Massimo Marzorati

Bacteria of the genus Asaia stably associate with Anopheles stephensi, an Asian malarial mosquito vector

Here, we show that an α-proteobacterium of the genus Asaia is stably associated with larvae and adults of Anopheles stephensi, an important mosquito vector of Plasmodium vivax, a main malaria agent in Asia. Asaia bacteria dominate mosquito-associated microbiota, as shown by 16S rRNA gene abundance, quantitative PCR, transmission electron microscopy and in situ-hybridization of 16S rRNA genes. In adult mosquitoes, Asaia sp. is present in high population density in the female gut and in the male reproductive tract. Asaia sp. from An. stephensi has been cultured in cell-free media and then transformed with foreign DNA. A green fluorescent protein-tagged Asaia sp. strain effectively lodged in the female gut and salivary glands, sites that are crucial for Plasmodium sp. development and transmission. The larval gut and the male reproductive system were also colonized by the transformed Asaia sp. strain. As an efficient inducible colonizer of mosquitoes that transmit Plasmodium sp., Asaia sp. may be a candidate for malaria control.

Asaia, a versatile acetic acid bacterial symbiont, capable of cross-colonizing insects of phylogenetically distant genera and orders

Bacterial symbionts of insects have been proposed for blocking transmission of vector-borne pathogens. However, in many vector models the ecology of symbionts and their capability of cross-colonizing different hosts, an important feature in the symbiotic control approach, is poorly known. Here we show that the acetic acid bacterium Asaia, previously found in the malaria mosquito vector Anopheles stephensi, is also present in, and capable of cross-colonizing other sugar-feeding insects of phylogenetically distant genera and orders. PCR, real-time PCR and in situ hybridization experiments showed Asaia in the body of the mosquito Aedes aegypti and the leafhopper Scaphoideus titanus, vectors of human viruses and a grapevine phytoplasma respectively. Cross-colonization patterns of the body of Ae. aegypti, An. stephensi and S. titanus have been documented with Asaia strains isolated from An. stephensi or Ae. aegypti, and labelled with plasmid- or chromosome-encoded fluorescent proteins (Gfp and DsRed respectively). Fluorescence and confocal microscopy showed that Asaia, administered with the sugar meal, efficiently colonized guts, male and female reproductive systems and the salivary glands. The ability in cross-colonizing insects of phylogenetically distant orders indicated that Asaia adopts body invasion mechanisms independent from host-specific biological characteristics. This versatility is an important property for the development of symbiont-based control of different vector-borne diseases.

A Novel Bacteroidetes Symbiont Is Localized in Scaphoideus titanus, the Insect Vector of Flavescence Dorée in Vitis vinifera

ABSTRACT Flavescence dorée (FD) is a grapevine disease that afflicts several wine production areas in Europe, from Portugal to Serbia. FD is caused by a bacterium, “Candidatus Phytoplasma vitis,” which is spread throughout the vineyards by a leafhopper, Scaphoideus titanus (Cicadellidae). After collection of S. titanus specimens from FD-contaminated vineyards in three different areas in the Piedmont region of Italy, we performed a survey to characterize the bacterial microflora associated with this insect. Using length heterogeneity PCR with universal primers for bacteria we identified a major peak associated with almost all of the individuals examined (both males and females). Characterization by denaturing gradient gel electrophoresis confirmed the presence of a major band that, after sequencing, showed a 97 to 99% identity with Bacteroidetes symbionts of the “Candidatus Cardinium hertigii” group. In addition, electron microscopy of tissues of S. titanus fed for 3 months on phytoplasma-infected grapevine plants showed bacterial cells with the typical morphology of “Ca. Cardinium hertigii.” This endosymbiont, tentatively designated ST1-C, was found in the cytoplasm of previtellogenic and vitellogenic ovarian cells, in the follicle cells, and in the fat body and salivary glands. In addition, cell morphologies resembling those of “Ca. Phytoplasma vitis” were detected in the midgut, and specific PCR assays indicated the presence of the phytoplasma in the gut, fat body and salivary glands. These results indicate that ST1-C and “Ca. Phytoplasma vitis” have a complex life cycle in the body of S. titanus and are colocalized in different organs and tissues.

