Genotype-by-genotype interkingdom cross-talk between symbiotic nitrogen fixing Sinorhizobium meliloti strains and Trichoderma species.

In the understanding of the molecular interaction between plants and their microbiome, a key point is to identify simplified models of the microbiome including relevant bacterial and fungal partners which could also be effective in plant growth promotion. Here, as proof-of-concept, we aim to identify the possible molecular interactions between symbiotic nitrogen-fixing rhizobia and soil fungi (Trichoderma spp.), hence shed light on synergistic roles rhizospheric fungi could have in the biology of symbiotic nitrogen fixation bacteria. We selected 4 strains of the model rhizobium Sinorhizobium meliloti and 4 Trichoderma species (T. velutinum, T. tomentosum, T. gamsii and T. harzianum). In an experimental scheme of 4 ×4 strains x species combinations, we investigated the rhizobia physiological and transcriptomic responses elicited by fungal spent media, as well as spent media effects on rhizobia-host legume plant (alfalfa, Medicago sativa L.) symbiosis. Fungal spent media had large effects on rhizobia, specific for each fungal species and rhizobial strains combination, indicating a generalized rhizobia genotype x fungal genotype interaction, including synergistic, neutral and antagonistic effects on alfalfa symbiotic phenotypes. Differential expression of a high number of genes was shown in rhizobia strains with up to 25% of total genes differentially expressed upon treatment of cultures with fungal spent media. Percentages over total genes and type of genes differentially expressed changed according to both fungal species and rhizobial strain. To support the hypothesis of a relevant rhizobia genotype x fungal genotype interaction, a nested Likelihood Ratio Test indicated that the model considering the fungus-rhizobium interaction explained 23.4% of differentially expressed genes. Our results provide insights into molecular interactions involving nitrogen-fixing rhizobia and rhizospheric fungi, highlighting the panoply of genes and genotypic interactions (fungus, rhizobium, host plant) which may concur to plant symbiosis.

Biocontrol potential of Pseudomonas protegens ML15 against Botrytis cinerea causing gray mold on postharvest tomato (Solanum lycopersicum var. cerasiforme).

Gray mold, caused by Botrytis cinerea is a major cause of post-harvest rot of fresh fruits and vegetables. The utilization of selected microorganisms as biocontrol agents is a promising alternative to effectively control gray mold on tomatoes. The current study was conducted to explore potential biocontrol mechanisms of the Pseudomonas strain to control infections on post-harvest tomatoes. Among the 8 tested bacterial isolates, Pseudomonas protegens ML15 demonstrated antagonistic activity to Botrytis cinerea. Moreover, P. protegens ML15 exhibited the production of siderophores, hydrogen cyanide, ammonia, exopolysaccharides, lipase, biosurfactant, 2,4-diacetylphloroglucinol, and several other antifungal compounds, such as 1-tetradecanol, cyclododecane, 2,4-di-tert-butylphenol, and 2-methyl-1-hexadecanol. A comprehensive genomic analysis of P. protegens ML15 unravels 18 distinct genetic regions with the potential for biosynthesizing secondary metabolites, known for their pivotal role in biocontrol responses against plant pathogens. In vivo, experiments showed that both culture suspension and cell-free supernatant of P. protegens ML15 significantly reduced fungal growth (53.0 ± 0.63%) and mitigated disease development (52.8 ± 1.5%) in cherry tomatoes at four days post-B. cinerea inoculation. During the infection, the application of P. protegens ML15 resulted in the augmentation of total antioxidant, phenolic content, and ascorbic acids content. Thus, our results suggested that P. protegens ML15's role as a biocontrol agent against B. cinerea-induced postharvest tomato decay achieved through the secretion of antifungal substances, induction of tomato defense responses, and inhibition of mycelial growth of B. cinerea. These findings provide a significant contribution to the ongoing search for alternative, eco-friendly methods of controlling gray mold in fresh products. The utilization of P. protegens ML15 as a biocontrol agent could help to reduce the reliance on chemical fungicides and promote sustainable agriculture practices.

