WO2019046726A1

WO2019046726A1 - Multispecies biofilm and reduction techniques

Info

Publication number: WO2019046726A1
Application number: PCT/US2018/049104
Authority: WO
Inventors: Bret M. GLEMBOCKI; Christopher SPANGENBERG; Dale Grinstead; Stephen Lyon; Xiaobao Li; Kenneth J. Roach
Original assignee: Diversey, Inc
Priority date: 2017-09-01
Filing date: 2018-08-31
Publication date: 2019-03-07

Abstract

A method of making a controlled growth multispecies biofilm is disclosed. The method may have at least two microbial species and have a primary growing step as a batch phase and a secondary growing phase as a continuously stirred tank reactor phase. Also disclosed is a method of treating a biofilm to inhibit the growth of microorganisms by adding a reactive species to inhibit the growth of at least one species within the biofilm.

Description

MULTISPECIES BIOFILM AND REDUCTION TECHNIQUES

CROSS-REFERENCE TO RELATED APLICATIONS

[0001] This claims priority to U.S. Provisional Patent Application No. 62/553,493, filed on September 1 , 2017, the entire contents of which are fully incorporated herein by reference.

TECHNICAL FI ELD

[0002] The present invention is in the technical field of biofilms. More particularly, the present invention relates to growth of biofilms and reduction techniques for inhibiting growth of biofilms.

BACKGROUND OF THE INVENTION

[0003] Bacteria in environmental settings exist as a biofilm greater than 90% of the time. This biofilm in the environment is nearly always comprised of multiple species. Biofilms are very difficult to control and antimicrobial agents are rarely tested for efficacy against biofilms. Most current lab test methods address planktonic bacteria. The few test methods that address bacteria in a biofilm are restricted to single species biofilms.

[0004] It would be very beneficial to have a biofilm growth method that produces a multiple species biofilm and would be representative of a more realistic setting in the controlled conditions of a lab. It would be necessary for the biofilm growth method to be reproducible, specifically for microbiological testing, the microorganism(s) need to be able to be grown to substantially the same levels under specific conditions. A mixed species biofilm containing a specific and predictable level of each species in the biofilm would need to be able to be reproducibly produced in order to be a valid test method.

BRIEF SUMMARY OF THE INVENTION

[0005] The present invention is directed to a method of making a controlled growth multispecies biofilm. The method may include adding at least two microbial species in a reactor. A first microbial species may be added before a second microbial species in the reactor. The method may also include primary growing of the at least two microbial species for 2 to 72 hours in the reactor. The reactor may be operated as a batch phase, with 60 mg/L to 20,000 mg/L of total organic carbon (TOC) in a growth medium at 5-43°C. The method may further include secondary growing of the at least two microbial species for 2 to 72 hours in the reactor. The reactor may be operated as a continuously stirred tank reactor phase, with 60 mg/L to 20,000 mg/L of TOC in a growth medium at 5-43°C.

[0006] The present invention is also directed to a method of treating a biofilm to inhibit the growth of at least one species. The method of treating may include adding a reactive species to the biofilm. The biofilm may have at least one species and the reactive species may be bacteria. The step of adding the reactive microorganism reduces the amount of at least one microbial species in the biofilm.

[0007] The present invention is further directed to a method of inhibiting a biofilm. The method of inhibiting a biofilm may include adding a reactive species to at least one microbial species capable of forming a biofilm. The reactive species may be bacteria. The step of adding the reactive microorganism reduces the amount of at least one microbial species capable of forming a biofilm.

BRIEF DESCRIPTION OF THE DRAWING

[0008] FIG. 1 is a flowchart describing the steps of the method of making a controlled growth multispecies biofilm.

DETAILED DESCRIPTION OF THE INVENTION

Definitions

[0009] While the following terms are believed to be well understood by one of ordinary skill in the art, the following definitions are set forth to facilitate explanation of the presently disclosed subject matter.

[0010] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which the presently disclosed subject matter belongs.

[0011] Following long standing patent law convention, the terms "a", "an", and "the" refer to "one or more" when used in the subject application, including the claims. Thus, for example, reference to "a formulation" includes a plurality of such formulations, and so forth. [0012] Unless indicated otherwise, all numbers expressing quantities of components, reaction conditions, and so forth used in the specification and claims are to be understood as being modified in all instances by the term "about." Accordingly, unless indicated to the contrary, the numerical parameters set forth in the instant specification and attached claims are approximations that can vary depending upon the desired properties sought to be obtained by the presently disclosed subject matter.

[0013] As used herein, the term "about", when referring to a value or to an amount of mass, weight, time, volume, concentration, percentage, and the like can encompass variations of, and in some embodiments, ±20%, in some embodiments ±10%, in some embodiments ±5%, in some embodiments ±1 %, in some embodiments ±0.5%, and in some embodiments ±0.1 %, ±0.01 %, from the specified amount, as such variations are appropriated in the disclosed package and methods.

[0014] For the recitation of numeric ranges herein, each intervening number there between with the same degree of precision is explicitly contemplated. For example, for the range of 6-9, the numbers 7 and 8 are contemplated in addition to 6 and 9, and for the range 6.0-7.0, the number 6.0, 6.1 , 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0 are explicitly contemplated.

[0015] As used interchangeably herein, the terms "microbial species,"

"microorganism" and "microbe" refer to any microscopic organism which may be single-celled or multicellular. For example, microbial species may include any species of bacteria, algae, fungi and protists.

[0016] As used interchangeably herein, the terms "facultative anaerobes" or

"facultative anaerobic species" refer to any organism or microorganism that makes ATP by aerobic respiration if oxygen is present, but can also switch to fermentation or anaerobic respiration if oxygen is absent.

[0017] As used herein, the term "growth medium" refers to any growth medium or culture medium that supports the growth of microorganisms or cells. The growth medium may be solid, liquid, or semi-solid and provides nutrients for the

microorganisms or cells. Concentrations of nutrients in growth medium may be referred to as a quantity in mg/L of nutrient concentration in a growth medium, or alternatively as mg/L of total organic carbon (TOC) in a growth medium. [0018] As used herein, the term "biofilm" refers to any group of microorganisms that are embedded in a matrix of polymeric material and other macromolecules. Biofilms may contain either single or multiple microbial species and readily adhere to a wide variety of surfaces. A "multispecies biofilm" refers to any biofilm that contains at least two microbial species.

