Evaluation of an immunomagnetic separation assay in combination with cultivation to improve Legionella pneumophila serogroup 1 recovery from environmental samples

Aims: Legionella isolation from environmental samples is often difficult because of the presence of heterotrophic‐associated bacteria that frequently overgrow when using standard culture (ISO 11731, 1998; NF T90‐431, 2003) methods. To improve Legionella pneumophila recovery from complex water samples (water from cooling towers, biofilms), we evaluated an immunomagnetic separation (IMS) assay using a monoclonal antibody raised against the lipopolysaccharide of Leg. pneumophila sg1 in combination with culture. Methods and Results: This study was conducted on 51 environmental specimens. The comparison between IMS‐culture and standard culture (ISO 11731, 1998; NF T90‐431, 2003) methods was made using ISO 17994, 2004 criteria for establishing equivalence between microbiological methods based on the upper and lower (X_H and X_L) values of the relative difference (95% confidence limit) and D as maximum acceptable deviation (value of the confidence limit). Conclusions: We found that the average performance of IMS culture was higher than the reference method. Significance and Impact of the Study: Thus, this IMS‐culture assay is particularly well adapted to the detection of Leg. pneumophila sg1 in environmental samples with high levels of interfering microflora.     

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. Methods and Results: All samples were processed by the ScanVIT test according to the manufacturer’s instructions and by a culture method (ISO 11731). ScanVIT detected significantly more positive samples, although concentrations were similar and a strong positive correlation between the two methods was observed (r = 0·888, P < 0·001). The new test was more accurate in identifying the co‐presence of Legionella pneumophila and Leg. non‐pneumophila. ScanVIT showed a slightly higher Legionella recovery from water samples artificially contaminated with Leg. pneumophila alone or together with Pseudomonas aeruginosa. Lastly, the inter‐laboratory comparison revealed that the ScanVIT test exhibits a lower variability than the traditional culture test (mean coefficient of variation 8·7 vs 16·1%). Conclusions: The results confirmed that the ScanVIT largely overlaps the reference method and offers advantages in terms of sensitivity, quantitative reliability and reduced assay time. Significance and Impact of the Study: The proposed method may represent a useful validated alternative to traditional culture for the rapid detection and quantification of Legionella spp. in water.     

Rapid detection of viable Legionella pneumophila in tap water by a qPCR and RT‐PCR‐based method

Aims

A molecular method for a rapid detection of viable Legionella pneumophila of all serogroups in tap water samples was developed as an alternative to the reference method (ISO). Legionellae are responsible for Legionnaires’ disease, a severe pneumonia in humans with high lethality.

Methods and Results

The developed method is based on a nutritional stimulation and detection of an increase in precursor 16S rRNA as an indicator for viability. For quantification, DNA was detected by qPCR. This method was compared to the ISO method using water samples obtained from public sports facilities in Switzerland. The sensitivity and specificity were 91 and 97%, respectively, when testing samples for compliance with a microbiological criterion of 1000 cell equivalents per l.

Conclusion

The new method is sensitive and specific for Leg. pneumophila and allows results to be obtained within 8 h upon arrival, compared to one week or more by the ISO method.

Significance and Impact of the Study

The method represents a useful tool for a rapid detection of viable Leg. pneumophila of all serogroups in water by molecular biology. It can be used as an alternative to the ISO method for official water analysis for legionellae and particularly when a short test time is required.     

