(Luận văn thạc sĩ) characterization of extended spectrum β lactamase producing escherichia coli in urban water environment in northern vietnam

VIETNAM NATIONAL UNIVERSITY, HANOI VIETNAM JAPAN UNIVERSITY NGUYEN BACH DUONG CHARACTERIZATION OF EXTENDED-SPECTRUM -LACTAMASE PRODUCING ESCHERICHIA COLI IN h URBAN WATER ENVIRONMENT IN NORTHERN VIETNAM MASTER’S THESIS VIETNAM NATIONAL UNIVERSITY, HANOI VIETNAM JAPAN UNIVERSITY NGUYEN BACH DUONG CHARACTERIZATION OF EXTENDED-SPECTRUM -LACTAMASE h PRODUCING ESCHERICHIA COLI IN URBAN WATER ENVIRONMENT IN NORTHERN VIETNAM MAJOR: ENVIRONMENTAL ENGINEERING CODE: 8520320.01 RESEARCH SUPERVISOR: Associate Prof Dr IKURO KASUGA Dr TAKEMURA TAICHIRO Hanoi, 2021 ACKNOWLEDGEMENT Doing science research is a long journey that I am so grateful that I have received a great deal of support and assistance over the last 12 months First and foremost, I would like to express my deepest thank to my supervisor – Associate Professor Kasuga Ikuro for his patient guidance, valuable advice, continuous support and encouragement His immense knowledge and varied experience have inspired me a lot during the research life at the graduate school I would also like to extend my deepest gratitude to my co-supervisor – Dr Takemura Taichiro for providing me the chance to carry out molecular biology experiments at NIHE-Nagasaki Friendship Laboratory His insightful suggestions have contributed greatly to this master’s thesis I must also thank all the staff at NIHE-Nagasaki Friendship Laboratory, especially My Hanh san, for their guidance and useful advice during the biological experiment h The experiment related to this master thesis would not have been done without the financial support from the Japan Agency for Medical Research and Development (AMED) via the project “Development of Integrated Surveillance for Antimicrobial Resistance” I would like to acknowledge lecturers at the Master’s Program in Environmental Engineering (Vietnam Japan University) for giving constructive criticism to improve the quality of my research Thanks also go to my classmates, my lab mates, as well as staffs at Vietnam Japan University, with whom I have the pleasure to work while doing the thesis Last but not least, my sincere thanks are given to my family, my friends for their profound belief in me I would not have been able to complete this master thesis without them Sincerely thank TABLE OF CONTENTS CHAPTER INTRODUCTION CHAPTER LITERATURE REVIEW 2.1 Antimicrobial resistance 2.1.1 Antimicrobials and antimicrobial resistance 2.1.2 Molecular genetics of antimicrobial resistance 2.1.3 Mechanisms of antimicrobial resistance .7 2.1.4 Strategies to control antimicrobial resistance 10 2.2 Antimicrobial resistance in the water environment 10 2.3 Wastewater treatment plants – hot spots of AMR 11 2.4 Occurrence of extended-spectrum -lactamase-producing Escherichia coli (ESBL E coli) 11 2.4.1 Extended-spectrum -lactamase-producing Escherichia coli 11 2.4.2 -lactam antibiotics .12 2.4.3 Extended-spectrum -lactamases 16 2.4.4 ESBL E coli in One Health 17 CHAPTER METHODS 25 h 3.1 Sampling .25 3.2 Quantification of E coli and ESBL E coli (cefotaxime-resistant E coli) 28 3.3 Antimicrobial susceptibility testing 33 3.4 Persistence of ESBL E