection by Mycoplasma pneumoniae, Legionella pneumophila, Streptococcus pneumoniae, Haemophilus influenzae, or Chlamydia pneumoniae; uncomplicated skin and soft tissue infections, abscesses and osteomyelitis, and puerperal fever related to infection by Staphylococcus aureus, coagulase-positive staphylococci (i.e., S. epidermidis, S. hemolyticus, etc.), S. pyogenes, S. agalactiae, Streptococcal groups C-F (minute-colony streptococci), viridans streptococci, Corynebacterium spp., Clostridium spp., or Bartonella henselae; uncomplicated acute urinary tract infections related to infection by S. saprophyticus or Enterococcus spp.; urethritis and cervicitis; sexually transmitted diseases related to infection by Chlamydia trachomatis, Haemophilus ducreyi, Treponema pallidum, Ureaplasma urealyticum, or Nesseria gonorrheae; toxin diseases related to infection by S. aureus (food poisoning and Toxic shock syndrome), or Groups A, S, and C streptococci; ulcers related to infection by Helicobacter pylori; systemic febrile syndromes related to infection by Borrelia recurrentis; Lyme disease related to infection by Borrelia burgdorferi; conjunctivitis, keratitis, and dacrocystitis related to infection by C. trachomatis, N gonorrhoeae, S. aureus, S. pneumoniae, S. pyogenes, H influenzae, or Listeria spp.; disseminated Mycobacterium avium complex (MAC) disease related to infection by Mycobacterium avium, or Mycobacterium intracellulare; gastroenteritis related to infection by Campylobacter jejuni; odontogenic infection related to infection by viridans streptococci; persistent cough related to infection by Bordetella pertussis; gas gangrene related to infection by Clostridium perfringens or Bacteroides spp.; skin infection by S. aureus, Propionibacterium acne; atherosclerosis related to infection by Helicobacter pylori or Chlamydia pneumoniae; or the like. The QS modulating molecule attached to surfaces as described herein can be used to treat any of these disorders.
In certain embodiments the disease or disorder that can be treated with QS modulating molecule attached surfaces as described herein include sepsis, pneumonia, lung infections from cystic fibrosis, otitis media, chronic obstructive pulmonary disease, a urinary tract infection, and/or combinations thereof. In other embodiments, the QS modulating molecule attached surfaces described herein can be used to reduce and/or eliminate a medical device-related infection. In further embodiments, the QS modulating molecule attached surfaces described herein can be used to treat a periodontal disease, such as gingivitis, periodontitis or breath malodor. In still further embodiments, the QS modulating molecule attached surfaces described herein can be used to treat infections, including but not limited to those infections caused by bacteria. In some embodiments, the bacteria are Gram-negative or Gram-positive bacteria. Non-limiting examples of diseases and/or disorders that can be treated and/or prevented with the QS modulating molecule attached surfaces include otitis media, prostatitis, cystitis, bronchiectasis, bacterial endocarditis, osteomyelitis, dental caries, periodontal disease, infectious kidney stones, acne, Legionnaire's disease, chronic obstructive pulmonary disease (COPD), and cystic fibrosis.
In one specific example, subjects with cystic fibrosis can display with an accumulation of biofilm in the lungs and digestive tract. Subjects afflicted with COPD, such as emphysema and chronic bronchitis, display a characteristic inflammation of the airways wherein airflow through such airways, and subsequently out of the lungs, is chronically obstructed. Infections, including biofilm-related disorders, also encompass infections on implanted/inserted devices, medical device-related infections, such as infections from biliary stents, orthopedic implant infections, and catheter-related infections (e.g., kidney, vascular, peritoneal, etc.). An infection can also originate from sites where the integrity of the skin and/or soft tissue has been compromised. Non-limiting examples include dermatitis, ulcers from peripheral vascular disease, burn injury, and trauma. All of these diseases and/or disorders can be treated using the QS modulating molecule attached surfaces as described herein.
A QS modulating molecule (e.g., an antagonist) attached surface as described herein can be used to inhibit QS, thereby inhibiting biofilm formation, biofilm streamer formation and/or virulence factor expression in the healthcare field, in waste water treatment facilities or to treat those microorganisms that up-regulate these traits in response to QS autoinducers. A QS modulating molecule (e.g., an agonist) attached surface as described herein can be used to promote QS thereby inhibiting biofilm formation, biofilm streamer formation and/or virulence factor expression in the healthcare field, in waste water treatment facilities or to treat those microorganisms that down-regulate these traits in response to QS autoinducers. Either of these types of QS modulating molecules could be used to alter QS-controlled traits in beneficial bacteria.
In a preferred embodiment, the QS modulator surface described herein attaches the QS modulating molecule to a surface through a chemical bond including, but not limited to, a covalent bond. Additionally, the QS modulator attached surfaces can be placed in a static environment or under pressure, such as in a fluid flow environment or under controlled pressure. The surface can be any material, e.g., glass, metals, including, but not limited to, stainless steel metals, silicon, plastic, polymers, metals, and/or ceramic materials.
In preferred embodiments, a surface can comprise a polymer, including, but not limited to, polyethylene, polypropylene, polystyrene, polyester, polyester PLA and other biosorbable plastics, polycarbonate, polyvinyl chloride, polyethersulfone, polyacrylate (e.g., Acrylic, PMMA), hydrogel (e.g., acrylate), polysulfone, polyetheretherketone, thermoplastic elastomers (e.g., TPE, TPU), thermoset elastomers, silicone, poly-p-xylylene (e.g., Parylene), fluoropolymers, or any combination thereof.
In other preferred embodiments, a surface can comprise a metal, including, but not limited to stainless steel, cobalt-base alloys, titanium, titanium-base alloys, and/or shape memory alloy.
In other preferred embodiments, a surface can comprise a ceramic material including, but not limited to, glass ceramics, calcium phosphate ceramics, and/or carbon-based ceramics. Moreover, the surface can have any shape such as, e.g., small particles, including but not limited to nanoparticles, and/or flat and/or curved surfaces as described herein.
More specifically, the surface can be circular, oval, square, rectangular, flat and/or irregularly shaped. In further embodiments, the surface may have a constant cross-sectional area and/or it may be variable (e.g., it may constrict in certain areas and/or expand in others). In other embodiments, the surface may change shape along its length. In still further embodiments, the surface may comprise depressions, gutter, groove and/or furrow. This depression may be shallow, deep, narrow and/or wide. In still further embodiments, the surface may be part of a larger device or machine. In other embodiments, the surface may be part of an implantable medical device. In still further embodiments, the surface may be part of machinery used in industrial processes. In some embodiments, the surface may be very small (i.e. just large enough for fluid and bacterial cells to flow through). In some embodiments, the surface may be very large (i.e. the large culverts and pools used in a waste water treatment facility.) In still further embodiments, the surface may be circular. In still further embodiments, the surface may be part of a pipe, a cooling tower, a medical device, and/or other industrial fluid handling machinery.
In some embodiments, the surface comprises at least one biofilm streamer promotion element. In further embodiments, the surface has at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90 and/or 100 or more biofilm streamer promotion elements. In further embodiments, the surface has between 20-100 biofilm streamer promotion elements.
In further embodiments, the surface is a curved channel, a channel with at least one turn, a channel with at least one corner, an edge projecting into the lumen of the channel, a mound projecting into the lumen of the channel, a channel with roughened surfaces, and/or one or more objects placed on the surface. In further embodiments, the surface has at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90 and/or 100 or more turns. In further embodiments, the surface has between 20-100 turns. In further embodiments, the surface has at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90 and/or 100 or more edges projecting onto the surface. In further embodiments, the surface has between 20-100 edges projecting onto the surface. In further embodiments, the surface has at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90 and/or 100 or more mounds projecting onto the surface. In further embodiments, the surface has between 20-100 mounds projecting onto the surface. In further embodiments, the surface has at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90 and/or 100 or more roughened surfaces. In further embodiments, the surface has between 20-100 roughened surfaces. In further embodiments, the surface has at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90 and/or 100 or more objects placed on the surface. In further embodiments, the surface has between 20-100 objects placed on the surface.
In further embodiments, the surface has at least 2 corners, at least 3 corners, at least 4 corners, at least 5 corners, at least 6 corners, at least 7 corners, at least 8 corners, at least 9 corners, at least 10 corners, at least 11 corners, at least 12 corners, at least 13 corners, at least 14 corners, at least 15 corners, at least 16 corners, at least 17 corners, at least 18 corners, at least 19 corners, at least 20 corners, at least 25 corners, at least 30 corners, at least 35 corners, and/or at least 40 corners or more. In further embodiments, the surface has between 20-40 corners. In still further embodiments, the surface has 1 turn about every 100 μm, every 200 μm, every 300 μm, every 400 μm, every 500 μm, every 600 μm, every 700 μm, every 800 μm, 900 μm, and/or every 1000 μm. Other dimensions could also be used.
Additionally, embodiments include a combination of any of these elements (e.g., turns, corners, edges, mounds, roughened surfaces, and/or objects) included on the surface.
In still further embodiments, the surface has NAFION® granules placed on the surface. In further embodiments, the surface comprises glass beads. In further embodiments, the surface comprises sand particles. In further embodiments, the surface comprises a welded polypropylene feed spacer mesh. In further embodiments, the surface is found on a stent. In still further embodiments, the surface is a bare-metal stent.
In some embodiments, the surface is subject to a laminar flow. In further embodiments, the flow of the fluid is characterized by a Reynolds number of less than 2000, of less than 1500, of less than 1000, of less than 750, of less than 500, of less than 400, of less than 300, of less than 200, of less than 100, of less than 50, of less than 25, of less than 10, of less than 5, of less than 4, of less than 3, of less than 2, and/or of less than 1. Other dimensions could also be used.
In some embodiments, the surface is subject to a turbulent flow. In further embodiments, the flow of the fluid is characterized by a Reynolds number of greater than 2000.
In some embodiments, the surface is subject to a shear stress. In further embodiments, the shear stress is characterized by a number between 0.01 and 100 Pa, between 0.01 and 90 Pa, between 0.01 and 80 Pa, between 0.01 and 70 Pa, between 0.01 and 60 Pa, between 0.01 and 50 Pa, between 0.01 and 40 Pa, between 0.01 and 30 Pa, between 0.01 and 20 Pa, between 0.01 and 10 Pa, between 0.02 and 10 Pa, between 0.03 and 10 Pa, between 0.04 and 10 Pa, between 0.05 and 10 Pa, between 0.06 and 10 Pa, between 0.07 and 10 Pa, between 0.08 and 10 Pa, between 0.09 and 10 Pa, between 0.1 and 10 Pa, between 0.02 and 100 Pa, between 0.03 and 100 Pa, between 0.04 and 100 Pa, between 0.05 and 100 Pa, between 0.06 and 100 Pa, between 0.07 and 100 Pa, between 0.08 and 100 Pa, between 0.09 and 100 Pa, between 0.1 and 100 Pa, between 0.1 and 90 Pa, between 0.1 and 80 Pa, between 0.1 and 70 Pa, between 0.1 and 60 Pa, between 0.1 and 50 Pa, between 0.1 and 40 Pa, between 0.1 and 30 Pa, between 0.1 and 20 Pa, between 0.02 and 90 Pa, between 0.03 and 80 Pa, between 0.04 and 70 Pa, between 0.05 and 60 Pa, between 0.06 and 50 Pa, between 0.07 and 40 Pa, between 0.08 and 30 Pa, and/or between 0.09 and 20 Pa. Other dimensions could also be used.
In the present invention, any linker can be used to attach the QS modulator to the surface. Examples of linkers are well known in the art, and can be synthesized in a variety of ways including, but not limited to, atom radical polymerization, reversible-addition fragmentation chain transfer polymerization, nitrous oxide-mediated polymerization, photo initiator-mediated polymerization, and can be selected based on the surface. For example, and in no way limiting, linkers can be selected from polyethylene glycol (PEGs), polyphosphazenes, polylactide, polyglycolide, polycaprolactone, poly(6-azidohexyl methacrylate), poly(2-bromoisobutyryloxyethyl methacrylate), poly(n-butyl methacrylate), poly(benzyl methacrylate), poly(cadmium methacrylate), poly(2-diethylaminoethyl methacrylate), poly(2,3-dihydroxypropyl methacrylate), poly(2-diisopropylaminoethyl methacrylate), poly(l-ethylene glycol dimethacrylate), poly(ethyl methacrylate), poly(3-ethyl-3-(methacryloyloxy methyloxetane), poly(ferrocenylmethyl methacrylate), poly(2-gluconamidoethyl methacrylate), poly(glycidyl methacrylate), poly(heptadecafluorodecyl methacrylate), poly(2-hydroxyethyl methacrylate), poly(2-hydroxylpropyl methacrylate), poly(isobutyl methacrylate), poly(isobornyl methacrylate), poly(2-lactobionamidoethyl methacrylate), poly(methacrylic acid), poly(methaacryloyladenosine), poly(3-O-methacryloly-di-Oisopropylidene-D-glucofuranose), poly(4-(10-methacryloydecyloxy)-4-pentylazobenzene), poly(2-methoxyethyl methacrylate), poly(2-(methacryloyloxy)ethyl succinate), poly(methyl methacrylate), poly(methacryloyluridine), poly(N-hydroxylsuccinimide methacrylate), poly(2-N-morpholinoethyl methacrylate), poly(octadecyl methacrylate), poly(poly(ethylene glycol) dimethacrylate, poly(poly(ethylene glycol) methacrylate, poly(poly(ethylene glycol)methyl ether methacrylate, poly(3-perylenylmethyl methacrylate, poly(2,2-dimethyl-1,3,-dioxolan-4-yl methyl methacrylate), poly(sprirobenzopyran methacrylate), poly(2-(tert-butylamino)ethyl methacrylate), poly(tert-butyl methacrylate), poly(trifluoroethyl methacrylate), poly(trimethylsilyl methacrylate), poly(3-(trimethoxylsilyl)propyl methacrylate), 2-(perfluoroalkyl)ethyl methacrylate, poly(2-(1-butylimidazolium-3-yl)ethyl methacrylate hexafluorophosphate, poly(carboxybetaine methacrylate), poly(l-ethyl 3-(2-methacryloyloxy ethyl) imidazolium chloride), poly(sodium methacrylate), poly(2-methacryloyloxyethyl phosphate), poly(2-(methacryloyloxy)ethyl phosphorylcholine), poly(sulfobetanine methacrylate), poly(2-sulfatoethyl methacrylate), poly(potassium 3-sulfopropyl methacrylate), poly(acrulic acid), poly(n-butyl acrylate), poly(2-bromoacetyloxyethyl acrylate), poly(2-(2-bromopropionyloxy)ethyl acrylate), poly(benzyl acrylate), poly(l1-(4-cyanophenyl-4-phenoxy)undecyl acrylate), poly(2-(dimethylamino)ethyl acrylate), poly(ethyl acrylate), poly(ethylene glycol diacrylate), poly(fluorescein acrylate), poly(l,6-hexanediol diacrylate), poly(heptadecafluorodecyl acrylate), poly(2-hydroxylethyl acrylate), poly(methyl acrylate), poly(octyl acrylate), poly(octadecyl acrylate), poly(poly(ethylene glycol)acrylate), poly(poly(glycol ethylene)acrylate succinyl fluorescein), poly(poly(glycol ethylene) methyl ether acrylate, poly(pentafluoropropyl acrylate), poly(tert-butyl acrylate), poly(trifluoroethyl acrylate), poly(trimethylsilyl acrylate), poly(triphenylamine acrylate), poly(N-(2-hydroxypropyl)methacrylamine, poly(methacrylamide), poly((3-methacryloylamino)propyl)-dimethyl-(3-sulfopropyl)ammonium hydroxide), poly(N-acryloyl glucosamine), poly(acrylamide), poly(potassium 2-acrylamido-2-methylpropane sulfonate), poly(carboxylbetane acrylamide), poly(N-cyclopropyl acrylamide), poly(N,N-dimethylacrylamide), poly(N-(3-(dimethylamino)propyl) acrylamide, poly(N-(3-dimethylamino)propyl) acrylamide methiodide), poly(N-hydroxylmethyl acrylamide), poly(N-N-methylenebisacrylamide), poly(methoxylethylacrylamide), poly(N-(6-(N-tert-butoxy-carbonylaminooxy)hexyl)acrylamide), poly(N-isopropyl acrylamide), poly(poly(ethylene glycol) methyl ether acrylamide), poly(acetoxystryrene), poly(4-chloromethylstyrene), poly(divinylbenzene), poly(4-(perfluoroalkyl)-oxymethylstyrene), poly(tert-butoxy-vinylbenzyl-polyglycidol), poly(4-methylstyrene), poly(N-octadecyl-N-(4-vinyl)-benzoyl-phenylalanineamide), polystyrene, poly(4-(poly(ethylene glycol) methyl ether styrene), poly(4-vinylaniline), poly(4-vinylbenzocyclobutene), poly(vinylquinoline), poly(4-styrenesulfonate), poly(4-vinylbenzoate), poly(l-(4-vinylbenzyl)-3-(butyl-imidazolium hexafluorophophate), poly(2-vinylpyridine), poly(3-vinylpyridine), poly(acrylonitrile), poly(itaconic acid), poly(maleic anhydride), poly(N-vinylimidazole), poly(N-vinyl-2-pyrrolidone), poly(N-vinyl-2-pyrrolidone), poly(m-isopropenyl-dimethyl-benzyl isocyanate), poly(2-vinyl-4,4-dimethyl azlactone), or any other combinations thereof. In some embodiments, combinations include any two or more of the aforementioned linkers attached at one end to a surface and at the other end with the QS modulating molecule. In other embodiments, combinations include any two or more of the aforementioned linkers arranged serially, e.g., a first linker having one end attached to a surface and another end attached to a second linker, the second linker having one end attached to the first linker and another end attached to a third linker, etc.). In other embodiments, any polymer architectures that consist of any combination of two or more of the aforementioned linkers including, but not limited to, end-functional linear polymers, di-end functional linear polymers, telechelic polymers, many-arm star polymers, copolymers, block polymers, dendritic polymers, branched polymers, gradient polymers, grafted polymers, microgel polymers, etc.
