A Comparison of Legionella pneumophila Occurrence in Hot Water Tanks and Instantaneous Devices in Domestic, Nosocomial, and Community Environments

The aim of this study was to compare the occurrence of L. pneumophila in hot water samples from hot water tanks and instantaneous devices. Tanks and devices were all operated by heat exchangers employed in the town's district heating system. Thirty-six out of 171 (21%) hot water samples tested positive for L. pneumophila isolation, with 14.6% belonging to serogroup 1 and 6.4% to serogroups 2–14. The proportion of L. pneumophila detected in hot water reservoirs (30%) was higher than that observed in hot water instantaneous devices (6.2%). Differences in L. pneumophila isolation reflected different temperatures registered at the faucet: ≤50°C for hot water from reservoir devices, and >60°C for hot water from instantaneous devices. These data emphasize the need to control temperature in hot water distribution devices, thus inhibiting the formation of biofilm and L. pneumophila colonization. Received: 4 April 2000 / Accepted: 31 May 2000     

<Emphasis Type="Italic">Legionella</Emphasis> pneumonia due to non-<Emphasis Type="Italic">Legionella pneumophila</Emphasis> serogroup 1: usefulness of the six-point scoring system

Background

Because of a limited number of reports, we aimed to investigate the clinical characteristics of patients with Legionella pneumonia due to non-Legionella pneumophila serogroup 1 and the diagnostic usefulness of the six-point scoring system for such patients compared with patients with pneumonia caused by L. pneumophila serogroup 1.

Methods

We retrospectively analysed patients diagnosed with Legionella pneumonia due to non-L. pneumophila serogroup 1 between March 2001 and June 2016. We examined the clinical characteristics, including symptoms, laboratory findings, radiologic findings, pneumonia severity, initial treatment and prognosis. We also calculated scores using the six-point scoring system in these patients. Furthermore, we compared the clinical characteristics and six-point scores between non-L. pneumophila serogroup 1 patients and L. pneumophila serogroup 1 patients among hospitalized community-acquired pneumonia patients enrolled prospectively between October 2010 and July 2016.

Results

Eleven patients had pneumonia due to non-L. pneumophila serogroup 1; their median age was 66 years and 8 patients (72.7%) were male. The most common pathogen was L. pneumophila serogroup 3 (6/11), followed by L. pneumophila serogroup 9 (3/11), L. pneumophila serogroup 6 (1/11) and L. longbeachae (1/11). Non-specific symptoms, such as fever and cough, were common. Six patients (54.5%) had liver enzyme elevation, but no patient developed hyponatraemia at <130 mEq/L. Nine patients (81.8%) showed lobar pneumonia and 7 patients (63.6%) manifested with consolidation and ground-glass opacity. Patients with mild to moderate severity comprised 10 (90.9%) by CURB-65 and 5 (45.5%) by the Pneumonia Severity Index. Of all patients, 4 were admitted to the intensive care unit and 3 died despite appropriate empiric therapy. The clinical characteristics were not significantly different between non-L. pneumophila serogroup 1 patients and L. pneumophila serogroup 1 patients (n?=?23). At a cut-off value of ≥?2 points, the sensitivity of the six-point scoring system was 54.5% (6/11) for non-L. pneumophila serogroup 1 patients and 95.7% (22/23) for L. pneumophila serogroup 1 patients.

Conclusions

Cases of non-L. pneumophila serogroup 1 pneumonia varied in severity from mild to severe and the clinical characteristics were often non-specific. The six-point scoring system was not useful in predicting such Legionella pneumonia cases.

     

Plasmids of serogroup 1 strains ofLegionella pneumophila

Twelve strains ofLegionella pneumophila were tested for the presence of plasmid DNA. Three strains, belonging to serogroup 1, had large plasmids of 83.8×10⁶ daltons, as determined by electron microscopy. A fourth strain, also from serogroup 1, had a similar large plasmid in addition to a smaller plasmid. Restriction analysis of plasmid DNA isolated from the strains with a single size plasmid indicated that the plasmids were structurally very similar. The biologic functions of these plasmids are yet to be determined.     

