Chlorine-resistant amoeba and application thereof

Abstract

The invention belongs to the technical field of microorganisms, and particularly relates to chlorine-resistant amoeba and application thereof. According to morphology and gene analysis, the amoeba is identified as Vermamoeba vermiform, and the vermamoeba vermiform is named as DW8S, and the vermamoeba vermiform is named as DW8S. The chlorine resistance test result shows that the amoeba DW8S can resist high-intensity chlorine disinfection treatment, when the CT value reaches 35 (mg.min) / L, the inactivation rate is less than 1-log, multiple culturable opportunistic pathogenic bacteria are carried in the spores of the amoeba DW8S, and the pathogenic bacteria still survive in the amoeba spores after chlorine disinfection. Besides, the amoeba DW8S is simple in culture condition and easy to store, and can be used as a novel water quality microbiological indicator, the indicator can reflect a new dimension of microbial pollution in a water system and also can indicate the effect of a disinfectant, and the amoeba DW8S shows a good application prospect in the aspect of guaranteeing water quality safety and has a wide application prospect. And a new tool is provided for future water quality monitoring and management.

Description

Technical Field

[0001] The present invention belongs to the field of microbial technology, and more specifically, relates to a chloroamoeba-resistant strain and application thereof. Background Art

[0002] Safe drinking water is the basic guarantee for human health and an important public health resource related to the national economy and people's livelihood. By making quantitative regulations on various factors related to human health in drinking water, the quality of drinking water can be effectively controlled and safe water can be provided for humans. However, there are many complex microbial communities in drinking water systems, such as bacteria, eukaryotes and viruses. These microbial communities not only exist in flowing tap water, but can also attach to other suspended matter or form biofilms on the inner wall of water pipes. Although the diversity of drinking water microbial communities has decreased significantly after the widespread use of disinfectants such as chlorine and other treatment steps, some microbial species show high resistance to disinfectants and survive in drinking water systems.

[0003] Amoeba is a type of unicellular heterotrophic eukaryotic organism that feeds on bacteria and is also the main eukaryotic organism group in the microbial community of drinking water systems. Infections with some amoeba groups can cause keratitis or meningitis, which seriously threatens human health. In addition, amoeba can also promote the spread of pathogenic bacteria. Studies have found that some conditional pathogens can resist the predation of amoeba and persist or even reproduce in cells. In addition, among these surviving bacteria, some bacteria have been identified to contain antibiotic resistance genes and mobile genetic elements, indicating that amoeba may act as a carrier to help the environmental spread of antibiotic resistance genes, posing a serious threat to human health. Studies have shown that amoeba can form hard and thick spores to avoid adverse environments and are highly resistant to disinfectants. Therefore, the presence of amoeba in tap water and its interaction with pathogenic bacteria pose a threat to drinking water safety that cannot be ignored, and also bring new challenges to the quality of drinking water.

[0004] Although studies have found that amoeba is widely present in drinking water systems and has been isolated and cultured, the understanding of amoeba's tolerance to disinfectants is very limited, and the biological resources of chlorine-resistant amoeba need to be further explored. Currently, most of the existing drinking water disinfection technologies use bacteria as microbial indicators to evaluate the effectiveness. Some studies have also tried to use the soil-derived amoeba model organism Dictyostelium discoideum to explore the inactivation efficiency of disinfectants on amoeba, but this experimental subject has great limitations. The differences in the living environment of amoeba will affect its mechanism of resisting chlorine disinfection.

[0005] Therefore, isolating and culturing amoeba in drinking water systems can effectively promote research on drinking water quality safety, which has important practical significance. Summary of the invention

[0006] The technical problem to be solved by the present invention is to overcome the different tolerance of amoeba obtained from different sources to disinfectants. At present, there are great limitations in using amoeba from soil as water quality microbial indicators, and it is impossible to effectively indicate the defects and shortcomings of water quality disinfection effects. A protozoan amoeba separated from a drinking water system and having high chlorine resistance is provided, a new model organism is provided for risk assessment of drinking water, and a theoretical basis and feasibility suggestions are provided for the optimization of drinking water disinfection technology and the control of chlorine-resistant amoeba.