Any specific chemistry can be used to form a chemical bond between a surface (e.g., plastic/glass/gold substrates) and a linker. For example, silanization, gold-sulfide bond formation, thiol-ene reactions, surface-initiated polymerization, etc. can be used to form a bond between a surface and a linker. In a preferred embodiment, the linker functionalized with silane moieties at one terminus can be covalently bound to the surface via hydroxyl moieties on the glass substrate using silanization. In yet another preferred embodiment, the linker functionalized with maleimide moieties at one terminus can be covalently bound to —SH— moieties on the glass substrate via thiol-ene reactions. In still another preferred embodiment, the linker can be covalently bound to the surface by using surface-initiated polymerization to catalyze chemical reactions via -SH- moieties on the glass substrate. In yet another preferred embodiment, the linker functionalized with thiol moieties at one terminus can be covalently bound to the surface on a gold substrate via gold-sulfide bond formation, wherein the glass surface has been coated with a gold substrate.
According to embodiments of the present invention, a variety of specific chemistries can be used to form a chemical bond between a surface and a linker and/or between a linker and a QS modulating molecule. Thus, a variety of synthetic chemical strategies are available to create surface-attached molecules for manipulation of QS. Examples of specific chemistries include, but are not limited to, biorthogonal reactions, click chemistry, thiol-ene reactions, gold-sulfide bond formation, esterification reactions, Grignard reactions, Michael reactions, ketone/hydroxylamine condensations, Staudinger ligations, strain-promoted alkyne-azide cycloadditions, photo-click cycloadditions, Diels-Alder cycloadditions, tetrazine-alkene/alkyne cycloadditions, Cu-catalyzed alkyne-azide cycloadditions, Pd-catalyzed cross coupling, strain promoted alkyne-nitrone cycloadditions, Cross-metathesis, Norbornene cycloadditions, Oxanorbornadiene cycloadditions, tetrazine ligations, tetrazole photoclick chemistry, or any other combinations of these chemistries.
The present invention also relates to a method of screening a test compound that can modulate (i.e. reduce/inhibit or promote) QS, biofilm formation, biofilm streamer formation, and/or virulence factor production by a microorganism, by contacting the surface comprising an attached QS modulating molecule (i.e., antagonist or agonist) with the test compound and by measuring the modulation (i.e., reduction/elimination or promotion) of QS, biofilm formation, biofilm streamer formation, growth, and/or morphology changes. This method includes contacting a composition comprising a test compound attached to a surface through a linker as described herein and monitoring either: (1) the reduction and/or elimination of QS, biofilm formation, biofilm streamer formation, virulence factor production, growth, and/or morphology/phenotypic changes; or (2) the promotion and/or increase of QS, biofilm formation, biofilm streamer formation, virulence factor production, growth, and/or morphology/phenotypic changes. For example, by determining the time until clogging (T) and the duration of the clogging transition (τ) or by imaging the formation, growth or morphology/phenotypic changes of the biofilm and/or biofilm streamer and/or production of virulence factors, Alternatively, by measuring the expression of a fluorescently tagged quorum-sensing-repressed molecule such as GFPmut2 (indicating that QS is off) and/or by measuring the expression of a second fluorescently tagged quorum-sensing-activated molecule such as mKate2 (indicating that QS is on), one can readily determine the ability of a test compound attached to a surface through a linker as described herein to inhibit or enhance biofilm and/or biofilm streamer growth. Other embodiments including a method of screening test compounds to identify compounds that can inhibit, promote or affect biofilm and/or biofilm streamer formation are also contemplated.
Bacterial organisms may be Gram-positive or Gram-negative. Gram-positive bacteria have a peptidoglycan coating covering the bacterial cell membrane. Thus, in some embodiments, the linker is of a sufficient length to traverse the peptidoglycan coating in order to interact with the receptor on the underlying cell membrane. In some embodiments, the peptidoglycan coating may have a thickness ranging from about 1 nm to about 200 nm, from about 1 nm to about 100 nm, from about 1 nm to about 50 nm, from about 15 nm to about 50 nm, from about 15 nm to about 30 nm, or from about 1 nm to about 15 nm.
The length of the linker may be designed to be longer than the peptidoglycan coating, so that the QS modulating molecule is able to interact with the receptor on the bacterial cell membrane. As the peptidoglycan coating thickness may vary between different species of bacteria, the length of the linker may vary depending on the bacterial organism targeted. Therefore, in some embodiments, the linker may have a length between 1 nm and about 200 nm, between about 1 nm to about 100 nm, between about 1 nm to about 50 nm, between about 15 nm to about 50 nm, between about 15 nm to about 30 nm, or between about 1 nm to about 15 nm. As an example, the linker PEG10000 is of a sufficient length to interact with cell membrane receptors for S. aureus.
In other embodiments, the linker may have a length greater than 1 nm, 2 nm, 3 nm, 4 nm, 5 nm, 6 nm, 7 nm, 8 nm, 9 nm, 10 nm, 11 nm, 12 nm, 13 nm, 14 nm, 15 nm, 16 nm, 17 nm, 18 nm, 19 nm, and 20 nm.
Gram-negative bacteria have an outer membrane layer covering a peptidoglycan layer. In some embodiments, the Gram-negative bacteria are treated with a reagent to make the outer membrane layer porous prior to coming into contact with the surface attached QS modulating molecules. In this embodiment, the length of the linker should be greater than the thickness of the outer membrane layer plus the thickness of the peptidoglycan layer, so that the QS modulatory molecule is able to interact with the receptor on the bacterial inner-cell membrane.
The peptidoglycan layer is an elastic mesh network with pores. In some embodiments, the pores may range in diameter from about 1 nm to 5 nm, from about 1 nm to about 10 nm, from about 1 nm to about 20 nm, or from about 1 nm to about 50 nm. Accordingly, the diameter of the linker may be designed to be smaller than the width of the peptidoglycan pores. Similarly, for Gram-negative bacteria that have been treated with a reagent to make the outer cell layer porous, the diameter of the linker may be desgined to be smaller than the diameter of the pore of the outer cell layer or of the peptidoglycan matrix, whichever is smaller. In some embodiments, the diameter of the linker is less than 5 nm, 6 nm, 7 nm, 8 nm, 9 nm or 10 nm.
The surface density of the QS modulating molecule is a factor in determining whether the QS modulating molecule can affect the behavior of the bacteria. In some embodiments, the average surface coverage density of the QS modulating molecule is between about 2×102 μm−2 and 2×104 μm−2. In preferred embodiments, the surface attached QS modulating molecule is 2.1×102 μm−2 or greater. In some embodiments, the average surface coverage density of the QS modulating molecule is between about 2×100 μm−2 and 2×102 μm−2 or between about 2×10−2 μm−2 and 2×100 μm−2.
In still other embodiments, the surface may comprise multiple types of QS modulating molecules e.g., two or more antagonists, two or more agonists, or a combination of antagonists and agonists. Each agonist or antagonist may be attached to the surface via a linker. For example, a first agonist may be tethered to the surface with a first type of linker and a second agonist may be tethered to the surface with a second type of linker. In some embodiments, two or more QS modulating molecules may be attached to the surface with the same type of linker. In other embodiments, a QS modulating molecule may be attached to the surface with the two or more types of linkers.
In still other embodiments, the surfaces having surface attached QS modulating molecules are stable. For example, the surfaces may be stored for up to 40 days, 50 days, 60 days, 70 days, or 80 days at about 4° C. without undergoing significant degradation.
In some embodiments, the QS modulating molecules are attached to the surface of a device (e.g., a medical device, a device to prevent biofouling, etc.). Due to the stability of the surface attached QS modulating molecules, the devices may be exposed to repeat infections in a serial manner and still retain the capability to modulate QS behavior of the bacteria. The surface attached QS modulating molecules are also resistant to degradation by host plasma components and to flow-generated stresses, e.g., from fluid entering and exiting the device. Thus, the surface attached QS modulating molecules described herein exhibit superior stability under a variety of conditions.
Embodiments of the present invention also relate to a method of detecting specific microorganisms that can respond to specific QS modulating molecules. An unknown microorganism that contacts the QS modulating molecule attached surface and responds to the surface will undergo a change in QS phenotype, and this alteration can be used to detect particular types of microorganisms. In one embodiment, a sample that contains an unknown bacterium that causes an infection in a patient in a healthcare setting can be introduced onto the QS modulating molecule-coated surface, and an alteration in a QS phenotype can be measured. In another embodiment, a sample that contains an unknown bacterium that causes contamination in a food processing setting can be introduced onto the QS modulating molecule-coated surface, and an alteration in a QS phenotype, such as byproduct production, or other traits can be measured. This application can be more rapid and provide lower detection limits than conventional microbial detection methodologies such as PCR verification techniques, immunological methods, and amplification methods in use today, which could be important to treat severely ill patients.
Thus, in preferred embodiments, the invention is described as a surface comprising a quorum sensing (“QS”) modulating molecule attached to the surface by a linker. In preferred embodiments: (a) the length of the linker is sufficient to traverse a bacterial cell's peptidoglycan layer, outer membrane layer or both; (b) the QS modulating molecule binds to a receptor on a cell membrane of a bacterial cell; (c) the linker has a diameter of less than 5 nm; (d) the linker has a length greater than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 nm; (e) the linker is a chemical bound; (f) the linker is selected from polyethylene glycol (PEGs), polyphosphazenes, polylactide, polyglycolide, polycaprolactone, or any other combinations thereof; (g) the linker is attached to the surface using one or more of the following types of chemical reactions: silanization, gold-sulfide bond formation, thiol-ene reactions, and surface-initiated polymerization; (h) the average surface coverage density of the QS modulating molecule is of a sufficient density to modulate QS; (i) the average surface coverage density of the QS modulating molecule is about 2.1×102 μm−2 or greater; (j) the QS modulating molecule comprises an antagonist of QS that alters QS-controlled phenotypes of biofilm production, biofilm streamer production, and/or virulence factor production; (k) the QS modulating molecule comprises an agonist of QS that alters QS-controlled phenotypes of biofilm production, biofilm streamer production and/or virulence factor production; (l) the QS modulating molecule retains modulating activity after being stored at about 4° C. for up to 40 days; (m) the QS modulating molecule remains bound to the surface via the linker after exposure to laminar fluid flow; (n) the QS modulating molecule exhibits modulating activity when exposed to a population of bacterial cells; (o) the QS modulating molecule exhibits modulating activity when reexposed to another population of bacterial cells; (p) the QS modulating molecule is a molecule selected from Tables 1A or 1B or its derivative molecules; (q) the QS modulating molecule is attached to the linker using one or more of the following types of chemical reactions: biorthogonal reactions, click chemistry, thiol-ene reactions, gold-sulfide bond formation, esterification reactions, Grignard reactions, Michael reactions, ketone/hydroxylamine condensations, Staudinger ligations, strain-promoted alkyne-azide cycloadditions, photo-click cycloadditions, Diels-Alder cycloadditions, tetrazine-alkene/alkyne cycloadditions, Cu-catalyzed alkyne-azide cycloadditions, Pd-catalyzed cross coupling, strain promoted alkyne-nitrone cycloadditions, Cross-metathesis, Norbornene cycloadditions, Oxanorbornadiene cycloadditions, tetrazine ligations, or tetrazole photoclick chemistry; (r) the surface comprises glass, metal, stainless metal, silicon, plastic, polymer, metal, or ceramic material or any combination thereof; (s) the surface is a small particle, a nanoparticle, a flat surface or a curved surface; or (t) any combination of (a)-(s).
In preferred embodiments, the bacterial cell is Gram-negative, Gram-positive or a mixture of Gram-negative and Gram-positve. In further preferred embodiments, the bacterial cell is exposed to a permeability agent that forms holes in the outer membrane layer of the bacterial cell prior to contacting the surface.
Additional preferred embodiments include (a) the polymer is selected from polyethylene, polypropylene, polystyrene, polyester, polyester PLA and other biosorbable plastics, polycarbonate, polyvinyl chloride, polyethersulfone, polyacrylate (e.g., Acrylic, PMMA), hydrogel (e.g., acrylate), polysulfone, polyetheretherketone, thermoplastic elastomers (e.g., TPE, TPU), thermoset elastomers, silicone, poly-p-xylylene (e.g., Parylene), fluoropolymers; (b) the metal is selected from stainless steel, cobalt-base alloy, titanium, titanium-base alloy, and/or shape memory alloy; and/or (c) the ceramic material comprises glass ceramic, calcium phosphate ceramic, and/or carbon-based ceramic.
In further preferred embodiments, the surface comprises a second QS modulating molecule attached the surface by a second linker. In these situations, preferred embodiments include, (a) the length of the second linker is sufficient to traverse the bacterial cell's peptidoglycan layer, outer membrane layer or both; (b) the second QS modulating molecule competitively binds to the receptor on the cell membrane of the bacterial cell; (c) wherein the second QS modulating molecule binds to a different receptor on the cell membrane; (d) the second linker has a diameter of less than 5 nm; (e) the second linker has a length greater than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 nm; (f) the second linker is a chemical bound; (g) the second linker is selected from polyethylene glycol (PEGs), polyphosphazenes, polylactide, polyglycolide, polycaprolactone, or any other combinations thereof; (h) the second linker is attached to the surface using one or more of the following types of chemical reactions: silanization, gold-sulfide bond formation, thiol-ene reactions, and surface-initiated polymerization; (i) the second linker is the same as the linker; (j) the second linker is different from the linker; (k) any combination of (a)-(j).
