Correlation ofLegionella pneumophila virulence with the presence of a plasmid

Legionella pneumophila is the causative agent of Legionellosis in man and considered an opportunistic intracellular Gram-negative bacterium that preferentially infects macrophages. The presence of a plasmid in these organisms was determined in cultures of the bacteria grown in vitro. A correlation was observed between the growth of virulent strains of theLegionella in murine macrophages and growth on standard buffered charcoal yeast extract agar supplemented with 0.1% -ketoglutarate (BCYE) agar medium rich in cysteine and widely used for growth of the bacteria in vitro. In contrast, the avirulent isolates of these strains grew well on supplemented Mueller-Hinton (SMH) agar utilized for differentiating virulent from avirulentLegionella. However, one virulent strain ofLegionella (the Iowa strain) was found to grow moderately well on the SMH agar. In addition, test strains ofLegionella that infect in vitro human monocytes were found to grow moderately well on the BCYE- agar, but did not grow on the SMH agar. Examination of these strains for plasmid DNA expression showed that extra chromosomal DNA-containing molecules were present in theL. pneumophila strains characterized as virulent for in vitro growth in macrophages. However, the avirulent strains that replicated in the human monocytes readily but only poorly in the permissive murine macrophages did not show evidence of similar plasmid DNA expression.     

Starvation/stationary-phase survival of <Emphasis Type="Italic">Rhodococcus erythropolis</Emphasis> SQ1: a physiological and genetic analysis

The adaptation of Rhodocccus erythropolis SQ1 to energy and carbon starvation was investigated in terms of both the capacity to survive starvation and the contribution of a nutrient-induced stationary phase to cross-protection to other types of environmental stress. It was found that R. erythropolis SQ1 survives for at least 43 days in LB and distilled water, and 65 days in chemically defined medium (CDM) containing high (1%) or low (0.1%) glucose. Furthermore, early stationary-phase R. erythropolis SQ1 grown in CDM 0.1% exhibited enhanced resistance to heat and oxidative stress compared with exponential-phase cells. A second objective of this study was to identify genetic elements involved in starvation/stationary-phase survival. A mutant bank of R. erythropolis SQ1 generated by random transposon insertion mutagenesis was screened; four mutants lost culturability when grown in CDM 1%. No drop in culturability was observed when these mutants were grown in CDM 0.1%. The DNA flanking transposon insertion could be recovered from three mutants. Transposon insertions were found in uvrB (UvrB, part of the DNA excision repair mechanism), between a putative guaB gene and another guaB-like gene, and between a gene encoding a putative phosphoglycerate mutase and putative thioredoxin/cytochrome c biogenesis genes. This represents a first study of the starvation/stationary-phase survival response of Rhodococcus, an organism of immense significance in environmental bioremediation and a number of industrial processes.     

Inhibition of growth ofLegionella species by heterotrophic plate count bacteria isolated from chlorinated drinking water

The ability of heterotrophic plate count bacterial strains isolated from chlorinated drinking water on low-nutrient media to inhibit the growth ofLegionella species was examined. Between 16% and 32% of these strains were able to inhibit the growth ofLegionella species when tested on buffered charcoal yeast extract agar. The exact proportion of inhibiting strains varied with the individualLegionella species. Two strains that inhibited the growth of severalLegionella species could also stimulate the growth of the same species when both the test strain and theLegionella species were grown on buffered charcoal yeast extract agar that lacked the essential amino acidl-cysteine.     

Comparative analyses of Legionella species identifies genetic features of strains causing Legionnaires’ disease

Background

The genus Legionella comprises over 60 species. However, L. pneumophila and L. longbeachae alone cause over 95% of Legionnaires’ disease. To identify the genetic bases underlying the different capacities to cause disease we sequenced and compared the genomes of L. micdadei, L. hackeliae and L. fallonii (LLAP10), which are all rarely isolated from humans.