[0019] As used herein, the term "lactic acid bacteria" refers to any gram-positive, non-spore forming cocci, coccobacilli or rods that ferment glucose primarily to lactic acid, or to lactic acid, carbon dioxide and ethanol.

[0020] As used herein, the term "reactive microorganism" refers to any

microorganism that in the presence of a biofilm will reduce the amount of at least one microorganism in the biofilm.

Controlled Growth Multispecies Biofilm

[0021] The disclosed invention is directed to a method of making a controlled growth multispecies biofilm. Successful development of a controlled growth multispecies biofilm in a laboratory reactor using the method disclosed will produce a sufficient quantity of the microorganisms present in the biofilm and allow for evaluation of the efficacy of biocides against specific microorganisms. The multispecies biofilm created from the method disclosed also enables the evaluation of materials or conditions that reduce one or more of microbial species in a multispecies biofilm. This method of making a controlled growth multispecies biofilm allows for the study of conditions that can favor growth of one species over other species present in a multispecies biofilm. These studies could be used to control the nature of

multispecies biofilms outside of laboratory tests. For example, the growth of an undesired microorganism (e.g. a pathogen or spoilage organism) could be inhibited. Conversely, the growth of a desirable microorganism (e.g. fermentation organism in food) could be promoted.

[0022] In some embodiments, the multispecies biofilm may have at least two microbial species. In other embodiments, the multispecies biofilm may have at least three microbial species. In some embodiments, the multispecies biofilm may have three microbial species. In some embodiments, the microbial species may be bacteria, fungi, or a combination thereof. In some embodiments, the microbial species may be an anaerobe. In some embodiments, the microbial species may be a facultative anaerobe. The microbial species may be selected from the group consisting of Aspergillus niger, Listeria innocua, Pseudomonas aeruginosa, and Staphylococcus aureus. In some embodiments, the microbial species may be Pseudomonas aeruginosa (P.a.) and Staphylococcus aureus (S.a.). In other embodiments, the microbial species may be P.a., S.a., and Listeria innocua (L.i.).

[0023] In some embodiments, the method may include adding at least two microbial species in a reactor. In other embodiments, the method may include adding at least three microbial species in a reactor. In some embodiments there may be a first microbial species and a second microbial species. In other embodiments, there may be a first microbial species, a second microbial species, and a third microbial species. A first microbial species may be added before a second microbial species in the reactor. A first microbial species may be added before a second microbial species and a third microbial species in the first reactor. A first microbial species may be added 10 minutes to 10 hours before a second microbial species in the reactor. A first microbial species may be added 10 minutes, 20 minutes, 30 minutes, 40 minutes, 50 minutes, 1 hour, 1 .5 hours, 2 hours, 2.5 hours, 3 hours, 3.5 hours, 4 hours, 4.5 hours, 5 hours, 5.5 hours, 6 hours, 6.5 hours, 7 hours, 7.5 hours, 8 hours, 8.5 hours, 9 hours, 9.5 hours, 10 hours, or any range between these values, before a second microbial species in the reactor. A first microbial species may be added 4 hours before a second microbial species in the reactor. In some embodiments, S.a. may be added before P.a. in the reactor. In some embodiments, S.a. may be added before L.i. in the reactor. In some embodiments, S.a. may be added 10 minutes to 10 hours before P.a. in the reactor. In some embodiments, S.a. may be added four hours before P.a. in the reactor.

[0024] The method may include primary growing of at least two microbial species. In some embodiments, the method may include primary growing of at least three microbial species. The step of primary growing may be for 2 to 72 hours in the reactor. The step of primary growing may be for 2 hours, 4 hours, 6 hours, 8 hours, 10 hours, 12 hours, 14 hours, 16, hours, 18 hours, 20 hours, 22 hours, 24 hours, 26 hours, 28 hours, 30 hours, 32 hours, 34 hours, 36 hours, 38 hours, 40 hours, 42 hours, 44 hours, 46 hours, 48 hours, 50 hours, 52 hours, 54 hours, 56 hours, 58 hours, 60 hours, 62 hours, 64 hours, 66 hours, 68 hours, 70 hours, 72 hours, or any range between these values. The step of primary growing may be at least 2 hours. The step of primary growing may be 24 hours. The step of primary growing may be operated as a batch phase in the reactor. In some embodiments, the method may include primary growing of at least three microbial species for 2 to 72 hours in the reactor, operated as a batch phase, with 200 mg/L to 60,000 mg/L of tryptic soy broth (TSB) growth medium at 5-43°C.

[0025] The reactor may have 60 mg/L to 20,000 mg/L of total organic carbon (TOC) in a growth medium during the step of primary growing. The reactor may have 60 mg/L, 100 mg/L, 150 mg/L, 200 mg/L, 500 mg/L, 750 mg/L, 1000 mg/L, 1500 mg/L, 2000 mg/L, 2500 mg/L, 3000 mg/L, 5000 mg/L, 7500 mg/L, 10,000 mg/L, 15,000 mg/L, 20,000 mg/L, of total organic carbon in a growth medium or any range between these values. The step of primary growing may use 200 mg/L of TSB as growth medium (in equivalent to 60 mg/L TOC). The step of primary growing may use 40,000 mg/L of TSB in a growth medium (in equivalent to 20,000 mg/L TOC).

[0026] The growth medium may be lysogeny broth (LB), minimal salts, tryptic soy broth, terrific broth, or combinations thereof. The growth medium may be any other media meeting the minimum growth requirements for each organism. The growth medium may be tryptic soy broth. The step of primary growing may use tryptic soy broth as the growth medium. The step of primary growing may use 200 mg/L of tryptic soy broth. The step of primary growing may use 40,000 mg/L of tryptic soy broth.