Rapid detection of total and viable Legionella pneumophila in tap water by immunomagnetic separation,double fluorescent staining and flow cytometry

We developed a rapid detection method for Legionella pneumophila (Lp) by filtration, immunomagnetic separation, double fluorescent staining, and flow cytometry (IMS‐FCM method). The method requires 120 min and can discriminate ‘viable’ and ‘membrane‐damaged’ cells. The recovery is over 85% of spiked Lp SG 1 cells in 1 l of tap water and detection limits are around 50 and 15 cells per litre for total and viable Lp, respectively. The method was compared using water samples from house installations in a blind study with three environmental laboratories performing the ISO 11731 plating method. In 53% of the water samples from different taps and showers significantly higher concentrations of Lp were detected by flow cytometry. No correlation to the plate culture method was found. Since also ‘viable but not culturable’ (VNBC) cells are detected by our method, this result was expected. The IMS‐FCM method is limited by the specificity of the used antibodies; in the presented case they target Lp serogroups 1–12. This and the fact that no Lp‐containing amoebae are detected may explain why in 21% of all samples higher counts were observed using the plate culture method. Though the IMS‐FCM method is not yet fit to completely displace the established plating method (ISO 11731) for routine Lp monitoring, it has major advantages to plating and can quickly provide important insights into the ecology of this pathogen in water distribution systems.     

Development of a new CARD-FISH protocol for quantification of Legionella pneumophila and its application in two hospital cooling towers

Aims: Open cooling towers are frequent sources of infections with Legionella pneumophila. The gold standard for the detection of Leg. pneumophila is based on cultivation lasting up to 10 days and detecting only culturable cells. Alternative fluorescence in situ hybridization (FISH) protocols have been proposed, but they result in faint fluorescence signals and lack specificity because of cross‐hybridization with other Legionella species. Our aim was thus to develop a new FISH protocol for rapid and specific detection of Leg. pneumophila in water samples. Methods and Results: A novel catalysed reporter deposition FISH (CARD‐FISH) protocol for the detection of Leg. pneumophila was developed, which significantly enhanced signal intensity as well as specificity of the probe through the use of a novel competitor probe. The developed protocol was compared with the culture method for monitoring the seasonal development of culturable and nonculturable Leg. pneumophila in two hospital cooling tower systems. Seasonal fluctuations of Leg. pneumophila concentrations detected via CARD‐FISH were related to the development of the total bacterial community in both cooling towers, with temperature and biocide as the main factors controlling this development. Conclusions: Our results clearly showed that the majority of the Leg. pneumophila cells were in a nonculturable state. Thus, detection of Leg. pneumophila with culture methods may underestimate the total numbers of Leg. pneumophila present. Significance and Impact of the Study: Rapid, sensitive and specific detection and quantification of Leg. pneumophila in water systems is prerequisite for reliable risk estimation. The new protocol significantly improves current methodology and can be used to monitor and screen for Leg. pneumophila concentrations in cooling towers or other water systems.     

MRCQuant- an accurate LC-MS relative isotopic quantification algorithm on TOF instruments

Background  

Relative isotope abundance quantification, which can be used for peptide identification and differential peptide quantification, plays an important role in liquid chromatography-mass spectrometry (LC-MS)-based proteomics. However, several major issues exist in the relative isotopic quantification of peptides on time-of-flight (TOF) instruments: LC peak boundary detection, thermal noise suppression, interference removal and mass drift correction. We propose to use the Maximum Ratio Combining (MRC) method to extract MS signal templates for interference detection/removal and LC peak boundary detection. In our method, MRCQuant, MS templates are extracted directly from experimental values, and the mass drift in each LC-MS run is automatically captured and compensated. We compared the quantification accuracy of MRCQuant to that of another representative LC-MS quantification algorithm (msInspect) using datasets downloaded from a public data repository.     

PCR methods for the rapid detection and identification of four pathogenic Legionella spp. and two Legionella pneumophila subspecies based on the gene amplification of gyrB

