Rapid detection of pathogenic Naegleria fowler in water distribution networks is critical for water utilities. Current detection methods rely on sampling drinking water followed by culturing and molecular identification of purified strains. This culture-based method takes an extended amount of time (days), detects both nonpathogenic and pathogenic species, and does not account for N. fowleri cells associated with pipe wall biofilms. In this study, a total DNA extraction technique coupled with a real-time PCR method using primers specific for N. fowleri was developed and validated. The method readily detected N. fowleri without preculturing with the lowest detection limit for N. fowleri cells spiked in biofilm being one cell (66% detection rate) and five cells (100% detection rate). For drinking water, the detection limit was five cells (66% detection rate) and 10 cells (100% detection rate). By comparison, culture-based methods were less sensitive for detection of cells spiked into both biofilm (66% detection for <10 cells) and drinking water (0% detection for <10 cells). In mixed cultures of N. fowleri and nonpathogenic Naegleria, the method identified N. fowleri in 100% of all replicates, whereastests with the current consensus primers detected N. fowleri in only 5% of all replicates. Application of the new method to drinking water and pipe wall biofilm samples obtained from a distribution network enabled the detection of N. fowleri in under 6 h, versus 3+ daysforthe culture based method. Further, comparison of the real-time PCR data from the field samples and the standard curves enabled an approximation of N. fowleri cells in the biofilm and drinking water. The use of such a method will further aid water utilities in detecting and managing the persistence of N. fowleri in water distribution networks.
The vanE operon was characterized from Enterococcus faecalis N00-410 (MIC of vancomycin ؍ 24 g/ml). The organization of the vanE operon was identical to that of the vanC1 operon from Enterococcus gallinarum, with protein identities ranging from 46 to 63%. An open reading frame located downstream of the vanE operon showed significant homology to a number of integrase genes, all of which are located downstream of the chromosomal GMP synthase gene guaA.In enterococci, normal peptidoglycan precursors have DAla-D-Ala termini that strongly bind vancomycin, whereas in vancomycin-resistant enterococci, alternate biosynthetic pathways lead to precursors with termini that bind vancomycin poorly, thus conferring resistance (4, 18). The vanA (3), vanB (9, 15), and vanD (5, 6, 14) genes code for D-Ala-D-Lac ligases and are responsible for the acquired intermediate-to highlevel resistance found mainly in Enterococcus faecalis and Enterococcus faecium. Intrinsic low-level vancomycin resistance is conferred by vanC1, vanC2, and vanC3, which code for D-Ala-D-Ser ligases found on the chromosomes of Enterococcus gallinarum, Enterococcus casseliflavus, and Enterococcus flavescens, respectively (7, 13). The acquired, nontransferable VanE DAla-D-Ser ligase found in E. faecalis also confers a low-level resistance phenotype (8). The function of the E. faecalis WCH9 putative vancomycin resistance gene vanG is unknown (11). The identification of the first vanE-containing E. faecalis isolated in Canada has recently been reported (17). In this report, we describe the characterization of the vanE resistance locus and the genes flanking this region.E. faecalis N00-410 (MIC of vancomycin ϭ 24 g/ml) (17) and E. faecium ATCC 19434 were grown at 35°C in brain heart infusion broth or cation-adjusted Mueller-Hinton broth. Induction studies were performed as previously described (5). Transfer experiments were attempted by liquid mating with selection on phenol red agar plates (Difco) containing 1% L-arabinose and 5 g of vancomycin/ml (11). Antimicrobial susceptibilities were determined by using Etest strips (AB Biodisk) or agar dilution according to NCCLS guidelines (12) for high levels of streptomycin and gentamicin. Genomic DNA was extracted from enterococci as previously described (5). An N00-410 DNA library (ϳ10-kb Sau3A fragments) in ZAP Express (Stratagene) was screened with a vanE PCR product generated with primers VANE1 and VANE2 (8). Labeling and detection of the probe were performed per the manufacturer's instructions (Amersham Pharmacia Biotech). The sequence was obtained by primer walking and by using the EZ::TNϽTET-1Ͼ insertion kit (Epicentre Technologies). Inverse PCR was carried out with primers vanRE-1 (5Ј-TCTCG GCTTTTCATGCATC-3Ј) and vanSE-DN1 (5Ј-GAATGAAA TTAATCATATTCG-3Ј) and with EcoRV-cut and -religated N00-410 DNA. Primers Eint-DN1 (5Ј-ATTCAAGGGATATT TTCAATAGC-3Ј) and guaDN-1 (5Ј-TTGCACATGTAAAC CGTATCG-3Ј) were used to amplify a 0.9-kb fragment overlapping the inverse PCR product. Homology searches were conducted with BLAST ...
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.
Although health risk due to discoloured water is minimal, such water continues to be the source of one of the major complaints received by most water utilities in Australia. Elevated levels of iron (Fe) and/or manganese (Mn) in bulk water are associated with discoloured water incidents. The accumulation of these two elements in distribution systems is believed to be one of the main causes for such elevated levels. An investigation into the contribution of pipe wall biofilms towards Fe and Mn deposition, and discoloured water events is reported in this study. Eight laboratory-scale reactors were operated to test four different conditions in duplicate. Four reactors were exposed to low Fe (0.05 mg l(-1)) and Mn (0.02 mg l(-1)) concentrations and the remaining four were exposed to a higher (0.3 and 0.4 mg l(-1) for Fe and Mn, respectively) concentration. Two of the four reactors which received low and high Fe and Mn concentrations were chlorinated (3.0 mg l(-1) of chlorine). The biological activity (measured in terms of ATP) on the glass rings in these reactors was very low (∼1.5 ng cm(-2) ring). Higher concentrations of Fe and Mn in bulk water and active biofilms resulted in increased deposition of Fe and Mn on the glass rings. Moreover, with an increase in biological activity, an increase in Fe and Mn deposition was observed. The observations in the laboratory-scale experiments were in line with the results of field observations that were carried out using biofilm monitors. The field data additionally demonstrated the effect of seasons, where increased biofilm activities observed on pipe wall biofilms during late summer and early autumn were found to be associated with increased deposition of Fe and Mn. In contrast, during the cooler months, biofilm activities were a magnitude lower and the deposited metal concentrations were also significantly less (ie a drop of 68% for Fe and 86% for Mn). Based on the laboratory-scale investigations, detachment of pipe wall biofilms due to cell death or flow dynamics could release the entrapped Fe and Mn into the bulk water, which could lead to a discoloured water event. Hence, managing biofilm growth on drinking water pipelines should be considered by water utilities to minimize accumulation of Fe and Mn in distribution networks.
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.
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