A strain of Lactobacillus brevis CQPC12 and its application in preparing products for preventing / treating livestock diarrhea

Abstract

The present invention provides a strain of Lactobacillus brevis CQPC12 and its use in the preparation of products for preventing / treating livestock diarrhea, belonging to the field of microbial technology. The preservation number of the Lactobacillus brevis CQPC12 provided by the present invention is CGMCC No.19339. The bacteria can alleviate the limited weight gain caused by diarrhea to a certain extent, show a lower diarrhea index, improve the damaged intestinal barrier, repair the intestinal mucosal barrier, and reduce the permeability of the intestinal mucosa, thereby alleviating diarrhea symptoms. In the treatment of diarrhea, CQPC12 shows better effects than enrofloxacin, and is non-toxic and has no side effects, can avoid the abuse of antibiotics, while improving feeding quality and reducing costs. CQPC12 can be used as a substitute for livestock antibiotics.

Description

Technical Field

[0001] The invention relates to the technical field of microorganisms, and in particular to a strain of Lactobacillus brevis CQPC12 and application thereof in preparing a product for preventing / treating livestock diarrhea. Background Art

[0002] Diarrhea is a common disease in livestock and poultry, which can lead to the death of a large number of livestock in severe cases. In most cases, livestock diarrhea is caused by pathogenic Escherichia coli, which affects the physiological functions of animals, leading to decreased production performance, and even death in severe cases, causing significant economic losses to farmers. At present, antibiotics are the main method for treating calf diarrhea, among which drugs such as gentamicin and oxytetracycline are widely used. However, the problem of bacterial resistance caused by the use of antibiotics has attracted increasing attention. Escherichia coli diarrhea is a zoonosis. Due to the widespread use of antibiotics in animal husbandry, the cross-transmission of resistance of pathogens between animals and plants has caused widespread public concern. Large-scale use of antibiotics may also affect the intestinal microbiota, leading to an imbalance in the flora, thereby reducing the body's immune capacity. Therefore, the need to develop green, efficient, and low-residue drugs or biological agents is becoming increasingly urgent.

[0003] With the development of microbial technology, the application of probiotics in animal husbandry has been gradually proven to solve the problem of diarrhea in livestock to a certain extent, regulate the intestinal environment of animals, promote animal health, and increase the economic benefits of farmers. As a natural biological preparation without toxic side effects, the types of probiotics that are actually used in animal husbandry are still limited. Therefore, the development and screening of more probiotics is of great significance and has broad application prospects. Summary of the invention

[0004] In view of this, the purpose of the present invention is to provide a strain of Lactobacillus brevis CQPC12 and its use in the preparation of products for preventing / treating livestock diarrhea. The Lactobacillus brevis CQPC12 provided by the present invention can effectively prevent or treat diarrhea symptoms, is non-toxic and has no side effects, can avoid the abuse of antibiotics, improve feeding quality, reduce costs, and has broad application prospects.

[0005] In order to achieve the above object, the present invention provides the following technical solutions:

[0006] A strain of Lactobacillus brevis CQPC12, the classification of the Lactobacillus brevis CQPC12 is named Lactobacillus brevis , deposited in the General Microbiology Center of China Microorganism Culture Collection on January 13, 2020, with the deposit number CGMCC No.19339, and the deposit address is the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0007] In the present invention, the Lactobacillus brevis CQPC12 preferably has one or more of the following characteristics:

[0008] (1) The colony is white or milky white in color, round in shape, with neat edges and a moist and smooth surface;

[0009] (2) Gram-positive bacteria;

[0010] (3) The bacterial morphology is long rod or short rod, and there is no budding reproduction.

[0011] In the present invention, the Lactobacillus brevis CQPC12 preferably has a 16S rDNA sequence that is 99% to 100% identical to SEQ ID No.1.

[0012] The present invention also provides a microbial preparation, comprising the Lactobacillus brevis CQPC12 described in any one of the above technical solutions.

[0013] The present invention also provides an application of the Lactobacillus brevis CQPC12 described in the above technical solution or the microbial preparation described in the above technical solution in the preparation of a product for relieving or treating diarrhea.

