Isabella Marchesi

Legionella Infection Risk from Domestic Hot Water

We investigated Legionella and Pseudomonas contamination of hot water in a cross-sectional multicentric survey in Italy. Chemical parameters (hardness, free chlorine, and trace elements) were determined. Legionella spp. were detected in 33 (22.6%) and Pseudomonas spp. in 56 (38.4%) of 146 samples. Some factors associated with Legionella contamination were heater type, tank distance and capacity, water plant age, and mineral content. Pseudomonas presence was influenced by water source, hardness, free chlorine, and temperature. Legionella contamination was associated with a centralized heater, distance from the heater point >10 m, and a water plant >10 years old. Furthermore, zinc levels of <20 μg/L and copper levels of >50 μg/L appeared to be protective against Legionella colonization. Legionella species and serogroups were differently distributed according to heater type, water temperature, and free chlorine, suggesting that Legionella strains may have a different sensibility and resistance to environmental factors and different ecologic niches.

Water ecology of Legionella and protozoan: environmental and public health perspectives.

Ecological studies on Legionella spp. are essential to better understand their sources in the natural environments, the mechanism of their entry into man-made water systems and the factors enabling their survival and growth in aquatic habitats. Legionella spp. exhibits peculiar and multiple strategies to adapt to stressful environment conditions which normally impair other germ survival. These strategies include the ability to enter in a viable but non-cultivable (VBNC) state, to multiply intracellularly within a variety of protozoa, such as amoebae, to survive as free organisms within biofilms and to be enhanced/inhibited by the presence of other aquatic bacteria. The host-parasite interaction has been shown to be central in the pathogenesis and ecology of L. pneumophila. The bacterial-protozoan interaction contributes to the amplification of Legionella population in water systems, represents a shelter against unfavourable environmental conditions, acts as a reservoir of infection and contributes to virulence by priming the pathogen to infect human cells. Legionella is able to survive as free organism for long periods within biofilms which are widespread in man-made water systems. Biofilm provides shelter and nutrients, exhibits a remarkable resistance to biocide compounds and chlorination, thus representing ecological niches for legionella persistence in such environments. Further knowledge on biofilm-associated legionellae may lead to effective control measures to prevent legionellosis. Lastly, new perspectives in controlling legionella contamination can arise from investigations on aquatic bacteria able to inhibit legionella growth in natural and artificial water systems.

Effectiveness of different methods to control legionella in the water supply: ten-year experience in an Italian university hospital

We report our ten-year experience of hyperchlorination, thermal shock, chlorine dioxide, monochloramine, boilers and point-of-use filters for controlling legionella contamination in a hospital hot water distribution system. Shock disinfections were associated with a return to pre-treatment contamination levels within one or two months. We found that chlorine dioxide successfully maintained levels at <100 cfu/L, whilst preliminary experiments gave satisfactory results with monochloramine. No contamination was observed applying point-of-use filters and electric boilers at temperatures of >58°C and no cases of nosocomial legionellosis were detected in the ten-year observation period. Our performance ranking in reducing legionella contamination was filter, boiler, chlorine dioxide, hyperchlorination and thermal shock. Chlorine dioxide was the least expensive procedure followed by thermal shock, hyperchlorination, boiler and filter. We suggest adopting chlorine dioxide and electric boilers in parallel.

Parameters predictive of Legionella contamination in hot water systems: association with trace elements and heterotrophic plate counts.

