SELECTIVE SALMONELLA OR E. COLI CULTIVATION METHOD, COMPOSITIONS AND USES

Abstract

The present disclosure provides rapid, convenient and sensitive means to detect low abundance target Gram negative bacteria such as Salmonella contamination from a range of samples, including food samples and environmental samples, through a single-step culture method and downstream sensitive detection.

Claims

1. A method for selectively culturing Salmonella or E. coli, the method comprising: culturing microorganisms present in a test sample in a culture medium, wherein the culture medium comprises an agent of Formula I ##STR00024## wherein A is selected from ##STR00025## and R is selected from substituted C.sub.5-20 aryl, with the proviso that when A is 2PY, then R is not 1,3-dimethylphenyl.

2. The method of claim 1 wherein the agent of Formula I is selected from the group consisting of 3PYaf, 4PYaf, 4PYam, 4PYcb, 4PYco, 4PYcq, 4PYeh, HDcb, HDce, HDcf and HDdb.

3. The method of claim 1, wherein the agent of Formula I is present in the culture media in a concentration of: at least 0.5 mg/L; and no more than 15 mg/L.

4. The method according to claim 1, wherein the E. coli is a Shiga toxin producing E. coli.

5. (canceled)

6. The method according to claim 1, wherein the culture medium further comprises an anti-bacterial agent, wherein the anti-bacterial agent comprises cefsulodin or novobiocin.

7. The method according to claim 1, wherein the culture medium further comprises at least one of: cefsulodin at a concentration of from 1 mg/L to 20 mg/L; novobiocin in an amount of from 2 mg/L to 40 mg/L; ferric chloride at a concentration of from 0.25 mg/L to 4 mg/L; a sulphite compound at a concentration of from 0.5 g/L to 4.0 g/L.

8. The method according to claim 1, wherein the test sample is a sample that comprises or is expected to comprise at least one or more cells other than a Salmonella or E. coli cell.

9. The method of claim 1, wherein the test sample comprises at least one of: a food product; a clinical sample; a therapeutic product; a swab; a cosmetic.

10. The method according to claim 1, wherein the test sample is not a pure culture of Gram negative bacteria.

11. The method according to claim 1, wherein the method comprises a single culture step.

12. The method of claim 1, wherein the test sample is cultured in a culture medium for: from 10 to 18 hours; less than 24 hours; or for 16 hours at a temperature from 30° C. to 44° C.

13. (canceled)

14. (canceled)

15. A culture medium for the selective growth of Salmonella cells or E. coli cells that may be present in a test sample, the culture medium comprising: an agent of Formula I as defined in claim 1; at least one of: cefsulodin; or novobiocin.

16. (canceled)

17. The culture medium according to claim 15, wherein the culture medium comprises at least one of: cefsulodin at a concentration of from 1 mg/L to 20 mg/L; or novobiocin in an amount of from 2 mg/L to 40 mg/L.

18. The culture medium according to claim 15, wherein the culture medium further comprises at least one of: ferric chloride; or a sulphite compound.

19. (canceled)

20. (canceled)

21. A composition comprising: an agent of Formula I as defined in claim 1; at least one of: cefsulodin; or novobiocin.

22. The composition according to claim 21 wherein the composition further comprises at least one of: ferric chloride; or a sulphite compound.

23. The composition according to claim 22, wherein the concentration of: the agent of Formula I is at least 1.00 mg/ml and no more than 1.50 mg/ml; novobiocin is at least 2.00 mg/ml and no more than 3.00 mg/ml; cefsulodin is at least 1.4 mg/ml and no more than 2.0 mg/ml; ferric chloride is from 10 mg/L to 10 g/L; the sulphite compound is at least 5 g/L and no more than 50 g/L.

24. The composition according to claim 21, wherein the composition is lyophilised.

25. A method for quantifying or detecting the presence or absence of Salmonella or E. coli in a test sample, the method comprising: selectively culturing microbial cells present in the test sample; and quantifying or detecting the presence or absence of the Salmonella or E. coli; wherein the test sample comprises a food product, a clinical sample, a swab, a cosmetic, raw meat, raw poultry, a swab taken from raw meat, a swab taken from raw poultry, non-microbial material, or liquid in which a sample substance is suspended.

26-34. (canceled)

35. A kit for the selective culture of Salmonella cells or E. coli cells that may be present in a test sample, wherein the kit comprises: a) an agent of Formula I as defined in claim 1; and cefsulodin, novobiocin, ferric chloride, or a sulphite compound; b) an agent of Formula I as defined in claim 1; cefsulodin; and novobiocin; or c) an agent of Formula I as defined in claim 1; cefsulodin; novobiocin; ferric chloride; and a sulphite compound.

36-39. (canceled)

40. The method of claim 1, wherein where A is 2PY, 3PY, 4PY PZ QN or HD, R is selected from the group consisting of: ##STR00026## ##STR00027## ##STR00028## ##STR00029## ##STR00030## ##STR00031## ##STR00032## ##STR00033## ##STR00034## ##STR00035## ##STR00036##

41. The method of claim 40, wherein the test sample comprises a food product, a clinical sample, a therapeutic product, a swab, a cosmetic, raw meat, raw poultry, a swab taken from raw meat, a swab taken from raw poultry, non-microbial material, or a suspension of any preceding sample in a liquid.

42. The culture medium of claim 18, wherein the concentration of ferric chloride is at least 0.25 mg/L and no more than 4 mg/L.

43. The culture medium of claim 18, wherein the concentration of the sulphite compound is at least 0.5 g/L and no more than 4.0 g/L.

44. The kit of claim 35, wherein the agent of Formula I is 4PYcq.

Description

FIGURE LEGENDS

[0912] FIG. 1

[0913] FIG. 1 illustrates the general structure of the LPS (O-antigen, core polysaccharide (oligosaccharide), lipid A) of certain bacteria of interest and shows a detailed illustration of the Salmonella LPS monomer including the species specific antibody binding epitope.