[0014] The present invention also provides an application of the Lactobacillus brevis CQPC12 described in the above technical solution or the microbial preparation described in the above technical solution in preparing a product for preventing or treating livestock diarrhea.

[0015] In the present invention, the livestock preferably includes but is not limited to cattle, sheep, and pigs.

[0016] In the present invention, the diarrhea preferably includes but is not limited to diarrhea caused by Escherichia coli.

[0017] Beneficial technical effects: The present invention provides a strain of Lactobacillus brevis CQPC12 and its use in the preparation of products for preventing / treating livestock diarrhea. The preservation number of the Lactobacillus brevis CQPC12 is CGMCC No.19339. The bacteria can alleviate the limited weight gain due to diarrhea to a certain extent, show a lower diarrhea index, improve the damaged intestinal barrier, repair the intestinal mucosal barrier, and reduce the permeability of the intestinal mucosa, thereby alleviating diarrhea symptoms. In the treatment of diarrhea, CQPC12 shows better effects than enrofloxacin, and is non-toxic and has no side effects. It can avoid the abuse of antibiotics, while improving feeding quality and reducing costs. CQPC12 can be used as a substitute for livestock antibiotics. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 This is the colony morphology of strain CQPC12;

[0019] Figure 2This is the Gram staining result of strain CQPC12;

[0020] Figure 3 It is the agarose gel electrophoresis diagram of PCR amplification products; wherein, M is 2000bp DNA Ladder; 0 is the negative control group; 12 represents the strain numbered CQPC12, and other numbers represent other strains obtained during bacterial separation. DETAILED DESCRIPTION

[0021] In order to better understand the present invention, the content of the present invention is further explained in conjunction with the following examples, but the content of the present invention is not limited to the following examples. The materials, reagents, etc. used in the examples and test examples of the present invention, unless otherwise specified, can be obtained from commercial channels; the methods used in the examples and test examples of the present invention, unless otherwise specified, are conventional methods.

[0022] Example 1 Isolation and identification of bacterial strains

[0023] Materials: Naturally fermented kimchi water was collected from farmers in Nan'an District, Chongqing. 40 mL of kimchi water was taken into sterile centrifuge tubes, placed in food sampling boxes, and stored in a 4°C refrigerator in the laboratory for later use.

[0024] 1.1 Isolation and purification of bacterial strains

[0025] Take 1 mL of kimchi water sample and dilute it 10 times to 10 with sterile saline. -6 , then take 10 -4 , 10 -5 , 10 -6 100 μL of the bacterial solution of 3 gradients was spread on the plate (MRS medium), cultured at 37℃ for 24h~48h, and the colony morphology was observed and recorded. The colonies with different morphologies on the plate were picked up and separated by streaking on the MRS medium plate. After culturing at 37℃ for 48h, the single colonies with different morphologies on the plate were picked up again and separated by streaking on the MRS medium plate. This was repeated 2 to 3 times until a pure single colony with consistent morphology was obtained.

[0026] 1.2 Preliminary identification of bacterial species

[0027] Pure colonies on the plate were picked and inoculated into 5 mL of MRS liquid medium and cultured at 37°C for 24 h.

[0028] Colony morphology Figure 1 As shown, the colonies of CQPC12 bacteria are mostly white or milky white in color, round in shape, with neat edges and a moist and smooth surface.

[0029] Take 1 mL of the above-mentioned bacterial culture medium into a sterile centrifuge tube, centrifuge at 4000 r / min for 10 min, discard the upper layer of culture medium, resuspend the bacterial pellet in sterile physiological saline and perform Gram staining microscopy. Gram staining microscopy is positive. Under 100x oil microscope, the cell morphology of CQPC12 strain is as follows: Figure 2 As shown, the cell morphology includes long rods and short rods, and budding reproduction does not exist.

[0030] In summary, CQPC12 was preliminarily identified as a lactic acid bacterium.

[0031] 1.3 Molecular identification

[0032] The purified suspected target strain was inoculated into MRS broth and cultured at 37°C for 18h~24h. DNA was extracted using a bacterial genomic DNA extraction kit (item number: K0512, purchased from Thermo Fisher Scientific, USA). The extracted DNA was numbered and stored in a -20°C freezer for later use.

