Legionella Pneumophila Strains Research Articles

ANALYSIS OF THE LEVEL AND RATE OF LEGIONELLA PNEUMOPHILA CONTAMINATION OF HOT WATER SUPPLY SYSTEMS IN THE THERAPEUTIC-AND-PROPHYLACTIC INSTITUTIONS OF MOSCOW

The specific features of Legionella colonization of water supply systems were studied in the large healthcare facilities (HCF) of Moscow. Examination of 16 buildings of 5 Moscow multidisciplinary HCFs, including their risk-group units, revealed the high level (68%) of Legionella pneumophila contamination of hot water supply systems. Among the Legionella pneumophila strains circulating in the hot water supply systems of Moscow HCFs, there was a preponderance of its serogroup 6 (44%), 5 (26%), and 1 (13%) strains. The findings suggest that it is necessary to arrange a set of prophylactic measures for microbiological monitoring and protection of water systems in the HCFs, which are aimed at preventing nosocomial legionellosis.

The N -Acylneuraminate Cytidyltransferase Gene, neuA , Is Heterogenous in Legionella pneumophila Strains but Can Be Used as a Marker for Epidemiological Typing in the Consensus Sequence-Based Typing Scheme

Sequence-based typing (SBT) is the internationally recognized standard method for genotyping Legionella pneumophila. To date all strains of serogroup 1 (SG1) and some of SGs 2 to 14 yield a seven-allele profile and can be assigned a sequence type (ST). However, for some strains belonging to SGs 2 to 14, the targeted region of the neuA gene could not be amplified using the published standard primers. We determined the DNA sequence of a neuA gene homolog located in the lipopolysaccharide synthesis locus of strain Dallas-1E. By using newly designed degenerate consensus primers based on the neuA homolog in strains Dallas-1E, Philadelphia-1, Paris, Lens, and Corby, we were able to obtain DNA sequences for all 48 non-SG1 strains which were untypeable by the standard method. Our data show that the neuA gene is present in all L. pneumophila strains but differs significantly in some non-SG1 strains at both the DNA and amino acid levels. The new primers can be used to amplify and sequence the neuA gene in all strains and can substitute for the standard primers. This offers the possibility of assigning an ST to all strains of L. pneumophila.

High-Throughput Typing Method To Identify a Non-Outbreak-Involved Legionella pneumophila Strain Colonizing the Entire Water Supply System in the Town of Rennes, France

Two legionellosis outbreaks occurred in the city of Rennes, France, during the past decade, requiring in-depth monitoring of Legionella pneumophila in the water network and the cooling towers in the city. In order to characterize the resulting large collection of isolates, an automated low-cost typing method was developed. The multiplex capillary-based variable-number tandem repeat (VNTR) (multiple-locus VNTR analysis [MLVA]) assay requiring only one PCR amplification per isolate ensures a high level of discrimination and reduces hands-on and time requirements. In less than 2 days and using one 4-capillary apparatus, 217 environmental isolates collected between 2000 and 2009 and 5 clinical isolates obtained during outbreaks in 2000 and 2006 in Rennes were analyzed, and 15 different genotypes were identified. A large cluster of isolates with closely related genotypes and representing 77% of the population was composed exclusively of environmental isolates extracted from hot water supply systems. It was not responsible for the known Rennes epidemic cases, although strains showing a similar MLVA profile have regularly been involved in European outbreaks. The clinical isolates in Rennes had the same genotype as isolates contaminating a mall's cooling tower. This study further demonstrates that unknown environmental or genetic factors contribute to the pathogenicity of some strains. This work illustrates the potential of the high-throughput MLVA typing method to investigate the origin of legionellosis cases by allowing the systematic typing of any new isolate and inclusion of data in shared databases.

Efficacy of Colloidal Silver-Hydrogen Peroxide and 2-Bromo-2-nitroporopane-1,3-diol Compounds Against Different Serogroups of Legionella pneumophila Strains