[0914] FIG. 2—ELISA values obtained from the primary enrichment broth for both the ISO test and the test of the present disclosure.

[0915] FIG. 2 illustrates ELISA buffered peptone water optical densities (450 nm) generated from various food samples artificially inoculated with low levels of multiple Salmonella serovars enriched using either an ISO 2-step enrichment protocol or a cefsulodin, novobiocin, 4PYcq lyophilised single step culture at 41.5° C., where an OD.sub.(450)≥0.20 is a presumptive positive result. The ELISA readings in both cases represent BPW samples taken at 20 hours and after heat treatment tested in our preferred ELISA system. Note the improved optical densities with the novel selective enrichment step compared to un-supplemented BPW.

[0916] Supplements (Abtek RK26S and SB29S) 1 ml added to 225 ml of BPW. Supplemented BPW @ 41.5° C. (20 hours). Food samples inoculated and left at 4 C for 48 hours before addition of media/stomaching/incubation at 41.5 C for 20 hours.

[0917] FIG. 3

[0918] FIG. 3 illustrates internal Method Comparison study data in multiple food categories and sample matrices containing competing microflora. In this unpaired study these samples were artificially inoculated with multiple Salmonella serovars (1-5 colony forming units) and enriched using either an ISO 2-step enrichment protocol; a selective cefsulodin, novobiocin, brilliant green lyophilised single step culture; or a selective cefsulodin, novobiocin, 4PYcq lyophilised single step culture. This data demonstrates the potential inhibitory effect of brilliant green supplement on the growth and recovery of Salmonella even in the presence of food matrixes resulting in an inferior performance to the Reference ISO 2-step enrichment protocol and our alternative supplement with 4PYcq. This is indicated by the 5 negative results seen with the brilliant green supplement compared to ISO and 4PYcq based supplement.

[0919] In contrast, within diverse food categories, the 4PYcq supplement shows superior performance over the brilliant green supplement, and performing at least as well as the ISO Reference method.

[0920] FIG. 4

[0921] FIG. 4 illustrates inclusivity study data where multiple Salmonella serovars were artificially inoculated into either Buffered Peptone Water (BPW) on its own, or BPW enriched with either a selective cefsulodin, novobiocin, brilliant green lyophilised supplement run in the absence or presence of an oatmeal food matrix; or a selective cefsulodin, novobiocin, 4PYcq lyophilised supplement run in the absence of an oatmeal food matrix. This data demonstrates the potentially toxic effect of brilliant green supplement and the requirement of a food matrix to allow growth of the majority of Salmonella serovars. In contrast, the 4PYcq supplement is showing superior performance through the inhibition of Gram positive bacteria thereby allowing the free growth of Salmonella even in the absence of an oatmeal food matrix.

[0922] FIG. 5

[0923] FIG. 5 illustrates exclusivity study data where multiple non-Salmonella bacterial species were artificially inoculated into Brain Heart Infusion broth at high levels in incubated for 20 hours before testing. This data demonstrates that the Salmonella One candidate test is highly specific and does not cross react with a diverse array of Gram negative bacterial species.

[0924] FIG. 6

[0925] FIG. 6 illustrates inclusivity study data where multiple Salmonella serovars were artificially inoculated into BPW enriched with a selective cefsulodin, novobiocin, 4PYcq lyophilised supplement at low levels. This data demonstrates that the Salmonella One candidate test is highly specific towards Salmonella and the selective cefsulodin, novobiocin, 4PYcq lyophilised supplement allows the free growth of Salmonella.

[0926] FIG. 7

[0927] FIG. 7 illustrates internal Method Comparison study data in multiple food categories and sample matrixes containing competing microflora. These samples are artificially inoculated with multiple Salmonella serovars and enriched using either an ISO 2-step enrichment protocol or a selective cefsulodin, novobiocin, 4PYcq lyophilised single step culture. This data demonstrates that within diverse sample matrixes a test according to the present disclosure is performing at least as well as the ISO Reference method, and is allowing the growth of Salmonella to levels high enough to be detected by the candidate test method and alternative cultural confirmation methods. Also in most cases the Salmonella were detected on the primary subcultures from the selective BPW which means basic confirmation of the positive ELISA can be done within 24 hours.

[0928] FIG. 8

[0929] Recovery of Salmonella from different spice and flavourings in BPW media alone.

[0930] FIG. 9

[0931] Common spice and flavourings sample analysis—effect of spice neutraliser

[0932] FIG. 10

[0933] Common spice and flavourings sample analysis—effect of the ‘spice neutraliser’ at fractional inoculation levels

[0934] In the presence of the ‘Spice neutraliser’, equivalence is observed between the different BAM reference methods (10-100 g/1:10-1:100 enrichment) and Solus One Salmonella test method (i.e. the one-step culture method) (25 g/1:10 enrichment).

[0935] Salmonella weren't detected in Tandoori masala using the BAM reference method, however the Solus Test method with spice neutraliser showed detection—this could be due to experimental variation.

[0936] This was at fractional inoculation level (2-8 cfu). Accordingly a method with the ‘Spice neutraliser’ supplement is capable of detecting Salmonella from spice/herb/flavouring samples, even when the Salmonella is present at fractional levels.

[0937] FIG. 11

[0938] Common spice and flavourings sample analysis—comparison of full strength vs half strength Solus supplement (4PYcq, Novobiocin and Cefsulodin)

[0939] Full strength: 5 mg/L 4PYcq, 8 mg/L Cefsulodin and 10 mg/L Novobiocin

[0940] Half strength 2.5 mg/L 4PYcq, 4 mg/L Cefsulodin and 5 mg/L Novobiocin

[0941] There is equivalence between full strength and half strength Solus supplement.

[0942] In the presence of the ‘Spice neutraliser’ supplement, equivalence is observed between the different BAM reference methods (10-100 g/1:10-1:100 enrichment) and the Solus One Salmonella test method (i.e. the one-step culture method) (25 g/1:10 enrichment) Un-spiked samples return low backgrounds. This was at a high inoculation level (40-92 cfu).

