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Sunbirds feed on tobacco tree nectar which contains toxic nicotine and anabasine secondary metabolites. Our aim was to understand the effect of nicotine and anabasine on the gut microbiota composition of sunbirds. Sixteen captive sunbirds were randomly assigned to two diets: artificial nectar either with (treatment) or without (control) added nicotine and anabasine. Excreta were collected at 0, 2, 4 and 7 weeks of treatment and samples were processed for bacterial culture and high-throughput amplicon sequencing of the 16S rRNA gene. The gut microbiome diversity of the treated and control birds changed differently along the seven-week experiment. While the diversity decreased in the control group along the first three samplings (0, 2 and 4 weeks), it increased in the treatment group. The microbiota composition analyses demonstrated that a diet with nicotine and anabasine, significantly changed the birds’ gut microbiota composition compared to the control birds. The abundance of nicotine- and anabasine- degrading bacteria in the excreta of the treated birds, was significantly higher after four and seven weeks compared to the control group. Furthermore, analysis of culturable isolates, including Lactococcus, showed that sunbirds’ gut-associated bacteria were capable of degrading nicotine and anabasine, consistent with their hypothesised role as detoxifying and nutritional symbionts.

Floral nectar harbors microbial communities which have significant impacts on its chemistry, volatiles, nutritional contents, and attractiveness for pollinators. Yet, fundamental knowledge regarding the structure and composition of nectar-associated microbiomes remains largely unknown. Especially elusive are the environmental factors and spatial effects that shape nectar-inhabiting microbial communities. The aim of this study was to explore and analyze the role of geographical and environmental factors affecting the composition and global distribution of floral nectar microbiota. We explored and compared the structure of bacterial communities inhabiting the floral nectar of the widely spread and invasive tobacco tree (Nicotiana glauca) in six continents: South and North America, Australia, Europe, Africa, and Asia, using 16S rRNA gene sequencing. Environmental abiotic data for each sampled plant was obtained from the Worldclim database and applied for inferring the effects of environmental conditions on bacterial community structure and diversity. Most abundant in the nectar were the Proteobacteria, Firmicutes, and Actinobacteria phyla, with Acinetobacter and Rosenbergiella (Proteobacteria) being the dominant bacterial genera that contributed most to the dissimilarities between sites. Acinetobacter and Rosenbergiella abundances were negatively correlated and significantly higher in the Mediterranean regions (Greece, Israel, and the Canary Islands) compared to Argentina and Australia. Temperature, site-elevation, rainfall, and density of vegetation were found to have significant effects on the structure and diversity of these bacterial communities in the nectar. Vegetation density was positively correlated with microbial diversity, while increased temperatures and elevation reduced the diversity and evenness of bacterial communities. Mantel's test showed that the similarity between the bacterial communities' composition significantly decreased as distances between them increased. We conclude that both geographical distance and local environmental abiotic conditions affect and shape the composition and diversity of nectar inhabiting bacterial communities.

Greywater (GW), domestic wastewater excluding the streams generated by toilets and kitchens, can serve as an alternative water source. The main options for GW reuse are toilet flushing and garden irrigation, both generating aerosols. These may transmit inhalable pathogens like Legionella and present a potential health risk. This study quantified the health risk that may arise from inhalation of Legionella-contaminated aerosols due to non-potable GW reuse. Data on Legionella concentrations in potable water and GW was collected. Then, Quantitative Microbial Risk Assessment (QMRA) was performed for two possible exposure scenarios: garden irrigation and toilet flushing. This was performed while considering Legionella seasonality. In order to determine the safety of GW reuse regarding Legionella transmission, the obtained results were compared with estimated tolerable risk levels of infection and of disease. Both limits were expressed as Disability-Adjusted Life Years index (DALY) being 10-4 and 10-5, respectively. The QMRA revealed that the annual risk associated with reuse of treated and chlorinated GW for garden irrigation and toilet flushing was not significantly higher than the risk associated with using potable water for the same two purposes. In all studied scenarios, the health risk stemming from reusing treated and chlorinated GW was acceptable regarding Legionella infection. In contrast, reuse of untreated or treated but unchlorinated GW should not be practiced, as these are associated with significantly higher health risks.

