Legionella pneumophila proliferates in freshwater environments at temperatures ranging from 25 to 45°C. To investigate the preference of different sequence types (ST) for a specific temperature ...range, growth of L. pneumophila serogroup 1 (SG1) ST1 (environmental strains), ST47, and ST62 (disease-associated strains) was measured in buffered yeast extract broth (BYEB) and biofilms grown on plasticized polyvinyl chloride in flowing heated drinking water originating from a groundwater supply. The optimum growth temperatures in BYEB were approximately 37°C (ST1), 39°C (ST47), and 41°C (ST62), with maximum growth temperatures of 42°C (ST1) and 43°C (ST47 and ST62). In the biofilm at 38°C, the ST47 and ST62 strains multiplied equally well compared to growth of the environmental ST1 strain and an indigenous L. pneumophila non-SG1 strain, all attaining a concentration of approximately 10
CFU/cm
Raising the temperature to 41°C did not impact these levels within 4 weeks, but the colony counts of all strains tested declined (at a specific decline rate of 0.14 to 0.41 day
) when the temperature was raised to 42°C. At this temperature, the concentration of Vermamoeba vermiformis in the biofilm, determined with quantitative PCR (qPCR), was about 2 log units lower than the concentration at 38°C. In columns operated at a constant temperature, ranging from 38 to 41°C, none of the tested strains multiplied in the biofilm at 41°C, in which also V. vermiformis was not detected. These observations suggest that strains of ST47 and ST62 did not multiply in the biofilm at a temperature of ≥41°C because of the absence of a thermotolerant host.
Growth of Legionella pneumophila in tap water installations is a serious public health concern. The organism includes more than 2,100 varieties (sequence types). More than 50% of the reported cases of Legionnaires' disease are caused by a few sequence types which are very rarely detected in the environment. Strains of selected virulent sequence types proliferated in biofilms on surfaces exposed to warm (38°C) tap water to the same level as environmental varieties and multiplied well as pure culture in a nutrient-rich medium at temperatures of 42 and 43°C. However, these organisms did not grow in the biofilms at temperatures of ≥41°C. Typical host amoebae also did not multiply at these temperatures. Apparently, proliferation of thermotolerant host amoebae is needed to enable multiplication of the virulent L. pneumophila strains in the environment at elevated temperatures. The detection of these amoebae in water installations therefore is a scientific challenge with practical implications.
Worldwide, over 90% of the notified cases of Legionnaires' disease are caused by Legionella pneumophila. However, the standard culture medium for the detection of Legionella in environmental water ...samples, Buffered Charcoal Yeast Extract (BCYE) agar of pH 6.9 ± 0.4 with or without antimicrobial agents incubated at 36 ± 1 °C, supports the growth of a large diversity of Legionella species. BCYE agar of elevated pH or/and incubation at elevated temperature gave strongly reduced recoveries of most of 26 L. non-pneumophila spp. tested, but not of L. pneumophila. BCYE agar of pH 7.3 ± 0.1, incubated at 40 ± 0.5 °C (BCYE pH 7.3/40 °C) was tested for selective enumeration of L. pneumophila. Of the L. non-pneumophila spp. tested, only L. adelaidensis and L. londiniensis multiplied under these conditions. The colony counts on BCYE pH 7.3/40 °C of a L. pneumophila serogroup 1 strain cultured in tap water did not differ significantly from those on BCYE pH 6.9/36 °C when directly plated and after membrane filtration and showed repeatability's of 13–14%. By using membrane filtration L. pneumophila was detected in 58 (54%) of 107 Legionella-positive water samples from premise plumbing systems under one or both of these culture conditions. The L. pneumophila colony counts (log-transformed) on BCYE pH 7.3/40 °C were strongly related (r2 = 0.87) to those on BCYE pH 6.9/36 °C, but differed significantly (p < 0.05) by a mean of – 0.12 ± 0.30 logs. L. non-pneumophila spp. were detected only on BCYE pH 6.9/36 °C in 49 (46%) of the samples. Hence, BCYE pH 7.3/40 °C can facilitate the enumeration of L. pneumophila and their isolation from premise plumbing systems with culturable L. non-pneumophila spp., some of which, e.g. L. anisa, can be present in high numbers.
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•More than 95% of the Legionnaires' Disease cases are caused by Legionella pneumophila.•Legionella pneumophila grows at 40 °C; most Legionella non-pneumophila species don't.•BCYE agar incubated at 36 °C supports growth of a variety of Legionella species.•BCYE agar of pH 7.3 incubated at 40 °C selects for Legionella pneumophila.