The contamination of hot water samples with Legionella spp. was studied in relation to temperature, total hardness, trace element concentrations (iron, zinc, manganese, and copper) and heterotrophic plate counts (HPC) at both 22 and 37 °C. Factor analysis and receiver operating characteristic (ROC) curves were used to establish the cut-off of water parameters as predictors for Legionella contamination. Legionella spp. was isolated in 194 out of 408 samples (47.5%), with Legionella pneumophila being the most common (92.8%). After multiple logistic regression analysis, the risk for legionellae colonisation was positively associated with Mn levels >6 μg l(-1), HPC at 22 °C >27 CFU l(-1), and negatively with temperature >55 °C and Cu levels >50 μg l(-1). Multiple regression analysis revealed that Legionella spp. counts were positively associated with Mn, HPC at 37 °C and Zn and negatively associated with temperature. Only 1 out of the 97 samples (1%) having a Mn concentration, an HPC at 22 °C and an HPC at 37 °C below the respective median values exhibited a Legionella spp. concentration exceeding 10(4) CFU l(-1)vs. 41 out of the 89 samples (46.1%) with the three parameters above the medians. Our results show a qualitative and quantitative relationship between Legionella spp., the Mn concentration and heterotrophic plate counts in hot water samples from different buildings, suggesting that these parameters should be included in a water safety plan. The role of manganese in biofilm formation and its possible involvement in the mechanisms favouring Legionella survival and growth in water niches should be investigated further.

Prevalence of anti-legionella antibodies among Italian hospital workers

This study evaluated the prevalence of anti-legionella antibodies in workers at hospitals with a long-term history of legionella contamination. The hospitals are located in Milan and Turin, northern Italy, and in Naples and Bari, southern Italy. Antibody prevalence and titres of healthcare workers, medical and dental students and blood donors were assessed. In total 28.5% of subjects were antibody positive, most frequently to L. pneumophila serogroups 7-14. Major differences were observed in seroprevalence and type of legionella antibody in persons from different geographic areas. Healthcare workers had a significantly higher frequency of antibodies compared with blood donors in Milan (35.4 vs 15.9%, P<0.001), whereas in Naples both groups exhibited high antibody frequency (48.8 vs 44.0%) and had a higher proportion of antibodies to legionella serogroups 1-6. Dental workers had a higher seroprevalence than office staff in Bari, but not in Turin, where daily disinfecting procedures had been adopted to avoid contamination of dental unit water. No association was found between the presence of antibodies and the presence of risk factors for legionellosis, nor with the occurrence of pneumonia and/or flu-like symptoms. In conclusion, the presence of legionella antibodies may be associated with occupational exposure in the hospital environment, but there was no evidence of any association with disease.

Control of Legionella contamination in a hospital water distribution system by monochloramine.

BACKGROUND We report the results of 1-year application of monochloramine to control Legionella pneumophila contamination in a hospital hot water distribution system. METHODS In the main building of the hospital, a device continuously distributing monochloramine was installed. Legionella pneumophila and Pseudomonas spp contamination was followed in comparison with 2 other water networks in the same building using chlorine dioxide. RESULTS Monochloramine significantly reduced the number of contaminated sites compared with baseline (from 97.0% to 13.3%, respectively), chlorine dioxide device I (from 100% to 56.7%, respectively), and device II (from 100% to 60.8%, respectively). No positive sample exceeded 10(4) colony-forming units/L versus 59.4% at baseline. CONCLUSION Monochloramine could represent a good alternative to chlorine dioxide in controlling legionellae contamination in public and private buildings.

Ecological behaviour of three serogroups of Legionella pneumophila within a model plumbing system

Three Legionella pneumophila strains isolated from water samples and belonging to serogroups (sgs) 1, 6 and 9 were analysed for their capacity to colonise an experimental model simulating a domestic hot water distribution system. Ecological factors that could influence the persistence of the sgs such as intracellular life within protozoan hosts and bacterial interference by the production of antagonistic compounds were also studied. Viable counts of L. pneumophila increased both in the planktonic and in the sessile phases. Sg 6 showed a marked prevalence during the whole experiment and exhibited the highest host infection efficiency. Sg 1 was significantly less represented, but showed the highest capacity to reproduce in the protozoan hosts. Sg 9 was poorly represented and less adapted to intracellular life. Among the 14 bacteria constantly isolated in the system, five (35.7%) produced antagonistic substances against Legionella, with differences according to the bacterial strain and L. pneumophila sgs.

