A method for detecting and isolating foodborne pathogenic Aeromonas

Abstract

The present invention discloses a method for detecting and separating foodborne pathogenic Aeromonas strains: comprising a special enrichment solution and a chromogenic culture medium, wherein the enrichment solution is composed of special peptone, bile salts and water; the chromogenic culture medium is composed of peptone, dextrin, maltose, betaine, bile salts, potassium chloride, potassium carbonate, magnesium sulfate, ampicillin, cefazolin, β-galactoside chromogenic substrate, bromothymol blue, agar, polyethylene glycol, etc. When used, the sample to be tested is first cultured in the enrichment solution, then streaked and inoculated on the chromogenic culture medium for culture, and the growth of characteristic colonies on the plate is directly observed to obtain the foodborne pathogenic Aeromonas strains. The enrichment solution and chromogenic culture medium described in the present invention are low in cost and simple in configuration, and the method can be specifically, simply and quickly used for the separation and identification of pathogenic Aeromonas, and has broad application prospects.

Description

Technical Field

[0001] The invention relates to a method for detecting and separating pathogenic Aeromonas, and in particular to the identification and separation of foodborne pathogenic Aeromonas, and belongs to the field of food safety microbial testing and monitoring. Background Art

[0002] Aeromonas bacteria are saprophytic bacteria widely distributed in natural aquatic environments. In the past, Aeromonas were considered to be only conditional pathogens to humans, but since the 1980s, a variety of pathogenic Aeromonas have been discovered. Case reports and clinical studies have confirmed that some species of Aeromonas can cause diarrhea, acute gastroenteritis, food poisoning, wound infection, cellulitis, and nosocomial infections such as peritonitis and sepsis in immunocompromised patients. There have also been several reports of deaths from acute Aeromonas infection. Aeromonas has become a new type of zoonotic pathogen and has attracted much attention.

[0003] At present, the research on Aeromonas in China is mainly carried out in clinical hospitals and aquaculture institutions, while there are relatively few studies on Aeromonas in food. Although a relatively systematic method for testing Aeromonas has been established, most of them are to directly culture and isolate suspected strains from diseased materials or water samples, and then identify whether they are Aeromonas through biochemical reactions. Aeromonas has not yet been included in the routine monitoring scope of food, and the relevant standards are limited to the detection of Aeromonas hydrophila and Aeromonas vernix. The separation medium used is usually MacConkey agar with ampicillin added or modified based on Xylose Lysine Desoxycholate Medium (XLD). MacConkey agar and XLD medium use the principle of acid production of sugars in bacterial fermentation medium, combined with acid-base indicators to isolate suspected strains. They are usually used to separate Enterobacteriaceae bacteria, and are not selective separation media for Aeromonas. The national standard GBT18652-2002 stipulates the inspection method for pathogenic Aeromonas hydrophila. RS culture medium is used to isolate the pathogen from the contaminated material, and the biochemical indexes of suspected Aeromonas hydrophila are determined. If the oxidase is positive, the AHM identification characteristics (the top is still purple, the bottom is light yellow; the bacteria grow in a brush-like manner along the puncture line, that is, the motility is positive; some strains are black on the top), indole is positive, Gram is negative, and sugar fermentation is positive, it is determined to be Aeromonas hydrophila. The above method is cumbersome and inefficient. At present, there is no special detection and separation method for foodborne pathogenic Aeromonas.

[0004] The colorimetric culture method is based on traditional culture medium. Through the bacterial biochemical identification technology of adding specific enzyme substrates, the culture, separation and identification are completed at one time. It is easy to operate, greatly shortens the detection time, and improves the accuracy through the characteristic identification of target bacteria. Therefore, it has broad application prospects and has been applied to the detection of bacteria such as Salmonella, Vibrio, Listeria, and pathogenic Escherichia coli O157 in many fields such as clinical medicine, food hygiene, and environmental protection. At present, there is no colorimetric culture medium for Aeromonas. Therefore, the field needs an efficient, rapid, low-cost, and easy-to-operate method for the separation and detection of Aeromonas. Summary of the invention

[0005] The present invention uses the principles of biochemistry, microbiology, inorganic, organic and analytical chemistry to screen and optimize different carbon sources, nitrogen sources, inorganic salts, vitamins, pH values ​​and other factors, and provides a method for detecting and separating pathogenic Aeromonas in food testing samples with high selectivity, short detection cycle, low cost and strong operability.