In preferred embodiments, the surface is placed in an environment, and preferably the environment is (a) static; (b) under pressure; (c) a flow environment; (d) under controlled pressure; and/or (e) an implantable medical device, part of machinery used in industrial processes, a culvert, a pool used in a waste water treatment facility, waste water treatment facility, a pipe, a cooling tower, a medical device, industrial fluid handling machinery, a wound, within the body, a medical process, an agricultural process, and/or machinery.
In further preferred embodiments, the invention is a method of modulating QS, biofilm formation, biofilm streamer formation, and/or a virulence factor production by a microorganism, wherein the method comprises contacting a microorganism with the surface described herein. For example, the method inhibits a pathogenic behavior of a microorganism and/or promotes a beneficial behavior of a microorganism.
Examples of microorganism that can be modulated include, but are not limited to bacteria, archaea, protozoa, fungi, and/or algae. Further examples of strains of bacteria include but are not limited to Abiotrophia, Achromobacter, Acidaminococcus, Acidovorax, Acinetobacter, Actinobacillus, Actinobaculum, Actinomadura, Actinomyces, Aerococcus, Aeromonas, Afipia, Agrobacterium, Alcaligenes, Alloiococcus, Alteromonas, Amycolata, Amycolatopsis, Anabaena, Anabaenopsis, Anaerobospirillum, Anaerorhabdus, Aphanizomenon, Arachnia, Arcanobacterium, Arcobacter, Arthrobacter, Atopobium, Aureobacterium, Bacillus, Bacteroides, Balneatrix, Bartonella, Bergeyella, Bifidobacterium, Bilophila, Bordetella, Borrelia, Brachyspira, Branhamella, Brevibacillus, Brevibacterium, Brevundimonas, Brucella, Burkholderia, Buttiauxella, Butyrivibrio, Calymmatobacterium, Camesiphon, Campylobacter, Capnocytophaga, Capnylophaga, Cardiobacterium, Catonella, Cedecea, Cellulomonas, Centipeda, Chlamydia, Chlamydophila, Chromobacterium, Chryseomonas, Chyseobacterium, Citrobacter, Clostridium, Collinsella, Comamonas, Corynebacterium, Coxiella, Cryptobacterium, Cyanobacteria, Cylindrospermopsis, Delftia, Dermabacter, Dermatophilus, Desulfomonas, Desulfovibrio, Dialister, Dichelobacter, Dolosicoccus, Dolosigranulum, Edwardsiella, Eggerthella, Ehrlichia, Eikenella, Empedobacter, Enterobacter, Enterococcus, Erwinia, Erysipelothrix, Escherichia, Eubacterium, Ewingella, Exiguobacterium, Facklamia, Filifactor, Flavimonas, Flavobacterium, Francisella, Fusobacterium, Gardnerella, Gemella, Globicatella, Gloeobacter, Gordona, Haemophilus, Hafnia, Hapalosiphon, Helicobacter, Helococcus, Hemophilus, Holdemania, Ignavigranum, Johnsonella, Kingella, Klebsiella, Kocuria, Koserella, Kurthia, Kytococcus, Lactobacillus, Lactococcus, Lautropia, Leclercia, Legionella, Leminorella, Leptospira, Leptospirae, Leptotrichia, Leuconostoc, Listeria, Listonella, Lyngbya, Megasphaera, Methylobacterium, Microbacterium, Micrococcus, Microcystis, Mitsuokella, Mobiluncus, Moellerella, Moraxella, Morganella, Mycobacterium, Mycoplasma, Myroides, Neisseria, Nocardia, Nocardiopsis, Nodularia, Nostoc, Ochrobactrum, Oeskovia, Oligella, Orientia, Paenibacillus, Pantoea, Parachlamydia, Pasteurella, Pediococcus, Peptococcus, Peptostreptococcus, Phormidium, Photobacterium, Photorhabdus, Phyllobacterium, Phytoplasma, Planktothrix, Plesiomonas, Porphyromonas, Prevotella, Propionibacterium, Proteus, Providencia, Pseudoanabaena, Pseudomonas, Pseudonocardia, Pseudoramibacter, Psychrobacter, Rahnella, Ralstonia, Rhodococcus, Rickettsia, Rochalimaea, Roseomonas, Rothia, Ruminococcus, Salmonella, Schizothrix, Selenomonas, Serpulina, Serratia, Shewenella, Shigella, Simkania, Slackia, Sphaerotilus, Sphingobacterium, Sphingomonas, Spirillum, Spiroplasma, Spirulina, Staphylococcus, Stenotrophomonas, Stomatococcus, Streptobacillus, Streptococcus, Streptomyces, Succinivibrio, Sutterella, Suttonella, Tatumella, Tissierella, Trabulsiella, Treponema, Trichodesmium, Tropheryma, Tsakamurella, Turicella, Umezakia, Ureaplasma, Vagococcus, Veillonella, Vibrio, Weeksella, Wolinella, Xanthomonas, Xenorhabdus, Yersinia, Yokenella. Acinetobacter baumannii, Actinobacillus actinomycetemcomitans, Actinobacillus pleuropneumoniae, Actinomyces bovis, Actinomyces Israelii, Bacillus anthracis, Bacillus ceretus, Bacillus coagulans, Bacillus liquefaciens, Bacillus popillae, Bacillus subtilis, Bacillus thuringiensis, Bacteroides distasonis, Bacteroides fragilis, Bacteroides thetaiotaomicron, Bacteroides vulgatus, Bartonella bacilliformis, Bartonella Quintana, Beneckea parahaemolytica, Bordetella bronchiseptica, Bordetella parapertussis, Bordetella pertussis, Borelia burgdorferi, Brevibacterium lactofermentum, Brucella abortus, Brucella canis, Brucella melitensis, Brucella suis, Burkholderia cepacia, Burkholderia mallei, Burkholderia pseudomallei, Campylobacter fetus, Campylobacter jejuni, Campylobacter pylori, Cardiobacterium hominis, Chlamydia pneumoniae, Chlamydia psittaci, Chlamydia trachomatis, Chlamydophila abortus, Chlamydophila caviae, Chlamydophila felis, Chlamydophila pneumonia, Chlamydophila psittaci, Chryseobacterium eningosepticum, Clostridium botulinum, Clostridium butyricum, Clostridium coccoides, Clostridium dijficile, Clostridium leptum, Clostridium tetani, Corynebacterium xerosis, Cowdria ruminantium, Coxiella burnetii, Edwardsiella tarda, Ehrlichia sennetsu, Eikenella corrodens, Elizabethkingia meningoseptica, Enterobacter aerogenes, Enterobacter cloacae, Enterococcus faecalis, Escherichia coli, Escherichia hirae, Flavobacterium meningosepticum, Fluoribacter bozemanae, Francisella tularensis, Francisella tularensis biovar Tularensis, Francisella tularensis subsp. Holarctica, Francisella tularensis subsp. nearctica, Francisella tularensis subsp. Tularensis, Francisella tularensis var. palaearctica, Fudobascterium nucleatum, Fusobacterium necrophorum, Haemophilus ducreyi, Haemophilus influenzae, Helicobacter pylori, Kingella kingae, Klebsiella mobilis, Klebsiella oxytoca, Klebsiella pneumoniae, Lactobacillus acidophilus, Lactobacillus casei, Lactobacillus hilgardii, Lactobacillus pentosus, Lactobacillus plantarum, Lactobacillus rhamnosus, Lactococcus lactis, Legionella bozemanae corrig., Legionella pneumophila, Leptospira alexanderi, Leptospira borgpetersenii, Leptospira fainei, Leptospira inadai, Leptospira interrogans, Leptospira kirschneri, Leptospira noguchii, Leptospira santarosai, Leptospira weilii, Leuconostoc lactis, Leuconostoc oenos, Listeria ivanovii, Listeria monocytogenes, Moraxella catarrhalis, Morganella morganii, Mycobacterium africanum, Mycobacterium avium, Mycobacterium avium subspecies paratuberculosis, Mycobacterium bovis, Mycobacterium bovis strain BCG, Mycobacterium intracellulare, Mycobacterium kansasii, Mycobacterium leprae, Mycobacterium marinum, Mycobacterium tuberculosis, Mycobacterium typhimurium, Mycobacterium ulcerans, Mycoplasma hominis, Mycoplasma mycoides, Mycoplasma pneumoniae, Neisseria gonorrhoeae, Neisseria meningitidis, Neorickettsia sennetsu, Nocardia asteroides, Orientia tsutsugamushi, Pasteurella haemolytica, Pasteurella multocida, Plesiomonas shigelloides, Propionibacterium acnes, Proteus mirabilis, Proteus morganii, Proteus penneri, Proteus rettgeri, Proteus vulgaris, Providencia akalifaciens, Providencia rettgeri, Pseudomonas aeruginosa, Pseudomonas mallei, Pseudomonas pseudomallei, Pyrococcus abyssi, Rickettsia akari, Rickettsia canadensis, Rickettsia canadensis corrig, Rickettsia conorii, Rickettsia montanensis, Rickettsia montanensis corrig, Rickettsia prowazekii, Rickettsia rickettsii, Rickettsia sennetsu, Rickettsia tsutsugamushi, Rickettsia typhi, Rochalimaea quintana, Salmonella arizonae, Salmonella choleraesuis subsp. arizonae, Salmonella enterica subsp. Arizonae, Salmonella enteritidis, Salmonella paratyphi, Salmonella typhi, Salmonella typhimurium, Selenomonas nominantium, Selenomonas ruminatium, Serratia marcescens, Shigella boydii, Shigella dysenteriae, Shigella flexneri, Shigella sonnei, Spirillum minus, Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus equi, Staphylococcus lugdunensis, Stenotrophomonas maltophila, Streptobacillus moniliformis, Streptococcus agalactiae, Streptococcus bovis, Streptococcus ferus, Streptococcus pneumoniae, Streptococcus pyogenes, Streptococcus viridans, Streptomyces ghanaenis, Streptomyces hygroscopicus, Streptomyces phaechromogenes, Treponema carateum, Treponema denticola, Treponema pallidum, Treponema pertenue, Vibrio cholerae, Vibrio parahaemolyticus, Vibrio vulnificus, Xanthomonas maltophilia, Yersinia enterocolitica, Yersinia pestis, Yersinia pseudotuberculosis, Zymomonas mobilis, or Fusospirochetes.
Examples of fungi include, but are not limited to Candida, Saccharomyces, or Cryptococcus.
The invention is further directed to use of the surfaces described herein to treat sepsis, pneumonia, infections from cystic fibrosis, otitis media, chronic obstructive pulmonary disease, a urinary tract infection, periodontal disease, gingivitis, periodontitis, breath malodor, treat infections, Gram-negative infections, Gram-positive infections, otitis media, prostatitis, cystitis, bronchiectasis, bacterial endocarditis, osteomyelitis, dental caries, periodontal disease, infectious kidney stones, acne, Legionnaire's disease, chronic obstructive pulmonary disease (COPD), cystic fibrosis, an accumulation of biofilm in the lungs or digestive tract, emphysema, chronic bronchitis, also encompasses infections on implanted/inserted devices, medical device-related infections, biliary stent infections, orthopedic implant infections, catheter-related infections, skin infections, dermatitis, ulcers from peripheral vascular disease, a burn injury, trauma, rosacea, skin infection, pneumonia, otitis media, sinusitus, bronchitis, tonsillitis, and mastoiditis related to infection by Streptococcus pneumoniae, Haemophilus influenzae, Moraxella catarrhalis, Staphylococcus aureus, Peptostreptococcus spp. or Pseudomonas spp.; pharynigitis, rheumatic fever, and glomerulonephritis related to infection by Streptococcus pyogenes, Groups C and G streptococci, Clostridium diptheriae, or Actinobacillus haemolyticum; respiratory tract infections related to infection by Mycoplasma pneumoniae, Legionella pneumophila, Streptococcus pneumoniae, Haemophilus influenzae, or Chlamydia pneumoniae; uncomplicated skin and soft tissue infections, abscesses and osteomyelitis, and puerperal fever related to infection by Staphylococcus aureus, coagulase positive staphylococci (i.e., S. epidermidis, S. hemolyticus, etc.), S. pyogenes, S. agalactiae, Streptococcal groups C-F (minute-colony streptococci), viridans streptococci, Corynebacterium spp., Clostridium spp., or Bartonella henselae; uncomplicated acute urinary tract infections related to infection by S. saprophyticus or Enterococcus spp.; urethritis and cervicitis; sexually transmitted diseases related to infection by Chlamydia trachomatis, Haemophilus ducreyi, Treponema pallidum, Ureaplasma urealyticum, or Nesseria gonorrheae; toxin diseases related to infection by S. aureus (food poisoning and Toxic shock syndrome), or Groups A, S, and C streptococci; ulcers related to infection by Helicobacter pylori; systemic febrile syndromes related to infection by Borrelia recurrentis; Lyme disease related to infection by Borrelia burgdorferi; conjunctivitis, keratitis, and dacrocystitis related to infection by C. trachomatis, N gonorrhoeae, S. aureus, S. pneumoniae, S. pyogenes, H influenzae, or Listeria spp.; disseminated Mycobacterium avium complex (MAC) disease related to infection by Mycobacterium avium, or Mycobacterium intracellulare; gastroenteritis related to infection by Campylobacter jejuni; odontogenic infection related to infection by viridans streptococci; persistent cough related to infection by Bordetella pertussis; gas gangrene related to infection by Clostridium perfringens or Bacteroides spp.; skin infection by S. aureus, Propionibacterium acne; atherosclerosis related to infection by Helicobacter pylori or Chlamydia pneumoniae; or the like.
The surfaces described herein can also be used in a method of screening for a compound that modulates QS, biofilm formation, biofilm streamer formation, and/or a virulence factor production by a microorganism, wherein the method comprises contacting a compound with any one of the surfaces described herein and measuring whether QS, biofilm formation, biofilm streamer formation, and/or a virulence factor production by a microorganism is either increased, decreased or maintained.
Embodiments are illustrated by way of example and are not limited to the figures of the accompanying drawings, in which like references indicate similar elements and in which:
To make the AIP-I amenable to surface attachment, we synthesized AIP-I containing an alkyne at the N-terminus (
For the surface modification work, we used silanization and maleimide-thiol chemistry to decorate the surface with PEG10000 polymers carrying azide moieties at one end (
To reinforce the above results, we show that S. aureus did not activate a QS response when introduced onto the identical surface lacking the triazole-AIP-I decoration (Surface-PEG10000) or that had not undergone the click reaction (Surface-PEG10000-azide) (
First, it was tested whether heterogeneity arises from variation in mKate2 fluorescent protein translation or from plasmid copy number effects. To do this, the agrP3 promoter driving mkate2 was replaced with the constitutive sarAP1 promoter on the plasmid (sarAP1-mkate2). The corresponding histogram depicting the normalized mKate2 fluorescence is narrower when sarAP1, rather than agrP3, is the promoter upstream of mkate2 (σ/μ=0.22 (±0.02), yellow histogram in
Next, it was tested whether heterogeneity arises from variation in copy numbers of genes encoding the QS-detection/response apparatus (agrP2-agrCA) from the plasmid. For this test, the agrP2-agrCA genes were introduced into the genome rather than having them expressed from a plasmid. The reporter gene, agrP3-mkate2, was expressed from the plasmid (
Lastly, it was tested whether heterogeneity arises from variation in copy number of the agrP3-mkate2 gene. For this assessment, both the agrP3-mkate2 gene and the agrP2-agrCA gene were introduced into the genome (
Taken together, these data indicate that heterogeneity from the reporter strain (
Each panel is discussed in more detail as follows:
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art, such as in the arts of peptide chemistry, cell culture, nucleic acid chemistry, and biochemistry. Standard techniques are used for molecular biology, genetic and biochemical methods (see, Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd ed., 2001, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.; Ausubel et al., Short Protocols in Molecular Biology (1999) 4th ed., John Wiley & Sons, Inc.), which are incorporated herein by reference.
In the present invention, a “microorganism” is defined as a bacterium, archaeon, protozoan, fungus, and/or alga.