Results

We show that these Legionella species possess different virulence capacities in amoeba and macrophages, correlating with their occurrence in humans. Our comparative analysis of 11 Legionella genomes belonging to five species reveals highly heterogeneous genome content with over 60% representing species-specific genes; these comprise a complete prophage in L. micdadei, the first ever identified in a Legionella genome. Mobile elements are abundant in Legionella genomes; many encode type IV secretion systems for conjugative transfer, pointing to their importance for adaptation of the genus. The Dot/Icm secretion system is conserved, although the core set of substrates is small, as only 24 out of over 300 described Dot/Icm effector genes are present in all Legionella species. We also identified new eukaryotic motifs including thaumatin, synaptobrevin or clathrin/coatomer adaptine like domains.

Conclusions

Legionella genomes are highly dynamic due to a large mobilome mainly comprising type IV secretion systems, while a minority of core substrates is shared among the diverse species. Eukaryotic like proteins and motifs remain a hallmark of the genus Legionella. Key factors such as proteins involved in oxygen binding, iron storage, host membrane transport and certain Dot/Icm substrates are specific features of disease-related strains.

Electronic supplementary material

The online version of this article (doi:10.1186/s13059-014-0505-0) contains supplementary material, which is available to authorized users.     

Diverse Legionella‐Like Bacteria Associated with Testate Amoebae of the Genus Arcella (Arcellinida: Amoebozoa)

Diverse species of Legionella and Legionella‐like amoebal pathogens (LLAPs) have been identified as intracellular bacteria in many amoeboid protists. There are, however, other amoeboid groups such as testate amoeba for which we know little about their potential to host such bacteria. In this study, we assessed the occurrence and diversity of Legionella spp. in cultures and environmental isolates of freshwater arcellinid testate amoebae species, Arcella hemispherica, Arcella intermedia, and Arcella vulgaris, via 16S rRNA gene sequence analyses and fluorescent in situ hybridization (FISH). Analysis of the 16S rRNA gene sequences indicated that A. hemispherica, A. intermedia, and A. vulgaris host Legionella‐like bacteria with 94–98% identity to other Legionella spp. based on NCBI BLAST search. Phylogenetic analysis placed Legionella‐like Arcella‐associated bacteria (LLAB) in three different clusters within a tree containing all other members of Legionella and LLAPs. The intracellular localization of the Legionella within Arcella hosts was confirmed using FISH with a Legionella‐specific probe. This study demonstrates that the host range of Legionella and Legionella‐like bacteria in the Amoebozoa extends beyond members of “naked” amoebae species, with members of the testate amoebae potentially serving an ecological role in the dispersal, protection, and replication of Legionella spp. in natural environments.     

Intracellular multiplication ofLegionella pneumophila in amoebae isolated from hospital hot water tanks

We studied the ability ofLegionella to multiply in potable water samples obtained from investigations of nosocomial legionellosis. AutochthonousLegionella multiplied in three of 14 hospital water samples after incubation at 35°C and 42°C. All three samples were from hot water tanks. Multiplication did not occur when a selected sample was filtered through a 0.45-m membrane and reinoculated with indigenousLegionella. We isolated bothLegionella pneumophila and one or more species of free-living amoebae, primarity members of theHartmannellidae, from each of these hot water tank samples. Amoebae from a total of six hot water tank samples were used for cocultivation studies withL. pneumophila. All amoebae supported multiplication ofLegionella in coculture at 35°C. Four of six isolates of amoebae supported multiplication oflegionella at 42°C, while none supported multiplication at 45°C. Gimenez staining and electron microscopy showed thatLegionella multiplied intracellularly in amoebae. Control of these amoebae in potable water may prevent colonization and multiplication ofLegionella in domestic hot water systems.     

Analysis of polyamine biosynthetic- and transport ability of human indigenous Bifidobacterium

Bifidobacteria are members of the human intestinal microbiota, being numerically dominant in the colon of infants, and also being prevalent in the large intestine of adults. In this study, we measured the concentrations of major polyamines (putrescine, spermidine, and spermine) in cells and culture supernatant of 13 species of human indigenous Bifidobacterium at growing and stationary phase. Except for Bifidobacterium bifidum and Bifidobacterium gallicum, 11 species contained spermidine and/or spermine when grown in Gifu-anaerobic medium (GAM). However, Bifidobacterium scardovii and Bifidobacterium longum subsp. infantis, which contain spermidine when grown in GAM, did not contain spermidine when grown in polyamine-free 199 medium. Of the tested 13 Bifidobacterium species, 10 species showed polyamine transport ability. Combining polyamine concentration analysis in culture supernatant and in cells, with basic local alignment search tool analysis suggested that novel polyamine transporters are present in human indigenous Bifidobacterium.