[0027] The step of primary growing may occur at a temperature of 5-43°C. The step of primary growing may occur at a temperature of 5°C, 6°C, 7°C, 8°C, 9°C, 10°C, 1 1 °C, 12°C, 13°C, 14°C, 15°C, 16°C, 17°C, 18°C, 19°C, 20°C, 21 °C, 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, 31 °C, 32°C, 33°C, 34°C, 35°C, 36°C, 37°C, 38°C, 39°C, 40°C, 41 °C, 42°C, 43°C or any range between these values. In some embodiments, the step of primary growing may occur at a temperature of 21 °C. In other embodiments, the step of primary growing may occur at a temperature of 42°C.

[0028] The reactor may have at least one baffle inside to promote mixing. The baffle promotes better flow of fluids and can also help to break the formation of a vortex in the reactor. The at least one baffle may be manual or automated. The at least one baffle may be a baffled stir bar that is magnetically driven. The at least one baffle in the reactor may have a speed of 50 RPM to 300 RPM. The at least one baffle in the reactor may have a speed of 50 RPM, 60 RPM, 70 RPM, 75 RPM, 80 RPM, 90 RPM, 100 RPM, 125 RPM, 150 RPM, 175 RPM, 200 RPM, 225 RPM, 250 RPM, 275 RPM, 300 RPM, or any range between these values. In some embodiments, during the step of primary growing, the at least one baffle in the reactor may have a speed of 125 RPM. In some embodiments, during the step of secondary growing, the at least one baffle in the reactor may have a speed of 125 RPM. Biofilm growth outside of a laboratory may experience turbulent flow across the biofilm growth surfaces. The turbulent flow may cause the biofilm to be more robust and have a more consistent growth surface. A speed of 125 RPM can ensure turbulent flow

characteristics and may help to develop a more consistent biofilm.

[0029] The reactor may be a bioreactor. The reactor may have a batch phase, fed batch phase, or continuous phase for growing microorganisms. The reactor may be at least one CDC biofilm reactor, at least one drip flow reactor, at least one rotating disk reactor, at least one annular reactor, at least one Robbins device, at least one modified Robbins device, at least one flow cell, or combinations thereof. The reactor may be a CDC biofilm reactor. The CDC biofilm reactor may have multiple

independent rods within the reactor. The independent rods may have removable coupons. These removable coupons are biofilm growth surfaces. The reactor may have up to 8 independent rods and each rod may have up to 3 removable coupons. The coupons may be constructed from polycarbonate, porcelain, mild steel, stainless steel, PVC, vinyl, glass, or combinations thereof.

[0030] The method may include secondary growing of at least two microbial species. In some embodiments, the method may include secondary growing of at least three microbial species. The step of secondary growing may be for 2 to 72 hours in the reactor. The step of secondary growing may be for 2 hours, 4 hours, 6 hours, 8 hours, 10 hours, 12 hours, 14 hours, 16, hours, 18 hours, 20 hours, 22 hours, 24 hours, 26 hours, 28 hours, 30 hours, 32 hours, 34 hours, 36 hours, 38 hours, 40 hours, 42 hours, 44 hours, 46 hours, 48 hours, 50 hours, 52 hours, 54 hours, 56 hours, 58 hours, 60 hours, 62 hours, 64 hours, 66 hours, 68 hours, 70 hours, 72 hours or any range between these values. The step of secondary growing may be for at least 2 hours. The step of secondary growing may be for 24 hours. The step of secondary growing may be operated as a continuously stirred tank reactor (CSTR) phase in the reactor. In some embodiments, the method may include secondary growing of at least three microbial species for 2 to 72 hours in the reactor, operated as a continuously stirred tank reactor phase, with 60 mg/L to 20,000 mg/L of TOC in a growth medium at 5-43°C.

[0031] The reactor may have 60 mg/L to 20,000 mg/L of total organic carbon (TOC) in a growth medium during the step of primary growing. The reactor may have 60 mg/L, 100 mg/L, 150 mg/L, 200 mg/L, 500 mg/L, 750 mg/L, 1000 mg/L, 1500 mg/L, 2000 mg/L, 2500 mg/L, 3000 mg/L, 5000 mg/L, 7500 mg/L, 10,000 mg/L, 15,000 mg/L, 20,000 mg/L, of total organic carbon in a growth medium or any range between these values. The growth medium may be lysogeny broth (LB), minimal salts, tryptic soy broth, terrific broth, or combinations thereof. The growth medium may be any other media meeting the minimum growth requirements for each organism. The step of secondary growing may use tryptic soy broth (TSB) as the growth medium. The step of secondary growing may use 4,000 mg/L of TSB.

[0032] The step of secondary growing may occur at a temperature of 5-43°C. The step of secondary growing may occur at a temperature of 5°C, 6°C, 7°C, 8°C, 9°C, 10°C, 1 1 °C, 12°C, 13°C, 14°C, 15°C, 16°C, 17°C, 18°C, 19°C, 20°C, 21 °C, 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, 31 °C, 32°C, 33°C, 34°C, 35°C, 36°C, 37°C, 38°C, 39°C, 40°C, 41 °C, 42°C, 43°C or any range between these values. In some embodiments, the step of secondary growing may occur at a temperature of 21 °C. The step of primary growing and the step of secondary growing may occur at a temperature of 21 °C. In other embodiments, the step of secondary growing may occur at a temperature of 42°C. The step of primary growing and the step of secondary growing may occur at a temperature of 42°C.

Method of Treating a Biofilm

[0033] The disclosed invention is also directed to a method of treating a biofilm to inhibit the growth of at least one microbial species. The method may include adding a reactive species to the biofilm, wherein the biofilm has at least one species and the reactive species is a bacteria and wherein adding the reactive species reduces the amount of at least one microbial species in the biofilm. In some embodiments, adding the reactive species may reduce the mean log density of at least one species in the biofilm. The reactive microorganism may reduce the mean log density by a factor of greater than two of at least one microbial species in the biofilm. The reactive species may reduce the mean log density by a factor of four of at least one microbial species in the biofilm.

[0034] In some embodiments, the microbial species in the biofilm may be facultative anaerobes. In some embodiments, the at least one species in the biofilm may be Aspergillus niger, Listeria innocua (Li.), Pseudomonas aeruginosa (P.a.),

Staphylococcus aureus (S.a.), or combinations thereof.

