The role of biofilms and protozoa in Legionella pathogenesis: implications for drinking water

Current models to study Legionella pathogenesis include the use of primary macrophages and monocyte cell lines, various free-living protozoan species and murine models of pneumonia. However, there are very few studies of Legionella spp. pathogenesis aimed at associating the role of biofilm colonization and parasitization of biofilm microbiota and release of virulent bacterial cell/vacuoles in drinking water distribution systems. Moreover, the implications of these environmental niches for drinking water exposure to pathogenic legionellae are poorly understood. This review summarizes the known mechanisms of Legionella spp. proliferation within Acanthamoeba and mammalian cells and advocates the use of the amoeba model to study Legionella pathogenicity because of their close association with Legionella spp. in the aquatic environment. The putative role of biofilms and amoebae in the proliferation, development and dissemination of potentially pathogenic Legionella spp. is also discussed. Elucidating the mechanisms of Legionella pathogenicity development in our drinking water systems will aid in elimination strategies and procedural designs for drinking water systems and in controlling exposure to Legionella spp. and similar pathogens.     

Introduction of a boost of Legionella pneumophila into a stagnant-water model by heat treatment

An environmentally representative stagnant-water model was developed to monitor the growth dynamics of Legionella pneumophila. This model was evaluated for three distinct water treatments: untreated tap water, heat-treated tap water, and heat-treated tap water supplemented with Pseudomonas putida, a known biofilm-forming bacterium. Bringing heat-treated tap water after subsequent cooling into contact with a densely formed untreated biofilm was found to promote the number of L. pneumophila by 4 log units within the biofilm, while the use of untreated water only sustained the L. pneumophila levels. Subsequent colonization of the water phase by L. pneumophila was noticed in the heat-treated stagnant-water models, with concentrations as high as 1 x 10(10) mip gene copies L(-1) stagnant water. Denaturing gradient gel electrophoresis in combination with clustering analysis of the prokaryotic community in the water phase and in the biofilm phase suggests that the different water treatments induced different communities. Moreover, boosts of L. pneumophila arising from heat treatment of water were accompanied by shifts to a more diverse eukaryotic community. Stimulated growth of L. pneumophila after heating of the water may explain the rapid recolonization of L. pneumophila in water systems. These results highlight the need for additional or alternative measures to heat treatment of water in order to prevent or abate potential outbreaks of L. pneumophila.     

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

Aims: 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. Methods and Results: 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. Conclusions: Identifying the presence of pathogenic legionellae may require the use of multiple host amoebae and incubation temperatures. Significance and Impact of the Study: 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.     

生物膜型污水脱氮系统中膜结构及微生物生态研究进展

生物膜法污水脱氮系统主要利用生物膜中脱氮功能微生物的代谢活动去除氮素,从而达到净化水质的目的,研究脱氮生物膜的微观结构和微生物生态是揭示生物膜脱氮机理从而提高脱氮效率的重要途径.本文综述了生物膜型污水脱氮系统类型、生物膜微观结构特征及其影响因素、生物膜型污水脱氮系统内氮素传质过程、脱氮机理和生物膜数学模型等方面的研究进展.另外,本文介绍了生物膜型污水脱氮系统内生物膜脱氮功能微生物分布特征,不同生物膜脱氮系统、底物、运行条件和时间对功能微生物群落影响,及新型脱氮功能微生物等方面的研究进展,为生物膜脱氮技术的深入研究提供参考.     

On the reproducibility of microcosm experiments - different community composition in parallel phototrophic biofilm microcosms

Phototrophic biofilms were cultivated simultaneously using the same inoculum in three identical flow-lane microcosms located in different laboratories. The growth rates of the biofilms were similar in the different microcosms, but denaturing gradient gel electrophoresis (DGGE) analysis of both 16S and 18S rRNA gene fragments showed that the communities developed differently in terms of species richness and community composition. One microcosm was dominated by Microcoleus and Phormidium species, the second microcosm was dominated by Synechocystis and Phormidium species, and the third microcosm was dominated by Microcoleus- and Planktothrix- affiliated species. No clear effect of light intensity on the cyanobacterial community composition was observed. In addition, DGGE profiles obtained from the cultivated biofilms showed a low resemblance with the profiles derived from the inoculum. These findings demonstrate that validation of reproducibility is essential for the use of microcosm systems in microbial ecology studies.     