[0943] FIG. 12

[0944] Comparison of single strength vs double strength ‘spice neutraliser’ in the Solus One Salmonella Test method

[0945] This data again shows that with half of the strength of the supplement (Half strength 2.5 mg/L 4PYcq, 4 mg/L Cefsulodin and 5 mg/L Novobiocin) plus the “spice neutraliser” (ferric chloride and a sulphite compound such as potassium sulphite), recovery and detection of Salmonella from onion powder, garlic powder and cinnamon is good.

[0946] However, again recovery from cloves and oregano is difficult, even in the presence of double strength “spice neutraliser” supplement.

[0947] FIG. 13

[0948] Relevance of individual spice neutraliser components

[0949] FIG. 14

[0950] BAM Reference Method

[0951] FIG. 15

[0952] Validation data, comparing a standard 2-step culture protocol, with a sulphite compound such as potassium sulphite and ferric chloride, to the single-culture method according to the disclosure, also comprising ferric chloride, a sulphite compound such as potassium sulphite and the selective supplement comprising 4PYcq, Novobiocin and Cefsulodin.

[0953] FIG. 16

[0954] FIG. 17

[0955] The chemical structure of some inhibitory components of common herbs and spices.

[0956] FIG. 18

[0957] Conditions.

[0958] FIG. 19

[0959] This table shows the growth of stressed Salmonella from non-selective and selective BPW from UHT milk as measured by ELISA. Solus One Salmonella Supplement recovers the Salmonella but inhibits non-Salmonella whereas the Brilliant Green supplement is showing an inhibitory effect.

[0960] FIG. 20

[0961] Data for the detection of E. coli O157 cultured using methods according to the disclosure. The enrichment for Solus One E. coli O157 uses BPW with the triple supplement (C-X, Novobiocin and Cefsulodin). In this instance, half strength supplement is used (2.22 mL combined supplement per Litre BPW broth, i.e. 2.5 mg/L 4PYcq, 4 mg/L Cefsulodin and 5 mg/L Novobiocin).

[0962] The data shows 75% recovery which is fractional recovery. The supplement allows a 2 cfu E. coli O157 inoculum to come through against a 1.68E+08 Gram −ve background.

[0963] The disclosure will be further understood with reference to the following non-limiting experimental examples.

EXAMPLES

Example 1—Sensitivity of Microbial Cells to Brilliant Green is Influenced by the Presence of Food and the Type of Food

[0964] The inventors surprisingly found that the standard prior art methods of culturing Salmonella, are relatively slow and inadequate for current testing needs, (FIG. 2).

[0965] The strength of the detection signal obtained from cultures of various Salmonella strains cultured from various food backgrounds was much more variable when the cells were cultured using the standard current ISO method which is a 2-step culture compared to a method according to the disclosure which involves the use of an agent of formula I such as 4PYcq rather than BG, and a single culture step (FIG. 3). Reduced variability in the ability of a particular method to detect the presence of Salmonella, whatever the background contaminants (e.g. food debris) or Salmonella strain is a particular advantage of the present disclosure.

Example 2—Culture with 4PYcg Allows Growth of Salmonella from Low Abundance Samples to Detectable Levels in 20 Hours Whereas Culture with BG does not

[0966] Furthermore, culture for 20 hours of low to moderate inoculum levels of pure Salmonella cultures with 8 mg/L BG resulted in insufficient recovery of Salmonella for detection using the ELISA method described herein (FIG. 4). However, 20 hour culture with 5 mg/L 4PYcq, the same concentration as used in the assays described herein and which results in sufficient suppression of competing microflora from a range of sample types, including for example dairy and probiotic foods of the same inoculums resulted in positive identification of the presence of Salmonella.

Example 3—Reduced Salt Levels During Exposure of the Sample to the Detection Member Significantly Increases Sensitivity

[0967]

TABLE-US-00001 TABLE 2 (signal) Conjugate Diluent Type Hepes 25 mM pH 7.0 with 3D11-HRP @ 0.06 μg/ml NaCl % 0% 0.2% 0.4% 0.6% 0.8% S. Enteritidis 2.662 3.752 3.376 2.401 1.577 10.sup.8 cfu/ml S. Enteritidis 1.626 2.389 1.083 0.599 0.308 10.sup.5 cfu/ml S. Typhimurium 3.513 4.458 3.923 2.872 2.246 10.sup.6 cfu/ml S. Typhimurium 1.016 1.978 0.940 0.531 0.324 10.sup.5 cfu/ml Blank value 0.067 0.073 0.095 0.045 0.038

TABLE-US-00002 TABLE 3 (signal to noise ratio) Conjugate Diluent Type Hepes 25 mM pH 7.0 with 3D11-HRP @ 0.06 μg/ml NaCl % 0% 0.2% 0.4% 0.6% 0.8% S. Enteritidis 40 52 36 53 42 10.sup.6 cfu/ml S. Enteritidis 24 33 11 13 8 10.sup.5 cfu/ml S. Typhimurium 52 61 42 63 59 10.sup.6 cfu/ml S. Typhimurium 15 27 10 12 9 10.sup.5 cfu/ml Blank value 0.067 0.073 0.095 0.045 0.038

[0968] Tables 2 and 3 show that an increase in sensitivity is seen when lower salt levels are present in the conjugate buffer. In particular, 0.2% NaCl (34.2 mM) conjugate buffer is optimal for detecting lower concentrations of S. Enteritidis and S. Typhimurium, such as at 1×10.sup.5 cfu/ml, ELISA plates used were coated directly with polyclonal anti-Salmonella antibody.

