Bacterial Microbiota of Rice Roots: 16S-Based Taxonomic Profiling of Endophytic and Rhizospheric Diversity, Endophytes Isolation and Simplified Endophytic Community

Rice is currently the most important food crop in the world and we are only just beginning to study the bacterial associated microbiome. It is of importance to perform screenings of the core rice microbiota and also to develop new plant-microbe models and simplified communities for increasing our understanding about the formation and function of its microbiome. In order to begin to address this aspect, we have performed a 16S rDNA taxonomic bacterial profiling of the rhizosphere and endorhizosphere of two high-yield rice cultivars\u2014Pionero 2010 FL and DANAC SD20A\u2014extensively grown in Venezuela in 2014. Fifteen putative bacterial endophytes were then isolated from surface-sterilized roots and further studied in vitro and in planta. We have then performed inoculation of rice seedlings with a simplified community composed by 10 of the isolates and we have tracked them in the course of 30 days in greenhouse cultivation. The results obtained suggest that a set was able to significantly colonize together the rice endorhizospheres, indicating possible cooperation and the ability to form a stable multispecies community. This approach can be useful in the development of microbial solutions for a more sustainable rice production

Microbe-assisted seedling crop improvement by a seaweed extract to address fucalean forest restoration

In the Mediterranean, Cystoseira sensu lato (s.l.) (Phaeophyceae) forests have sharply declined and restoration measures are needed to compensate for the loss. Assisted regeneration through the outplanting of seedlings grown ex-situ has proven to be a sustainable option. Optimizing mesocosm culture can maximize survival of the most critical embryonic stages and reduce long-term maintenance costs. Host-microbiome interactions could also play a crucial role in seedling development and welfare. In this context, we aimed to advance a cultivation protocol that stimulates the growth and fitness of Ericaria amentacea (Phaeophyceae) seedlings and identify the associated microbial biofilm communities. Seedlings were cultured in 6 treatments [i.e., filtered seawater (SW, C, Control), von Stoch-enriched SW (VS), VS + algal extract (VSA), algal extract-enriched SW: A1< A2< A3]. After the field, A2 seedlings had the highest cover (1372 ± 53.66 mm2), which was 1.8 and 1.9 times greater than in VS and VSA, respectively. The addition of the algal extract and nutrients significantly affected the structure and composition of the microbial community that shifted over time in each culture medium. We identified a treatment-specific microbial fingerprint. After the mesocosm phase, A2 was characterized by 4 unique taxa: Postechiella, Winogradskyella, Roseovarius and Arenibacter (Bacteria). Given the success of A2 seedlings, we propose the probiotic consortia candidates characterized by the unique treatment-taxa in conjunction with the shared taxon Psychroserpens (Bacteria, present in A1, A2, VSA, VS) and the reminder community. Within the holobiont concept, the effect of algal extract or nutrients on the algae and/or biofilm could have important consequences for tuning the overall interaction networks. Our study has shown that macroalgal restoration could benefit from both the use of commercial algal extract and tailored nutrient enrichment in ex-situ cultures and the identification of probiotic consortia candidates that promote seedling growth

pKBuS13, a KPC-2-Encoding Plasmid from Klebsiella pneumoniae Sequence Type 833, Carrying Tn4401b Inserted into an Xer Site-Specific Recombination Locus

Here, we report the first detection of a Klebsiella pneumoniae carbapenemase 2 (KPC-2)-producing Klebsiella pneumoniae strain belonging to sequence type 833 (ST833), collected in an Italian hospital from a patient coming from South America. Its bla KPC determinant was carried by a ColE1 plasmid, pKBuS13, that showed the Tn4401b::bla KPC-2 transposon inserted into the regulatory region of an Xer site-specific recombination locus. This interfered with the correct resolution of plasmid multimers into monomers, lowering plasmid stability and leading to overestimation of the number of plasmids harbored by a single host cell. Sequencing of the fragments adjacent to Tn4401b detected a region that did not have significant matches in databases other than the genome of a carbapenem-resistant Escherichia coli strain collected during the same year at a hospital in Boston. This is interesting in an epidemiologic context, as it suggests that despite the absence of tra genes and the instability under nonselective conditions, the circulation of pKBuS13 or of analogous plasmids might be wider than reported

