The aeroponic rhizosphere microbiome: community dynamics in early succession suggest strong selectional forces.

In the last decade there has been increased interest in the manipulation of rhizosphere microbial communities in soilless systems (hydroponics) through the addition of plant growth promoting microbes (PGPMs) to increase plant nutrition, lower plant stress response, and control pathogens. This method of crop management requires documenting patterns in communities living in plant roots throughout the growing season to inform decisions on timing of application and composition of the supplemental PGPM consortium. As a contribution to this effort, we measured changes in the bacterial community through early succession (first 26 days) in plant root biofilms growing in an indoor commercial aeroponic system where roots were sprayed with a mist of nutrient-amended water. By 12 days following seed germination, a root-associated community had established that was distinct from the source communities found circulating in the system. Successional patterns in the community over the following 2 weeks (12-26 days) included changes in abundance of bacterial groups that have been documented in published literature as able to utilize plant root exudates, release plant hormones, or augment nutrient availability. Six bacterial families/genera (Hydrogenophilaceae, Rhizobium, Legionellaceae, Methylophilus, Massilia, or Herbaspirillum) were the most abundant in each root sample, comprising 8-37% of the microbiome. Given the absence of soil-associated microbial communities in hydroponic systems, they provide an ideal design for isolating plant-microbial interactions and identifying key components possibly contributing to plant health.

The clinical presentation of Legionella arthritis reveals the mode of infection and the bacterial species: case report and literature review.

BACKGROUND: While Legionella is a common cause of pneumonia, extrapulmonary infections like arthritis are scarce. Here, we describe a case of monoarthritis due to Legionella bozemanii, with no history of pneumonia. We provide a literature review of the 9 previously published Legionella arthritis and highlight a dichotomous epidemiology suggesting different physiopathological pathways leading to joint infection. CASE PRESENTATION: A 56-year old woman under immunosuppressive treatment by oral and intra-articular corticosteroids, methotrexate, and tocilizumab for an anti-synthetase syndrome was hospitalized for worsening pain and swelling of the left wrist for 3 days. Clinical examination showed left wrist synovitis and no fever. The arthritis occurred a few days after an accidental fall on wet asphalt responsible for a cutaneous wound followed by a corticosteroid intra-articular injection. Due to both the negativity of conventional culture of articular fluid and suspicion of infection, 16S rRNA and specific PCRs were performed leading to the identification of L. bozemanii. Legionella-specific culture of the articular fluid was performed retrospectively and isolated L. bozemanii. The empiric antibiotic therapy was switched for oral levofloxacin and rifampin and the patient recovered after a 12-week treatment. CONCLUSION: We report a case of L. bozemanii monoarthritis in an immunosuppressed woman, following a fall on wet asphalt and intra-articular corticosteroid injection. The review of the literature found that the clinical presentation reveals the mode of infection and the bacterial species. Monoarthritis more likely occurred after inoculation in patients under immunosuppressive therapy and were associated with non-Legionella pneumophila serogroup 1 (Lp1) strains that predominate in the environment. Polyarthritis were more likely secondary legionellosis localizations after blood spread of Lp1, the most frequently found in pneumonia. In both settings, 16S rRNA and Legionella-specific PCR were key factors for the diagnosis.

A Systematic In Silico Analysis of the Legionellaceae Family for Identification of Novel Drug Target Candidates.

The family Legionellaceae consists of Gram-negative bacteria that are widely distributed in aquatic environments around the world. This family consists of a single genus, Legionella, that is recognized as an important cause of community-acquired pneumonia and hospital-acquired pneumonia. Legionella consists of intracellular pathogens, thus cellular pharmacokinetic and pharmacodynamic properties of an antibiotic against these bacteria as well as uptake and subcellular distribution into macrophages should be considered for a successful outcome of disease. Treatment strategies for Legionella infection require a combination of multiple antibiotics. Hence, because of the possible development of resistance to the drugs during therapy, a new alternative targeted therapy is yielding promising results. In this study, a comprehensive in silico target identification pipeline was performed on members of the family Legionellaceae to identify the best targets. Using a homology-based computational pipeline method, new drug targets were identified. Of 4,358 analyzed proteins, 18 proteins, including proteins involved in metabolism (amino acid, energy, and lipid metabolisms), cellular transport, cell division, and cell motility, were selected as the final putative drug targets. These proteins play an important role in the survival and propagation of Legionella infection. In conclusion, homology-based methods could improve the identification of novel drug targets and the drug discovery process, which can potentially be effective for the prevention and treatment of Legionella infections.

