Pediococcus acidilactici 36E-3 with antibacterial activity against multiple high virulence multi-drug resistant escherichia coli and application thereof

Abstract

The application discloses a Pediococcus acidilactici 36E-3 with bacteriostatic activity on multiple high-toxicity multi-drug resistant Escherichia coli and application thereof. The Pediococcus acidilactici 36E-3 is preserved in the Guangdong Microbial Culture Collection Center (GDMCC) on November 22, 2023, and the address is No. 59, Building 5, Institute of Microbiology, Guangdong Academy of Sciences, 100, Middle Junli Street, Guangzhou, with a postal code of 510070, and a preservation number of GDMCC No: 64049. The Pediococcus acidilactici 36E-3 of the application can simultaneously inhibit the growth of multiple high-toxicity diarrheal Escherichia coli, and carries a class II bacteriocin amino acid sequence, has a broad-spectrum bacteriostatic effect (also has obvious inhibitory effect on other common foodborne pathogenic bacteria), and has high application value in food pathogenic bacteria pollution prevention and control and healthy breeding of livestock and poultry.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, and specifically relates to a strain of Pseudococcus lactis 36E-3 that has high inhibitory activity against enterohemorrhagic Escherichia coli O157:H7 and other highly virulent multidrug-resistant pathogenic Escherichia coli, and its application. Background Technology

[0002] (1) Diarrheagenic Escherichia coli (DEC) is an important group of foodborne pathogens that can cause acute gastritis, persistent diarrhea, and other diseases in humans and animals. There are five important pathogenic types of this group of bacteria, including enteropathogenic E. coli (EPEC), enterotoxigenic E. coli (ETEC), Shiga toxin-producing E. coli (STEC), enteroinvasive E. coli (EIEC), and aggregater E. coli (EAEC). In the fields of foodborne diseases and clinical practice, the types that are most often reported are EPEC, STEC, EAEC, and EHEC O157:H7. These groups are the main pathogens causing diarrhea in the Chinese population during spring and summer, and are also common diarrhea pathogens in animals.

[0003] (2) Enterohemorrhagic Escherichia coli (E. coli) O157:H7 is a highly pathogenic serotype of the Shiga toxin-producing Escherichia coli STEC group. It can infect humans at low doses of 10-100 CFU, causing diseases such as hemorrhagic diarrhea, colitis, and hemolytic uremic syndrome. It is a foodborne pathogen under global surveillance. The typical pathogenicity of E. coli O157:H7 is that it can produce two Shiga toxins (Stx1 and Stx2; encoded by the stx1 and stx2 virulence genes, respectively). Shiga toxins are produced in the intestinal lumen, can cross epithelial cells and enter the blood circulation, causing damage to blood vessels in specific target tissues, such as the brain and kidneys. It has high pathogenicity and high mortality.

[0004] (3) Currently, foodborne illnesses caused by diarrheal Escherichia coli (including E. coli O157:H7) remain a significant public health problem in both developed and developing countries. Regarding the treatment of non-O157:H7 type diarrheal Escherichia coli infections, antibiotics such as amoxicillin and norfloxacin can be used as adjunctive therapy, but strains are prone to developing drug resistance, posing a potential threat to human health. For E. coli O157:H7 infections, effective treatment methods are still lacking both domestically and internationally. Multiple studies have confirmed that antibiotic use accelerates the production and release of Shiga toxin from E. coli O157:H7; therefore, antibiotic treatment is generally not recommended clinically. In animal husbandry, diarrhea caused by diarrheal Escherichia coli is a long-standing and persistent problem. The long-term use of antibiotics leads to the emergence of a large number of multidrug-resistant bacteria in animals and the farming environment, posing a significant threat to food, the environment, and human health.

