Biofilm Formation of Legionella pneumophila in Complex Medium under Static and Dynamic Flow Conditions
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This chapter analyzes biofilm formation of Legionella pneumophila in rich medium which supports extracellular proliferation of the bacteria. To investigate biofilm formation of Legionella under static conditions, a 1:1 mixture of L. pneumophila labeled with the fluorescent proteins EGFP or DsRed-Express was inoculated in a glass-bottom 35-mm petri dish. Biofilm formation was analyzed with an inverted confocal microscope (Axiovert 200M, 100 X oil objective Plan Neofluar; Zeiss). Biofilm formation of L. pneumophila in rich medium was also quantified by crystal violet staining in upright polystyrene microtiter plates or on polystyrene pins of "inverse" lids under static (no medium exchange) or quasi-static conditions (medium replaced twice a day). The L. pneumophila fliA mutant strain reproducibly accumulated 30% less biomass within 5 days, demonstrating that bacterial factors contribute to biofilm formation. It is noteworthy that mutants lacking rpoS or letA, both of which are required for the expression of transmissive (virulence) traits of L. pneumophila, were not affected in biofilm formation. A continuous-flow chamber system was set up to further test the hypothesis that plank-tonic cells are crucial for biofilm formation on surfaces and to noninvasively monitor in real-time the adherence and biofilm formation of L. pneumophila under dynamic flow conditions.Keywords:
Legionella
Crystal violet
Petri dish
Polystyrene
Legionella
Legionnaires' disease
Hot spring
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Legionella is an opportunistic waterborne pathogen which is transmitted to humans via inhalation of contaminated water droplets. In addition, Legionella pneumophila is considered ubiquitous bacterium which inherently resistant to chlorine in tap water. On the other side, recent researches revealed that SAEW is considered a novel sanitizer against a broad spectrum of bacteria and possesses nonselective antimicrobial properties. A total of 300 drinking water samples were collected from water tanks (100 samples from hotels, 100 samples from plants and 100 samples from residential units) at Alexandria and Cairo governments from January till July 2023. Results revealed that the incidence of Legionella species in drinking water samples from hotels, plants and residential units water tanks were 0 % (0/100) , 3 % (3/100) and 1% (1/100), respectively. Two isolates from plants water tanks were identified as Legionella pneumophila by RT PCR. The efficacy of slightly acidic electrolyzed water (SAEW) was assessed comparing to chlorine on inactivation and biofilm adherence strength of Legionella pneumophila isolates. The obtained results showed that SAEW was more effective on inactivation of the two Legionella pneumophila isolates than chlorine within shorter treatment duration, where 100 % inactivation of Legionella pneumophila achieved after 3 hrs and 48 hrs from SAEW and chlorine treatment, respectively. In addition, SAEW showed significant effect on the biofilm adherence strength of the two Legionella pneumophila isolates. This study underlines the highly antimicrobial effect of SAEW on inactivation of Legionella within short duration without any undesirable residual agents in water which may cause further water pollution.
Legionella
Tap water
Hand sanitizer
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ABSTRACT Garden soils were investigated as reservoirs and potential sources of pathogenic Legionella bacteria. Legionella bacteria were detected in 22 of 177 garden soil samples (12%) by amoebal coculture. Of these 22 Legionella -positive soil samples, seven contained Legionella pneumophila . Several other species were found, including the pathogenic Legionella longbeachae (4 gardens) and Legionella sainthelensi (9 gardens). The L. pneumophila isolates comprised 15 different sequence types (STs), and eight of these STs were previously isolated from patients according to the European Working Group for Legionella Infections (EWGLI) database. Six gardens that were found to be positive for L. pneumophila were resampled after several months, and in three gardens, L. pneumophila was again isolated. One of these gardens was resampled four times throughout the year and was found to be positive for L. pneumophila on all occasions. IMPORTANCE Tracking the source of infection for sporadic cases of Legionnaires' disease (LD) has proven to be hard. L. pneumophila ST47, the sequence type that is most frequently isolated from LD patients in the Netherlands, is rarely found in potential environmental sources. As L. pneumophila ST47 was previously isolated from a garden soil sample during an outbreak investigation, garden soils were investigated as reservoirs and potential sources of pathogenic Legionella bacteria. The detection of viable, clinically relevant Legionella strains indicates that garden soil is a potential source of Legionella bacteria, and future research should assess the public health implication of the presence of L. pneumophila in garden soil.
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Legionnaires' disease
Soil microbiology
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Legiolert® is a new culture method for quantification of Legionella pneumophila, which is the primary species associated with Legionnaires' disease. The test is based on a most probable number approach, and differs significantly from traditional culture methods by providing results at 7 days, rapid sample preparation and analysis, and objective interpretation of test results. In this study, we compared the performance of Legiolert with the U.S. Centers for Disease Control and Prevention (CDC) method for detection of L. pneumophila from non-potable samples, primarily comprising cooling tower waters. Our results demonstrated no significant difference between Legiolert and the CDC method for quantification of L. pneumophila. However, Legiolert showed a significant increase in sensitivity when water samples containing higher L. pneumophila concentrations were examined. Cooling tower waters often contain non-Legionella organisms (NLO) that interfere with traditional Legionella test methods, and we observed varying degrees of NLO interference on many CDC method plates. In contrast, Legiolert was resistant to NLO interference and produced a very low rate of false-positive results. Collectively, Legiolert is a sensitive and specific method for quantification of L. pneumophila from non-potable water that provides advantages over the CDC method.
