Table 1 Sampling
site locations and characteristics Code Site name (GISb map reference) check details Site characteristics C1 Coomera marina (-27.861672, 153.339089) Cattle/kangaroo feeding, house-boat mooring site C2 Santa Barbara (-27.855165, 153.350612) Well used park, BBQ, toilets and fishing, private houses about 100 m away C3 Sanctuary Cove (-27.851617, 153.362140) Canal estate, modern houses and apartments, modern infrastructure, commercial/light industrial area C4 Jabiru Island (-27.879057, 153.380685) Busy through road, disused sand mine, no houses, small park with toilets C5 Paradise Point (-27.886359, 153.396596) Public swimming area, mouth of river, LBH589 cell line much water traffic C6 Coombabah, Estuary (-27.896607, 153.366845) Established suburban area, bush island opposite b Global information system Water samples were collected in sterile bottles according to the sampling procedures described in the USEPA microbiology methods manual . The sampling depth for surface water samples were 6-12 inches below the water surface. Samples were transported in a cooler on ice packs to the laboratory where they were prepared for analyses immediately upon arrival and were tested within 6 h of collection for the presence of enterococci. Isolation
and identification of enterococci The environmental water samples were mixed thoroughly, and undiluted samples or a 1:10 dilution of water samples were filtered through 0.45 μm membrane filters (MilliporeCorporation, Interleukin-2 receptor Bedford, MA, USA), placed onto membrane-Enterococcus Indoxyl β-D-Glucoside Agar (mEI) (Becton-Dickinson, Sparks, MD, USA) according to the USEPA specifications . Triplicate samples were collected from each site and each sample was treated separately. The addition of Indoxyl-β-D-Glucoside, Nalidixic acid, 0.1 N NaOH, and Triphenyltetrazolium Chloride to mEI agar
(Difco) allowed for a single 24 h incubation period at 41°C . E. faecium ATCC 27270, E. faecalis ATCC 19433 and E. coli ATCC 25922 were used as positive and negative controls respectively to validate the mEI agar. Colonies producing a blue halo were typically observed for enterococci and counted, the result expressed as cfu/ml for each water sample. Statistical analysis The Mann-Whitney U-test at 5% significance level was performed to determine whether there was a significant increase of total enterococcal counts (cfu/ml) at each location after rainfall events. Identification of E. faecium and E. faecalis Typical colonies on the membranes were identified to the genus and species level by Gram-stain, catalase test, the ability to tolerate 6.5% NaCl and biochemical tests . The isolates identified as E. faecium and E.