Use of Host-Specific Bacteroidales 16S rRNA for Microbial Source Tracking of Environmental Water in Hanoi, Vietnam

VIETNAM NATIONAL UNIVERSITY VIETNAM-JAPAN UNIVERSITY - PHAM MINH NGOC USE OF HOST-SPECIFIC BACTEROIDALES 16S rRNA FOR MICROBIAL SOURCE TRACKING OF ENVIRONMENTAL WATER IN HANOI, VIETNAM MASTER THESIS Hanoi - 2019 VIETNAM NATIONAL UNIVERSITY VIETNAM-JAPAN UNIVERSITY - PHAM MINH NGOC USE OF HOST-SPECIFIC BACTEROIDALES 16S rRNA FOR MICROBIAL SOURCE TRACKING OF ENVIRONMENTAL WATER IN HANOI, VIETNAM PROGRAM: MASTER OF ENVIRONMENTAL ENGINEERING CODE: PILOT SUPERVISORS PROF HIROYUKI KATAYAMA ASSOC.PROF IKURO KASUGA Hanoi - 2019 TABLE CONTENT LIST OF TABLES ii LIST OF FIGURES iii ABBREVIATION .v ACKNOWLEDGMENTS vi INTRODUCTION vii CHAPTER 1: LITERATURE REVIEW 1.1 Fecal indicator bacteria (Escherichia coli) 1.2 Bacteroidales bacteria 1.3 Microbial source tracking method CHAPTER 2: MATERIAL AND METHODOLOGY .16 2.1 Material and Equipment 16 2.2 Methodology 16 CHAPTER 3: RESULTS AND DISCUSSIONS 33 3.1 Case study in Kanazawa 33 3.2 Case study in Hanoi .39 CHAPTER IV CONCLUSION 52 REFERENCE 53 i LIST OF TABLES Table 1.1 :The primer and probe sequence of host- specific Bacteroidales marker 10 Table 2.1: The information of sampling point for Pig-fecal source 18 Table 2.2: The information of sampling point for chicken-fecal source 18 Table 2.3: The information of sampling point for duck- fecal source 19 Table 2.4: The information of water sampling point 19 Table 2.5: The Primer and Probe of host-specific Bateroidales markers 29 Table 3.1: The information of sampling places in Kahokugata Lake .34 Table 3.2: Possitive ratios and mean concentrations of host-specific Bacteroidales markers in water sample 37 Table 3.3: Possitive ratios and mean concentrations of animal-specific Bacteroidales markers in fecal-source sample and water sample .47 ii LIST OF FIGURES Figure 1.1 Main orders of Bacteroidetes Figure 1.2: The concept of Microbial Source Tracking Figure 2.1 :The sampling point at the South of Hanoi 17 Figure 2.2: The process of culture E coli for fecal sample 23 Figure 2.3: The process of culture E coli for water sample .23 Figure 2.4: The steps of diluted standard solution 31 Figure 3.1: The sampling points at Kahokugata Lake 33 Figure 3.2: The concentration of E coli and total coliform of water sample 35 Figure 3.3: Relation between concentration of pig-specific assay and E coli bacteria in water sample 38 Figure 3.4: The concentration of E coli and Total coliform of Pig-fecal source 39 Figure 3.5: The concentration of E coli and Total coliform of Chicken-fecal source 40 Figure 3.6: The concentration of E coli and Total coliform of Duck-fecal source 41 Figure 3.7: The concentration of E coli and Total coliform in Day river 42 Figure 3.8: The Pig-2-Bac marker’s standard curve 43 Figure 3.9: The amplification plot of Pig-2-Bac .44 Figure 3.10: The Chicken/ Duck-Bac marker’s standard curve .45 Figure 3.11: The amplification plot of Chicken/Duck-Bac marker 46 Figure 3.12: Relation between concentrations of pig-specific Bacteroidales marker (Pig-2-Bac) and E coli bacteria throughout the fecal sample and water sample .49 iii LIST OF PICTURES Picture 2.1: The Duck farm .20 Picture 2.2: The Chicken farm 21 Picture 2.3: The Pig farm 21 Picture 2.4: One of the Day river's sampling point (R3) 22 iv ABBREVIATION 16S rRNA: 16S ribosomal RNA E coli: Escherichia coli FIB: Fecal Indicator Bacteria LD: Library-dependent LID: Library-independent MST: Microbial Source Tracking PCR: Polymerase Chains Reaction qPCR: Real-time Polymerase Chains Reaction v ACKNOWLEDGMENTS I would like to express my gratitude to all those who gave me the possibility to complete during my thesis I want to thank the Master of Environmental Engineering Lab of Vietnam-Japan University, Vietnam National University, and Nagasaki in National Institute of Hygiene and Epidemiology, Hanoi Besides, I want to thank the Microbial lab of professor Honda at Kanazawa University for giving me permission to carry out experiments to commence this thesis in the first instance Moreover, I appreciate the JICA company that supported me a lot in the Master program I am deeply indebted to my supervisor – Prof Katayama and Assoc Prof Kasuga for their enthusiastic instruction throughout my thesis time I also would like to thank all members in MEE laboratory for their help, support, interest and valuable hints Hanoi, May 2019 Student Phạm Minh Ngọc vi INTRODUCTION Vietnam is a developing country towards a sustainable development Therefore, the problem of environmental pollution is always an urgent issue and is concerned Currently, partly due to industrial development, tons of untreated wastewater are discharged directly into water inlet sluice, rivers, and lakes Pollutants such as organic substances and metals untreated penetrate directly into the water source In addition, in urban