In the present invention, “bacteria” are defined as any one of a large domain of single-celled prokaryotic microorganisms. As used herein, bacteria include any that are known to those of ordinary skill in the art and any that may be discovered. Preferred examples of bacteria are those known to be pathogenic to humans, animals or plants. Other preferred examples include those known to cause undesirable contamination and/or clogging of industrial flow systems. Still other preferred examples of bacteria include those known to infect implanted medical devices (e.g., pumps, stents, artificial joints, screws, rods, and the like). Further preferred examples of bacteria include those capable of forming biofilms and/or biostreamers or producing virulence factors. Further preferred examples include bacteria selected from the following genera: Abiotrophia, Achromobacter, Acidaminococcus, Acidovorax, Acinetobacter, Actinobacillus, Actinobaculum, Actinomadura, Actinomyces, Aerococcus, Aeromonas, Afipia, Agrobacterium, Alcaligenes, Alloiococcus, Alteromonas, Amycolata, Amycolatopsis, Anabaena, Anabaenopsis, Anaerobospirillum, Anaerorhabdus, Aphanizomenon, Arachnia, Arcanobacterium, Arcobacter, Arthrobacter, Atopobium, Aureobacterium, Bacillus, Bacteroides, Balneatrix, Bartonella, Bergeyella, Bifidobacterium, Bilophila, Bordetella, Borrelia, Brachyspira, Branhamella, Brevibacillus, Brevibacterium, Brevundimonas, Brucella, Burkholderia, Buttiauxella, Butyrivibrio, Calymmatobacterium, Camesiphon, Campylobacter, Capnocytophaga, Capnylophaga, Cardiobacterium, Catonella, Cedecea, Cellulomonas, Centipeda, Chlamydia, Chlamydophila, Chromobacterium, Chryseomonas, Chyseobacterium, Citrobacter, Clostridium, Collinsella, Comamonas, Corynebacterium, Coxiella, Cryptobacterium, Cyanobacteria, Cylindrospermopsis, Delftia, Dermabacter, Dermatophilus, Desulfomonas, Desulfovibrio, Dialister, Dichelobacter, Dolosicoccus, Dolosigranulum, Edwardsiella, Eggerthella, Ehrlichia, Eikenella, Empedobacter, Enterobacter, Enterococcus, Erwinia, Erysipelothrix, Escherichia, Eubacterium, Ewingella, Exiguobacterium, Facklamia, Filifactor, Flavimonas, Flavobacterium, Francisella, Fusobacterium, Gardnerella, Gemella, Globicatella, Gloeobacter, Gordona, Haemophilus, Hafnia, Hapalosiphon, Helicobacter, Helococcus, Hemophilus, Holdemania, Ignavigranum, Johnsonella, Kingella, Klebsiella, Kocuria, Koserella, Kurthia, Kytococcus, Lactobacillus, Lactococcus, Lautropia, Leclercia, Legionella, Leminorella, Leptospira, Leptospirae, Leptotrichia, Leuconostoc, Listeria, Listonella, Lyngbya, Megasphaera, Methylobacterium, Microbacterium, Micrococcus, Microcystis, Mitsuokella, Mobiluncus, Moellerella, Moraxella, Morganella, Mycobacterium, Mycoplasma, Myroides, Neisseria, Nocardia, Nocardiopsis, Nodularia, Nostoc, Ochrobactrum, Oeskovia, Oligella, Orientia, Paenibacillus, Pantoea, Parachlamydia, Pasteurella, Pediococcus, Peptococcus, Peptostreptococcus, Phormidium, Photobacterium, Photorhabdus, Phyllobacterium, Phytoplasma, Planktothrix, Plesiomonas, Porphyromonas, Prevotella, Propionibacterium, Proteus, Providencia, Pseudoanabaena, Pseudomonas, Pseudonocardia, Pseudoramibacter, Psychrobacter, Rahnella, Ralstonia, Rhodococcus, Rickettsia, Rochalimaea, Roseomonas, Rothia, Ruminococcus, Salmonella, Schizothrix, Selenomonas, Serpulina, Serratia, Shewenella, Shigella, Simkania, Slackia, Sphaerotilus, Sphingobacterium, Sphingomonas, Spirillum, Spiroplasma, Spirulina, Staphylococcus, Stenotrophomonas, Stomatococcus, Streptobacillus, Streptococcus, Streptomyces, Succinivibrio, Sutterella, Suttonella, Tatumella, Tissierella, Trabulsiella, Treponema, Trichodesmium, Tropheryma, Tsakamurella, Turicella, Umezakia, Ureaplasma, Vagococcus, Veillonella, Vibrio, Weeksella, Wolinella, Xanthomonas, Xenorhabdus, Yersinia, and Yokenella.
Further preferred examples include bacteria selected from the following species: Acinetobacter baumannii, Actinobacillus actinomycetemcomitans, Actinobacillus pleuropneumoniae, Actinomyces bovis, Actinomyces Israelii, Bacillus anthracis, Bacillus ceretus, Bacillus coagulans, Bacillus liquefaciens, Bacillus popillae, Bacillus subtilis, Bacillus thuringiensis, Bacteroides distasonis, Bacteroides fragilis, Bacteroides thetaiotaomicron, Bacteroides vulgatus, Bartonella bacilliformis, Bartonella Quintana, Beneckea parahaemolytica, Bordetella bronchiseptica, Bordetella parapertussis, Bordetella pertussis, Borelia burgdorferi, Brevibacterium lactofermentum, Brucella abortus, Brucella canis, Brucella melitensis, Brucella suis, Burkholderia cepacia, Burkholderia mallei, Burkholderia pseudomallei, Campylobacter fetus, Campylobacter jejuni, Campylobacter pylori, Cardiobacterium hominis, Chlamydia pneumoniae, Chlamydia psittaci, Chlamydia trachomatis, Chlamydophila abortus, Chlamydophila caviae, Chlamydophila felis, Chlamydophila pneumonia, Chlamydophila psittaci, Chryseobacterium eningosepticum, Clostridium botulinum, Clostridium butyricum, Clostridium coccoides, Clostridium dijficile, Clostridium leptum, Clostridium tetani, Corynebacterium xerosis, Cowdria ruminantium, Coxiella burnetii, Edwardsiella tarda, Ehrlichia sennetsu, Eikenella corrodens, Elizabethkingia meningoseptica, Enterobacter aerogenes, Enterobacter cloacae, Enterococcus faecalis, Escherichia coli, Escherichia hirae, Flavobacterium meningosepticum, Fluoribacter bozemanae, Francisella tularensis, Francisella tularensis biovar Tularensis, Francisella tularensis subsp. Holarctica, Francisella tularensis subsp. nearctica, Francisella tularensis subsp. Tularensis, Francisella tularensis var. palaearctica, Fudobascterium nucleatum, Fusobacterium necrophorum, Haemophilus ducreyi, Haemophilus influenzae, Helicobacter pylori, Kingella kingae, Klebsiella mobilis, Klebsiella oxytoca, Klebsiella pneumoniae, Lactobacillus acidophilus, Lactobacillus casei, Lactobacillus hilgardii, Lactobacillus pentosus, Lactobacillus plantarum, Lactobacillus rhamnosus, Lactococcus lactis, Legionella bozemanae corrig., Legionella pneumophila, Leptospira alexanderi, Leptospira borgpetersenii, Leptospira fainei, Leptospira inadai, Leptospira interrogans, Leptospira kirschneri, Leptospira noguchii, Leptospira santarosai, Leptospira weilii, Leuconostoc lactis, Leuconostoc oenos, Listeria ivanovii, Listeria monocytogenes, Moraxella catarrhalis, Morganella morganii, Mycobacterium africanum, Mycobacterium avium, Mycobacterium avium subspecies paratuberculosis, Mycobacterium bovis, Mycobacterium bovis strain BCG, Mycobacterium intracellulare, Mycobacterium kansasii, Mycobacterium leprae, Mycobacterium marinum, Mycobacterium tuberculosis, Mycobacterium typhimurium, Mycobacterium ulcerans, Mycoplasma hominis, Mycoplasma mycoides, Mycoplasma pneumoniae, Neisseria gonorrhoeae, Neisseria meningitidis, Neorickettsia sennetsu, Nocardia asteroides, Orientia tsutsugamushi, Pasteurella haemolytica, Pasteurella multocida, Plesiomonas shigelloides, Propionibacterium acnes, Proteus mirabilis, Proteus morganii, Proteus penneri, Proteus rettgeri, Proteus vulgaris, Providencia alcalifaciens, Providencia rettgeri, Pseudomonas aeruginosa, Pseudomonas mallei, Pseudomonas pseudomallei, Pyrococcus abyssi, Rickettsia akari, Rickettsia canadensis, Rickettsia canadensis corrig, Rickettsia conorii, Rickettsia montanensis, Rickettsia montanensis corrig, Rickettsia prowazekii, Rickettsia rickettsii, Rickettsia sennetsu, Rickettsia tsutsugamushi, Rickettsia typhi, Rochalimaea quintana, Salmonella arizonae, Salmonella choleraesuis subsp. arizonae, Salmonella enterica subsp. Arizonae, Salmonella enteritidis, Salmonella paratyphi, Salmonella typhi, Salmonella typhimurium, Selenomonas nominantium, Selenomonas ruminatium, Serratia marcescens, Shigella boydii, Shigella dysenteriae, Shigella flexneri, Shigella sonnei, Spirillum minus, Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus equi, Staphylococcus lugdunensis, Stenotrophomonas maltophila, Streptobacillus moniliformis, Streptococcus agalactiae, Streptococcus bovis, Streptococcus ferus, Streptococcus pneumoniae, Streptococcus pyogenes, Streptococcus viridans, Streptomyces ghanaenis, Streptomyces hygroscopicus, Streptomyces phaechromogenes, Treponema carateum, Treponema denticola, Treponema pallidum, Treponema pertenue, Vibrio cholerae, Vibrio parahaemolyticus, Vibrio vulnificus, Xanthomonas maltophilia, Yersinia enterocolitica, Yersinia pestis, Yersinia pseudotuberculosis, and Zymomonas mobilis.
Further preferred examples include bacteria selected from the class of bacteria known as Fusospirochetes.
In the present invention, “fungi” are defined as any one of a large domain of single-celled eukaryotic microorganisms such as yeasts. As used herein, fungi include any that are known to those of ordinary skill in the art and any that may be discovered. Preferred examples of fungi are those known to be pathogenic to humans, animals or plants. Other preferred examples include those known to cause undesirable contamination and/or clogging of industrial flow systems. Still other preferred examples of fungi include those known to infect implanted medical devices (e.g., pumps, stents, artificial joints, screws, rods, and the like). Further preferred examples of fungi include those capable of forming biofilms and/or biostreamers. Further preferred examples include fungi selected from the genera: Candida, Saccharomyces, and Cryptococcus.
In the present invention, an “autoinducer” is defined as a molecule that activates or represses the expression of QS regulated genes. An “agonist” is defined as a naturally produced or synthetic autoinducer molecule that activates the expression of QS regulated genes. An “antagonist” is defined as a naturally produced or synthetic autoinducer molecule that represses the expression of QS regulated genes. Both agonists and antagonists are QS modulating molecules.
In the present invention, “biofilms” are defined as sessile microorganism community, such as a bacterial and/or fungal communities, that occupies a surface. These biofilms can cause chronic and medical device-associated infections, clogging, and/or device failure. Biofilms are surface-associated assemblies of microorganisms, such as bacteria and/or fungi which are bound together by extracellular polymeric substances (4, 5). Biofilms are attached to the surface all along the edges, including the bottom edge, of the surface. Although bacterial biofilms are desirable in waste-water treatment (6), biofilms primarily cause undesirable effects such as chronic infections or clogging of industrial flow systems (1-3). Cells in biofilms display many behavioral differences from planktonic cells, such as a 1,000-fold increase in tolerance to antibiotics (7, 8), an altered transcriptome (9-11), and spatially heterogeneous metabolic activity (12, 13). Some of these physiological peculiarities of biofilm-dwelling cells may be due to strong gradients of nutrients and metabolites, which also affect biofilm morphology and composition (14, 15).
In the present invention, “biofilm streamers” are defined as biofilms that have been partially detached from the surface upon which the biofilm is growing. Under conditions of flow in the presence of available biofilm promotion element(s) (e.g., curves, corners, bends, etc.), the flow partially detaches the extra cellular matrix off of the substrate along with cells that were in it already and is suspended in the liquid attached only at its edges. The detached biofilm forms filaments or streamers in the flowing liquid. The streamer is then able to capture other flowing debris and cells in order to continue growing. Thus, biofilms grow by cellular division, while biofilm streamers grow both by cell division as well as cellular capture of passing cells in the flow.
In the present invention, “biofilm growth” is defined as the expansion of the surface-attached biofilm over time, whether through cell division or through attachment of additional cells to the surface from the surrounding environment. As used herein, this growth includes expansion laterally over available surfaces as well as expansion through thickening of the biofilm layer by layers of additional cells.
In the present invention, “biofilm morphology” is defined as the physical composition or shape of the biofilm. As used in the invention, biofilm morphology may change over time. These changes may be in the composition of the extracellular matrix, in the composition of microorganisms, such as bacteria and/or fungi in the biofilm, or in the shape of the biofilm. Biofilm growth would be an example of a change in biofilm morphology. Another example of a change in biofilm morphology would be the flow induced formation of biofilm streamers. A third example would be the inclusion or expulsion of different microbial species within the biofilm.
In the present invention, “biofilm streamer growth” is defined as the expansion of the biofilm streamer over time. As used herein, this expansion may be in the length of the biofilm streamer filaments and/or in the thickness of the biofilm streamer. This growth may be through cell division and/or through capture of additional cells, extracellular matrix, and/or debris from the surrounding liquid.
In the present invention, “biofilm streamer morphology” is defined as the physical composition and/or shape of the biofilm streamer. As used in the invention, biofilm streamer morphology may change over time. These changes may be in the extracellular matrix, in the composition of the microorganisms (e.g., bacteria and/or fungi) in the biofilm streamer and/or in the shape of the biofilm streamer. Biofilm streamer growth, flow induced formation and/or inclusion/exclusion of different microbial species are all examples of a change in biofilm streamer morphology.
In the present invention, “QS modulator attached surface” is defined as any surface that possesses or is attached to a molecule that modulates QS, and in turn, alters any QS phenotype including, but not limited to, a biofilm, a biofilm streamer, and/or a virulence factor production via a linker. The surface may be any suitable solid surface or solid porous surface as is known to one of ordinary skill in the art. In one example, and in no way limiting, the surface is a glass coverslip. In a further example, the surface can be glass, stainless steel, plastic, polymers, sand, wire mesh, bone, teeth, skin, or blood vessels. In other examples, the surface will line a channel, e.g., the tubing of a fluid handling system.
As used herein, a “QS antagonist attached surface” is defined as any substrate that possesses or is attached to a molecule that antagonizes (e.g., inhibits or reduces) QS, and in turn, alters any QS phenotype including, but not limited to, a biofilm, a biofilm streamer, and/or a virulence factor production via a linker. Examples of QS antagonists are described in U.S. Pat. No. 8,247,443, U.S. Pat. No. 8,568,756, or PCT/U.S. Ser. No. 14/56497 which are specifically incorporated by reference in their entirety. See, for example, the structures described in
Alternatively, a “QS agonist attached surface” is defined as any surface that possesses or is attached to a molecule that agonizes (e.g., promotes or increases) QS, and in turn, alters any QS phenotype including, but not limited to, a biofilm, a biofilm streamer, and/or a virulence factor production via a linker. Examples of QS agonists are described in U.S. Pat. No. 5,353,689 and PCT/US2014/051648 both of which are incorporated by reference in their entirety.
