33
Supporting Information for Exploration of the Antibiotic Resistome in a Wastewater Treatment Plant by a Nine-Year Longitudinal Metagenomic Study Xiaole Yin a , Yu Deng a , Liping Ma a,b , Yulin Wang a , Lilian Y.L. Chan c , Tong Zhang* , a,d a Environmental Microbiome Engineering and Biotechnology Laboratory, Department of Civil Engineering, The University of Hong Kong, Hong Kong, China. b Shanghai Key Laboratory for Urban Ecological Processes and Eco-Restoration, School of Ecological and Environmental Sciences, East China Normal University, Shanghai, China. c High Performance Computing Team, Information Technology Services, The University of Hong Kong, Hong Kong, China. d International Center for Antibiotic Resistance in the Environment, School of Environmental Science and Engineering, Southern University of Science and Technology, Shenzhen, China. * To whom correspondence should be addressed. E-mail address: [email protected] Figure S1. Dynamics of ARGs spanning 9 years in AS in the unit of copy of ARGs per copy of 16S rRNA. Figure S2. Dynamics of ARGs spanning 9 years in AS in the unit of PPM (ARG read per million reads). Figure S3. Seasonal coloring and PCoA analysis of all samples based on Bray-Curtis distance. Figure S4. Average abundances of ARGs in four consecutive time range (clustered by PCoA in Figure 1b) Figure S5. Profile of monthly variations of genes resistant to widely-used antibiotics types. Figure S6. Temporal variation of relative abundance of core resistome.

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Supporting Information for

Exploration of the Antibiotic Resistome in a Wastewater Treatment Plant by a Nine-Year Longitudinal Metagenomic Study

 Xiaole Yina, Yu Denga, Liping Maa,b, Yulin Wanga, Lilian Y.L. Chanc, Tong Zhang*, a,d

a Environmental Microbiome Engineering and Biotechnology Laboratory, Department of Civil Engineering, The University of Hong Kong, Hong Kong, China.

b Shanghai Key Laboratory for Urban Ecological Processes and Eco-Restoration, School of Ecological and Environmental Sciences, East China Normal University, Shanghai, China.

c High Performance Computing Team, Information Technology Services, The University of Hong Kong, Hong Kong, China.

d International Center for Antibiotic Resistance in the Environment, School of Environmental Science and Engineering, Southern University of Science and Technology, Shenzhen, China.

* To whom correspondence should be addressed. E-mail address: [email protected]

Figure S1. Dynamics of ARGs spanning 9 years in AS in the unit of copy of ARGs per copy of 16S rRNA.

Figure S2. Dynamics of ARGs spanning 9 years in AS in the unit of PPM (ARG read per million reads).

Figure S3. Seasonal coloring and PCoA analysis of all samples based on Bray-Curtis distance.

Figure S4. Average abundances of ARGs in four consecutive time range (clustered by PCoA in Figure 1b)

Figure S5. Profile of monthly variations of genes resistant to widely-used antibiotics types.

Figure S6. Temporal variation of relative abundance of core resistome.

Figure S7. A heatmap illustrating the abundance of core resistome in 9 years.

Figure S8. Dynamics of core resistome.

Figure S9. Variation of relative abundance of top 8 phyla or classes of proteobacteria.

Figure S10. Variation of relative abundance of 19 most abundant families.

Figure S11. Correlation analyses. a. RDA analysis of operational parameters with AS resistome; b. VPA analysis based on bacterial community, MGEs and AS resistome

Figure S12. Possibility of horizontal gene transfer on different ARGs-carrying species. Species in the same phyla/class were framed together. Numbers around center point stand for the total occurrence of each predicted ARGs-carrying species which were in proportion to the diameter of the corresponding pies. Potential pathogens were highlighted in red font.

Figure S13. Comparation of abundance of ARGs between this study with other ecotypes listed by (Li et al., 2015). Metagenomic datasets of these ecotypes were retrieved from (Li et al., 2015).

Figure S14. Occurrence patterns in other ecotypes of a list of genes which detected as core resistome in this study.

Figure S15. Temporal variation of the abundance of ereA gene and relevant hosts. b. Regression analysis showed close correlation between Geobacter with ereA gene.

Figure S16. Temporal variation of the abundance of qnrS gene and relevant hosts. b. Regression analysis showed close correlation between Vibrio anguillarum and qnrS gene.

Figure S17. Occurrence of emerging genotypes or genes related to superbugs.