Selenium interactions with essential and toxic elements in egg yolk from commercial and fortified eggs

The main objective of this work was to evaluate the interaction between selenium concentration in both commercial and Se-enriched eggs and other essential/toxic elements (Ca, Mg, Fe, Zn, Pb, and Cd), taking into account a possible synergic action of iodine. Commercial eggs were purchased from several sale points or directly from the producers (farmyard eggs). Fortified eggs were obtained by supplementing chickenfeed for 6 weeks with Se as sodium selenite (1.0 microg/g Se) or Se plus iodine (1.0 microg/g Se+3.7 microg/g I). Se in experimental egg yolks significantly increased over the basic value by 39% in the Se group and 61% in the Se+I group, suggesting that I addition may enhance Se absorption. Levels of Se in commercial yolks were identical in free-range, barn or battery eggs, but significantly lower in farmyard and higher in organic eggs where the Se content approximated that found in Se fortified eggs. A significant reduction in Cd was observed in Se+I treated yolks compared to both control and Se alone diet, thus suggesting a high sensitivity of Cd to the detoxifying effect of Se combined with I. Furthermore, Se+I supplementation was associated with a significant Zn reduction, a finding which needs clarification to avoid attempts to maximize one component affecting the levels of other essential elements.

Protozoa and human macrophages infection by Legionella pneumophila environmental strains belonging to different serogroups.

Three Legionella pneumophila strains isolated from municipal hot tap water during a multicentric Italian survey and belonging to serogroups 1, 6, 9 and the reference strain Philadelphia-1 were studied to determine the intracellular replication capability and the cytopathogenicity in human monocyte cell line U937 and in an Acanthamoeba polyphaga strain. Our results show that both serogroups 1 and Philadelphia-1 were able to multiply into macrophages inducing cytopathogenicity, while serogroup 6 and ever more serogroup 9 were less efficient in leading to death of the infected macrophages. Both serogroups 1 and 6 displayed a quite good capability of intracellular replication in A. polyphaga, although serogroup 1 was less cytopathogenic than serogroup 6. Serogroup 9, like Philadelphia-1 strain, showed a reduced efficiency of infection and replication and a low cytopathogenicity towards the protozoan. Our study suggests that bacterial pathogenesis is linked to the difference in the virulence expression of L. pneumophila serogroups in both hosts, as demonstrated by the fact that only L. pneumophila serogroup 1 shows the contextual expression of the two virulence traits. Serogroup 6 proves to be a good candidate as pathogen since it shows a good capacity for intracellular replication in protozoan.Three Legionella pneumophila strains isolated from municipal hot tap water during a multicentric Italian survey and belonging to serogroups 1, 6, 9 and the reference strain Philadelphia-1 were studied to determine the intracellular replication capability and the cytopathogenicity in human monocyte cell line U937 and in an Acanthamoeba polyphaga strain. Our results show that both serogroups 1 and Philadelphia-1 were able to multiply into macrophages inducing cytopathogenicity, while serogroup 6 and ever more serogroup 9 were less efficient in leading to death of the infected macrophages. Both serogroups 1 and 6 displayed a quite good capability of intracellular replication in A. polyphaga, although serogroup 1 was less cytopathogenic than serogroup 6. Serogroup 9, like Philadelphia-1 strain, showed a reduced efficiency of infection and replication and a low cytopathogenicity towards the protozoan. Our study suggests that bacterial pathogenesis is linked to the difference in the virulence expression of L. pneumophila serogroups in both hosts, as demonstrated by the fact that only L. pneumophila serogroup 1 shows the contextual expression of the two virulence traits. Serogroup 6 proves to be a good candidate as pathogen since it shows a good capacity for intracellular replication in protozoan.

Inter-laboratory validation of a rapid assay for the detection and quantification of Legionella spp. in water samples

Aims:  To compare the standard culture method with a new, rapid test (ScanVIT‐Legionella™) using fluorescently labelled gene probes for the detection and enumeration of Legionella spp. The new technique was validated through experiments conducted on both artificially and naturally contaminated water and through an inter‐laboratory comparison.