Predatory bacteria in combination with solar disinfection and solar photocatalysis for the treatment of rainwater.

The predatory bacterium, Bdellovibrio bacteriovorus, was applied as a biological pre-treatment to solar disinfection and solar photocatalytic disinfection for rainwater treatment. The photocatalyst used was immobilised titanium-dioxide reduced graphene oxide. The pre-treatment followed by solar photocatalysis for 120 min under natural sunlight reduced the viable counts of Klebsiella pneumoniae from 2.00 × 109 colony forming units (CFU)/mL to below the detection limit (BDL) (<1 CFU/100 µL). Correspondingly, ethidium monoazide bromide quantitative PCR analysis indicated a high total log reduction in K. pneumoniae gene copies (GC)/mL (5.85 logs after solar photocatalysis for 240 min). In contrast, solar disinfection and solar photocatalysis without the biological pre-treatment were more effective for Enterococcus faecium disinfection as the viable counts of E. faecium were reduced by 8.00 logs (from 1.00 × 108 CFU/mL to BDL) and the gene copies were reduced by ∼3.39 logs (from 2.09 × 106 GC/mL to ∼9.00 × 102 GC/mL) after 240 min of treatment. Predatory bacteria can be applied as a pre-treatment to solar disinfection and solar photocatalytic treatment to enhance the removal efficiency of Gram-negative bacteria, which is crucial for the development of a targeted water treatment approach.

Assessment of predatory bacteria and prey interactions using culture-based methods and EMA-qPCR.

Traditional culture-based enumeration methods were compared with the ethidium monoazide quantitative polymerase chain reaction (EMA-qPCR) technique to assess Bdellovibrio-and-like-organisms (BALOs) predator-prey interactions. Gram-negative [Pseudomonas spp. and Klebsiella pneumoniae (K. pneumoniae)] and Gram-positive [Staphylococcus aureus (S. aureus) and Enterococcus faecium (E. faecium)] organisms were employed as prey cells, while a Bdellovibrio bacteriovorus strain (PF13) was used as the predator. The co-culture experiments were also compared in diluted nutrient broth (DNB) and HEPES buffer. In both media, K. pneumoniae (maximum log reduction of 5.13) and Pseudomonas fluorescens (P. fluorescens) (maximum log reduction of 4.21) were sensitive to predation by B. bacteriovorus PF13 as their cell counts and gene copies were reduced during all the co-culture experiments, while the concentration of B. bacteriovorus PF13 increased. The concentration of B. bacteriovorus PF13 also increased in the presence of S. aureus (HEPES buffer) and E. faecium (DNB), indicating that the predator interacted with these Gram-positive prey in order to survive. Moreover, as no predator plaques were produced in the co-culture experiments with P. aeruginosa (DNB and HEPES buffer), S. aureus (DNB and HEPES buffer) and E. faecium (HEPES buffer), EMA-qPCR proved to be beneficial in monitoring the concentration of B. bacteriovorus. In conclusion, the cell counts and/or EMA-qPCR analysis for the HEPES buffer and DNB assays were successfully employed to monitor the predation of P. fluorescens and K. pneumoniae by B. bacteriovorus, while E. faecium was sensitive to predation in DNB and S. aureus was sensitive to predation in HEPES buffer.

Development and small-scale validation of a novel pigeon-associated mitochondrial DNA source tracking marker for the detection of fecal contamination in harvested rainwater.

The current study was aimed at designing and validating (on a small-scale) a novel pigeon mitochondrial DNA (mtDNA) microbial source tracking (MST) marker for the detection of pigeon fecal matter in harvested rainwater. The pigeon mtDNA MST marker was designed to target the mtDNA Cytochrome b gene by employing mismatch amplification mutation assay kinetics. The pigeon marker was validated by screening 69 non-pigeon and 9 pigeon fecal samples. The host-sensitivity of the assay was determined as 1.00 while the host-specificity of the assay was 0.96. Harvested rainwater samples (n=60) were screened for the prevalence of the marker with the mtDNA Cytochrome b marker detected in 78% of the samples. Bayes' theorem was applied to calculate the conditional probability of the marker detecting true pigeon contamination and the marker subsequently displayed a 99% probability of detecting true pigeon contamination in the harvested rainwater samples. In addition, the mtDNA Cytochrome b marker displayed high concurrence frequencies versus heterotrophic bacteria (78.3%), E. coli (73.3%), total coliforms (71.1%) and fecal coliforms (66.7%). This study thus validates that targeting mtDNA for the design of source tracking markers may be a valuable tool to detect avian fecal contamination in environmental waters.

Microbial source tracking markers associated with domestic rainwater harvesting systems: Correlation to indicator organisms.

Domestic rainwater harvesting (tank water) systems were screened for the presence of a panel of microbial source tracking (MST) markers and traditional indicator organisms. The indicator organisms were enumerated utilizing traditional culture-based methods, while the MST markers were quantified by quantitative PCR (qPCR). The indicators Escherichia coli (E. coli) and enterococci were also quantified using qPCR. Correlations and concurrence between these parameters were then investigated to determine which markers could be utilized to supplement traditional indicator analysis. Quantitative PCR analysis indicated that Bacteroides HF183, adenovirus, Lachnospiraceae and E. coli were detected and quantifiable in 100% of the tank water samples collected throughout the sampling period, while human mitochondrial DNA (mtDNA) was quantifiable in 90% of the tank water samples and Bifidobacterium adolescentis (B. adolescentis) and enterococci were quantifiable in 67% of the tank water samples, respectively. Significant positive correlations were recorded for Lachnospiraceae versus heterotrophic bacteria (p = 0.000), adenovirus versus E. coli (culturing) (p = 0.000) and heterotrophic bacteria (p = 0.024), the HF183 marker versus E. coli (qPCR) (p = 0.024) and B. adolescentis versus fecal coliforms (p = 0.037). In addition, 100% concurrence was observed for the HF183 marker, adenovirus and Lachnospiraceae versus E. coli (qPCR), enterococci (qPCR) and heterotrophic bacteria, amongst others. Based on the correlations and the concurrence analysis, the HF183 marker, Lachnospiraceae and adenovirus may be utilized to supplement indicator organism analysis for the monitoring of harvested rainwater quality.

Presence of microbial and chemical source tracking markers in roof-harvested rainwater and catchment systems for the detection of fecal contamination.

Microbial source tracking (MST) and chemical source tracking (CST) markers were utilized to identify fecal contamination in harvested rainwater and gutter debris samples. Throughout the sampling period, Bacteroides HF183 was detected in 57.5 % of the tank water samples and 95 % of the gutter debris samples, while adenovirus was detected in 42.5 and 52.5 % of the tank water and gutter debris samples, respectively. Human adenovirus was then detected at levels ranging from below the detection limit to 316 and 1253 genome copies/µL in the tank water and debris samples, respectively. Results for the CST markers showed that salicylic acid (average 4.62 µg/L) was the most prevalent marker (100 %) in the gutter debris samples, caffeine (average 18.0 µg/L) was the most prevalent in the tank water samples (100 %) and acetaminophen was detected sporadically throughout the study period. Bacteroides HF183 and salicylic acid (95 %) and Bacteroides HF183 and caffeine (80 %) yielded high concurrence frequencies in the gutter debris samples. In addition, the highest concurrence frequency in the tank water samples was observed for Bacteroides HF183 and caffeine (60 %). The current study thus indicates that Bacteroides HF183, salicylic acid and caffeine may potentially be applied as source tracking markers in rainwater catchment systems in order to supplement fecal indicator analyses.