WO2022141941A1 - Cronobacter standard strains containing specific molecular target, and detection and use thereof - Google Patents

Abstract

Provided are four Cronobacter standard strains, with deposit numbers of GDMCC60861, GDMCC60862, GDMCC60863 and GDMCC60864, respectively. The standard strains contain corresponding specific molecular targets (i.e., nucleotide sequences as shown in SEQ ID NOs: 1- 4). Also provided are primers for detecting the specific molecular targets. Further provided in the present invention is a cryoprotectant for Cronobacter strains.

Description

Standard strain of Cronobacter with specific molecular target and its detection and application technical field

The invention belongs to the technical field of bioengineering, in particular to the detection of molecular targets of Cronobacter and standard bacteria thereof.

Background technique

Cronobacter spp. (Enterobacter sakazakii) is a facultative anaerobic, gram-negative, non-spore-forming bacillus, an important food-borne pathogenic bacterium that can infect people of all ages , especially the immunocompromised elderly and newborn infants. Infection of infants and young children can cause serious diseases such as necrotizing enterocolitis, bacteremia, and meningitis, and the fatality rate is as high as 40% to 80%. There are 7 species of this genus and are widely present in the environment. The contamination rate in various foods is also high, especially in vegetables and cooked food. In recent years, reports of the detection of Cronobacter in infant food have often caused serious losses to dairy product manufacturers. Understanding the transmission rules and pathogenic evolution of this bacterium is the basis for effective prevention and control of food-borne diseases caused by this bacterium, and whether the appropriate standard strain is used determines the reliability of the research results.

At present, most of the standard strains used to study the evolutionary law and pathogenicity of the bacteria are from foreign clinical sources, which cannot well reflect the spread of the bacteria in food. China is an important food production and consumption country in the world. , the detection rate of Cronobacter has been at a high level, and the misjudgment rate of the bacteria is high, and there is still a lack of representative standard strains to study the genetic structure and transmission laws of the bacteria in China.

SUMMARY OF THE INVENTION

[Corrected 12.06.2021 according to Rule 26]
In order to solve the above-mentioned technical problems, the present invention provides 4 food isolates of Rhonobacter in China, the bacterial strains have typical physiological and biochemical characteristics of Rhonobacter, and can better reflect the genetic background of the bacteria in China, and can Make up for the vacancy in the availability of standard strains of non-foodborne Rhonobacter in China. The strain cro509C1 was isolated from the Haikou cold salad sample, the preservation number is: GDMCC 60861, and the classification name is: Cronobacter dublinensis; the strain cro611A3 is isolated from the Beihai hand-shredded chicken sample, and its preservation number is: GDMCC 60862, and the classification name is: Cronobacter sakazakii; Strain cro910B3 was isolated from Nanchang cold-mixed noodle sample, and its preservation number is: GDMCC 60863, and its classification name is: Cronobacter dublinensis; strain cro1537W is isolated from a pork head meat sample in Guangzhou, and its preservation number is: GDMCC 60864, and its classification name is: Cronobacter sakazakii . The rhonobacteria isolated above are all preserved in the Guangdong Provincial Microbial Culture Collection Center, address: Building 59, Yard, No. 100, Xianlie Middle Road, Guangzhou City, Guangdong Province, China, zip code: 510075, preservation date: October 27, 2019.

The technical scheme adopted in the present invention is:

The present invention provides specific molecular targets for detecting and identifying the four Cronobacter strains of the present invention, wherein the molecular targets are the nucleotide sequences shown in SEQ ID NOs: 1-4. The newly obtained Cronobacter in the present invention obtains the specific molecular target of the present invention through pan-genome analysis and comparison, and can specifically detect and identify 4 strains of Cronobacter in the present invention, which has strong specificity .

The present invention provides a specific molecular target for detecting Cronobacter, the molecular target is:

(a) any one or several nucleotide sequences shown in SEQ ID NOs: 1 to 4; or,

(b) The nucleotide sequence in (a) is substituted, deleted or added by one or several nucleotides and has more than 90% homology with the nucleotide in (a).

Preferably, PCR primers for amplification of the nucleotide sequence shown in SEQ ID NO: 1 include: upstream primers as shown in SEQ ID NO: 5 and downstream primers as shown in SEQ ID NO: 6; The PCR primers for the amplification of the nucleotide sequence shown in SEQ ID NO:2 include: the upstream primer shown in SEQ ID NO:7 and the downstream primer shown in SEQ ID NO:8; for SEQ ID NO:3 PCR primers for amplification of the nucleotide sequences shown include the upstream primer shown in SEQ ID NO:9 and the downstream primer shown in SEQ ID NO:10; for the nucleotide shown in SEQ ID NO:4 PCR primers for sequence amplification include an upstream primer set forth in SEQ ID NO:11 and a downstream primer set forth in SEQ ID NO:12.

