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Ralstonia insidiosa, a gram-negative waterborne bacteria able to survive and grow in any type of water source, can cause nosocomial infections, and are considered emerging pathogens of infectious diseases in hospital settings. In this study, we report an outbreak of R. insidiosa at our center related to contaminated heparinized syringes.The present study was conducted in a tertiary care university hospital in Turkey. An outbreak analysis was performed between September 2021 and December 2021. Microbiological samples were obtained from environmental sources and from patient blood cultures. Species identification was performed using matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS). To investigate the clonality of strains, all confirmed isolates were sent to the National Reference Laboratory and pulsed-field gel electrophoresis (PFGE) was used to perform molecular typing.Seventeen R. insidiosa isolates were identified from the blood cultures of 13 patients from various wards and intensive care units. Isolates from seven patient blood cultures and two heparinized blood gas syringes were characterized by PFGE. All isolates were found to belong to the same clone of R. insidiosa.R. insidiosa was identified as the cause of a nosocomial infection outbreak in our hospital, which was then rapidly controlled by the infection-control team. When rare waterborne microorganisms grow in blood or other body fluid cultures, clinicians and the infection-control team should be made aware of a possible outbreak.

Due to the constant increase in the number of plant diseases and the lack of available treatments, there has been a growing interest in plant extracts over the past few decades. Numerous studies suggest that plant extract molecules possess valuable antimicrobial activities, particularly against fungi and bacteria. This suggests that these biomaterials could potentially serve as attractive therapeutic options for the treatment of phytopathogen infections. In the present study, we investigated and analyzed the methanolic extract of Eryngium campestre L. whole plant extract using HPLC. The analysis revealed the presence of several polyphenolic constituents, with benzoic acid, catechol, quercetin, vanillic acid, resveratrol, naringenin, and quinol being the most abundant. The amounts of these constituents were determined to be 2135.53, 626.728, 579.048, 356.489, 323.41, 153.038, and 128.77 mg/kg, respectively. Furthermore, we isolated and identified different plant fungal and bacterial isolates from symptomatic potato plants, which were accessioned as Rhizoctonia solani (OQ880458), Fusarium oxysporum (OQ820156) and Fusarium solani (OQ891085), Ralstonia solanacearum (OQ878653), Dickeya solani (OQ878655), and Pectobacterium carotovorum (OQ878656). The antifungal activity of the extract was assessed using fungal growth inhibitions (FGI) at concentrations of 100, 200, and 300 µg/mL. The results showed that at the lowest concentration tested (100 µg/mL), the extract exhibited the highest effectiveness against R. solani with an FGI of 78.52%, while it was least effective against F. solani with an FGI of 61.85%. At the highest concentration tested, the extract demonstrated the highest effectiveness against R. solani and F. oxysporum, with FGIs of 88.89% and 77.04%, respectively. Additionally, the extract displayed a concentration-dependent inhibitory effect on all three bacterial pathogens. At the highest concentration tested (3000 µg/mL), the extract was able to inhibit the growth of all three bacterial pathogens, although the inhibition zone diameter varied. Among the bacterial pathogens, D. solani exhibited the highest sensitivity to the extract, as it showed the largest inhibition zone diameter at most of the extract concentrations. These findings highlight the potential of the E. campestre extract as a source of natural antimicrobial agents for controlling various plant pathogens. Consequently, it offers a safer alternative to the currently employed protective methods for plant disease management.

Bacterial wilt is one of the main diseases of Solanum spp., which caused by Ralstonia solanacearum (RS), formerly known as Pseudomonas solanacearum. Different concentrations of chitosan nanoparticles have been evaluated as one of the alternative methods of disease management in vitro and in vivo to reduce the risks of pesticide residues. Results in vitro experiment indicated that RS5 isolate was the most virulence one compared to RS1 and RS3. Increasing concentration of nano-chitosan, lead to increase inhibition zone, and this was observed at higher concentrations (100 and 200 µg/ml). In vivo results showed the highest concentration of spraying chitosan nanoparticles increase percentage reduction of disease incidence and severity, in effected potato and tomato plants. Recorded data of disease incidence and severity in treated potato plants were 78.93 % and 71.85 %, while on tomato plants were 81.64 % and 77.63 %, respectively compared to untreated infected potato plants were recorded 15.38 %, 20.87 %, and tomato plants were 20.98 % and 28.64 %. Results also revealed that 100 µg/ml of chitosan nanoparticles the lowest treatments used as soil amended curative treatments led to incease percentage reduction of disease incidence and severity, respectively on potato and tomato plants, but less than preventive treatment. The results registered that on potato plant were 54.93 % and 52.65 %, whilst recorded on tomato plants were 59.93 % and 56.74 %. Transmission electron microscopy (TEM) micrpgraphs illustrated that morphological of healthy R. solanacearum cells were undesirably stained with uranyl. The electron-dense uranyl acetate dye was limited to the cell surface slightly than the cytoplasm, which designated the integrity of the cell film of healthy cells. While bacterial cells treated with nano-chitosan, showed modification in the external shape, such as lysis of the cell wall and loss of cell flagella. Also, the result of using Random amplified polymorphic DNA (RAPD)-PCR observed that differences in treated Ralstonia solanancearum genotype by nano-chitosan compared to the genotype of the same untreated isolate.

Ralstonia causes wilt diseases by colonizing xylem vessels and disrupting water transport. The dogma is that bacterial biomass clogs vessels and reduces the flow of xylem sap due to Ralstonia abundance. However, the physiological mechanism of xylem disruption during bacterial wilt is untested. Using a tomato and Ralstonia pseudosolanacearum GMI1000 model, we visualized and quantified spatiotemporal dynamics of xylem disruption during bacterial wilt. First, we measured stomatal conductance of leaflets on mock-inoculated and wilt-symptomatic plants. Wilted leaflets had reduced stomatal conductance, as did turgid leaflets on the same petiole as wilted leaflets. Next, we used X-ray microcomputed tomography (X-ray microCT) and light microscopy to differentiate between mechanisms of xylem disruption: blockage by bacterial biomass, blockage by vascular tyloses, or sap displacement by gas embolisms. We imaged intact plant stems to quantify embolized vessels. Embolized vessels were rare, but infected plants with low bacterial populations had a nonsignificant trend of more vessel embolisms. To test whether vessels are clogged during bacterial wilt, we imaged excised stems after brief dehydration. Most vessels in mock-infected plants emptied their contents after excision, but nonconductive clogged vessels were abundant in infected plants by 2 days post infection. At wilt onset, when bacterial populations exceeded 5 × 108CFU/g stem tissue, approximately half of the vessels were clogged with electron-dense bacterial biomass. We found no evidence of tyloses in X-ray microCT reconstructions or from light microscopy of preserved stems. Therefore, bacterial blockage of vessels appears to be the principal cause of xylem disruption during Ralstonia wilt.[Formula: see text] The author(s) have dedicated the work to the public domain under the Creative Commons CC0 “No Rights Reserved” license by waiving all of his or her rights to the work worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law, 2022.

