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.     

Physical, chemical and microbiological water characteristics associated with the occurrence of Legionella in cooling tower systems

The occurrence of Legionella in water of 30 cooling tower systems was studied, as well as the relationship of its occurrence with the physical, chemical and microbiological characteristics of the waters. The samples were concentrated by membrane filtration and one part of the concentrate was acid washed. Three types of culture media, MWY, CCVC and BCYEα were used. Fourteen (47%) of the cooling systems were Legionella-positive. Numbers of Legionella in the positive systems varied between 50 and 490,000 cfu/l. Legionella pneumophila was the dominant isolate, and the serogroups 6 and 1 were the commonest ones. There was no statistically significant difference in mean water temperature between the Legionella-positive (27°C) and -negative (24°C) systems. The total number of bacteria (AODC) was lower in the Legionella-positive than in the negative systems. Also the nutrient concentrations were generally lower in the Legionella-positive cooling systems. The results suggest that different factors regulate the occurrence of Legionella than that of overall bacterial populations in the cooling tower systems.     

Legionella prevalence in hot spring recreation areas of Taiwan

Legionella is a bacterium ubiquitous to aquatic environments. Within the genus a few species are recognized as opportunistic potential human pathogens, especially the species Legionella pneumophila, which causes pneumonia legionellosis. Outbreaks of legionellosis are frequently reported by hotel guests and hospital patients, and are spread through inhaled aerosols of contaminated institutional water systems. Contaminations in hot tubs, spas and public baths are also possible. As a result, in this study, we investigated the distribution of Legionella at seven hot spring recreational areas throughout Taiwan. We gathered data on factors potentially associated with the pathogen's distribution, including environment, facility operation, and physical and microbiological water quality parameters. Spring water was collected from 91 sites and Legionella was detected in 21 (23%). The most frequently detected was L. pneumophila, followed by uncultured Legionella species, Legionella-like amoebal pathogen. Five species, L. bozemanii, L. dumoffi, L. feelei, L. lyticum and L. oakridgenesis, were all detected once. Legionella species were found in water temperatures ranging from 22 to 50 degrees C. Optimal pH appeared to be between 5.0 and 9.0. The prevalence of Legionella also coincided with the prevalence of indicator microorganisms. Legionella detection was not proportional to the frequency of cleaning. Results of this survey confirm the ubiquity of Legionella in Taiwan spring recreation areas. L. pneumophila, the organism responsible for the majority of legionellosis outbreaks, should be considered a potential public health threat in spa areas of Taiwan.     

VBNC Legionella pneumophila cells are still able to produce virulence proteins

Legionella pneumophila is the agent responsible for legionellosis. Numerous bacteria, including L. pneumophila, can enter into a viable but not culturable (VBNC) state under unfavorable environmental conditions. In this state, cells are unable to form colonies on standard medium but are still alive. Here we show that VBNC L. pneumophila cells, obtained by monochloramine treatment, were still able to synthesize proteins, some of which are involved in virulence. Protein synthesis was measured using ³⁵S-labeling and the proteomes of VBNC and culturable cells then compared. This analysis allowed the identification of nine proteins that were accumulated in the VBNC state. Among them, four were involved in virulence, i.e., the macrophage infectivity potentiator protein, the hypothetical protein lpl2247, the ClpP protease proteolytic subunit and the 27 kDa outer membrane protein. Others, i.e., the enoyl reductase, the electron transfer flavoprotein (alpha and beta subunits), the 50S ribosomal proteins (L1 and L25) are involved in metabolic and energy production pathways. However, resuscitation experiments performed with Acanthamoeba castellanii failed, suggesting that the accumulation of virulence factors by VBNC cells is not sufficient to maintain their virulence.     