The surfactant of Legionella pneumophila Is secreted in a TolC-dependent manner and is antagonistic toward other Legionella species

When Legionella pneumophila grows on agar plates, it secretes a surfactant that promotes flagellum- and pilus-independent "sliding" motility. We isolated three mutants that were defective for surfactant. The first two had mutations in genes predicted to encode cytoplasmic enzymes involved in lipid metabolism. These genes mapped to two adjacent operons that we designated bbcABCDEF and bbcGHIJK. Backcrossing and complementation confirmed the importance of the bbc genes and suggested that the Legionella surfactant is lipid containing. The third mutant had an insertion in tolC. TolC is the outer membrane part of various trimolecular complexes involved in multidrug efflux and type I protein secretion. Complementation of the tolC mutant restored sliding motility. Mutants defective for an inner membrane partner of TolC also lacked a surfactant, confirming that TolC promotes surfactant secretion. L. pneumophila (lspF) mutants lacking type II protein secretion (T2S) are also impaired for a surfactant. When the tolC and lspF mutants were grown next to each other, the lsp mutant secreted surfactant, suggesting that TolC and T2S conjoin to mediate surfactant secretion, with one being the conduit for surfactant export and the other the exporter of a molecule that is required for induction or maturation of surfactant synthesis/secretion. Although the surfactant was not required for the extracellular growth, intracellular infection, and intrapulmonary survival of L. pneumophila, it exhibited antimicrobial activity toward seven other species of Legionella but not toward various non-Legionella species. These data suggest that the surfactant provides L. pneumophila with a selective advantage over other legionellae in the natural environment.     

The microbial loop in a humic lake: seasonal and vertical variations in the structure of the different communities

Seasonal and vertical variations of the main microbial communities (heterotrophic bacteria, autotrophic picoplankton, auto- and heterotrophic nanoflagellates, ciliated protozoa and microalgae) and auto- and heterotrophic activities were estimated in a brown-colored humic and moderately acid lake in central France, the lake of Vassivière. The results demonstrated the dominant role of light in the vertical distribution of autotrophic and mixotrophic microorganisms which are confined to the 0–5 m layer during thermal stratification. The bacterial biomass was high throughout the water column probably because of the great availability of dissolved organic matter. Consequently, the predatory microzooplankton and particularly the various trophic groups of ciliated protozoa, were distributed in the water column according to the vertical distribution of the particular food resources (detritus, bacteria, algae). However, despite the great abundance of algae and bacteria, biomass of flagellated and ciliated protozoa was relatively weak. Most of the phytoplanktonic biomass was filamentous (Diatoms) or colonial (Cyanobacteria) and therefore almost probably difficult to ingest for algivorous microzooplankton. Regarding the low abundance of bacterivorous protozoa, the relation with the special physicochemical properties of this lake is discussed.

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Résumé Les variabilités saisonnière et verticale de l'abondance et de la biomasse des principles communautés de la boucle microbienne (bactéries hétérotrophes, picoplancton autotrophe, protozoaires flagellés auto- et hétérotrophes, protozoaires ciliés, microalgues et microcyanobactéries), et des activités auto- et hétérotrophes, ont été étudiées dans un lac à caractère humique et modérément acide du Massif Central Français, le lac de Vassivière.Le dénombrement des communautés de microorganismes a été réalisé en microscopie inversée et à épifluorescence après mise en oeuvre des fixations et des colorations adéquates. Les activités photosynthétique, photo- et chemohétérotrophes ont été mesurées à partir de l'assimilation de NaH¹⁴CO₃ et d'un mélange d'acides aminés tritiés grâce à une technique de double marquage. Les résultats obtenus mettent en évidence le rôle prépondérant du facteur lumineux dans la répartition verticale des microorganismes autotrophes et mixotrophes, dont l'essentiel de la biomasse est confiné dans la zone 0–5 m en période de stratification, alors que la biomasse bactérienne est élevée sur l'ensemble de la colonne d'eau en raison, sans doute, de la grande disponibilité en matiére organique dissoute. Consécutivement, le microzooplancton prédateur, et notamment les différents types trophiques de protozoaires ciliés, se répartit dans la colonne d'eau selon la distribution verticale des ressources nutritives particularies. Cependant, compte-tenu de l'abondance bactérienne et algae, la biomasse des protozoaires flagellés et ciliésest relativement peu importante. Concernant les espèces algivores, l'essentiel de la biomasse phytoplanctonique est de nature filamenteuse (Diatomées) ou coloniale (Cyanobactéries) est est donc sans doute difficilement ingérable pour le microzooplancton. Enfin, les relations entre les caractéristiques physico-chimiques spécifiques de ce lac et le faible développement des protozoaires bactérivores sont discutées.