Reduction of bioavailability and phytotoxicity effect of cadmium in soil by microbial-induced carbonate precipitation using metabolites of ureolytic bacterium Ochrobactrum sp. POC9.

The application of ureolytic bacteria for bioremediation of soil contaminated with heavy metals, including cadmium (Cd), allows for the efficient immobilization of heavy metals by precipitation or coprecipitation with carbonates. Microbially-induced carbonate precipitation process may be useful also in the case of the cultivation of crop plants in various agricultural soils with trace but legally permissible Cd concentrations, which may be still uptaken by plants. This study aimed to investigate the influence of soil supplementation with metabolites containing carbonates (MCC) produced by the ureolytic bacterium Ochrobactrum sp. POC9 on the Cd mobility in the soil as well as on the Cd uptake efficiency and general condition of crop plants (Petroselinum crispum). In the frame of the conducted studies (i) carbonate productivity of the POC9 strain, (ii) the efficiency of Cd immobilization in soil supplemented with MCC, (iii) crystallization of cadmium carbonate in the soil enriched with MCC, (iv) the effect of MCC on the physico-chemical and microbiological properties of soil, and (v) the effect of changes in soil properties on the morphology, growth rate, and Cd-uptake efficiency of crop plants were investigated. The experiments were conducted in soil contaminated with a low concentration of Cd to simulate the natural environmental conditions. Soil supplementation with MCC significantly reduced the bioavailability of Cd in soil with regard to control variants by about 27-65% (depending on the volume of MCC) and reduced the Cd uptake by plants by about 86% and 74% in shoots and roots, respectively. Furthermore, due to the decrease in soil toxicity and improvement of soil nutrition with other metabolites produced during the urea degradation (MCC), some microbiological properties of soil (quantity and activity of soil microorganisms), as well as the general condition of plants, were also significantly improved. Soil supplementation with MCC enabled efficient Cd stabilization and significantly reduced its toxicity for soil microbiota and plants. Thus, MCC produced by POC9 strain may be used not only as an effective Cd immobilizer in soil but also as a microbe and plant stimulators.

Fit-for-Purpose Assessment of QuEChERS LC-MS/MS Methods for Environmental Monitoring of Organotin Compounds in the Bottom Sediments of the Odra River Estuary.

Organotin compounds (OTCs) are among the most hazardous substances found in the marine environment and can be determined by either the ISO 23161 method based on extraction with non-polar organic solvents and gas chromatography analysis or by the recently developed QuEChERS method coupled to liquid chromatography-mass spectrometry (LC-MS/MS). To date, the QuEChERS LC/MS and ISO 23161 methods have not been compared in terms of their fit-for-purpose and reliability in the determination of OTCs in bottom sediments. In the case of ISO 23161, due to a large number of interferences gas chromatography-mass spectrometry was not suitable for the determination of OTCs contrary to more selective determination by gas chromatography with an atomic emission detector. Moreover, it has been found that the derivatization of OTCs to volatile compounds, which required prior gas chromatography determination, was strongly affected by the sediments' matrices. As a result, a large amount of reagent was needed for the complete derivatization of the compounds. Contrary to ISO 23161, the QuEChERS LC-MS/MS method did not require the derivatization of OTC and is less prone to interferences. Highly volatile and toxic solvents were not used in the QuEChERS LC-MS/MS method. This makes the method more environmentally friendly according to the principles of green analytical chemistry. QuEChERS LC-MS/MS is suitable for fast and reliable environmental monitoring of OTCs in bottom sediments from the Odra River estuary. However, determination of di- and monobutyltin by the QuEChERS LC-MS/MS method was not possible due to the constraints of the chromatographic system. Hence, further development of this method is needed for monitoring di- and monobutyltin in bottom sediments.

Lanthanide-Dependent Methanol Metabolism of a Proteobacteria-Dominated Community in a Light Lanthanide-Rich Deep Environment.

