Chlorine-resistant amoeba and application thereof

Abstract

The application belongs to the technical field of microorganisms and particularly relates to a chlorine-resistant amoeba and application thereof. The amoeba is identified as Vermamoeba vermiformis (earthworm-shaped Harmann amoeba) through morphological and genetic analysis and is named DW8S. The test result of chlorine resistance shows that the amoeba DW8S can resist high-intensity chlorine disinfection treatment, and when the CT value reaches 35 (mg·min) / L, the inactivation rate is less than 1-log, the amoeba DW8S carries a plurality of cultivable opportunistic pathogens in spores, and the pathogens still survive in the amoeba spores after chlorine disinfection. In addition, the culture condition of the amoeba DW8S is simple, the amoeba DW8S is easy to preserve, the amoeba DW8S can be used as a novel water quality microbial index, the index can not only reflect a new dimension of microbial pollution in a water system, but also indicate the good or bad of the effect of a disinfectant, and the index shows a good application prospect in guaranteeing water quality safety and provides a new tool for water quality monitoring and management in the future.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology. More specifically, it relates to a chloramphenicol-resistant strain and its applications. Background Technology

[0002] Safe drinking water is a fundamental guarantee of human health and an important public health resource related to national welfare. Quantifying various factors in drinking water that are relevant to human health can effectively control water quality and provide safe drinking water for humanity. However, drinking water systems contain a variety of complex microbial communities, such as bacteria, eukaryotes, and viruses. These microbial communities not only exist in flowing tap water but can also attach to other suspended solids or form biofilms on the inner walls of water pipes. Although the diversity of drinking water microbial communities has significantly decreased after the widespread use of disinfectants such as chlorine and other treatment steps, some microbial species have shown high resistance to disinfectants and survived in drinking water systems.

[0003] Amoeba are a group of single-celled heterotrophic eukaryotes that feed on bacteria and are a major eukaryotic group in the microbial community of drinking water systems. Infections with some amoebic groups can cause keratitis or meningitis, seriously threatening human health. Furthermore, amoebas can promote the spread of pathogenic bacteria. Studies have found that some opportunistic pathogens can resist amoeba predation and persist and even multiply within cells. Moreover, some of these surviving bacteria have been identified as containing antibiotic resistance genes and mobile genetic elements, indicating that amoebas may act as carriers to facilitate the environmental spread of antibiotic resistance genes, posing a serious threat to human health. Research shows that amoebas can form hard, thick spores to evade adverse environments and exhibit high resistance to disinfectants. Therefore, the presence of amoebas in tap water and their interaction with pathogenic bacteria pose a significant threat to drinking water safety and present new challenges to the improvement of drinking water quality.

[0004] Although existing studies have found that amoebae are widely present in drinking water systems and have been isolated and cultured, our understanding of amoebae's tolerance to disinfectants is very limited, and biological resources of chlorine-resistant amoebae need further exploration. Currently, most drinking water disinfection technologies use bacteria as microbial indicators for effectiveness assessment. Some studies have also attempted to use the soil-derived amoebae model organism *Dictyostelium discoideum* to explore the inactivation efficiency of disinfectants on amoebae. However, this experimental subject has significant limitations, as differences in the amoebae's living environment can affect their resistance to chlorine disinfection mechanisms.

[0005] Therefore, isolating and culturing amoebas in drinking water systems can effectively advance research on ensuring the safety of drinking water quality and has significant practical implications. Summary of the Invention

[0006] The technical problem to be solved by this invention is to overcome the fact that amoebae from different sources have different tolerances to disinfectants. Currently, the use of soil-derived amoebae as a microbial indicator of water quality has significant limitations and cannot effectively indicate the defects and deficiencies of water disinfection. This invention provides a protozoan amoebae isolated from drinking water systems with high chlorine tolerance, providing a new model organism for drinking water risk assessment, and providing a theoretical basis and feasible suggestions for the optimization of drinking water disinfection technology and the control of chlorine-tolerant amoebae.