[0035] The biofilm may be a multispecies biofilm comprising at least two microbial species. The multispecies biofilm may have at least two microbial species. The multispecies biofilm may have at least three microbial species. The multispecies biofilm may have at least four microbial species. The multispecies biofilm may have two, three or four microbial species.

[0036] In some embodiments, the at least two microbial species in the multispecies biofilm may be facultative anaerobes. The at least two microbial species in the multispecies biofilm may comprise Listeria innocua (L.i.), Pseudomonas aeruginosa (P.a.), Staphylococcus aureus (S.a.), or combinations thereof. The microbial species in the multispecies biofilm may be L.i. and S.a. The microbial species in the multispecies biofilm may be L.i. and P.a. The microbial species in the multispecies biofilm may be S.a. and P.a. The microbial species in the multispecies biofilm may be L.i., S.a. and P.a. The multispecies biofilm may have additional microbial species that may be well known to grow in combination with L.i., S.a., and P.a.

[0037] In some embodiments, adding the reactive species may reduce the amount of at least one microbial species in the multispecies biofilm. In some embodiments, adding the reactive species may reduce the amount of at least two microbial species in the multispecies biofilm. In some embodiments, adding the reactive species may reduce the mean log density of at least two microbial species in the multispecies biofilm. The reactive microorganism may reduce the mean log density by a factor of greater than two of at least two microbial species in the multispecies biofilm. The reactive species may reduce the mean log density by a factor of four of at least two microbial species in the multispecies biofilm. In other embodiments, adding the reactive species may reduce the mean log density of two microbial species in the multispecies biofilm. Adding the reactive species may reduce the mean log density of two microbial species in the multispecies biofilm by a factor of greater than two. Adding the reactive species may reduce the mean logio density of two microbial species in the multispecies biofilm by a factor of three (three logio reduction). Adding the reactive species may reduce the mean log density of two microbial species in the multispecies biofilm by a factor of four (four logio reduction).

[0038] The reactive species may be Acetoanaerobium, Aerococcaceae, Bacillaceae, Carnobacteriaceae, Enterococcaceae, Lactobacillaceae, Leuconostocaceae, Paenibacillaceae, Propionibacteriaceae, Streptococcaceae, or combinations thereof. In some embodiments, the reactive species may be Citrobacter freundii,

Lactobacillus acidophilus, Lactococcus lactis, Lactobacillus salivarius, Paenibacillus polymyxa, and Escherichia coli, or combinations thereof. In some embodiments the reactive species may be lactic acid bacteria.

[0039] The method may include making a controlled growth multispecies biofilm comprising the steps of adding at least two microbial species in a reactor, wherein a first microbial species is added before a second microbial species in the reactor; primary growing of the at least two species for 2 to 72 hours in the reactor, operated as a batch phase, with 60 mg/L to 20,000 mg/L of TOC in a growth medium at 5- 43°C; and secondary growing of the at least two microbial species for 2 to 72 hours in the reactor, operated as a continuously stirred tank reactor phase, with 60 mg/L to 20,000 mg/L of TOC in a growth medium at greater than 35°C. The microbial species may be selected from the group consisting of Aspergillus niger, Listeria innocua, Pseudomonas aeruginosa, or Staphylococcus aureus

[0040] The reactive species may be added to the multispecies biofilm during the step of primary growing, the step of secondary growing, after the step of primary growing, or after the step of secondary growing. The reactive species may be grown for 6 hours to 48 hours before being added to the multispecies biofilm. The reactive species may be grown for 24 hours before being added to the multispecies biofilm. The reactive species may be pipetted into the reactor during the method of making a controlled growth multispecies biofilm. The reactive species may be aseptically pipetted into the reactor. Once the reactive species is introduced into the

environment of the multispecies biofilm, the reactive species will begin to incorporate itself into the multispecies biofilm. [0041] The method of treating a biofilm to inhibit the growth of at least one microbial species may further comprise the step of putting the reactive species on an object before the step of adding the reactive species to the biofilm. The method may further include the step of transferring the reactive species from the object to the biofilm. The step of transferring the reactive species may occur after the step of putting the reactive species on an object.

[0042] The object may be an applicator tip, broom, brush, clean in place system, cloth, container, dispensing system, dosing and dispensing system, floor cleaning machine, mop, sponge, spray device, squeegee, towel, or combinations thereof. The object may be a mop, wherein the reactive species is in a solution in a container before being transferred to the mop. The mop may be dipped into the solution containing the reactive species and then transferred to the biofilm. The object may be a spray device that is attached to a container that contains the reactive species in a solution.

[0043] The biofilm may be on a surface. The surface may be an animal intestinal tract, animal skin, appliance surface, carpets, curtains, doors, door handles, drains, electronic device surface, filters, floors, floor care machines and components, countertop, furniture surface, food contact surface, grease traps, human intestinal tract, human skin, HVAC components, ducting, mattress surfaces, pipes, shower heads, sinks, tubing, walls, or combinations thereof. The filters may be air filters or water filters. The surface may be a floor. The surface may be a countertop. The surface may be a food contact surface. The food contact surface may be a pipe. The surface may be a non-food contact surface. The reactive microorganism may be sprayed onto the biofilm that is on a surface, for example, a floor or countertop.

[0044] The biofilm may be on surfaces in healthcare facilities. The surface may be a medical device, such as an MRI machine; a urinary tract catheter; a glucometer; an infusion pump; and an endoscope. The surface may be a patient bed and mattress. The surface may be in the bathroom and may be a shower head, a bath tub, a shower curtain, a sink, and a toilet.

Method of Inhibiting Biofilm Formation

[0045] The disclosed invention is also directed to a method of inhibiting biofilm formation. The method may include adding a reactive species to at least one microbial species capable of forming a biofilm, wherein the reactive species is bacteria. In some embodiments, adding the reactive species reduces the amount of at least one microbial species capable of forming a biofilm. In some embodiments, adding the reactive species may reduce the mean log density of at least one microbial species capable of forming a biofilm. The reactive microorganism may reduce the mean log density by a factor of greater than two of at least one microbial species capable of forming a biofilm.

