CN115896231A - Detection and separation method of food-borne pathogenic aeromonas - Google Patents

Abstract

The invention discloses a method for detecting and separating food-borne pathogenic aeromonas strains, which comprises the following steps: comprises two parts of special enrichment liquid and a chromogenic culture medium, wherein the enrichment liquid consists of special peptone, bile salt and water; the chromogenic culture medium consists of peptone, dextrin, maltose, betaine, bile salt, potassium chloride, potassium carbonate, magnesium sulfate, ampicillin, cefazolin, beta-galactoside chromogenic substrate, bromothymol blue, agar, polyethylene glycol and the like. When in use, a sample to be detected is firstly cultured in an enrichment medium, then streaked and inoculated to a chromogenic medium for culture, and the growth condition of characteristic colonies on a flat plate is directly observed, so that the food-borne pathogenic aeromonas strain can be obtained. The enrichment fluid and the chromogenic medium have low cost and simple configuration, and the method can be specifically, simply, conveniently and quickly used for separating and identifying the pathogenic aeromonas and has wide application prospect.

Description

Detection and separation method of food-borne pathogenic aeromonas

Technical Field

The invention relates to a method for detecting and separating pathogenic aeromonas, in particular to a method for identifying and separating food-borne pathogenic aeromonas, belonging to the field of food safety microorganism inspection and monitoring.

Background

The aeromonas is a saprophytic bacterium widely distributed in the natural aquatic environment, and the aeromonas is considered to be only a conditional pathogenic bacterium for human in the past, but a plurality of pathogenic aeromonas are discovered successively since the 80 s in the 20 th century. Case reports and clinical studies confirm that some strains in the genus of aeromonas can cause nosocomial infections such as diarrhea, acute gastroenteritis, food poisoning, wound infection, cellulitis, peritonitis and septicemia of patients with low immunity, and many cases of death caused by acute infection of aeromonas are reported. Aeromonas has become a new group of zoonotic pathogens and is of great interest.

At present, the research on aeromonas in China mainly comprises clinical hospitals, aquaculture and other institutions, and the research on aeromonas in food is relatively less. Although systematic methods for detecting Aeromonas have been established, most of them involve directly culturing and separating suspected strains from disease material or water sample, and then identifying whether the strains belong to the genus Aeromonas through biochemical reaction. Aeromonas has not been included in the conventional food monitoring range, and the relevant standard is limited to the detection of Aeromonas hydrophila and Aeromonas veronii. The isolation Medium used is usually MacconKa agar supplemented with ampicillin or modified on the basis of Xylose Lysine Desoxycholate Medium (XLD). The mecnkia agar and the XLD culture medium are used for separating out suspected strains by utilizing the principle of producing acid by saccharides in a bacterial fermentation culture medium and combining an acid-base indicator, are usually used for separating out enterobacteriaceae bacteria, and are not selective separation culture media aiming at aeromonas. The national standard GBT18652-2002 provides a method for testing pathogenic aeromonas hydrophila, which comprises the steps of carrying out pathogen separation on a polluted disease material by using an RS culture medium, carrying out biochemical index measurement on suspected aeromonas hydrophila, and judging as aeromonas hydrophila if the characteristics of oxidase positive and AHM identification (the top is purple, the bottom is faint yellow, the bacteria grow in a brush shape along a puncture line, namely transport power is positive, the tops of partial strains are black), indole positive, gram negative and sugar fermentation positive. The method is complex to operate and low in efficiency, and no special detection and separation method for food-borne pathogenic aeromonas exists at present.

The chromogenic culture medium method is based on the traditional culture medium, completes culture, separation and identification at one time by a bacterial biochemical identification technology of adding a specific enzyme substrate, is simple and convenient to operate, greatly shortens the detection time, and improves the accuracy by characteristic identification of target bacteria, so that the chromogenic culture medium method has a wide application prospect, and is applied to bacterial detection of salmonella, vibrio, listeria, pathogenic escherichia coli O157 and the like in multiple fields of clinical medicine, food sanitation, environmental protection and the like. At present, no chromogenic medium aiming at the aeromonas exists, so that a method which is efficient, rapid, low in cost and simple and convenient to operate is needed in the field for separating and detecting the aeromonas.