TABLE-US-00003 TABLE 4 Hepes 25 mM pH 7.0 3D11 HRP @ 0.04 μg/ml % NaCl 10 mM 0% 0.2% 0.4% 0.6% 0.8% dPBS Serrratia liquefaciens 0.070 0.102 0.089 0.052 0.046 0.040 Shigella sonnei 0.075 0.113 0.101 0.054 0.045 0.039 Citrobacter freundii 0.095 0.151 0.112 0.069 0.055 0.046 Escherichia coli 0.067 0.106 0.071 0.060 0.042 0.034 Klebsiella pneumoniae 0.065 0.109 0.067 0.061 0.040 0.035 S. Enteritidis 10.sup.7 cfu/ml 1.418 1.966 2.730 2.958 2.714 2.455 S. Enteritidis 10.sup.6 cfu/ml 2.657 3.762 3.041 2.345 1.424 1.155 S. Enteritidis 10.sup.5 cfu/ml 1.514 2.374 1.170 0.555 0.307 0.253 Blank value 0.066 0.124 0.085 0.073 0.042 0.036

[0969] Table 4 shows that lower levels of salt, for example, 0.2% NaCl (34.2 mM), intensifies the detection signal significantly for various strains of Salmonella. In this instance background signals increased with the fall in salt levels and there was evidence of a hook type effect.

Example 4—Solus One Salmonella Kit Instructions

[0970] An exemplary method is detailed below.

[0971] This method is for the next day detection of Salmonella in food. This method is adapted to the detection of all Salmonella both motile and non-motile.

[0972] 1. Introduction

[0973] Solus One Salmonella provides a negative or a presumptive positive result from a single enrichment step within 22 hours, of which the immunoassay is 2 hours

[0974] 2. Intended Use

[0975] The test method requires laboratory facilities plus qualified and trained personnel. Basic training is recommended to first time users and can be provided by Solus Scientific Solutions Ltd. Using the method includes compliance with Good Laboratory Practices (refer to EN ISO 7218).

[0976] 3. Reagents Provided

[0977] Kit components are supplied stabilised and ready to use at working concentration. Only the Washing Buffer Activator and Washing Buffer reagent require dilution. The activator must be first dissolved in 1440 ml of deionised (DI) water followed by the addition of the 60 ml concentrated Washing Buffer reagent to this solution.

[0978] Each kit contains sufficient material for 5×93 determinations, plus controls.

[0979] The kit expiry date is displayed on each product label. [0980] 5×96 well microplates (in breakable strip format). Wells are coated with antibodies against Salmonella spp. [0981] Negative Control (Green label). 10 ml in working dilution. Contains diluent with preservative. [0982] Positive Control (Red label). 10 ml in working dilution. Contains heat-killed Salmonella in diluent with preservative. [0983] Conjugate (Orange label). 60 ml in working dilution. Contains horseradish peroxidase-antibody conjugate in diluent with preservative. [0984] Substrate (Blue label). 60 ml in working dilution. Contains 3,3′5,5′-Tetramethylbenzidine (TMB), hydrogen peroxide and stabilisers. Solution should be clear or slightly faint blue. [0985] Stop Solution (Yellow label). 60 ml in working dilution. Contains 10% sulphuric acid. [0986] Washing Buffer Concentrate (25×). 5×60 ml. [0987] Washing Buffer Activator. 5×1 Sachet

[0988] 4. Materials and Equipment

[0989] Required but not Provided [0990] Refrigerator at 2-8° C. [0991] Deionised or distilled water [0992] Buffered Peptone Water (BPWV) according to ISO 6579 [0993] SALSUPPII—Solus One Salmonella Supplement [0994] 70% v:v Ethanol [0995] Measuring cylinder for 250 ml or 1 L [0996] Filter bags (e.g. Stomacher) [0997] Sterile 10 ml test tubes suitable for selective enrichment culture [0998] Stomacher (or similar apparatus) and bags [0999] 3 ml transfer pipettes (sterile) [1000] Test tube for sample boiling (e.g. 5 ml Poly propylene rimless test tubes 12×75 mm) [1001] Vortex mixer [1002] Timer [1003] Incubator or water bath at 41.5±1° C. [1004] Heating block or water bath (capable of heating to 85-100° C.) [1005] Pipettes and tips (1 ml; 0.1 ml) [1006] Microplate washer or squeeze bottle [1007] Microplate reader with 450 nm filter [1008] Autoclave for decontamination of samples

[1009] 5. Reagent Preparation

[1010] 5.1. Washing Buffer:

[1011] Prepare the following in a clean 2 litre vessel—

[1012] 5.1.1 Add the contents of a Washing Buffer Activator Sachet to 1440 ml DI or distilled water and mix until the activator has fully dissolved.

[1013] 5.1.2 Add 60 ml of the concentrated washing buffer reagent to the vessel containing 1440 ml of the dissolved activator solution.

[1014] 5.1.3 Transfer to a storage bottle and label the solution as appropriate.

[1015] 5.2. Culture Broth (growth medium): Prepare Buffered Peptone Water (BPW) ISO6579 following manufacturer's instructions. Allow to cool to room temperature before use in testing.

[1016] 5.3. Prepare the Solus One Salmonella Supplement by the addition of 15 ml 70% v:v Ethanol. Allow the contents to dissolve into solution, this typically takes around 30 minutes at room temperature. Add 4.44 ml supplement per 1 L (1 ml per 225 ml) of BPW. (In some embodiments, half-strength supplement can be used, for example in some circumstances where the sample is a spice or a spicey product, or where the target cells are E. coli for example E. coli O157. In these situations, use 2.22 ml supplement per 1 L BPW).

[1017] 6. Sample Preparation and Enrichment—Standard Method

[1018] Homogenise 25 g of the sample in 225 ml of supplemented BPW, and incubate for 20 to 22 hours at 41.5±1° C.

[1019] In the context of NF VALIDATION test portions weighing more than 25 g have not been tested. Refer to EN ISO 6579 for the specific preparations of the mother suspension for some foods.

[1020] Ensure that the bench processing time of supplemented BPW inoculated samples is kept to a minimum and transferred to the 41.5° C. incubator as soon as possible. This is important to avoid extensive growth of competing organisms.

[1021] When the incubation period in supplemented BPW is completed, carefully remove 1 ml, avoiding particulate and fatty matter (the use of a filter bag for this purpose is highly recommended), to a glass or polypropylene test tube.