[0046] The reactive species may be Acetoanaerobium, Aerococcaceae, Baciiiaceae, Carnobacteriaceae, Enterococcaceae, Lactobacillaceae, Leuconostocaceae, Paenibacillaceae, Propionibacteriaceae, Streptococcaceae, or combinations thereof. In some embodiments, the reactive species may be Citrobacter freundii,

[0047] Microbial species capable of forming a biofilm those species capable of growing and replicating while attached to another microbial cell or a surface. In some embodiments, the microbial species capable of forming a biofilm may be bacteria, fungi, or a combination thereof. In some embodiments, the microbial species capable of forming a biofilm may be an anaerobe. In some embodiments, the microbial species capable of forming a biofilm may be a facultative anaerobe. The microbial species capable of forming a biofilm may be selected from the group consisting of Aspergillus niger, Listeria innocua, Pseudomonas aeruginosa, and Staphylococcus aureus.

[0048] The method of inhibiting a biofilm may further comprise the step of putting the reactive species on an object before the step of adding the reactive species to at least one microbial species capable of forming a biofilm. The method may further include the step of transferring the reactive species from the object to the at least one microbial species capable of forming a biofilm. The step of transferring the reactive species may occur after the step of putting the reactive species on an object.

[0049] The object may be an applicator tip, broom, brush, clean in place system, cloth, container, dispensing system, dosing and dispensing system, floor cleaning machine, mop, sponge, spray device, squeegee, towel, or combinations thereof. The object may be a mop, wherein the reactive species is in a solution in a container before being transferred to the mop. The mop may be dipped into the solution containing the reactive species and then transferred to the biofilm. The object may be a spray device that is attached to a container that contains the reactive species in a solution.

[0050] The microbial species capable of forming a biofilm may be on a surface. The surface may be an animal intestinal tract, animal skin, appliance surface, carpets, curtains, doors, door handles, drains, electronic device surface, filters, floors, floor care machines and components, countertop, furniture surface, food contact surface, grease traps, human intestinal tract, human skin, HVAC components, ducting, mattress surfaces, pipes, shower heads, sinks, tubing, walls, or combinations thereof. The filters may be air filters or water filters. The surface may be a floor. The surface may be a countertop. The surface may be a food contact surface. The food contact surface may be a pipe. The surface may be a non-food contact surface. The reactive microorganism may be sprayed onto the microbial species capable of forming a biofilm that is on a surface, for example, a floor or countertop.

[0051] The microbial species capable of forming a biofilm may be on surfaces in healthcare facilities. The surface may be a medical device, such as an MRI machine; a urinary tract catheter; a glucometer; an infusion pump; and an endoscope. The surface may be a patient bed and mattress. The surface may be in the bathroom and may be a shower head, a bath tub, a shower curtain, and a toilet lid.

[0052] The microbial species capable of forming a biofilm may be in an industrial or commercial water system. The industrial or commercial water system may include cooling waters; food, beverage and industrial process waters; pulp and paper mill systems; brewery pasteurizers; sweetwater systems; air washer systems; oil field drilling fluids and muds; petroleum recovery processes; industrial lubricants; cutting fluids; heat transfer systems; gas scrubber systems; latex systems; clay and pigment systems; decorative fountains; water intake pipes; ballast water tanks; and ship reservoirs, among others.

[0053] While the foregoing written description of the invention enables one of ordinary skill to make and use what is considered presently to be the best mode thereof, those of ordinary skill will understand and appreciate the existence of variations, combinations, and equivalents of the specific embodiment, method, and examples herein. The invention should therefore not be limited by the above described embodiment, method, and examples, but by all embodiments and methods within the scope and spirit of the invention as claimed.

EXAMPLES

[0054] The following Examples provide illustrative embodiments. In light of the present disclosure and the general level of skill in the art, those of ordinary skill in the art will appreciate that the following examples are intended to be exemplary only and that numerous changes, modifications, and alterations can be employed without departing from the scope of the presently disclosed subject matter.

EXAMPLE 1

Growth of Single Species Biofilm

[0055] Pseudomonas aeruginosa (P.a.) and Staphylococcus aureus (S.a.) single species biofilm were grown in CDC reactors under the conditions described in Table 1 . The level of biofilm was quantified using a process as shown in FIG. 1 . FIG. 1 shows a process flow diagram that starts with a growth phase 200. The growth phase 200 was a 24 hour total batch phase followed by a 24 hour continuously stirred tank reactor (CSTR) phase using a CDC Biofilm Reactor (CBR). The batch phase reactor had a temperature of 21 °C and a nutrient level of 40,000 mg/L of tryptic soy broth and the CSTR phase had a temperature of 42°C and a nutrient level of 4,000 mg/L of tryptic soy broth (TSB). The CBR had several biofilm growth surfaces, known as "coupons", which are removable from the CBR. During the coupon removal 210, the coupons were removed and placed in a 50 ml tube with 40 ml of a buffer solution. Each tube containing a coupon was mixed 220 by using a sonication step: placing each tube in a sonic bath at 45(+/- 5) kHz for 30 seconds and then a vortex step: vortexing each tube at 2000-3000 rpm for 30 seconds. The mixing 220 step was repeated for a total of 3 vortex and 2 sonication steps. The tubes were swirled immediately and then a dilution step 230 was done where a dilution series for each tube was created. Each dilution from the dilution series was plated in duplicate on selective media, incubated, and the resulting colonies were counted. Mean log densities were the mean values of 3 separate borosilicate glass coupons from each CDC reactor. All mean log density values discussed below and in all tables are expressed as log base 10 organisms/cm², e.g. 8 mean log density would be 100,000,000 organisms/cm². When each organism was grown as a single species biofilm the mean log densities were near 8 under the appropriate

conditions. This example illustrates a different set of conditions for each organism to demonstrate log density values consistently near 8. A high log density of 8, allows for significant log reduction, so it is desired to start with a high level, such as 8 log density. It will be desired to show 5-6 log density reduction in testing of competitive exclusion, so starting at 8 will show that reduction can be achieved.