Cooling towers are considered as amplifier and disseminator sources for Legionella spp. despite preventive treatments. Information which was obtained from biocidal tests could improve the effectiveness of treatments. Therefore, the choice of appropriate biocides and the applying of biocides in correct dosages and contact times are important. Various oxidizing and non-oxidizing biocides have been investigated in vitro for their effectiveness against legionellae. Colloidal silver-hydrogen peroxide (CSHP) and 2-bromo-2-nitroporopane-1,3-diol (BNPD) biocides were selected as an example for oxidizing and non-oxidizing agents, respectively, in view of bactericidal action against different serogroups of L. pneumophila strains [serogroup 1 (S1) and serogroup 2-14 (S2)] which are isolated different cooling towers in the vicinity of Istanbul, Turkey and reference strain. In the current study, oxidizing biocide was found more effective than non-oxidizing biocide in terms of contact times, log reductions and recommended dosages. At the recommended concentrations for cooling towers (100ppm), while CSHP compound killed all strains in 3h contact time, BNPD compound killed S2 and reference strain in the same contact time, S1 strain after 6h contact time. The results of the present study showed that effective biocide applications can be achieved by pre-determining minimum inhibitory concentration (MIC) and minimum contact time of different biocides to kill target bacteria.

Differential growth of Legionella pneumophila strains within a range of amoebae at various temperatures associated with in-premise plumbing

The potential effect of in-premise plumbing temperatures (24, 32, 37 and 41°C) on the growth of five different Legionella pneumophila strains within free-living amoebae (Acanthamoeba polyphaga, Hartmannella vermiformis and Naegleria fowleri) was examined. Compared with controls that actively fed on Escherichia coli prey, when Leg. pneumophila was used as prey, strains Lp02 and Bloomington-2 increased in growth at 30, 32 and 37°C while strains Philadelphia-1 and Chicago 2 did not grow at any temperature within A. polyphaga. Strains Lp02, Bloomington-2 and Dallas 1E did not proliferate in the presence of H. vermiformis nor did strain Philadelphia-1 in the presence of N. fowleri. Yet, strain Bloomington-2 grew at all temperatures examined within N. fowleri, while strain Lp02 proliferated at all temperatures except 41°C. More intriguing, strain Chicago 2 only grew at 32°C within H. vermiformis and N. fowleri suggesting a limited temperature growth range for this strain. Identifying the presence of pathogenic legionellae may require the use of multiple host amoebae and incubation temperatures. Temperature conditions and species of amoeba host supported in drinking water appear to be important for the selection of human-pathogenic legionellae and point to future research required to better understand Legionella ecology.

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.

Comparative genome analysis of a large Dutch Legionella pneumophila strain collection identifies five markers highly correlated with clinical strains

BackgroundDiscrimination between clinical and environmental strains within many bacterial species is currently underexplored. Genomic analyses have clearly shown the enormous variability in genome composition between different strains of a bacterial species. In this study we have used Legionella pneumophila, the causative agent of Legionnaire's disease, to search for genomic markers related to pathogenicity. During a large surveillance study in The Netherlands well-characterized patient-derived strains and environmental strains were collected. We have used a mixed-genome microarray to perform comparative-genome analysis of 257 strains from this collection.ResultsMicroarray analysis indicated that 480 DNA markers (out of in total 3360 markers) showed clear variation in presence between individual strains and these were therefore selected for further analysis. Unsupervised statistical analysis of these markers showed the enormous genomic variation within the species but did not show any correlation with a pathogenic phenotype. We therefore used supervised statistical analysis to identify discriminating markers. Genetic programming was used both to identify predictive markers and to define their interrelationships. A model consisting of five markers was developed that together correctly predicted 100% of the clinical strains and 69% of the environmental strains.ConclusionsA novel approach for identifying predictive markers enabling discrimination between clinical and environmental isolates of L. pneumophila is presented. Out of over 3000 possible markers, five were selected that together enabled correct prediction of all the clinical strains included in this study. This novel approach for identifying predictive markers can be applied to all bacterial species, allowing for better discrimination between strains well equipped to cause human disease and relatively harmless strains.

Establishment of loop-mediated isothermal amplification method for detection of Legionella pneumophila

To establish a simple and rapid molecular detection for Legionella pneumophila. The loop-mediated isothermal amplification (LAMP) was applied for detection of Legionella pneumophila. A set of primers were designed to identify six special areas in mip gene of Legionella pneumophila. Genomic DNAs from 13 bacterial strains,including 8 Legionella pneumophila strains and 5 other bacterial strains were amplified by LAMP and general PCR method to evaluate the specificity and sensibility of LAMP. All positive tubes produced visible white precipitation, and no precipitation was observed in others. By adding smart green fluorescent dye, all Legionella pneumophila positive tubes presented a strong green fluorescence, while others showed weak fluorescence. The detection rate of LAMP was higher than that of general PCR. The detection limits were 576fg with genomic DNA of Legionella pneumophila,and 8 cfu/mL with positive water samples. LAMP detection of Legionella pneumophila is an effective and low-cost method with high specificity and sensitivity requiring no special equipment.