Development of an improved PCR-ICT hybrid assay for direct detection of Legionellae and Legionella pneumophila from cooling tower water specimens

A novelly improved polymerase chian reaction and immunochromatography test (PCR-ICT) hybrid assay comprising traditional multiplex-nested PCR and ICT, (a lateral-flow device) was developed for direct detection of Legionella bacteria from environmental cooling tower samples. The partial 16S rDNA (specific for Legionella spp.) and dnaJ (specific for Legionella pneumophila) genes from Legionella chromosome were first specifically amplified by multiplex-nested PCR, respectively, followed by detection using ICT strip. Reading of results was based on presence or absence of the two test lines on the strips. Presence of test line 1 indicated existence of Legionella spp. specific 16S rDNA and identified Legionella spp. Presence of test line 2 further indicated existence of dnaJ and thus specifically identified L. pneumophila. In contrast, for non-Legionellae bacteria no test line formation was observed. Results of direct detection of Legionella bacteria and L. pneumophila from water tower specimens by this assay showed 100% sensitivity, and 96.6% and 100% specificity, respectively compared with traditional culture, biochemical and serological identification methods. The PCR-ICT hybrid assay does not require sophisticated equipment and was proved to be practically useful in rapid and direct Legionellae detection from environmental water samples.     

Variable bacterial load of Legionella spp. in a hospital water system

Several approved protocols for the prevention of Legionella pneumonia base the type of intervention (to disinfect or not) on the level of contamination found (cfu/L). However, if the level of contamination by Legionella spp. of a water system fluctuates in a short period of time, inadequate sampling could lead to different decisions being made. To determine if there are significant variations in the bacterial count of Legionella spp., water samples were taken at different times from the same sites. Eight wards were selected from a large hospital in Southern Italy and a water sample was taken from 21 taps in each ward at the same time each day for 5 consecutive days.A Freidman test detected statistically significant differences in average Legionella spp. load over the 5 sampling days (p value < 0.001).This fluctuating load can have practical implications: the Italian Guidelines recommend disinfection only for a Legionella count > 10,000 cfu/L in hospitals without documented cases of disease. In the present study, the daily average loads varied, during the 5-day sampling period, above and below this cut-off (10,000 cfu/L). This means that the decision to disinfect or not would be different depending on which day the sampling was carried out. Our data suggest that, especially in health-care facilities, a single sampling would not give a realistic estimation of risk; therefore, even at lower levels of bacterial load, measures should be taken to reduce it further.     

A survey of Legionella pneumophila in water in 12 Canadian cities

A survey of 12 cities across Canada was conducted in order to determine the prevalence of Legionella species in potable water and cooling tower water within buildings. Legionellae were detected in 11.9% of the samples overall: 6.7% from potable water sources and 28.9% from cooling tower water. The maximum concentration of the organism was 45,000 l⁻¹ in one shower-water sample by culture methods. A significant difference in the isolation rate of Legionella pneumophila among cities was observed. The organism was isolated from waters at a temperature of 15–41°C and was most frequently isolated in the 20–29°C range. The concentration of free and total available chlorine in the water was not associated with legionellae recovery except that the organisms were never recovered when the free available chlorine residual exceeded 7.5 mg l⁻¹. Although L. pneumophila were in low concentrations or absent in most samples, the isolated organisms were usually serogroups 1 or 6, the same serogroups that are most often implicated in legionellosis cases in Canada.     

The use of Bacillus thuringiensis var. israelensis to control the nuisance fly Sylvicola fenestralis (Anisopodidae) in sewage filter beds

The effectiveness of using the microbial insecticide Bacillus thuringiensis var. israelensis (Bti) to control the nuisance fly Sylvicola fenestralis at Rossendale Sewage Works, Lancashire, was investigated. Following a provisional trial in 1987, the whole Works (28 filter beds) was treated with a two-stage application of Bti in 1988; the first application in late April was separated from the second in late May by about half the length of the Sylvicola life-cycle. The Bti treatment was timed to reduce fly nuisance in June, the month when most complaints (71%) had been received. For each application, 0.161. of the Bti preparation, containing nearly 10¹⁰ spores·cm⁻³, was applied per square metre of bed surface. An 80 and 98% kill of Sylvicola larvae was recorded following the first and second applications respectively. The lower kill achieved by the first application was believed to be due to insufficient mixing of the Bti with the settled sewage prior to dosing. Nuisance caused by the flies was considerably reduced; there were no complaints from the public during June, although a few were received in late July, early August. Sewage purification efficiency was not affected by Bti treatment. The maximum concentration of Bti spores in the Works' effluent was 7 × 10⁴ spores·cm⁻³. This concentration did not appear to affect the already impoverished macro-invertebrate fauna of the receiving river.     