Distribution of Sequence-Based Types of Legionella pneumophila Serogroup 1 Strains Isolated from Cooling Towers,Hot Springs,and Potable Water Systems in China

Legionella pneumophila serogroup 1 causes Legionnaires'' disease. Water systems contaminated with Legionella are the implicated sources of Legionnaires'' disease. This study analyzed L. pneumophila serogroup 1 strains in China using sequence-based typing. Strains were isolated from cooling towers (n = 96), hot springs (n = 42), and potable water systems (n = 26). Isolates from cooling towers, hot springs, and potable water systems were divided into 25 sequence types (STs; index of discrimination [IOD], 0.711), 19 STs (IOD, 0.934), and 3 STs (IOD, 0.151), respectively. The genetic variation among the potable water isolates was lower than that among cooling tower and hot spring isolates. ST1 was the predominant type, accounting for 49.4% of analyzed strains (n = 81), followed by ST154. With the exception of two strains, all potable water isolates (92.3%) belonged to ST1. In contrast, 53.1% (51/96) and only 14.3% (6/42) of cooling tower and hot spring, respectively, isolates belonged to ST1. There were differences in the distributions of clone groups among the water sources. The comparisons among L. pneumophila strains isolated in China, Japan, and South Korea revealed that similar clones (ST1 complex and ST154 complex) exist in these countries. In conclusion, in China, STs had several unique allelic profiles, and ST1 was the most prevalent sequence type of environmental L. pneumophila serogroup 1 isolates, similar to its prevalence in Japan and South Korea.     

Evaluation of Legionella pneumophila contamination in Italian hotel water systems by quantitative real‐time PCR and culture methods

Aims: This study was designed to define the extent of water contamination by Legionella pneumophila of certain Italian hotels and to compare quantitative real‐time PCR with the conventional culture method. Methods and Results: Nineteen Italian hotels of different sizes were investigated. In each hotel three hot water samples (boiler, room showers, recycling) and one cold water sample (inlet) were collected. Physico‐chemical parameters were also analysed. Legionella pneumophila was detected in 42% and 74% of the hotels investigated by the culture method and by real‐time PCR, respectively. In 21% of samples analysed by the culture method, a concentration of >10⁴ CFU l^?1 was found, and Leg. pneumophila serogroup 1 was isolated from 10·5% of the hotels. The presence of Leg. pneumophila was significantly influenced by water sample temperature, while no association with water hardness or residual‐free chlorine was found. Conclusions: This study showed a high percentage of buildings colonized by Leg. pneumophila. Moreover, real‐time PCR proved to be sensitive enough to detect lower levels of contamination than the culture method. Significance and Impact of the Study: This study indicates that the Italian hotels represent a possible source of risk for Legionnaires’ disease and confirms the sensitivity of the molecular method. To our knowledge, this is the first report to demonstrate Legionella contamination in Italian hotels using real‐time PCR and culture methods.     

Differences in the respiratory burst of human polymorphonuclear leukocytes induced by virulent and avirulent Legionella pneumophila Serogroup 1

Two strains of Legionella pneumophila of different virulence were examined for their influence on the metabolic oxidative activity of human polymorphonuclear leukocytes. The leukocytes exhibited decreased rates of oxygen consumption and diminished chemiluminescence activity following phagocytosis of a virulent strain of L. pneumophila serogroup 1. In contrast, phagocytosis of its multipassaged derivative rendered avirulent, was accompanied by increased rates of both oxygen consumption and chemiluminescence activity. Although no differences were observed in oxygen uptake induced by the virulent legionellae compared to leukocytes at rest, statistically significant differences were observed in the chemiluminescence responses. These observations were not unexpected, since the luminol-enhanced chemiluminescence assay, is more sensitive than the oxygen uptake assay. In spite of decreased metabolic activity of PMN in the presence of virulent legionellae, electron microscope studies showed higher numbers of intracellular L. pneumophila than the avirulent subtype. Thus, virulent and avirulent L. pneumophila can be differentiated on the basis of oxygen consumption and chemiluminescence assays.     

