Andrew Palmer

Evaluation of Bovine Feces-Associated Microbial Source Tracking Markers and Their Correlations with Fecal Indicators and Zoonotic Pathogens in a Brisbane, Australia, Reservoir

ABSTRACT This study was aimed at evaluating the host specificity and host sensitivity of two bovine feces-associated bacterial (BacCow-UCD and cowM3) and one viral [bovine adenovirus (B-AVs)] microbial source tracking (MST) markers by screening 130 fecal and wastewater samples from 10 target and nontarget host groups in southeast Queensland, Australia. In addition, 36 water samples were collected from a reservoir and tested for the occurrence of all three bovine feces-associated markers along with fecal indicator bacteria (FIB), Campylobacter spp., Escherichia coli O157, and Salmonella spp. The overall host specificity values of the BacCow-UCD, cowM3, and B-AVs markers to differentiate between bovine and other nontarget host groups were 0.66, 0.88, and 1.00, respectively (maximum value of 1.00). The overall host sensitivity values of these markers, however, in composite bovine wastewater and individual bovine fecal DNA samples were 0.93, 0.90, and 0.60, respectively (maximum value of 1.00). Among the 36 water samples tested, 56%, 22%, and 6% samples were PCR positive for the BacCow-UCD, cowM3, and B-AVs markers, respectively. Among the 36 samples tested, 50% and 14% samples were PCR positive for the Campylobacter 16S rRNA and E. coli O157 rfbE genes, respectively. Based on the results, we recommend that multiple bovine feces-associated markers be used if possible for bovine fecal pollution tracking. Nonetheless, the presence of the multiple bovine feces-associated markers along with the presence of potential zoonotic pathogens indicates bovine fecal pollution in the reservoir water samples. Further research is required to understand the decay rates of these markers in relation to FIB and zoonotic pathogens.

Toolbox Approaches Using Molecular Markers and 16S rRNA Gene Amplicon Data Sets for Identification of Fecal Pollution in Surface Water.

ABSTRACT In this study, host-associated molecular markers and bacterial 16S rRNA gene community analysis using high-throughput sequencing were used to identify the sources of fecal pollution in environmental waters in Brisbane, Australia. A total of 92 fecal and composite wastewater samples were collected from different host groups (cat, cattle, dog, horse, human, and kangaroo), and 18 water samples were collected from six sites (BR1 to BR6) along the Brisbane River in Queensland, Australia. Bacterial communities in the fecal, wastewater, and river water samples were sequenced. Water samples were also tested for the presence of bird-associated (GFD), cattle-associated (CowM3), horse-associated, and human-associated (HF183) molecular markers, to provide multiple lines of evidence regarding the possible presence of fecal pollution associated with specific hosts. Among the 18 water samples tested, 83%, 33%, 17%, and 17% were real-time PCR positive for the GFD, HF183, CowM3, and horse markers, respectively. Among the potential sources of fecal pollution in water samples from the river, DNA sequencing tended to show relatively small contributions from wastewater treatment plants (up to 13% of sequence reads). Contributions from other animal sources were rarely detected and were very small (<3% of sequence reads). Source contributions determined via sequence analysis versus detection of molecular markers showed variable agreement. A lack of relationships among fecal indicator bacteria, host-associated molecular markers, and 16S rRNA gene community analysis data was also observed. Nonetheless, we show that bacterial community and host-associated molecular marker analyses can be combined to identify potential sources of fecal pollution in an urban river. This study is a proof of concept, and based on the results, we recommend using bacterial community analysis (where possible) along with PCR detection or quantification of host-associated molecular markers to provide information on the sources of fecal pollution in waterways.

Public health implications of Acanthamoeba and multiple potential opportunistic pathogens in roof-harvested rainwater tanks