To define protection zones around groundwater abstraction wells and safe setback distances for artificial recharge systems in watertreatment, quantitative information is needed about the removal of ...microorganisms during soil passage. Column experiments were conducted using natural soil and water from an infiltration site with fine sandy soil and a river bank infiltration site with gravel soil. The removal of phages, bacteria, bacterial spores, and protozoan (oo)-cysts was determined at two velocities and compared with field data from the same sites. The microbial elimination rate (MER) in both soils was generally >2 log, but MER in the gravel soil was higher than that in the fine sandy soil. This was attributed to enhanced attachment, related to higher metal-hydroxides content. From the high sticking efficiencies (>1) and the low influence of flow rate on MER it was deduced that straining played a significant role in the removal of Escherichia coli and Cryptosporidium parvum oocysts in the gravel soil. Lower removal of oocysts than the 4-5 times smaller E. coli and spores in the fine sand indicates that the contribution of straining is variable and needs further attention in transport models. Thus, simple extrapolation of grain size and particle size to the extent of microbial transport underground is inappropriate. Finally, the low MER of indigenous E. coli and Clostridium perfringens observed in the soil columns as well as under field conditions and the second breakthrough peak found for Cryptosporidium and spores in the fine sandy soil upon a change in the feedwater pH indicate a significant role of detachment and retardation to microbial transport and the difficulty of extrapolation of quantitative column test results to field conditions.
The decimal elimination capacity (DEC) of slow sand filtration (SSF) for
Cryptosporidium parvum was assessed to enable quantitative microbial risk analysis of a drinking water production plant. A ...mature pilot plant filter of 2.56
m
2 was loaded with
C. parvum oocysts and two other persistent organisms as potential surrogates; spores of
Clostridium perfringens (SCP) and the small-sized (4–7
μm) centric diatom (SSCD)
Stephanodiscus hantzschii. Highly persistent micro-organisms that are retained in slow sand filters are expected to accumulate and eventually break through the filter bed. To investigate this phenomenon, a dosing period of 100 days was applied with an extended filtrate monitoring period of 150 days using large-volume sampling. Based on the breakthrough curves the DEC of the filter bed for oocysts was high and calculated to be 4.7
log. During the extended filtrate monitoring period the spatial distribution of the retained organisms in the filter bed was determined. These data showed little risk of accumulation of oocysts in mature filters most likely due to predation by zooplankton. The DEC for the two surrogates, SCP and SSCD, was 3.6 and 1.8
log, respectively.
On basis of differences in transport behaviour, but mainly because of the high persistence compared to the persistence of oocysts, it was concluded that both spores of sulphite-reducing clostridia (incl. SCP) and SSCD are unsuited for use as surrogates for oocyst removal by slow sand filters. Further research is necessary to elucidate the role of predation in
Cryptosporidium removal and the fate of consumed oocysts.
Nine novel biological stability parameters for drinking water have been developed recently. Here, we report data for these nine parameters in treated water from 34 treatment plants in the Netherlands ...to deduce guidance values for these parameters. Most parameters did not show a strong correlation with another biological stability parameter in the same sample, demonstrating that most parameters hold different information on the biological stability of drinking water. Furthermore, the novel biological stability parameters in treated water varied considerably between plants and five parameters in treated water were significantly lower for drinking water produced from groundwater than surface water. The maximum biomass concentration (MBC7), cumulative biomass potential (CBP14) from the biomass production potential test (BPP-W) and the total organic carbon concentration in treated water from groundwater were predictive parameters for HPC22 and Aeromonas regrowth in the distribution system. Guidance values of 8.6 ng ATP L−1, 110 d·ng ATP L−1 and 4.1 mg C L−1 were deduced for these parameters, under which the HPC22 and Aeromonas numbers remain at regulatory level. The maximum biomass growth (MBG7) from the BPP-W test, the particulate and/or high molecular organic carbon and the iron accumulation rate in treated water from surface water were predictive parameters for HPC22 and Aeromonas regrowth in the distribution system. Deduced guidance values for these biological stability parameters were 4.5 ng ATP L−1, 47 μg C L−1 and 0.34 mg Fe m−2 day−1, respectively. We conclude from our study that a multiple parameter assessment is required to reliable describe the biological stability of drinking water, that the biological stability of drinking water produced from groundwater is described with other parameters than the biological stability of drinking water produced from surface water, and that guidance values for predictive biological stability parameters were inferred under which HPC22 and Aeromonas regrowth is under control.