Legionella Contamination in Hot Water of Italian Hotels

A cross-sectional multicenter survey of Italian hotels was conducted to investigate Legionella spp. contamination of hot water. Chemical parameters (hardness, free chlorine concentration, and trace element concentrations), water systems, and building characteristics were evaluated to study risk factors for colonization. The hot water systems of Italian hotels were strongly colonized by Legionella; 75% of the buildings examined and 60% of the water samples were contaminated, mainly at levels of ≥10³ CFU liter⁻¹, and Legionella pneumophila was the most frequently isolated species (87%). L. pneumophila serogroup 1 was isolated from 45.8% of the contaminated sites and from 32.5% of the hotels examined. When a multivariate logistic model was used, only hotel age was associated with contamination, but the risk factors differed depending on the contaminating species and serogroup. Soft water with higher chlorine levels and higher temperatures were associated with L.pneumophila serogroup 1 colonization, whereas the opposite was observed for serogroups 2 to 14. In conclusion, Italian hotels, particularly those located in old buildings, represent a major source of risk for Legionnaires' disease due to the high frequency of Legionella contamination, high germ concentration, and major L. pneumophila serogroup 1 colonization. The possible role of chlorine in favoring the survival of Legionella species is discussed.     

Impact of Non-Legionella Bacteria on the Uptake and Intracellular Replication of Legionella pneumophila in Acanthamoeba castellanii and Naegleria lovaniensis

In aquatic environments, Legionella pneumophila survives, in association with other bacteria, within biofilms by multiplying in free-living amoebae. The precise mechanisms underlying several aspects of the uptake and intracellular replication of L. pneumophila in amoebae, especially in the presence of other bacteria, remain unknown. In the present study, we examined the competitive effect of selected non-Legionella bacteria (Escherichia coli, Aeromonas hydrophila, Flavobacterium breve, and Pseudomonas aeruginosa) on the uptake of L. pneumophila serogroup 1 by the amoebae Acanthamoeba castellanii and Naegleria lovaniensis. We also investigated their possible influence on the intracellular replication of L. pneumophila in both amoeba species. Our results showed that the non-Legionella bacteria did not compete with L. pneumophila for uptake, suggesting that the amoeba hosts took in L. pneumophila through a specific and presumably highly efficient uptake mechanism. Living and heat-inactivated P. aeruginosa best supported the replication of L. pneumophila in N. lovaniensis and A. castellanii, respectively, whereas for both amoeba species, E. coli yielded the lowest number of replicated L. pneumophila. Furthermore, microscopic examination showed that 100% of the A. castellanii and only 2% of the N. lovaniensis population were infected with L. pneumophila at the end of the experiment. This study clearly shows the influence of some non-Legionella bacteria on the intracellular replication of L. pneumophila in A. castellanii and N. lovaniensis. It also demonstrates the different abilities of the two tested amoeba species to serve as a proper host for the replication and distribution of the human pathogen in man-made aquatic environments such as cooling towers, shower heads, and air conditioning systems with potential serious consequences for human health.     

Detection of Legionella pneumophila serogroup 1 urinary antigen using an enhanced chemiluminescence ELISA

An enhanced chemiluminescence enzyme-linked immunosorbent assay has been developed for the detection of soluble antigen in the urine of patients with Legionnaires' disease (LD). In the assay antigen(s) in the urine samples are captured by a rabbit anti-L. pneumophila antibody coated onto microtitre strips. A fluorescein-isothiocyanate (FITC) conjugate of the same antibody is then added which binds to the captured antigen. Any immobilized FITC-labelled antibody is then detected with a horseradish peroxidase (HRP) conjugate of a monoclonal anti-FITC antibody. HRP activity is monitored after oxidation of luminol in the presence of H₂O₂ and iodophenol. The resulting luminescence is recorded using a camera luminometer. Urine specimens were available for testing from 31 patients with evidence of ongoing L. pneumophila serogroup 1 infection. A positive result was obtained in the cases of 12/12 specimens from culture-proven LD patients, and 16/19 specimens from patients with serological evidence of LD. Thus the sensitivity is estimated to be 28/31 (90%) The specificity was estimated using urine specimens from eight patients with non-L. pneumophila pneumonias of known aetiology. All eight specimens gave a negative result.     

