A. J. Watkinson

Antibiotic-resistant Escherichia coli in wastewaters, surface waters, and oysters from an urban riverine system

ABSTRACT The antibiotic resistance (AR) patterns of 462 Escherichia coli isolates from wastewater, surface waters, and oysters were determined. Rates of AR and multiple-AR among isolates from surface water sites adjacent to wastewater treatment plant (WWTP) discharge sites were significantly higher (P < 0.05) than those among other isolates, whereas the rate of AR among isolates from oysters exposed to WWTP discharges was low (<10%).

Novel method for rapid assessment of antibiotic resistance in Escherichia coli isolates from environmental waters by use of a modified chromogenic agar.

ABSTRACT We validated a novel method for screening Escherichia coli resistance to antibiotics in environmental samples using modified Difco MI agar (Becton Dickinson) impregnated with selected antibiotics (tetracycline, ampicillin, cephalexin, and sulfamethoxazole), termed MI-R. This method combines an existing rapid assessment technique for E. coli enumeration with clinical reference data for breakpoint analysis of antibiotic resistance and was developed to address issues encountered when clinical methods are used with environmental samples. Initial trials conducted using strains of E. coli with resistance to the selected antibiotics showed that this method was reproducible and accurate with respect to antibiotic resistance. Trials using wastewater effluent demonstrated the precision of the method, and the levels of resistance found in effluent were directly comparable to the levels of antibiotic resistance determined using the more traditional CLSI (formerly NCCLS) disk susceptibility test. All wastewater isolates growing on MI-R plates were confirmed to be resistant using the CLSI disk susceptibility test. Bacterial resistance to ampicillin (38% ± 4% overall), sulfamethoxazole, tetracycline (21% ± 3% overall), and ciprofloxacin (6% ± 1%) were found in wastewater effluent. A successful trial was also conducted with water collected from the Brisbane River, Australia. The levels of antibiotic resistance in E. coli ranged from 0 to 47% for ampicillin, from 0 to 24% for tetracycline, from 0 to 63% for sulfamethoxazole, and from 0 to 1% for ciprofloxacin, with the highest incidence of resistance associated with wastewater treatment plant discharges. This method has great potential for rapid and representative assessment of antibiotic resistance in E. coli and could allow increased sample analysis, resulting in greater confidence in spatial analysis in environmental studies.

Cattle-derived microbial input to source water catchments: an experimental assessment of stream crossing modification

The provision of safe drinking water is a global issue, and animal production is recognized as a significant potential origin of human infectious pathogenic microorganisms within source water catchments. On-farm management can be used to mitigate livestock-derived microbial pollution in source water catchments to reduce the risk of contamination to potable water supplies. We applied a modified Before-After Control Impact (BACI) design to test if restricting the access of livestock to direct contact with streams prevented longitudinal increases in the concentrations of faecal indicator bacteria and suspended solids. Significant longitudinal increases in pollutant concentrations were detected between upstream and downstream reaches of the control crossing, whereas such increases were not detected at the treatment crossing. Therefore, while the crossing upgrade was effective in preventing cattle-derived point source pollution by between 112 and 158%, diffuse source pollution to water supplies from livestock is not ameliorated by this intervention alone. Our findings indicate that stream crossings that prevent direct contact between livestock and waterways provide a simple method for reducing pollutant loads in source water catchments, which ultimately minimises the likelihood of pathogenic microorganisms passing through source water catchments and the drinking water supply system. The efficacy of the catchment as a primary barrier to pathogenic risks to drinking water supplies would be improved with the integration of management interventions that minimise direct contact between livestock and waterways, combined with the mitigation of diffuse sources of livestock-derived faecal matter from farmland runoff to the aquatic environment.

Reassessing the risk of microbial contamination from roosting cormorants in source water supply reservoirs

Abstract A previous water quality risk assessment of source water supply reservoirs in subtropical southeast Queensland (Australia) evaluated little black cormorants (Phalocrocorax sulcirostris) roosting on intake infrastructure as potentially posing an extreme risk of microbial contamination through direct deposition of fecal matter to the aquatic environment. To evaluate this risk rating, we assessed populations of little black cormorants occupying 3 intake structures across 2 reservoirs, enumerated Escherichia coli (E. coli) levels collected from fecal matter, and estimated a daily E. coli load to the reservoir for each population. Concurrently, we supplemented the existing routine monthly water quality monitoring program with targeted water sampling to measure E. coli concentrations in water at the 3 water intake points and at 2 sites without extant cormorant populations. Up to 3.9 × 1014 E. coli organisms were estimated to be produced per day by the largest population surveyed. Cormorants were present at intake sites and absent from reference sites; however, concentrations of E. coli were not significantly higher in water at intake sites compared with reference sites (p = 0.793 vs. p = 0.1069, respectively), and there was no significant relationship (p = 0.9671) between cormorant numbers and water column concentrations of E. coli. The inability to quantify significant differences in microbial concentrations among sites suggests a more intensive sampling regime is required to clarify the relative contribution of contamination sources. Populations of roosting cormorants in our study reservoirs are unlikely to pose an extreme risk to source water quality when compared to other catchment-based inputs that dominate microbial pollution.

Profiling the diversity of Cryptosporidium species and genotypes in wastewater treatment plants in Australia using next generation sequencing

Wastewater recycling is an increasingly popular option in worldwide to reduce pressure on water supplies due to population growth and climate change. Cryptosporidium spp. are among the most common parasites found in wastewater and understanding the prevalence of human-infectious species is essential for accurate quantitative microbial risk assessment (QMRA) and cost-effective management of wastewater. The present study conducted next generation sequencing (NGS) to determine the prevalence and diversity of Cryptosporidium species in 730 raw influent samples from 25 Australian wastewater treatment plants (WWTPs) across three states: New South Wales (NSW), Queensland (QLD) and Western Australia (WA), between 2014 and 2015. All samples were screened for the presence of Cryptosporidium at the 18S rRNA (18S) locus using quantitative PCR (qPCR), oocyst numbers were determined directly from the qPCR data using DNA standards calibrated by droplet digital PCR, and positives were characterized using NGS of 18S amplicons. Positives were also screened using C. parvum and C. hominis specific qPCRs. The overall Cryptosporidium prevalence was 11.4% (83/730): 14.3% (3/21) in NSW; 10.8% (51/470) in QLD; and 12.1% (29/239) in WA. A total of 17 Cryptosporidium species and six genotypes were detected by NGS. In NSW, C. hominis and Cryptosporidium rat genotype III were the most prevalent species (9.5% each). In QLD, C. galli, C. muris and C. parvum were the three most prevalent species (7.7%, 5.7%, and 4.5%, respectively), while in WA, C. meleagridis was the most prevalent species (6.3%). The oocyst load/Litre ranged from 70 to 18,055 oocysts/L (overall mean of 3426 oocysts/L: 4746 oocysts/L in NSW; 3578 oocysts/L in QLD; and 3292 oocysts/L in WA). NGS-based profiling demonstrated that Cryptosporidium is prevalent in the raw influent across Australia and revealed a large diversity of Cryptosporidium species and genotypes, which indicates the potential contribution of livestock, wildlife and birds to wastewater contamination.