This study investigated the occurrence and diversity of proteobacterial XoxF-type methanol dehydrogenases (MDHs) in the microbial community that inhabits a fossil organic matter- and sedimentary lanthanide (Ln3+)-rich underground mine environment using a metagenomic and metaproteomic approach. A total of 8 XoxF-encoding genes (XoxF-EGs) and 14 protein sequences matching XoxF were identified. XoxF-type MDHs were produced by Alpha-, Beta-, and Gammaproteobacteria represented by the four orders Methylococcales, Nitrosomonadales, Rhizobiales, and Xanthomonadales. The highest number of XoxF-EG- and XoxF-matching protein sequences were affiliated with Nitrosomonadales and Rhizobiales, respectively. Among the identified XoxF-EGs, two belonged to the XoxF1 clade, five to the XoxF4 clade, and one to the XoxF5 clade, while seven of the identified XoxF proteins belonged to the XoxF1 clade, four to the XoxF4 clade, and three to the XoxF5 clade. Moreover, the accumulation of light lanthanides and the presence of methanol in the microbial mat were confirmed. This study is the first to show the occurrence of XoxF in the metagenome and metaproteome of a deep microbial community colonizing a fossil organic matter- and light lanthanide-rich sedimentary environment. The presented results broaden our knowledge of the ecology of XoxF-producing bacteria as well as of the distribution and diversity of these enzymes in the natural environment.

Postdiagenetic Bacterial Transformation of Nickel and Vanadyl Sedimentary Porphyrins of Organic-Rich Shale Rock (Fore-Sudetic Monocline, Poland).

Nickel and vanadyl porphyrins belong to the so-called fossil geo- or sedimentary porphyrins. They occur in different types of organic matter-rich sediments but mostly occur in crude oils and their source rocks, oil shales, coals, and oil sands. In this study, we aimed to understand the process of bacterial transformation of geoporphyrins occurring in the subsurface shale rock (Fore-Sudetic Monocline, SW Poland). We studied these transformations in rock samples directly obtained from the field; in rock samples treated with bacterial strain isolated from shale rock (strain LM27) in the laboratory; and using synthetic nickel and vanadyl porphyrins treated with LM27. Our results demonstrate the following: (i) cleavage and/or degradation of aliphatic and aromatic substituents of porphyrins; (ii) degradation of porphyrin (tetrapyrrole) ring; (iii) formation of organic compounds containing 1, 2, or 3 pyrrole rings; (iv) formation of nickel- or vanadium-containing organic compounds; and (v) mobilization of nickel and vanadium. Our results also showed that the described bacterial processes change the composition and content of geoporphyrins, composition of extractable organic matter, as well as nickel and vanadium content in shale rock.

Sedimentary Cobalt Protoporphyrin as a Potential Precursor of Prosthetic Heme Group for Bacteria Inhabiting Fossil Organic Matter-Rich Shale Rock.

This study hypothesizes that bacteria inhabiting shale rock affect the content of the sedimentary cobalt protoporphyrin present in it and can use it as a precursor for heme synthesis. To verify this hypothesis, we conducted qualitative and quantitative comparative analyses of cobalt protoporphyrin as well as heme, and heme iron in shale rock that were (i) inhabited by bacteria in the field, (ii) treated with bacteria in the laboratory, and with (iii) bacterial culture on synthetic cobalt protoporphyrin. Additionally, we examined the above-mentioned samples for the presence of enzymes involved in the heme biosynthesis and uptake as well as hemoproteins. We found depletion of cobalt protoporphyrin and a much higher heme concentration in the shale rock inhabited by bacteria in the field as well as the shale rock treated with bacteria in the laboratory. Similarly, we observed the accumulation of protoporphyrin in bacterial cells grown on synthetic cobalt protoporphyrin. We detected numerous hemoproteins in metaproteome of bacteria inhabited shale rock in the field and in proteomes of bacteria inhabited shale rock and synthetic cobalt protoporhyrin in the laboratory, but none of them had all the enzymes involved in the heme biosynthesis. However, proteins responsible for heme uptake, ferrochelatase and sirohydrochlorin cobaltochelatase/sirohydrochlorin cobalt-lyase were detected in all studied samples.

