JHM column: Shu Longfei and Yang Xin team from Sun Yat-sen University: Amoeba can protect the bacteria in the body from the inactivation of drinking water disinfection

Click on the blue text above to follow our first author: He Zhenzhen Corresponding author: Shu Longfei, Yang Xin Correspondence unit: Sun Yat-sen University School of Environmental Science and Engineering Paper DOI: 10.
1016/j.
jhazmat.
2021.
126006 Picture Abstract Introduction Recently, the team of Shu Longfei and Yang Xin of Sun Yat-sen University The Journal of Hazardous Materials published a research paper entitled "Both viable and inactivated amoeba spores protect their intracellular bacteria from drinking water disinfection"
.

Traditional disinfection focuses on a single pathogen, but how the interaction between pathogens affects disinfection is unclear
.

We found that amoeba can protect the bacteria in its body from the inactivation of drinking water disinfection, indicating that microbial interaction will bring new challenges to drinking water biosafety
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Graphic guide COVID-19 has caused attention to biosafety and research on new pathogens.
Water-borne pathogens and related diseases are a major global public health threat.
However, most research focuses on a single source of infection, such as bacteria, Fungi, viruses and protists, the interaction between these microorganisms on the disinfection process has been widely ignored, one of which is the amoeba-bacterial interaction
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As one of the six major groups of protists, amoebas are widely distributed in water environments and are frequently detected in tap water systems
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Recent studies have found that amoeba has a complex interaction with other microorganisms.
Amoeba can serve as an environmental carrier for other microorganisms (such as bacteria, fungi and viruses), and most of the microorganisms that can be carried by amoeba are human pathogens.

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Amoeba has a certain tolerance to water disinfection treatment, but it is unclear to what extent the amoeba spores can protect the bacteria in the body from the inactivation of disinfection treatment
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In this study, the amoebic spores were treated with different concentrations of chlorine (1, 5, 20, and 50 mg/L) and different pH (5, 6.
5, and 9) (Figure 1)
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The results show that the amoebic spores are highly tolerated.
Low concentration (1mg/L) chlorine treatment cannot inactivate the amoebic spores.
After treatment for 3 minutes, the chlorine concentration is increased from 5 mg/L to 50 mg /L, the inactivation efficiency increased from 2-log to 4-log (Figure 1a)
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A CT value of 150 mg·min/L is required to achieve 4 log of amoebic spore inactivation (Figure 1b)
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Figure 1: Free chlorine inactivates amoebic spores at different concentrations (a, b, pH = 6.
5) and pH (c, initial chlorine concentration is 5mg/L)
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Reaction conditions: T = 25±1℃, the initial concentration of amoebic spores is about 5×105 spores/mL
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Before analyzing the protective effect of amoeba spores on bacteria in the body, this study first constructed an amoeba-Burkholderia system and confirmed it with confocal microscope, transmission electron microscope and scanning electron microscope (Figure 2)
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Figure 2: The morphology of amoebic spores (ad) and bacteria-containing amoebic spores (eh)
.

(A) Amoebic spores under an optical microscope; (b) Amoebic spores under a confocal microscope (calcium fluorescence staining shows the polysaccharide polymer cell wall of the spores); (c) Transmission electron microscope image of amoebic spores; ( d) Scanning electron microscope image of amoebic spores; (e) Amoebic spores containing Burkholderia (marked with rfp); (f) Amoebic spores and Burkholderia within the spores; (g ) Transmission electron microscopy images of Amoeba spores and Burkholderia in their bodies; (h) Scanning electron microscopy images of Amoeba spores carrying Burkholderia
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Ratio: a, e: 10µm; the rest are 2µm
.

Chlorine treatment, ultraviolet light (UV254), and chlorine dioxide disinfection respectively treat free bacteria and bacteria in amoebic spores.
The results show that amoebic spores have a significant sheltering effect on the bacteria in the body (Figure 3)
.

When bacteria are exposed to chlorine disinfection, they achieve 4-log inactivation at a CT value of 2.
1 mg·min/L and 6-log inactivation at a CT value of 4.
08 mg·min/L (Figure 3a), but they hide in The bacteria in the spores of amoeba showed strong resistance to chlorine.
When the CT value reached 40 mg·min/L, the inactivation rate was less than 3-log (Figure 3a)
.

The results of ultraviolet disinfection (Figure 3b) and chlorine dioxide treatment (Figure 3c) are similar: bacteria exposed to UV254 alone can achieve a 6-log inactivation effect at 20 J/m2, while hiding in amoeba at the same dose The bacteria in the spores can only achieve a 1-log reduction (Figure 3b); when the CT value of chlorine dioxide disinfection is 60 mg·min/L, the maximum inactivation rate of the bacteria in the amoebic spores is still less than 3-log , While the free bacteria were quickly inactivated at the first sampling point (Figure 3c)
.