Sulfur cycling and methanogenesis primarily drive microbial colonization of the highly sulfidic Urania deep hypersaline basin

Urania basin in the deep Mediterranean Sea houses a lake that is >100 m deep, devoid of oxygen, 6 times more saline than seawater, and has very high levels of methane and particularly sulfide (up to 16 mM), making it among the most sulfidic water bodies on Earth. Along the depth profile there are 2 chemoclines, a steep one with the overlying oxic seawater, and another between anoxic brines of different density, where gradients of salinity, electron donors and acceptors occur. To identify and differentiate the microbes and processes contributing to the turnover of organic matter and sulfide along the water column, these chemoclines were sampled at a high resolution. Bacterial cell numbers increased up to a hundredfold in the chemoclines as a consequence of elevated nutrient availability, with higher numbers in the upper interface where redox gradient was steeper. Bacterial and archaeal communities, analyzed by DNA fingerprinting, 16S rRNA gene libraries, activity measurements, and cultivation, were highly stratified and metabolically more active along the chemoclines compared with seawater or the uniformly hypersaline brines. Detailed analysis of 16S rRNA gene sequences revealed that in both chemoclines δ- and ε-Proteobacteria, predominantly sulfate reducers and sulfur oxidizers, respectively, were the dominant bacteria. In the deepest layers of the basin MSBL1, putatively responsible for methanogenesis, dominated among archaea. The data suggest that the complex microbial community is adapted to the basins extreme chemistry, and the elevated biomass is driven largely by sulfur cycling and methanogenesis.

Multiple symbiosis in the leafhopper Scaphoideus titanus (Hemiptera: Cicadellidae): details of transovarial transmission of Cardinium sp. and yeast-like endosymbionts.

Scaphoideus titanus is the insect vector of flavescence dorée (FD), a yellow disease of grapevines. Observations on adult females and nymphs of S. titanus showed that this insect is associated with a complex microbial community. Ultrastructural analysis showed that the fat body, salivary glands and ovary of the insect harbour microorganisms showing the brush-like structure typically observed in the genus Cardinium. In particular, it has been shown that these symbiotic bacteria are present both in the follicular cells and in the eggs. In addition, cells resembling bacteriocytes, harbouring numerous Cardinium symbionts in the cytoplasm, were observed in the apical portion of the ovary in adult females. These cells are likely responsible for bacterial transmission to the ovary. Optical microscopy showed that the fat body harbours an enormous population of yeast-like symbionts (YLSs). Ultrastructural observations showed that these symbionts are enclosed within specialized cells of the fat body and are also present in the ovary, where they are found in both the follicular cells and the eggs. There is thus evidence that both Cardinium and the YLSs are transovarially transmitted to the offspring. To our knowledge, S. titanus is the sole insect known to transmit two different kinds of symbionts to the eggs, a prokaryote and an eukaryote. Gene sequence analysis and in situ hybridization led to the identification of YLSs as members of the class Sordariomycetes (=Pyrenomycetes). Finally, ultrastructural observation of the midgut content revealed the presence, in both adult females and nymphs, of a complex microbial community, which include a phytoplasma-like microorganism, likely the agent of FD.