Table S1. Sampling dates since June of 2007 to December of 2015.

Table S2. Bacterial pathogen list (Li et al., 2015a)

Table S3. AS genomes used in analyzing the incidence of ARGs encountering MGEs.

Table S4. Significance of differences among the 4 clusters from Figure 1b. The numbers are the P values from the pair-wise Mann-Whitney test. Those values which are less than 0.05 are highlighted in red.

Table S5. Information of core resistome according to persistency and abundancy detected from 9 years

Figure S1. Dynamics of ARGs spanning 9 years in AS in the unit of copy of ARGs per copy of 16S rRNA.

Figure S2. Dynamics of ARGs spanning 9 years in AS in the unit of PPM (ARG read per million reads).

Figure S3. Seasonal coloring and PCoA analysis of all samples based on Bray-Curtis distance.

Figure S4. Average abundances of ARGs in four consecutive time range (clustered by PCoA in Figure 1b)

Figure S5. Profile of monthly variations of resistance genes to widely-used antibiotics types.

Figure S6. Temporal variation of relative abundance of core resistome

Figure S7. A heatmap illustrating the abundance of core resistome in 9 years.

Figure S8. Dynamics of core resistome (to be continued)

Figure S8. Dynamics of core resistome

Figure S9. Variation of relative abundance of top 8 phyla or classes of proteobacteria.

Figure S10. Variation of relative abundance of 19 most abundant families.

Figure S11. Correlation analyses. a. RDA analysis of operational parameters with AS resistome; b. VPA analysis based on bacterial community, MGEs and AS resistome

Figure S12. Possibility of horizontal gene transfer on different ARGs-carrying species. Species in the same phyla/class were framed together. Numbers around center point stand for the total occurrence of each predicted ARGs-carrying species which were in proportion to the diameter of the corresponding pies. Potential pathogens were highlighted in red font.

Figure S13. Comparation of abundance of ARGs between this study with other ecotypes listed by (Li et al., 2015). Metagenomic datasets of these ecotypes were retrieved from (Li et al., 2015).

Figure S14. Occurrence patterns in other ecotypes of a list of genes which detected as core resistome in this study.

Figure S15. Temporal variation of the abundance of ereA gene and relevant hosts. b. Regression analysis showed close correlation between Geobacter with ereA gene.

Figure S16. Temporal variation of the abundance of qnrS gene and relevant hosts. b. Regression analysis showed close correlation between Vibrio anguillarum and qnrS gene.

Figure S17. Occurrence of emerging genotypes or genes related to superbugs.

Table S1. Sampling dates since June of 2007 to December of 2015.

No.

Date

No.

Date

No.