[0033] The extracted DNA was subjected to PCR amplification, including 1 μL of upstream primer 27F (5'-AGA GTT TGA TCC TGGCTC AG-3', SEQ ID No.2), 1 μL of downstream primer 1495R (5'-CTA CGG CTA CCTTGT TAC GA-3', SEQ ID No.3), 12.5 μL of 2×Taq plus Buffer, 1 μL of template DNA, and the system was supplemented to 25 μL with sterile ddH2O. Sterile ultrapure water was used to replace template DNA as a negative control. The amplification conditions were: 94℃ 5min; 94℃ 30s, 55℃ 30s, 72℃ 1min, a total of 29 cycles, and finally 72℃ extension for 5min.

[0034] Take 5 μL of amplified product for agarose gel electrophoresis detection, the agarose concentration is 1.5%, the electrophoresis conditions are 110V, 45min. The successfully detected PCR product was sent to Beijing Qingke Biotechnology Co., Ltd. for sequencing, and the sequenced sequence was compared and analyzed using the BLAST (Basic Local Alignment Search Tool) program in NCBI.

[0035] The results of 16S rDNA amplification products were detected by 1.5% agarose gel. Figure 3 The lane with sterile ultrapure water as negative control had no bands, indicating that there was no contamination during the PCR amplification process. The length of the specific amplified fragment in lane 12 was about 1500 bp, which was consistent with the expected amplified fragment length.

[0036] The sequencing result of the 16S rDNA amplification product is shown in SEQ ID No. 1:

[0037]

[0038] As shown in Table 1, the CQPC12 bacteria were sequenced successfully. The CQPC12 bacteria is Lactobacillus brevis, and its homology with known lactic acid bacteria in the Gene Bank database is 99%.

[0039] Table 1 16S rDNA sequence analysis results of bacteria

[0040]

[0041] The bacteria was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on January 13, 2020, with the deposit number CGMCC No.19339.

[0042] 1.4 In vitro resistance screening

[0043] 1.4.1 Ability to tolerate 0.3% bile salts

[0044] Porcine bile salt was added to MRS-THIO medium (MRS broth containing 0.2% sodium thioglycolate) to make the concentration 0.3%, sterilized at 121℃ for 15min, and 5mL of activated bacteria were inoculated at 2% (v / v) into MRS-THIO medium without bile salt (0.0%) and MRS-THIO medium containing 0.3% bile salt, respectively. Blank medium (MRS-THIO medium without bacteria) was used as control. After culturing at 37℃ for 24h, the OD values ​​of the above different concentrations of culture medium were measured. 600nm The tolerance of the strain to bile salts was calculated according to the following formula:

[0045]

[0046] 1.4.2 Artificial gastric juice tolerance test

[0047] Preparation of artificial gastric juice: Artificial gastric juice is composed of 0.2% NaCl and 0.35% pepsin. The NaCl and pepsin required for the test are weighed according to the corresponding mass-to-volume ratio for preparation. The pH of the prepared artificial gastric juice is adjusted to 3.0 with 1 mol / L HCl, and then filtered with a 0.22 μm filter membrane for sterilization and set aside.

[0048] In the clean bench, 5 mL of the cultured bacterial culture was taken into a 10 mL sterile centrifuge tube, centrifuged at 3000 r / min for 10 min, the upper culture medium was discarded and the bacteria were collected, and an equal volume (5 mL) of sterile saline was added to mix to make a bacterial suspension, and then 1 mL of the bacterial suspension was mixed with 9 mL of artificial gastric juice with pH = 3.0. At this time, 1 mL of the above mixture was taken as the sample of artificial gastric juice treatment for 0 h, and the remaining 9 mL of the mixture was placed in a constant temperature water bath shaker (37 ° C, 150 r / min) and cultured for 3 h. The samples of 0 h and 3 h were diluted 10 times in a gradient, and the number of viable bacteria was determined by plate coating method with a suitable gradient. The samples were cultured at 37 ° C for 48 h on MRS solid culture medium, and the survival rate (%) was calculated according to the following formula:

[0049]

[0050] 1.4.3 In vitro resistance results

[0051] The results of the ability to tolerate 0.3% bile salts and the survival rate in artificial gastric juice are shown in Table 2. As shown in Table 2, the survival rate of CQPC12 Lactobacillus brevis in pH=3.0 artificial gastric juice reached 82.79%; the survival rate of CQPC12 Lactobacillus brevis in 0.3% bile salts was relatively high, reaching 68.18%, indicating that it has a strong ability to tolerate bile salts.

