COMPOSITIONS FOR EXPOSING FILM-FORMING MICROBES AND METHODS FOR USE OF THE COMPOSITIONS

Abstract

Current methods for detection of microbial contaminants on surfaces use swabbing/wiping to extract microbes for analysis. This removes easily transferable microbes but fails to extract microbes living in biofilms, which reduces sensitivity and may mask the true degree of contamination. The current disclosure provides an enzyme cocktail that disrupts the biofilm and improves the extraction of live microbes for analysis. Applicant's enzyme system is particularly useful for the application to a variety of surfaces, but particularly on a variety of food processing surfaces. Utilization of Applicant's enzyme cocktail makes possible the extraction of a representative sample of live microorganisms present on a surface, including film forming microorganisms, without affecting non-film forming microorganisms also present on a surface.

Claims

1. A method for disrupting a film formed by a colony of film-forming microbes on a surface of a manufactured article, the method comprising contacting the film with an enzyme cocktail for a time sufficient to disrupt the film and expose viable microbes for sampling.

2. The method of claim 1, where the enzyme cocktail includes an enzyme selected from the group consisting of dNase, collagenase, combinations thereof and at least one additional enzyme.

3. The method of claim 1, wherein the at least one additional enzyme includes an enzyme selected from the group consisting of proteases, polysaccharidases, and combinations thereof.

4. The method of claim 3, wherein the at least one additional enzyme is selected from the group consisting of alpha-amylase, Beta acetylhexosaminidase, polygalacturonase, Dextranase, Mutanase, Cellulase, Trypsin, Papain, Glutamyl endopeptidase, Actinidin, Proteinase K, and Salvinase.

5. The method of claim 1, further including removing viable microbes from the surface of a manufactured article for testing after contacting the film with the enzyme cocktail.

6. The method of claim 5, wherein the testing involves identifying the presence or absence of a specific microbe.

7. The method of claim 5, wherein the testing includes testing for a bacteria.

8. The method of claim 5, wherein the testing involves a test method selected from the group consisting of ATP luminescence, PCR, DNA sequencing, and aerobic plate count (APC), crystal violet assay, and BTG assay.

9. The method of claim 5, wherein the testing involves testing for bacteria selected from the group consisting of Campylobacter jejuni, Salmonella enterica, Listeria monocytogenes, Escherichia coli, Pseudomonas fluorescens, Acinetobacter johnsonii, Lactobacillus plantarum, and Serriatia proteamaculans.

10. The method of claim 5, wherein the testing involves testing for a fungus.

11. An enzyme cocktail comprising of water and at least one enzyme selected from the group consisting of dNase, collagenase, and combinations thereof and at least one additional enzyme, wherein the enzyme cocktail is configured for applying to a microbial film formed by one or more film-forming microbes on a surface of a manufactured article to release viable microbes from the film, and wherein the enzyme cocktail is configured for application to the surface of the manufactured article having any orientation.

12. The enzyme cocktail of claim 11, wherein the at least one additional enzyme is selected from the group consisting of alpha-amylase, Beta acetylhexosaminidase, polygalacturonase, Dextranase, Mutanase, Cellulase, Trypsin, Papain, Glutamyl endopeptidase, Actinidin, Proteinase K, and Salvinase.

13. The enzyme cocktail of claim 11 including at least two additional enzymes.

14. The enzyme cocktail of claim 13, wherein the at least two additional enzymes are selected from the group consisting of alpha-amylase, Beta acetylhexosaminidase, polygalacturonase, Dextranase, Mutanase, Cellulase, Trypsin, Papain, Glutamyl endopeptidase, Actinidin, Proteinase K, and Salvinase.

15. The enzyme cocktail of claim 11, further including an emulsifier configured to emulsify the enzyme cocktail.

16. The enzyme cocktail of claim 11, further including an agent to retard the evaporation of water.

17. The enzyme cocktail of claim 15, including at least one additional enzyme selected from the group consisting of alpha-amylase, Beta acetylhexosaminidase, polygalacturonase, Dextranase, Mutanase, Cellulase, Trypsin, Papain, Glutamyl endopeptidase, Actinidin, Proteinase K, and Salvinase.

18. A surface of a manufactured article including a biofilm thereon including at least one film-forming microbe, wherein the film has been degraded by contact with the enzyme cocktail of claim 11 exposing at least one viable microbe.