[1022] Heat the aliquot to 85-100° C. for 15-20 minutes in the test tube. After heating allow the sample to cool to room temperature. This may be accelerated by placing the test tubes in cold water for 5 minutes.

[1023] Post boiling some samples could contain coagulated protein and fat content which may cause pipetting issues on the Dynex instrument. To avoid instrument errors add a frit to the boiling tube and gently push the frit beneath the surface of the liquid.

[1024] The un-boiled supplemented BPW samples should be kept for verification until immunoassay results are obtained. These samples can be kept at 41.5±1° C. if the immunoassay test is to be carried out within 2 hours or at 2-8° C. for up to 72 hours prior to the immunoassay test.

[1025] 7. Immunoassay Procedure

[1026] 7.1. Take the test kit from storage at 2-8° C. one hour before use to allow the components to reach room temperature. Determine the number of wells required for the test. Take the required number of strips from the pouch and fit them to the frame provided. Unused strips should be returned to the pouch and stored at 2-8° C.

[1027] 7.2. Prepare Washing Buffer as detailed in section 5.1.

[1028] 7.3. Leave the first well in the strip empty to serve as a ‘blank’ for measuring the absorbance of the substrate.

[1029] 7.4. Pipette 0.1 ml of Negative Control (Green label) into the second well.

[1030] 7.5. Pipette 0.1 ml of Positive Control (Red label) into the third well.

[1031] 7.6. Pipette 0.1 ml of each boiled/cooled sample separately into consecutive wells in the strip. If there are wells left over at the end of a test strip the Positive or Negative Controls may be repeated.

[1032] 7.7. Incubate the plate (containing the strips) at 37±1° C. for 30 mins (±5 mins.).

[1033] 7.8. After incubation, aspirate the contents of the wells, removing as much of the liquid as possible. Wash the wells 5-7 times with Washing buffer ensuring complete filling and emptying of the wells through each wash cycle. The washing technique is critical to assay performance, hence it is recommended to use a microplate washer.

[1034] 7.9. Pipette 0.1 ml of Conjugate (Orange label) into all wells except the ‘blank’.

[1035] 7.10. Incubate the plate at 37±1° C. for 30 mins (±5 mins).

[1036] 7.11. Repeat the wash cycles as detailed in section 7.9.

[1037] 7.12. Pipette 0.1 ml of TMB Substrate (Blue label) into all wells, including the ‘blank’ well.

[1038] 7.13. Incubate the plate at room temperature for 30 mins. (±5 mins).

[1039] 7.14. After incubation stop the reaction by adding 0.1 ml of Stop Solution (Yellow label) to all wells including the ‘blank’ well. The Stop Solution will cause the blue colour in the wells to change to yellow.

[1040] 7.15. Read the optical densities within 10 minutes in a plate reader using a 450 nm filter. Inspect the wells before reading for air bubbles and if present burst with a needle. Zero the reader against the ‘blank’ well before the other wells are read.

[1041] 7.16. Do not use reference filter.

[1042] 8. Interpretation of Results

[1043] Results are expressed as optical density (OD450) measurements using microplate reader. Subtract the OD value of the blank well (usually A1) from all of the other results.

[1044] Assay Acceptance Criteria:

[1045] Negative Control <0.100

[1046] OD450

[1047] Positive Control >0.500

[1048] OD450

[1049] Samples with OD450 readings of less than 0.200 are considered negative in which case the analysis is complete, the results may be reported and the corresponding non-boiled aliquot of supplemented BPW broth may be discarded following local regulations/guidelines.

[1050] Sample wells with OD450≥0.200 are considered presumptive positive for Salmonella. Presumptive positive results must be verified using a recognised culture method.

[1051] 9. Confirmation of Positive Results from Salmonella Immunoassay

[1052] Samples with OD's≥0.200 are considered positive for Salmonella.

[1053] All samples identified as positive by the alternative method must be confirmed in one of the following ways.

[1054] The confirmation step must start from the (un-boiled) supplemented BPW samples stored at 41.5° C. or 2-8° C.:

[1055] Streak the supplemented BPW sample onto 1 agar plate (XLD or a chromogenic agar for Salmonella such as Colorex Salmonella from Chromagar). Incubate agar as specified by standard Salmonella cultural protocols and then perform confirmation tests e.g. Microgen latex test F42 or biochemical identification gallery directly on isolated colonies without purification step or by performing the tests described in the standardised methods (CEN or ISO). The F42 latex test uses polyclonal antibody to detect flagellar antigens. It is not adapted for the detection of non-motile Salmonella.

[1056] Or additionally subculture the supplemented BPW sample (0.1 ml+10 ml) in RVS broth incubated for 24 h+/−3 h at 41.5° c.+/−1° C. Streaking onto XLD or a chromogenic agar for Salmonella such as Colorex Salmonella from Chromagar). Incubate agar as specified by standard Salmonella cultural protocols and then perform confirmation tests e.g. Microgen latex test F42 or biochemical identification gallery directly on isolated colonies without purification step or by performing the tests described in the standardised methods (CEN or ISO).

[1057] In the event of discordant results (presumptive positive ELISA results, not confirmed by one of the means described above and in particular the latex test) the laboratory must follow the necessary steps to ensure the validity of the result obtained.

Example 5 Effect of Salt in the Wash Solution on the Detection of False Positives

[1058] The following components/steps may be used but other components/steps are also possible.

[1059] Materials

[1060] Culture Media:

[1061] Streptavidin Plates:

[1062] The wells of a 94 well streptavidin plate are coated in 2 μg/ml biotinylated capture antibody.

[1063] Conjugate Buffer:

[1064] 0.15 μg/ml conjugated 3D11 HRP

[1065] 0.06 μg/ml of HRP conjugated antibody with binding affinity for other Salmonella components

[1066] 25 mM HEPES

[1067] 34.22 mM NaCl

[1068] BSA 1%

[1069] 0.1% w/v Proclin 950

[1070] Potassium Ferricyanide 0.001%

[1071] Bovine gamma globulin 0.1%

[1072] It may also be suitable to supplement the conjugate buffer with detergent.

