Screening method

Abstract

A method for screening substances for their ability to reduce malodours from emanations from an animal, said method comprising determining the effect of said substances on the C-S lyase activity of bacteria that emit volatile sulphuric compounds (VSCs), by contacting a test substance with a sample comprising said bacteria or a supernatant obtainable from a culture of said bacteria in the presence of a substrate for a C-S lyase, detecting the levels of thiol production from said bacteria, and comparing the results with those obtained from similar bacteria in the absence of said substance.

Claims

1. A method for screening substances for their ability to reduce malodours from emanations from an animal, said method comprising determining the effect of said substances on the C-S lyase activity of bacteria present in an oral cavity of an animal that emit volatile sulphuric compounds (VSCs), by contacting a test substance with a sample comprising said bacteria or a supernatant obtainable from a culture of said bacteria in the presence of a substrate for a C-S lyase, detecting the levels of thiol production from said sample, and comparing the results with those obtained from said sample in the absence of said substance, wherein the method does not include lysis and wherein the bacteria are selected from selected from the group consisting of Escherichia coli DH5, Porphyromonas macacae (Genbank Accession No. JN713359), Porphyromonas macacae (Genbank Accession No. NZ_JRFB00000000) and Synergistales sp. (Genbank Accession No. JN713343).

2. A method according to claim 1 wherein the sample comprises live bacteria.

3. A method according to claim 1 wherein the C-S lyase is cystathione-β-lyase and the substrate is benzylcysteine.

4. A method according to claim 1 wherein levels of thiol production are detected using a fluorometric method.

5. A method according to claim 4 wherein levels of C-S lyase activity are determined by culturing live bacteria in the presence of both a substrate for the enzyme, and an indicator which reacts with a thiol to produce a fluorescent signal.

6. A method according to claim 5 wherein the indicator is a bimane dye derivative.

7. A method according to claim 6 wherein the bimane dye derivative is monobromobimane (3-(bromomethyl)-2,5,6-trimethyl-1H,7H-pyrazolo[1,2-α]pyrazole-1,7-dione).

8. A method according to claim 1 wherein a co-enzyme is added to the sample to catalyse the enzymatic reaction.

9. A method according to claim 8 wherein the co-enzyme is pyridoxal-5′-phosphate.

10. A method according to claim 1 wherein the animal is a companion animal.

11. A method according to claim 1 wherein the bacteria used is one which emits high levels of VSCs.

12. A method according to claim 11 wherein the bacteria is Escherichia coli DH5α.

13. A method according to claim 1 wherein the bacteria used has been identified as being present in plaque biofilms, saliva, or from the buccal region or dorsum part of the tongue, of one or more companion animals.

14. A method according to claim 13 wherein the bacteria used is identified as being one that emits a higher level of VSCs as compared to levels of VSCs of other bacteria identified as being present in the oral cavity.

15. A method according to claim 13 wherein the companion animal is a dog.

16. A method for screening substances for their ability to reduce malodours from emanations from an animal, said method comprising determining the effect of said substances on the C-S lyase activity of bacteria that emit volatile sulphuric compounds (VSCs), by contacting a test substance with a sample comprising said bacteria or a supernatant obtainable from a culture of said bacteria in the presence of a substrate for a C-S lyase, detecting the levels of thiol production from said sample, and comparing the results with those obtained from said sample in the absence of said substance, wherein said method is carried out using more than one bacterial strain selected from the group consisting of Escherichia coli DH5, Porphyromonas macacae (Genbank Accession No. JN713359), Porphyromonas macacae (Genbank Accession No. NZ_JRFB00000000) and Synergistales sp. (Genbank Accession No. JN713343), and does not include homogenisation and lysis.

17. A method according to claim 16 which further comprises selecting target substances for the treatment of malodour which inhibit VSC production from more than one of the bacterial strains tested.

18. A method according to claim 16 wherein the method is carried out in a combined culture.

Description

DETAILED DESCRIPTION

(1) The present method will now be particularly described by way of example. However, it will be apparent to one skilled in the art that the specific details are not required in order to practice the invention. The following descriptions of specific embodiments of the present invention are presented for purposes of illustration and description. They are not intended to be exhaustive of or to limit the invention to the precise forms disclosed. Many modifications and variations are possible in view of the above teachings. The embodiments are shown and described in order to best explain the principles of the invention and its practical applications, to thereby enable others skilled in the art to best utilize the invention and various embodiments with various modifications as are suited to the particular use contemplated. The examples refer to the accompanying diagrammatic drawings in which:

(2) FIG. 1 shows the results of an experiment to determine estimated mean VSC production per minute indicated by the fluorescence measures (mBB method). Annotated letters indicated Tukey groups from the pairwise comparisons where bacterial strains sharing no letters were significantly different (for example DH5α versus Neisseria animaloris JN713177).

