CN110177867B

CN110177867B - Organism Selective Growth Medium

Info

Publication number: CN110177867B
Application number: CN201780078646.3A
Authority: CN
Inventors: V·马思; D·格罗夫普赖斯
Original assignee: Beijing Baiketian Technology Co ltd
Current assignee: Beijing Baiketian Technology Co ltd
Priority date: 2016-12-19
Filing date: 2017-12-18
Publication date: 2023-11-21
Anticipated expiration: 2037-12-18
Also published as: CN110177867A; GB201621629D0; WO2018115829A1

Abstract

The present invention relates to selective and/or differential growth media for isolating vibrio cholerae and methods for isolating and/or identifying and/or selectively growing vibrio cholerae from a sample.

Description

Organism selective growth medium

Technical Field

The present invention relates to a selective and/or differential growth medium for isolating Vibrio cholerae (Vibrio cholerae) and a method for isolating and/or identifying and/or selectively growing Vibrio cholerae from a sample.

Introduction to the invention

Vibrio cholerae is one of the members of the Vibrionaceae (Vibrionaceae) family and is a facultatively anaerobic gram-negative bacterium. Most species of Vibrio are ubiquitous in estuary and marine environments, although Vibrio has been identified in fresh water, provided that there is a certain minimum level of sodium ions present ¹ 。

Of particular importance, ingestion of water or food contaminated with certain strains of Vibrio cholerae can lead to cholera, which can cause acute diarrhea, and if left untreated can lead to death within hours. Researchers estimated that 140 to 430 ten thousand cases annually worldwide and 28000 to 142000 people die from cholera ² . Providing safe drinking water and environmental sanitation is critical to prevent the transmission of this extremely deadly disease.

Vibrio cholerae can be serologically classified based on its Lipopolysaccharide (LPS) O antigen. In fact, more than two hundred serogroups of Vibrio cholerae exist in the aquatic environment, identified as Vibrio cholerae O1 to O200, based on the antigenicity of the O antigen. However, of these two hundred different serotypes, only the O1 and O139 strain subgroups were pathogenic ³ . Furthermore, pathogenic O1 serogroups are subdivided into two phenotypically distinct biotypes, eltolype and classical. These two biotypes can be further subdivided into two serotypes, the rice leaf type and the small Sichuan type. These serotypes are distinguished in agglutination and vibrio antibody tests based on their primary thermostable LPS thallus antigen. Cholera flora has a common antigen a, whereas serotypes are distinguished by type-specific antigens, B is a small sichuan type and C is a rice leaf type. It is widely believed that O139 originates from the Eertoto biotype, but loses the characteristic O1 cell antigen and at the same time gains the ability to produce polysaccharide capsules ⁴ 。

Prior to 1992, cholera was thought to be caused only by the leaf and small forms of rice, but in 1992 cholera caused by O139 appeared in india and bangladesh in epidemic proportions. However, despite the devastating disease caused by the O139 serogroup, the eltropism strain remains the globally dominant strain ³ . Other serogroups of Vibrio cholerae can cause diarrheaDiseases and other pathologies, but not epidemic cholera.

The vessel collects ballast water to adjust its stability. However, the discharge of ballast water at ports has become a cause of geographic migration of a large number of non-local and undesirable organisms (such as vibrio cholerae) that have a significant impact on human health worldwide. It is this risk of history proving that the International Maritime Organization (IMO) was forced to pass the international convention for ballast water and sediment control and management (International Convention for the Control and Management of Ships' Ballast Water and Sediments) (BWM) on 14 th 2004. BWM aims to prevent the spread of pathogenic organisms from one region to another by establishing standards and procedures for managing and controlling ship ballast water and sediment. In particular, rule D-2 of the convention establishes a numerical ballast water discharge standard for a particular microorganism. For Vibrio cholerae toxigenic (O1 and O139), this limit is less than 1 colony forming unit (cfu) per 100 ml or less than 1cfu per 1 gram (wet weight) zooplankton sample ⁵ . Compliance is required throughout the use of the vessel. Furthermore, typically the test cannot be performed on board the ship, but rather the sample needs to be sent to a laboratory. This can be particularly difficult in remote areas. In addition, laboratory testing is often slow, forcing the vessel to stay long in port, causing serious shipping disruption, and prohibitively expensive.

Thus, there is a need for a simple and effective test that is capable of rapidly detecting very small amounts (< 1CFU/ml or <1 CFU/g) of specifically virulent O1 or O139 vibrio cholerae in ballast water.

Once the pathogenic vibrio cholerae is released at the port, it can be rapidly transmitted through the faecal route, especially in areas of poor hygienic condition. In this regard, rapid bacteriological diagnosis is critical to preventing potential epidemic outbreaks. Also, there is a need for a rapid diagnostic test that can specifically identify pathogenic vibrio cholerae in a patient population.