The autoinducer synthase LqsA and putative sensor kinase LqsS regulate phagocyte interactions, extracellular filaments and a genomic island of Legionella pneumophila

The amoebae-resistant opportunistic pathogen Legionella pneumophila employs a biphasic life cycle to replicate in host cells and spread to new niches. Upon entering the stationary growth phase, the bacteria switch to a transmissive (virulent) state, which involves a complex regulatory network including the lqs gene cluster (lqsA-lqsR-hdeD-lqsS). LqsR is a putative response regulator that promotes host-pathogen interactions and represses replication. The autoinducer synthase LqsA catalyses the production of the diffusible signalling molecule 3-hydroxypentadecan-4-one (LAI-1) that is presumably recognized by the sensor kinase LqsS. Here, we analysed L. pneumophila strains lacking lqsA or lqsS. Compared with wild-type L. pneumophila, the DeltalqsS strain was more salt-resistant and impaired for the Icm/Dot type IV secretion system-dependent uptake by phagocytes. Legionella pneumophila strains lacking lqsS, lqsR or the alternative sigma factor rpoS sedimented more slowly and produced extracellular filaments. Deletion of lqsA moderately reduced the uptake of L. pneumophila by phagocytes, and the defect was complemented by expressing lqsA in trans. Unexpectedly, the overexpression of lqsA also restored the virulence defect and reduced filament production of L. pneumophila mutant strains lacking lqsS or lqsR, but not the phenotypes of strains lacking rpoS or icmT. These results suggest that LqsA products also signal through sensors not encoded by the lqs gene cluster. A transcriptome analysis of the DeltalqsA and DeltalqsS mutant strains revealed that under the conditions tested, lqsA regulated only few genes, whereas lqsS upregulated the expression of 93 genes at least twofold. These include 52 genes clustered in a 133 kb high plasticity genomic island, which is flanked by putative DNA-mobilizing genes and encodes multiple metal ion efflux pumps. Upon overexpression of lqsA, a cluster of 19 genes in the genomic island was also upregulated, suggesting that LqsA and LqsS participate in the same regulatory circuit.

Rapid discrimination of Legionella by matrix-assisted laser desorption ionization time-of-flight mass spectrometry

Molecular typing is an important tool in the surveillance and investigation of human Legionella infection outbreaks. In this study, two molecular typing methods, pulsed-field gel electrophoresis (PFGE) and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS), were used to discriminate 23 Legionella pneumophila strains. The usefulness of MALDI-TOF-MS was demonstrated. The MALDI-TOF-MS fingerprinting with filtered small acid-soluble molecules gave different molecular profiles among strains, and the clustal analysis with MALDI-TOF-MS showed a high discrimination of strains the same as that with PFGE. In addition, MALDI-TOF-MS data could be generated within a few hours after the initial culture, although PFGE analyses took several days to complete. Thus, MALDI-TOF-MS offers a simple and rapid discrimination technique that could aid in the tracking of fast-spreading outbreaks of Legionella.

Characterization of Legionella pneumophila isolates from patients in Japan according to serogroups, monoclonal antibody subgroups and sequence types

We collected 86 unrelated clinical Legionella pneumophila strains that were isolated in Japan during the period 1980-2008. Most (80.2%) belonged to serogroup 1, followed by serogroups 5, 3 and 2. Interestingly, the patients with L. pneumophila serogroup 1 had a significantly higher male-to-female ratio (12.4) than the patients with other L. pneumophila serogroups (2.0) (OR, 10.5; 95% CI, 2.5-44.5). When the serogroup 1 strains were analysed by monoclonal antibody (mAb) typing, the most prevalent subgroup was Benidorm (34.9% of all isolates). Moreover, 79.7% of the serogroup 1 isolates were bound by mAb 3/1, which recognizes the virulence-associated epitope. When all 86 isolates were subjected to sequence-based typing (SBT) using seven loci, they could be divided into 53 sequence types (STs). The ST with the most isolates (seven) was ST1, to which most isolates from patients and environments around the world belong. However, six of the seven ST1 isolates were isolated before 1994. Other major STs were ST306 (n=6), ST120 (n=5) and ST138 (n=5). All ST306 and ST138 isolates, except for one isolate (ST306), were suspected or confirmed to be derived from bath water, which suggests that these strains prefer bath habitats. The sources of all ST1 and ST120 isolates remain unclear. By combining the SBT and mAb data, the 86 isolates could be divided into 59 types (discrimination index, 0.984). This confirms the usefulness of this combination in epidemiological studies.