Biosynthesis of Tetrapyrrole Cofactors by Bacterial Community Inhabiting Porphyrine-Containing Shale Rock (Fore-Sudetic Monocline).

This study describes for the first time the comprehensive characterization of tetrapyrrole cofactor biosynthetic pathways developed for bacterial community (BC) inhabiting shale rock. Based on the genomic and proteomic metadata, we have detailed the biosynthesis of siroheme, heme, cobalamin, and the major precursor uroporphyrinogen III by a deep BC living on a rock containing sedimentary tetrapyrrole compounds. The obtained results showed the presence of incomplete heme and cobalamin biosynthesis pathways in the studied BC. At the same time, the production of proteins containing these cofactors, such as cytochromes, catalases and sulfite reductase, was observed. The results obtained are crucial for understanding the ecology of bacteria inhabiting shale rock, as well as their metabolism and potential impact on the biogeochemistry of these rocks. Based on the findings, we hypothesize that the bacteria may use primary or modified sedimentary porphyrins and their degradation products as precursors for synthesizing tetrapyrrole cofactors. Experimental testing of this hypothesis is of course necessary, but its evidence would point to an important and unique phenomenon of the tetrapyrrole ring cycle on Earth involving bacteria.

Incidents and risk factors of biliary complications after orthotropic liver transplantation.

ABSTRACT: Biliary complications (BC) especially stenosis and strictures are the most common complications after orthotropic liver transplantation (OLT) procedure in adult recipients. The intention of this study was analyzed BC in 273 patients after OLT for the last 4 years in our department.Retrospective study of 273 patients underwent cadaveric donor liver transplantation between January 2014 and December 2017. Most of them (n = 268) have anastomosed bile duct in end to end, rest of them (n = 5) underwent hepaticojejunostomy. Statistical analysis was performed using Fischer exact test and Student t test. A P value <.05 was considered significant.BC were developed in 48/273 transplants (17.6%). The most frequent was biliary stricture (n = 42, 87.5%) followed by bile leak (n = 4, 8.3%) and choledocholitiasis (n = 2, 4.2%). Treatment was usually using endoscopic retrograde cholangiopancreatography. Recipients with hypotension during and after OLT treated by norepinephrine have a higher index of BC.Self-expanding metal stents implantation seems to be more effective than repeated balloon dilatation of anastomotic strictures with subsequent plastic biliary stent placement and associated with similar complication rate. Good fluid management against inotropic therapy may reduce risk of BC.

Biostimulation of sulfate-reducing bacteria used for treatment of hydrometallurgical waste by secondary metabolites of urea decomposition by Ochrobactrum sp. POC9: From genome to microbiome analysis.

Sulfate-reducing bacteria (SRB) are key players in many passive and active systems dedicated to the treatment of hydrometallurgical leachates. One of the main factors reducing the efficiency and activity of SRB is the low pH and poor nutrients in leachates. We propose an innovative solution utilizing biogenic ammonia (B-NH3), produced by urea degrading bacteria, as a pretreatment agent for increasing the pH of the leachate and spontaneously stimulating SRB activity via bacterial secondary metabolites. The selected strain, Ochrobactrum sp. POC9, generated 984.7 mg/L of ammonia in 24 h and promotes an effective neutralization of B-NH3. The inferred metabolic traits indicated that the Ochrobactrum sp. POC9 can synthesize a group of vitamins B, and the production of various organic metabolites was confirmed by GC-MS analysis. These metabolites comprise alcohols, organic acids, and unsaturated hydrocarbons that may stimulate biological sulfate reduction. With the pretreatment of B-NH3, sulfate removal efficiency reached ~92.3% after 14 days of incubation, whereas SRB cell count and abundance were boosted (~107 cell counts and 88 OTUs of SRB) compared to synthetic ammonia (S-NH3) (~103 cell counts and 40 OTUs of SRB). The dominant SRB is Desulfovibrio in both S-NH3 and B-NH3 pretreated leachate, however, it belonged to two different clades. By reconstructing the ecological network, we found that B-NH3 not only directly increases SRB performance but also promotes other strains with positive correlations with SRB.