     

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.     

Abundance of Naked Amoebae in Sediments of Hiroshima Bay, Seto Inland Sea of Japan

ABSTRACT The present paper provides the first data on naked amoebae from sediments of Hiroshima Bay. Three stations in the inner part of the bay were sampled over a three-month period. Abundance of naked amoebae ranged from 1,019 to 45,561 cells/g dry sediment. Results indicate: (i) surface sediment populations in most cases were higher than subsurface populations; (ii) there was some evidence of temporal variation with counts generally increasing from March to May: and (iii) the site located near Hiroshima City had fewer amoebae on several occasions than the other two sites. There was a negative exponential relationship between acid-volatile sulfide concentration and abundance of amoebae. Most amoebae were small with the average size ranging from 6.6–14 μm. Morphotype 1, amoebae that extend lobose pseudopodia or subpseudopodia during normal locomotion, were dominant (40–100% of enumerated amoebae). Morphotypes 2 and 3 (limax amoebae) were found in lower numbers than the other two morphotypes. The proportion of amoebae occupied by Morphotype 4 (fan-shaped or discoidal-flattened amoebae) was higher at a lower total abundance.     

The role of dissolved organic matter bioavailability in promoting phytoplankton blooms in Florida Bay

The clear, shallow, oligotrophic waters of Florida Bay are characterized by low phytoplankton biomass, yet periodic cyanobacteria and diatom blooms do occur. We hypothesized that allochthonous dissolved organic matter (DOM) was providing a subsidy to the system in the form of bound nutrients. Water from four bay sites was incubated under natural light and dark conditions with enrichments of either DOM ( > 1 kD, 2×DOM) or inorganic nutrients (N+P). Samples were analyzed for bacterial numbers, bacterial production, phytoplankton biomass, phytoplankton community structure, and production, nutrients, and alkaline phosphatase (AP) activity. The influence of 2×DOM enrichment on phytoplankton biomass developed slowly during the incubations and was relatively small compared to nutrient additions. Inorganic nutrient additions resulted in an ephemeral bloom characterized initially as cyanobacterial and brown algae but which changed to dinoflagellate and/or brown algae by day six. The DIN:TP ratio decreased 10-fold in the N+P treatments as the system progressed towards N limitation. This ratio did not change significantly for 2×DOM treatments. In addition, these experiments indicated that both autotrophic and heterotrophic microbial populations in Florida Bay may fluctuate in their limitation by organic and inorganic nutrient availability. Both N+P and 2×DOM enrichments revealed significant and positive response in bioavailability of dissolved organic carbon (BDOC). Potential BDOC ranged from 1.1 to 35.5%, with the most labile forms occurring in Whipray Basin. BDOC at all sites was stimulated by the 2×DOM addition. Except for Duck Key, BDOC at all sites was also stimulated by the addition of N+P. BDOC was lower in the dry season than in the wet season (5.56% vs. 16.86%). This may be explained by the distinct chemical characteristics of the DOM produced at different times of year. Thus, both the heterotrophic and autotrophic microbial communities in Florida Bay are modulated by bioavailability of DOM. This has ramifications for the fate of DOM from the Everglades inputs, implicating DOM bioavailability as a contributing factor in regulating the onset, persistence, and composition of phytoplankton blooms.     