ITS2 metabarcoding analysis complements lichen mycobiome diversity data

Lichen thalli harbor complex fungal communities (mycobiomes) of species with divergent trophic and ecological strategies. The complexity and diversity of lichen mycobiomes are still largely unknown, despite surveys combining culture-based methods and high-throughput sequencing (HTS). The results of such surveys are strongly influenced by the barcode locus chosen, its sensitivity in discriminating taxa, and the depth to which public sequence repositories cover the phylogenetic spectrum of fungi. Here, we use HTS of the internal transcribed spacer 2 (ITS2) to assess the taxonomic composition and diversity of a wellcharacterized, alpine rock lichen community that includes thalli symptomatically infected by lichenicolous fungi as well as asymptomatic thalli. Taxa belonging to the order Chaetothyriales are the major components of the observed lichen mycobiomes. We predict sequences representative of lichenicolous fungi characterized morphologically and assess their asymptomatic presence in lichen thalli. We demonstrated the limitations of metabarcoding in fungi and show how the estimation of species diversity widely differs when ITS1 or ITS2 are used as barcode, and particularly biases the detection of Basidiomycota. The complementary analysis of both ITS1 and ITS2 loci is therefore required to reliably estimate the diversity of lichen mycobiomes

Genital Dysbiosis and Different Systemic Immune Responses Based on the Trimester of Pregnancy in SARS-CoV-2 Infection

Respiratory infections are common in pregnancy with conflicting evidence supporting their association with neonatal congenital anomalies, especially during the first trimester. We profiled cytokine and chemokine systemic responses in 242 pregnant women and their newborns after SARS-CoV-2 infection, acquired in different trimesters. Also, we tested transplacental IgG passage and maternal vaginal–rectal microbiomes. IgG transplacental passage was evident, especially with infection acquired in the first trimester. G-CSF concentration—involved in immune cell recruitment—decreased in infected women compared to uninfected ones: a beneficial event for the reduction of inflammation but detrimental to ability to fight infections at birth. The later the infection was acquired, the higher the systemic concentration of IL-8, IP-10, and MCP-1, associated with COVID-19 disease severity. All infected women showed dysbiosis of vaginal and rectal microbiomes, compared to uninfected ones. Two newborns tested positive for SARS-CoV-2 within the first 48 h of life. Notably, their mothers had acute infection at delivery. Although respiratory infections in pregnancy are reported to affect babies’ health, with SARS-CoV-2 acquired early during gestation this risk seems low because of the maternal immune response. The observed vaginal and rectal dysbiosis could be relevant for neonatal microbiome establishment, although in our series immediate neonatal outcomes were reassuring

Developmental stages and gut microenvironments influence gut microbiota dynamics in the invasive beetle Popillia japonica Newman (Coleoptera: Scarabaeidae)

Popillia japonica Newman (Coleoptera: Scarabaeidae) is a highly polyphagous invasive beetle originating from Japan. This insect is highly resilient and able to rapidly adapt to new vegetation. Insect?associated microorganisms can play important roles in insect physiology, helping their hosts to adapt to changing conditions and potentially contributing to an insect's invasive potential. Such symbiotic bacteria can be part of a core microbiota that is stably transmitted throughout the host's life cycle or selectively recruited from the environment at each developmental stage. The aim of this study was to investigate the origin, stability and turnover of the bacterial communities associated with an invasive population of P. japonica from Italy. Our results demonstrate that soil microbes represent an important source of gut bacteria for P. japonica larvae, but as the insect develops, its gut microbiota richness and diversity decreased substantially, paralleled by changes in community composition. Notably, only 16.75% of the soil bacteria present in larvae are maintained until the adult stage. We further identified the micro?environments of different gut sections as an important factor shaping microbiota composition in this species, likely due to differences in pH, oxygen availability and redox potential. In addition, P. japonica also harboured a stable bacterial community across all developmental stages, consisting of taxa well known for the degradation of plant material, namely the families Ruminococcacae, Christensenellaceae and Lachnospiraceae. Interestingly, the family Christensenallaceae had so far been observed exclusively in humans. However, the Christensenellaceae operational taxonomic units found in P. japonica belong to different taxonomic clades within this family

