Symbionts shape host innate immunity in honeybees.

The gut microbiome plays a critical role in the health of many animals. Honeybees are no exception, as they host a core microbiome that affects their nutrition and immune function. However, the relationship between the honeybee immune system and its gut symbionts is poorly understood. Here, we explore how the beneficial symbiont Snodgrassella alvi affects honeybee immune gene expression. We show that both live and heat-killed S. alvi protect honeybees from the opportunistic pathogen Serratia marcescens and lead to the expression of host antimicrobial peptides. Honeybee immune genes respond differently to live S. alvi compared to heat-killed S. alvi, the latter causing a more extensive immune expression response. We show a preference for Toll pathway upregulation over the Imd pathway in the presence of both live and heat-killed S. alvi. Finally, we find that live S. alvi aids in clearance of S. marcescens from the honeybee gut, supporting a potential role for the symbiont in colonization resistance. Our results show that colonization by the beneficial symbiont S. alvi triggers a replicable honeybee immune response. These responses may benefit the host and the symbiont, by helping to regulate gut microbial members and preventing overgrowth or invasion by opportunists.

Engineered symbionts activate honey bee immunity and limit pathogens.

Honey bees are essential pollinators threatened by colony losses linked to the spread of parasites and pathogens. Here, we report a new approach for manipulating bee gene expression and protecting bee health. We engineered a symbiotic bee gut bacterium, Snodgrassella alvi, to induce eukaryotic RNA interference (RNAi) immune responses. We show that engineered S. alvi can stably recolonize bees and produce double-stranded RNA to activate RNAi and repress host gene expression, thereby altering bee physiology, behavior, and growth. We used this approach to improve bee survival after a viral challenge, and we show that engineered S. alvi can kill parasitic Varroa mites by triggering the mite RNAi response. This symbiont-mediated RNAi approach is a tool for studying bee functional genomics and potentially for safeguarding bee health.

Detection and Characterization of Streptomycin Resistance (strA-strB) in a Honeybee Gut Symbiont (Snodgrassella alvi) and the Associated Risk of Antibiotic Resistance Transfer.

Use of antibiotics in medicine and farming contributes to increasing numbers of antibiotic-resistant bacteria in diverse environments. The ability of antibiotic resistance genes (ARG) to transfer between bacteria genera contributes to this spread. It is difficult to directly link antibiotic exposure to the spread of ARG in a natural environment where environmental settings and study populations cannot be fully controlled. We used managed honeybees in environments with contrasting streptomycin exposure (USA: high exposure, Norway: low exposure) and mapped the prevalence and spread of transferrable streptomycin resistance genes. We found a high prevalence of strA-strB genes in the USA compared to Norway with 17/90 and 1/90 positive samples, respectively (p < 0.00007). We identified strA-strB genes on a transferrable transposon Tn5393 in the honeybee gut symbiont Snodgrassella alvi. Such transfer of resistance genes increases the risk of the spread to new environments as honeybees are moved to new pollination sites.

Laribacter hongkongensis: clinical presentation, epidemiology and treatment. A review of the literature and report of the first case in Denmark.

BACKGROUND: Laribacter hongkongensis is an emerging pathogen related to gastroenteritis that can cause invasive and even fatal disease. The aim of this review is to describe the clinical presentation, epidemiology, treatment options and implications for the clinical microbiology laboratory. METHODS: We searched Pubmed using the term Laribacter hongkongensis with limitations human and language English, and identified 35 publications with eight reports on human cases. RESULTS: We describe our first case of prolonged, travel-related gastroenteritis where Laribacter hongkongensis was isolated as the sole pathogen. Our review suggests that L. hongkongensis causes non-bloody acute diarrhoea with potential for invasive disease, since three cases of bacteraemia and one case of dialysis related peritonitis have been described previously. L. hongkongensis has primarily been described in Asia, but reports from Europe, North America and Australia suggests a worldwide distribution. Broad culturing with subsequent identification by the MALDI-TOF is the current strategy for detection of L. hongkongensis. Phenotypic susceptibility testing is necessary to guide the treatment choice. Few resistance genes have been described in L. hongkongensis. CONCLUSION: L. hongkongensis should be considered a potential cause of acute and prolonged diarrhoea. Clinicians must be aware of the test methods in the local clinical microbiology laboratory, since L. hongkongensis is difficult to detect and easily overlooked.

