Application of flow cytometry to detection and characterization of Legionella spp

Flow cytometry, using fluorescein-bound specific antibodies and propidium iodide, was shown to be effective in detecting Legionella spp. in cooling tower waters. The procedure was quicker and less labor intensive than fluorescent microscopy. The use of these procedures also identified qualitative differences, perhaps related to infectivity, in Legionella populations.     

流式细胞术在细菌快速检测中的应用

流式细胞仪(Flow cytometer)是集应用流体学、光学、电子学、生物学、免疫学等多门学科和技术于一体的新型高科技仪器。它的核心技术是流式细胞术(Flow cytometry,FCM),该技术是利用流式细胞仪,使单个细胞或其他微小生物粒子处于快速直线流动状态,且逐个通过光束,从而对单个细胞或微粒进行多参数(数量、大小、核酸含量、细胞活性、特定菌群或物种等)定量分析和分选的检测技术,具有快速、灵敏、精确以及便于操作等突出优点。本文简要介绍流式细胞仪的原理,并论述流式细胞技术在实验室研究、工业生产、临床诊断、环境评估等领域的细菌快速检测应用。     

Isolation and characterization of Legionella spp. and Pseudomonas spp. from greenhouse misting systems

AIMS: Greenhouse misting systems used for watering plants produce fine aerosols. They are a possible cause for bacterial infections. This study investigates the colonization of greenhouse misting systems with Legionella spp. and Pseudomonas spp. and evaluates a possible health hazard. METHODS AND RESULTS: Between June and September 2003, a total of 80 water samples were collected in 20 different greenhouse systems in Germany, each tested on two different occasions. Each time, water was drawn at a central tap and at the outlet of spray nozzles. Sampled greenhouses were used to cultivate various plants and trees for commercial, recreational or scientific reasons, some of them in tropical conditions. Legionella spp. were detected in 10% of the systems (two systems), but only in low numbers. On the contrary, Pseudomonas spp. were recovered from 70% of the greenhouse watering systems (14 systems), occasionally at counts greater than 10,000 CFU per 100 ml. A random amplified polymorphic DNA polymerase chain reaction typing method was used to demonstrate that each colonized greenhouse had one or several individual strains of Legionella and Pseudomonas that could not be detected in any other system. CONCLUSIONS: This study demonstrates that aerosolizing greenhouse watering systems may be contaminated with Legionella or Pseudomonas which under certain circumstances could become a potential source of infection for workers and visitors. SIGNIFICANCE AND IMPACT OF THE STUDY: The study results indicate that greenhouse misting systems should be included in Legionella and Pseudomonas monitoring and control programs.     

A rapid detection method using flow cytometry to monitor the risk of Legionella in bath water

Legionella species are the causative agents of human legionellosis, and bathing facilities have been identified as the sources of infection in several outbreaks in Japan. Researchers in Japan have recently reported evidence of significant associations between bacterial counts and the occurrence of Legionella in bathing facilities and in a hot tub model. A convenient and quantitative bacterial enumeration method is therefore required as an indicator of Legionella contamination or disinfection to replace existing methods such as time-consuming Legionella culture and expensive Legionella-DNA amplification. In this study, we developed a rapid detection method (RDM) to monitor the risk of Legionella using an automated microbial analyzing device based on flow cytometry techniques to measure the total number of bacteria in water samples within two minutes, by detecting typical patterns of scattered light and fluorescence. We first compared the results of our RDM with plate counting results for five filtered hot spring water samples spiked with three species of bacteria, including Legionella. Inactivation of these samples by chlorine was also assessed by the RDM, a live/dead bacterial fluorescence assay and plate counting. Using the RDM, the lower limit of quantitative bacterial counts in the spiked samples was determined as 3.0 × 10³ (3.48 log) counts mL^− 1. We then used a laboratory model of a hot tub and found that the RDM could monitor the growth curve of naturally occurring heterotrophic bacteria with 1 and 2 days' delayed growth of amoeba and Legionella, respectively, and could also determine the killing curve of these bacteria by chlorination. Finally, samples with ≥ 3.48 or < 3.48 log total bacterial counts mL^− 1 were tested using the RDM from 149 different hot tubs, and were found to be significantly associated with the positive or negative detection of Legionella with 95% sensitivity and 84% specificity. These findings indicated that the RDM can be used for Legionella control at bathing facilities, especially those where the effectiveness of chlorine is reduced by the presence of Fe²⁺, Mn²⁺, NH₄⁺, skin debris, and/or biofilms in the water.     