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

[0008] Another objective of this invention is to provide a method for assessing the degree of microbial contamination in water.

[0009] Another object of the present invention is to provide the application of the aforementioned protozoan amoeba in evaluating the effectiveness of water disinfectants.

[0010] Another objective of this invention is to provide a method for evaluating the effectiveness of water disinfectants.

[0011] The above-mentioned objective of this invention is achieved through the following technical solution:

[0012] This invention protects a protozoan amoeba, namely Vermamoebavermiformis (Entamoeba harmanii) DW8S, which was deposited at the China Center for Type Culture Collection on July 2, 2024, with accession number CCTCC NO: C2024147.

[0013] Microorganisms are a general term for tiny organisms that are difficult to see directly with the naked eye. They include microorganisms with cellular structures such as bacteria, archaea, fungi, actinomycetes, protozoa, and algae, as well as organisms without complete cellular structures such as viruses, mycoplasma, and chlamydia. Free-living amoebae (FLA) are protozoa that are dispersed in various environments and belong to the microbial community (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, formerly known as Hartmannella vermiformis, is a type of free-living amoeba, a ubiquitous microorganism found in both natural and artificial habitats. As predators of other microorganisms, free-living amoebas help regulate microbial communities, maintain population sizes, promote microbial species diversity, and maintain the flow of energy and matter through food webs (Delafont, Vincent, et al. "Vermamoeba vermiformis: a free-living amoeba of interest." Microbial ecology 76.4 (2018): 991-1001.).

[0015] This invention yielded a *Vermamoeba vermiformis* DW8S strain isolated from a drinking water system. Compared to *Vermamoeba vermiformis* DW6, which was isolated simultaneously, DW8S exhibits stronger chlorine resistance. Disinfection experiments using sodium hypochlorite as a disinfectant revealed that even with a CT value of 35 mg / L, the inactivation rate was still less than 1-log. my country's *Standards for Drinking Water Quality* stipulates that the CT value for free chlorine disinfection should be 9.0 mg / L, and *Escherichia coli* should not be detected as a microbiological indicator. However, at this CT value, chlorine disinfection was ineffective in inactivating the isolated *Vermamoeba vermiformis* DW8S. Furthermore, these *Vermamoeba* spores carry various opportunistic pathogens and can maintain the persistent presence of endophytic bacteria within the spores after chlorine disinfection. By screening and isolating chlorine-resistant amoebas, we can more accurately grasp the composition of amoeba communities in water quality, providing a more accurate theoretical basis for the microbial safety assessment of water quality, especially drinking water, and providing technical support for the selection of disinfection technologies and process improvement in water supply plants, thereby better ensuring the biological safety of water quality.

[0016] Furthermore, the spores of the protozoan amoeba are spherical.

[0017] Furthermore, the spores of the protozoan amoeba have a diameter of 3–5 μm.

[0018] Furthermore, the spores of the protozoan amoeba contain opportunistic pathogens, including one or more of *Brucella pseudogrinnioni*, *Sphingospholipidobacterium polyphaga*, and *Oligotrophomonas rhizotrophus*.

[0019] Further, the isolation and culture of the amoebae includes the following steps: the water sample is enriched and concentrated through a 0.22 μm polycarbonate membrane, the filter membrane is removed and placed on a non-nutrient agar (NNA) medium coated with food bacterium Klebsiella pneumoniae (2.2 g potassium dihydrogen phosphate, 1.7 g dipotassium hydrogen phosphate, 15.0 g agar, 1.0 L water) and cultured at 21 °C. The movement of amoebae cells and the formation of spores on the plate are observed under a microscope. The amoebae spores on the plate are picked up using a sterile inoculation loop and transferred to a new non-nutrient agar plate coated with Klebsiella pneumoniae for further purification.

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

[0021] Furthermore, the water is drinking water.