areas, waste is thrown away in many places, causing sewage congestion around areas such as To Lich River, Nhue River and Day River which have the phenomenon of pollution and stinking generated by garbage In addition, in rural areas where livestock and poultry breeding, the water source was usually contaminated by human feces and animal feces Because waste treatment conditions in those areas have not been developed and backward, besides, it has small livestock Instead of collecting and treating according to the system, waste will normally be discharged directly into the surrounding environment such as groundwater and surface water (rivers and lakes) lead to the fecal pollution in environment water These are causes of the incidence of water-related diseases such as E coli infected diarrhea, dermatitis, or eye diseases They are increasing and likely to spread disease Hence, the pollution of the water environment is affecting directly to the health of people In Hanoi, one of the largest rivers flowing through many communes of Hanoi is the Day River This river starts from the Red River, and it goes through many communes which have many livestock and poultry farms (mainly pigs, chickens, ducks, and cows) that discharge livestock waste directly into that river area Therefore, the Day river has a lot of potential for being contaminated by the feces source However, the current fecal pollution in Day River is only determined by the FIB method, especially the use of E coli culture in the lab Therefore, the managers still not know exactly the pollution sources of there, whether the main source of pollution in the Day river basin comes from the treatment of livestock waste vii Currently in the world, in many developed countries, in addition to using the FIB method to assess water quality, the Microbial source tracking (MST) method has been developed to find sources of fecal contamination From there, managers can assess water quality and human health risks The MST method is now widely used with specific hosts of the 16S rRNA gene of Bacteroidales bacteria which were found unique in feces, rumen and other cavities of humans and animals, often in greater abundance than traditionally used coliform bacteria [1] In previous studies, manure contamination was found from the source of some animals, was based on the PCR endpoint method of the 16S rRNA gene of Bacteroides In addition, more advanced with the qPCR method determined an accurate concentration of pollutants according to the number of copies and combined with microbial source tracking method as a marker and identify sources of pollution [2] Microbial source tracking is a useful and important method and/or tool for environmental authorities to seek the source of possible outbreaks of animal diseases In Vietnam, a present outbreak of African pig cholera and past lessons of H5N1 chicken fever are firm evidence of the necessity of this research Therefore, this research topic on "Use of Host-Specific Bacteroidales 16S rRNA for Microbial Source Tracking (MST) of Environmental Water in Hanoi, Vietnam" which was to assess and detect the source of fecal pollution on Day River, which originates from pigs, chickens or ducks by application the method of biomarkers Using the specific host of the 16S rRNA Bacteroidales gene as a marker detect pollution sources by real-time PCR (qPCR) In addition, the study also assesses and compares the specificity and sensitivity of markers in Hanoi environment The study highlights the scientific significance and practicality of the topic The structure of this study including four main parts and it will be shown as below: CHAPTER 1: LITERATURE REVIEW 1.1 Fecal indicator bacteria 1.2 Bacteroidales bacteria 1.3 Microbial source tracking viii 3.2.2 Microbial source tracking’s result Figure 3.8: The Pig-2-Bac marker’s standard curve From the Fig 3.8, the Pig-2-Bac marker’s standard curve has equation being: , the coefficient R2 was 0.996 and the amplification efficiencies of this qPCR assay was 0.98 (Fig 3.9) 43 Figure 3.9: The amplification plot of Pig-2-Bac The amplification plot for the standard was presented by red color with series diluted from 106 to 101 copies/µl From the amplification plot, it was easily recognized the positive samples which appeared the plot by comparing with standard plot and negative control plot 44 Figure 3.10: The Chicken/ Duck-Bac marker’s standard curve For the Chicken/Duck-Bac, the standard curve with equation is: , the coefficient R2 was 0.994 and the amplification efficiencies of this qPCR assay was 1.02 (Fig 3.10 and 3.11) 45 Figure 3.11: The amplification plot of Chicken/Duck-Bac marker From Fig 3.8 and Fig 3.10, the quantification limits were 20 copies/reactions for both markers (Pig-2-Bac and Chicken/Duck-Bac) The result of positive samples and their concentration were detected by two markers as shown in table 3.3 below 46 Table 3.3: Possitive ratios and mean concentrations of animal-specific Bacteroidales markers in fecal-source sample and water sample Source No.of samples tested Pig-2-Bac Chicken/Duck-Bac No.of Conc.