A study of six potential opportunistic pathogens (Acanthamoeba spp., Legionella spp., Legionella longbeachae, Pseudomonas aeruginosa, Mycobacterium avium and Mycobacterium intracellulare) and an accidental human pathogen (Legionella pneumophila) in 134 roof-harvested rainwater (RHRW) tank samples was conducted using quantitative PCR (qPCR). All five opportunistic pathogens and accidental pathogen L. pneumophila were detected in rainwater tanks except Legionella longbeachae. Concentrations ranged up to 3.1×10(6) gene copies per L rainwater for Legionella spp., 9.6×10(5) gene copies per L for P. aeruginosa, 6.8×10(5) gene copies per L for M. intracellulare, 6.6×10(5) gene copies per L for Acanthamoeba spp., 1.1×10(5) gene copies per L for M. avium, and 9.8×10(3) gene copies per L for L. pneumophila. Among the organisms tested, Legionella spp. (99% tanks) were the most prevalent followed by M. intracellulare (78%). A survey of tank-owners provided data on rainwater end-uses. Fecal indicator bacteria (FIB) Escherichia coli and Enterococcus spp. were enumerated using culture-based methods, and assessed for correlations with opportunistic pathogens and L. pneumophila tested in this study. Opportunistic pathogens did not correlate well with FIB except E. coli vs. Legionella spp. (tau=0.151, P=0.009) and E. coli vs. M. intracellulare (tau=0.14, P=0.015). However, M. avium weakly correlated with both L. pneumophila (Kendalls tau=0.017, P=0.006) and M. intracellulare (tau=0.088, P=0.027), and Legionella spp. also weakly correlated with M. intracellulare (tau=0.128, P=0.028). The presence of these potential opportunistic pathogens in tank water may present health risks from both the potable and non-potable uses documented from the current survey data.

Seasonal Assessment of Opportunistic Premise Plumbing Pathogens in Roof-Harvested Rainwater Tanks

A seasonal study on the occurrence of six opportunistic premise plumbing pathogens (OPPPs) in 24 roof-harvested rainwater (RHRW) tanks repeatedly sampled over six monthly sampling events (n = 144) from August 2015 to March 2016 was conducted using quantitative qPCR. Fecal indicator bacteria (FIB) Escherichia coli (E. coli) and Enterococcus spp. were enumerated using culture-based methods. All tank water samples over the six events were positive for at least one OPPP (Legionella spp., Legionella pneumophila, Mycobacterium avium, Mycobacterium intracellulare, Pseudmonas aeruginosa, or Acanthamoeba spp.) during the entire course of the study. FIB were positively but weakly correlated with P. aeruginosa (E. coli vs P. aeruginosa τ = 0.090, p = 0.027; Enterococcus spp. vs P. aeruginosa τ = 0.126, p = 0.002), but not the other OPPPs. FIBs were more prevalent during the wet season than the dry season, and L. pneumophila was only observed during the wet season. However, concentrations of Legionella spp., M. intracellulare, Acanthamoeba spp., and M. avium peaked during the dry season. Correlations were assessed between FIB and OPPPs with meteorological variables, and it was determined that P. aeruginosa was the only OPPP positively associated with an increased antecedent dry period, suggesting stagnation time may play a role for the occurrence of this OPPP in tank water. Infection risks may exceed commonly cited benchmarks for uses reported in the rainwater usage survey such as pool top-up, and warrant further exploration through quantitative microbial risk assessment (QMRA).

Comparative enteric viruses and coliphage removal during wastewater treatment processes in a sub-tropical environment

Microbiological safety of reclaimed water is one of the most important issues in managing potential health risks related to wastewater recycling. Presence and removal of human adenovirus (HAdV), human polyomavirus (HPyV), human torque teno virus (HTtV) and somatic coliphage family Microviridae in three wastewater treatment plants (WWTP) in sub-tropical Brisbane, Australia was investigated. All three WWTPs employ activated sludge process with added on Bardenpho process for nutrient removal. HPyV, HAdV, HTtV and Microviridae were consistently detected in the influent (105 to 106 Genomic copies (GC) L-1) and secondary treated effluent (102 to 103GCL-1). The results of this study suggest that, under appropriate conditions, WWTPs with activated sludge process in sub-tropical climate could be an effective treatment barrier with >3 log10 removal of enteric virus. The geometric mean of pooled data for each virus from all sites showed the highest removal for HPyV (3.65 log10) and lowest for HAdV (2.79 log10) which was statistically significant (p=0.00001). Whereas, the removal rate of HTtV and Microviridae was identical (2.81 log10). A poor correlation between the presence of enteric virus in influent or effluent with routinely monitored physicochemical parameters suggests limited use of physicochemical parameters as predictors of enteric virus presence. High prevalence of HAdV in influent and effluent combined with comparatively low removal suggest that it could be used as a model microorganism for determining enteric virus removal efficacy. Additional tertiary treatment may be required prior to effluent reuse for non-potable purposes or discharge into the recreational waters to prevent exposure of people to health hazards.