19. The surface of the manufactured article of claim 18, comprising a surface selected from the group consisting of a plastic surface, a steel surface, and a coated surface.

20. A kit including an the enzyme cocktail of claim 11, an applicator for applying the enzyme cocktail to a film produced by one or more film-forming microbes on a surface of a manufactured article, and a tool configured for removing released and viable one or more film-forming microbes.

Description

DESCRIPTION

[0021] The extracellular polymeric substances of biofilms are composed primarily of polysaccharides, proteins, and extracellular DNA (eDNA). Certain enzymes have been found to break down individual components of extracellular polymeric substances, and enzyme cocktails have been formulated with various cleaning reagents (e.g. detergents, dispersing agents, wetting agents etc.) to produce enzymatic cleaners for cleaning surfaces with efficacy competitive with top chemical surface cleaners. However, such products are designed for killing microbes and are unsuitable for non-lethal extraction of microbes.

[0022] Enzymes having beneficial activity for inclusion in enzyme cocktails designed for the destruction of biofilms were determined by screening for biofilm disrupting activity using relevant bacteria that form biofilms on the surface of relevant manufactured articles and selecting the candidate combination that was the most effective against the biofilms produced by the greatest number of bacteria. Specific to this disclosure maintaining microbe viability, and the ability to collect microbes released by biofilm-disrupting effects from manufactured surfaces are also important aspects of the disclosure. The approach used in this disclosure considers that extracellular polymeric substances in biofilms can be composite fibers from a combination of microbes and that combining enzymes that attack distinct components of the biofilm may functionally synergize dissolution to break down fibers in the biofilm more effectively than mono-enzyme treatments.

Commercial anti-biofilm enzymes are not designed for maintaining microbe viability which is necessary for selecting suitable enzyme cocktails to destroy the biofilm without reducing the surface's functioning bacterial population and provide an accurate measurement of the surface's bacterial population. As a result, only enzymes that can destroy the biofilm's extracellular structure on manufactured surfaces while maintaining the viability of the released bacteria should be included in the enzyme cocktail which is the focus of this disclosure. For testing purposes, a specific group of biofilm forming bacteria were selected and a group of potential enzymes were similarly selected.

The Selection of Bacteria for Testing:

[0023] Thorough consideration of the bacteria selected for testing was important for the test design because the specific structural components of their biofilms can differ based on the bacterial system producing the biofilm. The utilization of an insufficient species breadth in the study would limit an enzyme cocktail's effectiveness, yet too large a panel risks would become excessively laborious and costly without gaining proportional value. In the case of food processing surfaces, consideration of non-pathogenic bacteria that typically form biofilms on such surfaces are important because they are known to resist cleaning and disinfection treatments and can potentially harbor and protect pathogens within their biofilms.

[0024] Dozens of microorganisms may be present in any food processing environment forming complex and poorly understood communities of microorganisms. In order to simulate this pattern in the test system, four (4) bacteria representing a resident community were included in the testing along with three (3) food borne pathogens (Table 1). Certain species were selected for their ability to cause food-borne illnesses. Other species were selected because they were commonly found to produce biofilms on surfaces of manufactured articles utilized in the food industry, but are not strictly considered pathogens (termed resident bacteria). The three (3) pathogens selected are dominant causes of food borne illnesses, are regularly isolated from food processing work surfaces, and are easily cultured using common culture practices. The bacteria selected were also generally guided by a focus on species associated with surfaces in contact with meat and poultry commodities because these are a dominant source of food borne illnesses. Table 1 provides a listing of the bacteria utilized in the applicant's disclosure research.

TABLE-US-00001 TABLE 1 Bacterial Species Utilized Foodborne Pathogens Resident Bacteria Escherichia coli O157:H7 Pseudomonas fluorescens Salmonella enterica Acinetobacter johnsonii Listeria monocytogenes Lactobacillus plantarum Serratia proteamaculans

Selection of Enzymes for Testing:

[0025] For this initial study, well established commercial enzymes are used for testing. Table 2 lists the selected enzymes and some characteristics associated with each of them.