Date

1

2-Jun-2007

34

20-Jul-2010

67

30-May-2013

2

5-Jul-2007

35

17-Aug-2010

68

26-Jun-2013

3

17-Aug-2007

36

15-Sep-2010

69

22-Jul-2013

4

19-Oct-2007

37

12-Oct-2010

70

28-Aug-2013

5

9-Nov-2007

38

18-Nov-2010

71

3-Oct-2013

6

14-Dec-2007

39

14-Dec-2010

72

31-Oct-2013

7

15-Jan-2008

40

12-Jan-2011

73

27-Nov-2013

8

20-Feb-2008

41

16-Feb-2011

74

31-Dec-2013

9

16-Apr-2008

42

17-Mar-2011

75

27-Jan-2014

10

21-May-2008

43

20-Apr-2011

76

4-Mar-2014

11

11-Jun-2008

44

18-May-2011

77

1-Apr-2014

12

18-Aug-2008

45

21-Jun-2011

78

29-Apr-2014

13

19-Sep-2008

46

21-Jul-2011

79

3-Jun-2014

14

8-Oct-2008

47

16-Aug-2011

80

27-Jun-2014

15

13-Nov-2008

48

19-Sep-2011

81

28-Jul-2014

16

15-Dec-2008

49

21-Oct-2011

82

5-Sep-2014

17

12-Jan-2009

50

17-Nov-2011

83

3-Oct-2014

18

13-Feb-2009

51

16-Dec-2011

84

29-Oct-2014

19

25-Mar-2009

52

11-Jan-2012

85

3-Dec-2014

20

17-Apr-2009

53

15-Feb-2012

86

6-Jan-2015

21

26-May-2009

54

24-Apr-2012

87

3-Feb-2015

22

9-Jun-2009

55

30-May-2012

88

2-Mar-2015

23

20-Jul-2009

56

27-Jun-2012

89

25-Mar-2015

24

24-Aug-2009

57

23-Jul-2012

90

28-Apr-2015

25

10-Sep-2009

58

27-Aug-2012

91

29-May-2015

26

19-Oct-2009

59

24-Sep-2012

92

25-Jun-2015

27

19-Nov-2009

60

22-Oct-2012

93

30-Jul-2015

28

15-Dec-2009

61

27-Nov-2012

94

4-Sep-2015

29

4-Jan-2010

62

20-Dec-2012

95

7-Oct-2015

30

25-Feb-2010

63

22-Jan-2013

96

2-Nov-2015

31

22-Mar-2010

64

21-Feb-2013

97

3-Dec-2015

32

20-Apr-2010

65

21-Mar-2013

33

18-May-2010

66

23-Apr-2013

Table S2. Bacterial pathogen list (Li et al., 2015a)

Abiotrophia defectiva

Anaerococcus prevotii

Bartonella henselae

Achromobacter piechaudii

Anaerococcus vaginalis

Bartonella quintana

Achromobacter xylosoxidans

Arcanobacterium bernardiae

Bergeyella zoohelcum

Acidaminococcus fermentans

Arcanobacterium heamolyticum

Bifidobacterium dentium

Acinetobacter baumannii

Arcanobacterium pyogenes

Bilophila wadsworthia

Acinetobacter calcoaceticus

Arcobacter butzleri

Bordetella avium

Acinetobacter haemolyticus

Arcobacter cryaerophilus

Bordetella bronchiseptica

Acinetobacter johnsonii

Anaplasma phagocytophila

Bordetella parapertussis

Acinetobacter junii

Bacillus anthracis

Bordetella pertussis

Acinetobacter lwoffii

Bacillus cereus

Borrelia brasiliensis

Acinetobacter radioresistens

Bacillus circulans

Borrelia burgdorferi

Actinobacillus actinomycetemcomitans

Bacillus coagulans

Borrelia caucasica

Actinobacillus equuli

Bacillus licheniformis

Borrelia crocidurae

Actinobacillus hominis

Bacillus mycoides

Borrelia duttonii

Actinobacillus lignieresii

Bacillus pumilus

Borrelia hermsii

Actinobacillus pleuropneumoniae

Bacillus sphaericus

Borrelia hispanica

Actinobacillus suis

Bacillus subtilis

Borrelia latyschewii

Actinobacillus ureae

Bacillus thuringiensis

Borrelia mazzottii

Actinomyces georgiae

Bacteroides caccae

Borrelia parkeri

Actinomyces gerencseriae

Bacteroides distasonis

Borrelia persica

Actinomyces israelii

Bacteroides eggerthii

Borrelia recurrentis

Actinomyces meyeri

Bacteroides forsythus

Borrelia turicatae

Actinomyces naeslundii

Bacteroides fragilis

Borrelia venezuelensis

Actinomyces neuii

Bacteroides galacturonicus

Brevibacillus brevis

Actinomyces odontolyticus

Bacteroides merdae

Brevundimonas diminuta

Actinomyces radingae

Bacteroides ovatus

Brevundimonas vesicularis

Actinomyces turicensis

Bacteroides pectinophilus

Brucella melitensis

Aerococcus viridans

Bacteroides splanchnicus

Burkholderia cepacia

Aeromonas caviae

Bacteroides stercoris

Burkholderia mallei

Aeromonas hydrophila

Bacteroides thetaiotaomicron

Burkholderia pseudomallei

Aeromonas sobria

Bacteroides uniformis

Campylobacter coli

Aeromonas veronii

Bacteroides ureolyticus

Campylobacter concisus

Alcaligenes odorans

Bacteroides vulgatus

Campylobacter curvus

Amycolatopsis orientalis

Bartonella bacilliformis

Campylobacter fetus

Anaerococcus lactolyticus

Bartonella elizabethae

Campylobacter gracilis

Table S2. Bacterial pathogen list (Li et al., 2015a) (continued)