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•Guidance values for novel biostability parameters for drinking water were deduced.•Treated water of 34 different treatment plants in the Netherlands were analysed.•Guidance values for TOC, MBC7 and CPB14 define biostable water from groundwater.•Guidance values for MBG7, PHMOC and FeAR define biostable water from surface water.•Multiple parameters are needed to reliable define biostability of drinking water.
Legionella pneumophila proliferates in freshwater environments at temperatures ranging from 25 to 45 degree C. To investigate the preference of different sequence types (ST) for a specific ...temperature range, growth of L. pneumophila serogroup 1 (SG1) ST1 (environmental strains), ST47, and ST62 (disease-associated strains) was measured in buffered yeast extract broth (BYEB) and biofilms grown on plasticized polyvinyl chloride in flowing heated drinking water originating from a groundwater supply. The optimum growth temperatures in BYEB were approximately 37 degree C (ST1), 39 degree C (ST47), and 41 degree C (ST62), with maximum growth temperatures of 42 degree C (ST1) and 43 degree C (ST47 and ST62). In the biofilm at 38 degree C, the ST47 and ST62 strains multiplied equally well compared to growth of the environmental ST1 strain and an indigenous L. pneumophila non-SG1 strain, all attaining a concentration of approximately 107 CFU/cm-2. Raising the temperature to 41 degree C did not impact these levels within 4 weeks, but the colony counts of all strains tested declined (at a specific decline rate of 0.14 to 0.41 day-1) when the temperature was raised to 42 degree C. At this temperature, the concentration of Vermamoeba vermiformis in the biofilm, determined with quantitative PCR (qPCR), was about 2 log units lower than the concentration at 38 degree C. In columns operated at a constant temperature, ranging from 38 to 41 degree C, none of the tested strains multiplied in the biofilm at 41 degree C, in which also V. vermiformis was not detected. These observations suggest that strains of ST47 and ST62 did not multiply in the biofilm at a temperature of greater than or equal to 41 degree C because of the absence of a thermotolerant host. IMPORTANCE Growth of Legionella pneumophila in tap water installations is a serious public health concern. The organism includes more than 2,100 varieties (sequence types). More than 50% of the reported cases of Legionnaires' disease are caused by a few sequence types which are very rarely detected in the environment. Strains of selected virulent sequence types proliferated in biofilms on surfaces exposed to warm (38 degree C) tap water to the same level as environmental varieties and multiplied well as pure culture in a nutrient-rich medium at temperatures of 42 and 43 degree C. However, these organisms did not grow in the biofilms at temperatures of greater than or equal to 41 degree C. Typical host amoebae also did not multiply at these temperatures. Apparently, proliferation of thermotolerant host amoebae is needed to enable multiplication of the virulent L. pneumophila strains in the environment at elevated temperatures. The detection of these amoebae in water installations therefore is a scientific challenge with practical implications.
In three separate distribution systems supplied with non-chlorinated drinking water produced from the same surface water, but with different microbial regrowth conditions, the spatiotemporal ...variation of biological stability and biofouling (biofilm and loose deposits) was determined. Main objective was to establish the cause(s) for differences in regrowth and to elucidate the mechanism of the observed differences in Aeromonas regrowth. Enhanced HPC22 and Aeromonas regrowth in two of these drinking water distribution systems (DWDSs) was related to (i) higher biopolymer concentration in the drinking water, (ii) increased biofouling with non-dissolved organic carbon and iron and (iii) the presence of Asellus aquaticus biomass in the loose deposits. The biofouling parameter iron accumulation rate was a good predictor for biofouling conditions in the biofilm and loose deposits. The observed association of A. aquaticus biomass in the loose deposits with Aeromonas regrowth in the drinking water suggests that release from the A. aquaticus biomass is a major mechanism for this regrowth. Complex interactions between biopolymers, iron and A. aquaticus as cause for the undesired regrowth needs further studies where the efficacy is assessed of biopolymer and Fe reduction and/or of removal of loose deposits (incl. A. aquaticus) by pipe flushing in the DWDS to control this regrowth.
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•Biopolymers and Fe biofouling promote regrowth in non-chlorinated drinking water.•Invertebrates in the food web of the DWDS are an essential part of the biofouling.•Technical Aeromonas standard potentially indicates the presence of A. aquaticus.•Regrowth control by biopolymers/Fe reduction and pipe flushing needs further study.
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