TABLE-US-00002 TABLE 2 Candidate enzymes and their characteristics Preliminary Enzymes Enzyme Type Enzyme Subtype/Activity DNase I Nuclease DNA degrading alpha-amylase Polysaccharidase α-1,4 endoglycocidase β-N- Polysaccharidase β-N- acetylglucosaminidase acetylglucosaminidase Polygalacturonase Polysaccharidase poly-alpha-1,4-galacturonide (PG) glycanohydrolase Dextranase Polysaccharidase β-1,6 glucanase Cellulase Polysaccharidase β-1,4 glucanase Trypsin Protease (serine) Cleaves at basic aa Papain (from papaya) Protease Cleaves at basic amino acids (cysteine) preceded by a hydrophobic aa Glutamyl Protease (serine) Cleaves at acidic aa endopeptidase Collagenase Protease Collagen cleavage (cysteine) Proteinase K Protease (serine) Cleaves at hydrophobic aa Savinase Protease (serine) A subtilisin, nonspecific cleavage
The selected enzymes represent three (3) enzyme classes: proteases, polysaccharidases, and nucleases. The selection focused on producing a diverse set of enzymatic activities within each of these main classes of enzymes because the bacteria selected encompass several genera and can have diverse biofilm compositions.

[0026] The disclosed research involves the testing of a set of commercially available (low cost) enzymes (listed in Table 2) against a panel of food-borne pathogens and resident bacteria (listed in Table 1) to identify an enzyme cocktail capable of disintegrating food industry relevant biofilms and increasing bacterial release/extraction from populated surfaces without substantial loss of viability. The enzyme cocktail developed is expected to be applicable for all current surface contamination monitoring systems used by the food industry, especially culture-based systems. Non-culture-based PCR or DNA sequencing systems will benefit as well because directly extracting DNA from surfaces may leave appreciable amounts of DNA bound to a surface, while extracting cells whole and then lysing and extracting the DNA in a testing tube is expected to result in higher recovery. If used with the ATP assay, ATP testing with and without Applicant's enzyme cocktail allows for specific detection of microbes associated with biofilms through the differential ATP levels providing a capability it currently lacks. Thus, the utilization of Applicant's enzyme cocktail is valuable to food processing safety managers in the food industry through its ability to allow contamination monitoring systems to detect and identify microbes on surfaces with higher sensitivity, accuracy, and reliability. This allows food processing surfaces to be better sanitized and reduce the incidence of food borne illnesses in the country and the world.

Applicant's Test Strategy:

[0027] Applicant's initial testing was carried out with combinations of two (2) enzymes rather than a single enzyme with a nuclease present in all enzyme combinations. DNase I, a specific commonly utilized DNA nuclease, was selected as a universal enzyme. Both DNase I and other DNA nucleases were compared and formulated to have equivalent activity and then used interchangeably in the research. Nuclease was tested against all seven (7) species at three (3) concentrations and then the most overall effective concentration was used in two (2) enzyme testing. Initial two (2) enzyme testing utilized traditional mono-species biofilms of the 7 species in Table 1. The nuclease was also always tested alone in all experiments allowing some determination of its contribution to the resulting antibiofilm activity. Non-nuclease enzymes were tested at 3 concentrations while the nuclease concentration was held constant. Anti-biofilm activity was measured using the Crystal Violet assay, an easy and ubiquitous assay used for measuring changes in biofilm magnitude in the biofilm research industry.
Two (2) enzyme combinations that showed especially high activity by the Crystal Violet were further evaluated. These enzyme combinations were tested for bacterial cell release from enzyme-treated biofilms. Post-enzyme exposure sample overlying solutions were collected and centrifuged to collect bacterial cells released by the enzyme treatment. The supernatants were removed, and the cell pellet resuspended in PBS and ATP luminescence was determined and compared to non-enzyme treated controls. Preferred enzyme pairs were expected to release more cells than untreated controls. Additionally, because maintaining extracted microbe viability was important, the ATP luminescence of the post-centrifugation cell free supernatants were also determined and compared to untreated controls. ATP is normally sequestered inside of cells so the presence of substantially more ATP in the cell free supernatant of enzyme treated biofilms indicates the enzyme treatment may have had toxic effects on the treated bacteria. Testing using a model quaternary ammonium substance (QAS) sanitizing agent at functionally active concentrations resulted in over twelve-fold higher cell-free ATP luminescence than untreated control biofilms in a representative species demonstrating the ability of the test to detect toxic effects on the bacteria.