[0006] The technical solution adopted by the present invention is: a method for detecting and isolating pathogenic Aeromonas, wherein the reaction system comprises a special enrichment solution and a chromogenic culture medium; wherein each 1000 mL of the enrichment solution comprises: 10-20 g of peptone, 2-5 g of bile salt, the remainder being water, and the pH being 8.5±0.2; each 1000 mL of the chromogenic culture medium comprises: 10-20 g of peptone, 5-12 g of dextrin, 0-2 g of betaine, 3-5 g of bile salt, 0-5 g of sodium thiosulfate, 0-1 g of ammonium ferric citrate, 3-5 g of potassium chloride, 1-2 g of potassium carbonate, 0-0.5 g of magnesium sulfate, 15-18 g of agar, PEG 0~2g, poloxamer 0~2g, 6-chloro-3-indole-β-D-galactoside 0.1~0.2g, bromothymol blue 0~0.1g, thymol blue 0~0.1g, ampicillin 2~8mg, cefazolin 2~8mg, the balance is water, pH is 7.6±0.2.

[0007] In some embodiments, the peptone in the enrichment solution is casein peptone without sodium chloride.

[0008] In some embodiments, the peptone in the chromogenic medium is fish peptone and trypsin.

[0009] In some embodiments, preferably, the bile salt is potassium deoxycholate, porcine bile potassium salt or ox bile potassium salt.

[0010] In some embodiments, preferably, the PEG is PEG4000.

[0011] In some embodiments, the enrichment solution preferably comprises, per 1000 mL of culture medium: 10 g of casein peptone without sodium chloride, 2 g of pig bile potassium salt, the remainder being water, and the pH is 8.5±0.2.

[0012] In some embodiments, the color development culture medium is preferably a culture medium comprising, per 1000 mL, 6 g fish peptone, 3 g trypsin, 12 g dextrin, 1 g betaine, 3 g ox bile potassium salt, 5 g potassium chloride, 1.5 g potassium carbonate, 0.1 g magnesium sulfate, 15 g agar, 1 g PEG4000, 0.1 g 6-chloro-3-indole-β-D-galactoside, 0.08 g bromothymol blue, 5 mg ampicillin, 5 mg cefazolin, the balance being water, and a pH of 7.6±0.2.

[0013] In some embodiments, the color development culture medium is preferably a culture medium comprising, per 1000 mL, 5 g fish peptone, 5 g trypsin, 10 g dextrin, 2 g maltose, 3 g pig bile potassium salt, 1.2 g potassium carbonate, 0.1 g magnesium sulfate, 15 g agar, 1 g poloxamer, 0.1 g 6-chloro-3-indole-β-D-galactoside, 0.04 g bromothymol blue, 0.04 g thymol blue, 5 mg ampicillin, 5 mg cefazolin, the balance is water, and the pH is 7.6±0.2.

[0014] The present invention uses casein peptone and potassium bile salt without sodium chloride to prepare an 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 Bacterial Systematic Taxonomy clearly points out that Aeromonas can be divided into mesophilic groups and psychrophilic groups. Mesophilic Aeromonas are mostly human pathogenic pathogens, and the optimal growth temperature range is 22°C~37°C. Psychrophilic Aeromonas are mostly aquatic animal pathogens, and the optimal growth temperature is 22°C~25°C. Therefore, when cultured at 36°C±1°C, pathogenic Aeromonas can multiply rapidly, while psychrophilic Aeromonas are inhibited.

[0015] The invention uses fish peptone and trypsin as main nutrient components in the color development medium, and the unique carbon-nitrogen ratio and protein molecular structure thereof can induce Aeromonas to produce a large amount of enzyme in a short time.

[0016] The present invention adds antibiotics to the color development medium, and the antibiotics can inhibit the growth of certain bacteria. The preferred ampicillin and cefazolin are both broad-spectrum antibiotics that can inhibit the growth of most bacteria. The pathogenic Aeromonas contains the metalloenzyme CphA, which can produce inducible β-lactamase and is resistant to carbapenem antibiotics.