[0022] This invention also protects a method for assessing the degree of microbial contamination in water, using the aforementioned protozoan amoeba as a microbial indicator for water quality. This method reflects a new dimension of microbial contamination in water quality by detecting the presence and quantity of this amoeba.

[0023] Furthermore, the water is drinking water.

[0024] This invention also protects the use of the protozoan amoeba in evaluating the effectiveness 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, and more preferably sodium hypochlorite.

[0027] This invention also protects a method for evaluating the effectiveness of a water disinfectant, using the aforementioned amoebae as a microbial indicator. This method assesses the disinfectant's effectiveness against microorganisms by monitoring the survival of the amoebae after disinfection treatment.

[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, and more preferably sodium hypochlorite.

[0030] Compared with existing technologies, this invention has the following advantages: This invention provides a highly chlorine-resistant protozoan amoebae, DW8S, isolated from a drinking water system. Morphological and genetic analysis identified this amoebae as *Vermamoeba vermiformis*. Chlorine resistance testing results show that the amoebae DW8S can resist high-intensity chlorine disinfection; when the CT value reaches 35 mg / L, the inactivation rate is less than 1-log, and its spores carry various culturable opportunistic pathogens, which remain viable within the amoebae spores after chlorine disinfection. Furthermore, the culture conditions for this amoebae DW8S are simple, and it is easy to preserve. It can serve as a novel microbial indicator for water quality, reflecting a new dimension of microbial contamination in water systems and demonstrating promising application prospects in ensuring water quality safety, providing a new tool for future water quality monitoring and management. Attached Figure Description

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

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

[0033] Figure 3 Phylogenetic tree constructed from the 16S rRNA gene sequence of the endosymbiotic bacteria of Entamoeba DW8S.

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

[0035] Figure 5 The inactivation characteristics curves of amoeba DW8S and DW6 are shown.

[0036] Figure 6 The graph shows the inactivation characteristics of endosymbiotic bacteria in the amoeba DW8S. Detailed Implementation

[0037] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but the embodiments do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in this technical field.

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

[0039] Example 1: Isolation and Purification of Amoeba

[0040] A 2L sample of tap water was collected and concentrated using a 0.22μm polycarbonate membrane. The membrane was then removed and inverted to cover a 200μL container coated with OD (October Spectrometry) solution. 600 Klebsiella pneumoniae was incubated on non-nutrient agar (NNA) medium (potassium dihydrogen phosphate 2.2 g, dipotassium hydrogen phosphate 1.7 g, agar 15.0 g, pure water 1.0 L) at 21°C for one week. The presence of amoebae was identified by observing the migration peaks and amoebae cells formed under a microscope. Amoebae spores were picked from the plates using a sterile inoculation loop and transferred to new non-nutrient agar plates coated with Klebsiella pneumoniae for further purification.

[0041] Example 2: Identification of Amoeba

[0042] (1) Morphological identification

[0043] The isolated and purified amoebae were placed on non-nutrient agar plates coated with 200 μL of Klebsiella pneumoniae with an OD600 value of 1.5 and incubated at 21°C for one week. During the incubation period, the plates were observed under a microscope and photographs were taken of the amoebae in the spore stage. The results are as follows: Figure 1As shown, the left side shows the sporophyte morphology of Entamoeba histolytica DW8S, and the right side shows the sporophyte morphology of Entamoeba histolytica DW6. Both types of entamoeba spores are spherical, with DW8S spores having a diameter of approximately 3-5 μm and DW6 spores having a diameter of approximately 6-8 μm. Morphological differences are minimal between individuals of the same entamoeba species.