A total of 25 gyrB gene sequences from 20 Legionella pneumophila subsp. pneumophila strains and five L. pneumophila subsp. fraseri strains were obtained and analyzed, and a multiplex PCR for the simultaneous detection of Legionella bozemanae, Legionella longbeachae, Legionella micdadei and Legioenella pneumophila, and two single PCRs for the differentiation of L. pneumophila subsp. pneumophila and L. pneumophila subsp. fraseri were established. The multiplex PCR method was shown to be highly specific and reproducible when tested against 41 target strains and 17 strains of other bacteria species. The sensitivity of the multiplex PCR was also analyzed and was shown to detect levels as low as 1 ng of genomic DNA or 10 colony-forming units (CFUs) per milliliter in mock water samples. Sixty-three air conditioner condensed water samples from Shanghai City were examined, and the result was validated using 16S rRNA sequencing. The data reported here demonstrate that the multiplex PCR method described is efficient and convenient for the detection of Legionella species in water samples. Twenty L. pneumophila subsp. pneumophila strains and five L. pneumophila subsp. fraseri strains were used for the validation of the two L. pneumophila subspecies-specific PCR methods, and the results indicated that the two PCR methods were both highly specific and convenient for the identification of L. pneumophila at the subspecies level.     

The natural alternative: protozoa as cellular models for Legionella infection

The severe pneumonia known as Legionnaires' disease occurs following infection by the Gram‐negative bacterium Legionella pneumophila. Normally resident in fresh‐water sources, Legionella are subject to predation by eukaryotic phagocytes such as amoeba and ciliates. To counter this, L. pneumophila has evolved a complex system of effector proteins which allow the bacteria to hijack the phagocytic vacuole, hiding and replicating within their erstwhile killers. These same mechanisms allow L. pneumophila to hijack another phagocyte, lung‐based macrophages, which thus avoids a vital part of the immune system and leads to infection. The course of infection can be divided into five main categories: pathogen uptake, formation of the replication‐permissive vacuole, intracellular replication, host cell response, and bacterial exit. L. pneumophila effector proteins target every stage of this process, interacting with secretory, endosomal, lysosomal, retrograde and autophagy pathways, as well as with mitochondria. Each of these steps can be studied in protozoa or mammalian cells, and the knowledge gained can be readily applied to human pathogenicity. Here we describe the manner whereby L. pneumophila infects host protozoa, the various techniques which are available to analyse these processes and the implications of this model for Legionella virulence and the pathogenesis of Legionnaires' disease.     

Icm/Dot‐dependent inhibition of phagocyte migration by Legionella is antagonized by a translocated Ran GTPase activator

The environmental bacterium Legionella pneumophila causes a severe pneumonia termed Legionnaires' disease. L. pneumophila employs a conserved mechanism to replicate within a specific vacuole in macrophages or protozoa such as the social soil amoeba Dictyostelium discoideum. Pathogen–host interactions depend on the Icm/Dot type IV secretion system (T4SS), which translocates approximately 300 different effector proteins into host cells. Here we analyse the effects of L. pneumophila on migration and chemotaxis of amoebae, macrophages or polymorphonuclear neutrophils (PMN). Using under‐agarose assays, L. pneumophila inhibited in a dose‐ and T4SS‐dependent manner the migration of D. discoideum towards folate as well as starvation‐induced aggregation of the social amoebae. Similarly, L. pneumophila impaired migration of murine RAW 264.7 macrophages towards the cytokines CCL5 and TNFα, or of primary human PMN towards the peptide fMLP respectively. L. pneumophila lacking the T4SS‐translocated activator of the small eukaryotic GTPase Ran, Lpg1976/LegG1, hyper‐inhibited the migration of D. discoideum, macrophages or PMN. The phenotype was reverted by plasmid‐encoded LegG1 to an extent observed for mutant bacteria lacking a functional Icm/Dot T4SS.Similarly, LegG1 promoted random migration of L. pneumophila‐infected macrophages and A549 epithelial cells in a Ran‐dependent manner, or upon ‘microbial microinjection’ into HeLa cells by a Yersinia strain lacking endogenous effectors. Single‐cell tracking and real‐time analysis of L. pneumophila‐infected phagocytes revealed that the velocity and directionality of the cells were decreased, and cell motility as well as microtubule dynamics was impaired. Taken together, these findings indicate that the L. pneumophila Ran activator LegG1 and consequent microtubule polymerization are implicated in Icm/Dot‐dependent inhibition of phagocyte migration.     