[1073] High Salt Wash Buffer:

[1074] 4.073 mM Tween® 20

[1075] 599 mM NaCl

[1076] Tris

[1077] Substrate:

[1078] This can be purchased as a proprietary solution: for example, Interchim TMB substrate product

[1079] code UP664782.

[1080] Stop Solution:

[1081] 0.1M H.sub.2SO.sub.4

[1082] Method

[1083] The test sample was cultured in a culture medium comprising 5 mg/L 4PYcq and 10 mg/L novobiocin and 8 mg/L cefsulodin. The culture medium was incubated at 41.5° C. for 20 hours. An aliquot of the test sample was heated to 85-100° C. for 15-20 minutes, and allowed to cool to room temperature prior to the assay. 100 μl of the aliquoted test sample was added to the sample wells. The samples were incubated for 30 mins at 37±1° C. for 30 mins with mild agitation for the first 5 mins. All wells are washed 5-7 times with the high salt wash buffer. 100 μl of 0.15 μg/ml conjugated 3D11 HRP and 0.06 μg/ml of HRP conjugated antibody with binding affinity for other Salmonella components is added to the sample well and also the positive and negative control wells and incubated at 37±1° C. for 30 mins. The wells are washed 5-7 times with the high salt wash buffer. 100 μl substrate was then added to the well and incubated at 37±1° C. for 30 mins, after which 100 μl stop solution is then added. Optical density (OD) was recorded from the sample wells and also the positive and negative controls using a plate reader using a 450 nm filter within 10 minutes of the stop solution being added, and calibrated against the blank well.

[1084] Samples with OD450 readings of less than 0.200 are considered negative in which case the analysis is complete, the results may be reported and the corresponding non-boiled aliquot of supplemented BPW broth may be discarded.

[1085] Assay Acceptance Criteria:

TABLE-US-00004 Negative Control OD450 <0.100 Positive Control OD450 >0.500

[1086] Results

TABLE-US-00005 Streptavidin Streptavidin Biotin anti-Salmonella coating Antibody concentration 2 μg/ml 2 μg/ml Anti-Salmonella 3D11 HRP Concentration 0.15 μg/ml 0.15 μg/ml Final Culture Wash buffer salt levels Result for 0.8 3.5% Presence of (137 mM) (599 mM) Salmonella Gaucho sirloin steak 0.010 0.010 − Wagu rump steak 0.020 0.022 − Lean Belgian blue beef 0.007 0.007 − rump steak Lean Italian buffalo 0.011 0.010 − rump steak Wild boar steak 0.064 0.031 − Venison haunch steaks 0.013 0.010 − Diced mixed game 0.255 0.060 − Buffalo quarter pounders 0.015 0.009 − Picanha rump joint 0.016 0.007 − Wild diced venison 0.110 0.021 − Turkey thigh mince 0.523 0.057 − Ostrich fillets 0.009 0.006 − Whole stuffed partridge 0.268 0.051 − Wood pigeon breasts 0.047 0.022 − Chicken livers 0.491 0.097 − Diced pheasant breast 0.100 0.050 − Pheasant thighs 0.195 0.039 − Turkey sausages 3.000 2.664 + Diced turkey breast 1.240 0.105 − Chicken wings 0.087 0.032 − Chicken drumstick 0.489 0.090 − Chicken thigh fillets 1.429 0.186 − Chicken thighs 0.030 0.007 − Chicken breast fillet 0.033 0.013 −

[1087] Table 5 shows that the presence of supplementary NaCl in the wash buffer reduced the signals of false positives compared to wash buffer when NaCl was added at physiological levels (0.8%).

[1088] Effect of MgCl.sub.2 in the Wash Buffer in Reducing the Frequency of False Positives

TABLE-US-00006 TABLE 6 Streptavidin Streptavidin Streptavidin Coating concentration of biotin-anti-Salmonella Ab 1.5 μg/ml 1.5 ug/ml 1.5 μg/ml Anti-Salmonella 3D11 HRP Concentration 0.1 μg/ml 0.1 μg/ml 0.1 μg/ml Wash buffer salt levels 0.8% NaCl 0.8% NaCl (136 mM) + (136 mM) + 0.8% 0.125% 0.0625% (136 mM) (13 mM) (6.6 mM) NaCl MgCl.sub.2 MgCl.sub.2 False +ve poultry 0.26 0.04 0.07 sample False +ve poultry 0.56 0.08 0.16 sample False +ve poultry 0.27 0.07 0.08 sample False +ve poultry 0.89 0.07 0.14 sample False +ve poultry 3.00 0.39 0.57 sample False +ve poultry 0.29 0.27 0.30 sample False +ve poultry 0.21 0.04 0.07 sample False +ve poultry 0.46 0.10 0.15 sample False +ve poultry 0.14 0.03 0.05 sample False +ve poultry 0.28 0.05 0.10 sample S. Ealing 1.10 0.65 0.72 S. Adelaide 1.30 0.74 0.80 S. arizonae 2.74 2.09 2.91 S. Bracknell 1.19 0.68 0.82 S. Tranaroa 3.00 3.00 3.00 S. Alachua 0.91 0.63 0.68 S. Poona 1.05 0.43 0.71 S. Mississippi 1.44 0.64 1.24 S. Redeny 3.00 3.00 3.00 S. Caracas 1.52 0.93 1.90 S. Nottingham 1.91 0.76 1.39 10.sup.6 cfu/ml S. Nottingham 0.17 0.08 0.13 10.sup.5 cfu/ml S. Enteritidis 3.00 2.95 3.00 10.sup.6 cfu/ml S. Enteritidis 2.09 0.32 0.61 10.sup.5 cfu/ml S. Typhimurium 3.00 2.03 3.00 10.sup.6 cfu/ml S. Typhimurium 1.11 0.27 0.48 10.sup.5 cfu/ml Negative control 0.02 0.03 0.03 Positive control 1.72 1.15 1.32

[1089] Table 6 shows that MgCl.sub.2 can be added to the wash buffer to reduce false positive signals although some signal loss for samples containing Salmonella was observed.