Life history responses of Daphnia magna feeding on toxic Microcystis aeruginosa alone and mixed with a mixotrophic Poterioochromonas species

The toxic effects of a mixotrophic golden alga (Poterioochromonas sp. strain ZX1) and a cyanobacterium Microcystis aeruginosa grazed by Poterioochromonas to a cladoceran were investigated through life history experiments using Daphnia magna. Poterioochromonas cultured in two ways (fed M. aeruginosa in an inorganic medium or grown in an organic medium) both induced starvation-like effects on D. magna, indicating that Poterioochromonas is neither acutely toxic nor a good food for D. magna. Despite a microcystin-LR content of 10⁻⁸ μg cell⁻¹ in M. aeruginosa, no toxins were accumulated in Poterioochromonas fed the cyanobacterium. The toxic effect of M. aeruginosa to D. magna was significantly reduced in the presence of Poterioochromonas, which may be performed in two ways: decrease M. aeruginosa cells ingestion of D. magna by grazing on M. aeruginosa; and decrease the toxicity of the medium by degrading the toxins released by M. aeruginosa. This study provides new information on the interactions between a cyanobacterium and its grazer under laboratory conditions and may increase our understanding of the ecological significance of such interactions in the aquatic food webs.     

Effects of nickel on daphnia magna during chronic exposure and alterations in the toxicity to generations pre-exposed to nickel

Daphnia magna was cultured at sublethal nickel concentrations. The mean life expectancy was significantly less when exposed to 40 ppb Ni. Statistically significant reductions of the number of offspring, maximum body length of adults and of neonates were caused by 80 ppb Ni. The length and brood size of primiparous animals were significantly less when exposed to 120 ppb Ni. The intrinsic rate of population growth (r) was inversely proportional to nickel concentrations. During seven successive generations exposed to 160 ppb Ni, both the mean life span and the length of primiparous animals decreased significantly with increasing time. The progeny of nickel pre-exposed generations exhibited no adaptation towards nickel except an altered reproduction pattern which induced an increase of r. After the transfer into nickel-free water, the progeny of nickel pre-exposed generations showed an increase of body length, mean life span, number of offspring, brood size and brood number as well as the maximum body length of neonates. However, the progeny of pre-exposed generations remained smaller than the progeny of an untreated generation.     

Cryptosporidium and Giardia detection in water bodies of Galicia, Spain

The objective of this study was to determine the mean concentration (per litre) of Cryptosporidium oocysts and Giardia cysts in recreational river areas (n = 28), drinking water treatments plants (DWTPs; n = 52) and wastewater treatment plants (WWTPs; n = 50) in Galicia (NW Spain). Water samples from rivers and from the influent (50–100 l) and the treated effluent (100 l) of the water plants were filtered using Filta-Max filters (IDEXX Laboratories, Inc., Westbrook, ME, USA). A total of 232 samples were processed and the (oo)cysts were concentrated, clarified by IMS and then detected by IFAT. The viability was determined by applying fluorogenic vital dye (PI).In the recreational areas, infective forms of Cryptosporidium and Giardia were detected in 16 (57.1%; 1–60 oocysts per litre) and 17 (60.7%; 1–160 cysts per litre) samples, respectively. In the water flowing into the water treatment plants, oocysts were detected in 21 DWTPs (40.4%; 1–13 oocysts per litre) and cysts were observed in 22 DWTPs (42.3%; 1–7 cysts per litre). In the effluents from the treatment plants, Cryptosporidium oocysts and Giardia cysts were identified in 17 DWTPs (32.7%; 1–4 oocysts per litre) and in 19 DWTPs (36.5%; 1–5 cysts per litre), respectively. The highest concentrations of (oo)cysts were found in the WWTPs; specifically, oocysts were detected in 29 (58.0%; 1–80 oocysts per litre) and cysts in 49 (98.0%; 2–14.400 cysts per litre) WWTP effluents. Cryptosporidium and Giardia were detected in 32 (64.0%; 1–120 oocysts per litre) and 48 (96.0%; 2–6.000 cysts per litre) WWTP effluents, respectively. The percentage viability of the (oo)cysts ranged between 90.0% and 95.0%. In all samples analysed. Moreover, it was found that the effluents from coastal WWTPs were discharged directly into the sea, while inland WWTPs were discharged directly into rivers. The concentrations of both enteropathogens detected in effluents from WWTPs therefore represent a significant risk to human and animal health.These results demonstrate the wide distribution of Cryptosporidium and Giardia in the environment, the ineffectiveness of treatments in DWTPs and WWTPs in reducing/inactivating both protozoa and the need to monitor the presence, viability and infectivity of Cryptosporidium and Giardia in water bodies. In conclusion, the findings suggest the need for better monitoring of water quality and identification of sources of contamination.     