The present invention also provides a group of Cronobacter spp., which are (a), (b), (c) or (d):

(a) strain cro509C1, containing the nucleotide sequence shown in SEQ ID NO: 1;

(b) strain cro611A3, containing the nucleotide sequence shown in SEQ ID NO:2;

(c) strain cro910B3, containing the nucleotide sequence as shown in SEQ ID NO:3;

(d) strain cro1537W, containing the nucleotide sequence as shown in SEQ ID NO:4;

Preferably, the strain cro509C1 further comprises a cefotaxime resistance gene.

Preferably, the strain cro910B3 further comprises a cefotaxime resistance gene.

Preferably, the strain cro1537W further comprises a cefotaxime resistance gene.

Preferably, the deposit number of the strain cro509C1 is: GDMCC 60861; the deposit number of the strain cro611A3 is GDMCC 60862; the deposit number of the strain cro910B3 is GDMCC 60863; the deposit number of the strain cro1537W is GDMCC 60864.

The invention also provides the application of the Cronobacter in the antibiotic resistance of the Cronobacter. Preferably, the antibiotic resistance of the strain cro509C1 is resistance to cefotaxime; the antibiotic resistance of the strain cro910B3 is resistance to cefotaxime; the antibiotic resistance of the strain cro1537W is resistance to cefotaxime Resistance to cefotaxime.

The invention also provides the application of the described Cronobacter in improving the accuracy of testing the color plate of Cronobacter.

The invention also provides a cryoprotectant for Cronobacter, comprising the following components in parts by weight: 0.1-3 parts of polydextrose, 5-10 parts of skim milk powder, 0.1-3 parts of sodium glycerophosphate, L-cysteine 0.2-4 parts of amino acid hydrochloride, 0.5-3 parts of phytic acid.

The reason why the above-mentioned components are selected in the present invention is that polydextrose and sodium glycerophosphate have strong hydrophilicity. The group forms hydrogen bonds to protect the integrity of cell membrane and protein structure and function. Skim milk powder can be wrapped in the outer layer of bacterial cells to protect the bacteria. L-cysteine hydrochloride and phytate are used as antioxidants. Reduce the activity of cellular oxidase during the process and long-term storage, and prevent the oxidative deterioration of lyophilized products.

Preferably, the following components are included in parts by weight: 2 parts of polydextrose, 8 parts of skim milk powder, 2 parts of sodium glycerophosphate, 1 part of L-cysteine hydrochloride, and 3 parts of phytic acid.

The beneficial effects of the present invention are: Cronobacter sakazakii cro611A3, cro1537W and Cronobacter dublinensis cro509C1, cro910B3 of the present invention have standard bacillus microscopic morphology and physiological and biochemical characteristics, Can be used to test the accuracy of Cronobacter chromogenic plates. Compared with other standard strains of this species, it is a strain that can reflect the genetic background in China at present, with stable genetics and special molecular markers, which can be used as reference strains for scientific research and detection quality control.

In addition, the inventor of the present invention provides a freeze-drying protective agent with the following advantages: good shape, beautiful appearance and good water solubility, can be completely dissolved within 1-2 seconds; freeze-drying survival rate can reach 85 % or more; it can be stored at -20°C for at least one year, which can ensure that its order of magnitude does not change, and can be used for long-term storage of quality control strains.

Description of drawings

Figure 1 is a diagram of Cronobacter gram staining and colony morphology;

Fig. 2 is the API 20E biochemical identification schematic diagram of Cronobacter strain;

Figure 3 is a PCR verification diagram of the unique molecular marker of Cronobacter dublini cro509C1;

Figure 4 is a PCR verification diagram of the unique molecular marker of Cronobacter sakazakii cro611A3;

Figure 5 is a PCR verification diagram of the unique molecular marker of Cronobacter dublini cro910B3;

Figure 6 is a PCR verification diagram of the unique molecular marker of Cronobacter sakazakii cro1537W;

Figure 7 is a graph showing the change of bacterial content within the shelf life of lyoprotectants with different compositions.

Detailed ways

In order to show the technical solutions, objects and advantages of the present invention more concisely and clearly, the technical solutions of the present invention are described in detail below with reference to specific embodiments and accompanying drawings.