Multiplication of Legionella pneumophila in HeLa Cells in the Presence of Cytoskeleton and Metabolic Inhibitors

A study has been carried out on the action of cytoskeleton and metabolic inhibitors on intracellular multiplication in HeLa cells of a virulent strain of Legionella pneumophila serogroup 6. The effects of the substances were separately tested on both penetration and intracellular multiplication of L. pneumophila. Only cytochalasin A and 2-deoxy-d -glucose (2dG) affected bacterial internalisation, whereas intracellular multiplication was inhibited by cytochalasins A, B, C, D and J (D being the most active) and by 2dG with a dose-response effect. The action of 2dG was counteracted by 50 mM glucose. Experiments carried out with cytochalasin D and a rhodamine-phalloidin conjugate showed the involvement of cytoskeletal elements in intracellular multiplication of Legionella; compounds acting on microtubules had no effect.     

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.     

Development of an indirect sandwich ELISA for detection of urinary antigen,using Legionella pneumophila PAL protein

Legionella pneumophila peptidoglycan-associated lipoprotein (PAL) protein is an extremely conserved antigen among Legionella species. In this study, rabbit and rat anti-PAL immunoglobulin G antibodies were produced by immunization with purified, recombinant PAL (r-PAL) protein of L. pneumophila serogroup 1 and used as capture and detection antibodies in the PAL antigen-based enzyme-linked immunosorbent assay (ELISA) to detect urinary PAL antigen. Urine samples were obtained from rats experimentally infected with L. pneumophila serogroup 1. The PAL antigen was measured in urine samples of 40 infected and 40 uninfected rats. After choosing the cut-off value of 0.192, the sensitivity and specificity of the PAL antigen-based ELISA were 87.5 and 97.5 %, respectively. The results obtained by PAL antigen base ELISA were compared with those obtained by Biotest. The PAL antigen was detected efficiently by both of the assays and all of the control human urine samples were negative by the ELISA test. The PAL antigen-based ELISA assay was relatively simple to perform, precise, highly sensitive and specific, and reproducible. Based on our data the PAL antigen-based ELISA described here is the first indirect sandwich ELISA for urinary antigen detection which could easily be applied for diagnosis of Legionnaires disease.     

Legionella pneumophila in the Environment: The Occurrence of a Fastidious Bacterium in Oligotrophic Conditions

Legionella pneumophila, a micro-organism encountered in aquatic environments, can cause serious intracellular infections among humans. Since the bacterium is ubiquitous in aquatic habitats, it appears to be impossible to prevent L. pneumophila from entering man-made water systems. However, many questions concerning the survival and/or growth in the environment, the partners and opponents of L. pneumophila remain unanswered. This review focuses on the factors governing the ecology of L. pneumophila, since there is considerable divergence and even contradiction in literature on its environmental requirements. A key question to be resolved is the discrepancy between the fastidious nature of L. pneumophila in axenic cultures (e.g. 400 mg l⁻¹ L-cysteine and 250 mg l^-1 ferric iron) and the nutritionally poor environments in which it is commonly detected. It is assumed that dense microbial communities, as occurring in sediments and biofilms – but not likely in surface and drinking water, – can provide the necessary growth requirements for L. pneumophila. However, most of the studies concerning L. pneumophila have led to the general opinion that the organism can only multiply in the aquatic environment as a parasite in certain protozoa. The discovery of the non-classical siderophore legiobactin also indicates that the iron requirement for survival and autonomous growth is not as high as has been assumed. It thus appears that in order to control Legionella in the environment, focus should be on the eradication of microbial hotspots in which L. pneumophila resides.     