Campylobacter hyointestinalis

Clostridium botulinum

Dichelobacter nodosus

Campylobacter jejuni

Clostridium butyricum

Edwardsiella hoshinae

Campylobacter lari

Clostridium chauvoei

Edwardsiella tarda

Campylobacter rectus

Clostridium difficile

Eggerthella lenta

Campylobacter sputorum

Clostridium fallax

Ehrlichia chaffeensis

Campylobacter upsaliensis

Clostridium histolyticum

Ehrlichia ewingii

Capnocytophaga canimorsus

Clostridium novyi

Eikenella corrodens

Capnocytophaga cynodegmi

Clostridium perfringens

Enterobacter aerogenes

Capnocytophaga gingivalis

Clostridium ramosum

Enterobacter amnigenus

Capnocytophaga ochracea

Clostridium septicum

Enterobacter asburiae

Capnocytophaga sputigena

Clostridium sordellii

Enterobacter cancerogenus

Cardiobacterium hominis

Clostridium sporogenes

Enterobacter cloacae

Cedecea davisae

Clostridium tertium

Enterobacter gergoviae

Cedecea lapagei

Clostridium tetani

Enterobacter hormaechei

Cedecea neteri

Collinsella aerofaciens

Enterobacter sakazakii

Cellulomonas cellulans

Comamonas testosteroni

Enterococcus avium

Cellulomonas turbata

Corynebacterium afermentans

Enterococcus casseliflavus

Centipeda periodontii

Corynebacterium amycolatum

Enterococcus durans

Chlamydia trachomatis

Corynebacterium argentoratense

Enterococcus faecalis

Chlamydophila pneumoniae

Corynebacterium bovis

Enterococcus faecium

Chlamydophila psittaci

Corynebacterium diphtheriae

Enterococcus flavescens

Chromobacterium violaceum

Corynebacterium jeikeium

Enterococcus gallinarum

Chryseobacterium balustinum

Corynebacterium kutscheri

Enterococcus hirae

Chryseobacterium meningosepticum

Corynebacterium macginleyi

Enterococcus mundtii

Citrobacter amalonaticus

Corynebacterium minutissimum

Enterococcus raffinosus

Citrobacter braakii

Corynebacterium propinquum

Erysipelothrix rhusiopathiae

Citrobacter farmeri

Corynebacterium pseudodiphthericum

Escherichia coli

Citrobacter freundii

Corynebacterium pseudotuberculosis

Eubacterium brachy

Citrobacter koseri

Corynebacterium striatum

Eubacterium combesii

Citrobacter rodentium

Corynebacterium ulcerans

Eubacterium contortum

Citrobacter sedlakii

Corynebacterium urealyticum

Eubacterium cylindroides

Citrobacter werkmanii

Corynebacterium xerosis

Eubacterium limosum

Citrobacter youngae

Coxiella burnetii

Eubacterium moniliforme

Clostridium baratii

Delftia acidovorans

Eubacterium multiforme

Clostridium bifermentans

Dermatophilus congolensis

Eubacterium nodatum

Table S2. Bacterial pathogen list (Li et al., 2015a) (continued)

Eubacterium rectale

Hafnia alvei

Leptospira meyeri

Eubacterium saburreum

Helicobacter cinaedi

Leptospira noguchii

Eubacterium saphenum

Helicobacter fennelliae

Leptospira santarosai

Eubacterium sulci

Helicobacter heilmannii

Leptospira weilii

Eubacterium tenue

Helicobacter pullorum

Leptotrichia buccalis

Ewingella americana

Helicobacter pylori

Listeria ivanovii

Fibrobacter intestinalis

Kingella denitrificans

Listeria monocytogenes

Filifactor alocis

Kingella kingae

Listeria seeligeri

Finegoldia magna

Klebsiella granulomatis

Listeria welshimeri

Fluoribacter bozemanae

Klebsiella ornithinolytica

Mannheimia haemolytica

Fluoribacter dumoffii

Klebsiella oxytoca

Megamonas hypermegale

Fluoribacter gormanii

Klebsiella pneumoniae

Megasphaera sp.

Francisella tularensis

Kluyvera ascorbata

Micromonas micros

Fusobacterium mortiferum

Kluyvera cryocrescens

Mogibacterium timidum

Fusobacterium necrophorum

Lactobacillus sp.