Legionella pneumophilasecretes an endoglucanase that belongs to the family-5 of glycosyl hydrolases and is dependent upon type II secretion

Examination of cell-free culture supernatants revealed that Legionella pneumophila strains secrete an endoglucanase activity. Legionella pneumophila lspF mutants were deficient for this activity, indicating that the endoglucanase is secreted by the bacterium's type II protein secretion (T2S) system. Inactivation of celA, encoding a member of the family-5 of glycosyl hydrolases, abolished the endoglucanase activity in L. pneumophila culture supernatants. The cloned celA gene conferred activity upon recombinant Escherichia coli. Thus, CelA is the major secreted endoglucanase of L. pneumophila. Mutants inactivated for celA grew normally in protozoa and macrophage, indicating that CelA is not required for the intracellular phase of L. pneumophila. The CelA endoglucanase is one of at least 25 proteins secreted by the type II system of L. pneumophila and the 17th type of enzyme effector associated with this pathway. Only a subset of the other Legionella species tested expressed secreted endoglucanase activity, suggesting that the T2S output differs among the different legionellae. Overall, this study represents the first documentation of an endoglucanase (EC 3.2.1.4) being produced by a strain of Legionella.

Immune responses to Legionella pneumophila in athymic nude rats.

Athymic nude rats (rnu) were immunized intraperitoneally with the attenuated Phil I strain (serogroup 1) of Legionella pneumophila. These rats developed a humoral immune response, including the IgG class, that was of comparable magnitude to that seen in their non-nude littermates. When the rats were challenged ip with a dose of the virulent strain LD-8 (also serogroup 1) that proved to be lethal in non-immune rnu-rats, none of them developed any signs of disease, but survived well for the observation period of 2 weeks. However, it was impossible by adoptive transfer of lymphocytes from immunized inbred PVG donors to protect non-immune syngeneic rats from the lethality of the LD-8. These results together with previous data (16) indicate that specific antibodies, present in the animal at the time of infection, protect effectively against a lethal dose of L. pneumophila in this experimental system.

Sequence-based and monoclonal antibody typing of Legionella pneumophila isolated from patients in Portugal during 1987-2008

The monoclonal antibodies and the sequence-based typing (SBT) are two methodologies widely used to characterise Legionella pneumophila strains serogroup 1 (sg1). In this study, we analysed the clinical strains received in two Portuguese laboratories since 1987, including the strains isolated in Portugal during the four years of the surveillance scheme for Legionnaires' disease implemented in 2004. In total, 63 clinical isolates of L. pneumophila sg1 were differentiated by SBT into 19 different sequence types. Ten of them were new in the SBT database of the European Working Group for Legionella Infections (EWGLI). As a result of the combination of the two methodologies, these strains were discriminated into 25 different profiles. This study enabled, for the first time in Portugal, not only to characterise the L. pneumophila sg1 clinical isolates, but also to create a database of Portuguese profiles for use in epidemiological surveillance efforts.

Legionella pneumophilaGoes Clonal—Paris and Lorraine Strain‐Specific Risk Factors

<i>Legionella pneumophila</i>Goes Clonal—Paris and Lorraine Strain‐Specific Risk Factors

Determination of Legionella pneumophila susceptibility to Melaleuca alternifolia Cheel (tea tree) oil by an improved broth micro-dilution method under vapour controlled conditions

The aim of this study was to determine the in vitro activity of Melaleuca alternifolia Cheel (tea tree) oil (TTO) against 22 strains of Legionella pneumophila of different serogroup and source of isolation. Both a standard broth micro-dilution method, with slight modifications, and a micro-atmosphere diffusion method were used. Furthermore, we have established a simple sealing procedure in the micro-dilution method to determine the antibacterial activity of TTO against Legionella in aqueous phase. The results showed that L. pneumophila, quite irrespective of serogroup and source of isolation, is exquisitely sensitive to TTO, with minimal inhibitory concentration (MIC) ranging from 0.125 to 0.5% v/v, and a bactericidal activity at 0.5% v/v. In addition, we show here that TTO vapours exert critical activity, that must be controlled for reproducible MIC determinations. Overall, our data suggest that TTO could be active as anti- Legionella disinfectant, for control of water system contamination, especially in spas, in small waterlines or in particular respiratory medical devices.