[0007] The purpose of the present invention is to provide the application of the protozoan amoeba as a water quality microbial indicator.

[0008] Another object of the present invention is to provide a method for evaluating the degree of microbial contamination in water.

[0009] Another object of the present invention is to provide the use of the protozoan amoeba in evaluating the effect of water disinfectants.

[0010] Another object of the present invention is to provide a method for evaluating the effectiveness of a water disinfectant.

[0011] The above-mentioned purpose of the present invention is achieved through the following technical solutions:

[0012] The present invention protects a protozoan amoeba, which is Vermamoebavermiformis (worm-like Hammann amoeba) DW8S, which has been deposited in the China Center for Type Culture Collection on July 2, 2024, and its preservation number is CCTCC NO: C2024147.

[0013] Microorganisms are a general term for tiny organisms that are difficult to see with the naked eye, including microorganisms with cell structures such as true bacteria, archaea, fungi, actinomycetes, protozoa, algae, as well as organisms without complete cell structures such as viruses, mycoplasmas, and chlamydia. Free-living amoebae (FLA) are protozoans scattered in different environments and belong to a type of microorganism (Marinho, Brenda Teixeira Scardini, et al. "First report of free-living amoebae in watercourses in southern Brazil: molecular diagnosis and phylogenetic analysis of Vermamoeba vermiformis, Naegleria gruberi, and Acanthamoeba spp." Journal of Water and Health 21.7(2023):972-980.;Abu Kwaik, Y. "The phagosome containing Legionella pneumophila within the protozoan Hartmannella vermiformis is surrounded by the rough endoplasmic reticulum." Applied and environmental microbiology 62.6(1996):2022-2028.).

[0014] Vermamoeba vermiformis was also called Hartmannella vermiformis before, belongs to a kind of in free-living amoeba, it is a common microorganism found in natural and artificial habitats. As a predator of other microorganisms, free-living amoeba helps to regulate microbial communities, maintain population size, promote the diversity of microbial species, and maintain the flow of energy and material through food webs (Delafont, Vincent, et al. "Vermamoeba vermiformis: a free-living amoeba of interest." Microbial ecology 76.4 (2018): 991-1001.).

[0015] The present invention obtains a strain of Vermamoeba vermiformis (vermiform Harman amoeba) DW8S isolated from a drinking water system. Compared with the vermiform Harman amoeba DW6 isolated at the same time, DW8S has a stronger chlorine resistance. Free chlorine disinfection experiments using sodium hypochlorite as a disinfectant found that when the CT value reached 35 (mg·min) / L, its inactivation rate was still less than 1-log. my country's "Sanitary Standards for Drinking Water" stipulates that the CT value of the free chlorine disinfection standard is 9.0 (mg·min) / L, and Escherichia coli should not be detected as a microbial indicator. However, at this CT value, chlorine disinfection has no inactivation effect on the isolated amoeba DW8S. In addition, the amoeba spores carry a variety of opportunistic pathogens and can maintain the continued existence of endophytes in the spores after disinfection with chlorine disinfectants. Through the screening and separation of chloramphenicol-resistant amoeba, we can more accurately understand the composition of amoeba communities in water quality, provide a more accurate theoretical basis for the microbial safety assessment of water quality, especially drinking water, and provide technical support for the selection of disinfection technology and process improvement of water supply plants, thereby better ensuring the biological safety of water quality.

[0016] Furthermore, the spore morphology of the protozoan amoeba is spherical.

[0017] Furthermore, the spore diameter of the protozoan amoeba is 3 to 5 μm.

[0018] Furthermore, the spores of the protozoan amoeba contain opportunistic pathogens, and the opportunistic pathogens include one or more of Brucella pseudogrignoli, Sphingobacterium multivorum, and Stenotrophomonas rhizophila.

[0019] Furthermore, the separation and cultivation of ameba comprises the following steps: enriching and concentrating a water sample through a 0.22 μm polycarbonate membrane, taking out the filter membrane and placing it on a nutrient-free agar (NNA) medium (2.2 g of potassium dihydrogen phosphate, 1.7 g of potassium dihydrogen phosphate, 15.0 g of agar, and 1.0 L of water) coated with food bacteria Klebsiella pneumoniae and cultivating it at 21° C., observing the movement of ameba cells and the formation of spores on the plate under a microscope, and using a sterile inoculation loop to pick up ameba spores on the plate and transfer them to a new nutrient-free agar plate coated with Klebsiella pneumoniae for further purification, thereby obtaining the product.