Necessity and Effect of Combating Legionella pneumophila in Municipal Shower Systems

The objective was to obtain research-based, holistic knowledge about necessity and effect of practiced measures against L. pneumophila in municipal shower systems in Stavanger, Norway. The effects of hot water treatment and membrane-filtering were investigated and compared to no intervention at all. The studies were done under real-world conditions. Additionally, a surveillance pilot study of municipal showers in Stavanger was performed. The validity of high total plate count (TPC) as an indication of L. pneumophila was evaluated. A simplified method, named “dripping method”, for detection and quantification of L. pneumophila was developed. The sensitivity of the dripping method is 5 colony-forming units of L. pneumophila/ml. The transference of L. pneumophila from shower water to aerosols was studied. Interviews and observational studies among the stakeholders were done in order to identify patterns of communication and behavior in a Legionella risk perspective. No substantial effects of the measures against L. pneumophila were demonstrated, except for a distally placed membrane filter. No significant positive correlation between TPC and L. pneumophila concentrations were found. L. pneumophila serogroup 2–14 was demonstrated in 21% of the 29 buildings tested in the surveillance pilot. Relatively few cells of L. pneumophila were transferred from shower water to aerosols. Anxiety appeared as the major driving force in the risk governance of Legionella. In conclusion, the risk of acquiring Legionnaires'' disease from municipal shower systems is evaluated as low and uncertain. By eliminating ineffective approaches, targeted Legionella risk governance can be practiced. Risk management by surveillance is evaluated as appropriate.     

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.     

Validation of IRS PCR,a molecular typing method,for the study of the diversity and population dynamics of Legionella in industrial cooling circuits

Legionella bacteria are ubiquitous in aquatic environments. Members of the species Legionella pneumophila are responsible for more than 98% of cases of Legionnaires' disease in France. Our objective was to validate a molecular typing method called infrequent restriction site PCR (IRS PCR), applied to the study of the ecology of Legionella and to compare this method with reference typing methods, pulsed‐field gel electrophoresis (PFGE) and sequence‐based Typing (SBT). PFGE and SBT are considered as gold methods for the epidemiological typing of Leg. pneumophila strains. However, these methods are not suitable to an ecological monitoring of Legionella in natural environments where a large number of strains has to be typed. Validation of IRS PCR method was performed by the identification of 45 Leg. pneumophila isolates from cooling circuits of thermal power plants by IRS PCR, PFGE and SBT. The parameters of each method were measured and compared to evaluate the effectiveness of IRS PCR. The results of this study showed that IRS PCR has a discriminating power similar or better than that of the reference methods and thus that, by its speed and low cost represents an appropriate tool for the study of the ecology of Legionella in cooling circuits.     

Induction of error-free DNA repair inEscherichia coli by heat shock or nutritional stress

Sterilization values were determined forLegionella pneumophila in chlorine-free, chlorine-demand-free water at elevated temperatures. These values were calculated from experimentally determined D values of 2500 min, 380 min, 13.93 min, 0.74 min, and 0.45 min at 45°C, 50°C, 55°C, 60°C, and 66°C, respectively. D values, Z value and temperature coefficient do not indicate unusual thermal resistance. Sterilization values, the minimum time required to eliminate an aquatic population ofL. pneumophila at a given test temperature, indicate that temperatures greater than about 65°C may not be necessary for efficient disinfection of potable quality water. These values and monitoring of time and temperature parameters can help predict the efficacy of in situ heat treatment of potable quality waters harboringL. pneumophila.     