L' ANTIBIOTICO RESISTENZA NELL' AMBIENTE MONITORAGGIO IN BATTERI ISOLATI DA AVIFAUNA

2001/2002Development of antibiotic resistance in bacteria is mainly due to the presence of resistance genes and to the selective pressure exerted by antibiotic use. Resistance can result from spontaneous mutations or acquisition of genes coding for a resistance mechanism. Antibacterial agents are used in agricultural techniques, in human and animai therapy and prevention of bacteria infections, and are added continuously to animai feeds to promote growth. As a result of exposure to antibiotics, the level of antibiotic resistance of bacteria belonging to the normal intestina! flora of human and animals increases. These bacteria not only constitute an enormous reservoir of resistance genes for potentially pathogenic bacteria, but also their level of resistance is considered to be a good indicator for the selection pressure exerted by antibiotic use in that population and for the resistance problems to be expected in pathogens. Indeed, reservoirs of antibiotic resistance in humans and in animals can interact in different ways: food and water are the most probable vectors of trasmission to the intestina! flora. Population of gulls (Larus cachinnans michahellis) has increased in North-East of Italy during the past decade. This increase has been attribuited to the ability of gulls to adapt to urban areas, in fact they are able to nest on roofs and feed of urban waste. Gulls ability to over fly large areas suggests that their feces may have a potential role in bacterial dissemination and more underhand antibiotic resistance in urban and rural environment. Transfer of antibiotic resistance genes between different species of bacteria c an be facilitated by mobile DNA elements, such as transposons and plasmids. Integrons have been identified on these mobile elements, and they often contain one or more genes that encode antibiotic resistance. Nine classes of integrases have been described, but class l is prevalent in Enterobacteriaceae. In this work l 02 cloacal swabs of Larus cachinnans michahellis were collected in natural reserve during spring in 2000 and 200 l. 214 strains of Enterobacteriaceae were isolated and identified as Proteus sp.(n = 89), E.coli (n = 84), Klebsiella sp.(n. = 18), Sa/monella sp. (n = 17) and Citrobacter sp. (n = 6). Also 162 Gram-positive bacteria strains were identified as Enterococcusfaecalis (n= 101) and Staphylococcus sp. (n= 61). Isolated avian strains were tested for susceptibility to a battery of antibiotics representing various classes of them. Gram-positive isolates showed low levels of resistance, but Enterobacteriaceae were resistant to a lot of antibiotics, especially to ampicillin (Sa/monella sp., E. coli and Proteus sp.), to tetracycline (Sa/monella sp., andE. coli), to streptomycin (Proteus sp., E. coli, Klebsiella sp. and Sa/monella sp.), to nalidixic acid (Proteus sp. andE. coli). The high resistance levels found in Gram-negative strains are very important if we consider the natural habitat of monitorated avifauna, and we could explain this fact as a result of the spread of environmental antibiotic contaminants with their consequent selection pressure and the dissemination of antibiotic resistance genes by horizontal transfer. Gram-negative avian strains were also screened for class l integrase using a specific probes which hybridizes to conserved regions of integron encoded gene intll. 25 of the 214 isolates were positive and showed to have class l integrons. Their sizes were detected by PCR with specific primers 5'CS-3'CS, flanking variable region of integron. Integrons ranged from 500 hp to 4800 bp. The incidence of class l integrons was correlated with multiple-drug resistances exhibited by avian Enterobacteriaceae to streptomycin, trimethoprimsulfamethoxazolo, tetracycline and chloramphenicol. The prevalent size ofintegrons was 1000 hp, 1600 hp and 1700 hp. Also 80 human clinical Enterobacteriaceae strains were screened for the integron presence, 16 strains showed to carry integrons ranged from 1000 pb to 2000 pb.The molecular characterization of integrons from avian and human strains, using different restriction endonucleases like Alul, Cfol, Mspl and Rsal, revealed the same types of integrons: 1000 pb integron, with the same restriction pattem, was found in 2 E. coli from Larus, in l E. coli and 5 Proteus sp. from human. An identica! 1600 pb integron was found in 6 avian strains (3 E. coli, l Klebsiella sp. and 2 Proteus sp.) and 3 Proteus sp. from human; 1700 pb integron was found in 2 E. coli from Larus and 4 E. coli from human. PFGE analysis ofthe strains from animals and humans carring the same types of integrons revelead different pattems. The presence of identica! integrons in different isolates from animals and humans implies horizontal transfer of the complete integron arrangement. 1000, 1600 and 1700 pb integrons were further characterized by sequencing. In integrons of l 000 pb sequence analysis identified a single aadA l gene cassette, integrons of 1600 pb contained dhfrl and aadA genes; integrons of 1700 pb contained d.frl7 and aadA5 genes. aadA, aadA l and aadA5 gene cassettes encoding an Aminoglicoside adenyltransferase and conferring resistance to streptomycin an d spectinomycin, dhfr an d dfr 17 gene cassettes encoding a dihydrofolate reductase and conferring resistance to trimethoprimXV Ciclo1967Versione digitalizzata della tesi di dottorato cartacea