Example 1 Isolation and culture of standard strains of Cronobacter

The collected food samples were thoroughly cut into pieces under sterile conditions, and 25 g of each sample was weighed and added to 225 mL of modified lauryl sulfate tryptose broth-vancomycin (modified lauryl sulfate tryptose broth-vancomycin edium, mLST- Vm) in a homogenizing bag, continuously homogenize for 1-2 min on a slap-type homogenizer to make a 1:10 uniform dilution. Pipette 1 mL of the 1:10 dilution with a pipette, add it to a test tube containing 9 mL of ST-Vm, shake well, and prepare a 1:100 dilution. In addition, prepare 10 incremental dilutions according to the above operation. For each increment, use a 1mL sterilized pipette tip. Inoculate three test tubes containing 9mLmLST-Vm for each dilution, inoculate 1mL in each tube, and set them at 37°C. Incubator for 8h-18h. In addition, the remaining mixed solution was placed in an incubator at 44°C ± 1°C and incubated for 18h-24h.

Dip a loop with an inoculation loop from all the test tubes or enrichment solutions that show growth, and culture it on the Enterobacter sakazakii chromogenic plate at 36 °C ± 1 °C for 24-48 hours. Observe the typical colony morphology, typical Cronau Bacilli are round, neat edges, translucent blue or blue-green colonies on the chromogenic medium; some Cronobacteria dublin are round, irregular edges, and surface on the chromogenic medium. Rough, dry, hard-textured blue or blue-green colonies, 1mm-5mm in diameter.

Pick suspicious colonies from each Sakazaki color plate containing suspected colonies, streak them on the Sakazaki color plate, and incubate at 36℃±1℃ for 18h-24h.

Suspicious colonies were picked from the secondary purified Sakazaki chromogenic plate, streaked on tryptic soy agar (TSA) plates, and cultured at 25℃±1℃ for 24-48h, and those producing yellow pigment were suspicious colonies.

Bacteria preservation: Under sterile conditions, the freshly cultured bacterial solution of BHI was added to a glycerol tube with a final concentration of 15%, stored in a -80°C refrigerator, and stored in a freeze-dried tube.

Example 2 Physiological and biochemical characteristics and serotype analysis of standard strains of Cronobacter

Staining microscopy: smear suspicious colonies, carry out Gram staining, and observe under oil microscope or phase contrast microscope. Cronobacter is a gram-negative non-bacillus, with pericytic flagella, about 3 μm in length and 1 μm in diameter. (figure 1)

Biochemical identification: Pick a single suspicious colony from pure culture and carry out the oxidase test. The colony with negative reaction of oxidase is identified by the AIP20E identification system (Figure 2).

Molecular identification: Cronobacter spp. carrying the fusA gene and biochemically identified as a strain of Enterobacter sakazakii, the fusA gene amplification test was carried out with the following primers, see (https://pubmlst.org/cronobacter/). The amplified products were sequenced, and the classification was determined by blast alignment analysis on NCBI.

The O antigen of Cronobacter strains was confirmed by multiplex PCR. For primers and amplification methods, refer to previous literature reports (

al. 2015, Applied and environmental microbiology 81:5574–5582). The primers used were synthesized by Shanghai Sangon Biological Co., Ltd. The primers were divided into 4 groups, Cronobacter sakazakii group: O1, O2, O3, O4, O6, O7; Cronobacter malonate group: O1, O2, O3, O4; Cronobacter dublini group: O1, O2, S. mogeni group: O1 and O2. The reaction system (25 μL) contained: 2×DS PCR mix, 12.5 μL; 0.5 μM upstream and downstream primers; ddH2O, 9.5 μL and genomic DNA, 1 μL. PCR conditions: (1) 95°C 5min, (2) 94°C 30s, (3) 53°C 30s, (4) 72°C 1min, (steps 2-4 30 cycles) (5) 72°C 5min, (6) 16°C∞. 10μL of PCR product was loaded on 2.0% agarose gel for electrophoresis separation (130V, 30min), using 50pb DNA ladder Marker.

Example 3 Drug susceptibility characteristics of standard strains of Cronobacter

Antibiotic susceptibility test: After the strain was activated on TSA plate, it was diluted with physiological saline to a final concentration of 1×10 ⁷ cfu/mL and spread on MH plate. After the bacterial liquid was dry, the antibiotic paper was attached to the surface of the medium, and the temperature was 37°C. Cultivated for 24h. The size of the inhibition zone was measured with a vernier caliper, accurate to 0.01 mm. The selected antibiotics are as follows: ampicillin (AMP, 10 μg), cefazolin (KZ, 30 μg), cefotaxime (KF, 30 μg), gentamicin (CN, 10 μg), tobramycin (TOB10), amyloid Kacin (AK30), Ampicillin Sulbactam (SAM10), Cefepime (FEP30), Ceftriaxone (CRO30), Ciprofloxacin (CIP, 5μg), Imipenem (IPM10), Trimethoprim pyridine-sulfamethoxazole (SXT, 25 μg), aztreonam (ATM30), chloramphenicol (C, 30 μg), tetracycline (TE30), amoxicillin-clavulanate (AMC30). Escherichia coli ATCC25922 and Cronobacter sakazakii ATCC 29544 were used as quality control strains.

