Vibrio cholerae O1 Inhabit Intestines and Spleens of Fish in Aquaculture Ponds.

Vibrio cholerae is the causative agent of cholera, an acute diarrheal disease that spreads locally and globally in epidemics and pandemics. Although it was discovered that fish harbor V. cholerae strains in their intestines, most investigations revealed non-toxic V. cholerae serogroups in fish. Due to the rarity of toxigenic V. cholerae serogroups, it is difficult to cultivate these strains from environmental samples. Hence, here we aimed to uncover evidence of the occurrence of toxigenic V. cholerae in the intestines and spleens of various fish species. By using molecular detection tools, we show that V. cholerae O1 and strains positive for the cholera toxin inhabit both healthy and diseased fish intestines and spleens, suggesting that fish may serve as intermediate vectors of toxigenic V. cholerae. No significant differences were found between the abundance of toxigenic V. cholerae (either O1 or cholera toxin positive strains) in the healthy and the diseased fish intestines or spleens. In conclusion, a variety of fish species may serve as potential vectors and reservoirs of toxigenic V. cholerae as they form a link between the other reservoirs of V. cholerae (chironomids, copepods, and waterbirds). Similarly, they may aid in the spread of this bacterium between water bodies.

Accumulating evidence suggests that some waterbird species are potential vectors of Vibrio cholerae.

Vibrio cholerae is the causative agent of cholera, a life-threatening diarrheal disease. Cholera causes epidemics and pandemics, but the ways this disease spreads worldwide is still unclear. This review highlights a relatively new hypothesis regarding the way V. cholerae can be globally dispersed. Copepods and chironomids are natural reservoirs of V. cholerae and are part of different fish species' diet. Furthermore, V. cholerae inhabits marine and freshwater fish species. Waterbird species feed on fish or on small invertebrates such as copepods and chironomids. Waterbirds have also been found to carry living copepods and/or chironomids internally or externally from one waterbody to another. All of the above points to the fact that some waterbird species might be vectors of V. cholerae. Indeed, we and others have found evidence for the presence of V. cholerae non-O1 as well as O1 in waterbird cloacal swabs, feces, and intestine samples. Moreover, hand-reared cormorants that were fed on tilapia, a fish that naturally carries V. cholerae, became infected with this bacterial species, demonstrating that V. cholerae can be transferred to cormorants from their fish prey. Great cormorants as well as other waterbird species can cover distances of up to 1,000 km/day and thus may potentially transfer V. cholerae in a short time across and between continents. We hope this review will inspire further studies regarding the understanding of the waterbirds' role in the global dissemination of V. cholerae.

Quorum-sensing signaling by chironomid egg masses' microbiota, affects haemagglutinin/protease (HAP) production by Vibrio cholerae.

Vibrio cholerae, the causative agent of cholera, is commonly isolated, along with other bacterial species, from chironomid insects (Diptera: Chironomidae). Nevertheless, its prevalence in the chironomid egg masses' microbiota is less than 0.5%. V. cholerae secretes haemagglutinin/protease (HAP) that degrades the gelatinous matrix of chironomid egg masses and prevents hatching. Quorum sensing (QS) activates HAP production in response to accumulation of bacterial autoinducers (AIs). Our aim was to define the impact of chironomid microbiota on HAP production by V. cholerae. To study QS signaling, we used V. cholerae bioluminescence reporter strains (QS-proficient O1 El-Tor wild-type and QS-deficient mutants) and different bacterial species that we isolated from chironomid egg masses. These egg mass isolates, as well as a synthetic AI-2, caused an enhancement in lux expression by a V. cholerae QS-deficient mutant. The addition of the egg mass bacterial isolate supernatant to the QS-deficient mutant also enhanced HAP production and egg mass degradation activities. Moreover, the V. cholerae wild-type strain was able to proliferate using egg masses as their sole carbon source, while the QS-deficient was not. The results demonstrate that members of the chironomid bacterial consortium produce external chemical cues that, like AI-2, induce expression of the hapA gene in V. cholerae. Understanding the interactions between V. cholerae and the insects' microbiota may help uncover the interactions between this pathogen and the human gut microbiota.

Virulence Traits of Environmental and Clinical Legionella pneumophila Multilocus Variable-Number Tandem-Repeat Analysis (MLVA) Genotypes.

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.IMPORTANCELegionella 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.