Bacterial pathogens in the Ralstonia solanacearum species complex (RSSC) infect the water-transporting xylem vessels of plants, causing bacterial wilt disease. Strains in RSSC phylotypes I and III can reduce nitrate to dinitrogen via complete denitrification. The four-step denitrification pathway enables bacteria to use inorganic nitrogen species as terminal electron acceptors, supporting their growth in oxygen-limited environments such as biofilms or plant xylem. Reduction of nitrate, nitrite, and nitric oxide all contribute to the virulence of a model phylotype I strain. However, little is known about the physiological role of the last denitrification step, the reduction of nitrous oxide to dinitrogen by NosZ. We found that phylotypes I and III need NosZ for full virulence. However, strains in phylotypes II and IV are highly virulent despite lacking NosZ. The ability to respire by reducing nitrate to nitrous oxide does not greatly enhance the growth of phylotype II and IV strains. These partial denitrifying strains reach high cell densities during plant infection and cause typical wilt disease. However, unlike phylotype I and III strains, partial denitrifiers cannot grow well under anaerobic conditions or form thick biofilms in culture or in tomato xylem vessels. Furthermore, aerotaxis assays show that strains from different phylotypes have different oxygen and nitrate preferences. Together, these results indicate that the RSSC contains two subgroups that occupy the same habitat but have evolved divergent energy metabolism strategies to exploit distinct metabolic niches in the xylem. IMPORTANCE Plant-pathogenic Ralstonia spp. are a heterogeneous globally distributed group of bacteria that colonize plant xylem vessels. Ralstonia cells multiply rapidly in plants and obstruct water transport, causing fatal wilting and serious economic losses of many key food security crops. The virulence of these pathogens depends on their ability to grow to high cell densities in the low-oxygen xylem environment. Plant-pathogenic Ralstonia can use denitrifying respiration to generate ATP. The last denitrification step, nitrous oxide reduction by NosZ, contributes to energy production and virulence for only one of the three phytopathogenic Ralstonia species. These complete denitrifiers form thicker biofilms in culture and in tomato xylem, suggesting they are better adapted to hypoxic niches. Strains with partial denitrification physiology form less biofilm and are more often planktonic. They are nonetheless highly virulent. Thus, these closely related bacteria have adapted their core metabolic functions to exploit distinct microniches in the same habitat.

Phytopathogenic bacteria secrete type III effector (T3E) proteins directly into host plant cells. T3Es can interact with plant proteins and frequently manipulate plant host physiological or developmental processes. The proper subcellular localization of T3Es is critical for their ability to interact with plant targets, and knowledge of T3E localization can be informative for studies of effector function. Here we investigated the subcellular localization of 19 T3Es from the phytopathogenic bacteria Ralstonia pseudosolanacearum and Ralstonia solanacearum. Approximately 45% of effectors in our library localize to both the plant cell periphery and the nucleus, 15% exclusively to the cell periphery, 15% exclusively to the nucleus, and 25% to other organelles, including tonoplasts and peroxisomes. Using tomato hairy roots, we show that T3E localization is similar in both leaves and roots and is not impacted by Solanum species. We find that in silico prediction programs are frequently inaccurate, highlighting the value of in planta localization experiments. Our data suggest that Ralstonia targets a wide diversity of cellular organelles and provides a foundation for developing testable hypotheses about Ralstonia effector function.

Metabolic engineering 68, 199-209 (2021). doi:10.1016/j.ymben.2021.10.006, Published by Academic Press, Orlando, Fla.

Phasin (PhaP), one of the polyhydroxyalkanoate granule-associated protein, enhances cell growth and polyhydroxybutyrate (PHB) biosynthesis by regulating the number and size of PHB granules. However, few studies have applied phasins to various PHB production conditions. In this study, we identified novel phasin genes from the genomic data of Arctic soil bacterium Pseudomonas sp. B14-6 and determined the role of phaP1Ps under different PHB production conditions. Transmission electron microscopy and gel permeation chromatography revealed small PHB granules with high-molecular weight, while differential scanning calorimetry showed that the extracted PHB films had similar thermal properties. The phasin protein derived from Pseudomonas sp. B14-6 revealed higher PHB production and exhibited higher tolerance to several lignocellulosic biosugar-based inhibitors than the phasin protein of Ralstonia eutropha H16 in a recombinant Escherichia coli strain. The increased tolerance to propionate, temperature, and other inhibitors was attributed to the introduction of phaP1Ps, which increased PHB production from lignocellulosic hydrolysate (2.39-fold) in the phaP1Ps strain. However, a combination of phasin proteins isolated from two different sources did not increase PHB production. These findings suggest that phasin could serve as a powerful means to increase robustness and PHB production in heterologous strains.

Pseudomonas oleovoransNCIMB 6576 and Ralstonia eutropha NCIMB 10442 were used for the production of Polyhydroxyalkanoates (PHA) from industrial waste cooking oils, the bacteria were cultured on tryptone soya broth (TSB) and Tryptone soya agar (TSA).P. oleovoransNCIMB6576 gave a better percentage PHB yield (8.2%) with PS oil as carbon source as compared to 6.45% with TS oil. However, a very low yield (0.64%) was recorded whenP. oleovoransNCIMB6576 was grown on TSB without the oils as carbon source. Ralstonia eutropha NCIMB 10442 gave an appreciable yield of 13.63% and 14.80% with PS and TS oil samples respectively as carbon source with negligible variation in the yields. The results obtained across all experiments were compared with one another. The SEM images from the PHB samples generated from the experiments shows that there is a slight difference in the surface morphologies of the PHB with respect to the oil samples as well as the different bacteria used in the experiment.

Marine macroalgae are potential renewable feedstocks for valuable biomaterials. Among them, alginate is a primary component in brown algae that can be nonenzymatically converted and enzymatically degraded by alginate lyases to 4-deoxy- l -erythro-5-hexoseulose uronic acid (DEH). Here, we constructed alginolytic enzyme complexes comprising two different alginate lyases for synergistic alginate degradation. The complexes showed good thermostability with 60% of the residual activity at high temperature (60 °C). Furthermore, they produced 0.85 and 0.18 mg/mL DEH from alginate and natural brown algae as substrates, respectively. The enzyme complex successfully decomposed brown algal biomass, resulting in a 3.15-fold improvement in DEH when compared to free enzymes. The Ralstonia eutropha strain with alginolytic enzyme complexes on the cell surface showed higher Polyhydroxybutyrate (PHB) production and produced 2.58 g/L PHB from alginate. After the use of alginate, remaining biomass such as fucoidan and laminaran can also be used in the future for high value ingredients in nutritional, medical device, skincare and dermatological products. These results demonstrate that it is possible to create more efficient strategies for producing biodegradable PHB and functional polysaccharides from brown algal substrates.

We share whole genome sequences of six strains from the Ralstonia solanacearum species complex, a diverse group of Betaproteobacteria that cause plant vascular wilt diseases. Using single-molecule real-time technology, we sequenced and assembled full genomes of Rs5 and UW700, two phylotype IA-sequevar 7 (IIA-7) strains from the southeastern United States that are closely related to the R. solanacearum species type strain, K60, but were isolated >50 years later. Four sequenced strains from Africa include a soil isolate from Nigeria (UW386, III-23), a tomato isolate from Senegal (UW763, I-14), and two potato isolates from the Madagascar highlands (RUN2474, III-19 and RUN2279, III-60). This resource will support studies of the genetic diversity, ecology, virulence, and microevolution of this globally distributed group of high-impact plant pathogens. [Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license .

