Field of the Invention The invention relates to a selective and/or differential growth medium for the isolation of toxigenic Vibrio cholerae, as well as methods for isolating and/or identifying and/or selectively growing toxigenic Vibrio cholerae from a sample.

Introduction

Vibrio cholerae is a member of the family Vibrionaceae, and is a facultative anaerobic, Gram-negative bacterium. Most Vibrio species are ubiquitous in estuarine and marine environments, although Vibrio have been identified in fresh water, provided there is a certain minimal level of sodium ions ¹ .

Of particular importance, ingestion of water or food contaminated with certain strains of V. cholerae cause Cholera, which causes acute diarrhoea, and which if left untreated, can cause death within hours. Researchers have estimated that there are 1.4 to 4.3 million cases, and 28 000 to 142 000 deaths worldwide due to cholera every year ² . The provision of safe drinking water and sanitation is crucial to prevent the transmission of this extremely virulent disease.

V. cholerae can be differentiated serologically on the basis of the O antigen of its lipoplysaccahride (LPS). Indeed, based on the antigenicity of the O antigen, more than two hundred serogroups, identified as 01 to O200 of V.cholerae exist in aquatic environments. However, of these two hundred different serotypes only a subset of 01 and 0139 strains are pathogenic ³ . Furthermore, the pathogenic 01 serogroup is subdivided into two phenotypically distinct biotypes, El Tor and classical. Both biotypes can be further subdivided into two serotypes, Inaba and Ogawa. These serotypes are differentiated in agglutination and vibriocidal antibody tests on the basis of their dominant heat-stable LPS somatic antigens. The cholerae group has a common antigen, A, while the serotypes are differentiated by type-specific antigens, B for Ogawa and C for Inaba. It is believed that 0139 is derived from the El Tor biotype, but has lost the characteristic 01 somatic antigen, while gaining the ability to produce a polysaccharide capsule ⁴ . Until 1992, cholera was believed to be caused only by Inaba and Ogawa, however, in 1992, cholera caused by 0139 emerged in epidemic proportions in India and Bangladesh. Nonetheless, although the disease caused by the 0139 serogroup was devastating, the EL Tor strain remains the dominant strand globally ³ . Other serogroups of V.cholerae can cause diarrheal disease and other pathology, but are not associated with epidemic cholera.

Ships collect ballast water to regulate their stability. However, the discharge of ballast water at port has been responsible for the geographical transfer of large numbers of non-native and undesirable organisms, such as V.cholerae, worldwide with obvious implications for human health. Such is the risk, evidenced in history, that the International Maritime Organisation (IMO) has adopted the International Convention for the Control and Management of Ships' Ballast Water and Sediments (BWM) on 14 February 2004. The BWM aims to prevent the spread of pathogenic organisms from one region to another by establishing standards and procedures for the management and control of ships' ballast water and sediments. In particular, regulation D-2 of the convention establishes numeric ballast water discharge standards for specific microorganisms. For toxigenic V.cholerae (01 and 0139) the limit is less than 1 colony forming unit (cfu) per 100 milliliters or less than 1 cfu per 1 gram (wet weight) zooplankton samples ⁵ . Compliance is required throughout the life of the ship. Moreover, often testing cannot be performed on the ship, but rather requires samples to be sent to laboratories. This can be particularly difficult in more remote locations. Furthermore, laboratory testing is typically slow forcing ships to have long stays at port with significant disruption to shipping at an enormous cost.

There therefore exists a need for a simple yet effective test capable of rapidly detecting very small numbers (<1 CFU/ml or <1 CFU/g) of specifically, toxigenic 01 or 0139 V.cholerae in ballast water.

Once pathogenic V.cholerae is released at ports, it can spread rapidly through the faecal-oral route, particularly in areas with poor sanitation. At this point, rapid bacteriologic diagnosis is essential to prevent a potential epidemic outbreak. Again, there exists a need for a rapid diagnostic test capable of specifically identifying pathogenic V.cholerae in a patient population. Currently, due to their rapid growth and characteristic morphology, V.cholerae are usually isolated and identified in a bacteriology laboratory, typically by plating a sample onto a selective medium. One such selective medium that has been routinely used is Thiosulfate-citrate-bile salts sugar agar (TCBS), where V.cholerae can be identified as a distinctive yellow colony. However, not only does this medium fail to distinguish between toxigenic and non-toxigenic V.cholerae, but the reliability of this medium has been questioned. Moreover, TCBS can also be used to isolate V parahaemolyticus meaning that definitive identification of pathogenic V.cholerae is not possible without further testing ⁴ . Subsequently, a number of alternative media have been suggested and developed, such as sodium dodecyl sulfate-polymyxin-sucrose (SPS) agar and CPC agar ⁵ . Subsequent testing can involve the use of the agglutination test with specific antisera or the use of the oxidase reaction, indole reaction, sugar fermentation reactions and gelatinase, lysine, arginine and ornithine decarboxylase reactions. Alternatively presence of specific bacteria can be detected using genetic methods such as PCR.

Therefore, there exists a need for a selective growth medium that is not only selective for Vibrio cholerae but even more specifically, for the pathogenic or toxigenic forms of V.cholerae. Moreover, there also exists a need for a selective growth medium that is effective at selectively and quickly growing (and therefore identifying) toxigenic forms of V.cholerae from samples where the potential numbers of bacteria are low, such as in ballast water or patients with cholera, and more particularly patients recovering from cholera. The present invention addresses this need.

Summary of the Invention

In one aspect of the invention, there is provided a selective and/or differential growth medium for the isolation of toxigenic Vibrio cholerae, the medium comprising

(a) a base medium;

(b) a salt;

(c) a source of nitrogen; and

(d) a dihydrofolate reductase (DHFR) inhibitor and/or a polymyxin. In one embodiment, the medium comprises between 1 and 50g/L of base medium, between 1 and 50g/L of a salt, between 1 and 50g/L of a source of nitrogen and between 35 and 300mg/L of DHFR and/or between 1 and 500mg/L of polymyxin. In one embodiment, the source of nitrogen is a peptone. Preferably, the peptone is bacto peptone.

In another embodiment, the dihydrofolate reductase inhibitor is an antibiotic, preferably trimethoprim.

In a further embodiment, the base medium is an agar base, preferably cellobiose polymyxin B colistin (CPC).

In one embodiment, the medium comprises 5g/L peptone, 15g/L sodium chloride, 32.54 g/L CPC agar and 175mg/L trimethoprim.

In another embodiment, the polymyxin is colistin.

In yet another embodiment, the medium further comprises a Gram positive antibacterial agent, preferably daptomycin. More preferably the medium comprises between 1 and 30mg/L of daptomycin.

In another aspect of the invention there is provided an agar plate comprising the medium described herein.

In a further aspect of the invention there is provided a method for isolating and/or identifying and/or selectively growing toxigenic cholera from a sample, the method comprising inoculating a sample into the medium described herein. In one embodiment, the method comprises

a. inoculating a sample into the growth medium;

b. incubating the growth medium to allow the growth of toxigenic cholerae; and c. detecting the presence of toxigenic cholera. Preferably, the sample is water, blood or stool. More preferably the water sample is ballast water.

In another aspect of the invention there is provided the use of a selective and/or differential growth medium as described herein for the isolation and/or identification and/or selective growth of toxigenic cholera from a sample.