Detection of novel Chlamydiae and Legionellales from human nasal samples of healthy volunteers.

Chlamydiae are intracellular bacterial parasites of eukaryotes, ranging from amoebae to humans. They comprise many novel members and are investigated as emerging pathogens. Environmental studies highlighted similarities between the ecologies of chlamydiae and legionellae, both groups being important agents of respiratory infections. Herein, we analyzed nasal samples from healthy persons, searching for the presence of amoebae, chlamydiae and legionellae. From a total of 25 samples, we recovered by PCR eight samples positive to chlamydiae and six samples positive to legionellae. Among these samples, four were positive to both organisms. The sequencing of 16S rDNAs allowed to identify (i) among Chlamydiae: Parachlamydia acanthamoebae, Chlamydophila psittaci, Chlamydophila felis, and members of Rhabdochlamydiaceae, Simkaniaceae and E6 lineage and (ii) among Legionellaceae: Legionella longbeachae, Legionella bozemanii and Legionella impletisoli. Unexpectedly, we also recovered Diplorickettsia sp. Amoebae collected from nasal mucosae, Acanthamoeba and Vermamoeba, were endosymbiont-free, and chlamydiae revealed refractory to amoeba coculture. This study shows common exposure to chlamydiae and legionellae and suggests open air activities like gardening as a probable additional source of infection.

Occurrence and diversity of Legionellaceae in polar lakes of the Antarctic peninsula.

Legionellaceae is a family of gram-negative, mesophilic, and facultative intracellular parasitic bacteria that inhabits freshwater environments. In this article, the Legionella population of water samples from the North and South Lake, located close to the Brazilian Scientific Station on King George Island, Keller Peninsula, Antarctica has been characterized. Culture onto selective medium and a independent-culture method were applied to the samples. In our attempt to isolate Legionella species from Antarctic lakes, we were able to obtain one L. pneumophila colony by an amoebic coculture procedure followed by plate culture onto a selective medium. In addition, results obtained from phylogenetic inference showed the presence of noncharacterized specimens of Legionella spp. These findings indicated the presence of legionellae in Antarctica and suggest that these bacteria can adapt to extreme conditions and open new possibilities for understanding the survival strategies of mesophilic Legionellaceae living in Antarctic environments. Furthermore, the isolation of these symbiotic bacteria in Antarctic lakes will allow future studies on cold-resistant mechanisms of legionellae in polar environments.

Characterization of members of the Legionellaceae family by automated ribotyping.

In order to implement a new and reliable method for characterizing different species of Legionella, a genetic fingerprinting study with an automated ribotyping system (RiboPrinter) was completed with members of this genus which were deposited at the American Type Culture Collection. The RiboPrinter examined the different patterns of EcoRI digestion fragments from the rRNA operons of 110 strains, representing 48 of the 49 described Legionella species as well as 70 serogroups of those species. Distinctive and consistent patterns were obtained for the type strains of the 48 species investigated. Legionella pneumophila subsp. fraseri and L. pneumophila subsp. pascullei each generated a specific pattern, whereas L. pneumophila subsp. pneumophila produced six different fingerprint patterns. No correlation seemed to exist between the ribotypes obtained and the 15 serotypes of L. pneumophila. For the other species, those with two known serogroups presented two distinctive patterns with the RiboPrinter with the exception of L. hackeliae and L. quinlivanii, which yielded only one pattern. We also encountered ribotypes for strains which were not identified to the species level. The ribotypes generated for these strains with the RiboPrinter did not match those generated for known type strains, suggesting the putative description of new serogroups or species. Although the automated system did not have sufficient discriminatory ability to serve as an epidemiological tool in a clinical setting, it appeared to be a powerful tool for general genomic analysis of the Legionella isolates (e.g., determination of new species) and assessment of the interrelationship among Legionella strains through the RiboPrinter database connection.

Three-dimensional structure of FEZ-1, a monomeric subclass B3 metallo-beta-lactamase from Fluoribacter gormanii, in native form and in complex with D-captopril.