[0017] The present invention adds a β-galactoside chromogenic substrate 6-chloro-3-indole-β-D-pyranogalactoside to the chromogenic culture medium, and bacteria that can express and produce β-galactosidase decompose the chromogenic substrate under the action of the enzyme to form pink colonies. Pathogenic Aeromonas has no β-galactosidase, but under the carbon-nitrogen ratio conditions of this culture medium, it can ferment dextrin and maltose to produce acid and form yellow colonies under the action of bromothymol blue; other bacteria are inhibited on the culture medium or are colorless or white colonies.

[0018] The present invention adds surfactants such as betaine, PEG, poloxamer and the like to the culture medium to play the roles of moisturizing, solubilizing, sustained release, solid dispersion and the like, so that each component of the culture medium is evenly dispersed and the shelf life of the plate is increased.

[0019] The method for detecting and isolating pathogenic Aeromonas described in the present invention comprises the following steps:

[0020] 1) Preparation of enrichment solution: Add the above enrichment solution components into deionized water, heat and stir to dissolve, adjust the pH to 8.5±0.2 with potassium carbonate, sterilize by high pressure at 121°C for 15 min, and divide into portions for use.

[0021] 2) Preparation of color development plate: Add all components of the above color development medium except antibiotics into deionized water, stir to dissolve, adjust the pH to 7.6±0.2, heat to boil, cool to 45℃~55℃, add antibiotics, mix well, pour into plates and set aside.

[0022] 3) Inoculation and culture: prepare a 1:10 sample solution and culture it at 36℃±1℃ for 12h~18h, then inoculate it onto a colorimetric plate and culture it at 30℃±1℃ for 18h~24h.

[0023] 4) Result analysis: Yellow colonies are pathogenic Aeromonas.

[0024] The beneficial effects of the present invention are:

[0025] The method for detecting and isolating pathogenic Aeromonas described in the present invention has the advantages of strong specificity, high sensitivity, easy operation, simple result judgment, etc. It is suitable for rapid screening and targeted monitoring of pathogenic Aeromonas in medical and health institutions, aquaculture institutions, and grassroots food hygiene monitoring units, and has broad application prospects. DETAILED DESCRIPTION

[0026] The following examples may enable those skilled in the art to more fully understand the present invention, but are not intended to limit the present invention in any way.

[0027] Example 1: Specificity test of the chromogenic culture medium of the present invention for pathogenic Aeromonas

[0028] 1) Solid plate preparation:

[0029] According to the formula, each 1000mL of the culture medium contains 6g of fish peptone, 3g of trypsin, 12g of dextrin, 1g of betaine, 3g of pig bile potassium salt, 5g of potassium chloride, 1.2g of potassium carbonate, 0.1g of magnesium sulfate, 15g of agar, 1g of PEG4000, 0.1g of 6-chloro-3-indole-β-D-galactoside, 0.08g of bromothymol blue, 5mg of ampicillin, and 5mg of cefazolin, and the balance is water, and the pH is 7.6±0.2 to prepare the culture medium. Add the components of the above-mentioned color development medium except the antibiotics to deionized water, stir to dissolve, and adjust the pH to 7.6±0.2. Heat to boil, wait until it cools to 45~55℃, add the corresponding antibiotics, operate aseptically, pour the plate, and set aside.

[0030] 2) Activation and cultivation of bacteria:

[0031] The tested bacteria include Aeromonas hydrophila ATCC7966, Aeromonas caviae ATCC15468, Aeromonas sobriae ATCC9071, Aeromonas veronii biotype ATCC35624, Aeromonas salmonicida ATCC7965, Aeromonas intermedia ATCC33907, Aeromonas jane ATCC49568, Aeromonas fountainii ATCC23309, Escherichia coli ATCC BAA-2452, Staphylococcus aureus ATCC25923, and Pseudomonas aeruginosa ATCC9027. ATCC is the abbreviation of American Type Culture Collection. Each strain of bacteria was biochemically verified before the test.