Background: Sunbirds (Cinnyris osea) feed on nectar of the tobacco tree (Nicotiana glauca), which contains the secondary metabolites nicotine and anabasine. These toxic pyridine-alkaloids presumably serve to deter nectar thieves and allow only specific pollinators to feed on nectar. The floral nectar contains low level of proteins and hence nectarivores may have problem balancing their nitrogen requirements. Our aim was to understand whether the gut microbiome of nectarivores contains bacteria that are able to degrade plant secondary metabolites and how the presence of secondary metabolites in nectar shapes the gut microbiome of nectarivores.Results: Sunbirds (n=16) that were held in captivity were randomly divided into control and treatment groups. The control group was fed with artificial nectar while the treatment group was fed with artificial nectar with the addition of nicotine and anabasine. Excreta were collected from each of the 16 birds on day 0 and at the end of weeks 2, 4 and 7. Nonmetric multidimensional scaling analysis (nMDS) demonstrated that the secondary metabolites significantly changed the gut microbiota composition and diversity of those sunbirds that were fed nectar with added pyridine alkaloids. Aeromonas, Enterobacteriaceae, Exiguobacterium, Klebsiella, Lactococcus, Leuconostoc, Rothia and Salmonella were found to contribute to the differences in gut microbiota between the control and treated birds (birds feed with Nicotine and Anabasine). Four weeks after starting to feed the birds with added nicotine and anabasine, we found that genera with the ability to degrade these pyridine-type metabolites proliferated from a total relative abundance of only 0.3% to 52.7% of the gut bacterial community.Conclusion: Sunbirds feed on Nicotiana nectar that contains toxic pyridine-alkaloids. Nonetheless, the symbiotic gut microbiota which is changing dynamically towards enabling the degradation of these secondary metabolites, supply the birds with nitrogen that enables the synthesis of essential amino acids. The current study contributes to our understanding of the complex relationships between plants, birds and their microbiomes.

Chironomids are aquatic insects that are known to be pollution tolerant. We have recently demonstrated that endogenous chironomid microbiota protects its host from toxic metals. Following these findings, we hypothesized that under different environmental conditions, a different bacterial consortium will evolve. Our aim was to explore the change in chironomid larval microbiota composition triggered by exposure to toxic copper and hexavalent chromium. Chironomid larvae were collected from the environment and treated in the laboratory with copper, hexavalent chromium, and no metal (control). After six days, the microbial composition of the surviving larvae was examined. We found a significant change in larval microbiota composition between the three treatments and for different copper concentrations. The abundance of specific taxa varied significantly between the treatments. At the genus level, the abundance of some genera (e.g. Yersinia, Acinetobacter) increased in the presence of copper, and some genera (e.g. Yersinia, Dysgonomonas, Delftia, Enterococcus) increased in the presence of hexavalent chromium, compared to the control. The change in the larval microbiota composition was rapid and metal-specific. We suggest that each larva hosts a consortium of bacterial species that can proliferate under a specific environmental change and thus, protect the insect under unstable environmental conditions.

Greywater (GW) is wastewater generated from domestic activities without the input of toilet and kitchen. Its reuse can help to reduce the overall potable water consumption in urban areas. In the current study, an extensive greywater treatment unit consisting of an attached growth unsaturated-flow bio-reactor, with “BIOROCK”® medium, was developed. Its performance was studied and the bacterial communities of the BIOROCK® biofilm were characterized through 454-pyrosequencing of 16S rRNA genes. The medium physical properties (porosity and high specific surface) made it possible to support highly diverse and relatively long-term stable bacterial communities that showed spatial variations in their relative abundance. The bacterial communities were dominated by genera previously associated with wastewater environments and that present some kind of degrading activities. Consequently, after treatment, water turbidity was reduced by 83 % and COD removal efficiency was 80 %, demonstrating that most of the biodegradable matter had been removed. Some pathogens were detected, but usually at low abundances. With some minor adjustments and the addition of a chlorination step, the treated greywater generated by this system can be used for non-potable purposes, such as irrigation and toilet flushing.

Legionella pneumophila cause human infections via inhalation of contaminated water aerosols, resulting in severe pneumonia. Legionella spp. prevalence was monitored in a drinking-water distribution system (DWDS) in Northern Israel. Five points (toilet faucets and showers) were sampled seasonally along a three years period. Toilet faucets and shower use, both generating aerosols, are known transmission routes for this pathogen and thus, present a potential health risk. Quantitative Microbial Risk Assessment (QMRA) was applied in order to assess the health risks posed by Legionella for these two exposure scenarios, while considering Legionella seasonality. The obtained results were compared with estimated tolerable risk levels of infection and of disease set by the USEPA and WHO. Both limits were expressed as Disability-Adjusted Life Years index (DALY) being 1 × 10−4 and 1 × 10−6, respectively. The QMRA revealed that the annual risk levels for both faucets and showers use exceeded the acceptable risk of infection with an average of 5.52 × 10−4 and 2.37 × 10−3 DALY'S per person per year, respectively. Annual risk levels were stable with no significant differences between the three years. Risk levels varied significantly between seasons by up to three orders of magnitude. Risk levels were highest during summer, autumn, and lowest during winter. The highest seasonal infection risk values were found in summer for both faucets and showers, which corresponded to 8.09 × 10−4 and 2.75 × 10−3 DALY'S per person per year, respectively. In conclusion, during summer and autumn there is a significant increase of the infection risk associated with exposure to Legionella-contaminated aerosols, in the studied water system. Public health assessment and prevention measures should focus on these seasons.