[0044] (2) Biological identification

[0045] The purified amoeba was transferred to a coating of 200 μL, OD 600 Klebsiella pneumoniae with a pH of 1.5 was cultured on SM / 5 agar plates (2.0 g glucose, 2.0 g peptone, 2.0 g yeast extract, 0.4 g magnesium sulfate heptahydrate, 1.9 g potassium dihydrogen phosphate, 1.0 g dipotassium hydrogen phosphate, 15.0 g agar, 1.0 L pure water) at 21 °C for five days, and its growth was then observed under a microscope. After amoeboid spore formation, a small number of amoeboid cells were picked and placed in 100 μL of sterile water containing 10% Chelex 100 (50-100 mesh), boiled for 10 min, and then incubated at -20 °C for 10 min, followed by centrifugation at 6000 rpm for 3 min. The supernatant was used for subsequent PCR reactions. Based on the amoeboid spore morphology, genus / species-specific primers and universal eukaryotic primers were selected to amplify the 18S rRNA gene of the isolated amoeboids by PCR. 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 (TaKaRaTaq) buffer. TM The reaction mixture consisted of 2 μL each of Version 2.0 plus dye and forward and reverse primers (10 μmol / L), 6 μL of amoebae DNA, and 15 μL of sterile water. The reaction program was as follows: 94℃ pre-denaturation for 3 min; 94℃ denaturation for 30 s, 60℃ annealing for 30 s, 72℃ extension for 1 min, repeated for 35 cycles; and 72℃ double extension for 10 min. PCR products were detected by 1.5% agarose gel electrophoresis and sequenced. Amoebae DW8S successfully amplified a 711 bp target band, and amoebae DW6 successfully amplified a 729 bp target band.

[0046] The 18S rRNA gene sequences of the isolated amoeba DW8S and DW6 (nucleotide sequence of DW8S shown in SEQ 1; nucleotide sequence of DW6 shown in SEQ 2) were subjected to BLAST homology alignment using the NCBI (National Center for Biotechnology Information) database (https: / / blast.ncbi.nlm.nih.gov). The 18S rRNA phylogenetic trees were constructed using FastTree and IQTree, respectively, with JTT+CAT and TN+F+I parameters selected. Bootstrapping analysis based on 1000 replicates was performed during phylogenetic tree construction to ensure the reliability of the results. The phylogenetic trees were plotted using the iTOL website (https: / / itol.embl.de / itol.cgi). The phylogenetic trees are shown below. Figure 2 As shown, the sequence alignment results indicate that DW8S and DW6 have 100% similarity to the 18S rRNA gene sequence of Vermamoeba vermiformis, and the 18S rRNA gene sequence similarity between DW8S and DW6 is 98.57%.

[0047] The purified amoebae isolate DW8S was transferred to a 200 μL container coated with OD200. 600 Klebsiella pneumoniae with a pH of 1.5 was incubated on SM / 5 agar plates at 21°C for five days, and its growth was then observed under a microscope. After amoeboid spore formation, the plates were washed with KK2 buffer (2.2 g potassium dihydrogen phosphate, 1.7 g dipotassium hydrogen phosphate, 1.0 L water), and the amoeboid spores were collected into 1.5 mL sterile centrifuge tubes. The collected amoeboid spore suspension was centrifuged at 2000 rpm for 5 min to remove the supernatant, and this process was repeated 5-6 times to remove unprepared food bacteria and bacteria attached to the amoeboid surface. The collected amoeboid spores were resuspended in 1 mL of KK2 buffer, and 10 μL was inoculated onto SM / 5 agar plates. Each amoeboid sample was prepared in six replicates. The inoculated plates were incubated 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 new SM / 5 agar plates for continuous culture and purification.