Simultaneous determination of isoniazid and p‐aminosalicylic acid by capillary electrophoresis using chemiluminescence detection

It was found that isoniazid (ISO) or p‐aminosalicylic acid (PAS) could enhance the chemiluminescence (CL) emission from Cu (II)‐luminol‐hydrogen peroxide system, and the increased chemiluminescence signals were proportional to their concentrations, respectively. Based on this phenomenon, a chemiluminescence method coupled to capillary electrophoresis (CE) was established for simultaneous determination of ISO and PAS. The CE conditions including running buffer and running voltage were investigated in detail. The effects of the pH of H₂O₂ solution and the concentrations of luminol, H₂O₂ and Cu (II) on the CL signal were also investigated carefully. Under the optimized conditions, the analysis could be accomplished within 10 min, with the limits of detection of 0.3 µg mL^–1 for ISO and 1.1 µg mL^–1 for PAS, corresponding to 7.2 and 26.4 pg per injection (24 nL), respectively. Finally, the method was validated by determining the two analytes in pharmaceutical preparation and spiked human serum samples. The results of pharmaceutical tablet analysis were in good agreement with the labeled amounts. The recoveries for ISO and PAS in human serum were in the range of 92–104% and 90–113%, respectively. Copyright © 2009 John Wiley & Sons, Ltd.     

Evasion of phagotrophic predation by protist hosts and innate immunity of metazoan hosts by Legionella pneumophila

Legionella pneumophila is a ubiquitous environmental bacterium that has evolved to infect and proliferate within amoebae and other protists. It is thought that accidental inhalation of contaminated water particles by humans is what has enabled this pathogen to proliferate within alveolar macrophages and cause pneumonia. However, the highly evolved macrophages are equipped with more sophisticated innate defence mechanisms than are protists, such as the evolution of phagotrophic feeding into phagocytosis with more evolved innate defence processes. Not surprisingly, the majority of proteins involved in phagosome biogenesis (~80%) have origins in the phagotrophy stage of evolution. There are a plethora of highly evolved cellular and innate metazoan processes, not represented in protist biology, that are modulated by L. pneumophila, including TLR2 signalling, NF‐κB, apoptotic and inflammatory processes, histone modification, caspases, and the NLRC–Naip5 inflammasomes. Importantly, L. pneumophila infects haemocytes of the invertebrate Galleria mellonella, kill G. mellonella larvae, and proliferate in and kill Drosophila adult flies and Caenorhabditis elegans. Although coevolution with protist hosts has provided a substantial blueprint for L. pneumophila to infect macrophages, we discuss the further evolutionary aspects of coevolution of L. pneumophila and its adaptation to modulate various highly evolved innate metazoan processes prior to becoming a human pathogen.     

Usefulness of real-time PCR as a complementary tool to the monitoring of Legionella spp. and Legionella pneumophila by culture in industrial cooling systems

Aims: This study was designed to evaluate the usefulness of quantification by real‐time PCR as a management tool to monitor concentrations of Legionella spp. and Legionella pneumophila in industrial cooling systems and its ability to anticipate culture trends by the French standard method (AFNOR T90‐431). Methods and Results: Quantifications of Legionella bacteria were achieved by both methods on samples from nine cooling systems with different water qualities. Proportion of positive samples for L. pneumophila quantified by PCR was clearly lower in deionized or river waters submitted to a biocide treatment than in raw river waters, while positive samples for Legionella spp. were quantified for almost all the samples. For some samples containing PCR inhibitors, high quantification limits (up to 4·80 × 10⁵ GU l^?1) did not allow us to quantify L. pneumophila, when they were quantified by culture. Finally, the monitoring of concentrations of L. pneumophila by both methods showed similar trends for 57–100% of the samples. Conclusions: These results suggest that, if some methodological steps designed to reduce inhibitory problems and thus decrease the quantification limits, could be developed to quantify Legionella in complex waters, the real‐time PCR could be a valuable complementary tool to monitor the evolution of L. pneumophila concentrations. Significance and Impact of the Study: This study shows the possibility of using real‐time PCR to monitor L. pneumophila proliferations in cooling systems and the importance to adapt nucleic acid extraction and purification protocols to raw waters.     