Moraxella (Branhamella) catarrhalis

Fusobacterium nucleatum

Legionella anisa

Moraxella (Branhamella) caviae

Fusobacterium periodonticum

Legionella birminghamensis

Moraxella (Branhamella) cuniculi

Fusobacterium ulcerans

Legionella cherrii

Moraxella (Branhamella) ovis

Fusobacterium varium

Legionella cincinnatiensis

Moraxella (Moxarella) atlantae

Gardnerella vaginalis

Legionella feeleii

Moraxella (Moxarella) bovis

Gemella morbillorum

Legionella hackeliae

Moraxella (Moxarella) lacunata

Gordonia amarae

Legionella jordanis

Moraxella (Moxarella) liquefaciens

Gordonia bronchialis

Legionella lansingensis

Moraxella (Moxarella) nonliquefaciens

Gordonia rubropertincta

Legionella longbeachae

Moraxella (Moxarella) osloensis

Gordonia sputi

Legionella oakridgensis

Moraxella lincolnii

Gordonia terrae

Legionella pneumophila

Morganella morganii

Granulicatella adjacens

Legionella rubrilucens

Mycobacterium abscessus

Haemophilus aphrophilus

Legionella sainthelensi

Mycobacterium africanum

Haemophilus ducreyi

Legionella tucsonensis

Mycobacterium asiaticum

Haemophilus haemolyticus

Legionella wadsworthii

Mycobacterium avium

Haemophilus influenzae

Leifsonia aquatica

Mycobacterium bovis

Haemophilus parahaemolyticus

Leptospira borgpetersenii

Mycobacterium celatum

Haemophilus parainfluenzae

Leptospira inadai

Mycobacterium chelonae

Haemophilus paraphrophilus

Leptospira interrogans

Mycobacterium conspicuum

Haemophilus segnis

Leptospira kirschneri

Mycobacterium fortuitum

Table S2. Bacterial pathogen list (Li et al., 2015a) (continued)

Mycobacterium genavense

Neisseria subflava

Porphyromonas levii

Mycobacterium gordonae

Neisseria weaveri

Porphyromonas macacae

Mycobacterium haemophilum

Neorickettsia sennetsu

Prevotella bivia

Mycobacterium kansasii

Nocardia asteroides

Prevotella buccae

Mycobacterium leprae

Nocardia brasiliensis

Prevotella buccalis

Mycobacterium malmoense

Nocardia caviae

Prevotella corporis

Mycobacterium marinum

Nocardia farcinica

Prevotella dentalis

Mycobacterium mucogenicum

Nocardia nova

Prevotella denticola

Mycobacterium peregrinum

Nocardia otitidiscaviarum

Prevotella disiens

Mycobacterium porcinum

Nocardia pseudobrasiliensis

Prevotella enoeca

Mycobacterium scrofulaceum

Nocardia transvalensis

Prevotella heparinolytica

Mycobacterium senegalense

Ochrobactrum anthropi

Prevotella intermedia

Mycobacterium shimoidei

Oligella ureolytica

Prevotella loescheii

Mycobacterium simiae

Oligella urethralis

Prevotella melaninogenica

Mycobacterium smegmatis

Orientia tsutsugamushi

Prevotella nigrescens

Mycobacterium szulgai

Paenibacillus alvei

Prevotella oralis

Mycobacterium tuberculosis

Paenibacillus macerans

Prevotella oris

Mycobacterium ulcerans

Pantoea agglomerans

Prevotella oulora

Mycobacterium xenopi

Pasteurella aerogenes

Prevotella ruminicola

Mycoplasma fermentans

Pasteurella caballi

Prevotella tannerae

Mycoplasma genitalium

Pasteurella canis

Prevotella veroralis

Mycoplasma hominis

Pasteurella dagmatis

Prevotella zoogleoformans

Mycoplasma pneumoniae

Pasteurella multocida

Propionibacterium acnes

Mycoplasma salivarium

Pasteurella pneumotropica

Propionibacterium avidum

Myroides odoratus

Pasteurella stomatis

Propionibacterium granulosum

Neisseria cinerea

Peptinophilus asaccharolyticus

Propionibacterium propionicus

Neisseria elongata

Peptococcus niger

Proteus mirabilis

Neisseria flava

Peptostreptococcus anaerobius

Proteus penneri

Neisseria flavescens

Photobacterium damselae

Proteus vulgaris

Neisseria gonorrhoeae

Plesiomonas shigelloides

Providencia alcalifaciens

Neisseria lactamica

Porphyromonas asaccharolytica

Providencia rettgeri

Neisseria meningitidis

Porphyromonas catoniae

Providencia stuartii

Neisseria mucosa

Porphyromonas circumdentaria

Pseudomonas aeruginosa

Neisseria perflava

Porphyromonas endodontalis

Pseudomonas alcaligenes

Neisseria sicca

Porphyromonas gingivalis

Pseudomonas fluorescens

Table S2. Bacterial pathogen list (Li et al., 2015a) (continued)