[0032] The above strains were activated and inoculated on Columbia blood agar plates. They were cultured at the corresponding optimal temperature for 18-24 hours until obvious colonies appeared. Three to five colonies were picked and dispersed in TSB liquid culture medium to prepare bacterial suspension. The turbidity of the bacterial suspension was adjusted to 0.5 McFarland turbidimetric standard. At this time, the concentration of the bacterial suspension was 1.5×10 8 CFU / mL, for future use.

[0033] 3) Inoculation: Take a loopful of the bacterial suspension prepared from the above 10 strains and streak it onto the two prepared colorimetric plates. Incubate at 30±1℃ for 18~24h and observe the color development of the colonies.

[0034] 4) Result analysis: Aeromonas grew well on the chromogenic plate, color developed within 18 hours, the colonies were yellow, the colony diameter was 1-2 mm, the edges were translucent, and non-target bacteria were inhibited or appeared as colonies of different colors; detailed results are shown in Table 1.

[0035] Table 1: Experimental results of Example 1

[0036]

[0037] The experimental results show that this method can be used to distinguish Aeromonas from other non-target bacteria.

[0038] Example 2: Growth characteristics test experiment of the foodborne pathogenic Aeromonas chromogenic culture medium of the present invention

[0039] 1) Solid plate preparation:

[0040] According to the formula, each 1000mL culture medium contains 5g of fish peptone, 5g of trypsin, 10g of dextrin, 2g of maltose, 3g of pig 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-β-D-galactoside, 0.04g of bromothymol blue, 0.04g of thymol blue, 5mg of ampicillin, and 5mg of cefazolin, and the balance is water, and the pH is 7.6±0.2 to prepare the culture medium. Add the components of the above-mentioned color development medium except the antibiotics to deionized water, stir to dissolve, and adjust the pH to 7.6±0.2. Heat to boil, wait until it cools to 45~55°C, add the corresponding antibiotics, operate aseptically, pour the plate, and set aside.

[0041] 2) Activation and cultivation of bacteria:

[0042] The tested bacteria include Aeromonas hydrophila ATCC7966, Aeromonas caviae ATCC15468, Aeromonas sobriae ATCC9071, Aeromonas veronii biotype ATCC35624, Aeromonas salmonicida ATCC7965, Aeromonas intermedia ATCC33907, Aeromonas jane ATCC49568, Aeromonas fountainii ATCC23309, Escherichia coli ATCC BAA-2452, Staphylococcus aureus ATCC25923, and Pseudomonas aeruginosa ATCC9027. ATCC is the abbreviation of American Type Culture Collection. Each strain of bacteria was verified by biochemical verification and drug sensitivity test before the test. The strain was activated according to the steps of GB4789.28-2013, and a 10-fold serial dilution of the bacterial suspension was prepared for standby use.

[0043] 3) Vaccination:

[0044] Select 0.1 mL of working bacterial suspension of appropriate dilution and evenly spread it on the chromogenic medium and reference plate TSA. Inoculate two plates for each dilution. The inoculation level of each plate is 20-200 CFU, culture at 30±1℃ for 18-24h, record the colony diameter and calculate the recovery rate.

[0045] 4) Result analysis:

[0046] Aeromonas colonies are yellow with a colony diameter of 3-4 mm, and non-target bacteria are inhibited or have colonies of different colors. The results of colony diameter d (mm) and recovery rate (PR) are shown in Table 2.

[0047] Table 2: Experimental results of Example 2

[0048]

[0049] As shown in Table 2, the Aeromonas chromogenic medium can effectively separate pathogenic Aeromonas, and the recovery rate (PR) of the target bacteria is much higher than the requirement of PR ≥ 0.5 in GB4789.28-2013.

[0050] Example 3: Isolation and detection of pathogenic Aeromonas in food samples according to the present invention

[0051] 1) Preparation of relevant culture medium:

[0052] Solid plate preparation: refer to Example 1 or Example 2 for aseptic operation, pour the plate and set aside;

[0053] Preparation of enrichment solution: Each 1000 mL of culture medium includes: 10 g of casein peptone without sodium chloride, 2 g of pig bile potassium salt, and the balance is water. Heat and stir to dissolve. Use potassium carbonate to adjust the pH to 8.5±0.2, sterilize at 121°C for 15 min, and divide into portions for use.

[0054] 2) Preparation of artificially contaminated samples: Samples (commercially available pork filling) were 2 The inoculation levels of CFU / 25g were respectively inoculated with Aeromonas hydrophila ATCC7966, Aeromonas caviae ATCC15468, Aeromonas sobriae ATCC9071, and Aeromonas veronii biotype veronii ATCC35624, which were numbered as sample 1, sample 2, sample 3, and sample 4, respectively; the sample without addition was numbered as sample 5.

[0055] 3) Sample processing: Add 5 samples into 225 ml of the prepared enrichment solution, mix evenly, and then incubate at 36℃±1℃ for 12~18h.

[0056] 4) Inoculation and culture: Inoculate the enriched culture medium onto the colorimetric culture medium, culture at 30℃±1℃ for 18~24h, and observe the phenomenon.

[0057] 5) Result analysis: The culture media of Example 1 and Example 2 can both be used to isolate and detect foodborne pathogenic Aeromonas. Pathogenic Aeromonas grows vigorously on the culture medium, and the colonies are yellow with transparent bacterial rings around them. Example 2 re-optimizes the formula and adds maltose as a carbon source. The color development time of Example 2 is earlier than that of Example 1, and the colony diameter is larger. Users can choose the corresponding formula culture medium according to specific needs. Detailed results are shown in Table 3.

[0058] Table 3: Experimental results of Example 3

[0059]

[0060] Although specific embodiments of the present invention have been described, it will be appreciated by those skilled in the art that various changes and modifications may be made to the present invention without departing from the scope or spirit of the present invention. Therefore, the present invention is intended to cover all such changes and modifications that fall within the scope of the claims and their equivalents.

Claims

1. A method for detecting and isolating foodborne pathogenic Aeromonas for non-disease diagnosis purposes, characterized in that: The following steps are involved: (1) Preparation of enrichment solution: add 10-20 g of peptone and 2-5 g of bile salt to 1 L of deionized water, heat and stir to dissolve, adjust the pH to 8.5±0.2 with potassium carbonate, sterilize by high pressure at 121°C for 15 min, and divide for use; wherein the peptone is casein peptone without sodium chloride; and the bile salt is potassium bile salt; (2) Preparation of color plate: add 10-20 g of peptone, 5-12 g of dextrin, 0-2 g of maltose, 0-2 g of betaine, 3-5 g of bile salt, 3-5 g of potassium chloride, 1-2 g of potassium carbonate, 0.1-0.5 g of magnesium sulfate, 2-5 g of color developer and substrate dispersant, and 15-18 g of agar to 1 L of deionized water, stir to dissolve, adjust the pH to 7.6±0.2, heat to boil, cool to 45°C~55°C, add 5-10 mg of ampicillin and 5-10 mg of cefazolin, mix well, pour on a plate, and set aside; wherein the peptone is a combination of fish peptone and trypsin; the bile salt is potassium bile salt; the color developer is a bacteria-specific enzyme reaction substrate and an acid-base indicator The bacterial specific enzyme reaction substrate is 6-chloro-3-indole-β-D-galactoside, and the content is 0.1-0.2 g / L; the acid-base indicator is a combination of bromothymol blue and thymol blue, the content of bromothymol blue is 0.04-0.1 g / L, and the content of thymol blue is 0.04-0.1 g / L; the substrate dispersant is a single or combined component of polyethylene glycol and poloxamer, the content of polyethylene glycol is 0-2 g / L, and the content of poloxamer is 0-2 g / L; (3) Inoculation and culture: Take 25g of the sample to be tested and add it to 225ml of the prepared enrichment solution. After homogenization, incubate the enrichment solution at 36℃±1℃ for 12~18h. Inoculate the enrichment solution onto the colorimetric plate and incubate it at 30℃±1℃ for 18h~24h. (4) Result analysis: Directly observe the characteristic colonies. Yellow colonies are pathogenic Aeromonas.

2. The method for detecting and isolating foodborne pathogenic Aeromonas for non-disease diagnosis purposes as claimed in claim 1, characterized in that: In step (4) result analysis, directly observe the growth of characteristic colonies, including whether colonies appear and whether the colonies that appear have a predetermined color.

Images