As used herein, “QS phenotype” or “morphology” or “trait” refers to any change in the bacterial colony/organism or in the constituents in the cells in the colony, including but not limited to, changes in appearance, e.g., an increase in streamer formation, a decrease in streamer formation, an increase in biofilm density, a decrease in biofilm density, etc. as well as other changes e.g., a change in gene expression, a change in mRNA production, a change in protein production, etc.
As used herein, “click chemistry” is a term to describe reactions that are high yielding, broadly applicable, create only byproducts that can be removed without chromatography, are stereospecific and generally simple to perform, and can be conducted in easily removable or benign solvents. In some embodiments, click chemistry allows generation of large libraries of compounds for screening in research. In one example, click chemistry enables covalent bond formation between molecule A with an azide group and with molecule B with an alkyne group. Click chemistry uses Cu catalysts to form triazoles by cycloaddition. A molecule with a PEG linker and an azide at one end may be reacted with another group (an anti-QS molecule) with an alkyne group attached at one end. Other methods are also possible, for example, the PEG linker can have an alkyne group attached, and the QS modulating molecules can possess azide groups at one end.
Thus, the inhibition of QS, biofilm, biofilm streamer, and/or a virulence factor production and/or morphology/phenotypic changes through the use of a QS antagonist may lead to either a decrease or an increase in overall virulence to the host depending on whether the microorganism relies on QS, biofilm, biofilm streamer, and/or a virulence factor production to promote infection. Similarly, the promotion of QS, biofilm, biofilm streamer, and/or a virulence factor production and/or morphology/phenotypic changes through the use of a QS agonist may lead to either a decrease or an increase in overall virulence to the host depending on whether the microorganism relies on QS, biofilm, biofilm streamer, and/or a virulence factor production to promote infection.
In the present invention, the surface can be made of any material. For example, glass, metals, including, but not limited to stainless metals, silicon, plastic, polymers, metals, and/or ceramic materials can be used.
Preferred examples of surfaces that can be used include, but are not limited to a surface comprising polymers, such as, for example, polyethylene, polypropylene, polystyrene, polyester, polyester PLA and other biosorbable plastics, polycarbonate, polyvinyl chloride, polyethersulfone, polyacrylate (e.g., Acrylic, PMMA), hydrogel (e.g., acrylate), polysulfone, polyetheretherketone, thermoplastic elastomers (e.g., TPE, TPU), thermoset elastomers, silicone, poly-p-xylylene (e.g., Parylene), fluoropolymers, a metal, including, but not limited to stainless steel, cobalt-base alloys, titanium, titanium-base alloys, and/or shape memory alloy, and/or a ceramic material including, but are not limited to glass ceramics, calcium phosphate ceramics, and/or carbon-based ceramics). Moreover, the surface can have any shape, such as, for example, small particles, including but not limited to nanoparticles, and/or flat and/or curved surfaces as described herein.
In the present invention, the surface may comprise a “biofilm promoting or biofilm streamer promotion element” as defined as any feature of the local environment in a flow system that, in the presence of pressure driven flow, serves as the site for biofilm formation, biofilm streamer formation or virulence factor production. For example, these biofilm streamer promotion elements may be roughened surfaces along the flow path, may be curves in the channel directing the flow, may be a turn in the channel directing the flow, may be a corner in the channel directing the flow, may be an edge or mound projecting into the lumen of the channel directing the flow, may be a constriction or expansion in the channel directing the flow, and/or may be provided by an object placed within the channel directing the flow.
In the present invention, the surface may comprise of a “channel” which is defined as a passage directing the flow of a fluid. As used in the invention, a channel may be an enclosed hollow tube. The cross section of the tube may be of any suitable geometry as is known by those of skill in the art. In one example the cross section is circular, oval, square, rectangular and/or irregularly shaped. The tube may have a constant cross-sectional area and/or it may be variable (e.g. it may constrict in certain areas and/or expand in others). The cross section of the channel may change shape along its length. In other examples, the channel may be a depression, gutter, groove and/or furrow. This depression may be shallow, deep, narrow and/or wide. In other examples, the channel may be provided by the gap between two parallel flat planar surfaces placed close together. In still other examples, the channel may be part of a larger device or machine or biological tissue or organ (e.g., lungs). The channel may be a flow conduit in an implantable medical device. The channel may also be a flow conduit in machinery used in industrial processes. The channel may be very small (i.e. just large enough for fluid and bacterial or fungal cells to flow through) or very large (i.e. the large culverts and pools used in a waste water treatment facility.)
In the present invention, “lumen” is defined as the area in a channel that is designed to direct the flow. It is defined as the interior of an enclosed hollow tube. It is the depressed area in a depression, gutter, groove or furrow. And, it is the gap between the adjacent parallel plates.
In the present invention, “circular” as applied to surface and/or a channel is defined as having a generally round cross sectional shape. As used in the present invention, circular does not require a perfectly circular cross section. In another example, circular means that the length of the diameter measured anywhere along the cross section of the channel is identical to that measured at any other point (i.e. it is perfectly circular).
In the present invention, “turn” is defined as a portion of a surface with a defined, discrete change in direction of the flow. This turn may be of any degree. In one example, the turn is a change in direction from about 210° to about 360°, more preferably from about 220° to about 350°, more preferably from about 230° to about 340°, more preferably from about 240° to about 330°. As used herein, a turn may be rounded or may be sharp. When a turn is sharp, it may result in a corner.
In the present invention, “corner” is defined as the point or area where two lines, edges, or sides of something meet. A corner may be an edge formed by a turn in the channel A corner may also be a raised point, for example, in the lumen of the channel For example, a pyramidal obstruction, placed in the lumen of the channel such that the base is against the surface of the channel and the tip is directed towards the center of the channel, would form a corner.
In the present invention, “edge” is defined as a line or line segment that is the intersection of two plane faces. An edge may be formed on a surface, such as in a channel along the inside surface of a turn. An edge may also be formed by a raised surface, for example, in the lumen of the channel For example, a raised wedge, placed on a surface, such as in the lumen of the channel, such that the base is against the surface of the channel and the raised edge of the wedge is directed towards the center of the channel, would form an edge. The edge formed by this wedge obstruction could be placed perpendicular to the fluid flow or parallel to the flow.
In the present invention, “mound” is defined as a raised area on a surface, such as for example, within the lumen of the channel, without any appreciable corners or edges. A used herein, a mound would be a generally curved obstruction on a surface. In one example, the mound is a raised circular bump. In another example, the mound may be formed by placing a half-cylinder (formed by cutting perpendicular to its circular faces) onto a surface. For example, a mound in a channel would be positioned such that a flat surface is placed against the surface of the channel with the circular surface faced towards the center of the channel A further example includes a cylinder placed such that the semicircular top and bottom are perpendicular to the flow or parallel to the flow.
In the present invention, “roughened surface” is defined as an irregular surface. It may be a surface that microscopically reduces to surfaces with many corners. In another example, it may a surface with distinct geometric and irregular deformities on a macroscopic level.
In the present invention, an “object” comprises a surface. In some examples, an object may include, for example, sand, gravel, granules and the like. In other examples, an object may include portions of medical devices or industrial fluid handling machinery. For example, an object may include filter support grids, filter mesh, stents, tubing or channel components for fluid handling, valves, pumps, and the like. These objects may be of any scale from miniature components of implantable medical devices to large scale fluid handling components of industrial cooling units or food processing machinery.
In the present invention, “NAFION® granules” are defined as amorphous particles of fluoropolymer first discovered in the late 1960s by DuPont. In one example, the granules are of the size and shape of sand. In another embodiment, the granules may be larger, including up to the size of grains of rice, stones or boulders. As used herein, one of ordinary skill in the art will recognize that additional fluoropolymers may be employed in the invention. In further examples, Teflon AF, Teflon FEP and CYTOP may each be used as part of the surface of the invention (47).
In the present invention, “welded polypropylene feed spacer mesh” is defined as mesh similar to that used in industrial reverse osmosis filters (48). As used herein, this mesh may be any porous mesh used in industrial, medical, or other fluid handling applications.
In the present invention, “stent” is defined as a mesh tube inserted into a natural passage/conduit in the body to prevent localized flow constriction.
In the present invention, “bare-metal stent” is defined as type of vascular stent without a coating (as used in drug-eluting stents, for example). Stents are made out of different types of fabrics, polymers, and other materials, such as for example, bare stainless steel or may be made of alloys (e.g., cobalt chromium).
In the present invention, “pipe” is defined as a generally rigid tube used to convey fluid or compressed gases. A pipe may have an inner diameter as small as 2 mm or as large as several feet. A pipe is made of glass, any number of metals, any number of plastics or other polymeric materials, or concrete. A pipe as used herein may be any that is known to one of ordinary skill in the art.
In the present invention, “cooling tower” is defined as a heat rejection device, which extracts waste heat to the atmosphere through the cooling of a water stream to a lower temperature. The type of heat rejection in a cooling tower is termed “evaporative” in that it allows a small portion of the water being cooled to evaporate into a moving air stream to provide significant cooling to the rest of that water stream.
In the present invention, “fluid” is defined as a liquid or a gas. In one example, the fluid is water, with or without the addition of other components. These additional components may include, but are not limited to nutrients and salts needed to support bacterial growth, bacteria, chemical or biochemical probes to assist with visualization of cells or extracellular components, test compounds, and compounds for selective growth of specific bacterial strains. In other embodiments, a fluid is a biological fluid such as, for example, blood.
In the present invention, “flow” or “fluid flow” is defined as movement of the fluid along a surface in a continuous stream.
In the present invention, “flow rate” is defined as the volume of a fluid moving along a surface per unit time.
In the present invention, “Reynolds number” is defined as a dimensionless quantity used to help predict similar flow patterns in different fluid flow situations. It is defined as the ratio of inertial forces to viscous forces and thus quantifies the relative importance of these two types of forces for given flow conditions. Reynolds numbers may be used to characterize different flow regimes within a similar fluid, such as laminar or turbulent flow. When a fluid is flowing through a surface, such as a closed channel such as a pipe or between two flat plates, either of two types of flow may occur depending on the velocity of the fluid: laminar flow or turbulent flow. Laminar flow tends to occur at lower velocities, below a threshold at which it becomes turbulent. A Reynolds number of less than 2320 is characteristic of laminar flow in a circular tube. A Reynolds number greater than 2320 is characteristic of turbulent flow in a circular tube.
In the present invention, “laminar flow” in a long straight surface is defined as a flow regime that occurs when a fluid flows in parallel layers, with no disruption between the layers. At low velocities, the fluid tends to flow without lateral mixing, and adjacent layers slide past one another like playing cards. For flow in a long straight surface, such as a long straight channel, there are no cross-currents perpendicular to the direction of flow, nor eddies or swirls of fluids. In laminar flow, the motion of the particles of the fluid is very orderly with all particles moving in straight lines parallel to the pipe walls. For flows in more complicated geometries, such as channels with bends and corners, the laminar flow is the time-independent motion for a steady pressure drop; the flow may be three-dimensional, i.e. the velocity may have all three components non-zero, but the flow remains steady (time independent) so long as the pressure drop is constant.
In the present invention, “turbulent flow” is defined as a flow regime characterized by chaotic property changes. This includes low momentum diffusion, high momentum convection, and rapid variation of pressure and velocity in space and time. In turbulent flow, unsteady vortices appear on many scales and interact with each other. Drag due to boundary layer friction increases. The structure and location of boundary layer separation often changes, sometimes resulting in a reduction of overall drag.
In the present invention, “shear stress” is defined as the force/area acting tangent to a surface. In an ordinary fluid such as water the shear stress is proportional to the fluid viscosity and proportional to the velocity gradient (as defined in standard textbooks).
In the present invention, “controlled pressure” is defined as pressure applied to a fluid moving through a channel such that the pressure drop along the channel is held constant. Thus, as resistance to flow in the pipe is increased, rather than continuing to apply increasing pressure to keep the flow rate constant, the flow rate is reduced such that the pressure remains constant. As used herein, a constant pressure includes pressure that varies. For example, the pressure may “pulse” at a given frequency, for example, but the average pressure will remain constant.
In the present invention, “time until clogging (T)” is defined as the time at which the fitted flow rate drops to half its initial value.
In the present invention, “duration of the clogging transition (τ)” is defined as the time period in which the fitted flow rate decreases from 76% to 27% of its initial value.
In the present invention, “test compound” is defined as any compound added to the test system for evaluation of its effect on QS, biofilm formation, biofilm streamer formation, and/or a virulence factor production. The effect of the test compound may be to inhibit (an antagonist) or to enhance (an agonist) QS, biofilm, biofilm streamer, and/or a virulence factor production and/or morphology changes. The inhibition of QS, biofilm, biofilm streamer, and/or a virulence factor production and/or morphology changes through the use of a QS antagonist may lead to either a decrease or an increase in overall virulence to the host depending on whether the microorganism relies on QS, biofilm, biofilm streamer, and/or a virulence factor production to promote infection. Similarly, the promotion of QS, biofilm, biofilm streamer, and/or a virulence factor production and/or morphology changes through the use of a QS agonist may lead to either a decrease or an increase in overall virulence to the host depending on whether the microorganism relies on QS, biofilm, biofilm streamer, and/or a virulence factor production to promote infection.
These compounds may be pharmaceutical compound, small molecules, or biological compounds. Some examples include peptides, proteins, peptidomimetics, antibodies, non-antibody specific binding molecules, such as adnectins, affibodies, avimers, anticalins, tetranectins, DARPins, mTCRs, engineered Kunitz-type inhibitors, nucleic acid aptamers and spiegelmers, peptide aptamers and cyclic and bicyclic peptides and small synthetic or natural organic molecules (Ruigrok et al. Biochem J. (2011) 436, 1-13; Gebauer et al., Curr Opin Chem Biol. (2009) (3):245-55.)
In the present invention, “antibody” refers to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e., molecules that contain an antigen binding site that immunospecifically binds an antigen. As such, the term antibody encompasses not only whole antibody molecules, but also antibody fragments as well as variants (including derivatives) of antibodies and antibody fragments. Examples of molecules which are described by the term “antibody” in this application include, but are not limited to: single chain Fvs, and fragments comprising or alternatively consisting of, either a VL or a VH domain. The term “single chain Fv” or “scFv” as used herein refers to a polypeptide comprising a VL domain of an antibody linked to a VH domain of an antibody. Antibodies of the invention include, but are not limited to, monoclonal, multispecific, human or chimeric antibodies or antibodies made in animals, single chain antibodies, Fab fragments, F9ab′) fragments, antiidiotypic (anti-Id) antibodies (including, e.g., anti-Idantibodies to antibodies of the invention), and epitope-binding fragments of any of the above. The immunoglobulin molecules of the invention can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) or subclass of immunoglobulin molecule.
As used herein, “average surface coverage density” refers to the number of QS modulating molecules attached to the surface per unit area. See also, Example 14.
As used herein, “Gram-positive” refers to a type of bacteria surrounded by a thick layer of peptidologlycan. Gram-positive bacteria include staphylococci (“staph”), streptococci (“strep”), pneumococci, and the bacterium responsible for diphtheria (Cornynebacterium diphtheriae) as well as anthrax (Bacillus anthracia). Gram-positive bacteria react with the Gram stain to turn dark blue or violet.
As used herein, “Gram-negative” refers to bacteria that have a cytoplasmic membrane, a thin peptidoglycan layer, and an outer membrane containing lipopolysaccharide. Gram-negative bacteria do not react with the Gram stain to turn dark blue or violet, instead these bacteria appear red or pink due to counterstain (usually safranin).
As used herein, “peptidoglycan layer” refers to an elastic polymeric mesh-like network found outside the bacterial cell membrane.
As used herein, “linker length” means the longitudinal length of the linker, usually in nm.
As used herein, “linker diameter” means the diameter or breadth of the linker.
As used herein, “permeability agent” means any agent capable of forming holes in the outer membrane layer of gram-negative bacteria. Examples include holins, endolysins, or bacteriocins50.