Disclosure of Invention

The invention provides a method for detecting and separating pathogenic aeromonas in a food detection sample, which has the advantages of high selectivity, short detection period, low cost and strong operability and is suitable for detecting and separating pathogenic aeromonas.

The technical scheme adopted by the invention is as follows: a detection and separation method of pathogenic aeromonas comprises a reaction system comprising a special enrichment medium and a chromogenic medium; wherein, every 1000mL enrichment liquid includes: 10 to 20g of peptone, 2 to 5g of bile salt and the balance of water, wherein the pH value is 8.5 +/-0.2; each 1000mL of the chromogenic medium comprises: 10 to 20g of peptone, 5 to 12g of dextrin, 0 to 2g of betaine, 3 to 5g of cholate, 0 to 5g of sodium thiosulfate, 0 to 1g of ammonium citrate, 3 to 5g of potassium chloride, 1 to 2g of potassium carbonate, 0 to 0.5g of magnesium sulfate, 15 to 18g of agar, 0 to 2g of PEG, 0 to 2g of poloxamer, 0.1 to 0.2g of 6-chloro-3-indole-beta-D-galactoside, 0 to 0.1g of bromothymol blue, 0 to 0.1g of thymol blue, 8mg of ampicillin 2, 8mg of ceftizoline 2 to 8mg, and the balance of water, wherein the pH is 7.6 +/-0.2.

In some embodiments, the peptone is sodium chloride-free casein peptone.

In some embodiments, the chromogenic medium the peptones are fish peptones and trypsin .

In some embodiments, preferably, the bile salt is potassium deoxycholate, potassium porcine bile salt or potassium bovine bile salt.

In some embodiments, preferably, the PEG is PEG4000.

In some embodiments, the enrichment fluid preferably comprises, per 1000mL of culture medium: 10g of casein peptone without sodium chloride, 2g of pig gall sylvite and the balance of water, wherein the pH value is 8.5 +/-0.2.

In some embodiments, the chromogenic medium preferably comprises, per 1000mL of medium: 6g of fish peptone, 5363 g of trypsin,g of dextrin, 12g of betaine, 3g of potassium taurocholate, 5g of potassium chloride, 1.5g of potassium carbonate, 0.1g of magnesium sulfate, 15g of agar, 4000 g of PEG, 0.1g of 6-chloro-3-indole-beta-D-galactoside, 0.08g of bromothymol blue, 5mg of ampicillin and 5mg of cefazolin, the balance of water, and the pH value is 7.6 +/-0.2.

In some embodiments, the chromogenic medium preferably comprises, per 1000mL of medium: 5g of fish peptone, 5363 g of trypsin,g of dextrin, 10g of maltose, 3g of swine bile potassium salt, 1.2g of potassium carbonate, 0.1g of magnesium sulfate, 15g of agar, 1g of poloxamer, 0.1g of 6-chloro-3-indole-beta-D-galactoside, 0.04g of bromothymol blue, 0.04g of thymol blue, 5mg of ampicillin and 5mg of cefazolin, the balance of water, and the pH value is 7.6 +/-0.2.

The invention uses casein peptone and potassium bile salt without sodium chloride to prepare the alkaline 0% NaCl enrichment solution, which can inhibit the growth of all gram-positive bacteria and most gram-negative bacteria in the sample, but the growth of aeromonas is not affected. The Bergey's Manual of bacteriological taxonomy clearly indicates that Aeromonas can be divided into Wen Qun and Leng Qun, wen Qun Aeromonas is mostly human pathogenic bacteria, the optimal growth temperature range is 22-37 ℃, leng Qun Aeromonas is mostly aquatic animal pathogenic bacteria, and the optimal growth temperature is 22-25 ℃. Therefore, when cultured at 36 +/-1 ℃, the pathogenic aeromonas can be rapidly propagated, and the Leng Qun aeromonas can be inhibited.

The invention uses fish peptone and trypsinas main nutrient components in the chromogenic culture medium, and the unique carbon-nitrogen ratio and the protein molecular structure can induce aeromonas to produce a large amount of enzyme in a short time.