Nitrification of ammonia in wastewater: Field observations and laboratory studies

Complete nitrification of ammonia in partially treated domestic wastewater was demonstrated in a mixed culture chemostat. The optimal detention time of water in the chemostat for maximal nitrification was 6–7 days. In the field, a significant number of nitrifying bacteria was present in a wastewater reservoir, as well as in samples of inflowing water from the preceding anaerobic overloaded oxidation pond.     

Responses of Cyperus involucratus Rottb. to nitrogen and phosphorus, with reference to wastewater reclamation

There is increasing use of aquatic plants in biosystems to ameliorate wastewaters. Cyperus involucratus Rottb. is a large emergent aquatic plant which may be useful in treating wastewater because it grows quickly, does not die back in winter, has little weed potential and grows in dryland situations. Many wastewaters contain high levels of N and P, and the capacity of C. involucratus to take up these nutrients was studied under glasshouse conditions. Maximum growth was attained between 84 and 168 mg NO₃-Nl⁻¹ and 1–5 mg PO₄-Pl⁻¹. At a low level of supplied NO₃-N, most of the N in plant organs was reduced-N; at levels of supplied NO₃-N above 84 mg l⁻¹, there was considerable accumulation of NO₃-N in plant organs. The accumulation of NO₃-N was greatest in culms where it made up over 50% of their N content when plants were grown on 350 mg NO₃-Nl⁻¹. Linear regression equations best described the relationships between dry matter and reduced-N contents in roots, culms and leaves. Over 60% of the total plant N and P accumulated in above ground organs, and concentrations of up to 0.53% P and 3.4% N (dry wt basis) were recorded in leaves. C. involucratus would be a suitable plant for wastewater reclamation on account of its high dry matter production, its ability to tolerate hyper-eutrophic levels of N and P, and the accumulation of most of the N and P in the harvestable portions of the plant.     

Influence of pH on bioactivity of cinnamon oil against Legionella pneumophila and its disinfection efficacy in hot springs

Cinnamon oil extracted from leaves of Cinnamomum osmophloeum has recently been proved as a promising antibacterial agent against Legionella pneumophila, an etiological agent of human pneumonia known as Legionnaires' disease. However, the pH effects on the efficacy of cinnamon oil against L. pneumophila and its applicability to recreational spring water remain unknown. We therefore determined the bactericidal activity of cinnamon oil at pH 3-10 in phosphate-buffered saline (PBS) and in four kinds of springs with various conductivity (259-5595 μs cm⁻¹) and pH (2.1-7.7) levels. Results show L. pneumophila cells were more susceptible to cinnamon oil at pH 8-10 than at pH 4-6 in PBS, which became more evident as increasing contact time from 10 to 60 min. An increase in concentration of cinnamon oil and contact time significantly increased the anti-L. pneumophila activity (P ≤ 0.001), indicating a consistent biocidal effect regardless of pH. Interestingly, this dose-response biocidal effect was also observed in spring waters. Moreover, L. pneumophila of 4 log CFU ml⁻¹ in spring waters was completely inactivated within 60 min by cinnamon oil at 300-750 μg ml⁻¹, with the highest inactivation in alkaline hydrogen carbonate spring. The great bioactivity of cinnamon oil demonstrates its potential to be used to control Legionella growth in recreational spring water and possibly other niches generally at basic pH, e.g., cooling towers.     