coli in oligotrophic water environment 37 3.5 Genotyping of ESBL-encoding genes .39 3.6 Statistical analysis 44 CHAPTER RESULT AND DISCUSSION 45 4.1 Occurrence of ESBL E coli 45 4.1.1 Validation of culture method 45 4.1.2 Occurrence of ESBL E coli in urban drainage 46 4.1.3 Occurrence of ESBL E coli in river water 48 4.1.4 Resistance ratios of ESBL E coli in water environments 49 4.2 Antimicrobial susceptibility of ESBL E coli .51 4.3 Genotyping of ESBL-encoding genes in ESBL E coli 54 4.4 Persistence of ESBL E coli in oligotrophic water environment 57 4.5 Removal of ESBL E coli by wastewater treatment plant 58 CONCLUSION 60 REFERENCE 61 APPENDIX 66 LIST OF TABLES h Table 2.1 Major mechanisms of resistance to antibiotic classes (Opal and Pop-Vicas, 2014) Table 2.2 Classification of cephalosporins (Nguyễn, 2011) 14 Table 2.3 Studies on the occurrence of ESBL-producing E coli in water environment .21 Table 3.1 Sampling points in Hanoi and Bac Ninh 25 Table 3.2 MALDI-TOF MS result interpretation 33 Table 3.3 Antibiotic disks used for susceptibility testing 34 Table 3.4 Criteria of susceptibility of E coli .36 Table 3.5 Primer set for multiplex PCR CTX-M group 1, 2, (Dallenne et al., 2010) .40 Table 3.6 Primer mixture CTX-M group 1, 2, 40 Table 3.7 PCR mixture for multiplex PCR 41 Table 3.8 Primer set for multiplex PCR CTX-M group 8/25 (Dallenne et al., 2010) 42 Table 3.9 PCR mixture for monoplex PCR 42 Table 4.1 Numbers of ESBL E coli isolates tested and percentages of isolates resistant to at least antibiotics 53 Table 4.2 Genotyping of ESBL E coli isolated from urban drainage .55 Table A1 Water quality in Hanoi samples 66 Table A2 Water quality in Bac Ninh samples 70 Table A3 Water quality in extended sampling sites (surface water) 72 Table A4 Water quality in extended sampling site (WWTP) 75 i LIST OF FIGURES h Figure 1.1 Transmission of AMR in One Health approach Figure 2.1 Antibiotics and its target sites on bacterial cells Figure 2.2 Horizontal gene transfer in bacteria Figure 2.3 lactam ring (i) and its subclasses: (ii) Penicillins, (iii) Cephalosporins, (iv) Carbapenems, and (v) Monocyclic -lactams 13 Figure 2.4 Prevalence of healthy people carrying intestinal ESBL E coli in six WHO regions (Bezabih et al., 2021) 18 Figure 2.5 Global trend on the presence of ESBL E coli in the intestine of healthy people (Bezabih et al., 2021) 19 Figure 3.1 Sampling sites in Hanoi and Bac Ninh 26 Figure 3.2 E coli appears as blue-green colony on TBX agar plate 31 Figure 3.3 Bruker MALDI Biotyper (Microflex LT/SH) 32 Figure 3.4 Result of species identification by MALDI-TOF MS 33 Figure 3.5 Growth of bacteria on the surface of agar plate after overnight incubation (16 hours) 36 Figure 4.1 Composition of isolates identified by MALDI-TOF MS 45 Figure 4.2 Abundance of ESBL E coli and total E coli and resistance ratios in different water samples in Hanoi and Bac Ninh (from Sep 2020 to May 2021) 47 Figure 4.3 Resistance ratios in different water samples in Hanoi and Bac Ninh 49 Figure 4.4 Resistance ratios in upstream and downstream water in Hanoi, Bac Ninh and other Northern provinces 50 Figure 4.5 Antibiotic resistance profile of ESBL E coli isolated from (i) Hanoi urban drainage; (ii) Bac Ninh urban drainge; (iii) WWTP