Laser flow cytometry and cancer chemotherapy: detection of intracellular anthracyclines by flow cytometry.

The intracellular distribution of important chemotherapeutic antibiotics belonging to the anthracycline group (e.g. adriamycin) can be detected by laser flow cytometry. The indirect method is based on the interference of these compounds with the binding of propidium iodide to the nuclear DNA. While in the direct method, the intracellular fluorescence of these antibiotics is excited and detected with a laser beam in a flow system. The present report demonstrates the use of these two methods for intracellular detection and quantitation of a number of important anthracyclines.     

Overview of diagnostic and detection methods for legionellosis and Legionella spp.

Since 1977, the diagnostic tools for Legionnaires' disease have been based on culture and serological investigations. Both methods require considerable time to produce results and have 'low' to 'reasonable' sensitivity. Since the introduction of urinary antigen tests in the mid 1990s, underdiagnosis has diminished and mortality has declined, thanks to early diagnosis. To obtain the most accurate diagnosis, culture, serological investigation, and urinary antigen testing should all be performed. In the last decade, advances in polymerase chain reaction technology allowed the development of assays detecting Legionella nucleic acids in clinical and environmental samples. Thus far, only widely varying results with relatively small series have been reported. Furthermore, these assays which are still labour intensive and complicated are not yet practicable for the average medical and/or environmental microbiological laboratory.     

Application of alkaline elution, Fast Micromethod and flow cytometry in detection of marine contamination.

DNA damage is an inescapable aspect of life in the biosphere. The presented investigations were an attempt to examine the response of a DNA damage as a biomarker of environmental quality in the mussels Mytilus galloprovincialis sampled at differently contaminated areas of Istrian coast, Northern Adriatic. The investigations were performed in order to get information about the genotoxic risk for marine organisms exposed to mixed environmental pollution, as well as the information about the presence of unknown mixture of genotoxic contaminants in the marine environment. Types of DNA damage detected are alkali-labile sites and single-strand breaks measured by Fast Micromethod, interstrand cross-links and DNA protein cross-links by alkaline filter elution and cell cycle disturbation by flow cytometry. The applicability of all three methods for marine quality control is discussed.     

LAMP-based method for a rapid identification of Legionella spp. and Legionella pneumophila

Legionella pneumophila is accounted for more than 80% of Legionella infection. However it is difficult to discriminate between the L. pneumophila and non-L. pneumophila species rapidly. In order to detect the Legionella spp. and distinguish L. pneumophila from Legionella spp., a real-time loop-mediated isothermal amplification (LAMP) platform that targets a specific sequence of the 16S rRNA gene was developed. LS-LAMP amplifies the fragment of the 16S rRNA gene to detect all species of Legionella genus. A specific sequence appears at the 16S rRNA gene of L. pneumophila, while non-L. pneumophila strains have a variable sequence in this site, which can be recognized by the primer of LP-LAMP. In the present study, 61 reference strains were used for the method verification. We found that the specificity was 100% for both LS-LAMP and LP-LAMP, and the sensitivity of LAMP assay for L. pneumophila detection was between 52 and 5.2 copies per reaction. In the environmental water samples detection, a total of 107 water samples were identified by the method. The culture and serological test were used as reference methods. The specificity of LS-LAMP and LP-LAMP for the samples detection were 91.59% (98/107) and 93.33% (56/60), respectively. The sensitivity of LS-LAMP and LP-LAMP were 100% (51/51) and 100% (18/18). The results suggest that real-time LAMP, as a new assay, provides a specific and sensitive method for rapid detection and differentiation of Legionella spp. and L. pneumophila and should be utilized to test environmental water samples for increased rates of detection.     

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.     