To improve understanding of the role of Ralstonia in cystic fibrosis (CF), whole genomes of 18 strains from clinical samples were sequenced using Illumina technology. Sequences were analysed by core genome Multi-Locus Sequence Typing, Average Nucleotide Identity based on BLAST (ANIb), RAST annotation, and by ResFinder. Phylogenetic analysis was performed for the 16S rRNA gene, and the OXA-22 and OXA-60 ß-lactamase families. The minimal inhibitory concentrations (MICs) were determined using broth microdilution. ANIb data for the 18 isolates and 54 strains from GenBank, supported by phylogenetic analysis, showed that 8 groups of clusters (A-H), as well as subgroups that should be considered as species or subspecies. Groups A-C contain strains previously identified as Ralstonia solanacearum and Ralstonia pseudosolanacearum. We propose that group A is a novel species. Group B and C are Ralstonia syzygii, Ralstonia solanacearum, respectively. Group D is composed of Ralstonia mannitolilytica and Group E of Ralstonia pickettii. Group F and G should be considered novel species. Group H strains belong to R. insidiosa. OXA-22 and OXA-60 family ß-lactamases were encoded by all strains. Co-trimoxazole generally showed high activity with low MICs (≤1 mg/l) as did ciprofloxacin (≤0.12 mg/l). MICs against the other antibiotics were more variable, but generally high. RAST annotation revealed limited differences between the strains, and virulence factors were not identified. The taxonomy of the genus Ralstonia is in need of revision, but sequencing additional isolates is needed. Antibiotic resistance levels are high. Annotation did not identify potential virulence factors.

Soil disease-suppressiveness depends on complex interactions among pathogens, native microbiota, and physicochemical properties, while these interactions remain understudied. Comparing field and microcosm experiments, we investigated the significance of these interactions in disease emergence or suppression using structural equation modelling (SEM) and receiver operating characteristic curve (ROC) analyses. We observed significant differences in the relative abundance of pathogenic and beneficial microbes, alpha and beta diversity indices between disease-conducive and -suppressive rhizosphere soils. The pathogenic (Ralstonia) and beneficial (Bacillus) taxa dominated disease-conducive and -suppressive rhizosphere soils, respectively. Moreover, the co-occurrences of Ralstonia with native microorganisms were positive and negative in the disease-conducive and -suppressive soils, respectively. These results suggest the supportive (Rudaea) and suppressive (Enterobacter, Bacillus) role of indigenous microbes in the invasion of soil and plant systems by Ralstonia. The SEM and ROC analysis predicted that Ralstonia invaded rhizospheric microbial networks and caused peanut wilt under high than low soil phosphorus conditions. Our results suggest the importance of soil phosphorus availability in altering the microbial interactions, thus leading to soil invasion by Ralstonia. Thus, we conclude by saying that feeding soil with high amounts of available phosphorus could deplete plant-beneficial microbes and increase the pathobiome abundance that may compromise plant health.

Continuous cropping of tomato (Solanum lycopersicum Mill.) causes soil degradation, accumulating Ralstonia solanacearum that induce Ralstonia wilt notably in plastic shed soils. Arbuscular mycorrhizal (AM) fungi play a crucial role in protecting hosts against such soil-borne pathogens, but comprehensive understanding of the soil–plant defense systems upon mycorrhization is not clear yet, especially at the later period of fruit production. The aim of this study was to investigate the underlining mechanisms in both soil and plant. A 10-week greenhouse pot experiment with four treatments, including control and inoculation with Funneliformis caledonium (Fc), R. solanacearum (Rs), and both strains (Rs + Fc), was carried out on a sterilized soil. Pots with two tomato plants each were randomly arranged with six replicates per treatment. The wilt severity; the tissue biomass and nutrient content; the root mycorrhizal colonization and total phenolic compounds; the leaf peroxidase (POD), polyphenol oxidase (PPO), and phenylalanine ammonia lyase (PAL) activities; and soil AM fungi and R. solanacearum abundances, soil pH, organic C and nutrient concentrations, and phosphatase activity were all tested. Both redundancy analysis (RDA) and structural equation modeling (SEM) were performed to illustrate plant overall performance among treatments and to elucidate the major influencing pathways of AM fungi. The additional inoculation with F. caledonium resulted in significant decreases of soil R. solanacearum abundance and Olsen-P concentration, as well as increases of soil pH, organic C concentration, and phosphatase activity, as compared to the soil only inoculated with R. solanacearum. Mycorrhizal inoculation also increased root total phenolic compound content, and leaf POD and PPO activities, but reduced shoot/root K ratio in plants under the attack of R. solanacearum, thereby alleviating Ralstonia wilt severity by 65.7% and yield loss by 46.5%. The RDA and SEM results revealed significant variation in plant overall performance among treatments, and the contribution of AM fungi in suppressing tomato Ralstonia wilt and yield damage particularly via ameliorating soil quality and alleviating plant metabolic pressure. This study verified the bio-protection of AM fungi in both soil and plant systems against tomato Ralstonia wilt. Mycorrhization shifted the soil environment and suppressed soil R. solanacearum population, and also modulated plant nutrient translocation, increased phenolic compounds synthetization, and activated defense enzymes. Through establishing the integrated defense systems in both rhizosphere and plant, AM fungi alleviated the severity of Ralstonia disease and ameliorated yield damage in tomato.

Polyhydroxybutyrate (PHB) is a biodegradable plastic with physical properties similar to petrochemically derived plastics. Here, Shewanella marisflavi BBL25 was engineered by inserting the pLW487 vector containing polyhydroxyalkanoates synthesis genes from Ralstonia eutropha H16. Under optimal conditions, the engineered S. marisflavi BBL25 produced 1.99 ± 0.05 g/L PHB from galactose. The strain showed high tolerance to various inhibitors and could utilize lignocellulosic biomass for PHB production. When barley straw hydrolysates were used as a carbon source, PHB production was 3.27 ± 0.19 g/L. In addition, PHB production under the microbial fuel cell system was performed to confirm electricity coproduction. The maximum electricity current output density was 1.71 mA/cm2, and dry cell weight (DCW) and PHB production were 11.4 g/L and 6.31 g/L, respectively. Our results demonstrated PHB production using various lignocellulosic biomass and the feasibility of PHB and electricity production, simultaneously, and it is the first example of PHB production in engineered Shewanella.

Sugarcane molasses was examined for the production of poly(3-hydroxybutyrate) [P(3HB)] and poly(3-hydroxybutyrate-co-lactate) [P(3HB-co-LA)] in recombinant Ralstonia eutropha strains expressing Mannheimia succiniciproducens sacC gene encoding β-fructofuranosidase, which can hydrolyze sucrose into glucose and fructose in the culture medium. When crude sugarcane molasses was added to the culture medium to support 20 g/L of sucrose in flask cultivation, the growth of R. eutropha NCIMB11599 expressing the sacC gene was significantly inhibited, which resulted in OD600 of 1.2 with P(3HB) content of 0.1wt%. The inhibition of cell growth due to the usage of the crude sugarcane molasses was relieved by pretreatment of sugarcane molasses with activated charcoal. Sugarcane molasses pretreated with activated charcoal could support the growth of R. eutropha NCIMB11599 expressing the sacC gene to OD600 of 87.2 with P(3HB) content of 82.5 wt% in batch fermentation when it was added to culture medium to support 20 g/L of sucrose. Also, R. eutropha 437–540 expressing Escherichia coli ldhA gene encoding lactate dehydrogenase along with the sacC gene produced P(3HB-co-6.2 mol%LA) with 29.1 wt% polymer content from sugarcane molasses in batch fermentation.