As used herein, the terms “a” or “an” are used, as is common in patent documents, to include one or more than one, independent of any other instances or usages of “at least one” or “one or more.”
As used herein, the term “or” is used to refer to a nonexclusive or, such that “A or B” includes “A but not B,” “B but not A,” and “A and B,” unless otherwise indicated.
As used herein, the term “about” is used to refer to an amount that is approximately, nearly, almost, or in the vicinity of being equal to or is equal to a stated amount, e.g., the state amount plus/minus about 5%, about 4%, about 3%, about 2% or about 1%.
S. aureus Agr QS is driven by an autoinducer peptide (called AIP) harboring a thiolactone ring and an exocyclic tail at the N-terminus. AIP is processed from the precursor peptide AgrD by AgrB and other proteases, and the AIP is secreted26,27. Extracellular AIP is detected by a cognate transmembrane-bound receptor histidine kinase, AgrC, that upon AIP binding, autophosphorylates and subsequently funnels a phosphoryl group to the partner response regulator, AgrA28,29 (
There are four S. aureus Agr allelic variants (I to IV) that make four AIPs differing only in a few amino acid residues. AIPs activate QS in the S. aureus cells that produce them, and they generally inhibit QS in heterologous S. aureus cells possessing different AIP variants8. In the S. aureus agr-I strain referenced herein, AIP-I is the native autoinducer.
TrAIP-II, a truncated AIP-II with the exocyclic tail replaced by an acetyl group, is a universal inhibitor for all four S. aureus Agr QS systems32. TrAIP-II competes with the cognate AIPs for binding to the receptor32. Unless otherwise indicated, AIP-I acts as an autoinducer agonist and TrAIP-II acts as a competitive antagonist to S. aureus agr-I (
The compositions described herein can be used to treat surfaces to modulate QS, biofilm formation, biofilm streamer formation, and/or virulence factor production or any other QS-controlled trait of interest. For example, the invention can reduce and/or prevent QS, biofilm formation, biofilm streamer formation, and/or virulence factor production on structures most susceptible to colonization and/or clogging by microorganisms by attaching a QS modulating molecule to the surface. In one embodiment, the QS modulator attached surface may be used to treat component parts or particular materials in microfluidic or other benchtop-sized assay systems. In another embodiment, the QS modulator attached surface may be used to treat industrial fluid handling systems or other areas where fluid is directed along channels. In a further embodiment, a QS modulator can be coated on any surface made of any material in medical devices or tools used in medicine. In a further embodiment, various types of QS modulator molecules and/or antibiotics and/or enzymes and/or antibodies can be simultaneously coated onto the same surface to target various microorganisms at the same time. In a further embodiment, the surface coated with a QS modulating molecule is resistant to multiple infections or colonization events. In a further embodiment, methods of screening for agonists or antagonists of QS, biofilm formation, biofilm streamer formation, and/or virulence factor production can be performed using a surface with a test compound linked to the surface. These screens may additionally be run in the presence of various antibiotics to detect effectors that enhance antibiotic inhibition. In a further embodiment, methods for detecting microorganisms can be performed using a surface coated with a QS modulator molecule.
For example, the QS modulator attached surface, and preferably a QS antagonist attached surface, has applications for any natural or artificial surface where the presence of a microorganism, such as, for example, S. aureus could be detrimental. These compositions have immediate applications for medical and health-care devices in which a microorganism, such as for example, S. aureus, colonizes. The QS modulating molecule attached surface as described herein are expected to inhibit biofilm formation, biofilm streamer formation and/or toxin synthesis of a microorganism by interfering with the QS regulatory network, thereby reducing the severity of infection and/or colonization caused in these devices. Furthermore, the QS antagonist attached surface are not prone to bacterial antibiotic-resistance, leading to improved treatment for the bacterial infections. The QS antagonist attached surface can be applied to other devices in which bacterial or other microorganism contamination is a concern. Examples include water supply lines, filters, stents, and intubation tubes, etc. In addition, the QS antagonist attached surface can affect a broad range of other Gram-positive and/or Gram-negative pathogens that use QS pathways to control virulence, including but not limited to, Staphylococcus epidermidis, Streptococcus pneumonia, Streptococcus mutans, and Streptococcus sanguinis.
More specifically, QS modulating molecule attached surface can be used in industrial settings, either in the presence or absence of antibiotics, to inhibit or prevent QS, biofilm formation, biofilm streamer formation, and/or virulence factor production and/or to remove antibiotic resistant bacteria, such as in a hospital or other public setting. For example, the QS antagonist attached surface can be used to remove biofilms that have grown on hospital surfaces, in moist and warm environments, such as showers, water and sewage pipes, cooling or heating water systems, (e.g., cooling towers), marine engineering systems, such as, for example, pipelines of the offshore oil and gas industry. The QS antagonist attached surface can also be used, for example, to remove and/or prevent bacterial adhesion to boat hulls, since once a biofilm of bacteria forms, it is easier for other marine organisms such as barnacles to attach. The QS antagonist attached surface can be used to reduce, for example, the time a boat is in dry dock for refitting and repainting, thereby increasing productivity of shipping assets, and useful life of the ships. The QS antagonist attached surface can also be used to remove biofilm production intentionally used to eliminate petroleum oil from contaminated oceans or marine systems, once the contamination is removed.
Additionally, the QS antagonist attached surface can be used to wash, rinse or swab floors and counters, such as in food preparation areas or medical facilities, as well as medical devices, including but not limited to, stents, catheters, intubation tubes, or ventilator equipment. Still further, the QS antagonist attached surface can be used as a handwash to help eliminate spread of virulent bacteria by health workers, patients and others.
The QS modulator attached surface can also be used to coat an implantable medical device, part of machinery used in industrial processes, a culvert, a pool used in a waste water treatment facility, waste water treatment facility, a pipe, a cooling tower, a medical device, industrial fluid handling machinery, a wound, within the body, a medical process, agricultural processes, and/or machinery
Particular species of bacteria may be especially problematic. For example, Pseudomonas aeruginosa is a pathogen that can survive in a wide range of environments. The bacterium is a public health threat because it causes a variety of secondary infections in humans, where those with burn wounds, cystic fibrosis, and implanted medical devices and cancer patients receiving chemotherapy are particularly at risk. With an outer membrane of low permeability, a multitude of efflux pumps, and various degradative enzymes to disable antibiotics, P. aeruginosa is difficult to treat. As with other common pathogenic bacteria, antibiotic-resistant strains are an increasing problem.
Thus, the QS modulator molecule attached surface invention can be used to treat bacterial infections (e.g., sepsis or infections from cystic fibrosis) in a patient, particularly those bacterial inventions cause by antibiotic resistant strains. In preferred embodiments, the bacterial infections are caused by P. aeruginosa and/or S. aureus.
The surface attached QS modulating molecule can be used to positively and negatively manipulate QS in bacteria such as S. aureus. For example, agonists or antagonists of QS can be attached to a surface via a linker, such as polyethylene glycol (PEG) polymers, which are known to be flexible, hydrophilic, and non-bulky. These surface-attached molecules can successfully be used to control bacterial QS, which has potential implications for the development of anti-biofouling or anti-colonization materials in industry and medicine, respectively. In S. aureus, Agr QS activation leads to the production of a battery of virulence factors that are reponsible for invasion and dissemination in host tissues10. Agr QS in S. aureus also activates the biofilm disassembly process42. Thus, precisely manipulating Agr QS using synthetic strategies can terminate virulence while not enabling biofilm formation. Due to resistance of S. aureus, there is an urgent medical need for the control of S. aureus.
Antagonist-coated surfaces can be used in scenarios such as acute infections, e.g. staphylococcal scalded skin syndrome and toxic shock syndrome where it is essential to halt production of exo-toxins. Reciprocally, autoinducer-coated surfaces can be used to treat chronic infections, e.g. pneumonia or medical device-related infections, where S. aureus biofilms are the major issue. Indeed, S. aureus cells residing in surface-bound biofilms are more resistant to antibiotics and host immune defenses than are their planktonic counterparts43. AIP-I-coated surfaces, by triggering biofilm dispersal and transitioning the S. aureus cells to the planktonic lifestyle, can render surfaces resistant to biofilm colonization and, furthermore, render the dispersing cells more susceptible to antibiotics and to host immune defenses.
The techniques provided herein for coating surfaces with pro- or anti-QS molecules and using them to influence bacterial behaviors are not limited to S. aureus. These strategies are generally applicable to any bacterial strain. For example, the Gram-positive bacterium Enterococcus faecalis causes life-threatening urinary tract infections, bacteremia, endocarditis, and meningitis in humans44. Pathogenicity of E. faecalis relies on the Fsr QS system, which is homologus to the S. aureus Agr QS system45. However, importantly, in the case of E. faecalis, activation of Fsr QS promotes both biofilm formation and virulence factor production45,46. Thus, surfaces harboring Fsr QS antagonists (i.e., ZBz1-YAA591147) can have a dual benefit in preventing biofilms and reducing exo-toxin production. Such dual benefits can also be imagined for other bacteria such as Listeria monocytogenes and Streptococcus pyogenes, as both pathogens possess Agr-type QS systems that activate biofilm formation and virulence factor expression at high cell density48. Moreover, the beneficial bacterium Lactobacillus plantarum, which is important in the dairy and fermented food industries, also has an Agr-type QS system called Lam that could be manipulated using surfaces in applications in food production49. Thus, any bacterial system capable of QS can be modulated based on the techniques provided herein.
Clearly, the techniques provided herein have the potential to be expanded to other systems with known ligands and with accessible cognate receptors. This method is especically useful for Gram-positive bacteria because they do not possess an outer membrane. Strategies for Gram-negative bacteria can be explored by exploiting surfaces coated with QS-manipulation compounds together with molecules that form pores in the outer membrane such as holins, endolysins, or bacteriocins50. Likewise, surface-attached QS molecules could be examined with other orthogonal approaches that exploit surface release of active compounds22. Present invention embodiments are not intended to be limited to the examples provided herein, and may be applicable in any bacterial system (or non-bacterial system) which utilizes QS.
Administration of the QS modulating attached surface to a subject is specifically contemplated. For example, a QS antagonist attached to a small particle/bead, such as, for example, a nanoparticle, can be administered to a patient to reduce or eliminate an infection.
Blocking virulence is one of the strategies contemplated to combat these bacteria. This approach provides less selective pressure for the spread of resistant mutants and leads to drug therapies that are effective over a greater time span compared to traditional antibiotics. Rather than preventing growth or killing the bacteria, an antivirulence approach prevents the expression of virulence traits. The bacteria that have been treated and are thus benign should then be more easily cleared by the host immune system.
The QS modulating molecule attached surface is resistant to multiple infections or colonization events by microorganisms. The surface can be simultaneously attached to various QS modulating molecules and/or antibiotics and/or enzymes.
Examples of QS modulating molecules are shown in Tables 1A-1B. Each of the references in Table 1A are included herein in their entirety.
Unless otherwise indicated, the compounds of PCT/US2014/056497 and U.S. Pat. No. 8,568,756 function as antagonists of QS to inhibit the QS pathway.
It is also expressly understood that the compounds referred to (and incorporated by reference) in PCT/US2014/056497 are limited to those that exhibit anti-pathogenic and anti-biofilm activity through inhibition of QS.
Unless otherwise indicated, the compounds of U.S. Pat. No. 8,535,689 and WO 2014/092751 function as agonists. Some QS systems, such as those found in cholera, work “in reverse” from other QS systems. For example, agonists of cholera QS receptors repress biofilm formation and pathogenicity, effectively functioning as inhibitors of bacterial infections.
Other molecules, including flavonoid compounds, function as antagonists of QS, and are shown in Table 1B.
It is expressly understood that present invention embodiments include both agonists and antagonists. In some systems, compounds act as antagonists with respect to the QS system to repress biofilm formation and pathogenicity, while in other systems, compounds act as agonists with respect to the QS system to repress biofilm formation and pathogenicity.
Any substrate can be used to conjugate a linker. For example, substrates can be any material, for example, but not limited to, polymers, metals, and/or ceramics. In this example, glass and gold substrates are used. Other examples of substrates are shown below in Table 2.
In some embodiments, linkers are chosen to be biocompatible and flexible. Linkers can be a variety of lengths and chemistries and they can contain different chemical moieties. The linker used in this study was Polyethylene Glycol (PEGs). Other examples of substrates are shown below in Table 3, but are in no way limiting.
Any specific chemistry can be used to make a chemical bond between a surface and a linker. Non-limiting examples include silanization, gold-sulfide bond formation, thiol-ene reactions, and surface-initiated polymerization.
In a preferred embodiment, the linker was covalently bound to the surface by using a free hydroxyl moiety on the glass substrate (e.g., see
In the example of thiol-ene reactions (see, e.g.,
In the example of the gold-sulfide bond formation reaction, commercially available gold-coated glass slides were submerged in acetone for 5 min and then dried. (See, e.g.,
In another example of thiol-ene reactions (See, e.g.,
In the example of the surface-initiated polymerization, RAFT (reversible-addition fragmentation chain transfer) polymerization was used, but in no way is limiting. The polymerization involved radical formation of a monomer in the presence of a RAFT agent such as a trithiocarbonate compound. In one preferred method, N-(2-hydroxylpropyl)methacrylamide (HPMA) was used and synthesized in the following way: 36 g (0.34 mol) of anhydrous sodium carbonate (Na2CO3) was suspended in 85 ml (freshly distilled) methylene chloride (CH2Cl2). The solution was cooled to −10° C. (using dry ice), and 25 ml (0.30 mol) of 1-aminopropan-2-ol was added. The suspension was maintained at −10˜° C., and (freshly distilled) 31.9 ml (0.29 mol) methacryloyl chloride was added drop-wise under cooling and vigorous stirring within 60 min. The reaction mixture was stirred for another 20˜30 min at 5˜15° C. 10 g of anhydrous sodium sulfate (Na2SO4) was added, the solid was filtered twice and the dry filtrate was concentrated to half volume under reduced pressure until formation of a crystallization seed. HPMA was obtained by crystallization from methylene chloride at −20° C. overnight, and purified by recrystallization from acetone.
In a preferred method, N-(Azido(PEG)) methacrylamide (AzPMA) was used and synthesized in the following way: 1.1 g (11 mmol) of Triethylamine (N(CH2CH3)3) was dried over MgSO4 and together with 1 g (1 ml, 4.53 mmol) of 11-Azido-3,6,9-trioxaundecan-1-amine, 3.4 mg of hydroquinone and dry 7.5 ml of methylene chloride (CH2Cl2) cooled to 0° C. in an ice-water bath. 1 mL (10 mmol) of methacryloyl chloride was added dropwise to the reaction mixture over 20 min followed by stirring at 0° C. for 1 hour and at room temperature for 14 hours. 3.6 ml of methylene chloride (CH2Cl2) was added and the mixture was washed twice with aqueous HCl (2 M), aqueous NaOH (2 M), and water (15 mL). The organic phase was dried over anhydrous MgSO4, and the solvent removed by evaporation to yield the final product AzPMA.
In another preferred method, surface-initiated polymerization using two types of monomers, HPMA and AzPMA, was performed using the following procedure: The glass substrate was coated with a thiol moiety by submersing it in 20% (3-Mercaptopropyl) trimethoxysilane in acetone for 60 min. The glass slide was rinsed with acetone twice, and subsequently rinsed with millipore water. The slide was transferred to a schlenk flask, and mixed with a solution of HPMA: 4-cyanopentanoic acid dithiobenzoate: 4,4-azobis(4-cyanovaleric acid)=800:3:1 in methanol. In this example, HPMA 71.59 mg (0.5 mmol) should be dissolved in 0.5 ml methanol with 0.5238 mg of 4-cyanopentanoic acid dithiobenzoate and 0.175 mg of 4,4-azobis(4-cyanovaleric acid). The sample with the solution was subsequently subjected to three-freeze-pump-thaw cycles. The polymerization was performed at 70° C. for 15-20 hours. 71.59 mg AzPMA was added to the flask, and the reaction was carried out for an additional 5 hours. After the reaction, the reacted slides were exposed to ethyl acetate 150 ml, and then to the mixture of methanol and ethyl acetate. The final glass slides had a polymer linker that contained p(HPMA)-p(AzPMA) with 100-200 mononers. (See, e.g.,
The length of PEG linkers can be varied. For example, in this example linkers in the 10-200 nm range were used. Mixing with a small amount of short PEG linkers is recommended when longer PEG linkers are used as the short linkers helps maintain the structural integrity of the longer linkers when attached to a surface.