Pseudomonas pseudoalcaligenes

Selenomonas artemidis

Streptococcus gordonii

Vibrio parahaemolyticus

Pseudomonas putida

Selenomonas dianae

Streptococcus intermedius

Vibrio vulnificus

Pseudomonas stutzeri

Selenomonas flueggei

Streptococcus milleri

Wolinella succinogenes

Pseudonocardia autotrophica

Selenomonas infelix

Streptococcus mitis

Yersinia bercovieri

Pseudoramibacter alactolyticus

Selenomonas noxia

Streptococcus mutans

Yersinia enterocolitica

Psychrobacter phenylpyruvicus

Serratia ficaria

Streptococcus pneumoniae

Yersinia frederiksenii

Rahnella aquatilis

Serratia marcescens

Streptococcus pyogenes

Yersinia intermedia

Ralstonia pickettii

Serratia odorifera

Streptococcus salivarius

Yersinia kristensenii

Rhodococcus equi

Serratia plymuthica

Streptococcus sanguis

Yersinia mollaretii

Rhodococcus erythropolis

Serratia proteamaculans

Streptococcus sobrinus

Yersinia pestis

Rhodococcus fascians

Serratia rubidaea

Streptococcus suis

Yersinia pseudotuberculosis

Rhodococcus rhodnii

Shigella boydii

Streptococcus uberis

Yersinia rohdei

Rhodococcus rhodochrous

Shigella dysenteriae

Sutterella wadsworthensis

Yersinia ruckeri

Rickettsia africae

Shigella flexneri

Suttonella indologenes

Rickettsia akari

Shigella sonnei

Tatlockia maceachernii

Rickettsia australis

Sphingomonas paucimobilis

Tatlockia micdadei

Rickettsia conorii

Spirillum minus

Tatumella ptyseos

Rickettsia felis

Staphylococcus aureus

Treponema carateum

Rickettsia honei

Staphylococcus epidermidis

Treponema pallidum

Rickettsia japonica

Staphylococcus haemolyticus

Tropheryma whippelii

Rickettsia massiliae

Staphylococcus hyicus

Tsukamurella inchonensis

Rickettsia prowazekii

Staphylococcus intermedius

Tsukamurella paurometabola

Rickettsia rickettsii

Staphylococcus lugdunensis

Tsukamurella pulmonis

Rickettsia sibirica

Staphylococcus saprophyticus

Tsukamurella tyrosinosolvens

Rickettsia typhi

Staphylococcus warneri

Ureaplasma urealyticum

Rothia dentocariosa

Stenotrophomonas maltophilia

Veillonella atypica

Ruminococcus productus

Streptobacillus moniliformis

Veillonella dispar

Saccharomonospora viridis

Streptococcus acidominimus

Veillonella parvula

Saccharopolyspora rectivirgula

Streptococcus agalactiae

Vibrio alginolyticus

Salmonella bongori

Streptococcus anginosus

Vibrio cholerae

Salmonella choleraesuis

Streptococcus bovis

Vibrio cincinnatiensis

Salmonella enteritidis

Streptococcus canis

Vibrio fluvialis

Salmonella typhi

Streptococcus constellatus

Vibrio furnissii

Salmonella typhimurium

Streptococcus criceti

Vibrio hollisae

Sebaldella termitidis

Streptococcus equi

Vibrio mimicus

Table S3. AS genomes used in analyzing the incidence of ARGs encountering MGEs.