The antibiotic is added into the chromogenic culture medium, the antibiotic can inhibit the growth of certain bacteria, and the preferred ampicillin and cefazolin are broad-spectrum antibiotics which can inhibit the growth of most bacteria. The pathogenic aeromonas contains metalloenzyme CphA, can generate inducible beta-lactamase and has drug resistance to carbapenem antibiotics.

According to the invention, the beta-galactoside chromogenic substrate 6-chloro-3-indole-beta-D-galactopyranoside is added into the chromogenic culture medium, so that the bacteria capable of expressing and producing beta-galactosidase can decompose the chromogenic substrate under the action of enzyme to form pink colonies. The pathogenic aeromonas does not contain beta-galactosidase, but can ferment dextrin and maltose to produce acid under the condition of the carbon-nitrogen ratio of the culture medium, and a yellow colony is formed under the action of bromothymol blue; other bacteria are inhibited or colorless, white colonies on the medium.

According to the invention, surfactants such as betaine, PEG and poloxamer are added into the culture medium, so that the effects of moisture retention, solubilization, slow release, solid dispersion and the like are achieved, the components of the culture medium are uniformly dispersed, and the shelf life of the plate is prolonged.

The invention relates to a method for detecting and separating pathogenic aeromonas, which comprises the following steps:

1) Preparing enriched liquid: adding the components of the enrichment broth into deionized water, heating, stirring for dissolving, adjusting pH to 8.5 + -0.2 with potassium carbonate, autoclaving at 121 deg.C for 15min, and packaging.

2) Preparation of color development plate: adding the components of the chromogenic culture medium except the antibiotics into deionized water, stirring for dissolving, adjusting the pH to 7.6 +/-0.2, heating for boiling, cooling to 45-55 ℃, adding the antibiotics, uniformly mixing, and pouring into a flat plate for later use.

3) Inoculating and culturing: 1.

4) And (4) analyzing results: yellow colonies appeared to be pathogenic aeromonas.

The invention has the beneficial effects that:

the method for detecting and separating the pathogenic aeromonas has the advantages of strong specificity, high sensitivity, easy operation, simple result judgment and the like, is suitable for quick screening and directional monitoring of the pathogenic aeromonas by medical health institutions, aquaculture institutions and food health monitoring basic units, and has wide application prospect.

Detailed Description

The following examples will allow one skilled in the art to more fully understand the present invention, but do not limit the invention in any way.

Example 1: the chromogenic medium of the invention is used for the specificity experiment of pathogenic aeromonas

1) Preparing a solid plate:

according to the formula, each 1000mL of culture medium contains 6g of fish peptone, 5363 g of trypsin,g of dextrin, 1g of betaine, 3g of swine bile potassium salt, 5g of potassium chloride, 1.2g of potassium carbonate, 0.1g of magnesium sulfate, 15g of agar, 4000 g of PEG, 0.1g of 6-chloro-3-indole-beta-D-galactoside, 0.08g of bromothymol blue, 5mg of ampicillin and 5mg of cefazolin, the balance of water, and the pH value is 7.6 +/-0.2. Adding the components of the chromogenic medium except the antibiotics into deionized water, stirring for dissolving, and adjusting the pH to 7.6 +/-0.2. Heating and boiling, cooling to 45-55 ℃, adding corresponding antibiotics, performing aseptic operation, and pouring the plate for later use.

2) Strain activation and culture:

the test bacteria include Aeromonas hydrophila ATCC7966, aeromonas caviae ATCC15468, aeromonas veronii temperate biotype ATCC9071, aeromonas veronii Vickers biotype ATCC35624, aeromonas salmonicida ATCC7965, aeromonas intermedia ATCC33907, aeromonas simplicissima ATCC49568, aeromonas spa ATCC23309, escherichia coli ATCC BAA-2452, staphylococcus aureus ATCC25923 and Pseudomonas aeruginosa ATCC9027. The ATCC is a shorthand for the American Type Culture Collection. Each strain is verified by biochemical rechecking before the test.