Wild waterfowl as potential vectors of Vibrio cholerae and Aeromonas species.

OBJECTIVE: To study the hypothesis that migratory waterfowl are possible disseminators of Vibrio cholerae and Aeromonas. METHODS: We monitored the presence of V. cholerae and Aeromonas in three wild waterfowl species. RESULTS: V. cholerae and Aeromonas species were isolated and identified from intestine samples of little egrets and black-crowned night herons. Only Aeromonas species were isolated from black-headed gulls. The majority of Aeromonas isolates were A. veronii. Twenty-three V. cholerae serogroups were identified. V. cholerae serogroup O1 was found in the intestine DNA extractions from four little egrets and black-crowned night herons; six birds carried cholera toxin subunit A gene. CONCLUSION: Wild waterfowl species may carry pathogenic V. cholerae O1 and non-O1 serogroups and Aeromonas species in their intestine. The migration of waterfowl is a potential mechanism for global distribution of V. cholerae and Aeromonas.

Temperature-Dependent Growth Modeling of Environmental and Clinical Legionella pneumophila Multilocus Variable-Number Tandem-Repeat Analysis (MLVA) Genotypes.

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.IMPORTANCELegionella 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.

From Microhabitat of Floral Nectar Up to Biogeographic Scale: Novel Insights on Neutral and Niche Bacterial Assemblies.

Microbial model systems are very useful in addressing macro-ecological questions. Two major theories exist to date, to explain the community structure of organisms: (1) the dispersal (neutral) assembly theory which predicts that community similarity decreases with increasing geographic distance, independent of any environmental variables, and (2) the niche assembly theory which predicts that the communities' compositions are more homogeneous among sites characterized by similar environmental conditions. Our study system offered a unique opportunity to investigate the relative role of environmental conditions and spatial factors in shaping community composition. We explored the bacterial community composition (BCC) of Nicotiana glauca floral nectar using the Illumina MiSeq technique at three spatial scales (plants, site, and region) and two taxonomic levels. Floral nectar samples were collected from 69 N. glauca plants at 11 different sites along a 200-km transect in Israel, along three biogeographic regions. A distance decay of BCC was found among all plants throughout Israel, but such pattern was not found among either sites or biogeographical regions. The BCC was also governed by environmental conditions in all examined scales (from the plant up to the biogeographical region). We also found that taxonomic resolution (89 and 97% sequence identity for clustering operational taxonomic units) affected the results of these BCC analyses. Hence, our study revealed that the BCC in N. glauca floral nectar is shaped by both the environmental conditions and the distance between plants, depending on the sampling scale under examination as well as by taxonomic resolution.

Izhakiella capsodis gen. nov., sp. nov., in the family Enterobacteriaceae, isolated from the mirid bug Capsodes infuscatus.

Gram-stain-negative, oxidase-negative, facultatively anaerobic, motile, rod-shaped, non-pigmented bacterial strains (N6PO6T, N8PO1 and N8PI1) were isolated from the mirid bug Capsodes infuscatus captured on Asphodelus aestivus plants. The 16S rRNA gene sequences of the strains shared 94.7-95.7 % similarity with species of the genus Pantoea and 95.6 % or less with species from other genera in the family Enterobacteriaceae. A polyphasic approach that included determination of phenotypic properties and phylogenetic analysis based on 16S rRNA, rpoB, gyrB and atpD gene sequences supported the classification of strains N6PO6T, N8PO1 and N8PI1 as representing a novel species of a new genus in the family Enterobacteriaceae. Strain N6PO6T, and the two reference strains of the novel species, grew at 1-37 °C, and in the presence of NaCl (up to 7.5 %, w/v) and sucrose (up to 60 %). Their major cellular fatty acids were C16 : 0, C17 : 0 cyclo, C18 : 1ω7c, summed feature 2 (C14 : 0 3-OH and/or iso-C16 : 1 I) and summed feature 3 (C16 : 1ω7c and/or iso-C15 : 0 2-OH). The DNA G+C content of strain N6PO6T was 49.9 mol%. On the basis of phenotypic properties and phylogenetic distinctiveness, the mirid bug isolates are classified as representing a novel species in a new genus Izhakiella, in the family Enterobacteriaceae, for which the name Izhakiella capsodis gen. nov., sp. nov. is proposed. The type strain of Izhakiella capsodis is N6PO6T ( = LMG 28430T = DSM 29293T).

Chironomid microbiome.

Chironomids are abundant insects in freshwater habitats. They undergo a complete metamorphosis of four life stages: eggs, larvae, and pupae in water, and a terrestrial adult stage. Chironomids are known to be pollution-tolerant, but little is known about their resistance mechanisms to toxic substances. Here we review current knowledge regarding the chironomid microbiome. Chironomids were found as natural reservoirs of Vibrio cholerae and Aeromonas spp. A stable bacterial community was found in the egg masses and the larvae when both culture-dependent and -independent methods were used. A large portion of the endogenous bacterial species was closely related to species known as toxicant degraders. Bioassays based on Koch's postulates demonstrated that the chironomid microbiome plays a role in protecting its host from toxic hexavalent chromium and lead. V. cholerae, a stable resident in chironomids, is present at low prevalence. It degrades the egg masses by secreting haemagglutinin/protease, prevents eggs from hatching, and exhibits host pathogen interactions with chironomids. However, the nutrients from the degraded egg masses may support the growth of the other microbiome members and consequently control V. cholerae numbers in the egg mass. V. cholerae, other chironomid endogenous bacteria, and their chironomid host exhibit complex mutualistic relationships.

Transfer of Pseudomonas flectens Johnson 1956 to Phaseolibacter gen. nov., in the family Enterobacteriaceae, as Phaseolibacter flectens gen. nov., comb. nov.

Pseudomonas flectens Johnson 1956, a plant-pathogenic bacterium on the pods of the French bean, is no longer considered to be a member of the genus Pseudomonas sensu stricto. A polyphasic approach that included examination of phenotypic properties and phylogenetic analyses based on 16S rRNA, rpoB and atpD gene sequences supported the transfer of Pseudomonas flectens Johnson 1956 to a new genus in the family Enterobacteriaceae as Phaseolibacter flectens gen. nov., comb. nov. Two strains of Phaseolibacter flectens were studied (ATCC 12775(T) and LMG 2186); the strains shared 99.8 % sequence similarity in their 16S rRNA genes and the housekeeping gene sequences were identical. Strains of Phaseolibacter flectens shared 96.6 % or less 16S rRNA gene sequence similarity with members of different genera in the family Enterobacteriaceae and only 84.7 % sequence similarity with Pseudomonas aeruginosa LMG 1242(T), demonstrating that they are not related to the genus Pseudomonas. As Phaseolibacter flectens formed an independent phyletic lineage in all of the phylogenetic analyses, it could not be affiliated to any of the recognized genera within the family Enterobacteriaceae and therefore was assigned to a new genus. Cells were Gram-negative, straight rods, motile by means of one or two polar flagella, fermentative, facultative anaerobes, oxidase-negative and catalase-positive. Growth occurred in the presence of 0-60 % sucrose. The DNA G+C content of the type strain was 44.3 mol%. On the basis of phenotypic properties and phylogenetic distinctiveness, Pseudomonas flectens Johnson 1956 is transferred to the novel genus Phaseolibacter gen. nov. as Phaseolibacter flectens gen. nov., comb. nov. The type strain of Phaseolibacter flectens is ATCC 12775(T) = CFBP 3281(T) = ICMP 745(T) = LMG 2187(T) = NCPPB 539(T).

Rosenbergiella nectarea gen. nov., sp. nov., in the family Enterobacteriaceae, isolated from floral nectar.

Gram-negative, rod-shaped, oxidase-negative, facultatively anaerobic, yellow-orange-pigmented and motile bacterial strains, designated 8N4(T), 9N2 and 10N3, were isolated from flower nectar of Amygdalus communis (almond) and Citrus paradisi (grapefruit). The 16S rRNA gene sequences of the strains shared highest sequence similarity of 97.0 % with that of Phaseolibacter flectens ATCC 12775(T) and lower similarity with sequences from other type strains of genera of the Enterobacteriaceae. A polyphasic approach that included determination of phenotypic properties and phylogenetic analysis based on 16S rRNA, gyrB, rpoB and atpD gene sequences supported the classification of strains 8N4(T), 9N2 and 10N3 within a novel species in a novel genus in the family Enterobacteriaceae. Strain 8N4(T), and the reference strains of the novel species, grew at 4-35 °C (optimum, 28-30 °C), with 0-5.0 % NaCl (optimum, 3 % NaCl) and with 0-60 % sucrose (optimum, 10-25 % sucrose). Their major cellular fatty acids were C16 : 0, C17 : 0 cyclo, C18 : 1ω7c and summed feature 3 (C16 : 1ω7c and/or iso-C15 : 0 2-OH). The DNA G+C content of strain 8N4(T) was 46.8 mol%. On the basis of phenotypic properties and phylogenetic distinctiveness, the floral nectar isolates are classified within a novel species in a new genus in the family Enterobacteriaceae, for which the name Rosenbergiella nectarea gen. nov., sp. nov. is proposed. The type strain of Rosenbergiella nectarea is 8N4(T) ( = LMG 26121(T) = DSM 24150(T)).

Variability of bacterial community composition on leaves between and within plant species.

The phyllosphere is one of the largest habitats for terrestrial microorganisms. To gain a better insight into the factors underlying the composition of bacterial communities inhabiting leaf surfaces we performed culture-dependent and independent (Denaturing Gradient Gel Electrophoresis) analyses on the bacteria associated with the leaves of three plant species: Amygdalus communis, Citrus paradisi, and Nicotiana glauca. We found that the culturable classes Bacilli and Actinobacteria were the predominant classes on the phyllosphere of all three plant species. In contrast to this consistency on the bacterial class level, we found a significant variation on the bacterial species-level based on the culturable methods. Although some variation was detected among individual plants within one plant species, the inter-specific variability exceeded the intra-specific variability. C. paradisi leaf surface had the highest predicted total species richness (Chao 2 and ICE) and the highest species diversity (ßw) among the three plant species. Our findings demonstrate that environmental conditions, mainly the plant species within a site, govern the bacterial community composition on leaf surfaces.

Aeromonashydrophila infection in tilapia triggers changes in the microbiota composition of fish internal organs.

Aeromonas hydrophila is a major pathogenic species that causes mass mortality in various freshwater fish species including hybrid tilapia, the main fish species in Israeli aquaculture. Our hypothesis was that A. hydrophila infection may cause changes in the microbiota composition of fish internal organs, and therefore we aimed to study the effect of A. hydrophila infection by injection or by net handling on the microbiota compositions of fish intestine, spleen, and liver. Significant differences in the microbiota composition were found between the internal organs of the diseased and the healthy fish in both experimental setups. Fusobacteriota was the most dominant phylum in the microbiota of healthy fish (∼70%, liver). Cetobacterium was the most abundant genus and relatively more abundant in healthy, compared to diseased fish. When A. hydrophila was inoculated by injection, it was the only pathogenic genus in the spleen and liver of the diseased fish. However, in the handling experiment, Vibrio was also detected in the diseased fish, demonstrating coinfection interactions. Based on these experiments, we conclude that indeed, A. hydrophila infection in tilapia causes changes in the microbiota composition of fish internal organs, and that fish net handling may trigger bacterial infection in freshwater aquaculture.

Structure of bacterial communities in diverse freshwater habitats.

The structures and dynamics of bacterial communities from raw source water, groundwater, and drinking water before and after filtration were studied in four seasons of a year, with culture-independent methods. Genomic DNA from water samples was analyzed by the polymerase chain reaction - denaturing gradient gel electrophoresis system and by cloning of the 16S rRNA gene. Water samples exhibited complex denaturing gradient gel electrophoresis genetic profiles composed of many bands, corresponding to a great variety of bacterial taxa. The bacterial communities of different seasons from the four sampling sites clustered into two major groups: (i) water before and after filtration, and (ii) source water and groundwater. Phylogenetic analyses of the clones from the autumn sampling revealed 13 phyla, 19 classes, and 155 operational taxonomic units. Of the clones, 66% showed less than 97% similarities to known bacterial species. Representatives of the phyla Proteobacteria, Bacteroidetes, and Actinobacteria were found at all four sampling sites. Species belonging to the phylum Firmicutes were an important component of the microbial community in filtered water. Representatives of Enterobacteriaceae were not detected, indicating the absence of fecal pollution in the drinking water. Differences were found in the bacterial populations that were sampled from the same sites in different seasons. Each water habitat had a unique bacterial profile. Drinking water harbors diverse and dynamic microbial communities, part of which may be active and resilient to chlorine disinfection. This study provides, for the first time, basic data for uncultivable drinking water bacteria in Israel.

Bacterial community composition associated with chironomid egg masses.

Chironomids (Diptera: Chironomidae) are the most widely distributed and often the most abundant insect in freshwater. They undergo a complete metamorphosis of four life stages, of which the egg, larva, and pupae are aquatic and the adult is terrestrial. Chironomid egg masses were found to be natural reservoirs of Vibrio cholerae and Aeromonas species. To expand the knowledge of the endogenous bacterial community associated with chironomid egg masses, denaturing gradient gel electrophoresis and clone analysis of 16S rRNA gene libraries were used in this study. Bacterial community composition associated with chironomid egg masses was found to be stable among different sampling periods. Cloned libraries of egg masses revealed that about 40% of the clones were related to bacteria known to degrade various toxicants. These findings were further supported when bacterial species that showed resistance to different toxic metals were isolated from egg masses and larval samples. Chironomids are found under a wide range of water conditions and are able to survive pollutants. However, little is known about their protective mechanisms under these conditions. Chironomid egg masses are inhabited by a stable endogenous bacterial community, which may potentially play a role in protecting chironomids from toxicants in polluted environments. Further study is needed to support this hypothesis.

Intestine and spleen microbiota composition in healthy and diseased tilapia.

Symbiotic bacteria within the gut microbiome of various organisms, including fish, provide the host with several functions that improve the immune system. Although the spleen plays an important role in the modulation of immune responses, the role of spleen microbiota in shaping the immune system is unclear. Our study aimed at understanding the relationship between fish health and microbiota composition in the intestine and spleen. Our model organism was the hybrid tilapia (Oreochromis aureus × Oreochromis niloticus). We sampled intestine and spleen from healthy and diseased adult tilapia and determined their microbiota composition by sequencing the 16S rRNA gene. Significant differences were found between the intestine and the spleen microbiota composition of healthy compared to diseased fish as well as between intestines and spleens of fish with the same health condition. The microbiota diversity of healthy fish compared to diseased fish was significantly different as well. In the intestine of healthy fish, Cetobacterium was the most abundant genus while Mycoplasma was the most abundant genus in the spleen. Vibrio was the most abundant genus in the intestine and spleen of diseased fish. Moreover, it seems that there is a co-infection interaction between Vibrio and Aeromonas, which was reflected in the spleen of diseased fish. While Vibrio, Aeromonas and Streptococcus were the probable pathogens in the diseased fish, the role of Mycoplasma as a pathogen of cultured hybrid tilapia remains uncertain. We conclude that the intestine and spleen microbiota composition is strongly related to the health condition of the fish.

Biological Warfare of the Spiny Plant Introducing Pathogenic Microorganisms into Herbivore's Tissues.

Recently, it has been proposed that plants which have spines, thorns, and prickles use pathogenic aerobic and anaerobic bacteria, as well as pathogenic fungi, for defense against herbivores, especially vertebrates. Their sharp defensive appendages may inject various pathogenic agents into the body of the herbivores by piercing the outer defensive layer of the skin in a type of biological warfare. Here, we review data regarding the various bacterial taxa found on spines, as well as the medical literature regarding infections by bacteria and fungi related to spine injuries. We also present new evidence that, concerning the microbial flora, spines belonging to the palm tree Washingtonia filifera are probably a different habitat than the nondefensive green photosynthetic leaf surfaces. In addition, many plant species have microscopic internal and external spines (raphids and silica needles) which can also wound large herbivores as well as insects and other small invertebrate herbivores that usually attack in between large spines, prickles, and thorns. The large spines and sharp microscopic structures may inject not only the microorganisms that inhabit them into the herbivore's tissues, but also those preexisting on the skin surface or inside the digestive system of the herbivores and on the surface of nonspiny plant parts. A majority of the spiny plants visually advertise their spiny nature, a characteristic known as aposematism (warning coloration). The pathogenic microorganisms may sometimes be much more dangerous than the physical wounds inflicted by the spines. In accordance, we suggest that the possible cooperation or even just the random association of spines with pathogenic microorganisms contributed to the evolution of aposematism in spiny plants and animals. The role of these sharp defensive structures in inserting pathogenic viruses into the tissues of herbivores was never studied systematically and deserves special attention.