Any specific chemistry can be used to make a chemical bond between a linker and a QS modulating molecule. For example, a bioorthogonal reaction can be used. This reaction is highly selective and has no side reactions. The chemistry is biocompatible and thus not toxic to living organisms, and the fast kinetic reactions make this process especially convenient. Many reported bioorthogonal reactions are known in the art and can be used to conjugate linkers to QS-modulating molecules, such as Staudinger Ligation and/or click chemistry. Additional reactions that are known in the art and that can be used include, but are not limited to: nitrone dipole cycloaddition, norbornene cycloaddition, tetrazine ligation, and/or quadricyclane ligation.
For example, we used the click chemistry method as one example. (See, e.g.,
First, the alkyne was attached to the QS autoinducer AIP (autoinducing peptide from S. aureus). All amino acid derivatives and resins were purchased from Novabiochem (San Diego, Calif.). All other chemical reagents were purchased from Sigma (St. Louis, Mo.). Analytical gradient reverse-phase high-performance liquid chromatography (HPLC) was performed on a Hewlett-Packard 1100 series instrument. Analytical HPLC was performed on a Vydac C18 column (particle size 5 μm, inner diameter 4.6 mm, length 150 mm) at a flow rate of 1 mL/min. Semi-preparative HPLC was performed routinely on the Vydac C18 column at a flow rate of 4 mL/min. All runs used linear gradients of 0.1% aqueous TFA (solvent A) vs 90% acetonitrile plus 0.1% TFA (solvent B). Mass spectrometry was performed on a Sciex API-100 single quadrupole electrospray mass spectrometer.
Autoinducer peptides (AIPs) were chemically synthesized as described in using standard solid-phase approaches. See, for example, George E A, Novick R P, Muir T W. Cyclic peptide inhibitors of staphylococcal virulence prepared by Fmoc-based thiolactone peptide synthesis. J Am Chem Soc. 2008;130(14): 4914-24. In particular, MBHA resin was preloaded with (tert-butoxycarbonyl) aminoacyl-3-mercaptopro-pionamide. Boc-protected amino acids were coupled with HBTU in DMF, and the deprotection was performed with neat TFA. HF-cleavage was used for releasing of the thioester and the global deprotection. The crude peptide product was precipitated and washed with chilled ethanol. After dissolving the peptide in 50% CH3CN/50% water/0.1% TFA, MBHA resin was removed through filtration. The crude peptide was purified with semi-preparative RP-HPLC and lyophilized. Cyclization was done by dissolving the peptide in 50% CH3CN/50% water and 0.1 M phosphate buffer at pH 7 and incubating at room temperature for 2 hours. After another semi-preparative RP-HPLC purification, the product was characterized with analytical RP-HPLC and mass spectrometry.
Next, an azide group attached to one end of the PEG linker that is fixed to the glass surface will react with the alkyne on the modified AIP (QS molecule) in the presence of Cu. Specifically, 1 mM Copper (II) Sulfate in water, 50 mM ascorbic acid and 10 μM alkyne AIP were used to treat the linker-attached substrates for 30 min-60 min at room temperature. Blowing nitrogen air and injection of water were used to remove unbound chemicals. Any autoinducers as described herein (agonist or antagonist) can be used for conjugation to the surfaces. As a proof of principle, we used S. aureus autoinducer (AIP) peptides, but the chemical reaction is identical for other QS molecules.
In this Example, Cu was used as a catalyst. There are also Cu-free ways to conduct the same chemical reaction which are well known in the art.
Methods for measuring QS, biofilm production, biofilm streamer production and/or virulence factor production have been reported in the literature and are herein incorporated by reference in their entirety. Kim MK et al. “Local and global consequences of/low on bacterial quorum sensing,” Nature Microbiology 1:15005 2016; Kim MK et al. “Filaments in curved streamlines: Rapid formation of Staphylococcus aureus biofilm streamers,” New J Phys. 2014 Jun 26;16(6):065024; Ng WL, et al., “Broad spectrum pro-quorum-sensing molecules as inhibitors of virulence in vibrios,” PLoS Pathog. 2012;8(6); and O′Loughlin CT, “A Quorum-Sensing Inhibitor Blocks Pseudomonas Aeruginosa Virulence And Biofilm Formation,” PNAS (2013) Oct 29;110(44):17981-6. Compositions of the invention can be tested in any of these published protocols.
QS Measurements
QS measurements at the transcriptional level were assessed using promoter fusion analysis. Promoters driving genes responsible for QS (e.g., in the case of P. aeruginosa, the lasl and rhll promoters were used. In the case of V. cholerae, the qrr4 and/or luxC promoters were used. In the case of S. aureus, the agrP3 promoters were used to measure QS activities. In all cases, the promoters were fused to genes encoding fluorescent proteins, luciferase, or the beta-lactamase enzyme, and/or an equivalent which can be quantitatively measured temporally and spatially using a microscopy or a spectrometer. Other promoters and/or reporter proteins could readily be used.
QS phenotypes are diverse, but in the context of healthcare settings, measuring pathogenic traits that are regulated by QS systems, such as virulence factor production and biofilm formation, are of interest. The following example assays may be used to quantitatively measure such traits.
Virulence Factor Production Measurements
Virulence factor production at the transcriptional level is assessed using promoter fusion analysis. Promoters driving genes responsible for virulence factors, e.g., in the case of P. aeruginosa, the lasAB and rhlAB promoters were used. In the case of V. cholerae, the ctxAB, toxT and hapA promoters were used. In the case of S. aureus, the hldBC and clfB promoters were used. In all cases, the promoters were fused to genes encoding fluorescent proteins, luciferase, or the beta-lactamase enzyme, or an equivalent which can be quantitatively measured temporally and spatially using microscopy or a spectrometer. The actual virulence factor (toxin, enzymes, etc.) can also be measured directly. Specifically, one can measure or verify the results from promoter-reporter fusions using enzyme-linked immunosorbent assay (ELISA) techniques, in which toxins from a sample are transferred to a membrane, and subsequently, antibodies that recognize the specific toxin are introduced. The antibodies are usually linked to an enzyme or a fluorophore that can be quantitatively measured.
Biofilm Production Analysis
One can measure the amount of biofilms formed using cells carrying a constitutively expressed fluorescent protein. Microscopy can be used to measure the 3D volumes or biomass.
Biofilms can also be measured using a conventional method. There are many commercially available stains that specifically bind to components of biofilms, such as the polysaccharide matrix and/or extracellular DNA. Subsequently, using microscopy, the amount of biomass can be quantified. Biofilms can also be measured in a commonly used microtiter plate assay and crystal violet staining.
The strains and plasmids used are listed in Table 4. Staphylococcus aureus strains include RN4220, RN9011, RN6390b, RN6911, and RN6607. Plasmids include pJL1111 and pRN7062. S. aureus strains MK12118 (RN6390b carrying pMK021; agrP3-gfpmut2, sarAP1-mkate2) and S. aureus MRSA strain MK13118 (BAA1680 carrying pMK021) were also used.
E. coli
S. aureus
DNA polymerase, dNTPs, and restriction enzymes were purchased from New England Biolabs (NEB, Ipswich, Mass.). DNA extraction and purification kits were acquired from Qiagen (Valencia, Calif.). DNA oligonucleotides were purchased from Integrated DNA Technologies (Coralville, Iowa). Sequences of plasmids were verified by Genewiz (South Plainfield, N.J.).
Plasmids carrying constitutively expressed fluorescent fusions were constructed by replacing the mkate2 gene from pMK014 (sarAP1-mkate2) with genes encoding different fluorescent proteins (gfpmut2, mturquoise2, and mko). To make these plasmids, the gfpmut2 gene was amplified by PCR from pMK02118 using primers MKF013/MKR013, the mturquoise2 gene51 was amplified by PCR from pDP428 using primers MKF011/MKR011 and the mko gene was amplified by PCR from pCN00552 using primers MKF014/MKR014. mkate2 was replaced with an amplified gene by overlap extension PCR cloning. These plasmids were called pMK012 (sarAP1-gfpmut2), pMK011 (sarAP1-mturquoise2), and pMK013 (sarAP1-mko).
The constitutively expressed reporter fusions were integrated into the S. aureus chromosome using a site-specific integration suicide vector, pJC1111, carrying a cadmium resistance cassette and a SaPI-1 attS sequence which integrates into the S. aureus chromosomal attachment site (attC) of pathogenicity island 1 (SaPI-1)53. This plasmid was integrated in single copy and maintained stably53. pJC1111 was digested using restriction enzymes NarI/SphI. The sarAP1-gfpmut2 gene was amplified by PCR from pMK012 using primers MKF031/MKR031 followed by digestion with NarI/SphI and ligation into digested pJC1111. This plasmid was called pMK032 (sarAP1-gfpmut2 in the suicide vector). The same procedure was used for other fluorescent genes, pMK031 (sarAP1-mturquoise2 in the suicide vector), and pMK033 (sarAP1-mko in the suicide vector). The plasmids were introduced into Escherichia coli DH5α using chemical transformation (New England Biolabs, Ipswich, Mass.) followed by selection with ampicillin. The plasmids were purified from E. coli, introduced by electroporation into S. aureus strain RN9011, which expresses the SaPI-1 integrase, and colonies containing the fusions integrated into the chromosome were selected with cadmium. Subsequently, the chromosomal integrants were transduced into S. aureus strain RN6911 using standard phage transduction techniques with phage 80α. These strains were called MK232 (sarAP1-gfpmut2 in the genome), MK231 (sarAP1-mturquoise2 in the genome), and MK233 (sarAP1-mko in the genome). Primers are shown in Table 5.
A plasmid carrying a transcriptional fusion to monitor S. aureus Agr QS activity was constructed by replacing the lacZ gene from vector pRN706254 (agrP3-lacZ) with the mkate2 gene. pRN7062 also harbored the genes encoding the Agr QS detection components agrCA under their native agrP2 promoter but driven in the opposite direction. To make this plasmid, the lacZ gene was removed from pRN7062 by digestion with EcoRI/NarI. The mkate2 gene was obtained from pMK014 by EcoRI/NarI digestion. The digested mkate2 gene was ligated into digested pRN7062. This plasmid was called pMK051 (agrP2-agrCA, agrP3-mkate2). This construct was first introduced into E. coli, purified, and subsequently introduced into S. aureus strain RN4220 using selection with erythromycin. Subsequently, using phage transduction, the plasmid was introduced into S. aureus strains MK232, MK231, and MK233. The resultant strains were called MK242 (sarAP1-gfpmut2 in the genome and pMK051), MK241 (sarAP1-mtor2 in the genome and pMK051), MK243 (sarAP1-mko in the genome and pMK051). To construct the S. aureus AagrBDCA strain harboring agrP3-mkate2 in a plasmid, the vector pMK00418 (agrP3-mkate2) was introduced into S. aureus strain MK232 (sarAP1-gfpmut2 in the genome of S. aureus AagrBDCA), leading to strain MK245.
Control strains were constructed to study heterogeneity of the Agr QS response. The first control strain had the agrP2-agrCA and agrP3-mkate2 genes inserted into the genome of RN6911, and harbored sarAP1-gfpmut2 in a plasmid. To make this strain, the agrP2-agrCA, and agrP3-mkate2 genes from pMK051 were amplified using primers MKF032/MKR032, and this fragment was inserted into the suicide vector pJC1111 by overlap extension PCR cloning. This plasmid was called pMK064 (agrP2-agrCA, agrP3-mkate2 in the suicide vector). The gene was integrated into the S. aureus strain RN6911 chromosome as described above. This strain was called MK264 (agrP2-agrCA, agrP3-mkate2 in the genome). The vector pMK012 (sarAP1-gfpmut2) was introduced into MK264, leading to strain MK265 (agrP2-agrCA, agrP3-mkate2 in the genome and pMK012). The second control strain was constructed by introducing pMK014 (sarAP1-mkate2) into strain MK232, leading to MK244 (sarAP1-gfpmut2 in the genome and pMK014). The third control strain had the agrP2-agrCA gene inserted into the genome of RN6911, and harbored agrP3-mkate2 in a plasmid. To construct this strain, the agrP2-agrCA gene from pMK051 was amplified using primers MKF033/MKR033, this fragment was inserted into the suicide vector pJC1111 by overlap extension PCR cloning. This plasmid was called pMK060 (agrP2-agrCA in the suicide vector). The gene was integrated into the S. aureus strain RN6911 chromosome as described above. This strain was called MK260 (agrP2-agrCA in the genome). The vector pMK00418 (agrP3-mkate2) was introduced into MK260, leading to strain MK261 (agrP2-agrCA in the genome and pMK004). Finally, a constitutively expressed mKO fluorescent reporter (sarAP1-mko in pMK013) into wild-type S. aureus agr-I (strain RN6390b) and wild-type S. aureus agr-II (strain RN6607) was used to measure the number of cells in biofilms on surfaces.
S. aureus RN6911 derivatives were grown overnight at 37° C. with shaking in Tryptic Soy Broth (TSB; Difco, Franklin Lakes, N.J.) with 10 μg/ml tetracycline and 10 μg/ml erythromycin to maintain plasmids, back-diluted 1:200, and re-grown for 3 h (to OD600˜0.05-0.1). S. aureus MK121, MK131, MK125, and MK126 were grown overnight at 37° C. with shaking in TSB with 10 μg/ml erythromycin, back-diluted 1:2000, and re-grown for 3 h (to OD600˜0.05-0.1).
AIP-I, AIP-II, and TrAIP-II derivatives were synthesized using a combined solid-phase/solution-phase approach. Linear peptide a-thioester precursors were generated using Fmoc-solid phase peptide synthesis employing a hydrazine linker system. The peptides were then cyclized in solution to create the thiolactone macrocyclic.
Transcription from fluorescence reporter genes was measured in S. aureus strain MK242. Overnight cultures were diluted 1:200 into fresh TSB with 10 μg/ml tetracycline and 10 μg/ml erythromycin, re-grown, and 90 μl of these cultures were distributed into wells of 96 well plates (MatTek, Ashland, Mass.), followed by addition of 10 μl of AIP-I and/or TrAIP-II and/or derivatives. Subsequently, 50 μl of mineral oil was added (Sigma, St. Louis, Mo.) to prevent evaporation. Using a Synergy 2 plate reader (Biotek, Winooski, Vt.), GFPmut2 and mKate2 levels were measured at 484 nm/528 nm and 588 nm/633 nm, respectively. Measurements were conducted with 15 min intervals at 37° C. with shaking. This assay was called “Solution Assay”.
Surface-PEG10000-azide, Surface-PEG400-azide, Surface-PEG10000, and Surface-PEG400 were fabricated as follows (see
The azides decorating the surface-attached PEG400 and PEG10000 were subjected to the click reaction to link molecules carrying alkyne groups, such as Alkyne-AIP-I, Alkyne-AIP-II, Alkyne-TrAIP-II, and Alkyne-dye. The solution for the click reaction contained: 1 mM CuSO4, 50 mM ascorbic acid, 5 mM Tris (3-hydroxypropyltriazolylmethyl)amine, and 100 μM alkyne molecule in 100 mM phosphate buffer (pH=7). Surface-PEG10000-azide and Surface-PEG400-azide were incubated with the click solution containing the alkyne molecules for 3 h. After rinsing the substrates three times with dimethyl sulfoxide (50% in water, Sigma) to remove any unreacted chemicals, the surfaces had become linked by the triazole rings to the AIP-I, AIP-II, TrAIP-II, or the dyes.
