SUPPLEMENTARY MATERIALS

Transcription

1 SUPPLEMENTARY MATERIALS Supplementary Table S1: Water sampling sites in Brisbane River and their characteristics Sampling sites GPS coordinates Site characteristics Suspected source of fecal pollution BR S ; ˮ E Rural Wildlife, waterfowls, recreational activities BR ˮ S ; ˮ E Rural Cattle, horses, septic tanks, wildlife BR ˮ S ; ˮ E Peri urban, tidally influenced Wastewater treatment plants, waterfowls BR ˮ S ; ˮ E Urban, tidally influenced Storm water drain BR ˮ S ; ˮ E Urban Recreational activities BR ˮ S ; ˮ E Urban Port, industrial activities, recreational activities

2 Supplementary Table S2: Target, primer/probe sequences and amplification conditions for real-time PCR assays used in this study Assays Target Primer or probe sequence (5-3 ) a Amplification conditions Universal bacteria 16S rrna F: ACT CCT ACG GGA GGC AGC AG R: ATT ACC GCG GCT GCT GG 5 min at 95 C, 40 cycles of 15 s at 95 C, 30 s at 53 C and 60 s at 60 C Sketa22 Oncorhynchus keta F: GGT TTC CGC AGC TGG G R: CCG AGC CGT CCT GGT CTA 10 min at 95 C, 40 cycles of 15 s at 95 C, 45 s at 63 C CowM3 Sialic acid-specific 9-Oacetylesterase secretory protein homolog P: FAM-AGT CGC AGG CGG CCA CCG T-TAMRA F: CCT CTA ATG GAA AAT GGA TGG TAT CT R: CCA TAC TTC GCC TGC TAA TAC CTT P: FAM-TTA TGC ATT GAG CAT CGA GGCC-TAMRA 10 min at 95 C followed by 40 cycles of 15 s at 95 C, and 1 min at 60 C Bacteroides HF183 16S rrna F: ATC ATG AGT TCA CAT GTC CCG R: TAC CCC GCC TAC TAT CTA ATG 95 C for 10 min, 45 cycles 95 C for 30 s, 53 C for 1 min, and 60 C for 1 min GFD Unclassified Helicobacter spp. F: TCG GCT GAG CAC TCT AGG G R: GCG TCT CTT TGT ACA TCC CA 10 min at 95 C, 40 cycles of 15 s at 95 C, 30 s at 57 C Bacteroides Horse 16S rrna F: CCAGCCGTAAAATAGTCGG R: CAATCGGAGTTCTTCGTG 95 C for 10 min, 45 cycles 95 C for 30 s, 59 C for 1 min, and 72 C for 1 min NGS 16S rdna V5-V6 hypervariable regions F: RGG ATT AGA TAC CC R: CGA CRR CCA TGC ANC ACC T 3 min at 95 C, 26 cycles of 50 s at 95 C, 30 s at 40 C, 60 s at 72 C, and 5 min at 72 C a F, forward primer; R, reverse primer; P, probe; FAM, 6-carboxyfluorescein; TAMRA, 6-carboxytetramethylrhodamine

3 Supplementary Table S3: Host-sensitivity and host-specificity values of the host-associated molecular markers tested in this study Markers No of targetsamplesamples Host-sensitivity No of target- Host-specificity References CowM (1) Bacteroides (2) HF183 GFD This study Bacteroides horse This study

4 2500 BR1a BR1b BR1c BR2a BR2b BR2c BR3b BR5a BR5b BR5c BR6c OTUs Sequence Reads Supplementary Figure S1: Rarefaction curves based on the numbers of operational taxonomic units (OTUs) for 11 Brisbane River water samples. Curves were produced at a cut-off of 0.03 for each sample.

5 WWTP 1 WWTP 2 WWTP 3 WWTP 4 WWTP 5 WWTP 6 WWTP 7 WWTP 8 WWTP 9 WWTP 10 WWTP 11 OTUs Sequence Reads Supplementary Figure S2: Rarefaction curves based on the numbers of operational taxonomic units (OTUs) for 11 human wastewater samples. Curves were produced at a cut-off of 0.03 for each sample.