Example 4 Multi-locus sequence (MLST) typing of standard strains of Cronobacter

The strain was sequenced through the whole genome, extracted seven gene sequences of atpD, fusA, glnS, gltB, gyrB, infB, pps, and uploaded the obtained sequence information to a special website (https://pubmlst.org/cronobacter/) to obtain the corresponding MLST type. Deleted genes were PCR amplified and sequenced according to standard operating procedures on the website. The identification information of the final Cronobacter standard strain is shown in Table 1:

Table 1. Information on standard strains of Cronobacter

Example 5 Characteristic sequence analysis of standard strains of Cronobacter

The whole genome sequences of 370 Cronobacter strains, including 4 Cronobacter standard strains, were selected for pan-genome analysis. The MP method was used to analyze, and the analysis results were processed by the local Perl script to obtain the core gene and non-core gene information of all strains.

Extract the unique non-core gene protein sequences (presumed unique genes) of cro509C1, cro611A3, cro910B3 and cro1537W strains, and align them back to the total protein library of Cronobacter and NCBI non-redundant protein database (NR) by local Blast respectively. Sequences that align to known Cronobacter proteins were removed, and what remained were genes specific to the strain.

Design primers to amplify the characteristic genes of cro509C1, cro611A3, cro910B3 and cro1537W strains. The primers are shown in Table 2. The reaction system (50 μL) contains: 2×DS PCR mix, 12.5 μL; 0.5 μM upstream and downstream primers; ddH2O, 9.5 μL and Genomic DNA, 1 μL. PCR conditions: (1) 98°C 2min, (2) 94°C 1min, (3) 60°C 1min, (4) 72°C 2min, (steps 2-4 30 cycles) (5) 72°C 5min, (6) 16°C∞. 10 μL of PCR products were loaded on a 1.5% agarose gel for electrophoresis separation (120V, 25min) using LD2000 DNA Marker. The results of the amplification are shown in Figures 3-6.

Table 2 Primers for amplifying the characteristic genes of cro509C1, cro611A3, cro910B3 and cro1537W strains

The present invention also provides a lyophilized protective agent for quantitative preservation of Cronobacter standard strains, and the components of the lyophilized protective agent in different embodiments and comparative examples are shown in Table 3:

Table 3: Components of each group of lyoprotectants

In the comparative example, the weight part of skim milk powder was increased to make up the total amount due to its lack of components.

The freeze-dried survival rate of the freeze-dried bacterial species (GDMCC 60864) of the protective agents of Examples 6 to 8 and Comparative Examples 1 to 3 will be used, and the specific method is as follows:

After the strains are recovered, they are cultured in the culture medium to the late logarithmic stage to the early stage of the stable stage. Select an appropriate amount of bacteria and add them to the protective agents in Examples 6-8 and Comparative Examples 1-3, mix them evenly, and distribute them into vials. , and sample for dilution count, which is the bacterial content A0 before freeze-drying. Transfer the half-stoppered vial into the freeze dryer for pre-freezing, the pre-freezing temperature is -40°C, the time is 3 hours, the main drying is turned on, the time is 20-25 hours, and then the analysis and drying stage is entered, and the time is 6 -8 hours, end drying, press the plug under vacuum and remove from the freeze dryer, carry out automatic capping, ensure the complete vacuum state of the sample, and store at -20 ℃ low temperature. Take the sample after freeze-drying and carry out dilution count, the count result is the bacterial content A after freeze-drying, and the freeze-drying survival rate is the percentage of A and A0, and the results are shown in Table 4:

Table 4: Comparison of lyophilized survival rates of lyoprotectants with different compositions

As can be seen from Table 4, the lyophilized protective agent of Example 8 is the best embodiment of the present invention, therefore, taking Example 8 as a comparison object, the lyophilized protective agents of Comparative Examples 1 to 3 were prepared, and the results are shown in Table 4 As shown, since Comparative Examples 1 to 3 respectively lack one of the components of the lyoprotectant of the present invention, the protective effects are all worse than those of the examples, thus indicating that the components in the lyoprotectant of the present invention are aggregated There is a synergistic effect among glucose, sodium glycerophosphate and phytate, and the effect of the protective agent of the present invention cannot be achieved without any one of them.