[0028] Additionally, multi-species biofilms were composed and tested using the bacteria listed in Table 1. It is well understood that in nature multi-species (MS) biofilms are the typical condition encountered. The complexity of such systems and our inability to understand and model these complex systems has limited most biofilm studies to mono-culture models. In vitro studies using two (2) or more species biofilms are more common in food safety research as it is appreciated that these complex systems are ubiquitous in the food processing environment, and dual species biofilms indicate that such biofilms can generally protect microbes from disinfectants and other stresses better than single-species biofilms. Applicant's enzyme cocktails (enzyme cocktail) developed using mono-culture biofilms face a greater challenge disrupting the more rugged multi-species (MS) biofilms.

[0029] Multi-species biofilms were screened by treatment with a sanitizing agent to identify species combinations that show heightened resistance. The multi-species biofilm with the most resistance to the sanitizer was used to screen three (3) enzyme combinations for anti-biofilm and bacterial release testing of the six (6) top performing two (2) enzyme combinations tested earlier. Non-nuclease enzymes were tested at two (2) different concentrations. Each three-enzyme combination was also compared to each of the two-enzyme combination core pair to try to identify synergy of the three-enzyme combination over the two-enzyme combination.

[0030] In addition, S. proteamaculans as a single species biofilm had a Crystal Violet % Ctr average value across all enzymes tested of just 78% Ctr (i.e., only an average 22% biofilm reduction), showing it was especially resistant to enzyme treatment. Therefore, three-enzyme combinations were also tested against single species biofilms of this species.

[0031] The results of the initial two enzyme combination testing using the Crystal Violet assay is given in Table 3 and are given as a percent of the untreated control Crystal Violet signal.

TABLE-US-00003 TABLE 3 Biofilm Reduction Due to Enzyme Cocktail Treatment in Model Species Enzyme 7-species Combined w/nuclease E. coli S. enterica L. monocytogenes S. proteamaculens A. JohnsonII P. fluorescens L. plantarum average Proteinase K 100%  100%  72%  0% 86% 19% 71% 64% Trypsin 50% 54% 92% 60% 29% 56% 57% Papain 100%  28% 100%  14% 60% 42% 27% 53% Savinase 25% 73% 90% 39% 85% 73% 89% 68% Collagenase (relacing 82% 62% 59% 79%  0% 20% 26% 47% Actinidin) Glutamyl (Glu-C) 22% 50% 36% endopeptidase Alpha amylase 70% 70%  0% 46% 31% 44% Dextranase  0% 18% 28.6%.sup.  38% 52% 69% 34% Dispersion B, (Beta  0%  0%  0%  0% acetylhexosaminidase Polygalacturonase 28.7 Cellulase  0% 10%  0%  0% 22.4%.sup.   0%  5%
Table 4 shows the results of the more comprehensive testing of select two-enzyme combinations which includes testing cell release from enzyme treatment as well as treatment toxicity.

TABLE-US-00004 TABLE 4a Cell Release and Toxicity Values for Select Enzyme Cocktails and Model Bacteria Cell Treatment Bacteria/EC Release Toxicity A. johnsonii, Proteinase K + Nuclease 448.1% 300.3 L. monocytogenes Papain + Nuclease 243.5% 74.9% L. monocytogenes Trypsin + Nuclease 293.7% 83.3% L. monocytogenes Savinase + Nuclease 65.8% 106.1% E. coli + Papain + Nuclease 173.5% 22.1% E. coli + Proteinase K + Nuclease 121.4% 21.4% E. coli + Collagenase + Nuclease 114.4% 40.0% S. enterica, + alpha amylase + Nuclease 391.6% 62.1% A. johnsonii, + Savinase + Nuclease 222.1% 57.4% A. johnsonii, + Dextranase + Nuclease 402.6% 58.9% S. proteamaculans + Dextranase + Nuclease 78.5% 37.1% L. plantarum + Savinase + Nuclease 191.4% 18.4% E. coli + alpha amylase + Nuclease 92.6% 66.9% E. coli + Collagenase + Nuclease 126.2% 65.1% S. proteamaculans + Collagenase + Nuclease 202.0% 56.7% S. proteamaculans + Trypsin + Nuclease 61.9% 86.6%
Cell release: Post-enzyme exposure sample overlying solutions were collected and centrifuged to pellet bacterial cells released by the enzyme treatment. The post-centrifugation supernatants were subsampled and removed, and the cell pellet resuspended in PBS and ATP luminescence was determined using the BacTiterGlo Microbial Cell Viability Assay (Promega) and compared to non-enzyme treated controls to give a percent of control value shown. Values over 100% were considered positively.
Treatment Toxicity: To measure cell toxicity the ATP luminescence of the post-centrifugation cell free supernatants were also determined using the BacTiterGlo Microbial Cell Viability Assay (Promega) and compared to untreated controls to give a percent control value shown. Values over 100% are viewed negatively as potential toxic effects of an enzyme treatment.

