Helen Y. Buse

Legionellae in engineered systems and use of quantitative microbial risk assessment to predict exposure

While it is well-established that Legionella are able to colonize engineered water systems, the number of interacting factors contributing to their occurrence, proliferation, and persistence are unclear. This review summarizes current methods used to detect and quantify legionellae as well as the current knowledge of engineered water system characteristics that both favour and promote legionellae growth. Furthermore, the use of quantitative microbial risk assessment (QMRA) models to predict potentially critical human exposures to legionellae are also discussed. Understanding the conditions favouring Legionella occurrence in engineered systems and their overall ecology (growth in these systems/biofilms, biotic interactions and release) will aid in developing new treatment technologies and/or systems that minimize or eliminate human exposure to potentially pathogenic legionellae.

Microbial diversities (16S and 18S rRNA gene pyrosequencing) and environmental pathogens within drinking water biofilms grown on the common premise plumbing materials unplasticized polyvinylchloride and copper

Drinking water (DW) biofilm communities influence the survival of opportunistic pathogens, yet knowledge about the microbial composition of DW biofilms developed on common in-premise plumbing material is limited. Utilizing 16S and 18S rRNA gene pyrosequencing, this study characterized the microbial community structure within DW biofilms established on unplasticized polyvinyl chloride (uPVC) and copper (Cu) surfaces and the impact of introducing Legionella pneumophila (Lp) and Acanthamoeba polyphaga. Mature (> 1 year old) biofilms were developed before inoculation with sterilized DW (control, Con), Lp, or Lp and A. polyphaga (LpAp). Comparison of uPVC and Cu biofilms indicated significant differences between bacterial (P = 0.001) and eukaryotic (P < 0.01) members attributable to the unique presence of several family taxa: Burkholderiaceae, Characeae, Epistylidae, Goniomonadaceae, Paramoebidae, Plasmodiophoridae, Plectidae, Sphenomonadidae, and Toxariaceae within uPVC biofilms; and Enterobacteriaceae, Erythrobacteraceae, Methylophilaceae, Acanthamoebidae, and Chlamydomonadaceae within Cu biofilms. Introduction of Lp alone or with A. polyphaga had no effect on bacterial community profiles (P > 0.05) but did affect eukaryotic members (uPVC, P < 0.01; Cu, P = 0.001). Thus, established DW biofilms host complex communities that may vary based on substratum matrix and maintain consistent bacterial communities despite introduction of Lp, an environmental pathogen.

Preferential colonization and release of Legionella pneumophila from mature drinking water biofilms grown on copper versus unplasticized polyvinylchloride coupons

Legionella occurrence in premise drinking water (DW) systems contributes to legionellosis outbreaks, especially in the presence of suitable protozoan hosts. This study examined L. pneumophila behavior within DW biofilms grown on copper (Cu) and unplasticized polyvinylchloride (uPVC) surfaces in the presence of Acanthamoeba polyphaga. One year-old DW biofilms were established within six CDC biofilm reactors: three each containing Cu or uPVC coupons. Biofilms were then inoculated with L. pneumophila (uPVC-Lp and Cu-Lp), or L. pneumophila and A. polyphaga (uPVC-Lp/Ap and Cu-Lp/Ap) and compared to sterile water inoculated controls (uPVC- and Cu-Control) over a 4 month period. L. pneumophila appeared more persistent by qPCR within Cu biofilms in the presence of A. polyphaga compared to uPVC biofilms with or without A. polyphaga, but maintained their cultivability in uPVC biofilms compared to Cu biofilms. Also, persistent shedding of L. pneumophila cells (assayed by qPCR) in the effluent water implied colonization of L. pneumophila within Cu-coupon reactors compared to no detection from uPVC-coupon reactor effluent 14 days after inoculation. Hence, L. pneumophila appeared to colonize Cu surfaces more effectively and may be shed from the biofilms at a greater frequency and duration compared to L. pneumophila colonized uPVC surfaces with host amoebae playing a role in L. pneumophila persistence within Cu biofilms.