Currently, vibrio cholerae is isolated and identified, typically by spreading the sample on a selection, usually in a bacteriology laboratory due to its rapid growth and characteristic morphologyOn sexual medium. One such common selective medium is thiosulfate-citrate-cholate agarose agar (TCBS), in which vibrio cholerae can be identified as a distinct yellow colony. However, the medium cannot distinguish between virulent and non-virulent vibrio cholerae, and the reliability of the medium has been questioned. In addition, TCBS can also be used for isolation of Vibrio parahaemolyticus, which means that clear identification of pathogenic Vibrio cholerae is not possible without further testing ⁴ . Subsequently, some alternative media have been proposed and developed, such as sodium dodecyl sulfate-polymyxin-sucrose (SPS) agar and CPC agar ⁵ . Subsequent tests may include agglutination tests involving specific antisera or the use of oxidase reactions, indole reactions, sugar fermentation reactions and gelatinase, lysine, arginine and ornithine decarboxylase reactions. Alternatively, the presence of a particular bacterium may be detected using genetic methods such as PCR.

Thus, there is a need for a selective growth medium that is selective not only for Vibrio cholerae, but even more specifically for pathogenic or toxic forms of Vibrio cholerae. In addition, there is a need for a selective growth medium that is effective in selectively and rapidly growing (and thereby identifying) a virulent form of Vibrio cholerae from a sample of lower potential bacterial count, such as in ballast water or in patients with cholera, and more particularly in patients recovering from cholera. The present invention addresses this need.

Disclosure of Invention

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

(a) A basal medium;

(b) A salt;

(c) A nitrogen source; and

(d) Dihydrofolate reductase (DHFR) inhibitors and/or polymyxins.

In one embodiment, the medium comprises between 1 and 50g/L of basal medium, between 1 and 50g/L of salt, between 1 and 50g/L of nitrogen source, and between 35 and 300mg/L of DHFR and/or between 1 and 500mg/L of polymyxin.

In one embodiment, the nitrogen source is peptone. Preferably, the peptone is Bacto peptone.

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

In other embodiments, the basal medium is an agar base, preferably cellobiose polymyxin B colistin (CPC).

In one embodiment, the medium comprises 5g/L peptone, 15g/L sodium chloride, 32.54g/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 daptomycin.

In another aspect of the invention, agar plates are provided that include the media described herein.

In yet another aspect of the invention, a method for isolating and/or identifying and/or selectively growing Vibrio cholerae produced from a sample is provided, the method comprising inoculating the sample into a culture medium as described herein.

In one embodiment, the method comprises

a. Inoculating the sample into a growth medium;

b. incubating the growth medium to allow growth of Vibrio cholerae; and

c. detecting the existence of the vibrio cholerae.

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 isolating and/or identifying and/or selectively growing vibrio cholerae virulence from a sample.

In yet another aspect, a method is provided for detecting the presence of at least one Colony Forming Unit (CFU) of Vibrio cholerae in a ballast water sample, the method comprising

a. Obtaining a sample of ballast water;

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

c. incubating the growth medium to allow growth of Vibrio cholerae;

and

d. detecting the presence of at least one CFU of Vibrio cholerae.

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 a medium as described herein, and detecting the presence of vibrio cholerae, wherein the presence of vibrio cholerae is indicative of a disease.

In one embodiment, the vibrio cholerae is vibrio cholerae O1 or vibrio cholerae O139. Preferably, vibrio cholerae O1 is an Eltols biotype or classical biotype.

The invention is further described in the following non-limiting drawings.

Drawings

FIG. 1 shows the use of a Speedy Breedy ^TM To detect the presence of Vibrio cholerae in the ballast water sample.

Step 1: waste bottles or barrels, pumps and manifolds are provided. By way of example only, EZ-Stream from Merck Millipore may be used ^TM And (3) a pump.

Step 2: pouring 100ml of ballast water into the filter bowl and covering the filter bowl with a cover;

step 3: removing the stopper from the cup bottom;

step 4: securing the cup to the manifold by pushing the cup downward;

step 5: opening the valve;

step 6: once all waste is in the waste bottle or bucket, the pump is turned back on and off;

step 7: the containers for the selection test (E.coli, enterococcus or Vibrio cholerae) were prepared by opening the lid and carefully placing it on the bench with the rubber side facing upwards.

Step 8: a pair of new gloves were put on and rubbed with an alcohol pad. The glove was allowed to dry.

Step 9: the index finger is slid under the thin filter and the sheet is doubled over (without touching the top side of the mesh of the sheet).

Step 10: the sheet continues to fold until it is thin enough to push through the Speedy Breedy ^TM The sample port of the container.

Step 11: the filter is pushed through the sample port, using the container lid if necessary.

Step 12: once the filter has entered the culture vessel, some sterile water is poured into the cup and 50ml of sterile water is drawn into a new sterile syringe.

Step 13: adding 50ml of water to the culture vessel through the sample port; and

step 14: placing the container into a speed Breedy ^TM And immediately begin testing.

Detailed Description

The invention will now be further described. In the following sections, the various 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 in the art. These techniques are fully described in the literature.

As used herein, the terms "pathogenic" and "toxigenic" are used interchangeably and refer to a strain, serotype, or biotype of vibrio cholerae that causes the symptoms associated with cholera disease. In one embodiment, the vibrio cholerae is selected from the group consisting of vibrio cholerae O1 and vibrio cholerae O139. In one embodiment, the vibrio cholerae produced O1 is of the eltolo type or classical type, and preferably is of the rice leaf serotype or the small Sichuan serotype.