[0052] Table 2 Survival rate of CQPC12 in pH 3.0 artificial gastric juice and 0.3% bile salt

[0053]

[0054] Example 2 Animal Experiment

[0055] 2.1 Experimental methods

[0056] 2.1.1 Experimental animals and strains

[0057] Fifty C57BL / 6 mice weighing 20±2g and aged 6 weeks were selected as experimental animals, half of which were male and half were female. The mice were allowed to eat and drink freely and were kept in the SPF barrier environment of the animal laboratory of Chongqing Engineering Laboratory for Functional Food Research and Development of Chongqing Second Normal University. They were fed SPF mouse feed purchased from Chongqing Tengxin Biotechnology Co., Ltd. and drinking laboratory pure water. The litter used was sterilized by high pressure and changed every three days. After changing the litter, the breeding room was disinfected with 84 disinfectant and irradiated with ultraviolet light once a week. Enteropathogenic Escherichia coli Escherichia coli EPEC (strain number: CICC 10663), purchased from China Industrial Microbiology Culture Collection Center.

[0058] 2.1.2 Main reagents and instruments

[0059] Mouse secretory immunoglobulin (sIgA) and polymeric immunoglobulin receptor (pIgR) ELISA kits, RNA extraction kits, reverse transcription kits, real-time fluorescence quantitative PCR kits, and real-time fluorescence quantitative PCR instrument were purchased from Thermo Fisher Scientific.

[0060] 2.1.3 Establishment and treatment of Escherichia coli diarrhea mouse model

[0061] Fifty mice were randomly divided into five groups, namely, normal group, model group, antibiotic (enrofloxacin) group, CQPC12 low-concentration gavage group and CQPC12 high-concentration gavage group, with 10 mice in each group. The mice in the normal group were intraperitoneally injected with 0.5 mL of normal saline, and the mice in the other groups were intraperitoneally injected with 0.5 mL of 1×10 8 After the mice showed diarrhea symptoms, the mice in the enrofloxacin group were intragastrically gavaged with 0.2 mL of enrofloxacin solution at a dose of 10 mg / kg, and the mice in the CQPC12 low-concentration gavage group (CQPC12L) and the CQPC12 high-concentration gavage group (CQPC12H) were intragastrically gavaged with 1×10 8 CFU / mL and 1×10 9 CFU / mL of CQPC12 bacterial suspension was used for continuous treatment for 7 days.

[0062] 2.1.4 Statistics of feed intake, body weight and diarrhea index

[0063] During the experiment, the clinical symptoms, body weight, and food intake of mice were recorded every day. On the third day of treatment, the mice were housed in single cages with filter paper as pads, and the diarrhea index was calculated.

[0064] Diarrhea index = loose stool rate × average loose stool grade

[0065] The loose stool rate is the ratio of the loose stool number of a single mouse to the total number of stools. The loose stool grade is assessed by the diameter of the loose stool stain on the filter paper: the stain diameter is less than 1.0 cm, which is graded as 1; the stain diameter is 1.0 cm to 1.9 cm, which is graded as 2; the stain diameter is 2 cm to 3 cm, which is graded as 3; the stain diameter is greater than 3 cm, which is graded as 4.

[0066] Sample collection: After 7 days of the experiment, the mice were dissected and the tissues 2 cm from the middle of the duodenum and ileum were taken. The dissolved contents were washed with saline and the mucosa of the duodenum and ileum were scraped and stored at -80°C for later use.

[0067] 2.1.5 Intestinal mucosal immunoglobulin levels

[0068] The ileal mucosa of mice was obtained, and the levels of sIgA and pIgR in the intestinal mucosa were determined according to the instructions of the kit.