Application of flow cytometry to algal physiology and phytoplankton ecology

Abstract Flow cytometry, originally developed for biomedical purposes, is now being put to extensive use in the study of microalgae. Successful applications to date include studies of cytochemistry, cell cycle dynamics and phytoplankton ecology. This review summarises recent work in a rapidly expanding field of research, and highlights those areas in which significant progress can be expected when the necessary equipment becomes more readily available.     

Presence and Persistence of Legionella spp. in Groundwater

Groundwater samples (111) from six different boreholes located in two geographical areas were examined for the presence of legionellae over a 7-year period. The number of Legionella isolates detected was generally low. The colonization of the aquifers was not uniform, and the persistence of Legionella was independent of the hydraulic pumps and the plumbing system present in the borehole. A total of 374 isolates identified by fatty acid methyl ester analysis belonged to Legionella pneumophila, L. oakridgensis, L. sainthelensi, and L. londiniensis. In area 1, L. oakridgensis constituted the major population detected, exhibiting only one random amplified polymorphic DNA (RAPD)-PCR profile. L. sainthelensi strains were less frequently isolated and also displayed a single RAPD profile, while L. pneumophila was only sporadically detected. In contrast, L. pneumophila comprised the vast majority of the isolates in area 2 and exhibited six distinct RAPD patterns, indicating the presence of different genetic groups; three L. londiniensis RAPD types were also detected. Two of the L. pneumophila and one of the L. londiniensis RAPD types were persistent in this environment for at least 12 years. The genetic structure of L. pneumophila groundwater populations, inferred from rpoB and dotA gene sequences, was peculiar, since the majority of the isolates were allied in a discrete group different from the lineages containing most of the type and reference strains of the three subspecies of L. pneumophila. Furthermore, gene exchange events related to the dotA allele could be envisioned.     

Presence and persistence of Legionella spp. in groundwater

Groundwater samples (111) from six different boreholes located in two geographical areas were examined for the presence of legionellae over a 7-year period. The number of Legionella isolates detected was generally low. The colonization of the aquifers was not uniform, and the persistence of Legionella was independent of the hydraulic pumps and the plumbing system present in the borehole. A total of 374 isolates identified by fatty acid methyl ester analysis belonged to Legionella pneumophila, L. oakridgensis, L. sainthelensi, and L. londiniensis. In area 1, L. oakridgensis constituted the major population detected, exhibiting only one random amplified polymorphic DNA (RAPD)-PCR profile. L. sainthelensi strains were less frequently isolated and also displayed a single RAPD profile, while L. pneumophila was only sporadically detected. In contrast, L. pneumophila comprised the vast majority of the isolates in area 2 and exhibited six distinct RAPD patterns, indicating the presence of different genetic groups; three L. londiniensis RAPD types were also detected. Two of the L. pneumophila and one of the L. londiniensis RAPD types were persistent in this environment for at least 12 years. The genetic structure of L. pneumophila groundwater populations, inferred from rpoB and dotA gene sequences, was peculiar, since the majority of the isolates were allied in a discrete group different from the lineages containing most of the type and reference strains of the three subspecies of L. pneumophila. Furthermore, gene exchange events related to the dotA allele could be envisioned.     

Sensitive detection of RNAs in single cells by flow cytometry.

A rapid and sensitive fluorescent in situ hybridization method has been developed to probe RNA contents of individual cells by flow cytometry. Fixed cells in suspension were hybridized with 5' end-fluorophore-labeled oligodeoxynucleotides complementary to defined regions of the RNA of interest and analyzed by flow cytometry. With this method, we monitored combinations of histone H4 mRNA, 18S rRNA and 28S rRNA levels in synchronized HeLa S3 cells by multicolor analysis. A fluorescence signal equivalent to 1800 copies of histone H4 mRNA per cell was detected with signal-to-background ratio of 5.4. If non-specific binding of the fluorophore-labeled probe can be reduced, as few as 100 copies of mRNA of the size of H4 could be detected in individual cells by flow cytometry.     

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.     