Diversification of Type VI Secretion System Toxins Reveals Ancient Antagonism among Bee Gut Microbes.

Microbial communities are shaped by interactions among their constituent members. Some Gram-negative bacteria employ type VI secretion systems (T6SSs) to inject protein toxins into neighboring cells. These interactions have been theorized to affect the composition of host-associated microbiomes, but the role of T6SSs in the evolution of gut communities is not well understood. We report the discovery of two T6SSs and numerous T6SS-associated Rhs toxins within the gut bacteria of honey bees and bumble bees. We sequenced the genomes of 28 strains of Snodgrassella alvi, a characteristic bee gut microbe, and found tremendous variability in their Rhs toxin complements: altogether, these strains appear to encode hundreds of unique toxins. Some toxins are shared with Gilliamella apicola, a coresident gut symbiont, implicating horizontal gene transfer as a source of toxin diversity in the bee gut. We use data from a transposon mutagenesis screen to identify toxins with antibacterial function in the bee gut and validate the function and specificity of a subset of these toxin and immunity genes in Escherichia coli Using transcriptome sequencing, we demonstrate that S. alvi T6SSs and associated toxins are upregulated in the gut environment. We find that S. alvi Rhs loci have a conserved architecture, consistent with the C-terminal displacement model of toxin diversification, with Rhs toxins, toxin fragments, and cognate immunity genes that are expressed and confer strong fitness effects in vivo Our findings of T6SS activity and Rhs toxin diversity suggest that T6SS-mediated competition may be an important driver of coevolution within the bee gut microbiota.IMPORTANCE The structure and composition of host-associated bacterial communities are of broad interest, because these communities affect host health. Bees have a simple, conserved gut microbiota, which provides an opportunity to explore interactions between species that have coevolved within their host over millions of years. This study examined the role of type VI secretion systems (T6SSs)-protein complexes used to deliver toxic proteins into bacterial competitors-within the bee gut microbiota. We identified two T6SSs and diverse T6SS-associated toxins in bacterial strains from bees. Expression of these genes is increased in bacteria in the bee gut, and toxin and immunity genes demonstrate antibacterial and protective functions, respectively, when expressed in Escherichia coli Our results suggest that coevolution among bacterial species in the bee gut has favored toxin diversification and maintenance of T6SS machinery, and demonstrate the importance of antagonistic interactions within host-associated microbial communities.

Formation of indigoidine derived-pigments contributes to the adaptation of Vogesella sp. strain EB to cold aquatic iron-oxidizing environments.

We investigated previously under explored cold aquatic environments of Andean Patagonia, Argentina. Oily sheens similar to an oil spill are frequently observed at the surface of water in creeks and small ponds in these places. Chemical analysis of a water sample revealed the occurrence of high concentrations of iron and the presence of a free insoluble indigoidine-derived pigment. A blue pigment-producing bacterium (strain EB) was isolated from the water sample and identified as Vogesella sp. by molecular analysis. The isolate was able to produce indigoidine and another derived-pigment (here called cryoindigoidine) with strong antifreeze properties. The production of the pigments depended on the cell growth at cold temperatures (below 15 °C), as well as on the attachment of cells to solid surfaces, and iron limitation in the media. The pigments produced by strain EB showed an inhibitory effect on the growth of diverse microorganisms such as Candida albicans, Escherichia coli and Staphylococcus aureus. In addition, pigmented cells were more tolerant to freezing than non-pigmented cells, suggesting a role of cryoindigoidine/indigoidine as a cold-protectant molecule. The possible roles of the pigments in strain EB physiology and its interactions with the iron-rich environment from which the isolate was obtained are discussed. Results of this study suggested an active role of strain EB in the investigated iron-oxidizing ecosystem.