In a further aspect there is provided a method of detecting the presence of at least one colony forming unit (CFU) of toxigenic Vibrio cholerea in a sample of ballast water, the method comprising

a. obtaining a sample of ballast water;

b. inoculating the sample into the growth medium as described herein c. incubating the growth medium to allow the growth of toxigenic cholerae; and

d. detecting the presence of at least one CFU of toxigenic cholera.

In a final aspect of the invention there is provided a method of diagnosing cholera in a patient, the method comprising obtaining a sample from said patient, inoculating the sample into the medium described herein and detecting the presence of toxigenic V. cholerae, wherein the presence of toxigenic V. cholerae is indicative of disease.

In one embodiment, the toxigenic cholera is Vibrio cholerae 01 or Vibrio cholerae 0139. Preferably, Vibrio cholerae 01 is the E1 Tor or classical biotype. The invention is further described in the following non-limiting figure.

Figures

Figure 1 shows a protocol for the use of Speedy Breedy™ to detect the presence of toxigenic cholerae in a sample of ballast water.

Step 1 : Set up the waste jar or bucket, pump and manifold. As an example only, the

EZ-Stream™ Pump from Merck Millipore may be used.

Step 2: Pour 100ml of Ballast Water into a filter cup and close the lid;

Step 3: Remove the plug from the bottom of the cup;

Step 4: Secure cup onto manifold by pushing it down; Step 5: Open the valve;

Step 6: Switch the pump back on and off again once all the waste is in the waste jar or bucket;

Step 7: Prepare the vessel for the chosen test (E.coli, Enterococci or toxigenic cholerae) by opening the lid and carefully placing it on the bench, rubber side up.

Step 8: Put on a fresh pair of gloves and wipe them with an alcohol wipe. Allow gloves to dry.

Step 9: Slip a forefinger under the thin, filter disc and (without touching the gridded upper side of the disc) fold the disc over in half.

Step 10: Continue to fold the disc until it is slim enough to push through the sample port of a Speedy Breedy™ vessel.

Step 1 1 : Push the filter through the sample port, use the lid of the vessel if necessary. Step 12: Once the filter is inside the culture vessel, pour some sterile water into a cup, draw up 50ml sterile water into a fresh, sterile syringe.

Step 13: Add the 50ml water to the culture vessel via the sample port; and

Step 14; Put the vessel into the Speedy Breedy™ and start the test immediately.

Detailed description of the Invention

The present invention will now be further described. In the following passages, different aspects of the invention are defined in more detail. Each aspect so defined may be combined with any other aspect or aspects unless clearly indicated to the contrary. In particular, any feature indicated as being preferred or advantageous may be combined with any other feature or features indicated as being preferred or advantageous.

The practice of the present invention will employ, unless otherwise indicated, conventional techniques of microbiology, tissue culture, chemistry and biochemistry which are within the skill of the art. Such techniques are explained fully in the literature. As used herein, the terms "pathogenic" and "toxigenic" are used interchangeably, and refer to the strain, serotype or biotype of V.cholerae responsible for producing the symptoms associated with the cholera disease. In one embodiment, a toxigenic V.cholerae is selected from the group comprising Vibrio cholerae 01 and Vibrio cholerae 0139. In one embodiment the toxigenic Vibrio cholera 01 is the E1 Tor or classical biotype and preferably either the Inaba or Ogawa serotype. In one aspect the invention relates to a selective and/or differential growth medium for the isolation or growth of toxigenic cholera, the medium comprising

(a) a base medium;

(b) a salt;

(c) a source of nitrogen; and

(d) a dihydrofolate reductase (DHFR) inhibitor and/or a polymyxin.

As used herein, the word "selective" means a growth medium that allows the growth of only a certain type of organism, while inhibiting the growth of other organisms. The word "differential" means a growth medium that can differentiate closely related organisms or a group of organisms.

In one embodiment, the base medium is an agar base. In a specific embodiment, the agar base is cellobiose polymyxin B colistin (CPC). Alternatively, the agar base is selected from TCBS (Thiosulfate-citrate-bile salts-sucrose agar), HiChrome Vibrio Agar, Vibrio Chromogenic Agar, or Gelatin taurocholate tellurite medium. In a further embodiment, the nutrient base does not comprise agar. For example, the base medium is one of the above, but without the addition of agar. Therefore, in another embodiment, the base medium is a nutrient broth or liquid nutrient medium.

In one embodiment, the CPC agar base comprises the following:

• Peptic digest of animal tissue, in the preferred range of 1 to 50g/L, more preferably, 5 to 30g/L, even more preferably 5 to 15g/L and most preferably 10g/L;

• Beef extract, in the preferred range of 1 to 50g/L, more preferably 1 to 40g/L, more preferably 1 to 30g/L, even more preferably 1 to 20g/L, even more preferably 1 to 10g/L, and most preferably 5g/L;

• A disaccharide, such as cellobiose in the preferred range of 1 to 50g/L, more preferably 1 to 40g/L, more preferably 1 to 30g/L, even more preferably 10 to

20g/L, and most preferably 15g/L;

• A salt such as sodium chloride in the preferred range of 1 to 50g/L, more preferably 1 to 40g/L, more preferably 1 to 30g/L, even more preferably 10 to 20g/L, and even more preferably 15 to 25 g/L and most preferably 20g/L; • Bromothymol Blue in the preferred range of 0.001 to 0.1 , more preferably, 0.01 to 0.09g/L and most preferably 0.04g/L.

• Cresol Red in the preferred range of .001 to 0.1 , more preferably, 0.01 to 0.09g/L and most preferably 0.04g/L.

Optionally, the CPC base may further comprise agar, in the preferred range of 1 to 50g/L, more preferably 1 to 40g/L, more preferably 1 to 30g/L, 10 to 20g/L, most preferably 15g/L. The base may or may not comprise polymyxin B and/or colistin. The skilled person would be able to produce the media as described above using known techniques in the art. Alternatively, the skilled person could obtain a pre- prepared medium from commercial sources, such as Sigma Aldrich™.

In one embodiment, the salt is a sodium salt, preferably sodium chloride. In another embodiment, the salt is a potassium salt, preferably potassium chloride.

In one embodiment the source of nitrogen is a peptone. The word "peptone" refers to a water-soluble mixture of polypeptides and amino acids that are formed by the partial hydrolysis of protein. As such, peptones are also a useful natural source of amino acids, peptides and proteins, and are most often obtained by enzymatic digestion or acid hydrolysis of natural products such as animal tissues, milk, plants or microbial cultures. A suitable peptone would be known to the skilled person. In a specific embodiment the peptone is a enzymatic digest of animal protein, such as Bacto ^RT peptone. In one embodiment, the peptone may have the following composition:

(% w/w)

Total Nitrogen 14.0

Amino Nitrogen 2.6

Sodium chloride 1.6

pH (2% solution)

6.2 ± 0.2 or alternatively,

(% w/w) Total Nitrogen 13.9

Amino Nitrogen 2.4

Sodium chloride 3.2

pH (2% solution) 7.0 ± 0.2

Alternative peptones that may be used include meat peptones, casein peptones, soy peptones, plant peptones and any other available peptone.

In a preferred embodiment, the growth medium comprises a dihydrofolate reductase (DHFR) inhibitor. Preferably, the DHFR is an antibiotic. Specifically in one embodiment, the antibiotic is Trimethoprim.