Polyhydroxybutyrate (PHB) is a biodegradable plastic that can be used as an alternative to petrochemical-based plastics. PHB is produced by various microorganisms such as Ralstonia, Halomonas, and Bacillus species. However, there are very few strains that produce PHB using xylose, an abundant and inexpensive carbon source. In this study, ten xylose-utilizing PHB producers isolated from South Korean marine environments were screened and characterized. Among these isolates, Bacillus sp. SM01, a newly identified strain, produced the highest amount of PHB using xylose. Under optimal conditions, the maximum dry cell weight (DCW) was 3.41 ± 0.09 g/L, with 62% PHB content, and Bacillus sp. SM01 showed Poly (3-hydroxybutyrate-co-3-hydroxyvalerate) copolymer production with propionate; however, the growth of Bacillus sp. SM01 was greatly inhibited by the presence of glucose. Co-culturing Bacillus sp. SM01 with Cupriavidus necator NCIMB 11599 resulted in increased DCW, PHB production, and utilization of glucose and xylose, the main sugar of lignocellulosic biomass, compared with the monoculture. Our results indicated that this co-culture system can be used to increase PHB production and overcome the limitation of sugar consumption associated with Bacillus sp. SM01 and C. necator.

Ralstonia solanacearum causes bacterial wilt of tomato and limits the crop production, and antagonistic microorganisms use to suppress the disease, of which Trichoderma and Pseudomonas species are the most effective agents to control bacterial wilt. In the present study, attempt was made to isolate these two microorganisms to evaluate their effectiveness to control R. solanacearum, the causal agent of bacterial wilt disease of tomato under greenhouse conditions. Thus R. solanacearum, Pseudomonas and Trichoderma spp. were isolated from wilted and healthy tomato plants grown from farmer's field in Ziway and Meki, Oromia Ethiopia. The virulence of the pathogen and the antagonistic effect of the bacteria and fungi were evaluated against R. solanacearum in vitro and in vivo condition. Based on the in vitro results the best two isolates were selected to show their antagonistic effect under greenhouse condition in single and combined designs. The result showed the pathogenicity test of the isolates were evaluated under greenhouse condition, and isolate AAURS1 showed highest virulence (75%) followed by isolate APPRCRS2 with pathogenicity of 50%. With regard to antagonism test, isolates AAURB20 and AAUTR23 showed the highest inhibition against R. solanacearum with inhibition zone of 16 and 15 mm, respectively. Among the treatments co-inoculation was more effective and reduced disease incidence by 13.33% and increased the bio-control efficacy by 72.22% when compared with individual treatment and negative control. The isolates significantly increased the plant height and dry weight by 72.33 cm and 12.18 g, respectively. Thus, the combined use of the biocontrol agents significantly reduced the incidence of tomato bacterial wilt disease. However, their performance should be evaluated using other yield parameters under field conditions to produce healthy tomato seedling to minimize the use of chemicals and reduce environmental pollution. Key words: Biocontrol, Pseudomonas, Ralstonia solanacearum, Trichoderma.

Background: Premature infants are considered high-risk subgroup for neonatal sepsis due to yet defective immune system, interventions practised and synergy of factors favoring multiple resistance of Gram-positive and Gram-negative pathogens to antimicrobial agents. Case presentation: We present a case of late-onset neonatal sepsis in a premature infant caused by an uncommon pathogen; a premature infant of extremely low birth weight had in his 4th week of life severe clinical deterioration with lethargy, fever, pallor, mottling, abdominal distention, tachycardia, and worsening respiratory impairment. Full septic screen was performed, broad-spectrum antibiotic therapy was initiated and supportive care per needs was provided. Blood cultures (endotracheal tube tip cultures) isolated meropenem- and gentamicin-resistant strain of rare pathogen Ralstonia mannitolilytica. Ralstonia spp. are aerobic, Gram-negative, lactose non-fermenting, oxidaseand catalase-positive bacilli, thriving in water and soil. Ralstonia spp. are identified only sporadically as causative agents of neonatal sepsis; to our knowledge, this is the second report of neonatal sepsis due to R. mannitolilytica in the literature so far. Our patient was eventually treated (per sensitivity pattern) with intravenous ciprofloxacin and recovered well from the infection. Conclusion: We intend to raise awareness among neonatologists with regard to early detection of unusual pathogens, the emergence of antibiotic resistance patterns, and the obligation for adherence to infection control policies.

Integrated bacteriophages (prophages) can impact host cells, affecting their lifestyle, genomic diversity, and fitness. However, many basic aspects of how these organisms affect the host cell remain poorly understood. Ralstonia solanacearum is a gram-negative plant pathogenic bacterium that encompasses a great diversity of ecotypes regarded as a species complex (R. solanacearum Species Complex - RSSC). RSSC genomes have a mosaic structure containing numerous elements, signaling the potential for its evolution through horizontal gene transfer. Here, we analyzed 120 Ralstonia spp. genomes from the public database to identify prophage sequences. In total, 379 prophage-like elements were found in the chromosome and megaplasmid of Ralstonia spp. These elements encode genes related to host fitness, virulence factors, antibiotic resistance, and niche adaptation, which might contribute to RSSC adaptability. Prophage-like elements are widespread into the complex in different species and geographic origins, suggesting that the RSSC phages are ancestrally acquired. Complete prophages belonging to the families Inoviridae, Myoviridae, and Siphoviridae were found, being the members of Inoviridae the most abundant. Analysis of CRISPR-Cas spacer sequences demonstrated the presence of prophages sequences that indicate successive infection events during bacterial evolution. Besides complete prophages, we also demonstrated 14 novel putative prophages integrated into Ralstonia spp. genomes. Altogether, our results provide insights into the diversity of prophages in RSSC genomes and suggest that these elements may deeply affect the virulence and host adaptation and shaping the genomes among the strains of this important pathogen.

To understand the diversities of diazotrophs and denitrifiers in red paddy soil under long-term fertilization conditions, nifH, nirK, and nosZ libraries were constructed by PCR–RFLP. nirK gene diversity proved to be lower than that of nosZ and nifH, and nirK and nosZ genes were more sensitive to different fertilization treatments than the nifH gene was. The 3 libraries were dominated by diverse microbes, including the Alpha, Beta, Gamma, and Delta subclasses of the Proteobacteria. Long-term addition of urea with straw mulch and azophoska increased the abundance of nonsymbiotic diazotrophs, which indicated that nonsymbiotic diazotrophs were responsible for the majority of the nitrogen-fixing ability in paddy soil. In addition, a potential link between nifH and nosZ was found due to the existence of nitrogen fixers, such as Bradyrhizobium and Ralstonia, in the nosZ library. The main chemical factors affecting the 3 genes were identified: pH was the most important factor of the nifH community; the nirK gene was more affected by pH and organic matter; available potassium and the carbon-to-nitrogen ratio significantly influenced the community structure of the nosZ gene.

The biosynthesis of polyhydroxyalkanoates from crude fish oil by bacteria isolated from soil samples was investigated. More than 200 bacterial colonies were isolated, among them 15 isolates were selected and tested for their abilities to synthesize PHA from crude fish oil. Eight strains were found to produce the poly-3-hydroxybutyrate (PHB) from crude fish oil with PHB content ranging from 7.4 to 50.1 wt %. The highest PHB producing bacterium (strain M91) was chosen for further studies. Based on the 16S rRNA gene sequences, strain M91 was classified into genus Ralstonia. In flask experiment, high cell dry weight (CDW) of 3.93 g/L and PHB concentration of 2.43 g/L were obtained by strain M91 when cultured in the medium containing 15 g/L crude fish oil. The CDW and PHB concentration were then increased to 5.32 and 2.73 g/L, respectively, when strain M91 was cultivated in 10-liter bioreactor. The production of PHB by Ralstonia M91 using crude fish oil as an inexpensive carbon source is expected to reduce the production cost and motivates further studies.