effluent 52 Figure 4.6 Image of gel electrophoresis of blaCTX-M group 1, group 2, and group 54 Figure 4.7 Result of genotyping blaCTX-M-type ESBL-encoding gene in ESBL E coli .55 Figure 4.8 Relationship of ESBL-encoding genes and number of antibiotics resistance .56 Figure 4.9 Log reduction of ESBL E coli and non-ESBL E coli in oligotrophic water with time 57 Figure 4.10 Concentration of E coli and ESBL E coli in influent and effluent of WWTP 58 Figure 4.11 Correlation of log reduction value of E coli and ESBL E coli without disinfection and with disinfection 59 ii LIST OF ABBREVIATIONS Ampicillin (antibiotic) ARB: Antimicrobial resistant bacteria ARGs: Antimicrobial resistance genes AMR: Antimicrobial resistance bla: gene encoding -lactamase bp: base pair CAZ: Ceftazidime (antibiotic) CFN: Cefdinir (antibiotic) CFU: Colony-forming unit CTX: Cefotaxime (antibiotic) CTX-M: Cefotaximase-Munich (-lactamase) CP: Chloramphenicol (antibiotic) CPR: Cefpirome (antibiotic) CLSI: Clinical and Laboratory Standards Institute E coli: Escherichia coli ESBL: Extended-spectrum -lactamase HGT: Horizontal gene transfer GES: Guiana extended-spectrum (-lactamase) GM: Gentamycin (antibiotic) h ABP: iii Imipenem (antibiotic) IZD: Inhibition zone diameter KM: Kanamycin (antibiotic) LVX: Levofloxacin (antibiotic) LRV: Log reduction value MDR: Multidrug resistance MPM: Meropenem (antibiotic) OXA: Oxacillin-hydrolyzing (-lactamase) PCR: Polymerase chain reaction SHV: Sulfhydryl-variable (-lactamase) ST: Sulfamethoxazole +Trimethoprim (antibiotic) TEM: Temoneira (-lactamase) TC: Tetracycline VEB: Vietnamese extended-spectrum -lactamase WHO: World Health Organization WWTP: Wastewater treatment plant h IPM: iv CHAPTER INTRODUCTION Antimicrobial resistance (AMR) – the ability of bacteria to fight against the antimicrobial medicines – is listed as one of ten global health issues that urgently needs tracking in 2021 (WHO, 2020) The global emergence and spread of AMR drives human to face the lack of available effective treatment for the infection caused by AMR bacteria AMR is so serious that it is predicted to bring about 10 million deaths in 2050 (O’Neill, 2014) AMR will continue remaining as a key challenge to human health in the years ahead To deal with AMR challenge, the United Nations (UN) has encouraged the application of the holistic approach – One Health This approach involved the collaborative work among specialized agencies working with the heath of human, animal, and environment Environment, especially the water environment, plays an important role in the emergence and transmission of AMR since it is not only a reservoir of AMR discharge from human and animal, but also a supply of water for agricultural irrigation, h and recreational activities (see in Figure 1.1) Figure 1.1 also indicates that wastewater treatment is a factor in discharging of AMR into the environment According to the Ministry of Natural Resources and Environment, only 13% of wastewater in Vietnam is treated while the remaining 87% is disposed directly into the environment (Bộ Tài nguyên Môi trường, 2018) Since AMR is a One Health problem, a multisectoral surveillance system, which is a powerful tool to provide the whole picture of AMR, is needed However, such