* No.of Conc.* possitive (mean ± possitive (mean ± samples SD) samples SD) (%) (%) Pig 12 11 (91.7) 7.73 ± 1.79 7(58) 5.01 ± 0.82 Chicken (12.5) 1.77 1(12.5) 5.53 Duck (22.2) 3.22 ± 1.29 6(66.67) 5.22 ± 1.77 Day River 6 (100) 4(66.7) Target 11 (91.7) 7(41.2) Non-target (17.6) 7(58) Sensitivity 91.7 41.2 82.4 41.7 86 41.4 (%) Specificity (%) Accuracy (%) Con.c: the concentration of the sample which has a concentration higher than LOQ value; *: unit, log10(copies/ml); ND: non-detected SD: standard deviation In the previous study such as B.Malla’s study at Nepal [40] and Mieszkin’s study in France [14], the Pig-2-Bac assay showed 100% sensitive when detecting the targethost fecal source Hence, this marker was checked in Hanoi environment, the result showed that Pig-specific Bacteroidales can detect almost target-host fecal source 47 sample which collected with 91.7% sensitivities The quantity value of Pig-2-Bac is 7.73 ± 1.79log10 (mean ± standard deviation) copies/g wet feces in pig, this result is higher than the value in the previous study [40] about 2log10 Likewise, comparing this result with the Mieszkin’s study[14], the concentration of E coli was 6.9 ± 0.7 log10 MNP/g of feces and the concentration of Pig-2-Bac was 8.6 ± 0.5 log10 copies/g, and in this case, it was 5.95 ± 1.2 log10 CFU/g and 7.73 ± 1.7 log10 copies/g, respectively As a result, Pig-2-Bac marker improved its specificity in this study with approximate 10 times E coli in both two study Moreover, the Pig-2-Bac marker has cross-reaction with the chicken fecal- source 12.5% (n=8, chicken fecal sample) and with the duck fecal-source 22.2% (n=9, duck fecal sample) However, according to the record in B.Malla’s research [40], this assay has cross-reaction with chicken fecal source 10% (n=10), besides, it has highly cross-reaction with duck fecal 50% (n=2) In other research, it also had cross-reaction with chicken 10.5% (n=19) [35] From that result, although it has cross-reaction with some different animal’s source (chicken and duck), its percentage is not high and not affect more for detecting unknown source in this study location (Hanoi) In addition, it can conclude that the Pig-2-Bac marker is specific to detect target fecal-source well in some different environment including Hanoi environment with 82.4% specificity Then, testing this marker for a drinking water source of Hanoi environment, it can detect all sample collected, and from that result, we know that Day river already had fecal contamination from pig source From Fig 3.12, the concentration of Pig-2-Bac marker was not significantly different from E coli concentration either in river sample However, it had significant differences in the target-host fecal sample (pig fecal sample) Since the Pig-2-Bac marker is specially designed for the pig source, hence, the concentration detected from pig farms were a range 107 to 1010 copies/g and concentrated in 109copies/g Its number is higher more than the concentration of E coli about 1001000 times Particularly, the concentration of marker for detected water sample was in a range of 102-105copies/ml and it was higher than the concentration of detected 48 by E coli about 100 times, it meant that the MST method with Pig-2-Bac marker is better for assessment pollution of fecal contamination by E coli and it was proved that pig-host assay has more sensitive Likewise, with the chicken and duck fecal samples, the concentration of Pig-2-Bac marker was significantly different from E coli Hence, it proved that the target-host fecal source marker is specific for Hanoi environment with a high level of correlation was found between the concentrations of the Pig-2-Bac marker and E coli concentration obtained by culture in pig waste E.coli (log10 cfu/g or ml) throughout the fecal-sample and water sample (R2=0.8) 10.00 8.00 6.00 4.00 y = 0.6566x + 0.1207 R² = 0.8013 2.00 0.00 0.00 2.00 4.00 6.00 8.00 10.00 12.00 Pig-2-Bac marker (log10 copies/g or ml) throughout the fecal-sample and water sample Figure 3.12: Relation between concentrations of pig-specific Bacteroidales marker (Pig-2-Bac) and E coli bacteria throughout the fecal sample and water sample River sample Chicken fecal sample Duck fecal sample Pig fecal sample Besides, based on the data in Table 3.2 and Table 3.3, comparing the result in Kanazawa and Hanoi, the Pig-2-Bac assay was shown 95% and 100% of sensitivity for the water sample, respectively Moreover, from the Fig 3.12, all water sample 49 was detected as a liner with the slope (0.65), while in Kanazawa’s case, almost water sample was detected with the slope was 0.78 which is higher than Hanoi’s case It meant that in both two cases if the slope is lower, the potential