6 Cat 1 Cat 2 Cat 3 Cat 4 Cat 5 Cat 6 Cat 7 Cat 8 Cat 9 Cat 10 Cat OTUs Sequence Reads Supplementary Figure S3: Rarefaction curves based on the numbers of operational taxonomic units (OTUs) for 11 cat fecal samples. Curves were produced at a cut-off of 0.03 for each sample.

7 Cattle WW 1 Cattle WW 2 Cattle WW 3 Cattle WW 5 Cattle WW 6 Cattle WW 7 Cattle WW 8 Cattle WW 9 Cattle WW 10 Cattle WW 11 Cattle WW OTUs Sequence Reads Supplementary Figure S4: Rarefaction curves based on the numbers of operational taxonomic units (OTUs) for 11 cattle wastewater samples. Curves were produced at a cut-off of 0.03 for each sample.

8 Dog 1 Dog 4 Dog 5 Dog 11 Dog 13 Dog 14 Dog 15 Dog 16 Dog 17 Dog 18 Dog OTUs Sequence Reads Supplementary Figure S5: Rarefaction curves based on the numbers of operational taxonomic units (OTUs) for 11 dog fecal samples. Curves were produced at a cut-off of 0.03 for each sample.

9 Horse 6 Horse 7 Horse 8 Horse 9 Horse 10 Horse 11 Horse 12 Horse 13 Horse 14 Horse 15 Horse OTUs Sequence Reads Supplementary Figure S6: Rarefaction curves based on the numbers of operational taxonomic units (OTUs) for 11 horse fecal samples. Curves were produced at a cut-off of 0.03 for each sample.

10 Kangaroo 1 Kangaroo 3 Kangaroo 4 Kangaroo 5 Kangaroo 8 Kangaroo 9 Kangaroo 10 Kangaroo 11 Kangaroo 12 Kangaroo 13 Kangaroo OTUs Sequence Reads Supplementary Figure S7: Rarefaction curves based on the numbers of operational taxonomic units (OTUs) for 11 kangaroo fecal samples. Curves were produced at a cut-off of 0.03 for each sample.

11 Supplementary Figure S8: Distribution of 15 most abundant order among individual samples from Brisbane River used for bacterial community comparisons. Asterisk (*) denotes orders designated incertae sedis

12 Supplementary Figure S9: Distribution of 15 most abundant order among individual samples from wastewater treatment plants (WWTPs) used for bacterial community comparisons.

13 Supplementary Figure S7: Rarefaction curves based on the numbers of operational taxonomic units (OTUs) for 11 kangaroo fecal samples. Curves were Supplementary Figure S10: Distribution of 15 most abundant order among individual samples from cat used for bacterial community comparisons.

14 Supplementary Figure S11: Distribution of 15 most abundant order among individual samples from cattle wastewater used for bacterial community comparisons. Asterisk (*) denotes orders designated incertae sedis

15 Supplementary Figure S12: Distribution of 15 most abundant order among individual samples from dog used for bacterial community comparisons.

16 Supplementary Figure S13: Distribution of 15 most abundant order among individual samples from horse used for bacterial community comparisons. Asterisk (*) denotes orders designated incertae sedis

17 Supplementary Figure S14: Distribution of 15 most abundant order among individual samples from kangaroo used for bacterial community comparisons. Asterisk (*) denotes orders designated incertae sedis

18 References (1) Ahmed W, Sritharan T, Palmer A, Sidhu JPS, Toze S Evaluation of bovine feces-associated microbial source tracking markers and their correlations with fecal indicators and zoonotic pathogens. Appl Environ Microbiol. 79: (2) Ahmed W, Masters N, Toze S Consistency in the host specificity and host sensitivity of the Bacteroides HF183 marker for sewage pollution tracking. Lett. Appl. Microbiol. 55: