Comparison of biofilm ecology supporting growth of individual Naegleria species in a drinking water distribution system.

Free-living amoebae (FLA) are common components of microbial communities in drinking water distribution systems (DWDS). FLA are of clinical importance both as pathogens and as reservoirs for bacterial pathogens, so identifying the conditions promoting amoebae colonisation of DWDSs is an important public health concern for water utilities. We used high-throughput amplicon sequencing to compare eukaryotic and bacterial communities associated with DWDS biofilms supporting distinct FLA species (Naegleria fowleri, N. lovaniensis or Vermamoeba sp.) at sites with similar physical/chemical conditions. Eukaryote and bacterial communities were characteristics of different FLA species presence, and biofilms supporting Naegleria growth had higher bacterial richness and higher abundance of Proteobacteria, Bacteroidetes (bacteria), Nematoda and Rotifera (eukaryota). The eukaryotic community in the biofilms had the greatest difference in relation to the presence of N. fowleri, while the bacterial community identified individual bacterial families associated with the presence of different Naegleria species. Our results demonstrate that ecogenomics data provide a powerful tool for studying the microbial and meiobiotal content of biofilms, and, in these samples can effectively discriminate biofilm communities supporting pathogenic N. fowleri. The identification of microbial species associated with N. fowleri could further be used in the management and control of N. fowleri in DWDS.

Elimination of Naegleria fowleri from bulk water and biofilm in an operational drinking water distribution system.

Global incidence of primary amoebic meningoencephalitis cases associated with domestic drinking water is increasing. The need for understanding disinfectant regimes capable of eliminating the causative microorganism, Naegleria fowleri, from bulk water and pipe wall biofilms is critical. This field study demonstrated the successful elimination of N. fowleri from the bulk water and pipe wall biofilm of a persistently colonised operational drinking water distribution system (DWDS), and the prevention of further re-colonisation. A new chlorination unit was installed along the pipe line to boost the free chlorine residual to combat the persistence of N. fowleri. Biofilm and bulk water were monitored prior to and after re-chlorination (RCl), pre-rechlorination (pre-RCl) and post-rechlorination (post-RCl), respectively, for one year. A constant free chlorine concentration of > 1 mg/L resulted in the elimination of N. fowleri from both the bulk water and biofilm at the post-RCl site. Other amoeba species were detected during the first two months of chlorination, but all amoebae were eliminated from both the bulk water and biofilm at post-RCl after 60 days of chlorination with free chlorine concentrations > 1 mg/L. In addition, a dynamic change in the biofilm community composition and a four log reduction in biofilm cell density occurred post-RCl. The pre-RCl site continued to be seasonally colonised by N. fowleri, but the constant free chlorine residual of > 1 mg/L prevented N. fowleri from recolonising the bulk and pipe wall biofilm at the post-RCl site. To our knowledge, this is the first study to demonstrate successful removal of N. fowleri from both the bulk and pipe wall biofilm and prevention of re-colonisation of N. fowleri in an operational DWDS. The findings of this study are of importance to water utilities in addressing the presence of N. fowleri and other amoeba in susceptible DWDSs.

Characterization of a Drinking Water Distribution Pipeline Terminally Colonized by Naegleria fowleri.

Free-living amoebae, such as Naegleria fowleri, Acanthamoeba spp., and Vermamoeba spp., have been identified as organisms of concern due to their role as hosts for pathogenic bacteria and as agents of human disease. In particular, N. fowleri is known to cause the disease primary amoebic meningoencephalitis (PAM) and can be found in drinking water systems in many countries. Understanding the temporal dynamics in relation to environmental and biological factors is vital for developing management tools for mitigating the risks of PAM. Characterizing drinking water systems in Western Australia with a combination of physical, chemical and biological measurements over the course of a year showed a close association of N. fowleri with free chlorine and distance from treatment over the course of a year. This information can be used to help design optimal management strategies for the control of N. fowleri in drinking-water-distribution systems.

Surface analysis of pilot distribution system pipe autopsies: The relationship of organic and inorganic deposits to input water quality.

Time-of-Flight Secondary Ion Mass Spectrometry (ToF-SIMS) surface analysis was conducted to characterise deposits in polyethylene pipes used in a novel pilot water distribution system (PDS). The system consisted of four (4) parallel distribution systems receiving water from different treatment processes, ranging from conventional coagulation through to an advanced membrane filtration system. After two years of operation, the distribution system was shut down and samples of pipe were collected for autopsy analysis. Inlet and outlet samples from each PDS were collected for purpose of comparison. ToF-SIMS was used to assess chemical differences in surface biofilm accumulation and particulate deposition, which resulted as a consequence of the treatment method and operational mode of each system. These data supplemented previously collected bacteriological and chemical water quality data. Results from the inorganic analysis of the pipes were consistent with corrosion and contamination events that occurred upstream in the corresponding treatment systems. Principal component analysis of data on organic constituents showed oxygen and nitrogen containing fragments were associated with the treatment inlet and outlet samples. These types of signals can often be ascribed to biofilm polysaccharides and proteins. A trend was observed when comparing samples from the same PDS, showing an association of lower molecular weight (MW) organic fragments with the inlet and higher MW organic fragments with the outlet samples.

Reduced Efficiency of Chlorine Disinfection of Naegleria fowleri in a Drinking Water Distribution Biofilm.

