Microbial Source Tracking in a Small Southern California Urban Watershed Indicates Wild Animals and Growth as the Source of Fecal Bacteria
By
Sunny C. Jiang*, Weiping Chu, Betty H. Olson, Jian-Wen He, Samuel Choi, Jenny Zhang, Joanne Y. Le, Phillip B. Gedalanga
Civil and Environmental Engineering
University of California
Irvine, California 92697
Phone: 949-824-5527
Email:
Supplementary Information
Material and Methods
PCR Detection and Confirmation of E. coli Toxin Genes:
For detection of host specific E. coli toxin genes, PCR primers, probes and annealing temperature are listed in Table 1. To differentiate the closely related gene marker between specific hosts, the –papG III from dog were aligned with human papG III to identify the allele polymorphisms. The human and dog papG III were differentiated by restriction enzyme PcilI, which yields two fragments of 69 and 371 bp, respectively, for human papG III but do not cut for dog papG III. Two restriction enzymes, PstI and AluI were used to confirm PCR amplicons of the cow toxin gene (LTIIa). PstI produces two fragments with sizes of 245 bp and 113 bp, and AluI produces two fragments with sizes of 222 bp and 136 bp. Southern blot hybridization was also used to confirm PCR amplicons for human and cow toxin genes using previously designed probes specifically for each toxin (Olson and Oshiro 1997; Khatib et al. 2002; Chern et al. 2004). Confirmation of the bird and rabbit toxin genes were performed using newly designed internal probe by southern blot hybridization. The internal probes and hybridization temperature are also listed in Table 1.
PCR Detection of Human Adenovirus and Enterovirus
Primers and probes for detection of adenoviruses and enteroviruses are listed in Table 2. Nested-PCR was performed following the protocol of Pina et al. (1998) for detection of adenoviruses with minor modifications by Jiang et al. (2001). These primer sets were shown to amplify multiple serotypes of adenoviruses including enteric adenoviruses serotypes 40 and 41 (Pina et al. 1998). Reverse transcription PCR for enteroviruses was performed essentially as described by Tsai et al. (1993) with a modification of the total reaction volume to 25 ml. Amplicons were confirmed by probing with internal oligonucleotide probes by southern transfer or in a dot-blotting format to increase the sensitivity of detection as well as confirmation of correct amplification products.
Quality Assurance and Quality Control Procedures
QA/QC of laboratory reagents and instruments were performed before sample processing and analyses. For E. coli and Enterocuccus spp. isolation, culture medium was purchased from credible commercial venders with QA/QC data on the medium and reagents. For viral concentration using tangential flow filtration system, the viral recovery rates were tested by viral seeding study using bacteriophage FHSIC. Average viral recovery with this system ranged from 50 to 80% (Chu et al. 2003). For viral extraction and purification, all reagents, test tubes and pippetts were tested independently three to five times to ensure the accuracy and free of contamination of the target organisms. At least one negative control was included during each extraction to ensure no cross-contamination during extraction procedure. For PCR and nested-PCR analysis, all assays were performed in a designated PCR hood. Working area was wiped with 1% bleach followed by 75% ethanol and radiated by UV radiation for 15 mins before each PCR set up. All PCR reagents, enzymes, instrument, pippettes and tubes were tested independently 3 to 5 times to ensure of accuracy and free of contaminants. At least one positive and one negative control were included in each PCR run to control for cross-contamination and false negative during PCR procedure. PCR amplicons were analyzed in a separate room to prevent amplicon contamination. Assays that did not satisfy this QA/QC requirement were repeated. Only results met the QA/QC requirements were reported here.
Table 1. Primers, probes and annealing or hybridization temperature for detection and confirmation of host specific E. coli toxin gene. The gene designation and GenBank accession numbers are included in the pretences.