[0020] The present invention also protects the use of the protozoan amoeba as a water quality microbial indicator.

[0021] Furthermore, the water quality is drinking water.

[0022] The present invention also protects a method for evaluating the degree of microbial contamination in water quality, using the protozoan amoeba as a water quality microbial indicator. The method reflects a new dimension of microbial contamination in water quality by detecting the presence or absence and the number of the amoeba.

[0023] Furthermore, the water quality is drinking water.

[0024] The present invention also protects the use of the protozoan amoeba in evaluating the effect of water disinfectants.

[0025] Preferably, the water disinfectant is a chlorine-containing disinfectant.

[0026] Preferably, the chlorine-containing disinfectant includes sodium hypochlorite, bleaching powder, sodium dichloroisocyanurate, trichloroisocyanuric acid or chlorine dioxide, more preferably sodium hypochlorite.

[0027] The present invention also protects a method for evaluating the effect of a water disinfectant, using the protozoan amoeba as a microbial indicator. The method evaluates the killing effect of the disinfectant on microorganisms by monitoring the survival of the amoeba after disinfection.

[0028] Preferably, the water disinfectant is a chlorine-containing disinfectant.

[0029] Preferably, the chlorine-containing disinfectant includes sodium hypochlorite, bleaching powder, sodium dichloroisocyanurate, trichloroisocyanuric acid or chlorine dioxide, more preferably sodium hypochlorite.

[0030] Compared with the prior art, the present invention has the following beneficial effects: The present invention provides a protozoan amoeba DW8S isolated from a drinking water system and having high chlorine resistance. Through morphological and genetic analysis, the amoeba was identified as Vermamoeba vermiformis. The test results of chlorine resistance show that the amoeba DW8S can resist high-intensity chlorine disinfection treatment. When the CT value reaches 35 (mg·min) / L, the inactivation rate is less than 1-log. Its spores also carry a variety of cultivable opportunistic pathogens, and the pathogens still survive in the amoeba spores after chlorine disinfection. In addition, the culture conditions of the amoeba DW8S are simple and easy to preserve. It can be used as a novel water quality microbial indicator. This indicator can not only reflect the new dimension of microbial pollution in the water system, but also show good application prospects in ensuring water quality safety, and provide a new tool for future water quality monitoring and management. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 Spore morphology of amoeba DW8S (left) and DW6 (right) isolated from tap water.

[0032] Figure 2Phylogenetic tree constructed for the 18S rRNA gene sequences of amoeba DW8S and DW6.

[0033] Figure 3 Phylogenetic tree constructed based on the 16S rRNA gene sequences of the endosymbiotic bacteria of amoeba DW8S.

[0034] Figure 4 Disinfectant decay curves for inactivation of amoeba DW8S and DW6.

[0035] Figure 5 This is the inactivation characteristic curve of amoeba DW8S and DW6.

[0036] Figure 6 This is the inactivation characteristic curve of symbiotic bacteria in Amoeba DW8S. DETAILED DESCRIPTION

[0037] The present invention is further described below in conjunction with the accompanying drawings and specific examples, but the examples do not limit the present invention in any form. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0038] Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

[0039] Example 1 Isolation and Purification of Amoeba

[0040] 2L of tap water was collected and concentrated by a 0.22μm polycarbonate membrane. The membrane was removed and flipped over a plate coated with 200μL of OD 600 The nutrient-free agar (NNA) medium (2.2 g potassium dihydrogen phosphate, 1.7 g potassium dihydrogen phosphate, 15.0 g agar, 1.0 L pure water) of Klebsiella pneumoniae with a value of 1.5 was incubated at 21°C for one week. The presence of ameba was identified by observing the migration front and amebic cells formed by ameba under a microscope. The ameba spores on the plate were transferred to a new nutrient-free agar plate coated with Klebsiella pneumoniae respectively using a sterile inoculation loop for further purification.