Response of antioxidant enzymes in freshwater fish populations (Leuciscus alburnoides complex) to inorganic pollutants exposure

Evidence is accumulating indicating the importance of antioxidant enzyme activity measurements in eco-toxicological studies, as they may constitute markers for exposure to a large variety of pollutants. Variation of antioxidant enzymes, superoxide dismutase (SOD) and glutathione S-transferases (GST) and the effect of heavy metals and selenium exposure on these enzymes were investigated in the livers of Iberian endemic minnows (Leuciscus alburnoides complex) captured in a copper (Cu) mining area. Higher hepatic levels of copper and selenium were always observed in fish captured at the polluted site relative to the reference area population, reflecting the environmental monitoring results. A seasonal fluctuation in zinc and selenium levels for both populations was also observed which could be related to gonad maturation. The activity of SOD did not show significant regional alterations, but a seasonal variation occurred presumably associated with the Leuciscus life cycle. The GST activity was higher in the fish population from the polluted area (except in spring) and GST variability was associated with selenium and copper levels when both regions were compared. The increased GST activity was probably a metabolic adaptation to the continuous exposure to higher levels of those elements.     

Wastewater treatment contributes to selective increase of antibiotic resistance among Acinetobacter spp.

The occurrence and spread of multi-drug resistant bacteria is a pressing public health problem. The emergence of bacterial resistance to antibiotics is common in areas where antibiotics are heavily used, and antibiotic-resistant bacteria also increasingly occur in aquatic environments. The purpose of the present study was to evaluate the impact of the wastewater treatment process on the prevalence of antibiotic resistance in Acinetobacter spp. in the wastewater and its receiving water. During two different events (high-temperature, high-flow, 31 °C; and low-temperature, low-flow, 8 °C), 366 strains of Acinetobacter spp. were isolated from five different sites, three in a wastewater treatment plant (raw influent, second effluent, and final effluent) and two in the receiving body (upstream and downstream of the treated wastewater discharge point). The antibiotic susceptibility phenotypes were determined by the disc-diffusion method for 8 antibiotics, amoxicillin/clavulanic acid (AMC), chloramphenicol (CHL), ciprofloxacin (CIP), colistin (CL), gentamicin (GM), rifampin (RA), sulfisoxazole (SU), and trimethoprim (TMP). The prevalence of antibiotic resistance in Acinetobacter isolates to AMC, CHL, RA, and multi-drug (three antibiotics or more) significantly increased (p < 0.01) from the raw influent samples (AMC, 8.7%; CHL, 25.2%; RA, 63.1%; multi-drug, 33.0%) to the final effluent samples (AMC, 37.9%; CHL, 69.0%; RA, 84.5%; multi-drug, 72.4%), and was significantly higher (p < 0.05) in the downstream samples (AMC, 25.8%; CHL, 48.4%; RA, 85.5%; multi-drug, 56.5%) than in the upstream samples (AMC, 9.5%; CHL, 27.0%; RA, 65.1%; multi-drug, 28.6%). These results suggest that wastewater treatment process contributes to the selective increase of antibiotic resistant bacteria and the occurrence of multi-drug resistant bacteria in aquatic environments.     

A toxicity warning monitor using the weakly electric fish, Gnathonemus petersi

A technically simple fish monitor was developed using the weakly electric fish, Gnathonemus petersi, as the test organism. The test system consists of a plexiglass test chamber (21., 27°C) with a flow-through of 4–5 times the chamber volume per hour. The spontaneous electric organ discharges (EOD) are recorded as activity signals. EODs, which have a potential of about 500 mV, last 0.3 ms and they can be converted to computer-compatible signals by a simple amplifier. The EOD-activity is analyzed by a low-cost desk-top computer. Two different modes of analysis which represent two levels of response sensitivity, are possible either alternatively or simultaneously. The toxicity response of Gnathonemus petersi was measured in experiments with the toxicants Hg²⁺, Cu²⁺, NaAsO₂ and CN⁻. The sensitivity to acutely toxic concentrations was found to be as high as that of trout or minnows.     

Light induced Nitrosomonas inhibition

The purpose of this study was to evaluate and verify the impact of light exposure on an enriched Nitrosomonas culture. Resting cells maintained under aerobic conditions without an exogenous ammonium-nitrogen source were fully inhibited within a 10 min period of ambient light contact (i.e. fluorescent and indirect natural room light). These cells were, however, protected against this inhibitive phenomenon during active respiration periods and during anoxic exposure conditions. Recovery, albiet partial, from such light induced inhibition was initiated after a “dark” contact period of approx. 2.5–3 h without exogenous ammonium presence. Research involving enriched or pure Nitrosomonas cultures (e.g. batch bioassay testing) should consequently be completed under environmental conditions designed to obviate this problem.