Accordingly, in a specific embodiment, the medium comprises

(a) CPC agar;

(b) Sodium chloride;

(c) Bacto peptone; and

(d) L-Trimethoprim.

In one embodiment, the medium comprises a base medium, a salt, and a source of nitrogen in the following ratios: 6.5: 3: 1 , wherein said source of nitrogen is in addition to any source of nitrogen that may be in the base medium.

In one embodiment, the medium comprises (a) a base medium in the preferred range of 1 to 50g/L, more preferably 1 to 40g/L, more preferably 1 to 30g/L, even more preferably 1 to 20g/L, even more preferably 1 to 10g/L, more preferably, 4 to 6g/L, and even more preferably, 4.1 , 4.2, 4.3, 4.4, 4.5, 4.6. 4.7, 4.8, 4.9, 5.0, 5.1 , 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9 or 6.0 g/L. In one specific embodiment the medium comprises 5g/L of base medium.

(b) a salt in the preferred range of 1 to 50g/L, more preferably 1 to 40g/L, more preferably 1 to 30g/L, more preferably 10 to 20g/L, more preferably, 14 to 16g/L, and even more preferably, 14.1 , 14.2, 14.3, 14.4, 14.5, 14.6, 14.7, 14.8, 14.9, 15.0, 15.1 , 15.2, 15.3, 15.4, 15.5, 15.6, 15.7, 15.8, 15.9 or 16.0g/L. In one specific embodiment the medium comprises 15g/L of salt. (c) a source of nitrogen in the preferred range of 1 to 50g/L, more preferably 10 to 40g/L or 20 to 50g/L, more preferably 25 to 40 g/L, more preferably 30 to 25 g/L, even more preferably, 32, 33 or 34 g/L. In one specific embodiment, the medium comprises 32.1 , 32.2, 32.3, 32.4, 32.5, 32.6, 32.7, 32.8, 32.9 or 40.0 g/L of a source of nitrogen. Even more preferably, the medium comprises

32.51 , 32.52, 32.53, 32.54, 32.55, 32.56, 32.57, 32.58, 32.59 or 32.60 g/L of a source of nitrogen. In one embodiment the medium comprises 32.54g/L of a source of nitrogen. In a specific embodiment, the medium comprises

• 5g/L of Bacto peptone

• 15g/L Sodium chloride; and

• 32.54g/L of CPC- Agar.

In a further embodiment, the medium comprises dihydrofolate reductase (DHFR) inhibitor in the preferred range of 35 to 250mg/L, even more preferably 100 to 350mg/L, even more preferably 55-250mg/L, and most preferably 55mg/L to 175mg/L. In one embodiment the medium comprises 175mg/L of DHFR inhibitor.

In one embodiment, the medium may comprise 5, 10, 15, 20, 25, 35, 45,55, 60, 65, 70, 7, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 205, 210, 215, 220, 225, 230 or 235 mg/L of a dihydrofolate reductase (DHFR). In one embodiment the amount of DHFR inhibitor may be 35mg/L or higher. In a further preferred embodiment, the amount of DHFR inhibitor may be at least 50mg/L. In another preferred embodiment, the amount of DHFR inhibitor may be at least 175mg/L. These concentrations will permit the growth of toxigenic V.cholerae in the growth medium while preventing or reducing the growth of closely related (e.g. non-toxigenic) strains. Such strains may include non-toxigenic V.cholerae (preferably 01 H 151220003), V.parahaemolyticus (preferably H1 51220010), Vmimicus (preferably H151220007), V. vulnificus (Ή 130860068), - V. cholerae Non-01 , 0139 (preferably H1 51220001), V.alginolyticus (preferably H15122001 1) and Vfluvialis ( preferably H151220008). The medium may be made up to 50ml using distilled water. Thus, in another aspect of the invention, there is provided a method for isolating and/or identifying and/or selectively growing toxigenic cholerae from a sample also containing at least one closely related strain of bacteria to V. cholerae, such as those described above, the method comprising inoculating a sample into the medium defined above and comprising between 5 and 300mg/L of a dihydrofolate reductase (DHFR) inhibitor, more preferably 35mg/L or above.

In another embodiment, the medium comprises dihydrofolate reductase (DHFR) inhibitor in the preferred range of 35 to 250mg/L, even more preferably 100 to 350mg/L, even more preferably 55-250mg/L, and most preferably 55mg/L to 175mg/L. In one embodiment the medium comprises 175mg/L of DHFR inhibitor. Preferably, during production of the medium, trimethoprim is added as a powder, encapsulated with the rest of the ingredients.

In a preferred embodiment of the above, the DHFR inhibitor is an antibiotic, and more preferably the antibiotic is Trimethoprim.

In another embodiment, the medium comprises a polymyxin. This may be as an alternative to or in addition to the DHFR inhibitor. In one embodiment, the polymyxin may be polymyxin E or colistin (colistin sulfate or colistimethate sodium (colistin methanesulfonate sodium, colistin sulfomethate sodium)). The medium may further comprise an additional antibiotic, such as Polymyxin B. In one embodiment, the medium comprises between 1 and 500mg/L, more preferably 1 to 400mg/L, more preferably 1 to 300mg/L, more preferably 50 to 150mg/L of polymyxin, preferably 100 to 130mg/L and more preferably 1 10 to 120mg/L, and even more preferably 108.8mg/L. In another embodiment, the medium comprises 1 to 10 mg/L of a further antibiotic, preferably another polymyxin such as Polymyxin B, and more preferably, between 1 and 50mg/L, more preferably between 1 and 40mg/L, more preferably between 1 and 30mg/L, even more preferably between 1 and 20mg/L, more preferably between 1 and 10mg/L and most preferably between 2 and 8mg/L, and more preferably between 4 and 6 mg/L and most preferably 4.26mg/L of antibiotic. In another embodiment, the selective and/or differential growth medium further comprises a Gram positive antibacterial agent. Preferably said agent does not affect the growth of Gram negative bacteria. In one embodiment, this agent is an antibiotic. Preferably, the antibiotic is Daptomycin. In another embodiment, the Gram positive antibacterial agent is selected from teicoplanin, quinupristin-dalfopristin, oxazolidinones and telavancin. In one embodiment, medium comprises a Gram positive antibacterial agent in the preferred range of 1 and 50mg/L, more preferably between 1 and 40mg/L, more preferably between 1 and 30mg/L, even more preferably between 1 and 20mg/L, more preferably between 1 and 10mg/L, even more preferably, 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10mg/L and most preferred 2mg/L.

In another embodiment, the selective and/or differential growth medium is a dry powder mix and comprises the following

- between 5 to 15% by weight, preferably 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14 or 15% by weight of a source of nitrogen as described herein, preferably bacto peptone;

between 10 and 40%, by weight preferably between 20 and 30% by weight, more preferably 21 , 22, 23, 24, 25, 26, 27, 28, 29 or 30% of a salt as described herein, preferably sodium chloride;

between 40 and 80% by weight of a base medium, preferably between 50 and 65% by weight, more preferably 50, 51 , 52, 43, 54, 55, 56, 57, 58, 59, 60, 61 , 62,

63, 64 or 65% by weight of a base medium as described herein, preferably CPC- Agar;

between 0.1 and 1 % by weight of a DHFR inhibitor, more preferably, 0.1 , 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8 or 0.9% by weight of a DHFR inhibitor as described herein, preferably trimethoprim; and

between 0.001 and 0.01 % by weight of a gram positive antibacterial agent, more preferably 0.001 , 0.002, 0.003, 0.004, 0.005, 0.006, 0.007, 0.008, 0.009 or 0.1 % by weight of a gram positive antibacterial agent as described herein, preferably daptomycin.