NCBI.Taxon.ID

Organism.Name

Ecotype

Pathogen

32002

Achromobacter denitrificans

AS

None

56780

Syntrophus aciditrophicus SB

AS

None

62977

Acinetobacter baylyi ADP1

AS

None

265072

Methylobacillus flagellatus KT

AS

None

272564

Desulfitobacterium hafniense DCB-2

AS

None

335543

Syntrophobacter fumaroxidans MPOB

AS

None

337191

Gordonia sp. KTR9

AS

None

338963

Pelobacter carbinolicus Bd1, GraBd1

AS

None

370438

Pelotomaculum thermopropionicum SI

AS

None

381666

Cupriavidus necator H16

AS

None

398578

Delftia acidovorans SPH-1

AS

None

402881

Parvibaculum lavamentivorans DS-1

AS

None

420662

Methylibium petroleiphilum PM1

AS

None

439375

Ochrobactrum anthropi ATCC 49188

AS

None

465721

Steroidobacter denitrificans DSM 18526

AS

None

479431

Nakamurella multipartita Y-104, DSM 44233

AS

None

479434

Sphaerobacter thermophilus 4ac11, DSM 20745

AS

None

522306

Candidatus Accumulibacter phosphatis Type IIA UW-1

AS

None

558173

Corynebacterium doosanense CAU 212, DSM 45436

AS

None

596154

Alicycliphilus denitrificans K601

AS

None

610130

Lachnoclostridium saccharolyticum WM1, DSM 2544

AS

None

635013

Thermincola potens JR

AS

None

643648

Syntrophothermus lipocalidus DSM 12680

AS

None

645991

Syntrophobotulus glycolicus FlGlyR, DSM 8271

AS

None

663278

Ethanoligenens harbinense YUAN-3

AS

None

675635

Pseudonocardia dioxanivorans CB1190

AS

None

706587

Desulfomonile tiedjei DCB-1, DSM 6799

AS

None

720554

Hungateiclostridium clariflavum EBR 45, DSM 19732

AS

None

742013

Delftia acidovorans Cs1-4

AS

None

760192

Haliscomenobacter hydrossis O, DSM 1100

AS

None

868595

Desulfotomaculum carboxydivorans CO-1-SRB, DSM 14880

AS

None

879274

Shinella sp. HZN7

AS

None

880072

Desulfobacca acetoxidans ASRB2, DSM 11109

AS

None

926569

Anaerolinea thermophila UNI-1

AS

None

927704

Selenomonas ruminantium lactilytica TAM6421

AS

None

980445

Desulfoglaeba alkanexedens ALDC

AS

None

983917

Rubrivivax gelatinosus IL144

AS

None

1030157

Sphingomonas sp. KC8

AS

None

1032480

Microlunatus phosphovorus NM-1

AS

None

1118963

Arthrobacter nitroguajacolicus Rue61a

AS

None

1150621

Sulfurospirillum multivorans DSM 12446

AS

None

1209989

Tepidanaerobacter sp. Re1

AS

None

1219035

Novosphingobium tardaugens NBRC 16725

AS

None

1325564

Nitrospira japonica

AS

None

1458492

Myroides sp. ZB35

AS

None

Table S4. Significance of differences among the 4 clusters from Figure 1b. The numbers are the P values from the pair-wise Mann-Whitney test. Those values which are less than 0.05 are highlighted in red.

ARGs

 

cluster1

cluster2

cluster3

cluster4

Count of Detected ARGs

cluster1

 

0.000455

0.307646

0.00322

cluster2

0.000455

 

0.001263

0.342295

cluster3

0.307646

0.001263

 

0.017629

cluster4

0.00322

0.342295

0.017629

 