The freeze-drying stability will be compared with the freeze-dried strains of the protective agents in Examples 6-8 and Comparative Examples 1-3, and the specific methods are as follows:

According to the above preparation method, different protective agents were used to prepare quantitative quality control bacteria, and they were stored at -20°C, and 3 bottles were extracted every month to check the bacterial content according to the aforementioned counting method. In order to better compare the effect of each protective agent in the long-term storage process, when preparing quantitative quality control bacteria, the number of viable bacteria before freeze-drying was calculated according to the freeze-drying survival rate of each protective agent, so that after each protective agent was freeze-dried, The bacterial content is about 3000cfu/bottle.

After 12 months of storage, the bacterial content protected by the freeze-drying protective agents of Examples 6 to 8 has no significant change, while the bacterial content protected by the freeze-drying protective agents of Comparative Examples 1 to 3 has a bacterial content after 2 months. Significantly decreased, and the bacterial content was close to 0 after 12 months.

The above-mentioned embodiments only represent several embodiments of the present invention, and the descriptions thereof are specific and detailed, but should not be construed as a limitation on the scope of the invention patent. It should be pointed out that for those of ordinary skill in the art, without departing from the concept of the present invention, several modifications and improvements can also be made, which all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention should be subject to the appended claims.

Claims (10)

1. A specific molecular target for detecting Cronobacter, characterized in that the molecular target is:

   (a) any one or several nucleotide sequences shown in SEQ ID NOs: 1 to 4; or,

   (b) The nucleotide sequence in (a) is substituted, deleted or added by one or several nucleotides and has more than 90% homology with the nucleotide in (a).
2. The primer for detecting the specific molecular target of claim 1, wherein the PCR primer for amplification of the nucleotide sequence as shown in SEQ ID NO:1 comprises: upstream as shown in SEQ ID NO:5 Primers and downstream primers as shown in SEQ ID NO:6; PCR primers for amplification of the nucleotide sequence as shown in SEQ ID NO:2 include: upstream primers as shown in SEQ ID NO:7 and as shown in SEQ ID Downstream primer set forth in NO:8; PCR primers for amplification of the nucleotide sequence set forth in SEQ ID NO:3 include the upstream primer set forth in SEQ ID NO:9 and the upstream primer set forth in SEQ ID NO:10 Downstream primers; PCR primers for amplification of the nucleotide sequence set forth in SEQ ID NO:4 include the upstream primer set forth in SEQ ID NO:11 and the downstream primer set forth in SEQ ID NO:12.
3. A Cronobacter spp., characterized in that it is (a), (b), (c), or (d):

   (a) strain cro509C1, containing the nucleotide sequence shown in SEQ ID NO: 1;

   (b) strain cro611A3, containing the nucleotide sequence shown in SEQ ID NO:2;

   (c) strain cro910B3, containing the nucleotide sequence as shown in SEQ ID NO:3;

   (d) strain cro1537W, containing the nucleotide sequence shown in SEQ ID NO:4.
4. The Cronobacter of claim 3, wherein the strain cro509C1 further comprises a cefotaxime resistance gene.
5. The Cronobacter of claim 3, wherein the strain cro910B3 further comprises a cefotaxime resistance gene.
6. The Cronobacter of claim 3, wherein the strain cro1537W further comprises a cefotaxime resistance gene.
7. Cronobacter as claimed in claim 3, is characterized in that, the deposit number of described strain cro509C1 is: GDMCC 60861; The deposit number of described strain cro611A3 is GDMCC 60862; The deposit number of described strain cro910B3 is GDMCC 60863; The deposit number of the strain cro1537W is GDMCC 60864.
8. The application of Cronobacter as claimed in claim 4 or 5 or 6 in the study of antibiotic resistance of Cronobacter. Preferably, the antibiotic resistance of the strain cro509C1 is resistance to cefotaxime; the antibiotic resistance of the strain cro910B3 is resistance to cefotaxime; the antibiotic resistance of the strain cro1537W is resistance to cefotaxime Resistance to cefotaxime.
9. The application of the Cronobacter as claimed in claim 3 in improving the accuracy of checking the color plate of Cronobacter.
10. A cryoprotectant for Cronobacter, comprising the following components by weight: 0.1-3 parts of polydextrose, 5-10 parts of skim milk powder, 0.1-3 parts of sodium glycerophosphate, L-cysteine Hydrochloride 0.2-4 parts, phytic acid 0.5-3 parts.

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