Evolution of host specialization in gut microbes: the bee gut as a model.

Bacterial symbionts of eukaryotes often give up generalist lifestyles to specialize to particular hosts. The eusocial honey bees and bumble bees harbor two such specialized gut symbionts, Snodgrassella alvi and Gilliamella apicola. Not only are these microorganisms specific to bees, but different strains of these bacteria tend to assort according to host species. By using in-vivo microbial transplant experiments, we show that the observed specificity is, at least in part, due to evolved physiological barriers that limit compatibility between a host and a potential gut colonizer. How and why such specialization occurs is largely unstudied for gut microbes, despite strong evidence that it is a general feature in many gut communities. Here, we discuss the potential factors that favor the evolution of host specialization, and the parallels that can be drawn with parasites and other symbiont systems. We also address the potential of the bee gut as a model for exploring gut community evolution.

Microbial gut diversity of Africanized and European honey bee larval instars.

The first step in understanding gut microbial ecology is determining the presence and potential niche breadth of associated microbes. While the core gut bacteria of adult honey bees is becoming increasingly apparent, there is very little and inconsistent information concerning symbiotic bacterial communities in honey bee larvae. The larval gut is the target of highly pathogenic bacteria and fungi, highlighting the need to understand interactions between typical larval gut flora, nutrition and disease progression. Here we show that the larval gut is colonized by a handful of bacterial groups previously described from guts of adult honey bees or other pollinators. First and second larval instars contained almost exclusively Alpha 2.2, a core Acetobacteraceae, while later instars were dominated by one of two very different Lactobacillus spp., depending on the sampled site. Royal jelly inhibition assays revealed that of seven bacteria occurring in larvae, only one Neisseriaceae and one Lactobacillus sp. were inhibited. We found both core and environmentally vectored bacteria with putatively beneficial functions. Our results suggest that early inoculation by Acetobacteraceae may be important for microbial succession in larvae. This assay is a starting point for more sophisticated in vitro models of nutrition and disease resistance in honey bee larvae.

Isolation of Laribacter hongkongensis from Little Egrets (Egretta garzetta) in Hangzhou, China.

AIMS: Laribacter hongkongensis is well adapted to diverse freshwater environments and is associated with human community-acquired gastroenteritis and traveller's diarrhoea. The study intended to investigate whether L. hongkongensis could be detected in Little Egrets (Egretta garzetta, a widespread aquatic bird) in Hangzhou, China. METHODS AND RESULTS: A total of 176 faecal specimens from three habitats in Hangzhou were obtained for this study. L. hongkongensis was isolated from 12 (6·8%) specimens. Identification of the 12 L. hongkongensis isolates was achieved through a combination of phenotypic characterization and 16S rRNA gene sequence analysis. CONCLUSIONS: The results illustrate that L. hongkongensis is present in the faeces of Little Egrets from three different habitats in Hangzhou, China. SIGNIFICANCE AND IMPACT OF THE STUDY: This study suggests the possibility that Little Egrets serve as mechanical carriers of L. hongkongensis, thus potentially transmitting the organism between bodies of water.

Detection of Deefgea chitinilytica in freshwater ornamental fish.

AIM: To identify and characterize six chitinolytic bacterial strains isolated from ornamental fish. METHODS AND RESULTS: Six different isolates of Deefgea chitinilytica were detected in healthy as well as diseased ornamental fish in Germany over a period of 2 years. Bacterial strains were identified using 16S rRNA partial gene sequencing and further characterized using different biochemical microtest systems and additional standard biochemical tests. CONCLUSION: We show that commercially available biochemical microtest systems are useful for identification of D. chitinilytica, supplemented by 16S rRNA partial gene sequencing. Furthermore, this study provides new information about the occurrence of D. chitinilytica, as this is the first isolation of D. chitinilytica from animals and first described isolation in Europe. SIGNIFICANCE AND IMPACT OF THE STUDY: Deefgea chitinilytica may be isolated regularly in fish diagnostic laboratories. Therefore, accurate identification of this bacterial species is important. Involvement of D. chitinilytica in opportunistic infections of aquatic organisms cannot be excluded and has to be further investigated.