[0069] 2.1.6 Expression levels of intestinal mucosal tight junction proteins

[0070] The total RNA was extracted from the duodenal mucosa of mice according to the kit instructions, and then reverse transcribed into cDNA after the concentration was determined. The intestinal tight junction protein was determined using a real-time fluorescence quantitative PCR kit. claudin-1 , occludin and ZO-1 Relative expression of mRNA. Primer information is shown in Table 3. All primers were synthesized by Beijing Qingke Biotechnology Co., Ltd. PCR reaction conditions: 95℃, 30s; 95℃, 5s, 60℃, 30s, 40 cycles; 95℃, 15s; 60℃, 60s; 95℃, 15s. GAPDH was used as the internal reference gene, and 2 -△△ct The relative expression of genes was calculated.

[0071] Table 3 Primer information

[0072]

[0073] 2.1.7 Data Statistical Analysis

[0074] The experimental data were analyzed by One-way ANOVA using SPSS 20.0 software, and multiple comparisons between groups were performed using Tukeytest and LSD method. The experimental results are expressed as mean ± standard deviation. P <0.05 indicated significant difference.

[0075] 2.2 Experimental Results

[0076] 2.2.1 Food intake of mice

[0077] As shown in Table 4, the food intake of mice in the normal group was significantly higher than that of the other groups, while that of the model group was significantly lower than that of the other groups ( P <0.05). The enrofloxacin group was slightly lower than the CQPC12H group, but significantly higher than the CQPC12L group ( P <0.05). The experimental results show that the antibiotic enrofloxacin and the probiotic CQPC12 can improve the feeding of diarrheal mice and facilitate the recovery of the mice, and the effect of CQPC12 is better than that of enrofloxacin.

[0078] Table 4 Average food intake of mice after treatment

[0079]

[0080] Note: Data in the same column with different letters on their shoulders indicate significant differences ( P <0.05); the same letters on shoulders indicate no significant difference ( P >0.05), the same below.

[0081] 2.2.2 Mouse weight

[0082] After intraperitoneal injection of E. coli, the weight of mice in the normal group continued to increase normally, while the weight of mice in the model group decreased (Table 5). Enrofloxacin and CQPC12 can inhibit the weight loss caused by intraperitoneal injection of E. coli, and CQPC12H has the best effect, while enrofloxacin is better than CQPC12L.

[0083] Table 5 Initial body weight, final body weight and average weight gain of mice

[0084]

[0085] 2.2.3 Diarrhea index of mice

[0086] The model group showed the highest loose stool rate and diarrhea index (Table 6). Enrofloxacin and CQPC12 could downregulate the high loose stool rate and diarrhea index caused by Escherichia coli, and the downregulation effect of CQPC12H was the most obvious.

[0087] Table 6 Loose stool rate and diarrhea index of mice after 3 days of treatment

[0088]

[0089] 2.2.4 Levels of immunoglobulins in the intestinal mucosa of mice

[0090] The normal group showed the highest sIgA and pIgR levels, and the sIgA and pIgR levels of the CQPC12H group were higher than those of the enrofloxacin group and the CQPC12L group (Table 7).

[0091] Table 7 Immunoglobulin levels in mouse intestinal mucosa

[0092]

[0093] 2.2.5 Expression levels of tight junction proteins in mouse intestinal tissue

[0094] Table 8 shows the normal group of mice claudin-1 , occludin and ZO-1 The relative expression of mRNA was significantly higher than that of other groups ( P <0.05), the above expression intensity of CQPC12H group was significantly higher than that of CQPC12L group ( P <0.05), slightly higher than the enrofloxacin group, but no significant difference ( P >0.05).

[0095] Table 8 Tight junction proteins in mouse intestinal tissue claudin-1 , occludin and ZO-1 Relative mRNA expression

[0096]

[0097] 2.3 Conclusion

[0098] The immune system of young animals is not yet fully developed, so they are more vulnerable to pathogens, which affects their growth and development. When young animals have diarrhea, it often leads to growth retardation, weight loss, and even death in severe cases. Changes in weight can intuitively reflect the body's recovery state. In this study, the weight of mice dropped significantly after intraperitoneal injection of bacterial liquid. After two days of drug treatment, the weight of the mice gradually recovered. The weight of mice in the normal group continued to increase, while the weight of mice in the model group increased slowly or even decreased. CQPC12 can alleviate the limited weight gain caused by diarrhea to a certain extent, showing the effect of treating diarrhea.