The objective of this study was to evaluate changes in the bacterial community in sugarcane silage, in distinct soil types along the storage period. We depicted the bacterial community associated with sugarcane, before and after ensiling, through a massive sequencing of the gene 16S rRNA using MiSeq platform. The ensilage process shifted the composition of the bacterial community from the heterofermentative lactic acid bacteria Leuconostoc to bacteria belonging to the genera Acinetobacter, Ralstonia and Novosphingobium. However, this shift did not convey statically significant differences in alfa diversity metrics. In addition, similarity percentage analysis showed that the bacterial Operational Taxonomic Units that were primarily responsible for the observed differences were Leuconostoc, Pseudomonas, Acinetobacter, Ralstonia, Fructobacillus, Novosphingobium, Lactobacillus, Burkholderia and Clostridium sensu stricto 1. The storage period was the most important factor responsible for changes in the bacterial community of silages. Results confirmed that the type of soil did not influence the dissimilarity found among samples.

Tomato is one of the most remunerable and widely grown vegetables in the world. The objective of this study was to evaluate tomato varieties for nematode and Ralstonia diseases resistance and productivity.

An astonishing variety of functions has been attributed to polyphosphate (polyP) in prokaryotes. Besides being a reservoir of phosphorus, functions in exopolysaccharide formation, motility, virulence and in surviving various forms of stresses such as exposure to heat, extreme pH, oxidative agents, high osmolarity, heavy metals and others have been ascribed to polyP. In this contribution, we will provide a historical overview on polyP, will then describe the key proteins of polyP synthesis, the polyP kinases, before we will critically assess of the underlying data on the multiple functions of polyP and provide evidence that – with the exception of a P-storage-function – most other functions of polyP are not relevant for survival of Ralstonia eutropha, a biotechnologically important beta-proteobacterial species.

Some pathogenic or symbiotic Gram-negative bacteria can manipulate the ubiquitination system of the eukaryotic host cell using a variety of strategies. Members of the genera Salmonella, Shigella, Sinorhizobium, and Ralstonia, among others, express E3 ubiquitin ligases that belong to the NEL family. These bacteria use type III secretion systems to translocate these proteins into host cells, where they will find their targets. In this review, we first introduce type III secretion systems and the ubiquitination process and consider the various ways bacteria use to alter the ubiquitin ligation machinery. We then focus on the members of the NEL family, their expression, translocation, and subcellular localization in the host cell, and we review what is known about the structure of these proteins, their function in virulence or symbiosis, and their specific targets. Ministerio de Ciencia e Innovación PID2019-106132RB-I00/AEI/10.13039/501100011033 Junta de Andalucía P20_00576 Universidad de Sevilla US-1380805

Tomato (Solanum lycopersicum) is one of the most economically important vegetable crops worldwide. Bacterial wilt (BW), caused by the Ralstonia solanacearum species complex, has been reported as the second most important plant pathogenic bacteria worldwide, and likely the most destructive. Extensive research has identified two major loci, Bwr-6 and Bwr-12, that contribute to resistance to BW in tomato; however, these loci do not completely explain resistance. Segregation of resistance in two populations that were homozygous dominant or heterozygous for all Bwr-6 and Bwr-12 associated molecular markers suggested the action of one or two resistance loci in addition to these two major QTLs. We utilized whole genome sequence data analysis and pairwise comparison of six BW resistant and nine BW susceptible tomato lines to identify candidate genes that, in addition to Bwr-6 and Bwr-12, contributed to resistance. Through this approach we found 27,046 SNPs and 5975 indels specific to the six resistant lines, affecting 385 genes. One sequence variant on chromosome 3 captured by marker Bwr3.2dCAPS located in the Asc (Solyc03g114600.4.1) gene had significant association with resistance, but it did not completely explain the resistance phenotype. The SNP associated with Bwr3.2dCAPS was located within the resistance gene Asc which was inside the previously identified Bwr-3 locus. This study provides a foundation for further investigations into new loci distributed throughout the tomato genome that could contribute to BW resistance and into the role of resistance genes that may act against multiple pathogens.

The phylogenetic relationships and distribution of Ralstonia pseudosolanacearum and Ralstonia solanacearum sequevars were analyzed in 108 strains of Solanaceae in three mesoregions of the Pernambuco State, Brazil. These mesoregions are characterized by a semiarid climate and account for 88.20% of the area of the state. Ralstonia pseudosolanacearum (phylotype I, biovar 3) was predominant in Agreste, and R. solanacearum (phylotype II, biovar 1) was predominant in Sao Francisco and Sertao. Two haplotypes of R. pseudosolanacearum were identified by analysis of the haplotype network of the partial egl gene sequences and were phylogenetically related to sequevars I-17 and I-18. Five haplotypes of R. solanacearum were identified; two haplotypes were related to sequevar IIA-50, and three haplotypes were related to new sequevars IIA-61, IIA-62, and IIA-63. In Brazil, sequevars I-17, IIA-61, IIA-62, and IIA-63 were detected exclusively in the semiarid climate of Pernambuco, unlike sequevars I-18 and IIA-50, which were detected in other regions of Brazil. Furthermore, R. pseudosolanacearum (phylotype I) and sequevar IIA-50 were detected in the Sertao mesoregion. Sequevar I-18, IIA-61, and IIA-62 strains were prevalent in the Agreste mesoregion, and sequevars I-17 and IIA-50 were prevalent in the Sao Francisco mesoregion. The virulence variability of the strains toward tomato was related to species and sequevars. Introduced R. pseudosolanacearum strains of sequevar I-18 had higher estimated virulence compared to that of the native R. solanacearum strains of sequevar IIA-50. This is the first study that analyzes the variability of Ralstonia spp. in the semiarid region of Brazil.

Ralstonia eutropha is a hydrogen-oxidizing chemolithotrophic bacterium that can synthesize poly[(R)-3-hydroxybutyrate] [P(3HB)], which is used as a biodegradable plastic. Various genes coding for carbonic anhydrase (CA), which are involved in CO2 fixation and metabolism, are present in the genome of R. eutropha. Although the significance of CA for CO2 metabolism in R. eutropha is well known, CAs have never been deliberately employed to enhance P(3HB) production from CO2. Thus, we investigated the gene dosage effect of the β-carbonic anhydrase (can) gene on P(3HB) accumulation in R. eutropha H16 under autotrophic and mixotrophic conditions. The multicopy plasmid-based dosage of the can gene showed a 1.5-fold increase in P(3HB) content in cells grown under nitrogen-source-limited autotrophic conditions. A similar result was observed under nitrogen-source-limited mixotrophic conditions in the presence of glycerol. Thus, these findings suggest a viable strategy to increase P(3HB) yield under autotrophic and mixotrophic culture conditions. Ralstonia eutropha is a hydrogen-oxidizing chemolithotrophic bacterium that can synthesize poly[(R)-3-hydroxybutyrate] [P(3HB)], which can be used as a biodegradable plastic. Various genes for carbonic anhydrase involved in CO2 fixation are present in the genome of R. eutropha. In this study, we investigated the gene dosage effect of the β-carbonic anhydrase (can) gene on P(3HB) accumulation in R. eutropha under autotrophic and mixotrophic conditions.

Microbial synthesis utilizes sustainable resources to produce valuable chemicals, as a potential alternative to petroleum-based chemical industry. Although metabolic engineering is an efficient method to enhance the biosynthesis efficacy of microorganisms, it requires complicated biological procedures. Herein, we report a facile intracellular catalysis system for augmenting the production of bio-based material in microorganism. Covalent linking of oligo(p-phenylenevinylene) (OPV) and cyclopentadienyl rhodium(III) bipyridine offers intracellular metal catalyst (OPV-Rh). The OPV-Rh complex displayed certain resistance to toxic biomolecules, which guaranteed its catalytic activity in complicated biological systems. With uptake by Gram-negative bacterium Ralstonia eutropha H16 (R. eutropha H16), the OPV-Rh complex promotes the transformation of intracellular NADP+ to NADPH, which further enhances the biosynthesis of polyhydroxybutyrate (PHB) by this microorganism. This work demonstrates that synthetic metal catalyst can be employed for regulating microbial biosynthesis intracellularly.