[0041] Example 2 Identification of Amoeba

[0042] (1) Morphological identification

[0043] The isolated and purified amoeba was placed on a non-nutrient agar plate coated with 200 μL of Klebsiella pneumoniae with an OD600 value of 1.5 and cultured at 21°C for one week. During the culture process, the plate was placed under a microscope for observation and photos of the amoeba in the spore stage were taken. The results are as follows: Figure 1As shown, the left side shows the sporophyte morphology of amoeba DW8S, and the right side shows the spore morphology of amoeba DW6. The spore morphology of the two amoebas is spherical, the spore diameter of DW8S is about 3-5μm, and the diameter of DW6 is about 6-8μm. There is little difference in morphology between individuals of the same amoeba.

[0044] (2) Biological identification

[0045] The purified amoebae were transferred to a plate coated with 200 μL of OD 600 The SM / 5 agar plate (glucose 2.0g, peptone 2.0g, yeast powder 2.0g, magnesium sulfate heptahydrate 0.4g, potassium dihydrogen phosphate 1.9g, dipotassium hydrogen phosphate 1.0g, agar 15.0g, pure water 1.0L) of Klebsiella pneumoniae with a value of 1.5 was cultivated at 21°C for five days, and its growth was observed under a microscope subsequently. After the formation of ameba spores, a small amount of ameba cells were picked in 100 μL of sterile water containing 10% Chelex100 (50-100 mesh), and after a boiling water bath for 10min, placed at -20°C for incubation for 10min, and then centrifuged at 6000rpm for 3min. The supernatant was taken for subsequent PCR reaction, and the 18SrRNA gene of the ameba separated was amplified by PCR using genus / species specific primers and eukaryotic universal primers according to the ameba spore morphology. The primers selected for DW8S were FLA-F (5'-CGGTAATTCCAGCTCCAATAGC-3') and FLA-R (5'-CAGGTTAAGGTCTCGTTCGTTAAC-3'). The primers selected for DW6 were SSU1 (5'-CGACTGGTTGATCCTGCCAGTAG-3') and SSU2 (5'-TCCTGATCCCTCCGCAGGTTCAC-3'). To amplify the 18S rRNA gene, the PCR reaction was performed in a 50 μL reaction system containing 20 μL of polymerase chain reaction mixture (TaKaRaTaq TM Version2.0plus dye), 2μL of upstream and downstream primers (10μmol / L), 6μL of amoeba DNA and 15μL of sterile water. The reaction procedure was: 94℃ pre-denaturation for 3min; 94℃ denaturation for 30s, 60℃ annealing for 30s, 72℃ extension for 1min, repeated 35 cycles; 72℃ re-extension for 10min. PCR products were detected and sequenced using 1.5% agarose gel electrophoresis. Amoeba DW8S successfully amplified a 711bp target band, and amoeba DW6 successfully amplified a 729bp target band.

[0046] The 18S rRNA gene sequences of the isolated amoeba DW8S and DW6 (the nucleotide sequence of DW8S is shown in SEQ1; the nucleotide sequence of DW6 is shown in SEQ2) were used for BLAST homology alignment using the NCBI (National Center for Biotechnology Information) database (https: / / blast.ncbi.nlm.nih.gov). The 18S rRNA phylogenetic tree was constructed using FastTree and IQTree, respectively, and the construction parameters were selected as JTT+CAT and TN+F+I, respectively. When constructing the phylogenetic tree, a bootstrap analysis based on 1000 replicates was performed to ensure the reliability of the results. The phylogenetic tree was drawn using the iTOL website (https: / / itol.embl.de / itol.cgi). The phylogenetic tree is shown in Figure 2 As shown, the sequence alignment results showed that the 18S rRNA gene sequence similarity between DW8S and DW6 and Vermamoeba vermiformis reached 100%, and the 18S rRNA gene sequence similarity between DW8S and DW6 was 98.57%.