Effect of flow regimes on the presence of Legionella within the biofilm of a model plumbing system

AIMS: Stagnation is widely believed to predispose water systems to colonization by Legionella. A model plumbing system was constructed to determine the effect of flow regimes on the presence of Legionella within microbial biofilms. METHODS AND RESULTS: The plumbing model contained three parallel pipes where turbulent, laminar and stagnant flow regimes were established. Four sets of experiments were carried out with Reynolds number from 10,000 to 40,000 and from 355 to 2,000 in turbulent and laminar pipes, respectively. Legionella counts recovered from biofilm and planktonic water samples of the three sampling pipes were compared with to determine the effect of flow regime on the presence of Legionella. Significantly higher colony counts of Legionella were recovered from the biofilm of the pipe with turbulent flow compared with the pipe with laminar flow. The lowest counts were in the pipe with stagnant flow. CONCLUSIONS: We were unable to demonstrate that stagnant conditions promoted Legionella colonization. SIGNIFICANCE AND IMPACT OF THE STUDY: Plumbing modifications to remove areas of stagnation including deadlegs are widely recommended, but these modifications are tedious and expensive to perform. Controlled studies in large buildings are needed to validate this unproved hypothesis.     

Perspectives and predictions on the microbial ecology of the hyporheic zone

1. Studies of hyporheic microbial ecology have suggested an important role for hyporheic microbial processes in stream ecosystem functioning. Using evidence from microbial communities in other aquatic habitats, some predictions are made concerning the diversity of microbial types and microbial processes likely to occur in the hyporheic zone, and the relative importance of these various types to the hyporheic ecosystem. 2. It is predicted that the biofilm growth form of interstitial micro-organisms will create a variety of microniches, allowing coexistence of a great diversity of microbial types, and promoting the activity of some otherwise poor competitors. It is further predicted that the confluence of reduced groundwaters and aerobic surface waters will favour chemolithotrophic processes in the hyporheic zone, but that these will contribute significantly to hyporheic production only if surface water is very low in dissolved organic carbon, or the groundwater is extremely reduced, such as by the influence of riparian wetlands. A variety of anaerobic respiratory pathways, such as nitrate, ferric ion, sulphate and even methanogenic respiration will be employed in the hyporheic zone, with biofilm dynamics permitting these to occur even in aerobic sediments. Anaerobic pathways may account for a significant proportion of total hyporheic organic matter mineralization. 3. The role of fungi in hyporheic dynamics is, as yet, almost completely unstudied. However, it is expected that they will be important in breaking down buried particulate organic matter (POM), which may account for a large proportion of total stream POM. 4. Physicochemical conditions in hyporheic sediments appear to be highly heterogeneous, and this heterogeneity may be very important in the cycling of certain nutrients, especially nitrogen, which involves a series of steps requiring different conditions. 5. Various new techniques are now available by which biofilm dynamics and in situ microbial processes may be measured. Studies are recommended of intact microbial communities both at the microscale of the biofilm and at the scale of the heterogeneities occurring in hyporheic sediments. Studies are needed that measure actual rates of microbial processes under in situ conditions.     

Analysis of the intestinal microflora: a renaissance

The ability of microbial ecologists to analyse the composition of complex bacterial communities has been greatly enhanced by the application of molecular methodologies. The use of these techniques should enable an accurate record of the identity and population dynamics of the inhabitants of the intestinal tract to be obtained, and should promote an improved comprehension of the relationship between the microflora and the human host. This, in turn, will lead to a new concept of the intestinal microflora of humans.     

一个细菌的致命之旅——嗜肺军团菌分泌系统及效应蛋白的研究进展

嗜肺军团菌是引起军团菌肺炎以及庞蒂亚克热的革兰氏阴性胞内病原细菌,嗜肺军团菌侵染宿主的主要特点是可以通过其IVB型毒力分泌系统,向宿主细胞内分泌超过150种的底物效应蛋白。通过这些效应蛋白的作用,嗜肺军团菌能够调整宿主细胞的胞内运输途径,改变内外环境来伪装自己的吞噬泡,干扰宿主的细胞周期,抑制宿主细胞的凋亡,从而有效逃避宿主细胞的防御功能,创造出理想的胞内增殖环境。最后,效应蛋白还可以帮助军团菌从宿主细胞中逃逸。目前,嗜肺军团菌已经成为"病原菌-宿主相互作用"的重要研究模型,其毒力分泌系统及其底物效应蛋白的功能也成为细胞微生物学的研究热点。对嗜肺军团菌分泌系统及效应蛋白的研究不仅能够帮助阐明病原细菌的致病机理,还有助于推动对宿主免疫机制的更深层次的研究。文章主要针对嗜肺军团菌的毒力分泌系统,尤其是IVB型分泌系统的结构和功能,以及底物效应蛋白的研究进展进行了综述,向读者展示出一个小小的细菌所拥有的那令人惊叹的、如此狡猾的生存策略和它精致的杀伤武器。     