Gulbenkiania indica sp. nov., isolated from a sulfur spring.

A novel bacterium, designated strain HT27(T), was isolated from a sulfur spring sample collected from Athamallik, Orissa, India, and was characterized by using a polyphasic approach. Cells were Gram-negative, strictly aerobic, rod-shaped and motile by means of a single polar flagellum. Strain HT27(T) was oxidase- and catalase-positive. Growth was observed at pH 5.0-11.0 and at 15-45 degrees C; the highest growth yield was observed at pH 7.5-8.0 and 30-37 degrees C. The G+C content of the genomic DNA of strain HT27(T) was 63 mol%. The major cellular fatty acids were C(16 : 1)omega7c (44.24 %), C(16 : 0) (27.65 %), C(18 : 1)omega7c (13.98 %), C(12 : 0) (2.60 %) and C(12 : 0) 3-OH (2.22 %). 16S rRNA gene sequence analysis indicated that strain HT27(T) clustered with the genus Gulbenkiania and showed 99.0 % similarity to Gulbenkiania mobilis E4FC31(T). However, the level of DNA-DNA relatedness between strain HT27(T) and G. mobilis E4FC31(T) was 30 %. On the basis of phenotypic and chemotaxonomic characteristics, 16S rRNA gene sequence analysis and DNA-DNA hybridization data, strain HT27(T) is considered to represent a novel species of the genus Gulbenkiania, for which the name Gulbenkiania indica sp. nov. is proposed. The type strain is HT27(T) (=DSM 17901(T) =JCM 15969(T)).

Aquitalea denitrificans sp. nov., isolated from a Korean wetland.

A novel bacterium, designated strain 5YN1-3(T), was isolated from wetland peat collected from Yongneup, Korea. The bacterium was facultatively anaerobic, Gram-negative, yellow-coloured, rod-shaped, mesophilic and motile with one polar flagellum. The strain grew optimally at 30 degrees C, at pH 6.0-9.0 and with 0-1 % NaCl (w/v). 16S rRNA gene sequence analysis showed the highest similarity to the sequence from Aquitalea magnusonii TRO-001DR8(T), with 98.7 % sequence similarity. However, strain 5YN1-3(T) showed DNA-DNA relatedness of 43 % (40 % in a reciprocal experiment) with A. magnusonii LMG 23054(T). The strain contained summed feature 3 (iso-C(15 : 0) 2-OH and/or C(16 : 1)omega7c) and C(16 : 0) as major cellular fatty acids. On the basis of DNA-DNA relatedness and physiological and biochemical characterization, strain 5YN1-3(T) should be assigned to a novel species of the genus Aquitalea, for which the name Aquitalea denitrificans sp. nov. is proposed. The type strain is 5YN1-3(T) (=KACC 12729(T) =DSM 21300(T)).

Vogesella perlucida sp. nov., a non-pigmented bacterium isolated from spring water.

A transparent, non-pigmented, Gram-negative, rod-shaped bacterium, designated strain DS-28(T), was isolated from water samples collected from a spring located in Tainan County, Taiwan. 16S rRNA gene sequence analysis indicated that the novel strain formed a monophyletic branch at the periphery of the evolutionary radiation occupied by the genus Vogesella; the only sole close neighbour of the novel strain was Vogesella indigofera ATCC 19706(T) (97.4 % 16S rRNA gene sequence similarity). The isolate was distinguished from V. indigofera on the basis of genotypic data, several phenotypic properties and an inability to produce characteristic blue-pigmented colonies on peptone agar. The fatty acid profile was slightly different from that reported for V. indigofera ATCC 19706(T). It was evident from the genotypic and phenotypic data that strain DS-28(T) represents a novel species of the genus Vogesella, for which the name Vogesella perlucida sp. nov. is proposed. The type strain is DS-28(T) (=BCRC 17730(T)=LMG 24214(T)).