In one aspect the invention relates to a selective and/or differential growth medium for isolating or growing Vibrio cholerae, the medium comprising

(a) A basal medium;

(b) A salt;

(c) A nitrogen source; and

(d) Dihydrofolate reductase (DHFR) inhibitors and/or polymyxins.

As used herein, the term "selective" means that the growth medium allows growth of only certain types of organisms while inhibiting growth of other organisms. The term "differential" means a growth medium that is capable of distinguishing between closely related organisms or a group of organisms.

In one embodiment, the basal medium is an agar base. In a particular embodiment, the agar base is cellobiose polymyxin B colistin (CPC). Alternatively, the agar base is selected from TCBS (thiosulfate-citrate-cholate-sucrose agar), hiChrome vibrio agar, vibrio chromogenic agar or gelatin taurocholate tellurite medium. In other embodiments, the nutritional base does not include agar. For example, the basal medium is one of the above without the addition of agar. Thus, in another embodiment, the basal medium is a broth or liquid nutrient medium.

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

pepsin digest of animal tissue, preferably in the 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, preferably in the 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;

disaccharides, such as cellobiose, preferably in the 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;

salts, such as sodium chloride, preferably in the range 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 25g/L, and most preferably 20g/L;

bromothymol blue, preferably in the range of 0.001 to 0.1, more preferably 0.01 to 0.09g/L, and most preferably 0.04g/L.

Cresol red, preferably in the 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 also comprise agar, preferably in the 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 include polymyxin B and/or colistin.

The skilled artisan will be able to produce media as described above using techniques known in the art. Alternatively, the skilled artisan may select from commercial sources (such as Sigma Aldrich ^TM ) A pre-prepared medium was obtained.

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

In one embodiment, the nitrogen source is peptone. The word "peptone" refers to a water-soluble mixture of polypeptides and amino acids formed by partial hydrolysis of proteins. Peptones are thus also a useful natural source of amino acids, polypeptides and proteins, and are most often obtained by enzymatic digestion or acid hydrolysis of natural products such as animal tissue, milk, plants or microbial cultures. Suitable peptones are known to the skilled worker. In particular embodiments, the peptone is an enzymatic digest of an animal protein, such as Bacto ^RT Peptone. In one embodiment, 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, the number of the cells to be processed,

(％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 peptone, casein peptone, soy peptone, plant peptone and any other useful peptone.

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

Thus, in a particular embodiment, the medium comprises

(a) CPC agar;

(b) Sodium chloride;

(c) Bacto peptone; and

(d) L-trimethoprim.

In one embodiment, the medium comprises basal medium, salt and nitrogen source in the ratio: 6.5:3:1, wherein the nitrogen source is other than any nitrogen source that may be present in the basal medium.

In one embodiment, the medium comprises

(a) The basal medium is preferably in the 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 6.0g/L. In a particular embodiment, the medium comprises 5g/L basal medium.

(b) The preferred range is 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 a particular embodiment, the medium comprises 15g/L salt.

(c) The nitrogen source is preferably in the range of 1 to 50g/L, more preferably 10 to 40g/L or 20 to 50g/L, more preferably 25 to 40g/L, more preferably 30 to 25g/L, even more preferably 32, 33 or 34g/L. In a particular embodiment, the medium comprises a nitrogen source of 32.1, 32.2, 32.3, 32.4, 32.5, 32.6, 32.7, 32.8, 32.9, or 40.0 g/L. Even more preferably, the medium comprises a nitrogen source of 32.51, 32.52, 32.53, 32.54, 32.55, 32.56, 32.57, 32.58, 32.59 or 32.60 g/L. In one embodiment, the medium includes 32.54g/L nitrogen source.

In a particular embodiment, the medium comprises

Bacto peptone 5g/L

15g/L sodium chloride; and

32.54g/L CPC-agar.

In other embodiments, the medium comprises a dihydrofolate reductase (DHFR) inhibitor, preferably in the 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 include 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 235mg/L of dihydrofolate reductase (DHFR). In one embodiment, the amount of DHFR inhibitor may be 35mg/L or greater. In other preferred embodiments, the amount of DHFR inhibitor can be at least 50mg/L. In another preferred embodiment, the DHFR inhibitor can be present in an amount of at least 175mg/L. These concentrations will allow the growth of Vibrio 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 vibrio cholerae (preferably O1H 151220003), parahaemolyticus (preferably H151220010), vibrio mimicus (preferably H151220007), vibrio vulnificus (H130860068), vibrio cholerae non-O1, O139 (preferably H1 51220001), vibrio alginolyticus (preferably H151220011) and vibrio fluvialis (preferably H151220008).

The medium can be made up to 50ml using distilled water.

Thus, in a further aspect of the invention, there is provided a method for isolating and/or identifying and/or selectively growing Vibrio toxigenic from a sample further comprising at least one bacterial strain closely related to Vibrio cholerae, such as those described above, the method comprising inoculating the sample into a medium as defined above and comprising between 5 and 300mg/L of a dihydrofolate reductase (DHFR) inhibitor, more preferably 35mg/L or more.

In another embodiment, the medium comprises a dihydrofolate reductase (DHFR) inhibitor, preferably in the 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, trimethoprim is added as a powder during the production of the medium, encapsulated with the other ingredients.