In another aspect of the invention, there is provided a selective and/or differential growth medium for the isolation of toxigenic cholerae, the medium comprising

(a) CPC media; and

(b) at least one polymyxin as defined above. In another aspect of the invention there is provided a method for the isolation and/or identification and/or selective growth of toxigenic cholerae from a sample, the method comprising inoculating a sample into the growth medium as described above. As used herein, "isolation" may refer to the identification or growth of at least one colony forming unit (CFU) of at least one type of toxigenic cholerae as described herein.

Specifically, the method may comprise

a. inoculating a sample into the growth medium as described herein;

b. incubating the growth medium to allow the growth of toxigenic cholerae; and c. detecting the presence of toxigenic cholerae.

In one embodiment, the sample is incubated for between 6 and 24 hours, preferably at least 6, at least 7, at least 8, at least 9, at least 10 or at least 12 or at least 24 hours and at a temperature between 10 and 43°C, preferably 37°C.

In one embodiment, the presence of toxigenic cholerae can be detected by counting the number of colonies of growth on the surface of agar plates. Such techniques would be well known to the skilled person. Alternatively, the presence or absence of growth can be measured by analysing the turbidity and/or colour of the broth in a bijou. Again, this is a standard microbiology technique.

In one embodiment, the sample is water. Preferably, the water is sea water or drinking water. In a specific embodiment the sample is ballast water. In an alternative embodiment the sample is a clinical sample, meaning that the sample is taken from a patient who has, is believed to have or is recovering from cholera. In a specific embodiment, the sample is a stool sample. We have previously described a system for monitoring the metabolism/growth of microorganisms, the system comprising a sealed chamber with a flexible diaphragm to provide sensitive pressure measurements of gas pressure in the headspace above a culture liquid. For details reference may be made, for example, to US2005/0170497 (incorporated by reference). Here we describe that such a system (called Speedy Breedy™) may be used in combination with the described growth medium to detect the presence of toxigenic cholerae. Accordingly, in one embodiment, the method further comprises

1. inoculating a sample, preferably 50ml of sample, into at least one container or vessel containing the growth medium, described above; and

2. loading the container(s) into the Speedy Breedy™ instrument.

An example of a detailed protocol for using the above system to detect the presence of toxigenic cholerae in ballast water is provided in Figure 1.

Preferably, the volume of the sample is 50ml, for example if the sample is water. Where the sample is being filtered prior to testing, any volume of water can be used and then the filter will be put into a vessel and topped up with 50ml sterile water. If a sample is being tested directly, 50ml is added to a Speedy Breedy™ vessel. Samples can also be diluted, but the eventual volume in the Speedy Breedy™ must always be 50ml. For example, 1 ml of a sample could be diluted with 49ml sterile water. For, e.g. a stool sample, 1g or so of sample can be mixed with 49ml sterile water for testing in Speedy Breedy™. For ballast water, a 100ml volume is filtered and the filter is put into a vessel and topped up with 50ml sterile water. If however, the sample is blood, sterile water can be added to the sample to take the total volume to 50ml.

In a further aspect of the invention there is provided an agar plate comprising a petri dish, and the growth medium as described above, wherein the base medium comprises agar and therefore is an agar base. Accordingly, in one embodiment there is provided an agar plate comprising an agar base, a salt, a source of nitrogen and a dihydrofolate reductase (DHFR) inhibitor and/or polymyxin, as defined above. Such an agar plate can be used to selectively grow toxigenic V. cholerae.

In another aspect of the invention there is provided the use of a selective and/or differential growth medium as described above for the isolation and/or identification and/or selective growth of toxigenic cholerae from a sample.

In another aspect of the invention there is provided a method of identifying the presence of toxigenic V.cholerae in ballast water, the method comprising obtaining a sample of ballast water, inoculating said sample into the growth medium as described herein and detecting the presence of toxigenic V.cholerae. Preferably the method is capable of detecting at least one colony forming unit (cfu) and preferably per 50ml or 100ml of ballast water. Alternatively, the method is capable of detecting at least one cfu per one gram weight of zooplankton sample.

In a further aspect of the invention there is provided a method of diagnosing cholera in a patient, the method comprising obtaining a sample from said patient, inoculating the sample into the medium described above, and detecting the presence of toxigenic V.cholerae. In an alternative embodiment, there is provided a method of diagnosing cholera in a patient, the method comprising inoculating a sample obtained from the patient into the medium described above, and detecting the presence of toxigenic V.cholerae. The presence of toxigenic cholerae in the sample may be indicative of disease. Preferably at least one colony forming unit is identified. In a final aspect of the invention, there is provided a method for distinguishing toxigenic 01 (preferably, the inaba serotype) from toxigenic 0139, the method comprising

a. inoculating a sample into the growth medium, wherein the growth medium comprises at least one polymyxin as described herein;

b. incubating the growth medium as described herein to allow the growth of toxigenic cholera 01 (preferably the inaba serotype); and

c. detecting the presence of toxigenic cholerae 01 (preferably the inaba serotype).

In the above described embodiment, we have shown that toxigenic cholerae 01 will grow in a medium described herein comprising a polymyxin, preferably colistin (also known as polymyxin E), whereas V.cholerae 0139 cannot grow. Accordingly, the growth of organisms on this medium indicates the presence of cholerae 01 and can be used to distinguish the serotype of cholerae in a sample. In one embodiment, the 01 toxigenic cholerae is the inaba serotype. A sample is defined above.

"and/or" where used herein is to be taken as specific disclosure of each of the two specified features or components with or without the other. For example "A and/or B" is to be taken as specific disclosure of each of (i) A, (ii) B and (iii) A and B, just as if each is set out individually herein. Where a range is described, it is intended that the endpoint of each range as well as any value falling between those endpoints are disclosed. For example, a range of 10 to 20mg/L includes 10 and 20 mg/L as well as any amount falling between 10 and 20mg/L.

Unless context dictates otherwise, the descriptions and definitions of the features set out above are not limited to any particular aspect or embodiment of the invention and apply equally to all aspects and embodiments which are described. Example 1

The panel of micro-organisms the media-antibiotic mixes were initially tested with are as follow: - Toxigenic V. cholerae 01 (inaba) H1 51220006

Toxigenic V. cholerae 0139 H151220002

Non-toxigenic V. cholerae 01 H151220003

V.parahaemolyticus H1 51220010

V.mimicus H151220007

- V. vulnificus H 130860068

V. cholerae Non-01 , 0139 H1 51220001

Toxigenic V. cholerae 01 (ogawa) H151220005

Toxigenic V. cholerae 0139 E099892

V.alginolyticus H15122001 1

- V.fluvialis H 151220008

Growth medium

The resulting recipe which has shown the best selectivity for toxigenic cholerae consists of a base medium, to which an antibiotic is added.

Base recipe

5g/L Bacto peptone (Oxoid LP0037)

15g/L Sodium chloride

32.54g/L CPC-Agar (Base) (Sigma 17134)*

Antibiotic 175mg/L Trimethoprim (Sigma 92131) and preferably, 2mg/L Daptomycin (Tocris 3917), to deter the growth of Gram positive organisms.