Occurrence of XoxF-type methanol dehydrogenases in bacteria inhabiting light lanthanide-rich shale rock.

This study analyzed the occurrence of lanthanide-dependent (XoxF type) methanol dehydrogenases in the bacterial community dominated by Proteobacteria inhabiting shale rock. In total, 22 sequence matches of XoxF were identified in the metaproteome of the community. This enzyme was produced by bacteria represented by orders such as Rhizobiales, Rhodobacterales, Rhodospiralles, Burkholderiales and Nitrosomonadales. Among the identified XoxF proteins, seven belonged to XoxF1 clade and 15 to XoxF5 clade. This study is the first to show the occurrence of XoxF proteins in the metaproteome of environmental lithobiontic bacterial community colonizing an underground rock rich in light lanthanides. The presented results broaden our understanding of the ecology of XoxF producing bacteria as well as the distribution and diversity of these enzymes in the natural environment.

Recurrence of Hepatocellular Carcinoma After Liver Transplantation: A Single-Center Experience.

BACKGROUND There is a worldwide increase in use of liver transplantation (LT) for treatment of hepatocellular carcinoma (HCC). We analyzed our experience with LT for HCC to determine long-term and recurrence-free survival, accuracy of imaging diagnosis of HCC compared to the explant pathology, recurrence rate of HCC, and predictors of recurrence. MATERIAL AND METHODS The whole explant was examined by the same pathologist and compared with the baseline diagnosis established according to clinical, laboratory, and radiological data. A group of patients with pathologically confirmed HCC was characterized, with special attention to etiology, survival, recurrence, and diagnostic accuracy of imaging techniques. RESULTS Among 718 patients transplanted from 2000 to 2018 in our center, HCC was found in 166 explanted livers. In 42 cases the clinical diagnosis of HCC was not accurate, being either false positive or negative; however, the specificity and sensitivity of CT/MRI in HCC recognition was 97.87% and 88.24%, respectively. Five- and 10-year survival was 81.27% and 66.57%, respectively, and it was inferior to the overall survival. The recurrence rate was 9.6% with a median time to recurrence of 14 months and a median survival time of 9 months. Poor differentiation of HCC and HCV etiology of the baseline disease, but not previous DAA treatment, were the risk factors of HCC recurrence. CONCLUSIONS Adherence to strictly defined selection criteria for LT in HCC patients guarantees the success of LT in HCC treatment.

Application of Fully Covered Self-Expandable Metallic Stents with and without Antimigration Waist Versus Repeated Plastic Biliary Stent Placement in Management of Anastomotic Biliary Strictures After Orthotopic Liver Transplantation.

BACKGROUND Standard methods for endoscopic retrograde cholangiopancreatography (ERCP) management of anastomotic strictures (AS) after OLT includes repeated balloon dilation of the stricture with subsequent insertion of a plastic biliary stent (PBS). In post-OLT patients not responding to standard endoscopic treatment, the placement of fully covered self-expanding metal stents (FCSEMS) is a valid alternative to surgical treatment. The aim of this study was to compare the results of new FCSEMS implantation with the standard ERCP stricture management protocol and with conventional FCSEMS insertion. MATERIAL AND METHODS This retrospective study involved 39 post-OLT patients with confirmed diagnosis of biliary AS. Enrolled subjects were divided into 2 groups: the FCSEMS group (study group) and the PBS group (control group). The study group was divided into 2 subgroups: the conventional FCSEMS group and the new-type FCSEMS group. RESULTS Stricture recurrence after PBS placement was observed in 36.36% of controls and in only 9.52% of study group members (P=0.170). Recurrence rates in patients after conventional FCSEMS and new type FCSEMS implantation was similar (10% vs. 9.09%; P=0.501). The applied treatment was successful in 82.61% of study group members and only 43.75% of controls (P=0.029). Success rates of conventional FCSEMS and new-type SEMS insertion did not differ significantly (81.82% vs. 83.33%, P=0.649). There was no statistically significant difference in complication rates between groups (P=0.879). CONCLUSIONS Implantation of FCSEMS is more effective than repeated balloon dilatation of AS with subsequent PBS placement and is they have similar complication rates. Application of new-type FCSEMS gives results comparable to conventional FCSEMS.