Respectively activating the strains, inoculating the activated strains to a Columbia blood agar plate, and culturing at a corresponding optimal temperature for 18 to 24h until obvious colonies appear. 3~5 colonies were picked and dispersed in TSB liquid medium to prepare a bacterial suspension. The turbidity of the bacterial suspension was adjusted to 0.5 McLeod standard, at which the concentration of the bacterial suspension was 1.5X 10 ⁸ CFU/mL, spare.

3) Inoculation: and (3) respectively taking a ring of bacterial suspension prepared from the 10 strains, streaking and inoculating the bacterial suspension on two prepared chromogenic plates, culturing at 30 +/-1 ℃ for 18 to 24h, and observing the chromogenic condition of bacterial colonies.

4) And (4) analyzing results: the growth of the aeromonas on the color development plate is good, the color development is carried out within 18h, the bacterial colony is yellow, the diameter of the bacterial colony is 1 to 2mm, the edge is semi-transparent, and non-target bacteria are inhibited or are bacterial colonies with different colors; the detailed results are shown in Table 1.

Table 1: experimental results of example 1

The experimental result shows that the method can well distinguish the aeromonas from other non-target bacteria.

Example 2: growth characteristic test experiment of food-borne pathogenic aeromonas chromogenic medium

1) Preparing a solid plate:

according to the formula, each 1000mL of culture medium comprises 5g of fish peptone,g of trypsin, 10g of dextrin, 2g of maltose, 3g of swine potassium salt, 1.2g of potassium carbonate, 0.1g of magnesium sulfate, 15g of agar, 1g of poloxamer, 0.1g of 6-chloro-3-indole-beta-D-galactoside, 0.04g of bromothymol blue, 0.04g of thymol blue, 5mg of ampicillin and 5mg of cefazolin, the balance of water, and the pH is 7.6 +/-0.2. Adding the components of the chromogenic medium except the antibiotics into deionized water, stirring for dissolving, and adjusting the pH to 7.6 +/-0.2. Heating and boiling, cooling to 45-55 ℃, adding corresponding antibiotics, performing aseptic operation, and pouring the plate for later use.

2) Strain activation and culture:

the test bacteria include Aeromonas hydrophila ATCC7966, aeromonas caviae ATCC15468, aeromonas veronii temperate biotype ATCC9071, aeromonas veronii Vickers biotype ATCC35624, aeromonas salmonicida ATCC7965, aeromonas intermedia ATCC33907, aeromonas simples ATCC49568, aeromonas spa ATCC23309, escherichia coli ATCC BAA-2452, staphylococcus aureus ATCC25923, pseudomonas aeruginosa ATCC9027.ATCC is a shorthand for American Type Culture Collection. Each strain is verified by biochemical rechecking and drug sensitivity test before test. The strains are activated according to the steps of GB4789.28-2013, and bacterial suspensions which are diluted by 10 times in series are prepared for standby.

3) Inoculation:

a working bacterial suspension of appropriate dilution, 0.1mL, is selected and evenly spread and inoculated on the chromogenic medium and the reference plate TSA. Two plates were inoculated at each dilution. The inoculation level of each plate is 20-200 CFU, the plate is cultured for 18 to 24h at the temperature of 30 +/-1 ℃, and the diameter of a colony is recorded and the recovery rate is calculated.

4) And (4) analyzing results:

the aeromonas colonies are yellow, the diameter of the colonies is 3 to 4mm, and non-target bacteria are inhibited or the colonies are different in color. The colony diameter d (mm) and recovery (PR) results are shown in Table 2.

Table 2: experimental results of example 2

As can be seen from Table 2, the aeromonas chromogenic medium can effectively separate pathogenic aeromonas, and the recovery rate (PR) of the target bacterium is far higher than the specification that PR is more than or equal to 0.5 in GB 4789.28-2013.

Example 3: the invention relates to the separation and detection of pathogenic aeromonas in food samples

1) Preparing a related culture medium:

preparing a solid plate: performing sterile operation according to example 1 or example 2, and pouring the plate for later use;

preparing enriched liquid: every 1000mL of medium included: heating, stirring and dissolving casein peptone 10g without sodium chloride, fel Sus Domestica potassium salt 2g, and water in balance, adjusting pH to 8.5 + -0.2 with potassium carbonate, autoclaving at 121 deg.C for 15min, and packaging.