In the preferred embodiments described above, the DHFR inhibitor is an antibiotic, and more preferably the antibiotic is trimethoprim.

In another embodiment, the medium comprises polymyxin. This may be an alternative or in addition to DHFR inhibitors. In one embodiment, the polymyxin may be polymyxin E or colistin (colistin sulfate or colistin sodium methane sulfonate (colistin sodium methane sulfonate, colistin sodium methane sulfonate)). The medium may also include additional antibiotics, 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 110 to 120mg/L, and even more preferably 108.8mg/L. In another embodiment, the medium comprises 1 to 10mg/L of other antibiotics, 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 6mg/L, and most preferably 4.26mg/L of antibiotics.

In another embodiment, the selective and/or differential growth medium further comprises a gram positive antibacterial agent. Preferably, the agent does not affect the growth of gram-negative bacteria. In one embodiment, the agent is an antibiotic. Preferably, the antibiotic is daptomycin. In another embodiment, the gram positive antibacterial agent is selected from teicoplanin (teicoplanin), quinupristin-dalfopritin (quinupristin-dalfopritin), oxazolidinones and telavancin (telavancin). In one embodiment, the culture medium comprises a gram-positive antibacterial agent, preferably in the 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 preferably 2mg/L.

In another embodiment, the selective and/or differential growth medium is a dry powder mixture and includes

-between 5 and 15% by weight, preferably 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15% by weight of a nitrogen source as described herein, preferably baco 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% by weight of a salt as described herein, preferably sodium chloride;

-between 40 and 80% by weight of a basal 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 basal 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 isolating Vibrio cholerae produced, the medium comprising

(a) CPC medium; and

(b) At least one polymyxin as defined above.

In another aspect of the invention, there is provided a method for isolating and/or identifying and/or selectively growing Vibrio cholerae toxigenic from a sample, the method comprising inoculating the sample into a 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 vibrio cholerae toxigenic as described herein.

In particular, the method may comprise

a. Inoculating a sample into a growth medium as described herein;

b. incubating the growth medium to allow growth of Vibrio cholerae; and

c. detecting the existence of the vibrio 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 ℃, preferably 37 ℃.

In one embodiment, the presence of Vibrio cholerae toxigenic can be detected by counting the number of colonies growing on the surface of an agar plate. Such techniques are well known to the skilled person. Alternatively, the presence or absence of growth may be measured by analyzing the turbidity and/or color of the culture broth in bijou. This is also a standard microbiological technique.

In one embodiment, the sample is water. Preferably, the water is seawater or potable water. In a particular 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 suffering from cholera, considered to suffer from cholera or recovering from cholera. In a particular embodiment, the sample is a fecal sample.

We have previously described a system for monitoring the metabolism/growth of microorganisms that includes a sealed chamber with a flexible membrane to provide a sensitive pressure measurement of the gas pressure in the headspace above the culture broth. Reference may be made in particular to, for example, US2005/0170497 (incorporated by reference). Here we describe such a system that can be used in conjunction with the growth medium to detect the presence of Vibrio cholerae toxigenic (called Speedy Breedey ^TM ). Thus, in one embodiment, the method further comprises

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

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

Fig. 1 provides an example of a detailed scheme for detecting the presence of vibrio cholerae in ballast water using the system described above.

Preferably, the volume of the sample is 50ml, for example if the sample is water. When the sample is filtered prior to testing, any volume of water may be used, and the filter element (cartridge) is then placed in the container and topped up with 50ml of sterile water. If the sample is tested directly, 50ml is added to the Speedy Breedy ^TM In a container. The sample may also be diluted, but Speedy Breedy ^TM Must always be 50ml. For example, 1ml of the sample may be diluted with 49ml of sterile water. For example, for fecal samples, about 1g of the sample may be mixed with 49ml of sterile water for use in a Speedy Breedy ^TM And (3) testing. For ballast water, a volume of 100ml was filtered and the filter was placed in a vessel and topped up with 50ml of sterile water. However, if the sample is blood, sterile water may be added to the sample to bring the total volume to 50ml。

In other aspects of the invention, there is provided an agar plate comprising a petri dish and a growth medium as described above, wherein the basal medium comprises agar and is thus an agar foundation. Thus, in one embodiment, there is provided an agar plate comprising an agar base, a salt, a nitrogen source, and a dihydrofolate reductase (DHFR) inhibitor and/or a polymyxin as defined above. Such agar plates can be used to selectively grow Vibrio cholerae, which is toxic.

In a further aspect of the invention there is provided the use of a selective and/or differential growth medium as described above for isolating and/or identifying and/or selectively growing Vibrio cholerae produced from a sample.

In another aspect of the invention, there is provided a method of identifying the presence of Vibrio cholerae in ballast water, the method comprising obtaining a sample of ballast water, inoculating the sample into a growth medium as described herein, and detecting the presence of Vibrio cholerae toxigenic. 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 gram weight of zooplankton sample.

In other aspects 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 above-described medium, and detecting the presence of vibrio cholerae. In an alternative embodiment, a method of diagnosing cholera in a patient is provided, the method comprising inoculating a sample taken from the patient into the above-described medium, and detecting the presence of vibrio cholerae. The presence of Vibrio cholerae in the sample is indicative of a disease. Preferably, at least one colony forming unit is identified.