The Effects of Sample Storage Conditions on the Microbial Community Composition in Hydraulic Fracturing Produced Water

The petroleum industry has an increasing interest in understanding the microbial communities driving biofouling and biocorrosion in reservoirs, wells, and infrastructure. However, sampling of the relevant produced fluids from subsurface environments for microbiological analyses is often challenged by high liquid pressures, workplace regulations, operator liability concerns, and remote sampling locations. These challenges result in infrequent sampling opportunities and the need to store and preserve the collected samples for several days or weeks. Maintaining a representative microbial community structure from produced fluid samples throughout storage and handling is essential for accurate results of downstream microbial analyses. Currently, no sample handling or storage recommendations exist for microbiological analyses of produced fluid samples. We used 16S rRNA gene sequencing to monitor the changes in microbial communities in hypersaline produced water stored at room temperature or at 4?°C for up to 7 days. We also analyzed storage at ?80?°C across a 3-week period. The results suggest ideal handling methods would include placing the collected sample on ice as soon as possible, but at least within 24?h, followed by shipping the samples on ice over 2–3?days, and finally, long-term storage in the ?20?°C or ?80?°C freezer.     

Identification and modelling of a PPM protein phosphatase fold in the Legionella pneumophila deAMPylase SidD

The intracellular parasitic bacterium Legionella pneumophila subverts host vesicle transport through reversible AMPylation of Rab1. The effector enzyme for deAMPylation is SidD. Here a complete PPM protein phosphatase fold catalytic domain in SidD is identified and modelled. The SidD model reveals insertions and deletions near the metal ion containing catalytic site which presumably determine its novel activity. It also sheds light on possible substrate binding residues and highlights the lack of an obvious group to act as general acid during reaction. Assignment of a PPM fold to SidD offers an important pointer towards identification of further deAMPylases.     

Acylated homoserine lactones in the environment: chameleons of bioactivity

Over the last 15 years, it has become increasingly apparent that a single class of compounds, the acylated homoserine lactones (AHLs), elicit effects on many levels of biological and ecological organization. Despite the fact that the distribution of AHL production in the prokaryotic phylogenetic tree is restricted to a small set of genera, representatives of these genera are abundant in the environment and are responsible for processes of much interest to humans. As well as driving interactions between clones, AHLs have been shown to mediate interactions between different species of bacteria and between bacteria and higher organisms, either through the phenotypes they regulate or directly through their own chemical behaviour. Understanding the biological activity of AHLs and the ecological consequences of these activities may provide us with an opportunity to manipulate the composition and function of complex biological assemblages. Ultimately, this broadens the biotechnological focus of AHL-based research beyond the attenuation of virulence in humans and plant pathogens.     

Ciliate communities of rotating biological contactor biofilms: a multivariate approach

Physical-chemical monitoring and characterization of ciliate communities from a full-scale rotating biological system (RBC) have been carried out for a year. RBC system operated efficiently in removing the organic matter, as the decrease of the BOD5 loading along the successive RBC units revealed. 55 species of ciliated protozoa were identified in the RBC biofilms. Differences in abundance, occurrence and type of species were found along the different units of the RBC system; the last RBC held a more stable and diverse ciliate community. The complexity of interspecific relationships among the ciliates has been outlined using multivariate methods (Cluster and Correspondence analysis). Correlation between ciliate species and physical-chemical conditions were obtained by regression analysis. Results show that 12 species of ciliates were related to an optimal efficiency in organic matter removal, Litonotus crystallinus being the most sensitive species. The presence and abundance of Litonotus crystallinus and Acineria uncinata was associated with a decrease in the organic factor. Metopus es was the only species related to a decrease in process efficiency due to its association with an increase in organic factor. The results provide statistical evidence of the use of certain ciliate species as reliable bioindicators in full-scale RBC wastewater treatment plants.