The beta-lactamases are involved in bacterial resistance to penicillin and related compounds. Members of the metallo-enzyme class are now found in many pathogenic bacteria and are thus becoming of major clinical importance. The structures of the Zn-beta-lactamase from Fluoribacter gormanii (FEZ-1) in the native and in the complex form are reported here. FEZ-1 is a monomeric enzyme, which possesses two zinc-binding sites. These structures are discussed in comparison with those of the tetrameric L1 enzyme produced by Stenotrophomonas maltophilia. From this analysis, amino acids involved in the oligomerization of L1 are clearly identified. Despite the similarity in fold, the active site of FEZ-1 was found to be significantly different. Two residues, which were previously implicated in function, are not present in L1 or in FEZ-1. The broad-spectrum substrate profile of Zn-beta-lactamases arises from the rather wide active-site cleft, where various beta-lactam compounds can be accommodated.

Presence of Legionellaceae in warm water supplies and typing of strains by polymerase chain reaction.

Outbreaks of Legionnaire's disease present a public health challenge especially because fatal outcomes still remain frequent. The aim of this study was to describe the abundance and epidemiology of Legionellaceae in the human-made environment. Water was sampled from hot-water taps in private and public buildings across the area of Göttingen, Germany, including distant suburbs. Following isolation, we used polymerase chain reaction in order to generate strain specific banding profiles of legionella isolates. In total, 70 buildings were examined. Of these 18 (26%) had the bacterium in at least one water sample. Legionella pneumophila serogroups 1, 4, 5 and 6 could be identified in the water samples. Most of the buildings were colonized solely by one distinct strain, as proven by PCR. In three cases equal patterns were found in separate buildings. There were two buildings in this study where isolates with different serogroups were found at the same time.

Distribution of mip-related sequences in 39 species (48 serogroups) of Legionellaceae.

The macrophage infectivity potentiator gene (mip) from Legionella pneumophila is a major virulence factor of the species. Thus, mip-detection by amplification has been proposed to assess the presence of L. pneumophila in clinical and environmental samples. The distribution of mip-related sequences within the Legionellaceae was studied by DNA amplification using mip-specific primers followed by Southern blot hybridization with an internal probe. Thirty-nine species (48 serogroups) of Legionellaceae were screened in this attempt. Using this approach, sequences related to mip were observed in 89% of the tested species including the most recently described L. fairfieldensis, L. lansingensis and L. shakespearei. In several cases, cloning and sequencing of the amplified products confirmed the high levels of similarity between the sequence found in non-pneumophila species with that of the L. pneumophila mip gene. This confirms previous reports that mip related genes are widespread among Legionellaceae and therefore specific detection of the species L. pneumophila cannot be based on mip-targeted amplification.

Intergenic 16S rRNA gene (rDNA)-23S rDNA sequence length polymorphisms in members of the family Legionellaceae.

A method based on PCR amplification of the 16S rRNA gene (rDNA)-23S rDNA intergenic regions was developed for the identification of species within the family Legionellaceae. The sizes of the PCR products varied from 1,353 to 350 bp. Strains of Legionella pneumophila were characterized as having products of approximately 900 and 530 bp, and L. birminghamensis had products of 1,390, 960, and 380 bp. Of the 38 species of legionellae examined, only 7 were indistinguishable (L. erythra from L. rubrilucens, L. anisa or L. cherrii from L. tucsonensis, and L. quateirensis from L. shakespearei). Two environmental isolates were identified as L. pneumophila. Strain LLAP-3, which was a symbiont of amoebae, could not be associated with any Legionella sp. studied.

Restriction fragment length polymorphism of rRNA genes for molecular typing of members of the family Legionellaceae.

Typing of Legionella pneumophila remains important in the investigation of outbreaks of Legionnaires' disease and in the control of organisms contaminating hospital water. We found that the discriminatory power of a nonradioactive ribotyping method could be improved by combining results obtained with four restriction enzymes (HindIII, NciI, ClaI, and PstI). Fifty-eight clinical and environmental L. pneumophila strains including geographically unrelated as well as epidemiologically connected isolates were investigated. Epidemiologically related strains had the same ribotypes independent of the combinations of enzymes used. Some strains belonging to the same serogroup were assigned to different ribotypes, and some ribotypes contained members of different serogroups, indicating, as others have found, that serogroup and genotype are not always related. The discriminatory power of the method was estimated by calculating an index of discrimination (ID) for individual enzymes and combinations thereof. The combined result with all four enzymes was highly discriminatory (ID = 0.97), but results for three enzymes also yielded ID values acceptable for epidemiological purposes. In addition, the testing of 27 type strains and 6 clinical isolates representing Legionella species other than L. pneumophila indicated that ribotyping might be of value for species identification within this genus, as previously suggested.

In situ identification of Legionellaceae using 16S rRNA-targeted oligonucleotide probes and confocal laser scanning microscopy.