(3) FIG. 2 shows the results of an experiment to determine estimated mean VSC production indicated by the lead acetate measures. Annotated letters indicate Tukey groups from the pairwise comparisons where bacterial strains sharing no letters were significantly different (for example Peptostreptococcus hiranonis JN713315 versus Neisseria zoodegmatis JN713518).

(4) FIG. 3 shows estimated means from the results illustrated in FIGS. 1 and 2, matched by bacterial strain.

(5) FIG. 4 shows mean lab measures for each bacterial strain tested with the estimated correlation and p-value for a significant difference from 0.

(6) FIG. 5 shows the results of an experiment to determine the estimated mean VSC produced by bacteria Porphyromonas macacae JN713359 with and without known active substances. Annotations show fold changes versus the control and p-values of significance.

(7) FIG. 6 shows the results of an experiment to determine estimated mean VSC produced by bacteria Fusobacterium nucleatum ATCC10953 with and without active substances. Annotations show fold changes versus the control and p-values of significance.

(8) FIG. 7 shows the results of an experiment to determine the estimated mean VSC produced by bacteria Porphyromonas macacae JN713359 with and without test substances in accordance with the screening method. Annotations show fold changes versus the control and p-values of significance.

(9) FIG. 8 shows the results of an experiment to determine the estimated mean VSC produced by bacteria Fusobacterium nucleatum ATCC10953 with and without test substances in accordance with the screening method Annotations show fold changes versus the control and p-values of significance.

(10) FIG. 9. Level of VSCs generated by canine oral and faecal strains. ATCC10953: Fusobacterium nucleatum: known bad breath causing bacteria in human, also isolated from canine oral cavity. JN713359: Porphyromonas macacae: a periodontal disease associated bacteria in canine oral cavity. ATCC19433(006): Enterococcus faecalis: canine faecal bacteria also found in human faeces, ATCC9689 (7276): Clostridium difficile another bacteria isolated from canine faeces, that has been isolated from elsewhere previously.

(11) FIG. 10 shows the results of an experiment to determine estimated mean VSC produced by bacteria Fusobacterium sp RMA1065 (AJ867040) with and without active substances. Annotations show fold changes versus the control and p-values of significance.

(12) FIG. 11 shows the results of an experiment to determine estimated mean VSC produced by E. coli bacteria (CP001855) with and without active substances. Annotations show fold changes versus the control and p-values of significance.

(13) FIG. 12 shows the results of an experiment to determine the estimated mean VSC produced by Fusobacterium sp RMA1065 (AJ867040) with and without test substances in accordance with the screening method. Annotations show fold changes versus the control and p-values of significance.

(14) FIG. 13 shows the results of an experiment to determine the estimated mean VSC produced by E. coli bacteria (CP001855) with and without test substances in accordance with the screening method. Annotations show fold changes versus the control and p-values of significance.

EXAMPLE 1

(15) Fluorescent Method for Detecting Hydrogen Sulphide Production and Identification of High VSC Emitting Strains from Oral Cavities of Dogs

(16) A range of bacterial samples obtained from isolates from the canine oral cavity as well as two type strains (F. nucleatum ATCC10953 and Escherichia coli DH5α) were used in the experiment and these are listed in Table 1 hereinafter. All bacteria strains were stored at −80 C°.

(17) TABLE-US-00001 TABLE 1 Genbank Accession Number Taxonomy JN713478 Bacteroides heparinolyticus JN713352, JN713353 Bergeyella_zoohelcum_COT-186 JN713168, JN713169, Campylobacter_sp. COT-011 JN713170, JN713171 JN713418 Capnocytophaga_sp._COT-254 JN713473 Chloroflexi_[G-1]_sp._COT-306 DH5 Escherichia_coli ATCC10953 Fusobacterium_nucleatum_ATCC10953 JN713356 Fusobacteriusp._COT-189 JN713237 Lachnospiraceae_bacterium_COT-073 JN713178 Moraxella_sp._COT-017 JN713497 Moraxella_sp._COT-328 JN713177 Neisseria_animaloris_COT-016 JN713254 Neisseria_sp._COT-090 JN713518 Neisseria_zoodegmatis_COT-349 JN713256 Pasteurella_dagmatis_COT-092 JN713216 Peptostreptococcaceae_bacterium_COT-047 JN713315 Peptostreptococcus_hiranonis_COT-148 JN713198 Peptostreptococcus_sp._COT-033 JN713350 Porphyromonadaceae_bacterium_COT-184 JN713276, JN713277 Porphyromonas_cangingivalis_COT-109 JN713184 Porphyromonas_gingivicanis_COT-022 JN713220, JN713221 Porphyromonas_gulae_COT-052 NZ_JRFB00000000 Porphyromonas_macacae COT-192 OH2631 JN713359 Porphyromonas_macacae_COT-192 JN713457 Porphyromonas_sp._COT-290 JN713343 Synergistales_bacterium_COT-178

(18) COT: Canine Oral Taxon, an identification system used at HOMINGS systems for microbial taxonomy. All strains used were isolated from canine oral cavities. Strain identification was made within 98% identity to the Genbank identifications and type strain mentioned above.