[0005] (4) The development of stable, efficient, and naturally safe biocontrol technologies has become a new hot topic in food safety and the development of human and animal health. Especially under the domestic and international "antibiotic ban" situation, lactic acid bacteria, as probiotics for pathogen control and regulation of human health, have attracted the attention of many food companies and animal breeding companies. Lactic acid bacteria can produce a variety of antibacterial active substances such as organic acids, bacteriocins, and sugars, which inhibit or kill pathogens without toxic side effects on humans and animals. Pediococcus lactis is a species of lactic acid bacteria, which has been proven to have a good inhibitory effect on Gram-positive and Gram-negative pathogens. In the past, there have been reports of different types of lactic acid bacteria inhibiting diarrhea-causing Escherichia coli, but they mainly target one pathogenic type or serotype of Escherichia coli, such as E. coli O157:H7 or ETECK88. There are few reports on probiotics that can simultaneously inhibit multiple highly virulent drug-resistant diarrhea-causing Escherichia coli (including E. coli O157:H7). Our team previously discovered in monitoring diarrhea-causing Escherichia coli in food, animal-derived, or clinical diarrhea samples that multiple pathogenic Escherichia coli species often exist in the same sample (e.g., simultaneous detection of EPEC and STEC). Therefore, it is necessary to screen for strains with multiple antibacterial effects for control, rather than targeting only a single serotype or pathogenic type. Furthermore, many previous studies on probiotic screening primarily used standard strains as indicator bacteria, lacking evaluation data on the antibacterial effects of strains isolated from actual samples (food strains, animal pathogenic strains, and clinical strains).

[0006] Based on the above shortcomings, this invention screened a strain of *Pediococcus lactis* 36E-3 that exhibits high inhibitory activity against multiple different types (serotypes) of diarrheal *Escherichia coli*. The indicator bacteria used are mainly derived from foodborne (highly virulent drug-resistant) strains, pathogenic animal isolates, and clinical isolates, making it more targeted and representative in practical applications. Compared with previously screened lactic acid bacteria, this invention's strain has higher application value in the prevention and control of diarrheal *E. coli* contamination and in the healthy breeding of livestock and poultry. Summary of the Invention

[0007] The purpose of this invention is to provide a strain of *Escherichia coli* 36E-3 that has antibacterial activity against a variety of highly toxic and drug-resistant diarrheal Escherichia coli (including *E. coli* O157:H7) and can produce class II bacteriocins, and its applications.

[0008] The *Pediococcus acidilactici* 36E-3 of this invention was deposited on November 22, 2023, at the Guangdong Provincial Microbial Culture Collection Center (GDMCC), located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, Guangdong Academy of Sciences, Postcode: 510070, with accession number GDMCC No: 64049.

[0009] A second objective of this invention is to provide the application of Pediococcus lactis 36E-3 in the preparation of antimicrobial agents.

[0010] Preferably, the antimicrobial agent is a microbial agent that is effective against Escherichia coli, Listeria monocytogenes, Salmonella, Cronobacter sakazakii, Bacillus cereus, or Staphylococcus aureus.

[0011] Preferably, the Escherichia coli is a diarrhea-causing Escherichia coli.

[0012] The third objective of this invention is to provide an antimicrobial agent, characterized in that it contains the above-mentioned Pediococcus lactis 36E-3 as an active ingredient.

[0013] A fourth object of the present invention is to provide an antibacterial agent, the protein sequence of which is shown in SEQ ID NO.2.

[0014] The present invention provides a gene encoding the above-mentioned antibacterial agent.

[0015] Preferably, the encoding gene is as shown in SEQ ID NO.3.

[0016] The Pediococcus acidilactici 36E-3 of this invention can simultaneously inhibit the growth of multiple highly virulent diarrhea-causing Escherichia coli, and carries the Bovicin_255_variant class II bacteriocin amino acid sequence, exhibiting broad-spectrum antibacterial effects (also showing significant inhibitory effects on other common foodborne pathogens). It has high application value in the prevention and control of foodborne pathogen contamination and in the healthy breeding of livestock and poultry.

[0017] Pediococcus acidilactici 36E-3 was deposited on November 22, 2023, at the Guangdong Provincial Microbial Culture Collection Center (GDMCC), located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, Guangdong Academy of Sciences, Postcode: 510070, Accession No: GDMCC No: 64049. Attached image description:

[0018] Figure 1 This shows the growth of *Pediococcus lactis* 36E-3 on MRS plates.

[0019] Figure 2 This is a microscopic image of *Pediococcus lactis* 36E-3 (Gram staining G+).