In a final aspect of the invention, there is provided a method for distinguishing between toxigenic O1 (preferably of the type She Xieqing) and toxigenic O139, the method comprising

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

b. incubating a growth medium as described herein to allow growth of Vibrio cholerae O1, preferably rice She Xieqing; and

c. the presence of Vibrio cholerae O1, preferably of the type She Xieqing, is detected.

In the above embodiment, we have shown that in the medium described herein comprising polymyxins, preferably colistin (also known as polymyxin E), vibrio cholerae O1, which is toxigenic, will grow, whereas Vibrio cholerae O139 cannot. Thus, the growth of microorganisms on this medium is indicative of the presence of Vibrio cholerae O1 and can be used to distinguish between the serotypes of Vibrio cholerae in a sample. In one embodiment, the O1-producing vibrio cholerae is a rice leaf serotype.

Samples are defined above.

As used herein, "and/or" should be considered as a particular disclosure of each of two particular features or components, with or without the other. For example, "a and/or B" should be considered a specific disclosure of each of (i) a, (ii) B, and (iii) a and B, as if each were individually listed herein.

In describing the ranges, it is intended that the endpoints of each range and any values falling between the endpoints are disclosed. For example, a range of 10 to 20mg/L includes 10 and 20mg/L, as well as any amount falling between 10 and 20 mg/L.

Unless the context indicates otherwise, the description and definition of the features above is not limited to any particular aspect or embodiment of the invention, and applies equally to all aspects and embodiments described.

Example 1

The panel of microorganisms used for the initial test medium-antibiotic mixture is as follows:

vibrio cholerae toxigenic O1 (rice leaf type) H151220006

Vibrio cholerae toxigenic O139H151220002

Vibrio cholerae O1H151220003 non-toxigenic

Vibrio parahaemolyticus H151220010

Vibrio mimicus H151220007

Vibrio vulnificus H130860068

Vibrio cholerae non-O1, O139H151220001

Vibrio cholerae O1 (Xiaochuan) H151220005

Vibrio cholerae toxigenic O139E099892

Vibrio alginolyticus H151220011

Vibrio fluvialis H151220008

Growth medium

The resulting formulation showing the best selectivity for Vibrio cholerae produced consists of basal medium with antibiotics added.

Basic formula

5g/L Bacto peptone (Oxoid LP 0037)

15g/L sodium chloride

32.54g/L CPC-agar (base) (Sigma 17134) ^*

Antibiotics

175mg/L trimethoprim (Sigma 92131)

And preferably 2mg/L daptomycin (Tocres 3917) to inhibit the growth of gram-positive organisms.

We have demonstrated that the above base formulation with trimethoprim and daptomycin at least 35mg/L, and more preferably 55mg/L and most preferably between 175-300mg/L, allows the growth of all of the Vibrio cholerae strains in this study while completely inhibiting the growth of E.coli, E.faecalis, P.aeruginosa, S.aureus and B.cereus, as well as Vibrio cholerae and other vibrio paravorus.

(composition of CPC-agar (base))

Pepsin 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

To date, trimethoprim used in the test was initially used as an additive, which was prepared by mixing with dH2O and filter-sterilizing through a 0.2um filter (up to VM122 medium). The test from VM123 medium was characterized by trimethoprim as the powder encapsulated with the remaining ingredients. Since trimethoprim is only partially soluble in water, this clearly causes a difference in the actual concentration of trimethoprim available in the medium, both when added as a liquid after filter sterilization and when added directly as a powder.

Our project progressively showed that the use of a concentration of trimethoprim with the basal medium listed above achieves unique selectivity for Vibrio cholerae toxigenic in the group of closely related organisms listed above. The number of combinations of media tested was distinguished using the letter "VM" as a prefix and tested in ascending numerical order. By the time culture medium VM97 was tested, the desired effect of trimethoprim was evident. The following are the test results of VM98-VM105, which investigated the effect of various concentrations of trimethoprim bound to basal medium on selection cultures in our test group.

Table 1: test Medium VM98 through VM105

Culture medium	mg/L trimethoprim
		VM98	5
VM99	10
		VM100	15
VM101	20
		VM102	25
VM103	35
		VM104	45
VM105	55

Table 2: results of VM98 through VM105

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The above results clearly show that increasing the concentration of trimethoprim in the medium effectively inhibited the growth of all strains except the strain of Vibrio cholerae, which is virulent. The results also show that absolute selectivity to Vibrio cholerae can be achieved once a 35mg/L trimethoprim concentration in CPC-PBS basis is reached.

Example 2

Increasing the concentration of trimethoprim in the CPC-PBS base gives a better opportunity to inhibit the growth of other gram negative strains, such as e.

Table 3: test Medium VM114 through VM118

Table 4: test Medium VM119 through VM122

These results show that even inclusion of 250mg/L trimethoprim allows good reproducible growth of all virulent vibrio cholerae in our group. Note that this level of trimethoprim when tested as a "solution" of trimethoprim in sterile deionized H2O, which was then filtered through a 0.2 μm filter-trimethoprim is known to be quite poorly soluble in water.