Bacterial weathering of fossil organic matter and organic carbon mobilization from subterrestrial Kupferschiefer black shale: long-term laboratory studies.

A large part of the organic carbon present in the lithosphere is trapped in fossil organic matter deposited in sedimentary rocks. Only specialized microorganisms are able to degrade it contributing to the return of the carbon to the global cycle. The role of bacteria in this process is not yet completely understood. In the present laboratory studies, subterrestrial organic-rich ∼256-million-year-old Kupferschiefer black shale was exposed to the activity of an indigenous consortium of lithobiontic bacteria for 365 days under aerobic conditions. An interdisciplinary research approach was applied, consisting of a detailed comparison of the chemical composition of extractable bitumens as well as resistant to extraction kerogen of the unweathered black shale to that of the bioweathered and chemically weathered, identification of mobilized organic compounds and spectrometry-based determination of proteomic composition of the bacterial biofilm. The oxidative bioweathering of bitumens and kerogen was confirmed. The mobilization of organic carbon in the form of oxidized organic compounds, such as monohydroxy and dihydroxy alcohols, aldehydes, monocarboxylic and dicarboxylic acids and esters due to the microbial activity, was documented. The enzymes crucial for the aerobic metabolism of aliphatic and aromatic hydrocarbons such as monooxygenases and dehydrogenases were identified in the epilithic biofilm inhabiting the black shale.

Shewanella sp. O23S as a Driving Agent of a System Utilizing Dissimilatory Arsenate-Reducing Bacteria Responsible for Self-Cleaning of Water Contaminated with Arsenic.

The purpose of this study was a detailed characterization of Shewanella sp. O23S, a strain involved in arsenic transformation in ancient gold mine waters contaminated with arsenic and other heavy metals. Physiological analysis of Shewanella sp. O23S showed that it is a facultative anaerobe, capable of growth using arsenate, thiosulfate, nitrate, iron or manganite as a terminal electron acceptor, and lactate or citrate as an electron donor. The strain can grow under anaerobic conditions and utilize arsenate in the respiratory process in a broad range of temperatures (10-37 °C), pH (4-8), salinity (0%-2%), and the presence of heavy metals (Cd, Co, Cr, Cu, Mn, Mo, Se, V and Zn). Under reductive conditions this strain can simultaneously use arsenate and thiosulfate as electron acceptors and produce yellow arsenic (III) sulfide (As2S3) precipitate. Simulation of As-removal from water containing arsenate (2.5 mM) and thiosulfate (5 mM) showed 82.5% efficiency after 21 days of incubation at room temperature. Based on the obtained results, we have proposed a model of a microbially mediated system for self-cleaning of mine waters contaminated with arsenic, in which Shewanella sp. O23S is the main driving agent.

Extracellular compounds produced by bacterial consortium promoting elements mobilization from polymetallic Kupferschiefer black shale (Fore-Sudetic Monocline, Poland).

Culture experiments employing Fe-deficient medium showed that a consortium of indigenous microorganisms isolated from Kupferschiefer black shale produced a mixture of extracellular compounds containing siderophores which could form complexes with a wide range of elements and were able to mediate element mobilization from polymetallic black shale. The mobilization of a diverse array of elements including a number of essential trace elements (Co, Cu, Mn, Mo, Zn) and toxic species (As) was shown. Since the bacteria used in this study were originally obtained from a subsurface copper deposit, these results highlight the potential importance of extracellular compounds in biogeochemical cycles of elements in underground environment and their ecological significance in promoting the uptake of essential trace metals and resistance to toxic elements.