Abbreviations: Put: putrescine; Spd: spermidine; Spm: spermine; GAM: Gifu anaerobic medium; BHI: brain-heart infusion     

Detection ofLegionella antibodies by enzyme-linked immunosorbent assay (ELISA) using whole cell and carbohydrate antigens

The systematic study ofLegionella as a human pathogen and a bacterium widely disseminated in the environment requires simplification of present methodology. We describe a highly sensitive enzyme-linked immunosorbent assay (ELISA) for the detection of serum antibodies that can also be used for the detection of antigen.Legionella pneumophila serogroups 1 and 3 (Philadelphia 2 and Bloomington 2),L. bozemanii (WIGA), andL. micdadei (TATLOCK) were grown in diphasic medium consisting of charcoal yeast extract agar (CYE) overlayed with yeast extract medium (YEM) for the production of whole cell antigen and CYE for the extraction of carbohydrate antigen. The whole cells were inactivated with 0.5% formalin. The carbohydrate was obtained from the supernatant of cells resuspended twice in phosphate buffered saline (PBS). The antigen was sterilized and concentrated by filtration and purified by chromatography through a Sepharose 4B column. The highest molecular weight fractions were used for chemical characterization, which confirmed the carbohydrate nature of the antigen, and for micro-ELISA. Titers ranging from 5×10³ to 3×10⁵ (inverse of serum dilutions) were obtained from rabbit sera collected after 1, 2, or 3 injections of whole cells. The titers were somewhat higher and more consistent with the higher of 2 antigen concentrations used (5 or 15g/ml protein or dry weight), and with the carbohydrate rather than the whole cell antigen. The reactions were serogroup and species specific and only low titers were obtained with some of the heterologous antigens. The sensitivity and specificity of the reactions were not diminished when as many as 4 antigens were mixed in the same well. Thus, the micro-ELISA can be used as a test of highly specific antigens as well as a screening test with mixtures of antigens. A preliminary test withLegionella containing water specimen concentrates and high-titer rabbit sera indicated that the micro-ELISA can also be used for the detection of antigen. This investigation appears to have paved the way for the simplification of the serological methodology for the study ofLegionella. On temporary leave from: Department of Microbiology, University of Maryland School of Medicine, 660 West Redwood Street, Baltimore, MD 21201.     

Phylogenetic Study of <Emphasis Type="Italic">Legionella</Emphasis> Species in Pristine and Polluted Aquatic Samples from a Tropical Atlantic Forest Ecosystem

Legionella species are ubiquitous bacteria in aquatic environments. To examine the effect of anthropogenic impacts and physicochemical characteristics on the Legionellaceae population, we collected water from two sites in the Itanhaém River system in the Atlantic Forest of Brazil. One sample was collected from an upstream pristine region, the other from a downstream estuarine region moderately affected by untreated domestic sewage. Cultures on a selective medium failed to isolate Legionella species. Culture-independent methods showed that water from the estuarine aquatic habitat contained DNA sequences homologous to the 16S ribosomal DNA gene of Legionella pneumophila and non-pneumophila species. In pristine water, only two sequences related to L. pneumophila were detected. The results suggest that salinity and anthropogenic factors, such as wastewater discharge, favor a diversity of Legionella species, whereas pristine freshwater selects for Legionella pneumophila.     

Inhibitory effects of silver ions on Legionella pneumophila grown on agar, intracellular in Acanthamoeba castellanii and in artificial biofilms

Aims: We undertook a series of experiments to investigate the susceptibility of Legionella pneumophila grown under extracellular and intracellular conditions and other water‐related bacteria to silver ions. Methods and Results: In this study, the antimicrobial effect of silver ions to intra‐ and extra‐cellular grown Legionella bacteria was investigated. The minimal inhibitory concentration (MIC) after 24 h exposure, leading to a 5 log reduction, was c. 64 μg l^?1 AgNO₃ for extracellular grown Legionella and other tested Gram‐positive and Gram‐negative bacteria. In contrast, the MIC for intracellularly grown Legionella was up to 4096 μg l^?1 AgNO₃ after 24 h. Furthermore, the heterotrophic bacteria grown within a biofilm model were killed at a concentration of 4–16 μg l^?1 AgNO₃. In contrast, biofilm‐associated Legionella were less sensitive (MIC 128–512 μg l^?1 AgNO₃). Conclusion: Intracellularly and biofilm‐grown legionellae are less sensitive against silver compared with agar‐grown bacteria. Significance and Impact of the Study: The reduced sensitivity of Legionella grown in amoebae might explain why the effect of silver decontamination requires an extended exposure in field trials.     