Results

Table 5: results of VM114 to VM118

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Table 6: results of VM119 through VM122

Example 3

The following test was performed using trimethoprim added as a dry powder. Daptomycin is also included. We developed Sp for selective detection of Vibrio cholerae O1 and O139eedy Breedy ^TM Testing (Speedy Breedy, as described above) ^TM Is a microbial respirometer system that is capable of performing a stand-alone, laboratory-free test on a sample to detect microorganisms.

Stage I of this project identified liquid media that specifically grew vibrio toxigenic O1 and O139 while inhibiting the growth of other closely related vibrios (including non-toxigenic vibrios) and other unrelated bacterial species.

Testing organisms

Vibrio cholerae toxigenic O1 (rice leaf type) H151220006

Vibrio cholerae toxigenic O139H151220002

Vibrio cholerae O1H151220003 non-toxigenic

Vibrio parahaemolyticus H151220010

Vibrio mimicus H151220007

Vibrio vulnificus (H130860068)

Vibrio cholerae non-O1, O139H151220001

Vibrio cholerae O1 (Xiaochuan) H151220005

Vibrio cholerae toxigenic O139E099892

Vibrio alginolyticus H151220011

Vibrio fluvialis H151220008

Composition of the Medium (VM 123)

5g/L Bacto peptone (Oxoid LP 0037);

15g/L sodium chloride;

32.54g/L CPC-agar (basal) (Sigma 17134); and

175mg/L trimethoprim (Sigma 92131)

2mg/L daptomycin (available from Tocres) was added as a filter sterilized starting material prepared in dH2O to all Speedy Breedy containing encapsulated VM123 ^TM In a container.

Results

Table 7: results of VM123 in mixture with non-toxigenic and toxigenic organisms

Example 4

The strain tested above was a strain closely related to Vibrio cholerae. While it is of paramount importance to design a selective medium that excludes closely related strains, it is also important to conduct tests on a wider range of microorganisms to ensure that the final medium excludes the growth of microorganisms that are highly diverse. In fact, marine and estuary waters often contain a large number of Pseudomonas species in addition to Flavobacterium (Flavobacterium) and Photobacterium (Photobacterium).

For this purpose, tests were also carried out to examine whether the basal medium+trimethoprim inhibited the growth of E.coli, P.aeruginosa, E.faecalis and S.aureus.

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

Introduction to the invention&Target object

55mg/L in our Vibrio cholerae medium base is considered the highest concentration of trimethoprim tested in medium batch VM98-VM105, which is fully selective for all Vibrio cholerae toxigenic tested in the Vibrio group.

In order to test the selectivity of the medium with respect to strains such as E.coli and enterococcus, the following experiments were set up.

Method

1. The following strains were grown on TSA plates and resuspended in sterile dH2O to give 100cfu per 10 μl (using od600=0.5=10) ⁸ cfu/ml, for calculation):

coli PHE Lenticule CRM09001H, batch 1233-14

Enterococcus faecalis PHE Lenticule CRM00775H, batch 1246-14

2. 4ml of: autoclaved 50% cpc agar (Sigma 17134) (filtered through filter paper) +50% peptone salt broth (peptone 3% salt broth=10 g/L baco peptone, 30g/L sodium chloride), to which 0.2 μm filtered trimethoprim in dH2O was added-poured into each of 9 sterile bijou bottles.

3. Each of the 3 bijous flasks was incubated with 100. Mu.l of E.coli suspension, each of the 3 bijous flasks was incubated with 100. Mu.l of enterococcus suspension, and each of the 3 bijous flasks was incubated with 100. Mu.l of sterile dH 2O.

4. Mu.l of each bacterial suspension plate was plated on TSA plates in duplicate.

5. Plates and bijous were incubated overnight at 37 ℃ and the number of colonies on each plate was recorded, as well as the presence or absence of growth (turbidity) and the color of the broth in each bijous.

Results (after 24 hours incubation)

Table 8: results of E.coli and enterococcus growth

These results indicate that our Vibrio cholerae medium containing 55mg/L trimethoprim does not support the growth of E.coli, but that enterococcus faecalis does grow in this medium. We see that in the above test, the concentration of trimethoprim in the experiment inhibited enterococcus growth 18hr before the test, after which growth was more pronounced. Vibrio cholerae at the same cfu level grew faster in this medium, and showed significant cloudy growth by 16 hr. This suggests that higher concentrations of trimethoprim (provided that it does not inhibit vibrio cholerae) can successfully inhibit the growth of enterococci. Taking into account the time difference between the growth of two organisms in the medium, we can configure a Speedy Breedy ^TM Protocol/test protocol such that it provides sufficient time for the growth of Vibrio cholerae, which is toxic, while other microorganisms do not.

Pseudomonas is not halophil, but our medium contains a total of 3.5% salts, 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 6November 2013: "No strains survived in 3%NaCI concentration").

Our results show that all of the Vibrio cholerae strains in our group grew well in the presence of 250mg/L trimethoprim (dissolved in sterile dH2O and filtered) in 50% CPC:50% peptone salt broth base (VM 122). The medium was also tested with E.coli, enterococci and Pseudomonas to check if the mix completely inhibited the growth of these bacteria. Strains (including Pseudomonas aeruginosa) were prepared as described above and tested in VM122 as described above.