We have shown that the basic recipe above, with trimethoprim at at least 35mg/L, although more preferably at 55mg/L, and most preferably between 175-300mg/L and 2mg/L daptomycin allows the growth of all the toxigenic cholerae strains in the study, while completely inhibiting the growth of E. coli, E. faecalis, P. aeruginosa, S. aureus and B. cereus as well as non-toxigenic cholerae and other Vibrios.

(Composition of CPC-Agar (Base))

Peptic digest of animal tissue 10g/L

Beef extract 5g/L

Cellobiose 15g/L

Sodium chloride 20g/L

Bromothymol Blue 0.04g/L

Cresol Red 0.04g/L

(optional)Agar 15g/L

The trimethoprim used in the testing to date was initially used as an additive that was prepared by mixing with dH20 and filter-sterilising through a 0.2um filter (up to VM 122 medium). Testing from VM123 mediums onwards features the trimethoprim as a powder, encapsulated with the rest of the ingredients. As trimethoprim is only partially soluble in water, this will no doubt cause a discrepancy in the actual concentration of trimethoprim available in the medium when added either as a liquid after filter- sterilisation, or directly as a powder. Our project progressively showed that the use of certain concentrations of trimethoprim with the base medium listed above achieved exclusive selectivity for toxigenic cholerae out of the panel of closely related organisms listed, also listed above. The number of media combinations tested was differentiated with the letters 'VM' as a prefix and tested in ascending numerical order. The desirable effect of trimethoprim was apparent by the time medium VM97 was tested. Below are test results for VM98-VM105, which investigated the effects of various concentrations of trimethoprim combined with the base medium on select cultures in our testing panel.

Table 1 : Test mediums VM98 to VM 105

Table 2: Results for VM98 to VM 105

Strain CFUS VM98 VM99 VM100 VM101 VM102 VM103 VM104 VM105

Growth Growth turbid, turbid,

Toxigenic duplicat duplicat

01 (inaba) Dupolicat e 1 = e 1 =

HI e 1 = browny- browny-

51220006 ~100CFU, Growt yellow, blue,

Duplicate h, Growth, duplicat Growth, Growth, Growth, duplicat Growth,

2 = ~150 turbid turbid e 2 = turbid turbid turbid e 2 = turbid

CFU yellow yellow yellow yellow yellow yellow yellow yellow

Growth Growth Growth Growth Growth Growth turbid, turbid, turbid, turbid, turbid, turbid,

Toxigenic duplicat duplicat duplicat duplicat duplicat duplicat

0139 Duplicate e 1 = e 1 = e 1 = e 1 = e 1 = e 1 =

H15122000 1 = ~200 yellow, yellow, browny- yellow, browny- browny-

2 CFU, Growt duplicat duplicat yellow, duplicat yellow, yellow,

Duplicate h, e 2 = e 2 = duplicat e 2 = duplicat Growth, duplicat

2 = -200 turbid browny- browny- e 2 = browny- e 2 = turbid e 2 =

CFU yellow yellow yellow yellow yellow yellow yellow yellow VMIO VMIO

VM98 VM99 VM101 VM102 VM104 VM105

Strain CFUS 0 3

Duplica

te 1 =

no

Duplica growth,

te 1 = duplicat

Non-toxigenic

~300 e 2 =

01 H151220003

CFU, not

Duplica turbid,

te 2 = slightly No

~400 lighter No No No No growt No No

CFU green growth growth growth growth h growth growth

Growth Growth Growth

Duplica turbid, turbid, Growth turbid, te 1 = duplicat duplicat turbid, duplica

-350 e 1 = e 1 = duplica te 1 =

Toxigenic 01 El- CFU, yellow, yellow, te 1 = yellow, Tor

Duplica duplicat Growt duplicat Growt yellow, duplica te 2 = e 2 = Growth h, e 2 = Growth h, duplica te 2 =

-350 brawny , turbid turbid brawny , turbid turbid te 2 = brawny

CFU -yellow yellow yellow -yellow yellow yellow purple -yellow

Duplica

te 1 =

growth

Duplica turbid,

Growth te 1 = yellow,

V.parahaemolyti Duplica turbid, growth Duplica

cus HI 51220010 te 1 = duplica Growt turbid, te 2 =

-75CFU te 1 = h, yellow, not

purple, turbid Duplica turbid,

Duplica Growth duplica yellow te 2 = slightly No

te 2 = , turbid te 2 = y- no lighter growt No No

-75 CFU yellow yellow green growth green h growth growth

Duplica

te 1 =

not

turbid,

slightly

Duplica ligher

V.mimicus

te 1 = green,

H151220007

-200 Duplica

CFU, te 2 =

Duplica not

te 2 = turbid, No

-300 yellow- No No No No growt No No

CFU green growth growth growth growth h growth growth

V.vulnificus

H15122009 up to Duplica

VM19 - this te 1 =

strain no longer -350

viable - so CFU,

H 130860068 Duplica

used instead for te 2 = No

Vm85-97 -250 No No No No No growt No No

CFU growth growth growth growth growth h growth growth Strain CFUS VM98 VM99 VM100 VM101 VM102 VM103 VM104 VM105

Duplicat

e 1 =

~150

Non-01,

CFU,

0139 HI

Duplicat

51220001

e 2 =

~100 No No No No No No No No

CFU growth growth growth growth growth growth growth growth

Duplicat

e 1 =

~150

Toxigenic 01

CFU,

(ogawa)

Duplicat

H151220005

e 2 = Growth, Growth Growth, Growth Growth Growth, Growth, Growth

~150 turbid , turbid turbid , turbid , turbid turbid turbid , turbid

CFU yellow yellow yellow yellow yellow yellow yellow yellow

Growth, Growth

Duplicat turbid turbid,

e 1 = duplicat duplicat

Toxigenic

~200 e 1 = e 1 =

0139

CFU, browny- yellow,

E099892

Duplicat Growth, greeny, duplicat

e 2 = turbid Growth duplicat Growth Growth e 2 = Growth, Growth

~150 browny- , turbid e 2 = , turbid , turbid browny- turbid , turbid

CFU greeny yellow yellow yellow yellow yellow yellow yellow

Duplicat

e 1 =

~100

V.alginolyticu

CFU,

s

Duplicat

H151220011

e 2 = Growth Growth

~100 turbid, turbid, No No No No No No

CFU yellow yellow growth growth growth growth growth growth

Duplicat

e 1 = Duplicat

-100 e 1 = no

V.fluvialis CFU, growth,

H151220008 Duplicat duplicat

e 2 = e 2 = not

-200 turbid, No No No No No No No

CFU yellow growth growth growth growth growth growth growth

The results above clearly show that increasing the concentration of trimethoprim in the medium effectively inhibits the growth of all strains, apart from the toxigenic cholerae strains. The results also show that once a concentration of 35mg/L of trimethoprim was reached in a CPC-PBS base, absolute selectivity for toxigenic cholerae was reached. Example 2

Concentrations of trimethoprim in a CPC-PBS base were increased to give chance of inhibiting the growth of other gram negative strains, such as E. coli.