Naegleria fowleri associated with biofilm and biological demand water (organic matter suspended in water that consumes disinfectants) sourced from operational drinking water distribution systems (DWDSs) had significantly increased resistance to chlorine disinfection. N. fowleri survived intermittent chlorine dosing of 0.6 mg/L for 7 days in a mixed biofilm from field and laboratory-cultured Escherichia coli strains. However, N. fowleri associated with an attached drinking water distribution biofilm survived more than 30 times (20 mg/L for 3 h) the recommended concentration of chlorine for drinking water. N. fowleri showed considerably more resistance to chlorine when associated with a real field biofilm compared to the mixed laboratory biofilm. This increased resistance is likely due to not only the consumption of disinfectants by the biofilm and the reduced disinfectant penetration into the biofilm but also the composition and microbial community of the biofilm itself. The increased diversity of the field biofilm community likely increased N. fowleri's resistance to chlorine disinfection compared to that of the laboratory-cultured biofilm. Previous research has been conducted in only laboratory scale models of DWDSs and laboratory-cultured biofilms. To the best of our knowledge, this is the first study demonstrating how N. fowleri can persist in a field drinking water distribution biofilm despite chlorination.

Assessing the impact of water treatment on bacterial biofilms in drinking water distribution systems using high-throughput DNA sequencing.

Biofilm control in drinking water distribution systems (DWDSs) is crucial, as biofilms are known to reduce flow efficiency, impair taste and quality of drinking water and have been implicated in the transmission of harmful pathogens. Microorganisms within biofilm communities are more resistant to disinfection compared to planktonic microorganisms, making them difficult to manage in DWDSs. This study evaluates the impact of four unique drinking water treatments on biofilm community structure using metagenomic DNA sequencing. Four experimental DWDSs were subjected to the following treatments: (1) conventional coagulation, (2) magnetic ion exchange contact (MIEX) plus conventional coagulation, (3) MIEX plus conventional coagulation plus granular activated carbon, and (4) membrane filtration (MF). Bacterial biofilms located inside the pipes of each system were sampled under sterile conditions both (a) immediately after treatment application ('inlet') and (b) at a 1 km distance from the treatment application ('outlet'). Bacterial 16S rRNA gene sequencing revealed that the outlet biofilms were more diverse than those sampled at the inlet for all treatments. The lowest number of unique operational taxonomic units (OTUs) and lowest diversity was observed in the MF inlet. However, the MF system revealed the greatest increase in diversity and OTU count from inlet to outlet. Further, the biofilm communities at the outlet of each system were more similar to one another than to their respective inlet, suggesting that biofilm communities converge towards a common established equilibrium as distance from treatment application increases. Based on the results, MF treatment is most effective at inhibiting biofilm growth, but a highly efficient post-treatment disinfection regime is also critical in order to prevent the high rates of post-treatment regrowth.

Drought to flood: a comparative assessment of four parallel surface water treatments during the 2010-2012 inflows to the Murray-Darling Basin, South Australia.

Four treatment processes; conventional coagulation, magnetic ion exchange (MIEX)/coagulation, with and without granular activated carbon (GAC), and membrane treatment combining microfiltration (MF) and nanofiltration (NF), were operated in parallel using the same source water from the Murray-Darling basin in South Australia. During the two year study, high levels of natural organic matter and turbidity arising from floods affecting the Murray-Darling basin in 2010-2012 challenged the four processes. The comparative study indicated that all four processes could effectively meet basic water quality guidelines of turbidity and colour despite challenging source water quality but that the more advanced treatments improved overall organic and bacterial removal. Interestingly, the high organics and turbidity arising from the floods resulted in improved treatment efficiency for all treatments incorporating coagulation to the extent that, despite flood conditions, treated water quality could remain comparatively constant provided that the process was operated and optimised effectively.

Comparison of drinking water treatment process streams for optimal bacteriological water quality.

Four pilot-scale treatment process streams (Stream 1 - Conventional treatment (coagulation/flocculation/dual media filtration); Stream 2 - Magnetic ion exchange (MIEX)/Conventional treatment; Stream 3 - MIEX/Conventional treatment/granular activated carbon (GAC) filtration; Stream 4 - Microfiltration/nanofiltration) were commissioned to compare their effectiveness in producing high quality potable water prior to disinfection. Despite receiving highly variable source water quality throughout the investigation, each stream consistently reduced colour and turbidity to below Australian Drinking Water Guideline levels, with the exception of Stream 1 which was difficult to manage due to the reactive nature of coagulation control. Of particular interest was the bacteriological quality of the treated waters where flow cytometry was shown to be the superior monitoring tool in comparison to the traditional heterotrophic plate count method. Based on removal of total and active bacteria, the treatment process streams were ranked in the order: Stream 4 (average log removal of 2.7) > Stream 2 (average log removal of 2.3) > Stream 3 (average log removal of 1.5) > Stream 1 (average log removal of 1.0). The lower removals in Stream 3 were attributed to bacteria detaching from the GAC filter. Bacterial community analysis revealed that the treatments affected the bacteria present, with the communities in streams incorporating conventional treatment clustering with each other, while the community composition of Stream 4 was very different to those of Streams 1, 2 and 3. MIEX treatment was shown to enhance removal of bacteria due to more efficient flocculation which was validated through the novel application of the photometric dispersion analyser.