Application of flow cytometry in chronobiology

A survey is given on flow cytometric techniques and their applications in chronobiology. Rapid automatic single cell measurements with a high rate (1,000-5,000 single cells/sec) enable the accumulation of large numbers of data at short intervals, thus obtaining important knowledge on circadian variations of cell proliferation in epidermis and other types of surface epithelium as well as hemopoiesis. In addition, the method has to some extent been used for monitoring cancer chemotherapy, and is thus available for the application in chronotherapy of malignancy. The introduction of rapid automatic techniques such as flow cytometry is a great advantage for any study on single cells and tissues where rhythmic variations in different functions have to be taken into consideration.     

Legionella spp. in Puerto Rico cooling towers.

Water samples from air conditioning cooling towers receiving different treatment protocols on five large municipal buildings in San Juan, P.R., were assayed for various Legionella spp. and serogroups by using direct immunofluorescence. Several water quality parameters were also measured for each sample. Guinea pigs were inoculated with water samples to confirm pathogenicity and recover viable organisms. Legionella pneumophila serogroups 1 to 6, L. bozemanii, L. micdadei, L. dumoffii, and L. gormanii were observed in at least one of the cooling towers. L. pneumophila was the most abundant species; its density reached 10(5) cells per ml, which is within the range that is considered potentially pathogenic to humans. A significantly higher density of L. pneumophila was observed in the cooling tower water that was not being treated with biocides. Percent respiration (INT) and total cell activity (acridine orange direct count) were inversely correlated with bacterial density. This study demonstrates that Legionella spp. are present in tropical air-conditioning cooling systems and that, without continuous biocide treatment, they may reach densities that present a health risk.     

Application of flow cytometry to studies of pathogenic free-living amoebae

Species of small, free-living amoebae of the genera Naegleria and Acanthamoeba can cause fatal amoebic meningoencephalitis. Previous investigations have shown that pathogenic amoebae are associated with thermally altered water. Flow cytometric techniques for identifying species of pathogenic and nonpathogenic amoebae from such water have been developed, using immunofluorescence and fluorescein-bound concanavalin A. Flow cytometry is accomplished with a cytofluorograph, in which cells are dispersed in a suspended carrier liquid and passed in front of a focused argon ion laser beam. Cells are then distinguished by the degree of scattered light (size) or fluorescence. Flow cytometry techniques have proven efficient for environmental samples, as indicated by the identification of pathogenic Naegleria fowleri and nonpathogenic Naegleri gruberi and Acanthamoeba castellanii isolated from the Savannah River Plant in South Carolina. Cytofluorographic analysis of environmental samples has several advantages over the current methods of isolation and classification of free-living amoebae. With this system, it is possible to rapidly identify species and quantitate mixtures of pathogenic amoebae in environmental samples. Cytofluorographic analysis of amoebic isolates reduces the time presently required to screen environmental sites for pathogenic amoebae. The cytofluorograph permits detection and species identification of nonthermophilic Naegleria spp. and Acanthamoeba spp. that could not easily be isolated for species identification by conventional methods. Other advantages of flow cytometry over fluorescent microscopy include a high degree of statistical precision due to the large numbers measured, high immunofluorescent titers, and elimination of subjectivity and fluorescence fading.     

Legionella spp. in Puerto Rico cooling towers

Water samples from air conditioning cooling towers receiving different treatment protocols on five large municipal buildings in San Juan, P.R., were assayed for various Legionella spp. and serogroups by using direct immunofluorescence. Several water quality parameters were also measured for each sample. Guinea pigs were inoculated with water samples to confirm pathogenicity and recover viable organisms. Legionella pneumophila serogroups 1 to 6, L. bozemanii, L. micdadei, L. dumoffii, and L. gormanii were observed in at least one of the cooling towers. L. pneumophila was the most abundant species; its density reached 10(5) cells per ml, which is within the range that is considered potentially pathogenic to humans. A significantly higher density of L. pneumophila was observed in the cooling tower water that was not being treated with biocides. Percent respiration (INT) and total cell activity (acridine orange direct count) were inversely correlated with bacterial density. This study demonstrates that Legionella spp. are present in tropical air-conditioning cooling systems and that, without continuous biocide treatment, they may reach densities that present a health risk.