Results

Table 9: results of E.coli ATCC11775, enterococcus faecalis and Pseudomonas aeruginosa growth

The above results indicate that none of the bacteria listed above grew in VM122 at 16 hours. This indicates that the increased concentration of trimethoprim in this medium (250 mg/L, added as stock solution of antibiotic in sterile dH2O filtered through a 0.2 μm filter) is a level sufficient to prevent growth of e.coli and enterococci at 16 hours. 16 hours is likely to be a sufficient test time for cholera testing because Vibrio cholerae produced grows well in VM122 at 16 hours. The growth of Pseudomonas in this medium was also completely inhibited, and this was probably due to the concentration of salts in the medium.

We have also found that at 17 hours VM122 was shown to be 10 in 4ml of culture broth ² The inoculum of staphylococcus aureus did not grow.

Table 10: results of Staphylococcus aureus

We next tested whether the culture medium with daptomycin added could also inhibit the growth of bacillus cereus.

Table 11: results of Bacillus cereus growth

Example 5:

the testing method comprises the following steps:

1. a slightly turbid suspension of each organism in sterile dH2O was prepared and the OD600 was measured.

Organisms used:

bacillus cereus (Selectrol disc NCTC 7464/ATCC 10876)

Staphylococcus aureus (NCTC 6571)

Coli (NCTC 9001)

Enterococcus faecalis (NCTC 775)

Pseudomonas aeruginosa (NCTC 10662)

2. OD600 0.5=10 was used ⁸ cfu/ml ^* The suspension was further diluted in sterile dH2O to give a concentration of approximately 100cfu/100 μl.

3. To each of the 12 vessels of each medium was added 50ml of sterile dH2O.

4. Batch 100002 of Vibrio cholerae medium (final formulation VM123, including daptomycin as a dry powder) was used in 5 parts per container of the package, and filled with 50ml of sterile dH2O per container.

5. For each bacteria, 100. Mu.l of 100cfu/100ml suspension was used and the vessel of each medium was inoculated in duplicate.

6. Mu.l of sterile dH2O used as diluent was added to each of the two remaining containers of each medium as a negative control.

7. Mu.l of each bacterial suspension plate for inoculation was plated on TSA plates, in duplicate and incubated at 37 ℃.

8. In Speedy belts ^TM In run speed Breedy ^TM The container was subjected to Vibrio cholerae regimen (24 hr).

9. The next day record Speedy Breedy ^TM Detection time, color of culture broth, and cfu plate count. (if necessary, incubating the plates of Staphylococcus aureus for at least 48 hr). * Note that bacillus cereus is a larger organism, and that od600=0.25 is close to 106cfu/ml.

Table 12: selectivity of growth Medium Using Speedy Breedy ^TM

It can be seen that the growth medium prevented the growth of all organisms tested. At 24hr, there were no apparent test results in the Speedy Breedy vibrio toxigenic vibrio cholerae test except for the vibrio toxigenic vibrio cholerae.

Further tests were established as described above at a target 10cfu per vessel using the following strains:

enterococcus faecalis (NCTC 775)

Enterococcus faecalis NCTC 12697/ACTC 29212

Bacillus cereus (Selectrol disc NCTC 7464/ATCC 10876)

Results

Table 13: results of enterococcus faecalis and Bacillus cereus growth

Summary

In any combination of basal medium+trimethoprim tested, the growth time of the Vibrio cholerae strain was very fast, at-7 hr in Speedy Breedy ^TM And 100cfu. Assume otherStrains will grow in the final medium, it is important that they grow slower (and are extremely slow) so that the test time can be configured to detect only Vibrio cholerae which is toxigenic.

EXAMPLE 6 CPC-agar and polymyxin

CPC-agar (Sigma 17134) was filtered through gauze (to remove agar) and then sterilized by filtration through a 0.2 μm filter. The following additives were added to the filter sterilized medium and the medium was encoded with the indicated VM numbers.

Concentration of additives: 25mg/L of trimethoprim, 108.8mg/L of colistin mesylate, 4.26mg/L of polymyxin B sulfate and 5g/L of sodium taurocholate.

Table 14: preparation of colistin culture medium

An inoculum suspension of cells was prepared for each organism by hanging bacterial growth from a blood agar plate and inoculating 5ml of 3% saline peptone culture and incubating on a shaking incubator at 37 ℃ until growth was sufficient, typically about 2-4 hours.

First, 1ml of this solution was dispensed into an Eppendorf cuvette (cuvette) and placed on a Eppendorf Biophotometer spectrophotometer to measure absorbance at a wavelength of 600 nm. McFarland densitometer DEN-1[Grant Instruments Ltd was used as a reference to achieve consistent cell densities among different strains while the McFarland standard set up was performed at 0.5, 1, 2, 3 and 4 MF-units. Once the measured absorbance is around 1,000, the solution is diluted and compared to McFarland standards until 0.5 MF-unit is reached. Physiological saline was used as a diluent because it was measured as 0.0 MF-unit without providing interference unlike 3% saline peptone, which was measured as 0.3 MF-unit.