The effects of salinity,crassulacean acid metabolism and plant age on the carbon isotope composition of <Emphasis Type="Italic">Mesembryanthemum crystallinum</Emphasis> L., a halophytic C<Subscript>3</Subscript>-CAM species

The carbon isotope composition of the halophyte Mesembryanthemum crystallinum L. (Aizoaceae) changes when plants are exposed to environmental stress and when they shift from C₃ to crassulacean acid metabolism (CAM). We examined the coupling between carbon isotope composition and photosynthetic pathway by subjecting plants of different ages to salinity and humidity treatments. Whole shoot ¹³C values became less negative in plants that were exposed to 400 mM NaCl in the hydroponic solution. The isotopic change had two components: a direct NaCl effect that was greatest in plants still operating in the C₃ mode and decreased proportionally with increasing levels of dark fixation, and a second component related to the degree of CAM expression. Ignoring the presumably diffusion-related NaCl effect on carbon isotope ratios results in an overestimation of nocturnal CO₂ gain in comparison to an isotope versus nocturnal CO₂ gain calibration established previously for C₃ and CAM species grown under well-watered conditions. It is widely taken for granted that the shift to CAM in M. crystallinum is partially under developmental control and that CAM is inevitably expressed in mature plants. Plants, cultivated under non-saline conditions and high relative humidity (RH) for up to 63 days, maintained diel CO₂ gas-exchange patterns and ¹³C values typical of C₃ plants. However, a weak CAM gas-exchange pattern and an increase in ¹³C value were observed in non-salt-treated plants grown at reduced RH. These observations are consistent with environmental control rather than developmental control of the induction of CAM in mature M. crystallinum under non-saline conditions.     

Legionella pneumophila Subversion of Host Vesicular Transport by SidC Effector Proteins

Tethering proteins play a key role in vesicular transport, ensuring that cargo arrives at a specific destination. The bacterial effector protein SidC and its paralog SdcA have been described as tethering factors encoded by the intracellular pathogen Legionella pneumophila. Here, we demonstrate that SidC proteins are important for early events unique to maturation of vacuoles containing Legionella and discover monoubiquitination of Rab1 as a new SidC‐dependent activity. The crystal structure of the SidC N‐terminus revealed a novel fold that is important for function and could be involved in Legionella adaptations to evolutionarily divergent host cells it encounters in natural environments.      

Detection of <Emphasis Type="Italic">Legionella</Emphasis> by quantitative-polymerase chain reaction (qPCR) for monitoring and risk assessment

Background

Culture and quantitative polymerase chain reaction (qPCR) assays for the detection of Legionella were compared on samples from a residential area before and after two interventions. A total of 84 samples were collected from shower hoses and taps as first flush samples and at constant temperature. Samples were grouped according to the origin of the sample, a) circulation water b) water from empty apartments c) water from shower hoses. The aims were to investigate the usefulness of qPCR compared to culture for monitoring remedial actions for elimination of Legionella bacteria and as a tool for risk assessment.

Results

In water collected from the apartments Legionella spp were detected by qPCR in the concentration range from LOQ to 9.6*10⁵GU/L while L. pneumophila were detected in a range from LOQ to 6.8*10⁵ GU/L. By culturing, the legionellae were detected in the range from below detection limit (> 10 CFU/L) to 1.6*10⁶ CFU/L. In circulating water and in first flush water from shower hoses, culture and qPCR showed the same tendencies. The overall correlation between the bacteria number detected by culture and the two developed qPCR assays (L. spp and L. pneumophila) was relatively poor (r² = 0.31 for culture and Legionella spp. assay, r² = 0.20 for culture and L. pneumophila assay).