Legionella pneumophila causes water-based infections resulting in severe pneumonia. Recently, we showed that different MLVA-8 (multilocus variable-number tandem-repeat analysis using 8 loci) genotypes dominated different sites of a drinking-water distribution system. Each genotype displayed a unique temperature-dependent growth behavior. Here we compared the pathogenicity potentials of different MLVA-8 genotypes of environmental and clinical strains. The virulence traits studied were hemolytic activity and cytotoxicity toward amoebae and macrophages. Clinical strains were significantly more hemolytic than environmental strains, while their cytotoxicity toward amoebae was significantly lower at 30°C. No significant differences were detected between clinical and environmental strains in cytotoxicity toward macrophages. Significant differences in virulence were observed between the environmental genotypes (Gt). Gt15 strains showed a significantly higher hemolytic activity. In contrast, Gt4 and Gt6 strains were more infective toward Acanthamoeba castellanii . Moreover, Gt4 strains exhibited increased cytotoxicity toward macrophages and demonstrated a broader temperature range of amoebal lysis than Gt6 and Gt15 strains. Understanding the virulence traits of Legionella genotypes may improve the assessment of public health risks of Legionella in drinking water. IMPORTANCE Legionella pneumophila is the causative agent of a severe form of pneumonia. Here we demonstrated that clinical strains were significantly more cytotoxic toward red blood cells than environmental strains, while their cytotoxicity toward macrophages was similar. Genotype 4 (Gt4) strains were highly cytotoxic toward amoebae and macrophages and lysed amoebae in a broader temperature range than to the other studied genotypes. The results can explain the relatively high success of Gt4 in the environment and in clinical samples; thus, Gt4 strains should be considered a main factor for the assessment of public health risks of Legionella in drinking water. Our findings shed light on the ecology, virulence, and pathogenicity potential of different L. pneumophila genotypes, which can be a valuable parameter for future modeling and quantitative microbial risk assessment of Legionella in drinking-water systems.

Vibrio cholerae is the cause of cholera, a devastating epidemic and pandemic disease. Despite its importance, the way of its global dissemination is unknown. V. cholerae is abundant in aquatic habitats and is known to be borne by copepods, chironomids and fishes. Our aim was to determine if fish-eating birds act as vectors in the spread of V. cholerae by consuming infected fish. We determined the existence of V. cholerae in the microbiome of 5/7 wild cormorants’ intestine. In three of these V. cholerae-positive wild cormorants, the presence of a gene for cholera toxin (ctxA) was detected. We subsequently tested eight captive, hand-reared cormorants, divided into two equal groups. Prior to the experiment, the feces of the cormorants were V. cholerae-negative. One group was fed exclusively on tilapias, which are naturally infected with V. cholerae, and the other was fed exclusively on goldfish or on koi that were V. cholerae-negative. We detected V. cholerae in the feces of the tilapia-fed, but not in the goldfish/koi-fed, cormorants. Hence, we demonstrate that fish-eating birds can be infected with V. cholerae from their fish prey. The large-scale movements of many fish-eating birds provide a potential mechanism for the global distribution of V. cholerae.

Legionella pneumophila causes waterborne infections resulting in severe pneumonia. High-resolution genotyping of L. pneumophila isolates can be achieved by multiple-locus variable-number tandem-repeat analysis (MLVA). Recently, we found that different MLVA genotypes of L. pneumophila dominated different sites in a small drinking-water network, with a genotype-related temperature and abundance regime. The present study focuses on understanding the temperature-dependent growth kinetics of the genotypes that dominated the water network. Our aim was to model mathematically the influence of temperature on the growth kinetics of different environmental and clinical L. pneumophila genotypes and to compare it with the influence of their ecological niches. Environmental strains showed a distinct temperature preference, with significant differences among the growth kinetics of the three studied genotypes (Gt4, Gt6, and Gt15). Gt4 strains exhibited superior growth at lower temperatures (25 and 30°C), while Gt15 strains appeared to be best adapted to relatively higher temperatures (42 and 45°C). The temperature-dependent growth traits of the environmental genotypes were consistent with their distribution and temperature preferences in the water network. Clinical isolates exhibited significantly higher growth rates and reached higher maximal cell densities at 37°C and 42°C than the environmental strains. Further research on the growth preferences of L. pneumophila clinical and environmental genotypes will result in a better understanding of their ecological niches in drinking-water systems as well as in the human body. IMPORTANCE Legionella pneumophila is a waterborne pathogen that threatens humans in developed countries. The bacteria inhabit natural and man-made freshwater environments. Here we demonstrate that different environmental L. pneumophila genotypes have different temperature-dependent growth kinetics. Moreover, Legionella strains that belong to the same species but were isolated from environmental and clinical sources possess adaptations for growth at different temperatures. These growth preferences may influence the bacterial colonization at specific ecological niches within the drinking-water network. Adaptations for growth at human body temperatures may facilitate the abilities of some L. pneumophila strains to infect and cause illness in humans. Our findings may be used as a tool to improve Legionella monitoring in drinking-water networks. Risk assessment models for predicting the risk of legionellosis should take into account not only Legionella concentrations but also the temperature-dependent growth kinetics of the isolates.