[0048] Purified bacterial cells were collected and placed in 100 μL of sterile water containing 10% Chelex 100 (50-100 mesh). After boiling for 10 min, the mixture was incubated at -20°C for 10 min, followed by centrifugation at 6000 rpm for 3 min. The supernatant was used for subsequent PCR reactions. Universal bacterial primers 27F (5'-AGAGTTTGATCMTGGCTCAG-3') and 1492R (5'-TACGGYTACCTTGTTAYGACTT-3') were used to amplify the 16S rRNA gene of the symbiotic bacteria. PCR was performed in a 50 μL reaction system containing 20 μL of polymerase chain reaction mixture (TaKaRa Taq). TM Version 2.0plus dye), 2 μL each of forward and reverse primers (10 μmol / L), 2 μL of amoeboid DNA, and 19 μL of sterile water were used. The reaction program was: 94℃ pre-denaturation for 3 min; 94℃ denaturation for 30 s, 55℃ annealing for 30 s, 72℃ extension for 1 min, repeated for 35 cycles; 72℃ double extension for 10 min. PCR products were detected by 1.5% agarose gel electrophoresis and sequenced. The isolated bacterial 16S rRNA gene sequence was BLAST homology compared using the NCBI (National Center for Biotechnology Information) database (https: / / blast.ncbi.nlm.nih.gov). Using MEGAX software, a phylogenetic tree was calculated and plotted 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 scores of branch nodes in the phylogenetic tree were estimated using a bootstrap test, with a total of 1000 tests performed. The phylogenetic tree is shown below. Figure 3 As shown, the results indicated that three endophytic bacteria, DW8SB1, DW8SB2, and DW8SB3, were isolated from DW8S. DW8SB1 showed 99.41% sequence similarity to the 16S rRNA gene of *Brucella pseudogrignonensis*, DW8SB2 showed 99.53% sequence similarity to the 16S rRNA gene of *Sphingobacterium multivorum*, and DW8SB3 showed 100% sequence similarity to the 16S rRNA gene of *Stenotrophomonas rhizophila*.

[0049] Example 3: Test for chlorine resistance of amoebae and their endophytic bacteria

[0050] The purified amoebae isolate was transferred to an SM / 5 agar plate coated with 200 μL of Klebsiella pneumoniae (OD600 value 1.5) and incubated at 21°C for five days. The plate was washed with KK2 buffer, and amoebae spores were collected into 1.5 mL sterile centrifuge tubes. The collected amoebae spore suspension was centrifuged at 2000 rpm for 5 min to remove the supernatant. This process was repeated 5-6 times to remove food bacteria and bacteria attached to the amoebae surface. The cells were resuspended in KK2 buffer to achieve a DW8S cell suspension concentration of 5 × 10⁻⁶ cells / mL. 5 1 spore / mL. A 5% sodium hypochlorite (NaOCl) solution was diluted to 1000 mg / L in ultrapure water to prepare a free chlorine stock solution. An appropriate amount of this stock solution was then added to the prepared DW8S cell suspension to bring the initial free chlorine concentration to 2 mg / L. Samples were collected every 5 minutes during disinfection. The residual chlorine concentration at each time point was determined using a residual chlorine analyzer (HACH, DR2800) via the diethyl-p-phenylenediamine (DPD) colorimetric method. Sodium thiosulfate (Na2S2O3) was immediately added to the collected samples to quench the residual chlorine; the molar ratio of chlorine to sodium thiosulfate was 1:3. The disinfected amoebae spores were serially diluted and added to 200 μL of OD200 solution. 600 Klebsiella pneumoniae with a concentration of 1.5 were mixed and spread on SM / 5 agar plates and incubated at 21°C. During incubation, the plates were observed periodically until predatory spots appeared, and the number of viable amoeba spores was calculated by recording the number of predatory spots. Bacteria within the amoeba spores were released by grinding and fragmentation, and counted using colony-forming units (CFU) after dilution and plating. Disinfectant decay curves were plotted based on residual chlorine concentrations at different time points. The CT value was expressed as the product of disinfectant concentration (mg / L) and reaction time (min), and calculated using linear regression of the disinfectant decay curves. Results are as follows: Figure 4 As shown, after 30 minutes of disinfection, the residual chlorine concentration in the Amoeba DW8S system was still greater than 1.0 mg / L, and the rate of chlorine reduction was slow. The calculated CT value 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 calculated CT value after 30 minutes of disinfection was only 9.819 (mg·min) / L.