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^* At a wavelength of 600nm

However, this method does not reach the required 100 cfu/100. Mu.l, since there is 10 at 0.5 MF-units ⁸ Cell density. Therefore, for the medium from VM2, the inoculated peptone culture broth was dispensed into a test tube of physiological saline once grown sufficiently. One point growth (i.e., 100. Mu.l) was added at a time and then measured on a McFarland densitometer, more broth was added to the saline until 0.5 MF-units were measured. The solution measured as 0.5 MF-units is equal to 108 cfu/100. Mu.l, so serial dilutions are required to reach 103 cfu/100. Mu.l. 5 dilutions 1:10 were performed (10 ^-1 To 10 ^-2 To 10 ^-3 To 10 ^-4 To 10 ^-5 )。

For each Vibrio species, at 10 ³ cfu/100. Mu.l (equal to 100 cfu) 100. Mu.l of the diluted solution was inoculated into two bijoux containing 2ml of VM medium. Duplicate controls were included, which contained 2ml VM medium +100 μl 3% saline peptone medium (not inoculated). 100 μl of the diluted solution (103 cfu/100 μl) was also used to inoculate Difco agar plates using a spread plate technique, which was used to count cfu after inoculation. The bijoux and plates were incubated for 16 hours at 37 ℃.

The next day cfu on Difco plates were counted and bijoux tubes were scored for growth (presence or absence). The color of the culture medium present in the incubator was recorded.

Results

Table 15: results of VM31 through VM39

As can be seen from the table above, the addition of at least colistin to the medium described herein allows the growth of toxigenic O1 (rice leaf type), but interestingly, does not allow the growth of toxigenic O139. Thus, the above-described culture medium can be used for selection between virulent strains of Vibrio cholerae, i.e.between the O1 and O139 serotypes, in particular the O1 rice leaf type.

General method

Medium specificity test-various Medium

1. Each medium was tested in duplicate with each strain listed using the following protocol:

vibrio cholerae toxigenic O1 (two different strains)

Vibrio cholerae toxigenic O139 (two different strains)

Vibrio cholerae (non-O1/non-O139)

Vibrio vulnificus

Vibrio parahaemolyticus

Vibrio mimicus

Vibrio alginolyticus

Vibrio fluvialis (V.fluvial)

2. A suspension of the cell inoculum in peptone (3%) medium was prepared and 100cfu/100 μl inoculum was used per bijou. Each vibrio species was inoculated in duplicate into 2ml aliquots of medium in bijou. A duplicate of 2ml control for each medium was included and "inoculated" with 100. Mu.l of peptone salt broth as a negative control.

3. Mu.l of the cell suspension was plated onto peptone agar (or other suitable agar) plates, in duplicate, and incubated as required for colony counting. At the end of the test cfu, temperature and time of plate incubation were recorded.

4. Following inoculation, bijou is incubated at an appropriate temperature for an appropriate period of time to enable observation of the presence or absence of growth. The time and temperature of incubation bijou, and the presence or absence of growth and any color change, were recorded.

Reference to the literature

1.Vibrio choleae:World Health Organisation；G.B.Nair,p119-142.

2.WHO；Cholera

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

4.Richard A Finkelstein；Chapter 24Cholera,Vibrio cholera O1and O139,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&I 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。

Claims

1. A selective and/or differential growth medium for isolating vibrio cholerae, said medium consisting of:

(e) A basal medium;

(f) A salt;

(g) A nitrogen source;

(h) Dihydrofolate reductase (DHFR) inhibitors and/or polymyxins; and

(j) Gram positive antibacterial agents;

the culture medium consists of 32.54g/L of basic culture medium, 15g/L of salt, 5g/L of nitrogen source, DHFR inhibitor and/or 1-500 mg/L of polymyxin and gram-positive antibacterial agent;

the vibrio cholerae is vibrio cholerae O1;

the basic culture medium is CPC-agar culture medium;

the polymyxin is colistin sodium methane sulfonate;

the DHFR inhibitor is an antibiotic; the antibiotic is trimethoprim;

the gram-positive antibacterial agent is daptomycin;

the content of trimethoprim is 175mg/L; the content of the daptomycin is 1-30 mg/L.

2. The medium of claim 1, wherein the nitrogen source is peptone.

3. The medium of claim 2, wherein the peptone is a bacto peptone.

4. The medium according to claim 1, wherein the Vibrio cholerae O1 is of the Eltolo type or classical type.

5. An agar plate comprising a culture medium as defined in any one of claims 1 to 4.

6. A method for isolating and/or identifying and/or selectively growing vibrio cholerae produced from a sample, the method comprising inoculating the sample into a culture medium as defined in any one of claims 1 to 4;

the sample is ballast water.

7. The method according to claim 6, wherein the method comprises:

d. inoculating a sample into the growth medium;

e. incubating the growth medium to allow growth of Vibrio cholerae; and

f. detecting the existence of the vibrio cholerae.

8. Use of a selective and/or differential growth medium as defined in any one of claims 1 to 4 for isolating and/or identifying and/or selectively growing vibrio cholerae from a sample;

the sample is ballast water.

9. A method of detecting the presence of at least one colony forming unit CFU of vibrio cholerae toxigenic in a sample of ballast water, the method comprising:

a. Obtaining a sample of ballast water;

b. inoculating the sample into a growth medium as defined in any one of claims 1 to 4;

c. incubating the growth medium to allow growth of Vibrio cholerae; and

d. detecting the presence of at least one CFU of Vibrio cholerae.