detection unknown sample is higher and more sensitive From this result above, it was more proved that this marker was not only completely suitable for environmental water of Kanazawa, but also for Hanoi’s environment too and it was very sensitive with the environment of Hanoi In the research of Kobayashi at Japan [38], the Chicken/ Duck- Bac marker detected 96% (n=26) and 61% (n=23) positive sample from the feces of wild ducks and chickens, respectively Nevertheless, checking this marker for Hanoi environment, it just detected 12.5% (n=8) for chicken fecal-source sample and 66.67% (n=9) for the sample of duck fecal Then the sensitivity and specificity of this assay are 41.2% and 41.7%, respectively The log concentration value of the positive samples was 5.53 log10 and 5.22 ± 1.77 log10 copies/g wet fecal in duck and chicken, respectively Furthermore, chicken/duck-bac highly cross-reacted with pig showing only 41.4% accuracy In the other hand, no cross-reactivity was observed with fecal-source samples of these hosts in Japan [38] As a consequence, Chicken/ Duck- Bac marker detected 66.7% samples (n=7) from an unknown source (Day river), notwithstanding, the percentage detected in water samples were likely from the source of pollution of pigs, not the source of pollution from ducks or chickens From that result, Chicken/Duck-Bac is not suitable for detect fecal contamination source in Hanoi environment 50 E.coli (log10 cfu/g or ml) throughout the fecalsample and water sample 10.00 8.00 6.00 4.00 2.00 y = 0.5476x + 1.8267 R² = 0.6336 0.00 0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 Chicken/Duck -Bac (log10 copies/g or ml) throughout the fecal-sample and water sample Figure 3.13: Relation between concentrations of chicken and duck-specific Bacteroidales marker (Chicken/Duck-Bac) and E coli bacteria throughout the fecal sample and water sample River sample Chicken fecal sample Duck fecal sample Pig fecal sample Base on Fig 3.13 below, the coefficient (R2) between the concentration of Chicken/Duck-Bac marker and E coli concentration obtained by cultured in duck waste is 0.6 From this result, it showed that Chicken/Duck-Bac marker was not significantly different from E coli concentration neither in almost duck fecal sample, river sample and almost pig fecal sample However, the specific marker for chicken and duck concentration was significantly different from E coli concentration in chicken sample From the above graph, this marker is not sufficiently reliable to assess the Hanoi environment 51 CHAPTER CONCLUSION In conclusion, the host-specificity of the pig, the chicken and the duck of Bacteroidales using target and non-target samples from South of Hanoi was considered to identify contamination source This study obtained the following conclusions: (1) two markers for pig host and host for chickens and ducks were tested for the environment of Hanoi, it found that pig markers gave higher sensitivity and specificity It is very suitable to apply in this environment (2) the marker of chicken and duck feces were not suitable to apply in Hanoi’s environment, because it responded to pig fecal samples One of the reasons is the difference between the breeding condition in two study location (Vietnam and Japan) Therefore, it does not have the ability to detect the chicken and duck- host feces contamination in Hanoi environment (3) comparing the ability and properties of pig markers in both two Hanoi and Kanazawa environments, it has demonstrated its ability to identify pollution sources and adaptability in both environments (4) The one of the drinking water sources from the South of Hanoi (Day river) had pollution from the pig-fecal contamination source In addition, since this is the first study on the use of microbial markers to mark sources of pollution in the Vietnamese environment, especially in Hanoi, there is not much information about suitable primers for the environment in Hanoi Therefore, this study once again proves the appropriate popularity of PCR primers for pigs in Southeast Asia as well as many countries in the world With this study, I hope it can continuously develop in the future with another host-specific to detect another source of contamination in the drinking water of Hanoi such as humanassociated, cow-associated, etc From that, the manager will have an 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OF HOST-SPECIFIC BACTEROIDALES 16S rRNA FOR MICROBIAL SOURCE TRACKING OF ENVIRONMENTAL WATER IN HANOI,. .. specific Bacteroidales marker 10 Table 2.1: The information of sampling point for Pig-fecal source 18 Table 2.2: The information of sampling point for chicken-fecal source 18 Table 2.3: The information... lessons of H5N1 chicken fever are firm evidence of the necessity of this research Therefore, this research topic on "Use of Host-Specific Bacteroidales 16S rRNA for Microbial Source Tracking (MST) of