Table 1

[0056] The results in Table 1 shows that P. a. was grown three times as a single species at an average mean log density of 8.537 under the conditions shown in reactors 1 -3. It is also shown that S.a. was grown three times as a single species with an average mean log density of 7.928. In conclusion, both P.a. and S.a. were reproducibly grown under controlled growth conditions with a high log density of 8. EXAMPLE 2

Comparison Testing Dual Species Biofilm Under Varied Conditions

[0057] P.a. - S.a. dual species biofilm were grown in CDC reactors under the conditions described in Table 2 below. The level of biofilm was quantified in as described earlier and in FIG. 1 . Mean log densities were the mean values of 5 separate borosilicate glass coupons from each CDC reactor. The conditions in reactor 1 yielded mean log densities near 8 for both organisms. The conditions used in reactor 1 were determined through a series of planned experimentation with selected sets of conditions that the CDC reactor was run under. For the purposes of determining an effect or influence from some additional factor this could be considered a preferred condition. A condition that yielded both organisms at relatively large cell density enabled the greatest ability to show statistically relevant impacts on the reduction of cell density.

Table 2

[0058] CDC reactor 2 demonstrated conditions that were found to have significantly reduced levels of both organisms. CDC reactor 3 demonstrated conditions that yielded a mean log density of approximately 8 for P.a. and less than 6 for S.a. This third example demonstrates conditions that were able to favor one organism over the other and still achieve a relatively large log density for at least one of the organisms. In conclusion, CDC reactor 1 yielded the desired mean log density for both species and was used for further studies.

EXAMPLE 3 Growth of Multispecies Biofilm with 3 Species

[0059] A tri-species biofilm using P.a., S.a., and Listeria innocua (L.i.) was grown in CDC reactors under the conditions described in Table 3. The level of biofilm was quantified in as described earlier and in Fig. 2. Mean log densities were the mean values of 5 separate borosilicate glass coupons from each CDC reactor. Reactors 1 - 4 were reproductions of the same reactor conditions and reactors 5-8 were reproductions of another set of conditions. Table 3 below lists the conditions and the mean log densities of each species after the growth experiment was completed.

Table 3

[0060] Both sets of conditions were found to be favorable conditions from earlier dual species experimentation. These two conditions yielded P.a. values that had a log density above 8 for all conditions and achieved mean log densities of approximately 7 for both S.a. and L.i. For the reactors that had a Batch Phase Concentration of 200 mg/L TSB the variation in Mean Log Density of S.a. was greater than that of the reactors that had a Batch Phase Concentration of 40000 mg/L TSB. These results suggest using the conditions that were used in reactors 5-8 may be more amenable to looking for significant impact on the biofilm species. EXAMPLE 4

[0061] S.a. single species biofilms were grown following the conditions specified in Table 4. To investigate the impact of competitive exclusion on this single species biofilm system, a second species of Lactoccocus lactis was added into the system at the same time as S.a. and the two species were grown under the same conditions as the S.a. single species biofilms. Table 4 gives the final log densities of S.a. in its single species and in a dual-species biofilm with Lactococcus lactis. Lactococcus lactis acts as a reactive species and reduces the mean log density of S.a.

Table 4

EXAMPLE 5

Competitive Exclusion from Additional Species

[0062] Example 3 depicted biofilm cell densities of each species that could be achieved over several reproductions of conditions in CDC reactors. To investigate the impact of competitive exclusion on this tri-species system a fourth species was added to the system at the same time point as P. a. and L.i. These fourth species impacted L.i. levels differently. The following species were used as the fourth species on the tri-species system using P. a., S.a., and L.i: Lactococcus lactis, lactobacillus acidophilus, lactobacillus salivarius, citrobacter freundii, and

paenibaciiius poiymyxa. Table 5 gives the final mean log density of L.i., S.a., and P.a. after the addition of the each test species which was the fourth species. These fourth species act as a reactive species which reduced the mean log density of the species in the tri-species system. Lactobacillus acidophilus had only a minor impact, if any, on the L.i. biofilm cell density. Paenibaciiius poiymyxa had some impact on L.i. biofilm cell density while Lactococcus lactis had a reduction in mean log density of over 4. In conclusion, the use of Lactococcus lactis when added to the 3 species model had a significant reduction in the log density of both Li. and S.a.

Table 5

[0063] The foregoing discussion discloses and describes merely exemplary embodiments of the invention. One skilled in the art will readily recognize from such discussion and from the accompanying drawings and claims, that various changes, modifications and variations can be made therein without departing from the spirit and scope of the invention as defined in the following claims.

[0064] For reasons of completeness, various aspects of the invention are set out in the following numbered clauses:

[0065] Clause 1 . A method of making a controlled growth multispecies biofilm comprising: adding at least two microbial species in a reactor, wherein a first microbial species is added before a second microbial species in the reactor; primary growing of the at least two microbial species for 2 to 72 hours in the reactor, operated as a batch phase, with 60 mg/L to 20,000 mg/L of total organic carbon (TOC) in a growth medium at 5-43°C; and secondary growing of the at least two microbial species for 2 to 72 hours in the reactor, operated as a continuously stirred tank reactor phase, with 60 mg/L to 20,000 mg/L of total organic carbon (TOC) in a growth medium at 5-43°C.

[0066] Clause 2. The method of clause 1 , wherein the at least two microbial species are selected from the group consisting of Aspergillus niger, Listeria innocua, Pseudomonas aeruginosa, and Staphylococcus aureus.

[0067] Clause 3. The method of clause 1 or 2, wherein the at least two microbial species comprise Staphylococcus aureus and Pseudomonas aeruginosa and wherein Staphylococcus aureus is added to the reactor 4 hours before

Pseudomonas aeruginosa.

[0068] Clause 4. The method of any one of clauses 1 -3, wherein the growth medium is tryptic soy broth.

[0069] Clause 5. The method of any one of clauses 1 -4, wherein the step of primary growing uses 200 mg/L of tryptic soy broth.

[0070] Clause 6. The method of any one of clauses 1 -4, wherein the step of primary growing uses 40,000 mg/L of tryptic soy broth.

[0071] Clause 7. The method of any one of clauses 1 -6, wherein the step of secondary growing uses 4,000 mg/L of tryptic soy broth.

[0072] Clause 8. The method of any one of clauses 1 -7, wherein the step of primary growing and the step of secondary growing occurs at a temperature of 21 °C.