[0047] Transfer the purified amoebae isolate DW8S to a 200 μL plate coated with OD 600 The SM / 5 agar plate of Klebsiella pneumoniae with a value of 1.5 was cultured at 21°C for five days, and then its growth was observed under a microscope. After the formation of amoeba spores, the plate was rinsed with KK2 buffer (2.2g potassium dihydrogen phosphate, 1.7g potassium dihydrogen phosphate, 1.0L water), and the amoeba spores were collected in a 1.5mL sterile centrifuge tube. The collected amoeba spore suspension was centrifuged at 2000rpm for 5min to remove the supernatant, and repeated 5-6 times to remove the food bacteria not preyed by amoeba on the plate and the bacteria attached to the surface of amoeba. The collected amoeba spores were resuspended with 1mL of KK2 buffer, and 10μL was inoculated on the SM / 5 agar plate. Each amoeba sample was paralleled for 6 times. The inoculated plate was cultured at 21°C for one week, and bacterial colonies of different colors, sizes and shapes were picked with a sterile inoculation loop and transferred to a new SM / 5 agar plate for continuous culture purification.

[0048] Purified bacterial cells were collected in 100 μL of sterile water containing 10% Chelex100 (50-100 mesh), incubated at -20°C for 10 min after boiling in a water bath for 10 min, and then centrifuged at 6000 rpm for 3 min. The supernatant was used for subsequent PCR reaction. Universal bacterial primers 27F (5'-AGAGTTTGATCMTGGCTCAG-3') and 1492R (5'-TACGGYTACCTTGTTAYGACTT-3') were used to amplify the 16S rRNA gene of symbiotic bacteria. The PCR reaction was carried out in a 50 μL reaction system, including 20 μL of polymerase chain reaction mixture (TaKaRa Taq TM Version 2.0plus dye), 2μL of upstream and downstream primers (10μmol / L), 2μL of amoeba DNA and 19μL of sterile water. The reaction procedure was: 94℃ pre-denaturation for 3min; 94℃ denaturation for 30s, 55℃ annealing for 30s, 72℃ extension for 1min, repeated 35 cycles; 72℃ extension for 10min. PCR products were detected and sequenced using 1.5% agarose gel electrophoresis. The isolated bacterial 16S rRNA gene sequence was subjected to BLAST homology comparison using the NCBI (National Center for Biotechnology Information) database (https: / / blast.ncbi.nlm.nih.gov). The phylogenetic tree was calculated and drawn using MEGAX software using the neighbor-joining (NJ) method based on the Kimura 2-parameter (K2) model and the maximum likelihood (ML) method based on the General Time Reversible (GTR) model. The confidence of the branch nodes of the phylogenetic tree was estimated by the bootstrap test, and a total of 1000 tests were performed. Figure 3 As shown, the results showed that three endophytic bacteria DW8SB1, DW8SB2 and DW8SB3 were isolated from DW8S. The 16S rRNA gene sequence similarity between DW8SB1 and Brucella pseudogrignonensis was 99.41%, the 16S rRNA gene sequence similarity between DW8SB2 and Sphingobacterium multivorum was 99.53%, and the 16S rRNA gene sequence similarity between DW8SB3 and Stenotrophomonas rhizophila was 100%.

[0049] Example 3: Testing of Chlorine Resistance of Amoeba and Its Endophytic Bacteria