Phthalates are widely used as plasticizers in many industrial products due to their chemical properties that confer flexibility and durability to building materials, lubricants, solvents, insect repellents, clothing, cosmetics, being widely distributed in the environment. Besides persistent, they are also considered endocrine-disrupting compounds (EDCs), causing a global concern about their release into the environment, once they can alter the reproductive and endocrine health of humans systems. Under natural conditions, photodegradation and hydrolysis rates of phthalates are often very slow; therefore, microbial degradation is a natural way to treat these pollutants. In this context, three bacterial consortia (CMS, GMS and GMSS) were isolated from environmental samples from the Santos Estuarine System (SES) and were able to grow on diethyl-phthalate (DEP) as an only carbon source. From the GMSS consortium, three different strains were isolated and identified as Burkholderia cepacia, Pseudomonas koreensis and Ralstonia pickettii by molecular and mass spectrometry (MALDI-TOF-Biotyper) techniques. Considering there are no reports about Ralstonia genus on phthalates degradation, this strain was chosen to proceed the kinetics experiments. Ralstonia pickettii revealed a great ability to degrade DEP (300 mg/L) in less than 24 h. This is the first report implicating R. pickettii in DEP degradation.

Objectives: In this study, we took advantage of nanotechnology to systematically investigate the antimicrobial activity of silver nanoparticles (AgNPs) against pathogenic microorganisms. This study aimed to synthesize AgNPs from Ralstonia sp. strain NS-7 and further characterization of synthesized AgNPs. Materials: The molecular characterization of isolated strain Ralstonia sp. NS-7 was done by 16S rRNA gene sequencing and the characterizations of synthesized AgNPs was achieved by UV-Visible spectroscopy, AFM, FTIR, HRTEM, SEM, EDS and XRD. Later on, the efficacy of previously synthesized AgNPs was assessed in vitro against pathogens, such as Escherichia coli, Enterococcus faecalis, Streptococcus pneumoniae and Staphylococcus aureus. Finding: The UVvisible spectrophotometric observation of synthesized AgNPs showed maximum absorbance at 420 nm, the AFM data revealed the polydispersity of spherical nanoparticles. Further, the FTIR analysis expressed a unique IR spectral band patterning and the HR-TEM and SEM analysis showed the size of biosynthesized AgNPs in the range of 14.72 nm to 47.32 nm. The analysis of phylogenetic tree of the strain NS-7 revealed the most sequence similarity with Ralstonia sp. strain PGNP6. Finally, the AgNPs represented a broad-spectrum antimicrobial activity against gram-positive and gram-negative bacteria. Application:The biological method for the synthesis of AgNPs is eco-friendly, economical, green and non-toxic. Synthesized AgNPs from Ralstonia sp. strain NS-7 could be used as an alternative source of antimicrobial for the management of pathogenic and multi-drug resistant microorganisms. Keywords: Ralstonia sp.; FTIR; HR-TEM; 16S rRNA gene sequencing; antimicrobial activity; AgNPs

The study aimed to explore the bacterial community composition and the functions of core microbiota in Eucommia ulmoides bark. The bark samples of E. ulmoides were collected from Wangcang Sichuan Province, Cili Hunan Province, and Zunyi Guizhou Province, in China, respectively. Through the high-throughput sequencing methods and techniques, the community composition, core microbiota, and function of the bacteria were studied. The bacterial community of E. ulmoides bark consisted of 9 phyla, 11 classes, 22 orders, 28 families, 31 genera, and 37 OTUs. At the genus level, the dominant genus was the unclassified bacteria of Cyanobacteria, with a relative abundance of 97.01%. The bacterial communities of E. ulmoides bark from different areas have their unique units except for the common microbiota. The core microbiota of bacteria included an unclassified genus of Cyanobacteria, an unclassified genus of Mitochondria, Pseudomonas, Sphingobium, Rhizobium, Novosphingobium, Enterobacter, Rhodococcus, Curtobacterium, and Ralstonia. FAPROTAX function prediction suggested that the core microbiota has a substantial potential for photoautotrophy, phototrophy, aerobic chemoheterotrophy, chemoheterotrophy. Ten taxa composed the core microbiota, and the majority of them were related to the pharmacologically active ingredients of E. ulmoides bark. The research provides a scientific basis for the biological marker of genuineness and microbial technology for improving the content of medicinal ingredients of E. ulmoides.

Ralstonia solanacearum species complex is the most destructive and economically important bacterial pathogen of many plant species around the world. It is a particular constraint to the production of the solanaceous vegetables such as tomato, chilli pepper and African eggplant in West Africa. Its broad host range, ability to survive in the soil for long periods and ability to sustain latent infection, make it difficult to control. Identification, characterization and mapping the distribution of the causal Ralstonia spp. strains are necessary to help design effective control strategies. Wilted tomato, African eggplant and pepper plant samples were collected from fields in different regions of Mali. The causal bacterial strains were isolated from the samples and all were identified as Ralstonia pseudosolanacearum. Multiplex PCR using four phylotype specific primer pairs identified the presence of phylotype I strains distributed across all the vegetable production areas whereas phylotype III strains were limited to areas of Sikasso, Koulikoro and Segou to the south and east of Bamako city. Phylogenetic analysis of part of the conserved endoglucanase virulence gene sequences revealed four sequevars with most phylotype I strains identified as sequevars 46 and 31 with only a few as sequevar 14 and one sequevar 18. The phylotype III strains were all in the sequevar 23–48 group. Pathogenicity testing of a selected subset of the strains isolated in Mali showed them all to be pathogenic to the susceptible tomato cultivar Roma. To our knowledge, this is the first study on the molecular diversity within the Ralstonia spp. complex in Mali. This information is important for developing wilt management strategies including breeding for resistance and specifying quarantine restrictions.

The tomato crop is confronted to numerous soilborne pathogens, including Ralstonia solanacearum, which considerably limits its production. In order to control this bacterium, a biological control approach has been considered by evaluating the efficacy of the NECO biopesticide against this bacteriosis. In vitro confrontations were carried out using a range of five concentrations of the biopesticide. In vivo, NECO solutions of 5 and 10 mL/L were incorporated into soil previously infested with R. solanacearum before transplanting tomato plants. Zones of bacterial growth inhibition were observed after the application of the NECO biopesticide. Results showed that the 20 mL/L concentration resulted in a higher inhibition rate. The biopesticide at the 10 mL/L concentration significantly reduced the incidence of bacterial wilt (54.05%) under in vivo conditions. The NECO biopesticide could be used as a control agent for Ralstonia solanacearum.