[0051] The inactivation rate of amoebae spores and intracellular bacteria is expressed as the logarithmic removal rate, calculated using the formula: n = log10(N0 / N t ), where N0 and N t The figures show the number of viable cells before and after disinfection, respectively. The inactivation characteristic curves of amoebae DW8S and DW6 are shown below. Figure 5As shown in the figure, DW8S exhibits significantly higher chlorine resistance than DW6. When the CT value reaches 35 mg / L, the inactivation rate is less than 1-log (1-log represents a 90% removal rate). Chlorine disinfection, however, shows better inactivation of DW6, achieving an inactivation rate of 4-log even with a CT value less than 10 mg / L. Further investigation into the inactivation effect of chlorine disinfection on the intraspore bacteria of Entamoeba histolytica DW8S revealed that the inactivation rate reached 2.5-log after the CT value reached 10 mg / L, but the inactivation effect did not increase with increasing CT value. The inactivation curves of the intraspore bacteria of Entamoeba histolytica DW8S are shown in the figure. Figure 6 As shown in the figure, this result indicates that the intraspores of amoebae DW8S can resist chlorine disinfection to a certain extent and survive and spread in the tap water system using amoebae as a carrier. Therefore, amoebae DW8S can serve as a novel microbiological indicator for drinking water quality. This indicator can not only reflect a new dimension of microbial contamination in the tap water system but also reflect the disinfection effect of disinfectants, demonstrating good application prospects in ensuring drinking water safety and providing a new tool for future water quality monitoring and management.

[0052] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. The application of protozoan amoeba in evaluating the effectiveness of water disinfectants, characterized in that, The water disinfectant is a chlorine-containing disinfectant; The protozoan amoeba is Vermamoeba vermiformis DW8S was deposited at the China Center for Type Culture Collection on July 2, 2024, with accession number CCTCC NO: C2024147. The spores of the protozoan amoeba contain opportunistic pathogens, including *Brucella pseudogrinnioni*. Brucella pseudogrignonensis Polysphingosine monocytogenes Sphingobacterium multivorum Oligotrophic rhizobia Stenotrophomonas rhizophila One or more of the following; The assessment was conducted by monitoring the survival of the amoeba after disinfection treatment to evaluate the disinfectant's effectiveness in killing microorganisms.

2. The application according to claim 1, characterized in that, The spores of the protozoan amoeba are spherical.

3. The application according to claim 1, characterized in that, The spores of the protozoan amoeba have a diameter of 3–5 μm.

4. The application according to claim 1, characterized in that, The chlorine-containing disinfectant is selected from sodium hypochlorite, bleaching powder, sodium dichloroisocyanurate, trichloroisocyanuric acid, or chlorine dioxide.

5. A method for evaluating the effectiveness of a water disinfectant, characterized in that, Using protozoan amoeba as a microbial indicator; The protozoan amoeba is Vermamoeba vermiformis DW8S was deposited at the China Center for Type Culture Collection on July 2, 2024, with accession number CCTCC NO: C2024147. The spores of the protozoan amoeba contain opportunistic pathogens, including *Brucella pseudogrinnioni*. Brucella pseudogrignonensis Polysphingosine monocytogenes Sphingobacterium multivorum Oligotrophic rhizobia Stenotrophomonas rhizophila One or more of the following; The evaluation method involves monitoring the survival of the amoeba after disinfection to assess the disinfectant's effectiveness in killing microorganisms. The water disinfectant is a chlorine-containing disinfectant.

6. The evaluation method according to claim 5, characterized in that, The spores of the protozoan amoeba are spherical.

7. The evaluation method according to claim 5, characterized in that, The spores of the protozoan amoeba have a diameter of 3–5 μm.

8. The evaluation method according to claim 5, characterized in that, The chlorine-containing disinfectant is selected from sodium hypochlorite, bleaching powder, sodium dichloroisocyanurate, trichloroisocyanuric acid, or chlorine dioxide.

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