Gulbenkiania mobilis gen. nov., sp. nov., isolated from treated municipal wastewater.

A bacterial strain (E4FC31(T)) isolated from treated municipal wastewater was characterized phenotypically and phylogenetically. Cells were Gram-negative, curved rods with a polar flagellum. The isolate was catalase-, oxidase- and arginine dihydrolase-positive, and able to grow between 15 and 45 degrees C and between pH 5.5 and 9.0. The predominant fatty acids were C(16 : 1)/iso-C(15 : 0) 2-OH and C(16 : 0), the major respiratory quinone was ubiquinone 8 and the G+C content of the genomic DNA was 63 mol%. 16S rRNA gene sequence analysis indicated that strain E4FC31(T) belonged to the class Betaproteobacteria and was a member of the family Neisseriaceae. Its closest phylogenetic neighbours were Aquitalea magnusonii and Chromobacterium violaceum (<94 % 16S rRNA gene sequence similarity). Phylogenetic analysis and phenotypic characteristics of strain E4FC31(T) suggest that it represents a novel species of a new genus, for which the name Gulbenkiania mobilis gen. nov., sp. nov. is proposed. The type strain of Gulbenkiania mobilis is E4FC31(T) (=DSM 18507(T)=LMG 23770(T)).

Protein secretion and secreted proteins in pathogenic Neisseriaceae.

Secreted proteins of pathogenic bacteria are often essential virulence factors. They are involved, for example, in the adherence of the bacteria to host cells or required to suppress the host's defence mechanisms. Until recently, only IgA1 protease had been studied in detail in the NeisseriaceaeNeisseria meningitidis and Neisseria gonorrhoeae. The availability of their genome sequences, however, has boosted research in this area. Here, we present a survey of the secretome of the pathogenic Neisseriaceae, based on the available genome sequences, and the current knowledge of the functions and structures of the secreted proteins. Of the six protein-secretion pathways that are widely disseminated among Gram-negative bacteria, three pathways appear to be present among the Neisseriaceae, i.e. the autotransporter-, the two-partner- and the type I-secretion mechanisms. Comparison of the predicted secretomes reveals a considerable flexibility. As compared with N. meningitidis and the nonpathogen N. lactamica, N. gonorrhoeae appears to have a considerably degenerated secretome, which may reflect its altered niche occupancy. The flexibility of the secretome may be enhanced by the presence of ORFs in the genomes potentially encoding fragments of secreted proteins. We hypothesize that these ORFs may substitute for the corresponding fragments in the full-length genes through genetic recombination, thereby changing the host-cell receptor specificity of the secreted protein.

Phylogenetic analysis of Alysiella and related genera of Neisseriaceae: proposal of Alysiella crassa comb. nov., Conchiformibium steedae gen. nov., comb. nov., Conchiformibium kuhniae sp. nov. and Bergeriella denitrificans gen. nov., comb. nov.

A phylogenetic analysis based on 16S rRNA gene sequences reveals that Alysiella filiformis belongs to the family Neisseriaceae. The genus Simonsiella is phylogenetically separated by the genera Kingella and Neisseria. The species Simonsiella crassa and A. filiformis show a close phylogenetic relationship, with the 16S rDNA sequence similarity and the DNA-DNA hybridization representing 98.7% and 35%, respectively. Therefore, S. crassa should be transferred from the genus Simonsiella to the genus Alysiella as Alysiella crassa comb. nov. Simonsiella steedae and Simonsiella sp. of cat origin show strong genetic affinities and are distantly related with the type species of Simonsiella, S. mulleri. Thus, a new genus, Conchiformibium is proposed; Conchiformibium steedae comb. nov. and Conchiformibium kuhniae sp. nov. are accommodated in this new genus. On the basis of the phylogenetic, phenotypic and chemotaxonomic distinction from the genus Neisseria, N. denitrificans should be reclassified, for which a new genus and new combination Bergeriella denitrificans are proposed.