TABLE-US-00005 TABLE 4b Validation of Toxicity Assay Using Known Lytic Product and Representative Bacteria Treatment Toxicity Treatment % Ctr Biofilm Lifestyle s. proteamaculans + 10 μg/ml BZAC 150.8% s. proteamaculans + 100 μg/ml BZAC 793.3% s. proteamaculans + 1 μg/ml BZAC 1250.5% Planktonic lifestyle s. proteamaculans + 10 μg/ml BZAC 284.9% s. proteamaculans + 100 μg/ml BZAC 724.9% s. proteamaculans + 1 μg/ml BZAC 859.3%
Table 4b shows application of the ATP luminescence Treatment Toxicity Assay applied to a model QAS sanitizer, Benzalkonium chloride, at three (3) different concentrations grown in either biofilm or planktonic lifestyle using S. proteamaculans as a model organism. In multi-species biofilm testing, single species biofilm controls with this species consistently show biofilm reduction by treatment with 100 ug/ml BZAC making it a good species for testing BZAC toxicity. With both growth lifestyles a clear dose response curve is evident.

Three Enzyme Combinations.

[0032]

TABLE-US-00006 TABLE 5 Top 5 Three-enzyme combinations in either multi-species, or S. proteamaculans biofilms (CVA-Crystal Violet Assay) CVA Cell Release Three enzyme combination treatment % Ctr % Ctr MS-biofilms Collagenase + Papain + Nuclease, best 4.50% 213.80% enzyme conc. Collagenase + Papain + Nuclease, 2 4.60% 215.00% concentration ave Trypsin + Papain + Nuclease, best enzyme 5.10% 481.80% conc. Trypsin + Papain + Nuclease, 2 5.70% 543.80% concentration ave Collagenase + alpha-Amylase + Nuclease, best 5.30% 149.30% enzyme conc. Collagenase + alpha-Amylase + Nuclease, 2 5.80% 198.50% concentration ave Collagenase + Dextranase + Nuclease, best 5.70% 254.50% enzyme conc. Collagenase + Dextranase + Nuclease, 2 6.90% 281.40% concentration ave Trypsin + Dextranase + Nuclease, best 7.20% 507.10% enzyme conc. Trypsin + Dextranase + Nuclease, 2 8.30% 544.40% concentration ave S. proteamaculans Biofilms Collagenase + alpha-Amylase + Nuclease, 29.10% 89.20% best enzyme conc. Collagenase + alpha-Amylase + Nuclease, 37.20% 162.50% 2 concentration ave Collagenase + Savinase + Nuclease, best 31.80% 120.70% enzyme conc. Collagenase + Savinase + Nuclease, 2 49.60% 94.70% concentration ave Collagenase + Dextranase + Nuclease, 45.90% 205.70% best enzyme conc. Collagenase + Dextranase + Nuclease, 2 54.60% 228.90% concentration ave Collagenase + Papain + Nuclease, best 56.80% 150.80% enzyme conc. Collagenase + Papain + Nuclease, 2 61.80% 150.70% concentration ave Savinase + Trypsin + Nuclease, best 77.60% 56.90% enzyme conc. Savinase + Trypsin + Nuclease, 2 85.40% 58.70% concentration ave
Table 5 illustrates the top five (5) three-enzyme combinations for either the multi-species biofilms, or S. proteamaculans biofilms. Surprisingly, multi-species biofilms proved to be more vulnerable to enzyme combinations than S. proteamaculans biofilms. Nevertheless, multiple 3 enzyme combinations showed high activity against this resistant species. It is also noteworthy that collagenase is one component of 7 of the 10 three (3) enzyme combinations given in Table 5 making it an important member of the enzyme cocktail developed.

[0033] While applicant's disclosure has been summarized with reference to specific embodiments above, it will be understood that modifications and alterations in the embodiments disclosed may be made by those practiced in the art without departing from the spirit and scope of the invention. All such modifications and alterations are intended to be covered.