Acinetobacter rudis sp. nov., isolated from raw milk and raw wastewater.

Two bacterial strains, G30(T) and A1PC16, isolated respectively from raw milk and raw wastewater, were characterized using a polyphasic approach. Chemotaxonomic characterization supported the inclusion of these strains in the genus Acinetobacter, with Q-8 and Q-9 as the major respiratory quinones, genomic DNA G+C contents within the range observed for this genus (38-47 mol%) and C(16:0), C(18:1)ω9c and C(16:1)ω7c/iso-C(15:0) 2-OH as the predominant fatty acids. The observation of 16S rRNA gene sequence similarity lower than 97% with other Acinetobacter species with validly published names led to the hypothesis that these isolates could represent a novel species. This hypothesis was supported by comparative analysis of partial sequences of the genes rpoB and gyrB, which showed that strains G30(T) and A1PC16 did not cluster with any species with validly published names, forming a distinct lineage. DNA-DNA hybridizations confirmed that the two strains were members of the same species, which could be distinguished from their congeners by several phenotypic characteristics. On the basis of these arguments, it is proposed that strains G30(T) and A1PC16 represent a novel species, for which the name Acinetobacter rudis sp. nov. is proposed. The type strain is strain G30(T) (=LMG 26107(T) =CCUG 57889(T) =DSM 24031(T) =CECT 7818(T)).

Molecular analysis of bacterial communities in raw cow milk and the impact of refrigeration on its structure and dynamics.

The impact of refrigeration on raw cow milk bacterial communities in three farm bulk tanks and three dairy plant silo tanks was studied using two methods: DGGE and cloning. Both methods demonstrated that bacterial taxonomic diversity decreased during refrigeration. Gammaproteobacteria, especially Pseudomonadales, dominated the milk after refrigeration. Farm samples and dairy plant samples differed in their microbial community composition, the former showing prevalence of Gram-positive bacteria affiliated with the classes Bacilli, Clostridia and Actinobacteria, the latter showing prevalence of Gram-negative species belonging to the Gammaproteobacteria class. Actinobacteria prevalence in the farm milk samples immediately after collection stood at about 25% of the clones. A previous study had found that psychrotolerant Actinobacteria identified in raw cow milk demonstrated both lipolytic and proteolytic enzymatic activity. Thus, we conclude that although Pseudomonadales play an important role in milk spoilage after long periods of cold incubation, Actinobacteria occurrence may play an important role when assessing the quality of milk arriving at the dairy plant from different farms. As new cooling technologies reduce the initial bacterial counts of milk to very low levels, more sensitive and efficient methods to evaluate the bacterial quality of raw milk are required. The present findings are an important step towards achieving this goal.

Direct Evidence That Sunbirds' Gut Microbiota Degrades Floral Nectar's Toxic Alkaloids.

Orange-tufted sunbirds (Cinnyris osea) feed on the nectar of the tobacco tree (Nicotiana glauca) which contains toxic pyridine alkaloids characterized by high concentrations of anabasine and much lower concentrations of nicotine. We aimed at determining whether the gut microbiota of sunbirds harbors bacterial species that enable the birds to cope with these toxic alkaloids. An in vivo experiment that included 12 birds showed that inducing dysbiosis in sunbirds' guts by the addition of sulfamethoxazole and trimethoprim, significantly reduced the birds' ability to degrade anabasine (n = 3) compared to control birds (n = 3) with undisturbed microbiota. Sunbirds whose gut bacterial communities were altered by the antibacterial agents and who were fed with added nicotine, also showed a lower percentage of nicotine degradation (n = 3) in their excreta compared to the sunbirds with undisturbed microbiota (n = 3), though this difference was not significant. In an in vitro experiment, we studied the ability of Lactococcus lactis, Enterobacter hormaechei, Chryseobacterium gleum, Kocuria palustris, and Methylorubrum populi that were isolated from sunbirds' excreta, to degrade anabasine and nicotine. By using gas chromatography-mass spectrometry (GC-MS) analysis, we successfully demonstrated, for the first time, the ability of these species to degrade the focal secondary metabolites. Our findings demonstrate the role of gut bacteria in detoxifying toxic secondary metabolites found in the N. glauca nectar. The degradation products may supply the birds with nitrogen which is scarce in nectar-rich diets. These findings support another role of bacteria in mediating the interactions between plants and their pollinators.