Bacteria of the family Legionellaceae form a monophyletic group within the gamma-subclass of Proteobacteria. Based on comparative sequence analysis we constructed two oligonucleotide probes complementary to regions of 16S rRNA characteristic for Legionellaceae. Probe specificities were tested by whole-cell or dot-blot hybridization against 14 serogroups of Legionella pneumophila, 22 different Legionella spp. and 72 non-legionellae reference strains. Using optimized conditions both probes hybridized to all tested strains of L. pneumophila. Probes LEG226 and LEG705 hybridized to 71% and 90% of the Legionella species tested, respectively. With the exception of Methylomonas alba none of the non-target strains showed complete sequence homology within the target molecule. In a preliminary evaluation the results of classical techniques employing selective media, immunofluorescence and the probe assay were in good accordance for routine environmental and clinical isolates. L. pneumophila suspended in drinking water at approximately 10(3)-10(4) c.f.u. ml-1 could be rapidly detected by a combination of membrane filtration on polycarbonate filters and whole-cell hybridization. Even after incubation for 1 year a proportion of the released cells was still detectable. In situ hybridization also facilitated visualization of Legionella spp, cells in model biofilms. A combination of in situ hybridization and confocal laser scanning microscopy (CLSM) was used to analyse the three-dimensional arrangement of L. pneumophila within cells of the ciliated protozoan Tetrahymena pyriformis. Whole-cell probing with 16S rRNA-targeted oligonucleotides could, in the future, complement established techniques like immunofluorescence and PCR in ecological and epidemiological studies of Legionellaceae.

Legionellaceae in the potable water of Nova Scotia hospitals and Halifax residences.

Water was cultured from 39 of 48 hospitals (7 Halifax hospitals and 32 non-Halifax hospitals) in the province of Nova Scotia and from 90 residences (74 private dwellings, 16 apartments) in Halifax to determine the frequency of legionella contamination. Six of seven Halifax hospitals had Legionellaceae isolated from their potable water compared with 3 of 32 non-Halifax hospitals (P < 0.0001). Overall, 19 of 59 (32%) of the water samples from Halifax hospitals were positive for legionellae compared with 5 of 480 (1%) samples from non-Halifax hospitals (P < 0.0000). Five of the six positive Halifax hospitals had Legionella pneumophila serogroup 1 and 1 had L. longbeachae serogroup 2 recovered from their potable water. Legionella contamination was associated with older, larger (> or = 50 beds) hospitals with total system recirculation. These hospitals also had water with a higher pH and calcium content but lower sodium, potassium, nitrate, iron and copper content. Fourteen of the 225 (6.2%) water samples from Halifax residences were positive for legionellae -8% (6/74) of the single family dwellings were positive, compared with 25% (4/16) apartments. The positivity rate of 15.7% for the 19 electric hot-water heaters in Halifax homes was not significantly different from the 32% positivity for Halifax hospitals. L. longbeachae accounted for 2 of the 14 isolates of legionellae from Halifax homes.

Immunophilins: structure-function relationship and possible role in microbial pathogenicity.

Immunophilins are housekeeping proteins present in a wide variety of organisms. Members of two protein superfamilies, cyclophilins (Cyps) and FK506-binding proteins (FKBPs) belong to this class of immunophilins. Despite the fact that the amino acid sequences of Cyp and FKBPs do not exhibit noticeable homology to each other, proteins of both classes are able to ligate immunosuppressive peptide derivatives. Cyps form complexes with the cyclic undercapeptide cyclosporin A and FKBPs are able to bind FK506 as well as rapamycin, both of which have a pipecolyl bond within their structure. In a ligand-bound form, immunophilins interfere with signal transduction in T cells. In addition, immunophilins have peptidyl prolyl cis-trans isomerase (PPlase) activity and are able to accelerate the rate of conformational events in proline-containing polypeptides. Microorganisms produce proteins that exhibit extensive sequence homologies to cyclophilins and FKBPs of higher organisms and which have considerable PPlase catalytic activity. While cyclophilins seem to be present in most if not all microbial species investigated, FKBPs are produced by yeasts as well as by a number of pathogenic bacteria, such as Legionella pneumophila, Chlamydia trachomatis and Neisseria meningitidis. The Mip protein of L. pneumophila is a virulence factor that plays an essential role in the ability of the bacteria to survive and multiply in phagocytic cells. Some results are summarized on the structure and putative functions of immunophilins and place special emphasis on the contribution of these polypeptides to the virulence of pathogenic microorganisms.