[0073] Clause 9. The method of any one of clauses 1 -7, wherein the step of primary growing and the step of secondary growing occurs at a temperature of 42°C.

[0074] Clause 10. The method of any one of clauses 1 -9, wherein the reactor comprises at least one member selected from the group consisting of a CDC biofilm reactor, a drip flow reactor, a rotating disk reactor, an annular reactor, a Robbins device, a modified Robbins device, and a flow cell.

[0075] Clause 1 1 . The method of any one of clauses 1 -10, wherein the reactor is a CDC biofilm reactor. [0076] Clause 12. The method of any one of clauses 1 -1 1 , wherein the step of primary growing occurs for at least 2 hours.

[0077] Clause 13. The method of any one of clauses 1 -12, wherein the step of secondary growing occurs for at least 2 hours.

[0078] Clause 14. A method of treating a biofilm to inhibit the growth of at least one population of microorganisms comprises adding a reactive species to the biofilm, wherein the biofilm has at least one microbial species and the reactive species is a bacteria and wherein adding the reactive species reduces the amount of at least one microbial species in the biofilm.

[0079] Clause 15. The method of clause 14, wherein the biofilm is a multispecies biofilm and has at least two microbial species.

[0080] Clause 16. The method of clause 15, wherein adding the reactive species reduces the amount of at least two microbial species in the multispecies biofilm.

[0081] Clause 17. The method of clauses 15 or 16, wherein adding the reactive species reduces the mean log density of two microbial species in the multispecies biofilm.

[0082] Clause 18. The method of clause 17, wherein adding the reactive species reduces the mean log density of two microbial species in the multispecies biofilm by a factor of greater than two.

[0083] Clause 19. The method of clause 17, wherein adding the reactive species reduces the mean log density of two microbial species in the multispecies biofilm by a factor of four.

[0084] Clause 20. The method of any one of clauses 14-19, wherein the reactive species comprises at least one member selected from the group consisting of Acetoanaerobium, Aerococcaceae, Bacillaceae, Carnobacteriaceae,

Enterococcaceae, Lactobacillaceae, Leuconostocaceae, Paenibacillaceae,

Propionibacteriaceae, and Streptococcaceae.

[0085] Clause 21 . The method of any one of clauses 14-20, wherein the reactive species comprises at least one member selected from the group consisting of Citrobacter freundii, Lactobacillus acidophilus, Lactococcus lactis, Lactobacillus salivarius, Paenibacillus polymyxa, and Escherichia coli. [0086] Clause 22. The method of any one of clauses 15-21 , wherein the multispecies biofilm comprises Listeria innocua and Staphylococcus aureus.

[0087] Clause 23. The method of any one of clauses 14-22, further comphsing a step of putting the reactive species on an object before the step of adding the reactive species to the biofilm.

[0088] Clause 24. The method of clause 23, further comphsing the step of transferring the reactive species from the object to the biofilm.

[0089] Clause 25. The method of clause 23 or 24, wherein the object comprises at least one member selected from the group consisting of an applicator tip, broom, brush, clean in place system, cloth, container, dispensing system, dosing and dispensing system, floor cleaning machine, mop, sponge, spray device, squeegee, and towel, or combinations thereof.

[0090] Clause 26. The method of any one of clauses 14-25, wherein the biofilm is on a surface.

[0091] Clause 27. The method of clause 26, wherein the surface comprises at least one member selected from the group consisting of animal intestinal tract, animal skin, appliance surface, carpets, curtains, doors, door handles, drains, electronic device surface, filters, floors, floor care machines and components, countertop, furniture surface, food contact surface, grease traps, human intestinal tract, human skin, HVAC components, ducting, mattress surfaces, pipes, shower heads, sinks, tubing, walls, or combinations thereof.

[0092] Clause 28. A method of inhibiting biofilm formation comprising adding a reactive species to at least one microbial species capable of forming a biofilm wherein the reactive species is bacteria.

[0093] Clause 29. The method of clause 28, wherein adding the reactive species reduces the amount of at least one microbial species capable of forming a biofilm.

[0094] Clause 30. The method of clause 28 or 29, wherein adding the reactive species reduces the mean log density of at least one microbial species capable of forming a biofilm. [0095] Clause 31 . The method of any one of clauses 28-30, wherein adding the reactive species reduces the mean log density of at least one microbial species capable of forming a biofilm by a factor of greater than two.

[0096] Clause 32. The method of any one of clauses 28-31 , wherein the reactive species comprises at least one member selected from the group consisting of Acetoanaerobium, Aerococcaceae, Bacillaceae, Carnobacteriaceae,

Enterococcaceae, Lactobacillaceae, Leuconostocaceae, Paenibacillaceae,

Propionibacteriaceae, and Streptococcaceae.

[0097] Clause 33. The method of any one of clauses 28-32, wherein the reactive species comprises at least one member selected from the group consisting of Citrobacter freundii, Lactobacillus acidophilus, Lactococcus lactis, Lactobacillus salivarius, Paenibacillus polymyxa, and Escherichia coli.

[0098] Clause 34. The method of any one of clauses 28-33, wherein the at least one microbial species capable of forming a biofilm comprises Aspergillus niger, Listeria innocua, Pseudomonas aeruginosa, Staphylococcus aureus or a combination thereof.

[0099] Clause 35. The method of any one of clauses 28-34, further comprising a step of putting the reactive species on an object before the step of adding the reactive species to the at least one microbial species capable of forming a biofilm.

[00100] Clause 36. The method of clause 35, wherein the object comprises at least one member selected from the group consisting of an applicator tip, broom, brush, clean in place system, cloth, container, dispensing system, dosing and dispensing system, floor cleaning machine, mop, sponge, spray device, squeegee, and towel, or combinations thereof.

Claims

CLAIMS What is claimed is:

1 . A method of making a controlled growth multispecies biofilm comprising:

adding at least two microbial species in a reactor, wherein a first microbial species is added before a second microbial species in the reactor; primary growing of the at least two microbial species for 2 to 72 hours in the reactor, operated as a batch phase, with 60 mg/L to 20,000 mg/L of total organic carbon (TOC) in a growth medium at 5-43°C; and

secondary growing of the at least two microbial species for 2 to 72 hours in the reactor, operated as a continuously stirred tank reactor phase, with 60 mg/L to 20,000 mg/L of total organic carbon (TOC) in a growth medium at 5-43°C.