[1090] Effect of KCl in the Wash Buffer on Assay Specificity

TABLE-US-00007 Plate coated with anti-Salmonella biotin-polyclonal Ab at 1.5 μg/ml Streptavidin Streptavidin 3D11 HRP Concentration 0.10 μg/ml 0.10 μg/ml 50 μl MgCl.sub.2 Sample additive to 100 μl of sample 3.75% 3.75% (394 mM) (394 mM) Wash buffer NaCl Concentration 0.8% NaCl 0.8% NaCl (137 mM) (137 mM) Wash buffer KCl Concentrations 2.75% KCl 0% (370 mM) False positive poultry 0.09 0.06 sample False positive poultry 0.23 0.09 sample False positive poultry 0.17 0.08 sample False positive poultry 0.41 0.20 sample False positive poultry 0.31 0.11 sample False positive poultry 0.19 0.08 sample False positive poultry 0.25 0.07 sample False positive poultry 0.37 0.10 sample False positive poultry 0.61 0.19 sample False positive poultry 0.44 0.12 sample False positive poultry 0.27 0.24 sample S. Enteritidis 3.00 3.00 1 × 10.sup.7 cfu/ml S. Enteritidis 3.00 3.00 1 × 10.sup.8 cfu/ml S. Enteritidis 3.00 3.00 1 × 10.sup.5 cfu/ml S. Enteritidis 0.72 0.63 1 × 10.sup.4 cfu/ml S. Typhimurium 3.00 3.00 1 × 10.sup.7 cfu/ml S. Typhimurium 3.00 3.00 1 × 10.sup.6 cfu/ml S. Typhimurium 2.25 3.00 1 × 10.sup.5 cfu/ml S. Typhimurium 0.32 0.28 1 × 10.sup.4 cfu/ml

[1091] Table 8 shows the effect of adding supplementary KCl to the wash buffer has a similar effect to NaCl on reducing false positive signals. In this assay 50 μl of the MgCl.sub.2 sample additive as added to 100 μl of sample prior to incubation.

Example 6—Preferred Use of Streptavidin Coated Assay Plates

[1092]

TABLE-US-00008 TABLE 7 Passively coated Streptavidin Anti-Salmonella Coating Antibody concentration 8 ug/ml 2 ug/ml Anti-Salmonella 3D11 HRP Concentration 0.1 ug/ml 0.1 ug/ml 50 μl Sample Additive (+100 μl sample) MgCl.sub.2 3.75% MgCl.sub.2 3.75% (394 mM) (394 mM) Wash Buffer salt levels 3.5% NaCl 3.5% NaCl (599 mM) (599 mM) False +ve poultry sample 0.05 0.03 False +ve poultry sample 0.10 0.05 False +ve poultry sample 0.04 0.03 False +ve poultry sample 0.13 0.04 False +ve poultry sample 0.14 0.08 False +ve poultry sample 0.10 0.05 False +ve poultry sample 0.17 0.08 False +ve poultry sample 0.07 0.03 False +ve poultry sample 0.10 0.05 False +ve poultry sample 0.15 0.06 False +ve poultry sample 0.07 0.05 False +ve poultry sample 0.19 0.06 False +ve poultry sample 0.06 0.03 False +ve poultry sample 0.08 0.04 False +ve poultry sample 0.19 0.06 False +ve poultry sample 0.15 0.06 False +ve poultry sample 0.07 0.04 Salmonella +ve poultry 1.31 2.58 sample Salmonella +ve poultry 0.21 0.41 sample Salmonella +ve poultry 1.70 2.34 sample Salmonella +ve poultry 3.00 3.00 sample Salmonella +ve poultry 3.00 3.00 sample Salmonella +ve poultry 1.78 3.00 sample Salmonella +ve poultry 0.43 0.99 sample Salmonella +ve poultry 1.12 2.43 sample Salmonella +ve poultry 0.77 1.09 sample Salmonella +ve poultry 0.55 1.04 sample Salmonella +ve poultry 0.14 0.48 sample Salmonella +ve poultry 0.09 0.12 sample Salmonella Enteritidis 10.sup.7 3.00 3.00 cfu/ml Salmonella Enteritidis 10.sup.6 3.00 3.00 cfu/ml Salmonella Enteritidis 10.sup.5 0.85 1.20 cfu/ml Salmonella Typhimurium 10.sup.7 3.00 3.00 cfu/ml Salmonella Typhimurium 10.sup.6 3.00 3.00 cfu/ml Salmonella Typhimurium 10.sup.5 0.39 0.76 cfu/ml

[1093] Table 7 shows that using biotinylated polyclonal capture antibody bound to a streptavidin coated plate increases the signal of samples containing Salmonella and also improves assay specificity, compared to the same capture antibody passively bound the polystyrene plate.

Example 7—Use of Alternative Monoclonal Antibody to Detect Salmonella

[1094]

TABLE-US-00009 Plate coated with anti-Salmonella biotin-polyclonal Ab at 1.5 μg/ml Streptavidin Streptavidin Alternative Anti-Salmonella LPS HRP Conjugate concentration 0.20 μg/ml 0.20 μg/ml Wash buffer Salt Levels 0.8% 3.5% (137 mM) (599 mM) False +ve poultry sample 0.30 0.09 False +ve poultry sample 0.49 0.10 False +ve poultry sample 0.24 0.10 False +ve poultry sample 0.26 0.09 False +ve poultry sample 0.98 0.08 False +ve poultry sample 0.38 0.10 False +ve poultry sample 1.29 0.10 False +ve poultry sample 0.72 0.12 False +ve poultry sample 0.19 0.11 False +ve poultry sample 0.17 0.09 False +ve poultry sample 0.33 0.10 False +ve poultry sample 1.90 0.08 False +ve poultry sample 0.29 0.08 False +ve poultry sample 0.83 0.10 False +ve poultry sample 0.28 0.09 False +ve poultry sample 0.85 0.09 False +ve poultry sample 1.20 0.10 False +ve poultry sample 0.43 0.09 False +ve poultry sample 0.27 0.09 S. Enteritidis 10.sup.7 cfu/ml 3.00 2.54 S. Enteritidis 10.sup.6 cfu/ml 3.00 2.71 S. Enteritidis 10.sup.5 cfu/ml 3.00 0.53 S. Typhimurium 10.sup.7 cfu/ml 3.00 3.00 S. Typhimurium 10.sup.6 cfu/ml 3.00 1.91 S. Typhimurium 10.sup.5 cfu/ml 2.62 0.32

[1095] Table 9 shows that a similar monoclonal to 3D11 can also be used in the present disclosure to detect Salmonella cells.