[0020] Figure 3 This is the result of sequence alignment of the bacteriocin gene of Pediococcus lactis 36E-3. Detailed implementation method:

[0021] The following embodiments are further illustrations of the present invention, but not limitations thereof.

[0022] Example 1: Isolation and Identification of Pediococcus lactis

[0023] (1) Strain isolation

[0024] The *Pediococcus acidilactici* 36E-3 described in this invention was isolated from Cairo cheese in Africa and exhibits typical characteristics of *Lactobacillus*. The strain 36E was revived in MRS broth for 48 hours, streaked onto MRS plates, and incubated at 37°C for 24-48 hours. On MRS plates, this strain presents as round, smooth-surfaced, convex-center, and regularly edged, milky-white colonies with a diameter of 1.5-2 mm. Figure 1 ).

[0025] Gram staining: Gram staining microscopic examination revealed that *Pediococcus lactis* strain 36E was Gram-positive, appearing as short rods under the microscope, without flagella or spores. Figure 2 ).

[0026] Its physicochemical characteristics are: catalase-negative, oxidase-negative, capable of utilizing D-xylose, galactose, glucose, fructose, mannose, cellobiose, and trehalose, and able to grow at 45℃. For details on its bacterial morphology, please refer to the appendix. Figure 1 .

[0027] (2) Strain identification

[0028] Straw strain 36E-3 was streaked onto an MRS plate and incubated at 37°C for 48 hours. Single colonies were picked and plated onto a MAID-TOF target plate. Protein proteometry was performed according to the instructions, and the protein sequences were compared with the database.

[0029] Furthermore, molecular identification of the strain was performed using 16S rDNA as the target gene. Lactic acid bacteria DNA was extracted using a bacterial genomic DNA extraction kit (Meiji Biotechnology, Guangzhou), and PCR amplification was performed using 16S rDNA primers, as follows:

[0030] 27F: 5'-AGA GTT TGATCC TGG CTCAG-3'

[0031] 1492R-1: 5'-TAC GAC TTAACC CCAATC GC-3'

[0032] or 1492R-2: 5'-GGT TAC CTT GTTACGACT T-3'

[0033] PCR reaction system (25 μL): 12.5 μL of 2X PCR mixture (Angco Biotech, Guangzhou), 1 μL each of 10 μM forward and reverse primers, 8.5 μL of deionized water, and 2 μL of DNA template. PCR amplification parameters: 94℃ pre-denaturation for 5 min, 94℃ denaturation for 1 min, 56℃ annealing for 1 min, 72℃ extension for 2 min; 30 cycles; 72℃ extension for 7 min. The amplified products were sent to BGI Genomics for bidirectional sequencing. After sequence assembly, BLAST comparison analysis was performed in the NCBI database. The 16S rDNA sequence of strain 36E-3 is shown in SEQ ID No. 1. The similarity between the strain and *Pediococcus acidilactici* in the NCBI database is >97%, and strain 36E-3 is identified as *Pediococcus acidilactici*. MAID-TOF mass spectrometry identification results are *Pediococcus acidilactici* (>99%).

[0034] The *Pediococcus acidilactici* 36E-3 of this invention was deposited on November 22, 2023, at the Guangdong Provincial Microbial Culture Collection Center (GDMCC), located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, Guangdong Academy of Sciences, Postcode: 510070, with accession number GDMCC No: 64049.

[0035] Example 2: Inhibitory effect of Pyrococcus 36E-3 fermentation broth on highly virulent, multidrug-resistant Escherichia coli

[0036] Pietrococcus 36E-3 was revived in MRS broth for 48 hours, and 20 strains of highly virulent, multidrug-resistant Escherichia coli were used. 8 CFU was used as the test strain, and the antibacterial effect of 36E-3 was evaluated using the Oxford cup method. The results showed that 36E-3 had a good antibacterial effect against 20 highly virulent multidrug-resistant bacteria (Table 1).

[0037] Table 1. Inhibition effect of *Pediococcus lactis* 36E-3 bacterial suspension on highly virulent, multidrug-resistant *Escherichia coli*.