Conclusion

Detection by qPCR was suitable for monitoring changes in the concentration of Legionella but the precise determination of bacteria is difficult. Risk assessment by qPCR only on samples without any background information regarding treatment, timing, etc is dubious. However, the rapid detection by qPCR of high concentrations of Legionella - especially Legionella pneumophila - is valuable as an indicator of risk, although it may be false positive compared to culture results. On the other hand, the detection of a low number of bacteria by qPCR is a strong indication for the absence of risk.

     

Enterotoxin A production in Staphylococcus aureus: inhibition by glucose

In this study, we investigated the relationship between carbohydrate metabolism and repression of staphylococcus enterotoxin A (SEA) in Staphylococcus aureus 196E and a pleiotrophic mutant derived from strain 196E. The mutant, designated at strain 196E-MA, lacked a functional phosphoenolpyruvate phosphotransferase system (PTS). The mutant produced acid, under aerobic conditions, from only glucose and glycerol. The parent strain contained an active PTS, and aerobically produced acid from a large number of carbohydrates. Prior growth in glucose led to repression of SEA synthesis in the parent strain; addition to the casamino acids enterotoxin production medium (CAS) led to more severe repression of toxin synthesis. The repression was not related to pH decreases produced by glucose metabolism. When S. aureus 196E was grown in the absence of glucose, there was inhibition of toxin production as glucose level was increased in CAS. The inhibition was related to pH decrease and was unlike the repression observed with glucose-grown strain 196E. The inhibition of SEA synthesis in mutant strain 196E-MA was approximately the same in cells grown with or without glucose and was pH related. Repression of SEA synthesis similar to that seen with glucose-grown S. aureus 196E could not be demonstrated in the mutant. In addition, glucose-grown S. aureus 196E neither synthesized -galactosidase nor showed respiratory activity with certain tricarboxylic acid (TCA) cycle compounds. Glucose-grown strain 196E-MA, however, did not show supressed respiration of TCA cycle compounds; -galactosidase was not synthesized because the mutant lacked a functional PTS. Cyclic adenosine-3, 5-monophosphate did not reverse the repression by glucose of SEA or -galactosidase synthesis in glucose-grown S. aureus 196E. An active PTS appears to be necessary to demonstrate glucose (catabolite) repression in S. aureus.Abbreviations SEA staphylococcal enterotoxin A - SEB staphylococcal enterotoxin B - SEC staphylococcal enterotoxin C - PTS phosphoenolpyruvate phosphotransferase system - CAS casamino acids salts medium - TCA tricarboxylic acid cycle     

Effects of competition and N and P supply on carbon isotope discrimination and <Superscript>15</Superscript>N-natural abundance in four grassland species

The effect of interspecific competition and element additions (N and P) on four grassland species (Poa pratensis, Lolium perenne, Festuca valida, Taraxacum officinale) grown under field conditions was studied. Two grasses (L. perenne, F. valida) grown in monoculture (absence of competition) showed lower carbon isotope discrimination (¹³C) and enriched ¹⁵N values. Nitrogen addition (as urea) had inconsistent effects on species ¹³C while caused enrichment of ¹⁵N of P. pratensis and F. valida but strong depletion of ¹⁵N of T. officinale. Phosphorous had no significant effect on ¹³C but depleted ¹⁵N of all species.     

Replication of <Emphasis Type="Italic">Legionella pneumophila</Emphasis> in Floating Biofilms

Biofilms are a major source of human pathogenic Legionella pneumophila in aquatic systems. In this study, we investigated the capacity of L. pneumophila to colonize floating biofilms and the impact of Acanthamoeba castellanii on the replication of biofilm-associated Legionella. Biofilms were grown in Petri dishes and consisted of Aeromonas hydrophila, Escherichia coli, Flavobacterium breve, and Pseudomonas aeruginosa. Six hours following inoculation, Legionella were detected in floating biofilms in mean concentrations of 1.4 × 10⁴ cells/cm² (real-time polymerase chain reaction) and 8.3 × 10² CFU/cm² (culture). Two-way analysis of variance tests and fluorescent in situ hybridization clearly proved that increased biofilm-associated L. pneumophila concentrations were the result of intracellular replication in A. castellanii. Forty-eight hours after the introduction of A. castellanii in the Petri dishes, 90 ± 0.8% of the amoebae (infection rate) were completely filled with highly metabolic active L. pneumophila (mean infection intensity).     