[1096] Selective Growth of Salmonella

[1097] ISO Buffered Peptone Water

TABLE-US-00010 Formulation g/L Enzymatic digest of casein 10 Sodium chloride 5.0 Disodium hydrogen phosphate anhydrous 3.6 Potassium dihydrogen phosphate 1.5

[1098] The above medium is made selective to prevent the overgrowth of the enrichment with competing flora allowing more unrestricted growth of Salmonella.

TABLE-US-00011 Selective supplement mg/L 4PYcq 5.0 Novobiocin 10.0 Cefsulodin 8

[1099] See FIG. 3

Example 8—Recovery and Detection of Salmonella from Test Samples that Comprise Spices, Herbs and Flavourings

[1100] Microbes, such as Salmonella, are notoriously difficult to recover from samples of spices, flavourings and herbs due, in part, to the phenolic compounds that are present in spices, flavourings and herbs. Recovery is typically performed by diluting out the spices and flavourings from the sample. This requires a suitably high initial inoculum level of microbes so that sufficient microbial cells are present in the diluted sub-sample which is taken for recovery and testing. At least for this reason, current methods for recovering and detecting microbes such as Salmonella from spices, flavourings and herbs, are unreliable and inaccurate and are not suitable for routine high-throughput, rapid testing.

[1101] FIG. 8 shows that culturing Salmonella cells from various spices, flavourings and herbs in BPW media alone in a single step, as described herein, is difficult, if not possible.

[1102] However, FIG. 9A shows that the inclusion of the selective supplement comprising 5 mg/L 4PYcq, 8 mg/L Cefsulodin and 10 mg/L Novobiocin as described herein, allows the recovery of Salmonella cells from a variety of different spices, herbs and flavourings. However, the recovery of Salmonella present in garlic granules and cinnamon in a single culture step was not sufficient to allow detection.

[1103] The inventors have surprisingly found that the inclusion of an additional 2 agents, ferric chloride and a sulphite compound such as potassium sulphite, in addition to the 4PYcq/Cefsulodin/Novobiocin supplement, not only allows recovery of Salmonella from these samples (which is achieved with the 4PYcq/Cefsulodin/Novobiocin supplement), but provides results which show equivalence with the results obtained from the BAM reference method (10-100 g/1:10-1:100 enrichment). See FIGS. 9A and 9B.

[1104] The data provided in FIGS. 9A and 9B are derived from samples that were inoculated with a relatively high level of Salmonella (FIG. 9A—77-114cfu; FIG. 9B—38 cfu).

[1105] FIG. 10 provides data derived from fractional inoculation levels (2-8 cfu). In this instance the cells are cultured in the 4PYcq/Cefsulodin/Novobiocin supplement at half the concentration of that used previously, i.e. at a working concentration of 2.5 mg/L 4PYcq, 4 mg/L Cefsulodin and 5 mg/L Novobiocin. Both the standard concentration (5 mg/L 4PYcq, 8 mg/L Cefsulodin and 10 mg/L Novobiocin) and the half concentration (2.5 mg/L 4PYcq, 4 mg/L Cefsulodin and 5 mg/L Novobiocin) have been shown to be equivalent in their ability to recover Salmonella from spices, herbs and flavourings (see FIG. 11).

[1106] FIG. 10 shows that even at fractional inoculation levels, the inclusion of ferric chloride and potassium sulphite (the “spice neutraliser” supplement) allows the recovery and detection of Salmonella from spices, herbs and flavourings.

[1107] Recovery and detection of Salmonella from cloves and oregano is however more difficult, and a higher initial inoculation level may be required. This is true even when the “spice neutraliser” composition is used at double strength (FIG. 12).

[1108] FIG. 13 shows the relevance of each of the components of the “spice neutraliser” (ferric chloride and potassium sulphite).

[1109] With spices/flavourings from the allium family, the potassium salt component in Solus's ‘Spice neutraliser’ is sufficient for recovery of Salmonella.

[1110] For flavouring and pure products just containing onion, the presence of the Solus supplement is sufficient.

[1111] For different spices such as Tandoori masala, we require both the iron and potassium salts in the complete ‘spice neutraliser’ to get effective recovery of Salmonella. See for example FIG. 10 which shows that recovery from Tandoori masala is good when cultured in the presence of both agents of the “spice neutraliser”, i.e. in the presence of ferric chloride and potassium sulphite.

[1112] FIG. 15 shows validation data, comparing a standard 2-step culture protocol, with potassium sulphite and ferric chloride, to a single-culture method according to the disclosure, also comprising ferric chloride, potassium sulphite and the selective supplement comprising 4PYcq, Novobiocin and Cefsulodin.

[1113] FIG. 17 shows the structures of some anti-microbial components of some spices.

Example 9—Methods and Compositions According to the Disclosure are Suitable for the Culture and Detection of Gram Negative Bacteria Other than Salmonella

[1114] Half-strength supplement in BPW was used to culture E. coli O157 from 375 g beef samples (ground mince & raw beef trim)/1:4 enrichment. Culture after only 8-10 hours shows equivalence to some of the molecular methods currently available. See FIG. 20.