(19) The C-S lyase activity was measured in order to quantitate VSC production by the bacteria. This was conducted by using monobromobiname, a fluorescent probe for thiol. The bacteria were washed and resuspended in Tris buffer (pH 8.5) at OD=1.0. The 250 μL reaction mix contained 4 mM benzylcysteine, 100 μM monobromobimane, 10 μM pyridoxal 5′-phosphate, 50 μL of the bacteria suspension and was made up to 250 μL with tris buffer (pH 8.5). The mix was incubated anaerobically for 45 minutes at 37 C°. The C-S lyase activities were determined based on fluorescence at 490 nm (em) upon excitation at 385 nm (ex).

(20) All measurements were performed in biological duplicate with each repeated in technical triplicates, creating total of 6 data points per strain. Blank measurements (containing buffer instead of crude extract) were used to correct the values derived from the substrate. The standard curve was measured using Benzyl mercaptan at 8-128 μM. The protein concentration of the bacteria suspension was determined by means of a commercially available BCA kit in accordance with the manufacturer's method. The specific C-S lyase activity was calculated by dividing the increase in fluorescence equivalent to Benzyl mercaptan concentration by the total protein content of the suspension in mg and expressed as unit which was defined as “nM Benzyl mercaptan release/μg protein/minute”.

(21) The results are shown in FIG. 1.

(22) An additional experiment was carried out using a different hydrogen sulfide method. In this case, bacteria cultured in Brian Heart Infusion media (BHI) were pelleted at 2000×g for 10 minutes then transferred to fresh BHI at pH 8.5 at OD600=0.5. Three mL of BHI suspension was added to a 7 mL bijoux vial.

(23) Fresh BHI was used as the negative control. All samples were conditioned in an anaerobic cabinet at 38 C° for at least half an hour prior to the following steps. A 50 μL of 0.05% (w/w) cysteine solution was then added to the samples. In this case, a lead acetate strip was placed so as to hang from the lid of bijoux vial in order to avoid contact with the solution. Samples were incubated for one hour anaerobically. Lead acetate strips were then removed from the vials and visually measured for their colour intensity in a scale of 0-10 with 10 being the most intense black.

(24) Results are shown in FIG. 2.

(25) Analysis of the two lab measurements of VSC production indicate clear patterns in the produced quantities from the bacterial strains, FIG. 1 and FIG. 2. For both measurements significant differences were found between certain pairs of bacteria. For example, DH5α versus Neisseria animaloris JN713177 in the fluoroscopy data. Comparing the two lab measures, FIG. 4, indicates significant correlation between the two although with a different order of bacteria when sorted by production, FIG. 3.

(26) As a result, six bacteria, which were either high VSC emitters and/or isolable from various areas of a dog's mouth were selected for further work. These were Escherichia coli DH5, Porphyromonas macacae (JN713359 and NZ_JRFB00000000), Synergistales sp. (JN713343), Peptostreptococcus hiranonis (JN713315), Fusobacterium nucleatum (OH65) and Neisseria zoodegmatis (JN713518). Three of these Escherichia coli DH5, Porphyromonas macacae (JN713359 and NZ_JRFB00000000) and Synergistales sp. (JN713343) were particularly useful as they were the highest emitters with a similar level of statistical significance.

EXAMPLE 2

(27) Proof of Principle for Screening Method Using mBB with Known Actives

(28) Two test strains, Fusobacterium nucleatum (ATCC10953) and Porphyromonas macacae (JN713359), were selected for a proof of concept experiment. Known active ingredients in amounts summarised in Table 2 below, were tested using mBB method described above in Example 1. Each active ingredient was added to the bacteria resuspension immediately before its addition in the well. The following active ingredients were tested for this proof of concept experiment.

(29) TABLE-US-00002 TABLE 2 Final Reported Active concentration used concentration Chlorohexidine (CHX) 0.12% w/w 0.12% w/w DL-Propargylglycine (PAG) 100 μM 40 μM O-Carboxylmethyl- 20 μM 10 μM hydroxylamine hemihydrochloride(AOAA) Aminoevulinic acid (AVG) 5 μM 1 μM

(30) The selection of actives was based on the cost and volatility. The results are shown in FIGS. 5 and 6 respectively. These results show that the each of the active ingredients tested reduced the VSC production, confirming that this method provides an indicator of activity.