Humans (STh, M34916)Outer (amplicon size 241 bp)
Forward 5’-TGTATTGTCTTTTTCACC-3’
Reverse 5’-CATCATCAGAATCAGAAC-3’
Annealing Temp: 45°C
Inner (amplicon size 166 bp)
Forward 5’-CSCTCAGGATGCTAAACCAG-3’
Reverse 5’-TTAATAGCACCCGGTACAAGC-3’
Annealing Temp: 55°C
Probe
5’-GTGGTCCTGAAAGCATGAATAGTAGC-3’
Hybridization temp: 54°C
Birds (tsh, AF218073)
Outer (amplicon size 481 bp)
Forward 5’-CAACCTCTAACGGAAGTACC-3’
Reverse 5’-ATAAACGGGCCTCTATCACG-3’
Annealing Temp: 57°C
Inner (amplicon size 197 bp)
Forward 5’-CAGGCGGACAATAAAGGACAGG-3’
Reverse 5’-AGCACGGCACCATAATCTGC-3’
Annealing Temp: 62°C
Probe
5’-CGACCGTGGTACTGACTGATGAGC-3’
Hybridization temp: 58°C
Cow (LTIIa, M17894)
Outer (amplicon size 359 bp)
Forward 5’-GGGTGTGCATTTCAGCGAC-3’
Reverse 5’-CGTCCACCCGGAATATACCA-3’
Annealing Temp: 61°C
Inner (amplicon size 204 bp)
Forward 5-gcatggagaaagagatgagc-3’
Reverse 5’-CTTACCACATAGATCCCACG-3’
Annealing Temp: 56°C
Probe
5’-CTGATGACTGGCATTCTGTCTGGTCAGGTATATGCTGG-3’
Hybridization Temp: 58°C
Dog (papG III, AF237474)
Outer (amplicon size 336 bp)
Forward 5’-ATTTTGCATGGCTGGTTGTT-3’
Reverse 5’-AAGGGATTTTGTAGCGTCCA-3’
Annealing temp: 58°C
Inner (amplicon size 106 bp)
Forward 5’-GGATTTGCATGGGATGAAGT-3’
Reverse 5’-ATCTGGCGTTCAGGGTAACA-3’
Annealing Temp: 56°C
Confirmation method: Restriction enzyme Digest (PcilI)
Rabbits (ralG, U84144)
Outer (amplicon size 344 bp)
Forward 5’-TGGTAAATCTGATGCGTTCCAAGCTTCAGG-3’
Reverse 5’-CAATCATGTTGACC GCTGTAGAACCACTCAAGCC-3’
Annealing Temp: 68°C
Inner (amplicon size 204 bp)
Forward 5’-TAGCATTTCTTTTAGTGGTGCAGAC-3’
Reverse 5’-ATCGGATGAGGACCACCATA-3’
Annealing Temp: 61°C
Probe
5’-ACAAACAAAGTACCGAGC-3’
Hybridizaton temp: 60°C
Table 2. Primers and probes for detection and confirmation of human viruses.
Forward, 5’-CCTCCGGCCCTGAATG-3’
Reverse, 5’-ACCGGATGGCCAATCCAA-3’
Probe, 5’-TACTTTGGGTGTCCGTGTTTC-3’
Adenovirus
Outer primers (amplicon size 301bp)
Forward: 5’-GCCGCAGTGGTCTTACATGCACATC-3’
Reverse: 5’-CAGCACGCCGCGGATGTCAAAGT-3’
Inner primers (amplicon size 143bp)
Forward:5’-GCCACCGAGACGTACTTCAGCCTG-3’
Reverse: 5’-TTGTACGAGTACGCGGTATCCTCGCGGTC-3’
Reference Cited
Chern EC, Tsai YL, Olson BH (2004) Occurrence of genes associated with enterotoxigenic and enterohemorrhagic Escherichia coli in agricultural waste lagoons. Applied and Environmental Microbiology 70: 356-362
Chu W, Choi S, Jiang S (2003) Concentration of viruses from aquatic environments by CentraMate TFF systems and Centricon Plus-80 Abstracts of the General Meeting of the American Society for Microbiology, Washington D. C.
Jiang S, Noble R, Chu W (2001) Human adenoviruses and coliphages in urban runoff-impacted coastal waters of Southern California. Applied and Environmental Microbiology 67: 179-184.
Khatib LA, Tsai YL, Olson BH (2002) A biomarker for the identification of cattle fecal pollution in water using the LTIIa toxin gene from enterotoxigenic Escherichia coli. Appl Microbiol Biotechnol 59: 97-104
Olson BH, Oshiro RK (1997) Occurrence of STh Toxin Gene In Wastewater.
Coliforms and E.coli: Problem or Solution? The Royal Society of Chemistry. Cambridge
Pina S, Puig M, Lucena F, Jofre J, Girones R (1998) Viral pollution in the environment and in shellfish: Human adenovirus detection by PCR as an index of human viruses. Applied and Environmental Microbiology 64: 3376-3382.
Tsai YL, Sobsey MD, Sangermano LR, Palmer CJ (1993) Simple method of concentrating enteroviruses and hepatitis A virus from sewage and ocean water for rapid detection by reverse transcriptase-polymerase chain reaction. Appl Environ Microbiol 59: 3488-3491.