surveillance is still lacking To tackle this problem, the World Health Organization (WHO) has developed the Tricycle protocol for surveillance of a single bacteria that possesses a specific resistant mechanism, which is extended-spectrum -lactamaseproducing Escherichia coli (ESBL E coli) The name “Tricyle” implies the idea that the data of ESBL E coli will be collected in three sectors: human, food chain (animal), and the environment Clearly, ESBL E coli does not represent the overall situation of AMR in the world since there still exists several infectious microorganisms and other resistant traits However, ESBL E coli were selected as the target of the surveillance protocol based on the following reasons (World Health Organization, 2021): (i) Existence of great variation in the rate of ESBL E coli colonization in humans and among countries, as well as the prevalence over time (ii) Existence of ESBL E coli among farm animals (iii) Existence of proof that some of human deaths that are linked to ESBL E coli caused by either antibiotic use in food production or by ESBL E coli in the environment (iv) Interventions that aim to decrease antibiotics use or exposure in human and animals have been accompanied with the decline in in ESBL E coli occurrence rates (v) ESBL production is an important resistant mechanism that makes critically important antimicrobials ineffective h Figure 1.1 Transmission of AMR in One Health approach The research on ESBL E coli in Vietnam to date has tended to focus on the occurrence in human and animal rather than in the water environment Only papers reported the occurrence of ESBL E coli in the pig farm and slaughterhouse REFERENCE Amarasiri, M., Sano, D., Suzuki, S., 2020 Understanding human health risks caused by antibiotic resistant bacteria (ARB) and antibiotic resistance genes (ARG) in water environments: Current knowledge and questions to be answered Crit Rev Environ Sci Technol 50, 2016–2059 https://doi.org/10.1080/10643389.2019.1692611 Bezabih, Y.M., Sabiiti, W., Alamneh, E., Bezabih, A., Peterson, G.M., Bezabhe, W.M., Roujeinikova, A., 2021 The global prevalence and trend of human intestinal carriage of ESBL-producing Escherichia coli in the community J Antimicrob Chemother 76, 22–29 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18, 128–135 https://doi.org/10.1017/S1466252317000160 44 Yamashita, N., Katakawa, Y., Tanaka, H., 2017 Occurrence of antimicrobial resistance bacteria in the Yodo River basin, Japan and determination of betalactamases producing bacteria Ecotoxicol Environ Saf 143, 38–45 https://doi.org/10.1016/j.ecoenv.2017.04.053 65 APPENDIX Table A1 Water quality in Hanoi samples Sample site City Sampling date GPS ID TL1.09 640 29.7 1.34E+05 5.2E+05 22/10/2020 TL1.10 920 25.1 1.88E+05 1.6E+06 7/11/2020 TL1.11 750 29.8 1.47E+05 4.5E+05 TL1.12 830 25.6 3.46E+05 2.0E+06 TL1.01 990 22.7 2.53E+05 1.4E+06 23/2/2021 TL1.02 930 23.5 1.68E+05 1.5E+06 15/3/2021 TL1.03 910 24.0 1.55E+05 1.3E+06 15/4/2021 TL1.04 530 26.1 9.73E+04 8.2E+05 12/5/2021 TL1.05 530 30.8 2.90E+04 4.5E+05 KN2.09 670 31.1 1.65E+05 1.2E+06 h 8/9/2020 10/12/2020 To Lich River Hanoi Hanoi EC Temp ESBL E coli E coli (S/cm) (°C) (CFU/100mL) (CFU/100mL) 23/1/2021 8/9/2020 21°01'12.5"N 105°48'05.4"E 20°59'27.9"N 105°51'46.2"E 66 Kim Nguu River: Before WWTP KN2.10 890 26.5 2.19E+05 1.7E+06 7/11/2020 KN2.11 820 27.7 2.85E+05 1.3E+06 10/12/2020 KN2.12 840 26.3 3.91E+05 3.2E+06 23/1/2021 KN2.01 484 24.2 4.17E+05 2.2E+06 23/2/2021 KN2.02 1000 24.5 2.73E+05 2.4E+06 15/3/2021 KN2.03 860 26.4 2.77E+05 2.9E+06 16/4/2021 KN2.04 660 27.2 5.33E+05 5.6E+06 13/5/2021 KN2.05 700 29 3.97E+05 3.9E+06 8/9/2020 KN3.09 410 29.7 1.60E+04 9.3E+04 22/10/2020 KN3.10 770 26.9 2.67E+00 4.4E+00 7/11/2020 KN3.11 600 23.9 1.11E+04 4.7E+04 10/12/2020 KN3.12 730 25.3 KN3.01 700 23.2 1.75E+03 1.0E+04 23/2/2021 KN3.02 700 24.8 8.23E+03 6.9E+04 15/3/2021 KN3.03 650 24.2 8.57E+03 7.8E+04 16/4/2021 KN3.04 850 26.7 4.53E+03 4.3E+04 h 22/10/2020 Kim Nguu River: After WWTP Hanoi 23/1/2021 20°58'33.6"N 105°51'55.2"E 67 13/5/2021 KN3.05 540 29.6 5.87E+03 6.3E+04 10/9/2020 N1.09 190 31.3 2.67E+02 6.7E+02 N1.10 160 27.1 3.60E+01 5.8E+02 7/11/2020 N1.11 255 29.5 2.17E+03 1.0E+04 14/12/2020 N1.12 165 24.1 8.13E+01 3.4E+02 23/1/2021 N1.01 215 22.5 7.97E+03 3.8E+04 22/2/2021 N1.02 239 22.5 3.17E+01 2.1E+02 N1.03 472 25.9 1.40E+02 9.4E+02 15/4/2021 N1.04 660 27.2 1.20E+02 8.2E+02 12/5/2021 N1.05 303 28.9 5.03E+02 6.8E+03 10/9/2020 N2.09 497 33.1 3.40E+04 2.5E+05 7/10/2020 N2.10 263 27.00 1.43E+04 1.5E+05 N2.11 720 26.7 5.27E+04 3.7E+05 14/12/2020 N2.12 780 28.5 1.18E+05 7.2E+05 23/1/2021 N2.01 650 21.6 5.17E+04 3.9E+05 7/10/2020 Nhue River: Upstream Hanoi Nhue River: Downstream Hanoi 7/11/2020 21°05'31.7"N 105°46'18.2"E h 20/3/2021 21°05'21.8"N 105°46'14.2"E 21°01'51.4"N 105°45'30.8"E 68 22/2/2021 N2.02 478 24.8 1.93E+04 1.8E+05 20/3/2021 N2.03 720 25.8 5.27E+04 5.7E+05 15/4/2021 N2.04 850 26.7 9.50E+04 7.4E+05 12/5/2021 N2.05 412 29.8 1.53E+04 1.4E+05 h 69 Table A2 Water quality in Bac Ninh samples Sample site Ngu Huyen Khe River: Upstream City Bac Ninh Sampling date GPS ID EC Temp ESBL E coli E coli (S/cm) (°C) (CFU/100mL) (CFU/100mL) 21/9/2020 21°04'26.6"N 105°51'23.2"E HK1.09 244 30.3 3.0E+02 4.3E+03 11/11/2020 21°04'26.5"N 105°51'23.7"E HK1.11 530 24.3 1.8E+03 2.1E+04 18/1/2021 HK1.01 251 21 1.0E+01 7.0E+01 11/3/2021 HK1.03 500 22.3 4.0E+04 5.1E+05 HK1.05 h Ngu Huyen Khe River: Downstream Bac Ninh WWTP: Influent Bac Ninh Bac Ninh 21/9/2020 21°11'44.6"N 106°02'44.2"E HK3.09 129 31.4 2.8E+03 2.5E+04 11/11/2020 21°11'44.6"N 106°02'44.2"E HK3.11 1200 26 2.7E+03 2.5E+04 18/1/2021 HK3.01 486 19.6 4.1E+02 3.3E+03 11/3/2021 HK3.03 386 24 5.7E+03 8.3E+04 21/9/2020 21°11'22.7"N 106°04'01.9"E BI.09 680 29.9 3.6E+04 1.7E+05 11/11/2020 21°11'22.7"N 106°04'01.9"E BI.11 870 25.4 5.2E+04 3.5E+05 BI.01 1020 19.4 5.4E+04 2.9E+05 18/1/2021 70 11/3/2021 Bac Ninh WWTP: Effluent Bac Ninh BI.03 950 26 4.0E+04 5.1E+05 21/9/2020 21°11'22.7"N 106°04'01.9"E BE.09 530 28.3 5.5E+03 8.0E+04 11/11/2020 21°11'22.7"N 106°04'01.9"E BE.11 840 21.4 1.3E+04 8.3E+04 18/1/2021 BE.01 1020 19.4 3.2E+04 2.3E+05 11/3/2021 BE.03 720 22.3 7.0E+03 8.3E+04 h 71 Table A3 Water quality in extended sampling sites (surface water) Sampling date GPS ID Nhue River Hanoi 10/9/2020 21°05'21.8"N 105°46'14.2"E N1.09 190 31.3 2.7E+02 6.7E+02 Nhue River Hanoi 10/9/2020 21°01'51.4"N 105°45'30.8"E N2.09 497 33.1 3.4E+04 2.5E+05 Nhue River Hanoi 10/9/2020 20°57'34.2"N 105°47'28.4"E N3.09 510 32.8 5.0E+04 6.3E+05 Nhue River Hanoi 10/9/2020 20°54'45.0"N 105°48'07.2"E N4.09 510 33.4 2.9E+04 2.7E+05 Nhue River Hanoi 10/9/2020 20°52'08.5"N 105°49'50.3"E N5.09 510 32.1 1.3E+04 1.1E+05 Cau Bay River Hanoi 28/9/2020 21°01'45.9"N 105°55'20.2"E CB1.09 500 30 2.9E+04 3.1E+05 Cau Bay River Hanoi 28/9/2020 21°00'04.7"N 105°55'43.1"E CB2.09 374 33.6 2.3E+04 1.8E+05 Day River Hanoi 14/9/2020 21°06'49.5"N 105°36'24.0"E D1.09 391 31.2 0.0E+00 6.0E+02 Day River Hanoi 14/9/2020 21°02'20.4"N 105°39'35.5"E D2.09 247 31.2 2.7E+03 4.3E+04 Day River Hanoi 14/9/2020 20°58'54.1"N 105°40'46.8"E D3.09 520 31.9 2.0E+02 1.9E+03 Day River Hanoi 14/9/2020 20°53'36.2"N 105°43'24.1"E D4.09 520 31.9 2.0E+01 6.8E+02 Day River Hanoi 14/9/2020 20°48'45.8"N 105°45'13.6"E D5.09 520 31.9 6.0E+01 1.1E+03 Day River Hanoi 14/9/2020 20°48'29.2"N 105°42'30.2"E D6.09 520 31.9 1.4E+02 2.2E+03 Duong River Hanoi 21/9/2020 21°04'40.7"N 105°54'32.1"E DU.09 162 26.9 2.3E+01 8.4E+02 h City Sample site EC Temp ESBL E coli E coli (uS/cm) (°C) (CFU/100mL) (CFU/100mL) 72 Bac Ninh 21/9/2020 21°04'26.6"N 105°51'23.2"E HK1.09 244 30.3 3.0E+02 4.3E+03 Ngu Huyen Khe River Bac Ninh 21/9/2020 1°09'51.8"N 105°57'26.5"E HK2.09 295 30.3 1.7E+01 1.8E+02 Ngu Huyen Khe River Bac Ninh 21/9/2020 21°11'44.6"N 106°02'44.2"E HK3.09 129 31.4 2.8E+03 2.5E+04 Cau River Bac Ninh 21/9/2020 21°12'17.3"N 106°05'27.7"E C1.09 198 30.4 1.2E+02 1.1E+03 Cau River Bac Ninh 21/9/2020 21°11'55.7"N 106°07'27.0"E C2.09 203 30.7 1.5E+02 9.4E+02 Cau River Bac Ninh 21/9/2020 21°11'14.0"N 106°10'00.4"E C3.09 191 31.2 1.6E+01 1.1E+03 Bac Hung Hai Hung River Yen 28/9/2020 20°58'18.7"N 105°55'11.4"E HY1.09 141 31.9 4.9E+01 1.0E+03 Bac Hung Hai Hung River Yen 28/9/2020 20°57'23.8"N 105°59'35.5"E HY2.09 198 29.5 1.1E+03 1.3E+04 Bac Hung Hai Hung River Yen 28/9/2020 20°53'45.1"N 106°00'55.0"E HY3.09 254 32.4 6.3E+02 9.3E+03 Ninh Binh Vân River Vung Tram River 27/9/2020 20°13'40.8"N 105°58'03.0"E NB.09 420 30.6 4.5E+02 3.2E+03 Ninh Binh h Ngu Huyen Khe River 73 Nam Dinh 27/9/2020 20°25'13.2"N 106°10'49.4"E ND1.09 610 32.5 4.2E+01 3.1E+02 Chanh River An Duyên Bridge Nam Dinh 27/9/2020 20°24'08.7"N 106°07'51.5"E ND3.09 144 31.6 5.6E+02 3.5E+03 Trà Lý River Thai Binh 27/9/2020 20°28'08.3"N 106°22'06.6"E TB1.09 149 30.4 5.2E+01 3.0E+02 Vĩnh Trà river Kỳ Đồng Bridge Thai Binh 27/9/2020 20°26'19.9"N 106°19'43.0"E TB2.09 262 30.2 1.3E+04 1.6E+05 Sam Bridge Thai Binh 27/9/2020 20°25'20.1"N 106°19'47.2"E TB3.09 315 32.1 2.0E+03 1.8E+04 h Nam Dinh River -Do Quan Bridge 74 Table A4 Water quality in extended sampling site (WWTP) Sampling date Sample site City Yen So WWTP: Influent Hanoi Yen So WWTP: Effluent Hanoi Bay Mau WWTP: Influent Hanoi Bay Mau WWTP: Effluent Hanoi Vinh Niem WWTP: Influent Hai Phong 30/12/2020 Vinh Niem WWTP: Effluent Hai Phong 30/12/2020 27/10/2020 GPS ID EC Temp (uS/cm) (°C) ESBL E coli E coli (CFU/100mL) (CFU/100mL) YSI.10 810 17.3 2.1E+05 7.6E+05 YSE.10 590 16.1 7.1E+02 2.2E+03 BMI.10 770 19.0 1.8E+06 4.7E+06 BME.10 640 22.3 0.0E+00 0.0E+00 20°49'14.5"N 106°40'31.9"E HPI 1960 22.5 7.7E+04 4.6E+05 20°49'14.5"N 106°40'31.9"E HPE 3700 20.8 1.0E+02 7.4E+02 20°58'39.1"N 105°51'51.1"E 27/10/2020 20°58'39.1"N 105°51'51.1"E 27/10/2020 21°00'58.4"N 105°50'35.8"E h 27/10/2020 21°00'58.4"N 105°50'35.8"E 75