Impact of drinking water conditions and copper materials on downstream biofilm microbial communities and Legionella pneumophila colonization

This study examined the impact of pipe materials and introduced Legionella pneumophila on downstream Leg. pneumophila colonization and microbial community structures under conditions of low flow and low chlorine residual.

Counting Legionella cells within single amoeba host cells.

ABSTRACT Here we present the first attempt to quantify Legionella pneumophila cell numbers within individual amoeba hosts that may be released into engineered water systems. The maximum numbers of culturable L. pneumophila cells grown within Acanthamoeba polyphaga and Naegleria fowleri were 1,348 (mean, 329) and 385 (mean, 44) CFU trophozoite−1, respectively.

Exposure to synthetic gray water inhibits amoeba encystation and alters expression of Legionella pneumophila virulence genes.

ABSTRACT Water conservation efforts have focused on gray water (GW) usage, especially for applications that do not require potable water quality. However, there is a need to better understand environmental pathogens and their free-living amoeba (FLA) hosts within GW, given their growth potential in stored gray water. Using synthetic gray water (sGW) we examined three strains of the water-based pathogen Legionella pneumophila and its FLA hosts Acanthamoeba polyphaga, A. castellanii, and Vermamoeba vermiformis. Exposure to sGW for 72 h resulted in significant inhibition (P < 0.0001) of amoebal encystation versus control-treated cells, with the following percentages of cysts in sGW versus controls: A. polyphaga (0.6 versus 6%), A. castellanii (2 versus 62%), and V. vermiformis (1 versus 92%), suggesting sGW induced maintenance of the actively feeding trophozoite form. During sGW exposure, L. pneumophila culturability decreased as early as 5 h (1.3 to 2.9 log10 CFU, P < 0.001) compared to controls (Δ0 to 0.1 log10 CFU) with flow cytometric analysis revealing immediate changes in membrane permeability. Furthermore, reverse transcription-quantitative PCR was performed on total RNA isolated from L. pneumophila cells at 0 to 48 h after sGW incubation, and genes associated with virulence (gacA, lirR, csrA, pla, and sidF), the type IV secretion system (lvrB and lvrE), and metabolism (ccmF and lolA) were all shown to be differentially expressed. These results suggest that conditions within GW may promote interactions between water-based pathogens and FLA hosts, through amoebal encystment inhibition and alteration of bacterial gene expression, thus warranting further exploration into FLA and L. pneumophila behavior in GW systems.

Screening-level assays for potentially human-infectious environmental Legionella spp.

In spite of the fact that various Legionella species are isolated from nonclinical water settings, there is no standard method to determine whether environmental legionellae may be infectious to humans. Here we provide a screening-level approach based on an in vivo murine (A/J mouse) model and three in vitro proliferation assays using Acanthamoeba polyphaga, and THP-1 human and J774 murine macrophage cell lines to identify potentially human-infectious legionellae. As an initial demonstration the infectivity potential of three clinical (Legionella pneumophila, L, longbeacheae, and L. micdadei) and three environmental (L. dumoffii, L. maceachernii, and L. sainthelensi) legionellae were evaluated. A/J mice were intranasally infected and by 6 h post infection (p.L), there were significant bacterial titers in the lungs. L. pneumophila, L. dumoffii, and L. micdadei densities were higher than L. longbeacheae, L. maceacherni, and L. sainthelensi at 24 h p.i. However, only L. pneumophila and L. micdadei persisted in the lungs after 48 h, indicating that the other isolates were rapidly cleared. Results from the in vitro assays showed that only L. pneumophila significantly multiplied within A. polyphaga, THP-1 and J774 cells after 72 h, but lysis of any of the in vitro hosts also flagged the strains for potential concern (e.g. L. dumoffii and L. micdadei). The results demonstrate the value of using multiple approaches to assess the potential level of pathogenicity of Legionella strains isolated from different environmental matrices.