Bacterial endosymbiont localization in Hyalesthes obsoletus, the insect vector of Bois Noir in Vitis vinifera

ABSTRACT One emerging disease of grapevine in Europe is Bois noir (BN), a phytoplasmosis caused by “Candidatus Phytoplasma solani” and spread in vineyards by the planthopper Hyalesthes obsoletus (Hemiptera: Cixiidae). Here we present the first full characterization of the bacterial community of this important disease vector collected from BN-contaminated areas in Piedmont, Italy. Length heterogeneity PCR and denaturing gradient gel electrophoresis analysis targeting the 16S rRNA gene revealed the presence of a number of bacteria stably associated with the insect vector. In particular, symbiotic bacteria detected by PCR with high infection rates in adult individuals fell within the “Candidatus Sulcia muelleri” cluster in the Bacteroidetes and in the “Candidatus Purcelliella pentastirinorum” group in the Gammaproteobacteria, both previously identified in different leafhoppers and planthoppers. A high infection rate (81%) was also shown for another symbiont belonging to the Betaproteobacteria, designated the HO1-V symbiont. Because of the low level of 16S rRNA gene identity (80%) with the closest relative, an uncharacterized symbiont of the tick Haemaphysalis longicornis, we propose the new name “Candidatus Vidania fulgoroideae.” Other bacterial endosymbionts identified in H. obsoletus were related to the intracellular bacteria Wolbachia pipientis, Rickettsia sp., and “Candidatus Cardinium hertigii.” Fluorescent in situ hybridization coupled with confocal laser scanning microscopy and transmission electron microscopy showed that these bacteria are localized in the gut, testicles, and oocytes. As “Ca. Sulcia” is usually reported in association with other symbiotic bacteria, we propose that in H. obsoletus, it may occur in a bipartite or even tripartite relationship between “Ca. Sulcia” and “Ca. Purcelliella,” “Ca. Vidania,” or both.

Bacterial diversity and reductive dehalogenase redundancy in a 1,2-dichloroethane-degrading bacterial consortium enriched from a contaminated aquifer

BackgroundBacteria possess a reservoir of metabolic functionalities ready to be exploited for multiple purposes. The use of microorganisms to clean up xenobiotics from polluted ecosystems (e.g. soil and water) represents an eco-sustainable and powerful alternative to traditional remediation processes. Recent developments in molecular-biology-based techniques have led to rapid and accurate strategies for monitoring and identification of bacteria and catabolic genes involved in the degradation of xenobiotics, key processes to follow up the activities in situ.ResultsWe report the characterization of the response of an enriched bacterial community of a 1,2-dichloroethane (1,2-DCA) contaminated aquifer to the spiking with 5 mM lactate as electron donor in microcosm studies. After 15 days of incubation, the microbial community structure was analyzed. The bacterial 16S rRNA gene clone library showed that the most represented phylogenetic group within the consortium was affiliated with the phylum Firmicutes. Among them, known degraders of chlorinated compounds were identified. A reductive dehalogenase genes clone library showed that the community held four phylogenetically-distinct catalytic enzymes, all conserving signature residues previously shown to be linked to 1,2-DCA dehalogenation.ConclusionsThe overall data indicate that the enriched bacterial consortium shares the metabolic functionality between different members of the microbial community and is characterized by a high functional redundancy. These are fundamental features for the maintenance of the communitys functionality, especially under stress conditions and suggest the feasibility of a bioremediation treatment with a potential prompt dehalogenation and a process stability over time.

Response of 1,2-dichloroethane-adapted microbial communities to ex-situ biostimulation of polluted groundwater.