(31) The results from validation using known actives also showed that the method will identify agents that target the underlying causes of oral malodour and steers away from masking agents. Chlorohexidine (CHX) is a widely prescribed antiseptic in the form of an oral rinse after oral surgery whereas the other three tested actives are known inhibitors for VSC producing enzymes, i.e. cystathionine β synthase (CBS) and cystathionine γ lyase (CSE). Only CHX has been reported to have activity as an antiseptic or disinfectant. This demonstrates that the method is capable of identifying and evaluating enzyme inhibitors as well as antimicrobials.

(32) Another noteworthy point is that the mBB method in particular leverages the enzyme degradation of amino acid to VSC, therefore it will be able to screen out masking agents.

EXAMPLE 3

(33) Use of Screening Method to Test a Range of Substances as VSC Inhibitors

(34) The method of Example 2 was then repeated using a range of substances of unknown activity. These are listed in the following Table 3.

(35) TABLE-US-00003 TABLE 3 Active Comments Vitamin C The optimal pH for the target enzyme is in the (ascorbic acid) alkaline range. The impact on addition of food grade acid and lowering pH may impact on enzyme activity Tea polyphenol Green tea extract is included in many products (catechin) active in breath freshening. Polyphenols and catechins have previously been reported as having anti-VSC- activity Thymol An active anti-microbial in commercial mouthwash and thus often used as the positive control for personal care/food grade oral care products where comparison with prescription antiseptic such as chlorhexidine may not be relevant. Magnolia Bark known food grade anti-microbial natural product Extract (MBE) (see for example U.S Pat. No. 8,012,514) Chlorhexidine The gold standard antiseptic for oral microbiology

(36) Of the above, Chlorhexidine (CHX) acted as a positive control, and buffer (Neg control) and 1% DMSO as negative controls. 1% DMSO was also used to dissolve the actives.

(37) MBE at both concentrations showed inhibitory activities with p<0.01.

(38) The results are shown in FIGS. 7 and 8. The analysis of the bacterial VSC production, under several active ingredients, indicated significant reductions in production for all ingredients aside from DMSO, ascorbic acid and catechin. For example with strain Porphyromonas macacae JN713359 the 0.12% CHX treatment resulted in only 25% the quantity of VSC produced as the control.

(39) FIG. 7 shows the results from canine oral bacteria and FIG. 8 with human oral bacteria often used as the gold standard for bad breath causing bacteria (Fusobacterium nucleatum). FIG. 8 confirms that the method of the invention works with human oral species and thus validates results from canine oral bacteria.

EXAMPLE 4

(40) Demonstration of Similar VSC Production from Faecal Bacteria

(41) The method of Example 2 was repeated using two canine faecal bacteria, alongside two canine oral bacteria. The results are shown in FIG. 9. These results show that VSCs are produced, albeit in lower levels, by faecal bacteria and that therefore the method of the invention may be used to screen substances that could impact on faecal malodour also.

(42) In such cases, any substances tested would either be required to administered orally, and so be resistant to degradation in the stomach, and/or be formulated with for example an enteric coating, to protect the substance as it passes through the gut of the companion animal.

(43) In addition, the method of example 2 was repeated using two strains of fecal bacteria isolated from canine faeces.

(44) In particular, canine faeces were suspended in nutrient broth to form a faecal slurry. Dilutions of the slurry were inoculated on varied type of agar plates and incubated in anaerobic conditions. Individual bacterial colonies were selected and re-streaked on the same type of agar for isolation, and propagated. Isolated bacterial species were then stored by suspending in BHI/glycerol solution and frozen at −80° C. Strains were then raised by taking a loop of inoculum from the frozen vial, steaking on Columbia blood agar and incubating in anaerobic conditions for a minimum of 24 hours. Species are then identified by 16S sequencing for selection.

(45) Two faecal isolates that generated VSC were identified as fusobacterium sp. RMA 1065 (genbank accession number AJ867040) and as an E. coli strain (genbank accession number CP001855. The results obtained when these bacteria were subjected to the method of Example 2 are shown in FIGS. 10 and 11 respectively. Again, these results show that the each of the active ingredients tested reduced the VSC production, confirming that this method provides an indicator of activity.

(46) The same two strains were then tested using the method of Example 3. Results are shown in FIGS. 12 and 13. The analysis of the bacterial VSC production, under several active ingredients, indicated significant reductions in production for all ingredients aside from DMSO, ascorbic acid and catechin as before. Thus, the faecal bacteria appear to respond in a similar fashion to the oral bacteria tested.