Uruburuella suis gen. nov., sp. nov., isolated from clinical specimens of pigs.

Five strains of an unusual Gram-negative, catalase-positive, oxidase-positive, coccobacillus-shaped bacterium isolated from the lungs and heart of pigs with pneumonia and pericarditis were characterized by phenotypic and molecular genetic methods. On the basis of cellular morphology and biochemical criteria, the isolates were tentatively assigned to the family Neisseriaceae, although they did not appear to correspond to any recognized genus or species. Comparative 16S rRNA gene sequencing showed that the five unidentified strains were phylogenetically highly related to each other and represent a hitherto unknown subline within the family Neisseriaceae. On the basis of both phenotypic and phylogenetic evidence, it is proposed that the unknown isolates from pigs be classified as a novel genus and species within the family Neisseriaceae, for which the name Uruburuella suis gen. nov., sp. nov. is proposed. The type strain of U. suis is 1258/02(T) (=CCUG 47806(T)=CECT 5685(T)).

Chitinibacter tainanensis gen. nov., sp. nov., a chitin-degrading aerobe from soil in Taiwan.

Five strains with strong chitinolytic activity were isolated from a soil sample collected from southern Taiwan. The strains shared more than 92% DNA-DNA similarity, indicating membership of the same genospecies. This close relationship was supported by high similarities in fatty acid composition and biochemical characteristics. A 16S rRNA gene sequence analysis indicated that the isolates were members of the class 'Betaproteobacteria', in which they formed an individual subline of descent that was distantly related (<94% similarity) to lineages defined by Formivibrio citricus DSM 6150T and Iodobacter fluviatilis DSM 3764T. On the basis of the phylogenetic and phenotypic distinctness of these novel chitin-degrading organisms, a new genus, Chitinibacter, is proposed, with Chitinibacter tainanensis (type strain, S1T=BCRC 17254T=DSM 15459T) as the type species.

The role of Branhamella ovis, Mycoplasma conjunctivae and Chlamydia psittaci in conjunctivitis of sheep.

Smooth (S) and rough (R) types of Branhamella ovis were used alone and in conjunction with Mycoplasma conjunctivae and Chlamydia psittaci in attempts to produce conjunctivitis in lambs. This was only successful with one S-type previously isolated from a clinical case of conjunctivitis. In experimental mixed infection, B. ovis contributed to the severity of ovine keratoconjunctivitis attributable to infection with Mycoplasma conjunctivae. When B. ovis and C. psittaci were inoculated into the conjunctival sac mild conjunctivitis was produced but C. psittaci could not be re-isolated.

Isolation and characterization of group EF-4 bacteria from various lesions in cat, dog and badger.

Bacteria of Group EF-4 were isolated from local purulent lesions in 7 dogs and in 5 cats, from pneumonic lungs in 2 additional cats and from internal organs in a badger. Results of characterization were compared with those of a human strain of Group EF-4a isolated from an infected dog-bite wound. Lungs of 2 necropsied cats showed a severe focal necrotizing pneumonia in the various lobes. Organs of the badger were typical for a case of septicemia. The organisms were isolated mostly in pure culture. Colonies were up to 1.5 mm in size, round, entire, more or less yellowish pigmented and non hemolytic. Culture smelled slightly like popcorn. In conventional biochemical tests, 12 isolates as well as the human strain were shown to belong to Group EF-4a and the 3 remaining strains to EF-4b. Belonging to Group EF-4 was confirmed by assimilation tests with the gallery ATB 32 GN of BioMerieux. However some differences to other reports were observed when compared with results obtained with similar methods. Results of this study seem to indicate that bacteria of Group EF-4 are important in veterinary medicine not only from an epidemiological but also from an etiological point of view.