2) Preparing an artificial pollution sample: samples (commercial pork filling) were measured according to 10 ² CFU/25g of inoculation level is respectively inoculated with aeromonas hydrophila ATCC7966, aeromonas caviae ATCC15468, aeromonas veronii temperate biotype ATCC9071 and aeromonas veronii vickers biotype ATCC35624, and the numbers of sample 1, sample 2, sample 3 and sample 4 are respectively numbered; the no additive sample was numbered as sample 5.

3) Sample treatment: adding 5 samples into prepared 225ml enrichment medium, homogenizing and mixing uniformly, and carrying out enrichment culture at 36 +/-1 ℃ for 12 to 18h.

4) Inoculating and culturing: respectively inoculating the enrichment medium on a chromogenic medium, culturing at 30 +/-1 ℃ for 18 to 24h, and observing the phenomenon.

5) And (4) analyzing results: example 1 and example 2 both media were applied to the isolation and detection of food-borne pathogenic aeromonas. Pathogenic aeromonas thrives on the culture medium, the colony is yellow, and transparent fungus rings exist around the colony. Example 2 the formula is re-optimized, maltose is added as a carbon source, the color development time of example 2 is earlier than that of example 1, the colony diameter is larger, and a user can select a culture medium with the corresponding formula according to specific requirements. The detailed results are shown in Table 3.

Table 3: experimental results of example 3

While particular embodiments of the present invention have been described, those skilled in the art will recognize that many changes and modifications may be made thereto without departing from the scope or spirit of the invention. Accordingly, it is intended to embrace all such changes and modifications that fall within the scope of the appended claims and equivalents thereof.

Claims (6)

1. A method for detecting and separating food-borne pathogenic aeromonas is characterized in that a reaction system comprises a special enrichment medium and a chromogenic medium; wherein, the enrichment fluid includes: 10-20g/L peptone, 2-5g/L bile salt and the balance of water, wherein the pH value is 7.2 +/-0.2; the chromogenic medium comprises: 10-20g/L peptone, 5-12g/L dextrin, 0-2g/L maltose, 0-2g/L betaine, 3-5g/L cholate, 3-5g/L potassium chloride, 1-2g/L potassium carbonate, 0-0.5 g/L magnesium sulfate, 5-10mg/L ampicillin, 5-10mg/L ceftazolin, 2-5g/L developer and substrate dispersant, 15-18g/L agar, and 7.6 +/-0.2 pH (potential of Hydrogen) as the balance.

2. The special enrichment broth for the isolation and detection of food-borne pathogenic aeromonas according to claim 1, wherein the peptone is casein peptone without sodium chloride; the bile salt is potassium bile salt, potassium deoxycholate, pig bile salt, and ox bile salt, and can be single or combined.

3. The chromogenic medium for the isolation and detection of food-borne pathogenic aeromonas according to claim 1, wherein said peptone is fish peptone, trypsin , alone or in combination; the bile salt is potassium bile salt, potassium deoxycholate, pig bile salt, and ox bile salt, and can be single or combined.

4. The chromogenic medium for isolating and detecting food-borne pathogenic aeromonas according to claim 1, wherein the chromogenic agent is a bacterial specific enzyme reaction substrate, an acid-base indicator, alone or in combination; the content of 6-chloro-3-indole-beta-D-galactoside in a bacterial specific enzyme reaction substrate is 0 to 0.2g/L; and the acid-base indicator contains 0-0.1g/L of bromothymol blue and 0-0.1g/L of thymol blue.

5. The chromogenic medium for isolating and detecting food-borne pathogenic aeromonas according to claim 1, wherein the substrate dispersant is polyethylene glycol (PEG) and poloxamer alone or in combination, the PEG content is 0 to 2g/L, and the poloxamer content is 0 to 2g/L.

6. The method for detecting and isolating food-borne pathogenic aeromonas sp according to claim 1, wherein the result analysis directly observes characteristic colony growth including the presence of colonies and the presence of colonies with established color.