The Ptr1 (Pseudomonas tomato race 1) locus in Solanum lycopersicoides confers resistance to strains of Pseudomonas syringae pv. tomato expressing AvrRpt2 and Ralstonia pseudosolanacearum expressing RipBN. Here we describe the identification and phylogenetic analysis of the Ptr1 gene. A single recombinant among 585 F2 plants segregating for the Ptr1 locus was discovered that narrowed the Ptr1 candidates to eight nucleotide-binding leucine-rich repeat protein (NLR)-encoding genes. From analysis of the gene models in the S. lycopersicoides genome sequence and RNA-Seq data, two of the eight genes emerged as the strongest candidates for Ptr1. One of these two candidates was found to encode Ptr1 based on its ability to mediate recognition of AvrRpt2 and RipBN when it was transiently expressed with these effectors in leaves of Nicotiana glutinosa. The ortholog of Ptr1 in tomato and in Solanum pennellii is a pseudogene. However, a functional Ptr1 ortholog exists in Nicotiana benthamiana and potato, and both mediate recognition of AvrRpt2 and RipBN. In apple and Arabidopsis, recognition of AvrRpt2 is mediated by the Mr5 and RPS2 proteins, respectively. Phylogenetic analysis places Ptr1 in a distinct clade compared with Mr5 and RPS2, and it therefore appears to have arisen by convergent evolution for recognition of AvrRpt2.

The fuel acclimation process offers flexibility in microbial fuel cell (MFC) power generation behavior. Different concentrations (50–200 mg/L) of phenol were used for adapting the activated sludge (AS), obtained from a local petroleum wastewater treatment plant and Ralstonia eutropha pure culture. Anodic biomass capable of oxidizing phenol substrate, using either AS inoculum microbial consortium or R. eutropha in the MFC system, has been a reflection of growth supportive functionality of phenol and 150 mg/L as initial concentration was used in the experiments. For both types of inocula. The results of phenol and COD removals obtained for closed system configuration were compared with those under open circuit condition. The current production by AS and R. eutropha was improved through phenol acclimation process. The highest power density (PD) using either AS or R. eutropha was 11 and 5.8 mW/m2, respectively. In terms of using olive oil mill wastewater as the anodic substrate, the behavior of phenol-acclimated R. eutropha was better than that of the synthetic type of wastewater, and the PD value was 7.8 mW/m2.

A preliminary study was conducted to demonstrate fate of the bacterial storage compound poly-beta-hydroxybutyrate (PHB) once ingested and degraded in vivo in crustaceans. The 2% supplementation of C-13-labeled Ralstonia eutropha DSM545 containing 75% PHB on dry weight in postlarval whiteleg shrimp, Litopenaeus vannamei resulted in consistent enrichment of two fatty acids (containing 14-24 carbons [C] in length) pentadecanoic acid (15:0) and palmitic acid (16:0) in all lipid fractions. This could indicate that C-13 signal may not only originate from the PHB but partly from the structural components of the bacterial cell. There was a trend of lower C-13 enrichment in various lipid fractions of the postlarvae as compared to the diet. These findings may have important implications as to potential of PHB as energy source rather than as building block. There was a rapid transfer of C-13 in the postlarvae as seen by the significant C-13 enrichment in the tissues 4 hr after feeding with labeled bacterial cells. Overall, the assimilation of PHB in crustacean tissues is demonstrated for the first time. Our results indicate that PHB once ingested is rapidly assimilated in the tissues and could probably act as an energy source rather than as a building block.

A structured survey of the cystic fibrosis pathogens Achromobacter, Pandoraea and Ralstonia species from thirteen sentinel hospitals throughout England was undertaken by Public Health England. One isolate per patient of these genera collected from CF patients during the seven-month survey period in 2015 was requested from participating hospitals. Species-level identification was performed using nrdA/gyrB sequence cluster analysis, and genotyping by pulsed-field gel electrophoresis. In total, 176 isolates were included in the survey; 138 Achromobacter spp. (78.4%), 29 Pandoraea spp. (16.5%) and 9 Ralstonia spp. (5.1%). Novel Achromobacter and Pandoraea clusters were identified. High levels of antimicrobial resistance were found, particularly among Pandoraea isolates. Genotyping analysis revealed considerable diversity, however one geographically-widespread cluster of A. xylosoxidans isolates from six hospitals was found, in addition to two other clusters, both comprising isolates from two hospitals, either derived from the same region (A. xylosoxidans), or from hospitals within the same city (P. apista).

Brown rot of potatoes caused by Ralstonia solanacearum is common in developing countries. Historically, brown rot losses in the United States have been restricted to southern states. This trend will most likely change with new Ralstonia introductions and temperature variability. A cold tolerant Ralstonia strain was used in this study to compare resistance between 18 wild accessions from eight species, nine accessions from the International Potato Center (CIP), and seven common US cultivars. Testing was done at both 18 °C and 30 °C. Of the wild potato species tested, S. commersonii and S. microdontum were the most resistant. CIP accessions varied from moderately resistant to very resistant. Four of the US cultivars were also moderately resistant. With certain accessions temperature played a role in resistance with high temperatures usually increasing severity of disease and cool temperatures promoting the occurrence of asymptomatic plants. The resistance observed appears to be partial, multigenic resistance.

This paper presents a control architecture suitable for autotrophic cultivations in a stirred tank reactor feeding a gas mixture of H2/CO2/O2. No metabolic information about the cultivated strain is required for the gas phase controller, which can also be used as part of the overall control for the entire process. A gain-scheduled MIMO-PI controller connected to an adaptive feedforward disturbance rejection is proposed. The latter exploits the estimated gas transfer rates of a Kalman filter solely based on a description of the non-biotic processes occurring. Thus, without further knowledge of the autotrophic strain, experiments can be run to generate data and to develop a process model including strain-specific biological details. The wild-type strain H16 of Ralstonia eutropha and two mutants, HF805 and H798, are used to demonstrate how this can be done.

The scope of this study is the management of insects that carry the cause of banana blood disease (BBD), Ralstonia solanacearum Phylotype IV. The objectives of this study are: (1) to study the diversity and density of visitor insect populations to the Kepok banana flower, and (2) to identify insects in the Kepok banana flower that act as carriers of R. solanacearum Phylotype IV, and the population density of these bacteria was carried by each insect. Sampling of banana plantations is done based on pusposive sampling method. Insect collection uses a modified insect net, and insect collection uses modified insect nets, and insects were morphologically identified. This bacterial isolation was carried out based on the spread method on NA + TZC media. Inoculation of bacterial isolates was carried out by injection method on the tip of an mature Kepok banana. Density of insects visitors banana flower per tree in South Minahasa and Minahasa regencies are as follows: Oscinella sp. (15.50 and 18.08 individuals), Aphis mellifera (0.50 and 1.58), Chelisoches morio (0.28 and 0.20 individuals, and Dolichoderus sp. (1.44 and 6.21 individuals). All insects on the Kepok banana flower in South Minahasa and Minahasa carry Ralstonia solanacearum Phylotype IV. Oscinella sp., Aphis mellifera, Chelisoches morio, and Dolichoderus sp. in both districts it brought 17,636.39 and 75,533.33 CFU / ml, 15,666.67 and 17,400.00 CFU / ml, 113.33 and 2,667.67 CFU / ml, and 2,400.00 CFU / ml and 21,133.33 CFU / ml.

Banana Blood disease is a bacterial wilt caused by Ralstonia syzygii subsp. celebesensis and is an economically important disease in Indonesia and Malaysia. Transmission of this pathogen is hypothesized to occur through insects mechanically transferring bacteria from diseased to healthy banana inflorescences and other pathways involving pruning tools, water movement, and root-to-root contact. This study demonstrates that the ooze from the infected male bell and the sap from various symptomatic plant parts are infective, and the cut surfaces of a bunch peduncle, petiole, corm, pseudostem, and the rachis act as infection courts for R. syzygii subsp. celebesensis. In addition, evidence is provided that R. syzygii subsp. celebesensis is highly tool transmissible, that the bacterium can be transferred from the roots of a diseased plant to the roots of a healthy plant and transferred from the mother plant to the sucker. We provide evidence that local dispersal of Blood disease occurs predominantly through mechanical transmission by insects, birds, bats, or human activities from diseased to healthy banana plants and that long-distance dispersal occurs through the movement of contaminated planting material. Disease management strategies to prevent crop losses associated with this emerging disease are discussed based on our findings.