[0099] Food intake can reflect the body's development and pathological recovery to a certain extent. The average daily food intake of mice in the CQPC12 group was significantly higher than that in the model group, but lower than that in the normal group, indicating that the physical recovery of mice with diarrhea was improved after treatment with CQPC12. Mouse diarrhea models are often used when studying the efficacy of anti-diarrhea drugs. The diarrhea index can intuitively reflect the severity of diarrhea in mice. The lower the diarrhea index, the lighter the diarrhea in mice, indicating that the drug treatment is more effective. CQPC12 showed a lower diarrhea index, suggesting that CQPC12 plays a role in inhibiting diarrhea caused by Escherichia coli.

[0100] Immunoglobulins in the intestinal mucosa play an important role in resisting the invasion of foreign pathogens. Secretory immunoglobulin A (sIgA) can be transported across cells through polymeric immunoglobulin A (pIgA), secreted outside the cell and enter the intestinal cavity to neutralize harmful substances such as toxins and pathogens. The level of intestinal mucosal immunoglobulin in the model group mice was significantly lower than that in the CQPC12 treatment group and the control group, indicating that under the influence of Escherichia coli, the intestinal mucosa of mice was damaged and the immune function of the intestinal mucosa decreased. The level of intestinal mucosal immunoglobulin in mice treated with CQPC12 was significantly higher than that in the model group, indicating that CQPC12 can effectively promote the secretion of sIgA and pIgR in the intestinal mucosa. sIgA is an important defense factor in the mucosal immune system, and pIgR, as a transport receptor for sIgA, can maintain the balance of the mucosal surface, resist the invasion of external pathogens, and promote the repair of the intestinal mucosa. CQPC12 can significantly enhance this process. Intestinal epithelial cells play a vital role in maintaining the stability and balance of the intestinal environment. claudin-1 , occludin and ZO-1As key proteins of intestinal epithelial cell tight junctions, they have an important impact on intestinal barrier function. When the intestine is invaded by bacteria and other microorganisms, these proteins play a protective role. The experimental results showed that tight junction proteins in the intestinal tissue of the model group mice claudin-1 , occludin and ZO-1 The relative expression level of mRNA in the CQPC12 group was significantly lower than that in the other groups treated with drugs, indicating that when the intestinal mucosal barrier of mice is damaged, the relative expression of tight junction proteins in the intestinal tissue decreases, leading to increased intestinal mucosal permeability and thus diarrhea. This study showed that after CQPC12 treatment, tight junction proteins claudin-1 , occludin and ZO-1 The relative expression of mRNA of CQPC12 was increased, and it was able to repair intestinal villus damage, indicating that CQPC12 can improve the damaged intestinal barrier, repair the intestinal mucosal barrier, and reduce the permeability of the intestinal mucosa, thereby alleviating the diarrhea symptoms of mice. In particular, enrofloxacin, as an antibiotic veterinary drug, is often used to treat diarrhea. CQPC12 shows better effects than enrofloxacin, and is non-toxic and has no side effects. It can avoid the abuse of antibiotics, while improving feeding quality and reducing costs. CQPC12 can be used as a substitute for livestock antibiotics.

[0101] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A strain of Lactobacillus brevis ( Lactobacillus brevis ) CQPC12, characterized in that The Lactobacillus brevis CQPC12 was deposited in the General Microbiology Center of the China Microorganism Culture Collection Administration on January 13, 2020, with the deposit number CGMCC No.19339, and the deposit address is the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

2. A microbial preparation, characterized in that: It comprises the Lactobacillus brevis CQPC12 described in claim 1.

3. Use of the Lactobacillus brevis CQPC12 according to claim 1 or the microbial preparation according to claim 2 in the preparation of a product for relieving or treating diarrhea.

4. Use of the Lactobacillus brevis CQPC12 of claim 1 or the microbial preparation of claim 2 in the preparation of a product for preventing or treating diarrhea in livestock.

5. The use according to claim 4, characterized in that: The livestock include but are not limited to cattle, sheep, and pigs.

6. The use according to claim 4, characterized in that: The diarrhea includes but is not limited to diarrhea caused by Escherichia coli.

Images