[0038]

[0039] Note: Size of the inhibition zone ("+" 6-9mm, "++" 10-13mm, "+++" 14-18mm, "++++" >18mm)

[0040] Antibiotics: Cephalothin (KF, 30μg), Cefotaxime (CTX, 30μg), Ampicillin (AMP, 10μg), Amoxicillin-clavulanic acid (AMC, 30μg), Amikacin (AK, 30μg), Gentamicin (GEN, 10μg), Kanamycin (K, 30μg), Streptomycin (S, 25μg), Norfloxacin (NOR, 10μg), Ciprofloxacin (CIP, 5μg), Nalidixic acid (NA, 30μg), Tetracycline e (TE, 30 μg), chloramphenicol (C, 30 μg), trimethoprim-sulfamethoxazole (SXT, 25 μg), colistin (COL, 2 μg / mL), and florfenicol (FFC, 30 μg)

[0041] Example 3: Inhibitory effect of Pediococcus lactis supernatant on pathogenic bacteria from various sources.

[0042] Lactococcus 36E-3 was revived in MRS broth for 48 hours, then sterilized by filtration through a 0.22 filter membrane and set aside. 10 5CFU samples containing various pathogenic bacteria (including diarrhea-causing Escherichia coli, Listeria monocytogenes, Salmonella, Cronobacter sakazakii, Bacillus cereus, Staphylococcus aureus, etc.) were spread on NA or BHA plates. Using the Oxford cup method, 200 μL of Pediococcus lactis 36E-3 fermentation broth was inoculated and co-cultured at 37°C for 24 h. The size of the inhibition zone was measured to assess the inhibition effect.

[0043] The results (Table 2) showed that *Pediococcus lactis* 36E-3 exhibited inhibitory effects against foodborne pathogenic *Escherichia coli*, animal pathogenic strains, clinical strains of *Escherichia coli*, and other common foodborne pathogens, classifying it as a broad-spectrum antibacterial lactic acid bacterium. A key advantage of this strain is its strong antibacterial activity against both Gram-positive and Gram-negative bacteria, making it particularly suitable for the control of common pathogens in food and animal husbandry.

[0044] Table 2 Antibacterial spectrum of sterile supernatant containing *Pediococcus lactis* 36E-3

[0045]

[0046]

[0047] Note: Size of the inhibition zone ("+" 6-9mm, "++" 10-13mm, "+++" 14-18mm, "++++" >18mm)

[0048] Example 4: Bacteriocin prediction (acid neutralization and proteinase K treatment assay)

[0049] Lactococcus 36E-3 was inoculated into 5 ml LMR broth and incubated at 36°C for 48 h. The pH of the supernatant was adjusted to 7.0 with 1 mol / L sodium bicarbonate, and the supernatant was sterilized by filtration through a 0.22 μm membrane. Using the Oxford cup method, 200 μL of the sterile supernatant was inoculated onto a 10 μL plate. 5 CFU was cultured on NA or BHA plates for Gram-positive bacteria (Listeria monocytogenes, Bacillus cereus, Staphylococcus aureus, Bacillus cereus) and Gram-negative bacteria (Escherichia coli, E. coli O157:H7, Salmonella) at 37°C for 48 h, with untreated supernatant as a control. The antibacterial effect of the acid-neutralized lactic acid bacteria fermentation supernatant on pathogenic bacteria was observed. Additionally, the effect of antibacterial substances was observed through high-temperature treatment, with untreated supernatant as a control. To further investigate the antibacterial activity, the fermentation supernatant was treated with neutral proteinase K at the optimal pH value, with a final enzyme concentration of 5 mg / mL, and incubated at 37°C for 0.5 h. The antibacterial activity of the treated fermentation supernatant was determined using the Oxford cup method, with untreated fermentation broth as a control.

[0050] The results (Table 3) showed that the inhibition zone did not change significantly after heat treatment, indicating that the antibacterial substance is heat-resistant. The supernatant still exhibited antibacterial activity after acid neutralization, indicating that other antibacterial active substances were present in the supernatant besides the acidic antibacterial substance. The inhibition zone decreased in size after proteinase K treatment, suggesting the presence of antibacterial proteins (bacteriocins) in the supernatant (Table 4).

[0051] Table 3. Antibacterial effect of 36E-3 after acidification.