Table 3: Test medium VM114 to VM118

These results showed that even including 250mg/L of trimethoprim allows good reproducible growth of all the toxigenic cholerae in our panel. Please note that this is the level of trimethoprim when tested as a 'solution' of trimethoprim in sterile deionised H20 that was then filtered through a 0.2uM filter - trimethoprim is known to be rather insoluble in water

Results

Table 5: Results for VM1 14 to VM1 18

For VM

114-118 -

CFUs VM114 VM115 VM116 VM117 VM118 Growth, Growth,

Duplicate turbid, turbid,

1 Duplicate 1 Duplicate 1

Toxigenic 01

-600CFU = yellow, = yellow,

(inaba) HI

Duplicate Growth, duplicate 2 duplicate 2 Growth 51220006

2 turbid, Growth, turbid

-700CFU greeny purple/bro turbid, purple/bro greeny brown wn yellow wn brown

Growth,

Duplicate Growth, turbid,

1 turbid, Duplicate 1

Toxigenic

-500CFU Duplicate 1

0139

Duplicate = greeny purple/bro

H151220002

2 Growth, Growth, brown, wn, Growth,

-400CFU turbid, turbid, duplicate 2 duplicate 2 turbid, yellow yellow = yellow = yellow yellow

Duplicate

1 =

Non-toxigenic

-lOOCFU, No 01 No growth No growth No growth No growth

Duplicate growth

H151220003

2

-200CFU

Growth,

Duplicate turbid,

Toxigenic 01

1 Duplicate 1 Growth,

(Originally

-600CFU = greeny turbid,

thought it was

Duplicate brown, Duplicate 1

non-tox 0139)

2 duplicate 2 = greeny

H151220004

-600CFU Growth, brown, Growth, Growth, purple/bro turbid, duplicate 2 turbid, turbid, wn yellow = yellow yellow yellow

Duplicate

1 =

V.parahaemol

-350CFU

yticus HI

Duplicate

51220010

2 no

-250CFU no growth no growth no growth no growth growth

Duplicate

1 -

V.mimicus -500CFU

H151220007 Duplicate

2 no

-400CFU no growth no growth no growth no growth growth V.vulnificus

H15122009 up

Duplicate

to VM19 - this

1

strain no

-200CFU

longer viable -

Duplicate

so

2

H 130860068

-400CFU

used instead

for Vm85-97 no no growth no growth no growth no growth growth

Duplicate

1

Non-01, 0139 -300CFU

HI 51220001 Duplicate

2 no

-300CFU no growth no growth no growth no growth growth

Growth Growth Growth

Duplicate

turbid, turbid, turbid,

1

Toxigenic 01 duplicate 1 duplicate 1 duplicate 1

-300CFU

(ogawa) = greeny = greeny = greeny

Duplicate

H151220005 Growth, brown, brown, brown, Growth,

2

turbid, duplicate 2 duplicate 2 duplicate 2 turbid,

-200CFU

yellow = yellow = yellow = yellow yellow

Growth,

Duplicate turbid,

1 Duplicate 1

Toxigenic -400CFU = yellow,

0139 E099892 Duplicate duplicate 2

2 Growth, Growth, Growth, Growth,

-500CFU turbid, purple/bro turbid, turbid, turbid, yellow wn yellow yellow yellow

This result

has been

Duplicate disregarde

1 d, due to

V.alginolyticu -300CFU incongruen

s H151220011 Duplicate cy with the

2 surroundin

-400CFU g results

for this no no growth no growth strain. no growth growth

Duplicate

V.fluvialis 1

H151220008 -400CFU no

Duplicate no growth no growth no growth no growth growth

Table 6: Results for VM 1 19 to VM 122

Example 3 The following tests were conducted using trimethoprim added as a dry powder. Daptomycin was also included. We developed a Speedy Breedy™ test for the selective detection of toxigenic Vibrio cholerae 01 and 0139 (as described above, Speedy Breedy™ is a microbial respirometer system which enables the contained, lab-free testing of samples for the detection of micro-organisms).

Phase I of the project identified liquid media that specifically grow toxigenic V. cholerae 01 and 0139, while inhibiting the growth of other closely related Vibrios (including non- toxigenic V. cholerae) and other unrelated species of bacteria.

Test Organisms

Toxigenic V. cholerae 01 (inaba) H 1 51220006

Toxigenic V. cholerae 0139 H 151220002

Non-toxigenic V. cholerae 01 H151220003

- V.parahaemolyticus H1 51220010

V.mimicus H151220007

V. vulnificus (H 130860068)

V. cholerae Non-01 , 0139 H1 51220001

Toxigenic V. cholerae 01 (ogawa) H 151220005

- Toxigenic V. cholerae 0139 E099892

V.alginolyticus H15122001 1

V.fluvialis H 151220008

Composition of media (VM123)

5g/L Bacto peptone (Oxoid LP0037);

15g/L Sodium Chloride;

32.54g/L CPC-Agar (Base) (Sigma 17134); and

175mg/L of trimethoprim (Sigma 92131)

2mg/L of daptomycin (purchased from Tocris) will be added to all the Speedy Breedy™ vessels containing encapsulated VM123, as a filter-sterilised stock, prepared in dH20.

Results Table 7: Results for VM 123 and a mixture of non- and toxigenic organisms

Strain Duplicate Medium Time to Colours CFU/100ul Neg

No. Detection of Control (TTD) broths

at end

of test

E099892 1 VM123 6 hours Yellow 1 144 Salt (Toxigenic V. 12 peptone cholera 0139) minutes spread plate done - no growth duplicate 1

2 VM123 8 hours Yellow 1760 Salt

17 peptone minutes spread plate done - no growth duplicate 2

H 151220002 1 VM123 7 hours Yellow 336 Salt (Toxigenic V. 55 peptone cholera 0139) minutes spread plate done - no growth duplicate 1

2 VM123 8 hours 6 Yellow 216 Salt

minutes peptone spread plate done - no growth duplicate 2

H 151220005 1 VM123 9 hours 9 Yellow 664 Salt (Toxigenic V. minutes peptone cholera 01 spread Ogawa) plate done

- no growth duplicate 1

2 VM123 9 hours Yellow 563 Salt

35 peptone minutes spread plate done - no growth duplicate 2

H 151220006 1 VM123 12 hours Yellow 1503 Salt (Toxigenic V. 34 peptone cholera 01 minutes spread Inaba) plate done

- no growth duplicate 1

2 VM123 10 hours Yellow 956 Salt

9 minutes peptone spread plate done - no growth duplicate 2

Control (salt 1 VM123 N/A Green No growth Salt peptone) peptone spread plate done - no growth duplicate 1

2 VM123 N/A Green No growth Salt peptone spread plate done - no growth duplicate 2

Example 4 The strains tested above are ones closely related to toxigenic Vibrio cholerae. While it is paramount in designing a selective medium that closely related strains are ruled out, it is also important that tests are carried out on a wider range of microbes, to ensure that the final medium excludes the growth of widely different microbes. Indeed, marine and estuarine waters, in general, contain a significant number of Pseudomonas species, in addition to Flavobacterium and Photobacterium.

For this reason, tests were also carried out to check whether the base medium + trimethoprim would inhibit the growth of Escherichia coli, Pseudomonas aeruginosa, Enterococcus faecalis and Staphylococcus aureus.

Bactest selectivity test using 50% CPC + 50% Peptone salt broth + 55mg/L trimethoprim.

Introduction & Aims

55mg/L in our cholerae medium base was seen to be the highest concentration of trimethoprim trialled in media batch VM98-VM105 that gave full selectivity for all the toxigenic cholerae tested in the panel of Vibrios.