Microalgae fiber optic biosensors for herbicide monitoring using sol–gel technology

Three microalgal species (Dictyosphaerium chlorelloides (D.c.), Scenedesmus intermedius (S.i.) and Scenedesmus sp. (S.s.)) were encapsulated in silicate sol–gel matrices and the increase in the amount of chlorophyll fluorescence signal was used to quantify simazine. Influence of several parameters on the preparation of the sensing layers has been evaluated: effect of pH on sol–gel gelation time; effect of algae density on sensor response; influence of glycerol (%) on the membrane stability. Long term stability was also tested and the fluorescence signal from biosensors remained stable for at least 3 weeks. D.c. biosensor presented the lowest detection limits for simazine (3.6 μg L⁻¹) and the broadest dynamic calibration range (19–860 μg L⁻¹) with IC₅₀ 125 ± 14 μg L⁻¹. Biosensor was validated by HPLC with UV/DAD detection. The biosensor showed response to those herbicides that inhibit the photosynthesis at photosystem II (triazines: simazine, atrazine, propazine, terbuthylazine; urea based herbicides: linuron). However, no significant increases of fluorescence response was obtained for similar concentrations of 2,4-D (hormonal herbicide) or Cu(II). The combined use of two biosensors that use two different genotypes, sensitive and resistant to simazine, jointly allowed improving microalgae biosensor specificity.     

Soluble NSF attachment protein receptor molecular mimicry by a Legionella pneumophila Dot/Icm effector

Upon infection, Legionella pneumophila uses the Dot/Icm type IV secretion system to translocate effector proteins from the Legionella‐containing vacuole (LCV) into the host cell cytoplasm. The effectors target a wide array of host cellular processes that aid LCV biogenesis, including the manipulation of membrane trafficking. In this study, we used a hidden Markov model screen to identify two novel, non‐eukaryotic s oluble N SF a ttachment protein re ceptor (SNARE) homologs: the bacterial Legionella SNARE effector A (LseA) and viral SNARE homolog A proteins. We characterized LseA as a Dot/Icm effector of L. pneumophila, which has close homology to the Qc‐SNARE subfamily. The lseA gene was present in multiple sequenced L. pneumophila strains including Corby and was well distributed among L. pneumophila clinical and environmental isolates. Employing a variety of biochemical, cell biological and microbiological techniques, we found that farnesylated LseA localized to membranes associated with the Golgi complex in mammalian cells and LseA interacted with a subset of Qa‐, Qb‐ and R‐SNAREs in host cells. Our results suggested that LseA acts as a SNARE protein and has the potential to regulate or mediate membrane fusion events in Golgi‐associated pathways.     

Legionella Confirmation Using Real-Time PCR and SYTO9 Is an Alternative to Current Methodology

The currently accepted culture techniques for the detection of Legionella spp. in water samples (AS/NZS 3896:1998 and ISO 11731 standard methods) are slow and laborious, requiring from 7 to 14 days for a result. We describe a fully validated rapid confirmation technique that uses real-time PCR incorporating the intercalating dye SYTO9 for the direct identification of primary cultures, significantly decreasing turnaround time and allowing faster remedial action to be taken by the industry.     

Length–weight relationships of three caught flatfish using shrimp trawler in Motaf fishing grounds,Bushehr province (Persian Gulf)

This study provides length–weight relationships (LWRs) for three flatfish species: Cynoglossus bilineatus (Lacepède, 1802), Solea elongata (Day, 1877), and Pseudorhombus elevatus (Ogilby, 1912) from Motaf fishing grounds (latitude 27°00′ to 28°03′N; longitude 51°41′ to 52°11′E), Bushehr province, Iran. A total of 103 fish specimens were collected using shrimp trawler with 40 mm (stretched mesh size) at depths of 10–30 m between August and September 2017. The LWRs for fish species were W = 0.0019L^3.353 for C. bilineatus: W = 0.0093L^3.174 for S. elongata, and W = 0.006L^3.181 for P. elevatus, respectively.