Ralstonia pseudosolanacearum causes bacterial wilt in ginger, reducing ginger production worldwide. We sequenced the whole genome of a highly virulent phylotype I, race 4, biovar 3 Ralstonia pseudosolanacearum strain GRsMep isolated from a severely infected ginger field in India. R. pseudosolanacearum GRsMep genome is organised into two replicons: chromosome and megaplasmid with a total genome size of 5,810,605 bp. This strain encodes approximately 72 effectors which include a combination of core effectors as well as highly variable, diverse repertoire of type III effectors. Comparative genome analysis with GMI1000 identified conservation in the genes involved in the general virulence mechanism. Our analysis identified type III effectors, RipBJ and RipBO as present in GRsMep but absent in the reported genomes of other strains infecting Zingiberaceae family. GRsMep contains 126 unique genes when compared to the pangenome of the Ralstonia strains that infect the Zingiberaceae family. The whole-genome data of R. pseudosolanacearum strain will serve as a resource for exploring the evolutionary processes that structure and regulate the virulence determinants of the strain. Pathogenicity testing of the transposon insertional mutant library of GRsMep through virulence assay on ginger plants identified a few candidate virulence determinants specific to bacterial wilt in ginger.

Background Inflammatory bowel diseases (IBDs), such as ulcerative colitis (UC), are characterized by a disturbance of the normal gut microbiota and contribute to the development of chronic kidney disease (CKD). The incidence of CKD is higher in individuals with UC, but the causal link is unknown. Therefore, we investigated the role of gut microbiota in decreasing renal function in patients with UC Methods We performed 16S ribosomal DNA sequencing using ileocecal mucosal samples from nine individuals with UC and CKD (UC+CKD), 29 individuals with UC only, and 12 healthy controls. We also analyzed the operational taxonomic units, microbial diversity, and correlation with renal function. Co-culture assay using kidney organoids and Caco-2 cells was performed. Figure 1. The taxonomic composition of the gut microbiota of the study population and comparison of the 10 most abundant genera and species in the UC + CKD, UC, and control groups. Results Bacterial species diversity was significantly decreased in the UC+CKD group compared to that in the other groups based on Shannon and inverse Simpson indexes. At the genus level, Ralstonia had significantly greater abundance in the UC+CKD and UC groups compared to the control group. At the species level, unclassified Ralstonia species and Citrobacter portucalensis showed higher abundance in the UC+CKD group compared to the other groups. The relative abundance of Ralstonia showed a negative correlation with the estimated glomerular filtration rate (eGFR), but a positive correlation with the serum uric acid level. Ralstonia pickettii represented a negative correlation with eGFR, and induced damaging changes to kidney organoids in co-culture assay. Figure 2. The correlation between the relative abundance of Ralstonia with renal function (indicated by eGFR) and serum uric acid level. Effects of Ralstonia pickettii on Caco-2 cells and kidney organoids. (A) mRNA expression of pro-inflammatory cytokine genes in Caco-2 cells treated with and without Ralstonia pickettii, reflecting the inflammation seen in ulcerative colitis. Conclusion Gut microbial community profiles of the UC+CKD group are different from those of the UC group. Furthermore, Ralstonia pickettii contributes in decreasing renal function in patients with UC.

Introduction: The incidence of infection by Ralstonia is increasing. Several reports describing infection by these bacteria in immunocompromised patients have been published. In this study, we reported a case of Ralstonia pickettii infection in a patient with normal immunity. Case Presentation: A woman presented with fever after thyroid surgery. We identified R. pickettii in her blood culture using 16S rRNA gene sequencing. The patient’s condition improved clinically upon treatment with levofloxacin. Conclusions: Our report highlights the potential of Ralstonia to cause sepsis in patients with normal immunity and emphasizes the importance of blood culture testing when a hospitalized patient has an unexplained high fever.

The bacterium Ralstonia syzygii subsp. celebesensis causes Blood disease of banana, a vascular wilt of economic significance in Indonesia and Malaysia. Blood disease has expanded its geographic range in the last 20 years and is an emerging threat to Southeast Asian banana production. Many aspects of the disease cycle and biology are not well understood, including the ability of different parts of the female and male inflorescence of banana to act as infection courts. This study confirms that the banana varieties of Cavendish, and Kepok ‘Kuning’ are susceptible to Blood disease and that an inoculum concentration of 102 CFU/ml of R. syzygii subsp. celebesensis is adequate to initiate disease after pseudostem inoculation. Data show that infection occurs through both the male and female parts of a banana inflorescence and the rachis when snapped to remove the male bell. The infection courts are the female flowers, the male bell bract scar, the male bell flower cushion, the snapped rachis, and deflowered fingers. The location of these infection courts concurs with the dye studies demonstrating that dye externally applied to these plants parts enters the plant vascular system. Thus, the hypothesis is supported that infection of R. syzygii subsp. celebesensis occurs through open xylem vessels of the male and female parts of the banana inflorescence.

The Brazilian ethanol industry is one of the most important in the global market, however these important industrial activities have been generating significant amounts of vinasse and its management has become costly for distilleries. In this study, the aim was to evaluate concentrated and in natura vinasse as basal culture media for biotechnological processes. Different bacteria and processes were assessed: L-threonine production by E. coli THR14, with glucose as carbon source; PHB production by halophilic strain Halomonas sp. HG03, with sucrose as carbon source; and PHB biosynthesis by R. eutropha L359PCJ, which used glycerol from vinasse as carbon source. Strains were evaluated firstly in shake flasks cultivations using vinasse-based media. E. coli THR14 had no statistical difference for biomass and L-threonine concentrations among control and vinasse-based treatments (up to 50% v v-1 of in natura vinasse). Halomonas sp. HG03 and R. eutropha L359PCJ were cultivated in mineral media diluted by in natura (50% and 75% v v-1) and concentrated (50% and 75% v v-1) vinasses. Higher vinasse concentrations resulted in higher cellular growth rather than PHB accumulation for both bacteria. In vinasse-based treatments, Halomonas sp. HG03 had PHB content between 19.6 – 75.2% and R. eutropha L359PCJ, 48.4 – 68.5%. 50% (v v-1) of concentrated vinasse was the most attractive condition for PHB production by both bacteria. Further experiments in CSTR bioreactors used this nutritional condition and R. eutropha L359PCJ had PHB content of 66.3%, concentrations of residual cell dry weight (rCDW) = 9.4 g L-1 and PHB = 18.6 g L-1, with YX/S = 0.16 g gGLYCEROL-1, YP/S = 0.32 g gGLYCEROL-1 and 0.25 gPHB Lh-1. Halomonas sp. HG03 had PHB content of 45.7%, rCDW = 9.8 g L-1, PHB = 8.3 g L-1 and YX/S = 0.18 g gSUCROSE-1, YP/S = 0.16 g gSUCROSE-1 and 0.12 gPHB Lh-1. Finally, cost reductions of PHB production by R. eutropha L359PCJ with concentrated vinasse-based medium were evaluated in silico by using SuperPro Designer. As a partial source of glycerol and other nutrients for PHB production by R. eutropha L359PCJ, vinasse reduced overall production costs by 13%. Simulated processes that used concentrated vinasse-based media combined with improvements of PHB productivity and higher cellular densities had production costs between US$ 3.9 – 7.5/kgPHB and 2.6 – 7.3 years of payback time.