The Legionella pneumophila icm locus: a set of genes required for intracellular multiplication in human macrophages

Legionella pneumophila, the causative agent of Legionnaires’ disease and related pneumonias, infects, replicates within and eventually kills human macrophages. A key feature of the intracellular lifestyle is the ability of the organism to replicate within a specialized phagosome which does not fuse with Iysosomes or acidify. Avirulent mutants that are defective in intracellular multiplication and host-cell killing are unable to prevent phagosome–Iysosome fusion. In a previous study, a 12kb fragment of the L. pneumophila genome containing the icm locus (intracellular multiplication) was found to enable the mutant bacteria to prevent phagosome-Iysosome fusion, to multiply intracellularly and to kill human macrophages. The complemented mutant also regained the ability to produce lethal pneumonia in guinea-pigs. In order to gain information about how L. pneumophila prevents phagosome-Iysosome fusion and alters other intracellular events, we have studied the region containing the icm locus. This locus contains four genes, icmWXYZ, which appear to be transcribed from a single promoter to produce a 2.1–2.4kb mRNA. The deduced amino acid sequences of the Icm proteins do not exhibit significant similarity to other proteins of known sequence, suggesting that they may carry out novel functions. The icmX gene encodes a product with an apparent signal sequence suggesting that it is a secreted protein. The icmWXYZ genes are located adjacent to and on the opposite strand from the dot gene, which is also required for intracellular multiplication and the ability of L. pneumophila to modify organelle traffic in human macrophages. Five L. pneumophila Icm mutants that had been generated with transposon Tn903dIIlacZ were found to have Inserted the transposon within the icmX, icmY, icmZ and dot genes, confirming their role in the ability of the organism to multiply intracellularly.     

Genomic Characterization of a Large Outbreak of Legionella pneumophila Serogroup 1 Strains in Quebec City, 2012

During the summer of 2012, a major Legionella pneumophila serogroup 1 outbreak occurred in Quebec City, Canada, which caused 182 declared cases of Legionnaire''s disease and included 13 fatalities. Legionella pneumophila serogroup 1 isolates from 23 patients as well as from 32 cooling towers located in the vicinity of the outbreak were recovered for analysis. In addition, 6 isolates from the 1996 Quebec City outbreak and 4 isolates from patients unrelated to both outbreaks were added to allow comparison. We characterized the isolates using pulsed-field gel electrophoresis, sequence-based typing, and whole genome sequencing. The comparison of patients-isolated strains to cooling tower isolates allowed the identification of the tower that was the source of the outbreak. Legionella pneumophila strain Quebec 2012 was identified as a ST-62 by sequence-based typing methodology. Two new Legionellaceae plasmids were found only in the epidemic strain. The LVH type IV secretion system was found in the 2012 outbreak isolates but not in the ones from the 1996 outbreak and only in half of the contemporary human isolates. The epidemic strains replicated more efficiently and were more cytotoxic to human macrophages than the environmental strains tested. At least four Icm/Dot effectors in the epidemic strains were absent in the environmental strains suggesting that some effectors could impact the intracellular replication in human macrophages. Sequence-based typing and pulsed-field gel electrophoresis combined with whole genome sequencing allowed the identification and the analysis of the causative strain including its likely environmental source.     

Molecular Determination of Infection Source of a Sporadic Legionella Pneumonia Case Associated with a Hot Spring Bath

To determine the infection source of a sporadic Legionella pneumonia case associated with a hot spring bath, we used five molecular methods, including repetitive element polymerase chain reaction (rep-PCR), arbitrarily primed PCR (AP-PCR), ribotyping, restriction endonuclease analysis (REA), and macrorestriction endonuclease analysis (MREA) by pulsed-field gel electrophoresis. L. pneumophila serogroup (SG) 3 strain EY 3702, isolated from an intratracheal specimen of a 71-year-old Japanese female who developed pneumonia after nearly drowning in a hot spring spa bath, produced rep-PCR and AP-PCR fingerprints identical to those of L. pneumophila SG 3 strains EY 3768 and EY 3769 isolated from the bath water. Four epidemiologically unrelated L. pneumophila SG 3 strains showed different rep-PCR or AP-PCR fingerprints from those of the three EY strains (EY 3702, 3768, and 3769). The three EY strains were also genotypically indistinguishable by ribotyping with EcoRI and PstI, by REA with EcoBI or HindIII, and by MREA with NotI. Based on these results, we identified the bath water of the hot spring spa as the source of infection of this patient, even though the viable number of the organisms in the bath water was low (3 CFU/100 ml) when determined 27 days after her nearly drowning.     