Intragenic tandem repeat variation between Legionella pneumophila strains

BackgroundBacterial genomes harbour a large number of tandem repeats, yet the possible phenotypic effects of those found within the coding region of genes are only beginning to be examined. Evidence exists from other organisms that these repeats can be involved in the evolution of new genes, gene regulation, adaptation, resistance to environmental stresses, and avoidance of the immune system.ResultsIn this study, we have investigated the presence and variability in copy number of intragenic tandemly repeated sequences in the genome of Legionella pneumophila, the etiological agent of a severe pneumonia known as Legionnaires' disease. Within the genome of the Philadelphia strain, we have identified 26 intragenic tandem repeat sequences using conservative selection criteria. Of these, seven were "polymorphic" in terms of repeat copy number between a large number of L. pneumophila serogroup 1 strains. These strains were collected from a wide variety of environments and patients in several geographical regions. Within this panel of strains, all but one of these seven genes exhibited statistically different patterns in repeat copy number between samples from different origins (environmental, clinical, and hot springs).ConclusionThese results support the hypothesis that intragenic tandem repeats could play a role in virulence and adaptation to different environments. While tandem repeats are an increasingly popular focus of molecular typing studies in prokaryotes, including in L. pneumophila, this study is the first examining the difference in tandem repeat distribution as a function of clinical or environmental origin.

Long-term persistence of a single Legionella pneumophila strain possessing the mip gene in a municipal shower despite repeated cycles of chlorination

The ability of Legionella pneumophila to colonise domestic water systems is a primary cause of outbreaks of Legionnaire's disease in humans. World Health Organization guidelines recommend that drinking water is chlorinated to between 0.2 and 1mg/L [Chlorine in drinking-water. Guidelines for drinking-water quality, 2nd edn. Geneva: World Health Organization; 1996], but L. pneumophila is repeatedly isolated from chlorinated water systems, indicating that this treatment is not effective at preventing colonisation. Current UK guidelines recommend a one-off treatment of 20-50mg/L of free chlorine to remove the bacteria. In this study we report on the persistence of L. pneumophila serogroup 1 in a domestic shower system despite repeated cycles of chlorination at 50mg/L for 1h exposure time, over the course of two and a half years. Persisting isolates were subjected to in-vitro phenotypic analyses and polymerase chain reaction analysis for the toxin-encoding mip gene. Random amplified polymorphic DNA typing was also performed to determine whether the isolates recovered on different occasions were the same strain. We found that seven isolates of L. pneumophila recovered over a two-and-a-half year period are the same genetically defined strain, indicating that the bacteria can persist despite repeated cycles of chlorination after each successive isolation.

Comparison of two molecular methods used for subtyping of Legionella pneumophila 1 strains isolated from a hospital water supply

The results of the pulsed-field gel electrophoresis and the sequence-based typing (using the loci flaA, pilE, asd, mip, mompS and proA) were compared for subtyping of Legionella pneumophila 1 strains isolated from a hospital water supply. Molecular typing was carried out on 61 isolates (38% of the positive samples) selected on space and temporal criteria in order to follow the evolution of the water-system colonization. For all the 61 isolates, the sequence of the amplified mip gene fragment identified Legionella pneumophila strain Wadsworth. Genotype testing by PFGE analysis showed three different patterns, correspondent to three SBT types according to the allelic profiles. Both PFGE and SBT indicated the circulation and the persistence in the hospital potable water-system of three types randomly distributed in space and time. The two molecular methods adopted showed a 100% concordance, although a low degree of genetic heterogeneity characterized the isolates. The electrophoretic patterns were sufficiently unambiguous to consider PFGE a highly discriminatory typing method, but the SBT technique besides accurately characterizing isolates, was able to identify Legionella strains through analysis of the mip gene. A typing method with this level of discriminatory power has great potential for assisting in epidemiological studies.

Use of three molecular methods for investigations of Legionnaires' disease in a hospital in Milan

Molecular typing, associated with epidemiological surveying, can be of particular importance in identifying sources of infection, demonstrating the link between clinical and environmental isolates.1 Various techniques have been developed in recent years for the molecular typing of micro-organisms; those most used for analysing strains of Legionella pneumophila include:2 pulsed-field gel electrophoresis (PFGE),3,4 amplified fragment length polymorphism (AFLP)1,5 and sequence-based typing (SBT).6 However, because of the low incidence of legionellosis, there are relatively few isolates from both clinical cases and associated environmental strains with which to work.