Total abundance

cluster1

0.818736

3.16E-14

7.02E-05

cluster2

0.818736

3.30E-10

0.000532

cluster3

3.16E-14

3.30E-10

0.000135

cluster4

7.02E-05

0.000532

0.000135

aminoglycoside

cluster1

1.31E-07

5.86E-19

1.32E-11

cluster2

1.31E-07

4.31E-08

3.71E-05

cluster3

5.86E-19

4.31E-08

0.94552

cluster4

1.32E-11

3.71E-05

0.94552

bacitracin

cluster1

2.13E-05

0.85181

0.005188

cluster2

2.13E-05

1.68E-06

1.33E-08

cluster3

0.85181

1.68E-06

0.004079

cluster4

0.005188

1.33E-08

0.004079

beta.lactam

cluster1

2.44E-09

1.86E-06

1.32E-11

cluster2

2.44E-09

0.024939

0.002337

cluster3

1.86E-06

0.024939

4.09E-08

cluster4

1.32E-11

0.002337

4.09E-08

chloramphenicol

cluster1

6.67E-10

5.86E-19

1.32E-11

cluster2

6.67E-10

4.80E-09

0.119496

cluster3

5.86E-19

4.80E-09

8.25E-06

cluster4

1.32E-11

0.119496

8.25E-06

fosfomycin

cluster1

0.827717

0.123212

0.812021

cluster2

0.827717

0.127117

0.71118

cluster3

0.123212

0.127117

0.332092

cluster4

0.812021

0.71118

0.332092

fosmidomycin

cluster1

0.339077

0.376661

0.33057

cluster2

0.339077

0.323475

0.036637

cluster3

0.376661

0.323475

0.05612

cluster4

0.33057

0.036637

0.05612

kasugamycin

cluster1

0.033024

0.000247

0.043438

cluster2

0.033024

0.554874

0.904001

cluster3

0.000247

0.554874

0.647833

cluster4

0.043438

0.904001

0.647833

MLS

cluster1

0.007098

0.000305

2.66E-05

cluster2

0.007098

0.673056

0.044784

cluster3

0.000305

0.673056

0.038112

cluster4

2.66E-05

0.044784

0.038112

polymyxin

cluster1

0.006051

0.014271

0.000351

cluster2

0.006051

0.59287

0.044784

cluster3

0.014271

0.59287

0.005701

cluster4

0.000351

0.044784

0.005701

quinolone

cluster1

2.73E-08

1.04E-13

3.96E-10

cluster2

2.73E-08

0.728687

0.598552

cluster3

1.04E-13

0.728687

0.732494

cluster4

3.96E-10

0.598552

0.732494

Table S4. Significance of differences among the 4 clusters from Figure 1b. The numbers are the P values from the pair-wise Mann-Whitney test. Those values which are less than 0.05 are highlighted in red. (continued)

rifamycin

cluster1

1.30E-08

7.79E-07

0.00055

cluster2

1.30E-08

0.004701

0.010577

cluster3

7.79E-07

0.004701

0.646118

cluster4

0.00055

0.010577

0.646118

sulfonamide

cluster1

1.61E-05

1.35E-14

0.014784

cluster2

1.61E-05

0.000257

0.101454

cluster3

1.35E-14

0.000257

2.65E-06

cluster4

0.014784

0.101454

2.65E-06

tetracycline

cluster1

3.22E-10

2.34E-18

1.32E-11

cluster2

3.22E-10

0.021341

0.019174

cluster3

2.34E-18

0.021341

0.604536

cluster4

1.32E-11

0.019174

0.604536

vancomycin

cluster1

0.001495

4.73E-08

1.86E-07

cluster2

0.001495

0.517275

0.16274

cluster3

4.73E-08

0.517275

0.171818

cluster4

1.86E-07

0.16274

0.171818

Table S5. Information of core resistome according to persistency and abundancy detected from 9 years

ARG subtypes

Occurrence frequency (%)

Average abundance

(Copy of ARGs per cell)

Relative abundance (%)

aminoglycoside__aadA

100

0.008257

3.37

aminoglycoside__aph(3'')-I

100

0.005744

2.34

aminoglycoside__aph(6)-I

100

0.004243

1.73

bacitracin__bacA

100

0.031070

12.68

beta-lactam__class A beta-lactamase

100

0.003326

1.36

chloramphenicol__floR

100

0.003257

1.33

fosmidomycin__rosA

100

0.001377

0.56

fosmidomycin__rosB

100

0.001055

0.43

MLS__ereA

100

0.004958

2.02

MLS__macA

96

0.000692

0.28

MLS__macB

100

0.003216

1.31

multidrug__acrB

100

0.007931

3.24

multidrug__adeJ

100

0.000357

0.15

multidrug__ceoB

98

0.000158

0.06

multidrug__mdtB

100

0.000663

0.27

multidrug__mdtC

99

0.000387

0.16

multidrug__mdtF

100

0.000634

0.26

multidrug__mexB

100

0.002494

1.02

multidrug__mexD

100

0.000428

0.17

multidrug__mexF

100

0.002082

0.85

multidrug__mexI

100

0.001577

0.64

multidrug__mexW

100

0.004074

1.66

multidrug__multidrug_ABC_transporter

100

0.002327

0.95

multidrug__multidrug_transporter

100

0.009492

3.87

multidrug__qacEdelta1

100

0.006008

2.45

multidrug__smeB

97

0.000215

0.09

multidrug__smeE

96

0.000120

0.05

polymyxin__arnA

100

0.000561

0.23

rifamycin__ADP-ribosylating transferase_arr

100

0.003623

1.48

sulfonamide__sul1

100

0.023627

9.64

sulfonamide__sul2

99

0.002698

1.10

sulfonamide__sul4

99

0.002514

1.03

tetracycline__tet32

96

0.000218

0.09

tetracycline__tetC

96

0.002511

1.02

tetracycline__tetG

100

0.002959

1.21

tetracycline__tetM

98

0.000476

0.19

tetracycline__tetO

99

0.000372

0.15

tetracycline__tetracycline_resistance_protein

96

0.000635

0.26

tetracycline__tetW

99

0.000618

0.25

unclassified__transcriptional regulatory protein CpxR

100

0.005376

2.19

vancomycin__vanR

97

0.001088

0.44