Effect of temperature and colonization of Legionella pneumophila and Vermamoeba vermiformis on bacterial community composition of copper drinking water biofilms.

It is unclear how the water‐based pathogen, Legionella pneumophila (Lp), and associated free‐living amoeba (FLA) hosts change or are changed by the microbial composition of drinking water (DW) biofilm communities. Thus, this study characterized the bacterial community structure over a 7‐month period within mature (> 600‐day‐old) copper DW biofilms in reactors simulating premise plumbing and assessed the impact of temperature and introduction of Lp and its FLA host, Vermamoeba vermiformis (Vv), co‐cultures (LpVv). Sequence and quantitative PCR (qPCR) analyses indicated a correlation between LpVv introduction and increases in Legionella spp. levels at room temperature (RT), while at 37°C, Lp became the dominant Legionella spp. qPCR analysis suggested Vv presence may not be directly associated with Lp biofilm growth at RT and 37°C, but may contribute to or be associated with non‐Lp legionellae persistence at RT. Two‐way PERMANOVA and PCoA revealed that temperature was a major driver of microbiome diversity. Biofilm community composition also changed over the seven‐month period and could be associated with significant shifts in dissolved oxygen, alkalinity and various metals in the influent DW. Hence, temperature, biofilm age, DW quality and transient intrusions/amplification of pathogens and FLA hosts may significantly impact biofilm microbiomes and modulate pathogen levels over extended periods.

Development of an Escherichia coli K12-specific quantitative polymerase chain reaction assay and DNA isolation suited to biofilms associated with iron drinking water pipe corrosion products.

A quantitative polymerase chain reaction assay (115 bp amplicon) specific to Escherichia coli K12 with an ABI(TM) internal control was developed based on sequence data encoding the rfb gene cluster. Assay specificity was evaluated using three E. coli K12 strains (ATCC W3110, MG1655 & DH1), 24 non-K12 E. coli and 23 bacterial genera. The biofilm detection limit was 10(3) colony-forming units (CFU) E. coli K12 mL(-1), but required a modified protocol, which included a bio-blocker Pseudomonas aeruginosa with ethylenediaminetetraacetic acid buffered to pH 5 prior to cell lysis/DNA extraction. The novel protocol yielded the same sensitivity for drinking water biofilms associated with Fe3O4 (magnetite)-coated SiO2 (quartz) grains and biofilm-surface iron corrosion products from a drinking water distribution system. The novel DNA extraction protocol and specific E. coli K12 assay are sensitive and robust enough for detection and quantification within iron drinking water pipe biofilms, and are particularly well suited for studying enteric bacterial interactions within biofilms.

Electrophoretic mobility of Legionella pneumophila serogroups 1 to 14

Legionella pneumophila (Lp) is ubiquitous in the aquatic environment and can persist within drinking water distribution systems (DWDS) enabling these systems to serve as a potential source of human infections. Bacterial surface charge, deduced from electrophoretic mobility (EPM), is a well-recognized contributor to microorganism mobility, adherence and interactions with their surrounding environment. In this study, the EPM of 32 Lp strains representing serogroup (sg) 1 to 14 were measured, in 9.15 mM KH2PO4 at pH 8, to understand cell surface properties that may influence their occurrence within DWDS. EPM measurements indicated the charge of Lp varied widely between serogroups with five distinct clusters, from least to most negatively charged: (i) sg1 to 3, 5, and 12; (ii) sg6, 8, and 10; (iii) sg9 and 13; (iv) sg7, 11, and 14; and (v) sg4. The EPM of sg1 and 4 strains were pH dependent; however, values were constant between pH 6 and 9, a range typical of drinking water, suggesting that EPM differences between Lp serogroups could impact their survival within DWDS. Understanding the ecological importance of Lp surface properties (e.g. in mobility, colonization, resistance to disinfectants, etc.) within DWDS would aid in mitigation of health risks associated with this water-based pathogen.