Figure 3: Chlorine (a), ultraviolet light (b) and chlorine dioxide (c) inactivate bacteria in amoeba spores
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Reaction conditions: T = 25±1℃, pH = 6.
5, the initial concentration of Cl2 and ClO2 is 5 mg/L, the initial concentration of amoebic spores is about 5 × 105 spores/mL, and the initial concentration of bacteria in the spores is about 2 × 106 CFU/mL, the initial concentration of bacteria is about 5 × 106 CFU/mL
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In this study, the amoebic spores and the amoebic spores carrying bacteria were treated by chlorine, chlorine dioxide and ultraviolet light respectively, and the inactivation effect of the conventional water disinfection treatment on the amoebic spores was analyzed, and the effect of the bacteria carrying bacteria on the amoeba spores was further discussed.
The impact of Miba spore tolerance (Figure 4)
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The results showed that both chlorine dioxide and UV254 disinfection had better inactivation effects on amoebic spores than chlorine disinfection, and the carrying bacteria had little effect on the tolerance of amoebic spores (Figure 4)
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Interestingly, this study found that although some amoebic spores are inactivated under a certain exposure dose, they can still protect the bacteria in the body from disinfection and inactivation
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For example, when the UV254 disinfection dose was increased from 50 to 200 J/m2, the inactivation rate of amoeba spores increased sharply from 2-log to 5-log, but the inactivation rate of bacteria in the amoebas hardly changed (Figure 3 and Figure 4)
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The same phenomenon also exists in the results of chlorine and chlorine dioxide treatment
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These results raise a new potential drinking water safety problem, that is, inactivated microorganisms can still protect the microorganisms in their cells from disinfection
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Figure 4: Chlorine (a), ultraviolet light (b) and chlorine dioxide (c) inactivate the spores of amoeba that carry and non-stain
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Reaction conditions: T = 25±1℃, pH = 6.
5, the initial concentration of Cl2 and ClO2 is 5 mg/L, and the initial concentration of amoebic spores is about 5 × 105 spores/mL
.

Comparison of transmission electron microscopy and scanning electron microscopy showed that there was no difference in the morphology of amoeba spores and bacteria in their bodies before and after inactivation (Figure 5).
After three treatments (Cl2, ClO2 and UV254), all spores remained intact, and no spore damage or damage was seen.
Broken
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Interestingly, according to the previous results (Figure 4), most spores are actually inactivated, but their structure remains intact, which may explain why inactivated amoebic spores can still protect their intracellular bacteria
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Finally, the image shows that the structure of the bacteria in the amoeba spores is also intact, which shows that the amoeba does play a protective role in the disinfection process
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Figure 5: Ultrastructural analysis of amoebic spores and intracellular bacteria before and after three kinds of disinfection (Cl2, ClO2, UV254)
.

Reaction conditions: T = 25±1℃, pH = 6.
5, the initial concentration of Cl2 and ClO2 is 5 mg/L, the initial concentration of amoebic spores is about 5 × 105 spores/mL, and the initial concentration of bacteria in the spores is about 2 × 106 CFU/mL, the initial concentration of bacteria is about 5 × 106 CFU/mL
.

The CT values ​​of Cl2 and ClO2 at the treatment end points were 40.
6 and 88.
5 mg·min/L, respectively, and the dose of UV254 was 192.
6 J/m2
.

Summary This study shows that amoebic spores have a significant sheltering effect on the bacteria carried in the body, and the current disinfection dose of drinking water system is limited to the inactivation of bacteria hidden in amoebic spores
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But so far, it is not clear whether the disinfection method can induce the formation of amoebic spores and cysts, but it is very likely that, like other environmental stressors, disinfectants (such as chemical fungicides) may be in the drinking water system.
Induce the formation of spores
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Therefore, this kind of shelter will undoubtedly pose a major threat to drinking water safety and public health
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Future research should conduct a large-scale investigation of the distribution of amoebas in the drinking water system
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And given that many pathogenic amoebas are thermophilic, it is particularly important to assess the impact of global warming on the distribution of pathogenic amoebas in drinking water systems
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Future research should also better describe the microbial communities that live in various amoebas, and should be committed to developing new technologies to deal with this hidden danger of drinking water safety
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Introduction of the main author Corresponding author: Shu Longfei, doctoral supervisor, associate professor of the School of Environmental Science and Engineering, Sun Yat-sen University
.

Ph.
D.
graduated from ETH Zurich
.

Mainly focus on amoeba to carry out environmental ecology related research, including: environmental microbial ecology and evolution, soil microbiological ecology, and drinking water microbial safety
.

Published more than 30 papers in mainstream journals such as The ISME Journal, eLife, PLOS Biology, Molecular Ecology, Functional Ecology, Environmental Science & Technology, Journal of Hazardous Materials, Applied and Environmental Microbiology, and hosted the National Natural Science Foundation of China.
Seven domestic and foreign projects including the Life Science Foundation
.

Corresponding author: Yang Xin, female, doctoral supervisor, professor of School of Environmental Science and Engineering, Sun Yat-sen University
.

He graduated from the Department of Environmental Science of Nankai University and entered the Hong Kong University of Science and Technology in 2002 for further studies.
In 2007, he received a Ph.
D.
from the Department of Civil and Environmental Engineering of the Hong Kong University of Science and Technology
.

Mainly engaged in drinking water safety and control water pollution, including disinfection of disinfection by-products are formed and migration and transformation technology and control technology and control mechanism, trace contaminants and the like
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Presided over more than ten projects including the National Natural Science Foundation Outstanding Youth Fund and Guangdong Outstanding Youth Fund
.

More than 90 SCI papers have been published in important international academic journals, and 5 papers have been selected as ESI highly cited papers
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He is a member of the China Youth Committee of the International Water Association, a member of the Environmental Chemistry Branch of the Chinese Society of Environmental Sciences, a guest editor of Water Science & Technology: Water Supply, an associate editor of Bulletin Environmental Contamination and Toxicology Asia, and a member of the editorial board of the Journal of Hazardous Materials
.

The first author: He Zhenzhen, a postgraduate student of the School of Environmental Science and Engineering, Sun Yat-sen University, email: hezhzh8@mail2.
sysu.
edu.
cn
.

Contribution: The team of Shu Longfei and Yang Xin of Sun Yat-Sen University
.

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