[0050] The purified amoeba isolate was transferred to an SM / 5 agar plate coated with 200 μL of Klebsiella pneumoniae with an OD600 value of 1.5 and cultured at 21°C for five days. The plate was rinsed with KK2 buffer and the amoeba spores were collected in a 1.5 mL sterile centrifuge tube. The collected amoeba spore suspension was centrifuged at 2000 rpm for 5 min and the supernatant was removed. This was repeated 5-6 times to remove food bacteria and bacteria attached to the surface of the amoeba. The cells were resuspended in KK2 buffer to a concentration of 5 × 10 5 spores / mL. Dilute 5% sodium hypochlorite (NaOCl) solution in ultrapure water to 1000 mg / L to prepare a free chlorine stock solution. Then add an appropriate amount of the stock solution to the prepared DW8S cell suspension so that the original free chlorine concentration is 2 mg / L. Samples were collected every 5 minutes during the disinfection process. The residual chlorine concentration at each time point was determined using a residual chlorine analyzer (HACH, DR2800) using the diethyl-p-phenylenediamine (DPD) colorimetric method. Sodium thiosulfate (Na2S2O3) was immediately added to the collected samples to quench the residual chlorine, and the molar ratio of chlorine to sodium thiosulfate was 1:3. The disinfected amoeba spores were gradient diluted and mixed with 200 μL, OD 600 The Klebsiella pneumoniae with a CT value of 1.5 was mixed and spread on SM / 5 agar plates and cultured at 21°C. During the culture process, observations were made regularly until predation spots appeared, and the number of live spores of amoeba was calculated by recording the number of predation spots. The bacteria in the amoeba spores were released by grinding and crushing, and the colony forming units (CFU) were counted by dilution and spreading. The disinfectant attenuation curve was drawn according to the residual chlorine concentration at different time points. The CT value was expressed as the product of the disinfectant concentration (mg / L) and the reaction time (min), and was calculated by linear regression using the disinfectant attenuation curve. The results are shown in Figure 4 As shown in the figure, the residual chlorine concentration in the amoeba DW8S system was still greater than 1.0 mg / L after 30 minutes of residual chlorine disinfection, and the residual chlorine reduction rate was slow. The CT value calculated after 30 minutes of disinfection was 36.405 (mg·min) / L. However, in the DW6 system, the residual chlorine was rapidly consumed within 10 minutes, and the CT value calculated after 30 minutes of disinfection was only 9.819 (mg·min) / L.

[0051] The inactivation rate of amoeba spores and intracellular bacteria is expressed as logarithmic removal rate, calculated as follows: n = log10 (N0 / N t ), where N0 and N t The inactivation characteristic curves of amoeba DW8S and DW6 are shown in Figure 5As shown. The chlorine resistance of DW8S is much higher than that of DW6. When the CT value reaches 35 (mg·min) / L, the inactivation rate is less than 1-log (1-log means 90% removal rate). Chlorine disinfection has a better inactivation effect on DW6, and a 4-log inactivation rate can be achieved when the CT value is less than 10 (mg·min) / L. Further investigation of the inactivation effect of chlorine disinfection on bacteria within the spores of amoeba DW8S found that after the CT value reached 10 (mg·min) / L, the inactivation rate of bacteria within the spores could reach 2.5-log, but the inactivation effect did not increase with the increase of the CT value. The inactivation curve of bacteria within the spores of amoeba DW8S is shown in Figure 6 This result shows that the intraspore bacteria of Amoeba DW8S can resist chlorine disinfection to a certain extent, and survive and spread in the tap water system with Amoeba as a carrier. Therefore, Amoeba DW8S can be used as a novel microbial indicator of drinking water quality. This indicator can not only reflect the new dimension of microbial contamination in the tap water system, but also reflect the disinfection effect of disinfectants. It shows good application prospects in ensuring drinking water safety and provides a new tool for future water quality monitoring and management.

[0052] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. A protozoan amoeba, characterized in that The protozoan amoeba is Vermamoebavermiformis DW8S, which was deposited in the China Center for Type Culture Collection on July 2, 2024, and its deposit number is CCTCCNO: C2024147.

2. The protozoan amoeba according to claim 1, characterized in that: The spore morphology of the protozoan amoeba is spherical.

3. The protozoan amoeba according to claim 1, characterized in that: The spore diameter of the protozoan amoeba is 3 to 5 μm.

4. The protozoan amoeba according to claim 1, characterized in that: The spores of the protozoan amoeba contain opportunistic pathogens, which include one or more of Brucella pseudogrignolifolia, Sphingobacterium multivorum, and Stenotrophomonas rhizophila.

5. Use of the protozoan amoeba described in any one of claims 1 to 4 as a water quality microbial indicator.

6. A method for evaluating the degree of microbial contamination in water, characterized in that: The protozoan amoeba described in any one of claims 1 to 5 is used as a water quality microbial indicator.

7. Use of the protozoan amoeba described in any one of claims 1 to 4 in evaluating the effect of water disinfectants.

8. The use according to claim 7, characterized in that: The water disinfectant is a chlorine-containing disinfectant.

9. A method for evaluating the effect of a water disinfectant, characterized in that: The protozoan amoeba described in any one of claims 1 to 4 is used as a microbial indicator.

10. The evaluation method according to claim 9, characterized in that: The water disinfectant is a chlorine-containing disinfectant.

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