In order to test the selectivity of this medium against strains such as E. coli and Enterococcus the following experiment was set up.

Method

1. The following strains were cultured on TSA plates and resuspended in sterile dH20 to give l OOcfu per 10ul (using OD600 = 0.5 = 10 ⁸ cfu/ml, for calculations): - E. coli PHE Lenticule CRM09001 H, Batch 1233-14

- Enterococcus faecalis PHE Lenticule CRM00775H, Batch 1246-14

2. Pour 4ml of: autoclaved 50% CPC agar (Sigma 17134) (filtered through filter paper) + 50% Peptone Salt broth (Peptone 3% Salt Broth = 10g/L Bacto peptone, 30g/L Sodium chloride), to which is added 0.2um filtered trimethoprim in dH20 - into each of 9 sterile, bijou bottles.

3. Inoculate each of 3 of the bijous bottles with 100ul of the E. coli suspension, each of 3 of the bijous with 100ul of the Enterococcus suspension and each of 3 of the bijous with 100ul sterile dH20.

4. Spread-plate 100ul of each bacterial suspension onto TSA plates, in duplicate.

5. Incubate the plates and bijous at 37°C overnight and record the number of colonies on each plate and presence or absence of growth (turbidity) and colour of the broth in each bijou.

Results (after 24 hours incubation)

Table 8: Results for E.coli and Enterococcus growth

These results show that the growth of E. coli is not supported by our cholerae medium containing 55mg/L trimethoprim, but Enterococcus faecalis does grow in this medium. We saw, in the test above, that the concentration of trimethoprim in this experiment inhibited the growth of Enterococcus for the first 18hr of the test, after which growth was more apparent. The same cfu levels of cholerae grow much faster in this medium, with turbid growth evident by 16hr. This indicates that a higher concentration of trimethoprim (providing this does not inhibit toxigenic cholerae) may successfully inhibit the growth of Enterococcus. Given the time difference in the growth of the two organisms in this media, we can configure the Speedy Breedy™ protocol/ test protocol, such that it allows sufficient time for the growth of toxigenic cholerae, but not of other organisms.

Pseudomonas, is not a halophile and our medium contains a total of 3.5% salt, which may be sufficient to inhibit the growth of Pseudomonas ('Effect of Salinity on Growth and PGPR Activity of Pseudomonads', Deshwal et al, Journal of Academia and Industrial Research (JAIR), Volume 2, Issue 6 November 2013: "No strains survived in 3% NaCI concentration").

Our results show that all the toxigenic cholerae strains in our panel grow well in the presence of 250mg/L trimethoprim (dissolved in sterile dH20 and filtered) in the 50%CPC: 50% peptone salt broth base (VM122). This medium was also tested with E. coli, Enterococcus and Pseudomonas to check whether this mix fully inhibits the growth of these bacteria. The strains were prepared as above (P. aeruginosa was included) and tested in VM122, as described above Results

Table 9: Results for E.coli ATCC11775, E.faecalis and P. aeruginosa growth

at 16 hours at 24hr at 3 days

Colo Colo Colo cfu Turbidi ur of Turbidi ur of Turbidi ur of

Colony inoculu Replic ty/ brot ty/ brot ty/ brot

Strain counts m ate Growth h Growth h Growth h

Wispy

36 and

36 (Only Dark Dark growth >60

the Gree Gree not Yello (coloni

value 1 None n None n turbid w es

from

E. coli were

the

ATCC smudg

countab

11775 ed, so

le plate

not

is

possibi

consider

e to Wispy

ed here)

count) Dark Dark growth

Gree Gree not Yello

2 None n None n turbid w

Enterococ Dark Dark

cus Gree Gree Yello faecalis 1 None n None n Turbid w

NCTC 6 and 6 6

12697/ Dark Dark

ATCC Gree Gree Yello 29212 2 None n None n Turbid w

Wispy

Dark Dark growth

P. Gree Gree not Gree aeruginos 54 and 1 None n None n turbid n

47

a ATCC 40

9027 Wispy

Dark Dark growth Gree Gree not Gree

2 None n None n turbid n

Dark Dark Dark Gree Gree Gree

1 None n None n None n dH20 1 and 0 0.5

Dark Dark Dark Gree Gree Gree

2 None n None n None n The results above show that, at 16 hours, none of the bacteria listed above grew in VM122. This suggests that the increased concentration of trimethoprim in this medium 5 (250mg/L, added as a stock of the antibiotic in sterile, dH20, filtered through a 0.2uM filter)) is at a sufficient level to prevent the growth of E. coli and Enterococcus at 16 hours. 16 hours is most likely a sufficient test time for the cholera test, as toxigenic cholera grows well in VM122 in 16 hours. The growth of Pseudomonas has also been entirely inhibited in this medium and this is most likely due to the concentration of salt 10 in the medium.

We also found that at 17 hours. VM 122 shows no growth of a 10 ² inoculum of S. aureus in 4ml of broth.

15 Table 10: Results for S. aureus

at 17 hours at 24hr at 48hr

Colon cfu Colou Colou Colou y inoculu Replicat Turbidity r of Turbidity r of Turbidity r of

Strain counts m e / Growth broth / Growth broth / Growth broth

S.

Slight,

aureu 804

Dark Dark wispy Dark s and 740

1 None Green None Green growth Green

ATCC 676

6538

Dark Dark Dark

2 None Green None Green None Green

Dark Dark Dark

0 and 1 None Green None Green None Green dH20 0

0

Dark Dark Dark

2 None Green None Green None Green We next tested whether a medium with the addition of Daptomycin could also inhibit the growth of Bacillus cereus.

Table 11 : Results for Bacillus cereus growth

Example 5:

Test method:

1. Prepare a lightly cloudy suspension of each organism in sterile dH20 and measure the OD600.

Organisms used:

Bacillus cereus (Selectrol disc NCTC 7464/ATCC 10876)

Staphylococcus aureus (NCTC 6571)

E. coli (NCTC 9001)

E. faecalis (NCTC 775)

P. aeruginosa (NCTC 10662)

2. Using OD600 0.5 = 10 ⁸ cfu/ml*, dilute the suspensions further in sterile dH20 to give concentrations approximating 100cfu/100ul.

3. Add 50ml sterile dH20 to each of 12 vessels of each medium.

4. Batch 100002 of Toxigenic cholerae medium (final recipe VM123, including daptomycin as dry powder) was used at 5 capsules per vessel, topped up with 50ml sterile dH20 per vessel.

5. Inoculate vessels of each medium in duplicate, using 100ul of 100cfu/100ml suspension, for each bacterium. Add 100ul of the sterile dH20 used as diluent to each of the two remaining vessels of each medium to serve as negative controls.

Spread-plate 100ul of each bacterial suspension used for the inoculations onto TSA plates, in duplicate and incubate at 37°C.

Run the Speedy Breedy™ vessels in Speedy Breedies™, using the Toxigenic cholerae protocol (24hr).

The following day, record the Speedy Breedy™ time to detections, colours of broths and cfu plate counts. (Incubate the plates of S. aureus for at least 48hr, if necessary). *Please note that B. cereus is a relatively large organism and an OD600 = 0.25 approximates 106 cfu/ml.