[0052]

[0053] Table 4. Changes in inhibition zones after proteinase K treatment.

[0054]

[0055] Example 5: Acid and bile salt resistance test of Pediococcus lactis 36E-3

[0056] Acid resistance test: Lactic acid bacteria were revived in MRS broth for 48 hours to prepare 10 8 CFU / mL bacterial suspension was prepared by adding 1 mL of the suspension to 9 mL of MRS liquid medium at pH 2.0 and incubating at 37°C for 18–24 h. Bacterial growth was observed by measuring the absorbance at 600 nm.

[0057] Bile salt tolerance test: Lactic acid bacteria were revived in MRS broth for 48 hours to prepare 10 8 CFU / mL bacterial suspension was used. 1 mL of the bacterial suspension was added to 9 mL of MRS liquid medium containing 3% bile salts. After incubation at 37℃ for 18-24 h, the absorbance at 600 nm was measured to observe bacterial growth.

[0058] The results showed that *Pediococcus lactis* 36E-3 exhibits acid and bile salt tolerance, and can grow and survive in broth containing 0.3% bile salts and pH 2.0.

[0059] Example 6: Results of sequence alignment of the bacteriocin gene of Pediococcus lactis 36E-3

[0060] The bacteriocin gene sequence is as follows: NCBI genome BLAST alignment shows that this gene is annotated as a hypothetical protein expressing Bovicin_255_variant class II bacteriocins. The sequence matches 52.273% of existing sequences in the BAGEL4 bacteriocin database. Figure 3 ).

[0061] bacteriocin gene sequence:

[0062] TGCGTGCCAAACAATATCGTTATTACACTGCTAATGGCTATGAATATCGTGATAGTAAAGGACATTGGCACTACGTAGTTACTAAATCTCCATTTGAAGCTGCATTTGGTGTGACATTGCATGGCTGGGAAGGCGCACTTGGTGGTTCATGGAAATCTGGGCATGAAAAATAGG(SEQ ID NO.3)

[0063] Related:

[0064] MRAKQYRYYTANGYEYRDSKGHWHYVVTKSPFEAAFGVTLHGWEGALGGSWKSGHEK(SEQ IDNO.2)

[0065] 36E-316srDNA data (SEQ ID No.1)

[0066]

[0067] Example 7 Characteristic recognition target of Pediococcus lactis strain 36E-3

[0068] To further protect strain 36E-3, specific recognition sequences for this strain were screened. The specific steps were as follows: Whole genome sequences of *Pediococcus lactis* and other *Lactobacillus* strains were downloaded from the NCBI database and subjected to pan-genome analysis. All genome sequences were annotated using Prokka v1.11. After annotation, the gff annotation files for all strains were copied and pan-genome analysis was performed. The generated `gene_presence_absence.csv` file is the pan-genome matrix file. The presence / absence profiles of all genes in the pan-genome matrix file were converted to 1 / 0 format to identify characteristic target sequences for *Pediococcus lactis* strain 36E-3. Blast alignment with the NCBI database showed that this target fragment exists only in strain 36E-3 and is absent in the genome sequences of other strains. The nucleotide sequence of the molecular target is shown in SEQ ID NO.4.

[0069] Characteristic DNA sequence of Pediococcus lactis 36E-3 (SEQ ID No. 4)

[0070] > / L.36E-3_00504

[0071]

Claims

1. Pediococcus acidilactici ( Pediococcus acidilactici ) 36E-3 was deposited at the Guangdong Provincial Center for Microbial Culture Collection on November 22, 2023, with accession number: GDMCC No: 64049.

2. The application of the *Pediococcus lactis* 36E-3 according to claim 1 in the preparation of antimicrobial agents, wherein the antimicrobial agent is an antimicrobial agent against *Escherichia coli*, *Listeria monocytogenes*, *Salmonella*, *Cronobacter sakazakii*, *Bacillus cereus* and *Staphylococcus aureus*.

3. The application according to claim 2, characterized in that, The Escherichia coli mentioned is a diarrhea-causing Escherichia coli.

4. An antibacterial microbial agent, characterized in that, It contains the active ingredient of Pediococcus lactis 36E-3 as described in claim 1.

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