Bacteria of the genus Legionella cause water-based infections resulting in severe pneumonia. Here we analyze and compare the bacterial microbiome of sputum samples from pneumonia patients in relation to the presence and abundance of the genus Legionella. The prevalence of Legionella species was determined by culture, PCR, and Next Generation Sequencing (NGS). Nine sputum samples out of the 133 analyzed were PCR-positive using Legionella genus-specific primers. Only one sample was positive by culture. Illumina MiSeq 16S rRNA gene sequencing analyses of Legionella-positive and Legionella-negative sputum samples, confirmed that indeed, Legionella was present in the PCR-positive sputum samples. This approach allowed the identification of the sputum microbiome at the genus level, and for Legionella genus at the species and sub-species level. 42% of the sputum samples were dominated by Streptococcus. Legionella was never the dominating genus and was always accompanied by other respiratory pathogens. Interestingly, sputum samples that were Legionella positive were inhabited by aquatic bacteria that have been observed in an association with amoeba, indicating that amoeba might have transferred Legionella from the drinking water together with its microbiome. This is the first study that demonstrates the sputum major bacterial commensals and pathogens profiles with regard to Legionella presence.

Bacteria of the genus Legionella cause water-based infections, resulting in severe pneumonia. To improve our knowledge about Legionella spp. ecology, its prevalence and its relationships with environmental factors were studied. Seasonal samples were taken from both water and biofilm at seven sampling points of a small drinking water distribution system in Israel. Representative isolates were obtained from each sample and identified to the species level. Legionella pneumophila was further determined to the serotype and genotype level. High resolution genotyping of L. pneumophila isolates was achieved by Multiple-Locus Variable number of tandem repeat Analysis (MLVA). Within the studied water system, Legionella plate counts were higher in summer and highly variable even between adjacent sampling points. Legionella was present in six out of the seven selected sampling points, with counts ranging from 1.0 × 10(1) to 5.8 × 10(3) cfu/l. Water counts were significantly higher in points where Legionella was present in biofilms. The main fraction of the isolated Legionella was L. pneumophila serogroup 1. Serogroup 3 and Legionella sainthelensis were also isolated. Legionella counts were positively correlated with heterotrophic plate counts at 37 °C and negatively correlated with chlorine. Five MLVA-genotypes of L. pneumophila were identified at different buildings of the sampled area. The presence of a specific genotype, "MLVA-genotype 4", consistently co-occurred with high Legionella counts and seemed to "trigger" high Legionella counts in cold water. Our hypothesis is that both the presence of L. pneumophila in biofilm and the presence of specific genotypes, may indicate and/or even lead to high Legionella concentration in water. This observation deserves further studies in a broad range of drinking water systems to assess its potential for general use in drinking water monitoring and management.

The COP9 signalosome (CSN) is a eukaryotic protein complex, which regulates a wide range of biological processes mainly through modulating the cullin ubiquitin E3 ligases in the ubiquitin-proteasome pathway. The CSN possesses a highly conserved deneddylase activity that centers at the JAMM motif of the Csn5 subunit but requires other subunits in a complex assembly. The classic CSN is composed of 8 subunits (Csn1-8), yet in several Ascomycota, the complex is smaller and lacks orthologs for a few CSN subunits, but nevertheless contains a conserved Csn5. This feature makes yeast a powerful model to determine the minimal assemblage required for deneddylation activity. Here we report, that Csi1, a diverged S. cerevisiae CSN subunit, displays significant homology with the carboxyl terminal domain of the canonical Csn6, but lacks the amino terminal MPN(-) domain. Through the comparative and experimental analyses of the budding yeast and the mammalian CSNs, we demonstrate that the MPN(-) domain of the canonical mouse Csn6 is not part of the CSN deneddylase core. We also show that the carboxyl domain of Csn6 has an indispensable role in maintaining the integrity of the CSN complex. The CSN complex assembled with the carboxyl fragment of Csn6, despite its lack of an MPN(-) domain, is fully active in deneddylation of cullins. We propose that the budding yeast Csi1 is a functional equivalent of the canonical Csn6, and thus the composition of the CSN across phyla is more conserved than hitherto appreciated.