Legionella
Potable water
Legionnaires' disease
Cooling tower
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Legionella
Legionnaires' disease
Cross-reactivity
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Legionella anisa is one of the most frequent species of Legionella other than Legionella pneumophila in the environment and may be hospital acquired in rare cases. We found that L. anisa may mask water contamination by L. pneumophila, suggesting that there is a risk of L. pneumophila infection in immunocompromised patients if water is found to be contaminated with Legionella species other than L. pneumophila.
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Legionnaires' disease
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ABSTRACT Duopath Legionella (Merck KGaA, Darmstadt, Germany) is a new immunochromatographic assay for the simultaneous identification of cultured L. pneumophila and Legionella species other than L. pneumophila . In tests of 89 L. pneumophila strains and 87 Legionella strains other than L. pneumophila representing 41 different species, Duopath and a widely used latex agglutination assay detected L. pneumophila with 100% and 98% accuracy, respectively, whereas the percentages differed significantly for other Legionella spp. (93% versus 37% [ P < 0.001]). Since many countries’ regulations require the identification of Legionella spp. in water and environmental samples, the use of Duopath Legionella to comply with those regulations could contribute to significantly fewer false-negative results.
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Legionnaires' disease
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Legionella pneumophila, a Gram-negative bacterium, is the causative agent of legionellosis. Traditionally, culture methods are normally used to detect Legionella species in different types of water (e.g. surface or tap water, circulating systems, air conditioners and their cooling devices). In this study the PCR conditions to detect Legionella were optimised based on the EnviroAmp Legionella kit (Perkin-Elmer) which is no longer commercially available. The PCR is very sensitive and specific in indicating the presence or absence (no quantification with classical PCR) of Legionella spp in general and more specifically L. pneumophila. To identify L. pneumophila. DNA sequences from the mip (macrophage infectivity potentiator) gene were amplified. The mip gene is conserved and specific for L. pneumophila although mip-like genes are also present in other Legionella spp. The PCR techniques were able to detect small amounts of Legionella in tap water samples. Cooling water, however, often contained PCR-inhibiting substances that could result in false negative PCR results for Legionella.
Legionella
Tap water
Infectivity
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ABSTRACT The dynamics of Legionella spp. and of dominant bacteria were investigated in water from a cooling tower plant over a 9-month period which included several weeks when Legionella pneumophila proliferated. The structural diversity of both the bacteria and the Legionella spp. was monitored by a fingerprint technique, single-strand conformation polymorphism, and Legionella spp. and L. pneumophila were quantified by real-time quantitative PCR. The structure of the bacterial community did not change over time, but it was perturbed periodically by chemical treatment or biofilm detachment. In contrast, the structure of the Legionella sp. population changed in different periods, its dynamics at times showing stability but also a rapid major shift during the proliferation of L. pneumophila in July. The dynamics of the Legionella spp. and of dominant bacteria were not correlated. In particular, no change in the bacterial community structure was observed during the proliferation of L. pneumophila. Legionella spp. present in the cooling tower system were identified by cloning and sequencing of 16S rRNA genes. A high diversity of Legionella spp. was observed before proliferation, including L. lytica, L. fallonii , and other Legionella -like amoebal pathogen types, along with as-yet-undescribed species. During the proliferation of L. pneumophila, Legionella sp. diversity decreased significantly, L. fallonii and L. pneumophila being the main species recovered.
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Legionella, a waterborne pathogen, is the main cause of Legionnaires' disease. Therefore, timely and accurate detection and differentiation of Legionella pneumophila and non-Legionella pneumophila species is crucial. In this study, we develop an easy and rapid recombinase polymerase amplification assay combined with EuNPs-based lateral flow immunochromatography (EuNPs-LFIC-RPA) to specifically distinguish Legionella pneumophila and non-Legionella pneumophila. We designed primers based on the mip gene of Legionella pneumophila and the 5S rRNA gene of non-Legionella pneumophila. The recombinase polymerase amplification reaction could go to completion in 10 min at 37°C, and the amplification products could be detected within 5 min with EuNPs-LFIC strips. Using a florescent test strip reader, the quantitative results were achieved by reading the colored signal intensities on the strips. The sensitivity was 1.6 × 101 CFU/ml, and a linear standard linear curve plotted from the test strip reader had a correlation coefficient for the determination of Legionella pneumophila (R2 = 0.9516). Completed concordance for the presence or absence of Legionella pneumophila by EuNPs-LFIC-RPA and qPCR was 97.32% (κ = 0.79, 95% CI), according to an analysis of practical water samples (n = 112). In short, this work shows the feasibility of EuNPs-LFIC-RPA for efficient and rapid monitoring of Legionella pneumophila and non-Legionella pneumophila in water samples.
Legionella
Recombinase Polymerase Amplification
Legionnaires' disease
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