Rapid Detection and Enumeration of Legionella pneumophila in Hot Water Systems by Solid-Phase Cytometry

A new method for the rapid and sensitive detection of Legionella pneumophila in hot water systems has been developed. The method is based on an IF assay combined with detection by solid-phase cytometry. This method allowed the enumeration of L. pneumophila serogroup 1 and L. pneumophila serogroups 2 to 6, 8 to 10, and 12 to 15 in tap water samples within 3 to 4 h. The sensitivity of the method was between 10 and 100 bacteria per liter and was principally limited by the filtration capacity of membranes. The specificity of the antibody was evaluated against 15 non-Legionella strains, and no cross-reactivity was observed. When the method was applied to natural waters, direct counts of L. pneumophila were compared with the number of CFU obtained by the standard culture method. Direct counts were always higher than culturable counts, and the ratio between the two methods ranged from 1.4 to 325. Solid-phase cytometry offers a fast and sensitive alternative to the culture method for L. pneumophila screening in hot water systems.     

Characterization of a spontaneous avirulent mutant of <Emphasis Type="Italic">Legionella pneumophila</Emphasis> Serogroup 6: Evidence of DotA and flagellin involvement in the loss of virulence

The pathogenesis of Legionella pneumophila mainly resides in its ability to inhibit the phagosome-lysosome fusion, which normally prevents the killing of the host cells. In order to characterize the molecular alterations that occurred in a spontaneous avirulent mutant of Legionella pneumophila serogroup 6, named Vir-, we investigated the ability of the mutant to adhere to and multiply in the WI26VA4 alveolar epithelial cell line and in human macrophages, when compared to its parental strain, Vir+. We also determined the colocalization of bacteria with LAMP-1 to gain an insight into the phagosome-lysosome fusion process. Additionally, we determined the flagellin expression and dotA nucleotide sequencing. We observed a lack of expression of flagellin and an in-frame mutation in the dotA. gene. The data obtained strongly suggest the loss of virulence of the mutant could probably be due to the absence of flagellin and the dysfunctional type IV secretion System, resulting from the DotA protein being severely compromised.     

Legionella pneumophila has two 60-kilodalton heat-shock proteins

Legionella pneumophila is a thermotolerant bacterium. To learn more about the thermal adaptation of this organism, we studied the properties of the Legionella 60-kDa heat-shock protein (MopA, GroEL-analog, HtpB, Lp-Hsp60) in L. pneumophila and in an Escherichia coli strain containing the cloned gene. Lp-Hsp60 was found in both cytosol and membrane fractions; however, Lp-Hsp60 in the membrane fraction of L. pneumophila was slightly larger than Lp-Hsp60 in the cytosol. In contrast, both membrane-associated and cytosolic Lp-Hsp60 in the E. coli clone were similar in size to the smaller cytosolic Lp-Hsp60 of L. pneumophila. While peptide mapping suggests there are differences between the two proteins, the larger membrane-associated Lp-Hsp60 and the smaller cytosolic LP-Hsp60 shared Legionella-specific and E. coli GroEL cross-reacting epitopes, and the sequence of their first 20 N-terminal amino acids was identical. Further, Southern blot analysis of EcoRI-digested chromosomal DNA from several strains of L. pneumophila showed two fragments reacting with an htpAB-operon probe. In summary, L. pneumophila contains two Hsp60 proteins, and possibly two hsp60 genes.