The microbial community of a groundwater system contaminated by 1,2-dichloroethane (1,2-DCA), a toxic and persistent chlorinated hydrocarbon, has been investigated for its response to biostimulation finalized to 1,2-DCA removal by reductive dehalogenation. The microbial population profile of samples from different wells in the aquifer and from microcosms enriched in the laboratory with different organic electron donors was analyzed by ARISA (Amplified Ribosomal Intergenic Spacer Analysis) and DGGE (Denaturing Gradient Gel Electrophoresis) of 16S rRNA genes. 1,2-DCA was completely removed with release of ethene from most of the microcosms supplemented with lactate, acetate plus formate, while cheese whey supported 1,2-DCA dehalogenation only after a lag period. Microbial species richness deduced from ARISA profiles of the microbial community before and after electron donor amendments indicated that the response of the community to biostimulation was heterogeneous and depended on the well from which groundwater was sampled. Sequencing of 16S rRNA genes separated by DGGE indicated the presence of bacteria previously associated with soils and groundwater polluted by halogenated hydrocarbons or present in consortia active in the removal of these compounds. A PCR assay specific for Desulfitobacterium sp. showed the enrichment of this genus in some of the microcosms. The dehalogenation potential of the microbial community was confirmed by the amplification of dehalogenase-related sequences from the most active microcosms. Cloning and sequencing of PCR products indicated the presence in the metagenome of the bacterial community of a new dehalogenase potentially involved in 1,2-DCA reductive dechlorination.

Arabinoxylans, inulin and Lactobacillus reuteri 1063 repress the adherent-invasive Escherichia coli from mucus in a mucosa-comprising gut model

The microbiota that colonises the intestinal mucus may particularly affect human health given its proximity to the epithelium. For instance, the presence of the adherent-invasive Escherichia coli (AIEC) in this mucosal microbiota has been correlated with Crohn’s disease. Using short-term screening assays and a novel long-term dynamic gut model, which comprises a simulated mucosal environment (M-SHIME), we investigated how (potential) pro- and prebiotics may repress colonisation of AIEC from mucus. Despite that during the short-term screening assays, some of the investigated Lactobacillus strains adhered strongly to mucins, none of them competed with AIEC for mucin-adhesion. In contrast, AIEC survival and growth during co-culture batch incubations was decreased by Lactobacillus rhamnosus GG and L. reuteri 1063, which correlated with (undissociated) lactic acid and reuterin levels. Regarding the prebiotics, long-chain arabinoxylans (LC-AX) lowered the initial mucin-adhesion of AIEC, while both inulin (IN) and galacto-oligosaccharides (GOS) limited AIEC survival and growth during batch incubations. L. reuteri 1063, LC-AX and IN were thus retained for a long-term study with the M-SHIME. All treatments repressed AIEC from mucus without affecting AIEC numbers in the luminal content. As a possible explanation, L. reuteri 1063 treatment increased lactobacilli levels in mucus, while LC-AX and IN additionally increased mucosal bifidobacteria levels, thus leading to antimicrobial effects against AIEC in mucus. Overall, this study shows that pro- and prebiotics can beneficially modulate the in vitro mucosal microbiota, thus limiting occurrence of opportunistic pathogens among those mucosal microbes which may directly interact with the host given their proximity to the epithelium.

Identification of Molecular Markers to Follow Up the Bioremediation of Sites Contaminated with Chlorinated Compounds

The use of microorganisms to clean up xenobiotics from polluted ecosystems (soil and water) represents an ecosustainable and powerful alternative to traditional remediation processes. Recent developments in molecular-biology-based techniques have led to rapid and sensitive strategies for monitoring and identifying bacteria and catabolic genes involved in the degradation of xenobiotics. This chapter provides a description of recently developed molecular-biology-based techniques, such as PCR with degenerate primers set, real-time quantitative PCR (qPCR), reverse transcription PCR (RT-PCR), southern blot hybridization, and long-range PCR, used to give a picture of the catabolically relevant microorganisms and of the functional genes present in a polluted system. By using a case study of a groundwater aquifer contaminated with 1,2-dichloroethane (1,2-DCA), we describe the identification of microorganisms potentially involved in the 1,2-DCA dehalorespiration (Dehalobacter sp. and Desulfitobacterium sp.) and a complete new gene cluster encoding for a 1,2-DCA reductive dehalogenase. The application of these techniques to bioremediation can improve our understanding of the inner mechanisms to evaluate the feasibility of a given treatment and provide us with a method to follow up bacteria and catabolic genes involved in the degradation of contaminants during the activities in situ.