Discovery of a nonclassical siderophore, legiobactin, produced by strains of Legionella pneumophila

The mechanisms by which Legionella pneumophila, a facultative intracellular parasite and the agent of Legionnaires' disease, acquires iron are largely unexplained. Several earlier studies indicated that L. pneumophila does not elaborate siderophores. However, we now present evidence that supernatants from L. pneumophila cultures can contain a nonproteinaceous, high-affinity iron chelator. More specifically, when aerobically grown in a low-iron, chemically defined medium (CDM), L. pneumophila secretes a substance that is reactive in the chrome azurol S (CAS) assay. Importantly, the siderophore-like activity was only observed when the CDM cultures were inoculated to relatively high density with bacteria that had been grown overnight to log or early stationary phase in CDM or buffered yeast extract. Inocula derived from late-stationary-phase cultures, despite ultimately growing, consistently failed to result in the elaboration of siderophore-like activity. The Legionella CAS reactivity was detected in the culture supernatants of the serogroup 1 strains 130b and Philadelphia-1, as well as those from representatives of other serogroups and other Legionella species. The CAS-reactive substance was resistant to boiling and protease treatment and was associated with the <1-kDa supernatant fraction. As would also be expected for a siderophore, the addition of 0.5 or 2.0 microM iron to the cultures repressed the expression of the CAS-reactive substance. Interestingly, the supernatants were negative in the Arnow, Csáky, and Rioux assays, indicating that the Legionella siderophore was not a classic catecholate or hydroxamate and, hence, might have a novel structure. We have designated the L. pneumophila siderophore legiobactin.     

Water-use efficiency and carbon isotope discrimination of <Emphasis Type="Italic">Acacia ampliceps</Emphasis> and <Emphasis Type="Italic">Eucalyptus camaldulensis</Emphasis> at different soil moisture regimes under semi-arid conditions

Acacia ampliceps Maslin and Eucalyptus camaldulensis Dehnh. were grown for one year in lysimeters at three soil moisture regimes: 100 % (well-watered), 75 % (medium-watered) and 50 % (low-watered) of total plant available water. Biomass yield of both species increased with increase in soil moisture. Water-use efficiency (WUE) of E. camaldulensis decreased and that of A. ampliceps increased markedly with decrease in available soil moisture. A. ampliceps showed 4 – 5 times more biomass yield than E. camaldulensis grown at similar soil moisture. A. ampliceps showed almost 5, 9 and 12 times higher WUE than E. camaldulensis under low-, medium- and well-watered treatments, respectively. Significant negative correlation of ¹³C with WUE (r = –0.99) was observed in A. ampliceps. In contrast, ¹³C of E. camaldulensis showed a significant positive correlation with WUE (r = 0.82).     

Evaluation of the usefulness of a new direct immunofluorescence assay (ScanVIT‐Legionella™) for monitoring hospital water systems contaminated with Legionella spp.

Aims: To compare the efficiency of the ScanVIT‐Legionella^? test (Vermicon, Munich, Germany) vs a conventional culture method for the quantification of Legionella spp. in hospital water samples in daily hospital practice. Methods and Results: The detection of Legionella spp. takes place on a cultivated filter brought into contact with dye‐marked gene probes. The results are analysed under fluorescence microscopy. Bacteria that light up green belong to the genus Legionella; those that light up both green and red belong to the species Legionella pneumophila. Our results showed that the ScanVIT test has a sensitivity of 90%; agreement between the two methods was 82%. In the 48 samples that tested positive with both methods, the Legionella concentration detected by the culture method was consistently higher. A statistically significant difference between the results obtained with the two test methods emerged at the Wilcoxon test (P < 0·001). Conclusion: The ScanVIT test may be recommended for investigating the presence of Legionella by qualitative testing. Significance and Impact of the Study: Given the simplicity of colony identification by fluorescence, the ScanVIT test can be used in laboratories where staffs are not experienced in identifying typical colonies of Legionella.