2. The method of claim 1 , wherein the at least two microbial species are

selected from the group consisting of Aspergillus niger, Listeria innocua, Pseudomonas aeruginosa, and Staphylococcus aureus.

3. The method of claim 1 , wherein the at least two microbial species comprise Staphylococcus aureus and Pseudomonas aeruginosa and wherein

Staphylococcus aureus is added to the reactor 4 hours before Pseudomonas aeruginosa.

4. The method of claim 1 , wherein the growth medium is tryptic soy broth.

5. The method of claim 1 , wherein the step of primary growing uses 200 mg/L of tryptic soy broth.

6. The method of claim 1 , wherein the step of primary growing uses 40,000 mg/L of tryptic soy broth.

7. The method of claim 1 , wherein the step of secondary growing uses 4,000 mg/L of tryptic soy broth.

8. The method of claim 1 , wherein the step of primary growing and the step of secondary growing occurs at a temperature of 21 °C.

9. The method of claim 1 , wherein the step of primary growing and the step of secondary growing occurs at a temperature of 42°C.

10. The method of claim 1 , wherein the reactor comprises at least one member selected from the group consisting of a CDC biofilm reactor, a drip flow reactor, a rotating disk reactor, an annular reactor, a Robbins device, a modified Robbins device, and a flow cell.

1 1 . The method of claim 1 , wherein the reactor is a CDC biofilm reactor.

12. The method of claim 1 , wherein the step of primary growing occurs for at least 2 hours.

13. The method of claim 1 , wherein the step of secondary growing occurs for at least 2 hours.

14. A method of treating a biofilm to inhibit the growth of at least one population of microorganisms comprises adding a reactive species to the biofilm, wherein the biofilm has at least one microbial species and the reactive species is a bacteria and wherein adding the reactive species reduces the amount of at least one microbial species in the biofilm.

15. The method of claim 14, wherein the biofilm is a multispecies biofilm and has at least two microbial species.

16. The method of claim 15, wherein adding the reactive species reduces the amount of at least two microbial species in the multispecies biofilm.

17. The method of claim 15, wherein adding the reactive species reduces the mean log density of two microbial species in the multispecies biofilm.

18. The method of claim 17, wherein adding the reactive species reduces the mean log density of two microbial species in the multispecies biofilm by a factor of greater than two.

19. The method of claim 17, wherein adding the reactive species reduces the mean log density of two microbial species in the multispecies biofilm by a factor of four.

20. The method of claim 14, wherein the reactive species comprises at least one member selected from the group consisting of Acetoanaerobium, Aerococcaceae, Bacillaceae, Carnobacteriaceae, Enterococcaceae,

Lactobacillaceae, Leuconostocaceae, Paenibacillaceae,

Propionibacteriaceae, and Streptococcaceae.

21 . The method of claim 14, wherein the reactive species comprises at least one member selected from the group consisting of Citrobacter freundii,

Lactobacillus acidophilus, Lactococcus lactis, Lactobacillus salivarius, Paenibacillus polymyxa, and Escherichia coli.

22. The method of claim 15, wherein the multispecies biofilm comprises Listeria innocua and Staphylococcus aureus.

23. The method of claim 14, further comprising a step of putting the reactive species on an object before the step of adding the reactive species to the biofilm.

24. The method of claim 23, further comprising the step of transferring the

reactive species from the object to the biofilm.

25. The method of claim 23, wherein the object comprises at least one member selected from the group consisting of an applicator tip, broom, brush, clean in place system, cloth, container, dispensing system, dosing and dispensing system, floor cleaning machine, mop, sponge, spray device, squeegee, and towel, or combinations thereof.

26. The method of claim 14, wherein the biofilm is on a surface.

27. The method of claim 26, wherein the surface comprises at least one member selected from the group consisting of animal intestinal tract, animal skin, appliance surface, carpets, curtains, doors, door handles, drains, electronic device surface, filters, floors, floor care machines and components, countertop, furniture surface, food contact surface, grease traps, human intestinal tract, human skin, HVAC components, ducting, mattress surfaces, pipes, shower heads, sinks, tubing, walls, or combinations thereof.

28. A method of inhibiting biofilm formation comprising adding a reactive species to at least one microbial species capable of forming a biofilm wherein the reactive species is bacteria.

29. The method of claim 28, wherein adding the reactive species reduces the amount of at least one microbial species capable of forming a biofilm.

30. The method of claim 28, wherein adding the reactive species reduces the mean log density of at least one microbial species capable of forming a biofilm.

31 . The method of any one of claim 28, wherein adding the reactive species

reduces the mean log density of at least one microbial species capable of forming a biofilm by a factor of greater than two.

32. The method of any one of claim 28, wherein the reactive species comprises at least one member selected from the group consisting of Acetoanaerobium, Aerococcaceae, Bacillaceae, Carnobacteriaceae, Enterococcaceae,

Lactobacillaceae, Leuconostocaceae, Paenibacillaceae,

Propionibacteriaceae, and Streptococcaceae.

33. The method of any one of claim 28, wherein the reactive species comprises at least one member selected from the group consisting of Citrobacter freundii, Lactobacillus acidophilus, Lactococcus lactis, Lactobacillus salivarius, Paenibacillus polymyxa, and Escherichia coli.

34. The method of any one of claim 28, wherein the at least one microbial species capable of forming a biofilm comprises Aspergillus niger, Listeria innocua, Pseudomonas aeruginosa, Staphylococcus aureus or a combination thereof.

35. The method of any one of claim 28, further comprising a step of putting the reactive species on an object before the step of adding the reactive species to the at least one microbial species capable of forming a biofilm.

36. The method of claim 35, wherein the object comprises at least one member selected from the group consisting of an applicator tip, broom, brush, clean in place system, cloth, container, dispensing system, dosing and dispensing system, floor cleaning machine, mop, sponge, spray device, squeegee, and towel, or combinations thereof.