Table 12: Selectivity of growth medium using Speedy Breedy

As can be seen, the growth medium prevented the growth of all organisms tested. No detection is evident in the Speedy Breedy toxigenic cholerae test of anything other than toxigenic cholerae at 24hr. A further test was set up as above using the following strains at target 10cfu/ vessel:

E. faecalis (NCTC 775)

E. faecalis NCTC 12697/ ACTC 29212

Bacillus cereus (Selectrol disc NCTC 7464/ATCC 10876)

Results

Table 13: Results fot E.faecalis and B.cereus growth

Summary

The growth time of toxigenic cholerae strains in any combination of base medium + trimethoprim tested is very fast, with detection of -100 cfu detected in Speedy Breedy™ at ~7hr. Should other strains grow in the final medium, it is important that they grow more slowly (and radically so), such that the test time can be configured to pick up toxigenic cholerae only.

Example 6 CPC-Agar and a polymyxin CPC-Agar (Sigma 17134) was filtered through muslin (to remove the agar), then filter- sterilised through 0.2um filters. The following additives were added to the filter- sterilised media and the media coded with the VM numbers shown. Concentration of additives: trimethoprim 25mg/L, colistimethate sodium 108.8mg/L, polymyxin B sulphate 4.26mg/L, sodium taurocholate 5g/L.

Table 14: Colistin medium preparations

The inoculum suspension of cells were prepared for each organism by taking a sweep of the bacterial growth from the blood agar plate and inoculating a 5ml 3% salt peptone broth and incubating at 37°C on a shaking incubator until growth was sufficient, typically around 2-4 hours. Initially, 1 ml of this solution was dispensed into an Eppendorf cuvette and placed on the Eppendorf Biophotometer spectrophotometer at a wavelength of 600nm to measure the absorbance. The McFarland Densitometer DEN-1 [Grant Instruments Ltd] was used as a reference to achieve to achieve a uniform cell density across the different strains, alongside the McFarland Standard Set at 0.5, 1 , 2, 3, and 4MF-units. Once the absorbance measured around 1 ,000 the solution was diluted down and compared to the McFarland Standards until 0.5 MF-units was attained. Saline was used as the diluent as it offered no interference by measuring 0.0 MF-units, unlike the 3% salt peptone which measured 0.3 MF-units.

*at wavelength of 600nm

McFarland standards are used as a reference to adjust the turbidity of bacterial suspensions so that the number of bacteria will be within a given range. However, the method didn't attaint the 100 cfu/100ul that was required because at 0.5 MF-units there was 10 ⁸ cell density. Therefore, for mediums VM2 onwards, the inoculated peptone broth once growth ws sufficient, was dispensed into a tube of saline. The growth was added a little at a time (i.e. 100μΙ) and then measured on the McFarland densitometer, adding more broth into the saline until it measured 0.5 MF- units. A solution measuring 0.5MF-units equates to 108 cfu/100ul, thus a serial dilution was required to achieve 103 cfu/100ul. A 1 : 10 dilution was performed 5 times (10 ^"1 to 10 ^"2 to 10 ^"3 to 10 ^"4 to 10 ^"5 ).

For each Vibrio species two bijoux containing 2ml VM medium were inoculated with 100ul of the diluted solution at 10 ³ cfu/100ul, which equated to 100 cfu. Duplicate controls were included which contained 2ml VM medium + 100ul 3% salt peptone broth (non-inoculated). 100ul of the diluted solution (103 cfu/100ul) was also used to inoculate a Difco agar plate using the spread plate technique, this was used to count the cfu after incubation. The bijoux and plates were incubated for 16 hours at 37°C. The following day, the cfu on the Difco plate was counted and the bijoux tubes were scored for growth (presence or absence). In an incubator was present in the medium the colour of the broths was recorded. Results

Table 15: Results for VM31 to VM39

Toxigenic Non- Non- V.parahaemolyticus

Toxigenic 01 0139 toxigenic toxigenic

(inaba) 01 0139

VM31 Growth yellow Growth NT Growth Growth (yellow) 70cfu

(very cloudy) (yellow) yellow

300cfu 200cfu (very

cloudy)

VM32 Growth No growth NT No growth No growth 70 cfu

(yellow) 200cfu

duplicate

2 not as

yellow) 300cfu

VM33 Growth No growth NT No growth No growth 70cfu (yellow)300cfu 200 cfu

VM34 Growth No growth NT No growth No growth 70cfu

(yellow) 200cfu

300cfu

VM35 No growth No growth NT No growth No growth 70cfu

300cfu 200cfu

VM36 Growth No growth NT No growth No growth 70cfu

(yellow) 200cfu

300cfu

VM37 Growth No growth NT No growth No growth 70 cfu

(yellow 200cfu

duplicate 2

not as yellow)

300/cfu VM38 No growth No growth NT No growth No growth 70cfu 300cfu 200cfu

VM39 Growth No growth NT No growth No growth 70cfu

(yellow) 200cfu

300cfu

It can be seen from the above table that addition of at least colisitin to the medium described herein permits the growth of toxigenic 01 (inaba) but interestingly, not toxigenic 0139. Accordingly, the above described medium can be used to select between toxic strains of Vibrio cholerea, that is between the 01 and 0139 serotypes, particularly 01 inaba.

General methods

Medium specificity test - various media

1. Test each of the media with each of the strains listed, in duplicate, using the protocol below: - toxigenic Vibrio cholerae 01 (two different strains)

- toxigenic Vibrio cholerae 0139 (two different strains)

- Vibrio cholerae (non-01/non-0139)

- Vibrio vulnificus

- Vibrio parahaemolyticus

- Vibrio mimicus

- Vibrio alginolyticus

- Vibrio fluvialis

2. Prepare the inoculum suspensions of cells in Peptone Salt (3%) broth and use 100cfu/100ul inoculum per bijou. Inoculate 2ml aliquots of the media in bijous, in duplicate, with each Vibrio species. Include duplicate, 2ml controls of each medium, 'inoculated' with 100ul Peptone Salt broth, as negative controls.

3. Plate 100ul of the cell suspensions used onto Peptone Salt agar (or other appropriate agar) plates in duplicate and incubate as appropriate for enumeration of colonies. Record the cfu, temperature and time the plates were incubated for, at the end of the test. Incubate the bijous after inoculation at an appropriate temperature for an appropriate length of time in order to be able to observe the presence or absence of growth. Record the time and temperature the bijous were incubated for and the presence or absence of growth and any colour changes.

References

1. Vibrio choleae: World Health Organisation; G.B. Nair, p1 19-142.

2. WHO; Cholera

3. Nelson EJ et al. Cholera transmission: the host, pathogen and bacteriophage dynamic. Nat Rev Microbiol. 2009 Oct; 7(10).

4. Richard A Finkelstein; Chapter 24 Cholera, Vibrio cholera 01 and 0139, and other Pathogenic Vibrios; Medical Microbiology, 4 ^th edition, 1996.

5. IMO website "International Convention for the Control and Management of Ships' Ballast Water and Sediments (BWM)".

6. Legal Briefing; UK P&l Club, February 2015.

7. Massad G & Oliver J.D., New Selective and Differential Medium for Vibrio cholera and Vibrio vulnifucus, Applied and Environmental Microbiology, Sept. 1987, p.2262-2264.

8. Trimethoprim and enterococci in urinary tract infections: new perspectives on an old issue', Tegmark Wisell et al, Journal of Antimicrobial Chemotherapy, Volume 62, Issue 1 , p. 35-40.