To investigate long-term surface stability, the Surface-PEG10000-triazole-AIP-I and the Surface-PEG10000-triazole-dye were stored at 4° C. for 40 days in a wet container covered with aluminum foil to prevent desiccation and exposure to light.
The modified surfaces were characterized using a Fourier transform infrared (FTIR) spectrometer, fluorophore labeling, or atomic force microscopy (AFM) as follows: An FTIR spectra for the Surface-PEG10000-azide was obtained as an averaged signal by scanning the sample 128 times at 4 cm−1 resolution using a Thermo Nicolet Nexus 670 FTIR spectrometer (Thermo Electron Corp., Waltham, Mass.) equipped with a liquid nitrogen-cooled MCT/A detector (
To characterize the Surface-PEG10000-azide, Alexa Fluor 555 dye functionalized with an alkyne moiety (Thermo Fisher, Mass.) was used. The Surface-PEG10000 was used as the negative control. The surfaces were treated with the click solution containing the Alexa Fluor 555 alkyne dye. After washing away the unreacted dyes, the surfaces were imaged by confocal microscopy (
AFM was used to measure the heights of the polymer brushes on the Surface-PEG10000-azide and the Surface-PEG400-azide. The surfaces were allowed to dry via evaporation in air. AFM topographical images of the boundaries between the PEG-attached regions and the unmodified regions were obtained in air using a Bruker Dimension Icon AFM (Bruker Biosciences, Billerica, Mass.) equipped with AFM tips (Bruker Biosciences) in Scanasyst-air peak force tapping mode. The scan regions were 2×2 μm2. The heights of surface-attached polymers were measured as the difference between the unmodified region and the PEG-attached region. Several regions from two independently fabricated surfaces were scanned.
The chemically modified surfaces were bonded to microfluidic chambers (400 μm×100 μm×2 cm) using an epoxy glue (Fisher Scientific). The assembled chambers were inoculated with the S. aureus agr-I reporter strain cultures, and cells were allowed to settle onto surfaces for 10 min, after which sterile M9 medium containing 10 μg/ml erythromycin, 0.5% glucose, 0.5% casamino acids, 5 mM NaCl, 50 mM MgSO4, and 0.1 mM CaCl2 was flowed steadily into the devices for 30 min to remove planktonic cells. After this step, chambers were placed on the microscope and the fluorescent reporter output measured. In experiments containing autoinducer or antagonist in solution, molecules were added in the wash medium. In experiments containing human blood plasma (Biological Specialty Corp, Colmar, Pa.), 100% blood plasma solution was initially flowed into the chambers containing the chemically modified surfaces for 10 min. Subsequently, S. aureus agr-I reporter cells were seeded for 10 min, and then 20% or 50% blood plasma diluted in the above medium was flowed into the chambers for 30 min to remove the planktonic cells. After this step, chambers were placed on the microscope and the fluorescent reporter output measured.
Chambers containing modified surfaces were seeded with wild-type S. aureus strains harboring constitutively expressed mko on a plasmid, and the cells were allowed to settle onto the surfaces for 10 min, after which sterile TSB containing 3% NaCl and 10 μg/ml erythromycin was flowed steadily into the devices for 8 h. This procedure was sufficient to produce three-dimensional S. aureus biofilms of 10-30 μm thickness. Flow was terminated for 10 h, and subsequently, planktonic cells were removed by steady flow for 10 min. The remaining biofilms were measured by confocal microscopy. Cells in biofilms covering a surface area of 225×225 μm2 were imaged at six different regions for each surface type in n=3 independent experiments.
Imaging was performed using a Nikon Eclipse Ti inverted microscope (Melville, N.Y.) fitted with a Yokogawa CSU X-1 confocal spinning disk scanning unit (Biovision Technologies, Exton, Pa.) and DU-897 X-9351 camera (Andor, Concord, Mass.). Laser lines at 445, 488, 543, and 592 nm were used to excite the mTurquoise2, GFPmut2, mKO, and mKate2 fluorescent proteins, respectively. Laser lines at 488, 543, and 592 nm were used to excite Alexa Fluor 488 fluorophore, Alexa Fluor 555 fluorophore, and Alexa Fluor 594 fluorophore, respectively. In order to obtain single-cell resolution, both a 100× oil objective with N.A. 1.4 (Nikon, Melville, N.Y.) and a 1.5× lens placed between the CSU X-1 and the Nikon microscope side port were used. Consequently, the magnification of 0.1 μm per pixel in the XY plane was obtained. For single-cell analysis, custom code was written in Matlab. Briefly, the area of an individual cell was recognized and segmented using a watershed-based algorithm. In this process, cells were removed if they were on the edge of the image or if they were smaller than 30% of the average cell size, suggesting that they were out of focus. In the area of an individual cell, both the constitutive GFPmut2 fluorescence and the QS controlled mKate2 fluorescence were measured, subtracted from background signals and summed. The normalized QS output was calculated as the QS controlled mKate2 intensity divided by the constitutively expressed GFPmut2 intensity in individual cells. In each experiment, images of many regions on the surfaces were taken to include 1000-4000 individual cells. Each replicate was performed using independent bacterial cultures and independent surfaces at room temperature. Identical procedures were performed for the strains harboring different constitutive fluorescent proteins such as mTurquoise2 and mKO (
The coverage density of “clicked” azides on the Surface-PEG10000-azide was quantified based on fluorophore labeling. The alkyne-dye, the Alkyne-AIP-I, and the Alkyne-TrAIP-II were assumed to have identical reactivity in the click reaction, rendering the same surface coverage density. Thus, surface coverage density can be quantified using fluorophore labeling as a proxy for surface-attached QS compounds. In the fluorophore labeling assay, the surface-attached azides underwent the click reaction with 100 μM Alexa Fluor 555 alkyne fluorophore. After conjugation, the total intensity of fluorescence from the surface was measured (at 10% laser power), subtracted from the background signal, and divided by the average single-molecule intensity. The average single-molecule intensity was obtained by measuring the intensity of single Alexa Fluor 555 molecules as follows: the Surface-PEG10000-azide was treated with 0.1−10 nM Alexa Fluor 555 alkyne fluorophore, and this low concentration of alkyne-dye produced individually discernable fluorescent spots (20-300 in a 51×51 μm2) (
Surface-attached compounds were used to positively and negatively manipulate S. aureus Agr QS. Copper-catalyzed azide-alkyne cyclo-addition click chemistry was used to attach active compounds to surfaces33. Polyethylene glycol (PEG) polymers decorated with azide groups were selected as the surface linkers because PEG is flexible, hydrophilic, and non-bulky.
To make AIP-I amenable to surface attachment, AIP-I containing an alkyne at the N-terminus was synthesized (
Table 6. EC50 values (from n=3 experiments) for AIP-I and its derivatives and IC50 values (from n=4 experiments) for TrAIP-II and its derivatives when AIP-I is present at 100 nM (i.e., the EC95).
For the surface modification work, silanization and maleimide-thiol chemistry was used to decorate the surface with PEG10000 polymers carrying azide moieties at one end (
To reinforce the above results, it was also shown that S. aureus did not activate a QS response when introduced onto the identical surface lacking the triazole-AIP-I decoration (Surface-PEG10000) or that had not undergone the click reaction (Surface-PEG10000-azide) (
By changing particular features of the tethered molecules for the surface-attached AIP-I to bind AgrC-I and activate Agr QS in S. aureus, it could be determined which features of these molecules were responsible for activity. Presumably, AgrC-I diffuses freely in the membrane35. In S. aureus, the plasma membrane is covered by a 15-30 nm peptidoglycan layer with 4-5 nm diameter pores36. Peptidoglycan is considered to be an elastic mesh-network that can expand, contract, and tolerate transport of globular molecules of up to 100 kDa36.
Initially, it was determined how altering the length of the polymer attaching the AIP-I to the surface influenced the S. aureus Agr QS response. AIP-I was attached with two different surface linkers: Surface-PEG10000-azide and Surface-PEG400-azide. Using atomic force microscopy, the heights of the linkers were measured to be 26.5 nm (±2.5) and 1.9 nm (±0.2), respectively. The calculated Flory radius of PEG10000 in solution was previously determined to be ˜10 nm34, indicating that the surface-attached PEG10000 exists in an extended configuration due to local crowding. This configuration suggests that the terminal functional groups are exposed and thus could properly position attached AIP-I molecules to interact with the AgrC-I receptors. Indeed, the azide moieties on both polymers were amenable to the click reaction using alkyne-functionalized dyes (Surface-PEG10000-azide,
To examine how surface coverage density of PEG10000-triazole-AIP-I affects activation of AgrC-I-directed QS, the number of reacted azides in a unit area on the Surface-PEG10000-azide following the click reaction was measured. To do this, a fluorescent dye harboring an alkyne was joined to the Surface-PEG10000-azide (
It was investigated whether a QS antagonist, when immobilized on a surface, could interfere with Agr QS signal transduction. A clickable QS antagonist was synthesized, the Alkyne-TrAIP-II (
QS inhibition by surface-bound TrAIP-II was examined. Introduction of the S. aureus reporter strain to the Surface-PEG10000-azide in the presence of AIP-I in solution at its EC50 (30 nM) resulted in activation of Agr QS (
It was investigated whether surface-attached pro- and anti-QS molecules could control S. aureus behaviors in the more natural and clinically-relevant context of S. aureus strains that are capable of producing AIP-I37, unlike the reporter strain used above. For this analysis, the fluorescent QS reporter genes were introduced into wild-type S. aureus agr-I (RN6390b) and S. aureus MRSA agr-I. In the presence of the Surface-PEG10000-azide, S. aureus agr-I activated Agr QS in response to the accumulation of endogenously produced AIP-I (
Next, it was explored how the modified surfaces influence S. aureus biofilm colonization dynamics. A constitutively expressed mKO fluorescent reporter was introduced into the wild-type S. aureus agr-I strain and the strain was grown on the Surface-PEG10000-azide, the Surface-PEG10000-triazole-AIP-I, and the Surface-PEG10000-triazole-TrAIP-II (
To expand these results to other surfaces, gold and polydimethylsiloxane (PDMS) surfaces were chemically modified with the dye-triazole-PEG moieties (
Having a reasonable shelf lifetime was also desirable for chemically coated surfaces in medical and industrial applications. The long-term stability of the Surface-PEG10000-triazole-dye and the Surface-PEG10000-triazole-AIP-I as a proxy for modified surface longevity was examined. Irrespective of the presence or absence of S. aureus cells, the intensity of the surface-attached dye did not change for 15 h (
To further explore issues pertaining to medical applications of modified surfaces, QS analysis in the presence of human blood plasma was performed because S. aureus frequently causes bacteremia. Having blood plasma present throughout the experiment did not alter the ability of the AIP-I-coated surface to stimulate S. aureus QS (
2-chloro-trityl chloride resin was first derivatized with hydrazine monohydrate in dimethylformamide (DMF) for activation. Amino acids were coupled through standard Fmoc-based solid phase peptide synthesis. Note that the cysteine was protected with—StBu (or tert-butyl-thiolate). 4-pentynoic acid (5 equiv.) was activated by N,N′-diisopropyl carbodiimide (DIC, 20 equiv.) in 10 ml of CH2Cl2 under N2 for 30 min before coupling to the peptide on resin for another 2 hS10. The peptide was cleaved from the resin by stirring with a cleavage cocktail (95% TFA, 2.5% TIPS, and 2.5% H2O) at room temperature for 2 h. The post-cleavage supernatant was precipitated with cold ether. The resulting pellet was washed twice with cold ether, dried under N2 and purified by preparative reverse-phased high performance liquid chromatography (RP-HPLC) on a C18 column.
The cleaved and purified hydrazide peptide was subjected to oxidation and thioester formation at the C-terminusS11. In the oxidation step, the peptide was dissolved in degassed oxidation buffer (6M guanidine hydrochloride and 100 mM phosphate at pH=3) to a final concentration of 5 mM before reaction with 20 equiv. NaNO2 for 20-30 min in a −10° C. ice-salt water bath. Next, the system was brought back to room temperature and mixed with 100 mM MESNa dissolved in thiol buffer (6M guanidine hydrochloride and 100 mM phosphate) for in situ thioesterification. The pH was adjusted to 6. After running for 2 h, the reaction products were purified by semi-preparative RP-HPLC. The thioester precursor was lyophilized and redissolved in cyclization buffer (50 mM TCEP, 100 mM phosphate of pH 7, 25% ACN) for cysteine deprotection and thiolactone formationS12. The reaction was allowed to run for 2 h at room temperature, followed by purification through semi-preparative RP-HPLC and characterization through analytical RP-HPLC and ESI-MS (
For generation of PEG330-triazole-AIP-I and PEG330-triazole-TrAIP-II, the CuAAC click reaction was performed to incorporate the O-(2-azidoethyl)heptaethylene glycol onto the Alkyne-AIP derivatives. The alkyne-AIPs and PEG330-azide were brought to solution in 100 mM phosphate buffer at pH 7 at 200 μM and 400 μM, respectively. Copper catalyst and ligands were added at final concentrations of 0.10 mM CuSO4 and 0.50 mM Tris(3-hydroxypropyltriazolylmethyl)amine, and subsequently treated with 5 mM sodium ascorbate. The reaction was mixed well and carried out for 1 h. At the end of the reaction, the system was quenched with 2 mM EDTA before purification by semi-prep RP-HPLC and characterization through analytical RP-HPLC and ESI-MS.
All Alkyne-AIPs and PEG330-triazole-AIPs were quantified by 1H-NMRS13. This was a simple approach to determine sample concentration using a standard capillary and an external calibration sample of known concentration. First, 150 μM of DSS was dissolved in D20 as the reference material for the standard capillary. Next, 3 mM glycine dissolved in D2O was used as the calibration sample. Finally, the peptide samples in deuterated DMSO (DMSO-d6) were prepared.
All 1H NMR were performed on a 500 MHz NMR. The NMR spectrum of the glycine calibration sample containing the DSS standard capillary was acquired first, followed by that of the peptide sample containing the same capillary. The intensities of the peaks of interest and of the reference capillary in the samples were determined through MNova (MestreLab) data processing software. In this study, peaks in the corresponding to aromatic protons (from Tyr in AIP-I and Phe in TrAIP-II) were integrated.
The concentration of any sample of interest was determined using equation PI I is the integral (or sum of integrals) of the peak(s) of interest, #P is the number of protons that contribute to the peaks of interest per molecule, and C is the overall concentration of the material in solution. The calibration material is glycine, where the peak of interest corresponds to the two protons at the alpha position. The capillary material is DSS with the peak of interest arising from the nine silicon-shielded methyl protons.
The derivation of equation [1] rested on the assumption that each individual proton in the sample contributes the same amount of area to the peak integral, and that #Pcapillary×Ccapillary is constant for both the calibration sample and the sample of interest because the same capillary insert is used for the two samples.
This application is a Continuation in Part of PCT/US16/29213 filed on Apr. 25, 2016, which claims priority to U.S. 62/152,896 filed on Apr. 26, 2015, and this application also claims priority to U.S. 62/459,482 filed on Feb. 15, 2017 and U.S. 62/478,485 filed on Mar. 29, 2017, all which are incorporated by reference in their entirety.
This invention was made with government support under Grant No MCB-1119232, Grant No. MCB-1344191 and Grant No. MCB-0948112 awarded by the National Science Foundation and Grant No. GM-065859 awarded by the National Institutes of Health. The government has certain rights in the invention.
| Number | Date | Country | |
|---|---|---|---|
| 62478485 | Mar 2017 | US | |
| 62459482 | Feb 2017 | US | |
| 62152896 | Apr 2015 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | PCT/US16/29213 | Apr 2016 | US |
| Child | 15793090 | US |