Gut microbial community response to herbicide exposure in a ground beetle

Gut microbiota plays a key role in physiological processes of insects, including nutritional metabolism, development, immunity and detoxification. Environmental stressors such as herbicides, used to optimize and improve crop yields, may interfere with the mutualistic relationships causing negative consequences for the host health. Dinitroaniline herbicides, for example pendimethalin, are used worldwide in pre-emergence application to control grass and some broadleaf weeds. They target microtubules to arrest cell division and inhibit the development of roots and shoots. Effects of a pendimethalin-based herbicide were assessed on the gut microbial community of Pterostichus melas italicus Dejean, 1828 (Coleoptera, Carabidae). The exposure effect was tested in vivo by using a recommended field rate (4 L per ha, 330 gL 121 of active ingredient) and evaluating the variability of responses in 21 days, corresponding to the half-life of pendimethalin. The 16S rRNA sequencing data showed that the gut lumen was dominated by Proteobacteria, Firmicutes, Fusobacteria, Tenericutes and Bacteroidetes. The exposure interfered with the bacterial community richness and diversity associated with the gut from 2 days after the treatment. The differential abundance analyses highlighted a shift involving Lactobacillaceae, Streptomycetaceae, Neisseriaceae, Ruminococcaceae and Enterobacteriaceae. An increase in species such as Enterobacter sp., Pseudomonas sp., Pantoea sp and Paracoccus sp. involved in the herbicide degradation was also recorded after 21 days of exposure. Phylogenetic investigation of communities by reconstruction of unobserved states (PICRUSt) analysis indicated that the exposure has effects on the most predicted functional categories of gut microbiota related to metabolic function including carbohydrate, amino acid and lipid metabolism. These results demonstrate that pendimethalin can impact microbial communities associated with generalist predators inhabiting croplands leading to severe implications for the species\u2019 ecological role. Understanding the effects of herbicides such as pendimethalin on ground beetles may help to protect beneficial soil insects that have a crucial role in the ecosystem services

Bacterial Microbiota of Rice Roots: 16S-Based Taxonomic Profiling of Endophytic and Rhizospheric Diversity, Endophytes Isolation and Simplified Endophytic Community

Rice is currently the most important food crop in the world and we are only just beginning to study the bacterial associated microbiome. It is of importance to perform screenings of the core rice microbiota and also to develop new plant-microbe models and simplified communities for increasing our understanding about the formation and function of its microbiome. In order to begin to address this aspect, we have performed a 16S rDNA taxonomic bacterial profiling of the rhizosphere and endorhizosphere of two high-yield rice cultivars—Pionero 2010 FL and DANAC SD20A—extensively grown in Venezuela in 2014. Fifteen putative bacterial endophytes were then isolated from surface-sterilized roots and further studied in vitro and in planta. We have then performed inoculation of rice seedlings with a simplified community composed by 10 of the isolates and we have tracked them in the course of 30 days in greenhouse cultivation. The results obtained suggest that a set was able to significantly colonize together the rice endorhizospheres, indicating possible cooperation and the ability to form a stable multispecies community. This approach can be useful in the development of microbial solutions for a more sustainable rice production

Insights into the Oral Bacterial Microbiota of Sows

The investigation of bacterial microbiota represents a developing research field in veterinary medicine intended to look for correlations between animal health and the balance within bacterial populations. The aim of the present work was to define the bacterial microbiota of the oral cavity of healthy sows, which had not been thoroughly described so far. In total, 22 samples of oral fluid were collected and analyzed by 16S-rRNA gene sequencing. CLC Genomics Workbench 20.0 (QIAGEN Digital Insights, Aarhus, Denmark) was then used to examine the results. The predominant orders were Lactobacillales, Clostridiales, and Corynebacteriales. Lactobacillaceae, Corynebacteriaceae, Moraxellaceae, Aerococcaceae, and Staphylococcaceae were the most represented families. As regards the most abundant genera, Lactobacillus, Corynebacterium, Acinetobacter, Staphylococcus, Rothia, Aerococcus, and Clostridium can be pointed out as the bacterial core microbiota. Sows were also divided into “gestating” and “lactating” groups, and mild differences were found between pregnant and lactating sows. The data herein described represent an original contribution to the knowledge of the porcine bacterial microbiota. Moreover, the choice of sows as experimental animals was strategic for identifying the adult microbial community. These data provide a basis for further studies on the oral bacterial microbiota of pigs