Calcium modulation of bacterial wilt disease on potato.

Ralstonia solanacearum species complex (RSSC) is a phytopathogenic bacterial group that causes bacterial wilt in several crops, being potato (Solanum tuberosum) one of the most important hosts. The relationship between the potato plant ionome (mineral and trace elements composition) and the resistance levels to this pathogen has not been addressed until now. Mineral content of xylem sap, roots, stems and leaves of potato genotypes with different levels of resistance to bacterial wilt was assessed in this work, revealing a positive correlation between divalent calcium (Ca) cation concentrations and genotype resistance. The aim of this study was to investigate the effect of Ca on bacterial wilt resistance, and on the growth and virulence of RSSC. Ca supplementation significantly decreased the growth rate of Ralstonia pseudosolanacearum GMI1000 in minimal medium and affected several virulence traits such as biofilm formation and twitching motility. We also incorporate for the first time the use of microfluidic chambers to follow the pathogen growth and biofilm formation in conditions mimicking the plant vascular system. By using this approach, a reduction in biofilm formation was observed when both, rich and minimal media, were supplemented with Ca. Assessment of the effect of Ca amendments on bacterial wilt progress in potato genotypes revealed a significant delay in disease progress, or a complete absence of wilting symptoms in the case of partially resistant genotypes. This work contributes to the understanding of Ca effect on virulence of this important pathogen and provides new strategies for an integrated control of bacterial wilt on potato. IMPORTANCE: Ralstonia solanacearum species complex (RSSC) includes a diverse group of bacterial strains that cause bacterial wilt. This disease is difficult to control due to pathogen aggressiveness, persistence, wide range of hosts, and wide geographic distribution in tropical, subtropical, and temperate regions. RSSC causes considerable losses depending on the pathogen strain, host, soil type, environmental conditions, and cultural practices. In potato, losses of $19 billion per year have been estimated for this pathogen worldwide. In this study, we report for the first time the mineral composition found in xylem sap and plant tissues of potato germplasm with different levels of resistance to bacterial wilt. This study underscores the crucial role of calcium (Ca) concentration in the xylem sap and stem in relation to the resistance of different genotypes. Our in vitro experiments provide evidence of Ca's inhibitory effect on the growth, biofilm formation, and twitching movement of the model RSSC strain R. pseudosolanacearum GMI1000. This study introduces a novel element, the Ca concentration, which should be included into the integrated disease control management strategies for bacterial wilt in potatoes.

Phenotypic and genetic characterization of a lipolytic Ralstonia mannitolilytica isolate from petroleum-contaminated soil in Iraq.

BACKGROUND: Lipases play a crucial role in various industrial applications, and microbial lipases, particularly those from bacteria, possess significant properties. With increasing concerns about the environmental and health impacts of hydrocarbons from pipelines and refineries, there is a growing need to mitigate the risks associated with these compounds. METHODS: In this study, 40 bacterial isolates were recovered from contaminated soil samples collected from multiple refineries across Iraq. Using the Vitek system, bacterial isolates were identified up to the species level, revealing that only 12 isolates exhibited lipase-producing capabilities. RESULTS: Among the lipase-producing isolates, Ralstonia mannitolilytica demonstrated the highest extracellular lipase activity, as determined by an olive oil plate assay supplemented with rhodamine B. Confirmation of the species identity was achieved through 16S rRNA gene sequencing, with the obtained sequence deposited under accession number LC772176.1. Further sequence analysis revealed single nucleotide polymorphisms (SNPs) in the genome of Ralstonia mannitolilytica strain H230303-10_N19_7x_R2 (CP011257.1, positions 1,311,102 and 1,311,457). Additionally, the presence of the lipase gene was confirmed through amplification and sequencing using a thermocycler PCR. Sequence analysis of the gene, aligned using Geneious Prime software, identified SNPs (CP010799, CP049132, AY364601, CP011257, and CP023537), and a phylogenetic tree was constructed based on genetic characterization. CONCLUSION: Our findings highlight the potential of Ralstonia mannitolilytica as a promising candidate for lipase production and contribute to our understanding of its genetic diversity and biotechnological applications in hydrocarbon degradation and industrial processes.

Characterization of Ralstonia insidiosa C1 isolated from Alpine regions: Capability in polyhydroxyalkanoates degradation and production.

This study ventures into the exploration of potential poly-3-hydroxybutyrate (PHB) degradation in alpine environments. PHB-degrading bacteria were identified in both campus soil, representing a residential area, and Mt. Kurodake soil, an alpine region in Hokkaido, Japan. Next-generation sequencing analysis indicated that the campus soil exhibited higher microbial diversity, while Ralstonia insidiosa C1, isolated from Mt. Kurodake soil, displayed the highest proficiency in PHB degradation. R. insidiosa C1 efficiently degraded up to 3% (w/v) of PHB and various films composed of other biopolymers at 14 °C. This bacterium synthesized homopolymers using substrates such as 3-hydroxybutyric acid, sugars, and acetic acid, while also produced copolymers using a mixture of fatty acids. The analysis results confirmed that the biopolymer synthesized by strain C1 using glucose was PHB, with physical properties comparable to commercial products. The unique capabilities of R. insidiosa C1, encompassing both the production and degradation of bioplastics, highlight its potential to establish a novel material circulation model.

Complete sequence of carbapenem-resistant Ralstonia mannitolilytica clinical isolate co-producing novel class D ß-lactamase OXA-1176 and OXA-1177 in Japan.

In 2020, the Ralstonia mannitolilytica strain JARB-RN-0044 was isolated from a midstream urine sample of an elderly hospitalized patient in Japan and was highly resistant to carbapenem (i.e., imipenem, meropenem, and doripenem). Whole-genome sequencing revealed that the complete genome consists of two replicons, a 3.5-Mb chromosome and a 1.5-Mb large non-chromosomal replicon which has not been reported in R. mannitolilytica, and referred to as the "megaplasmid" in this study based on Cluster of Orthologous Group of proteins functional analysis. The strain JARB-RN-0044 harbored two novel OXA-60 and OXA-22 family class D ß-lactamase genes blaOXA-1176 and blaOXA-1177 on the megaplasmid. Cloning experiments indicated that Escherichia coli recombinant clone expressing blaOXA-1176 gene showed increased minimum inhibitory concentrations (MICs) of imipenem, meropenem, and doripenem, indicating that blaOXA-1176 gene encodes carbapenemase. In contrast, E. coli recombinant clone expressing blaOXA-1177 gene showed increased MICs of piperacillin and cefazolin, but not of carbapenem. Interestingly, the 44.6 kb putative prophage region containing genes encoding phage integrase, terminase, head and tail protein was identified in the downstream region of blaOXA-1176 gene, and comparative analysis with some previously reported R. mannitolilytica isolates revealed that the prophage region was unique to strain JARB-RN-0044. The existence of a highly carbapenem-resistant R. mannitolilytica isolate may raise human health concerns in Japan, where the population is rapidly aging.IMPORTANCERalstonia mannitolilytica is an aerobic non-fermenting Gram-negative rod commonly found in aquatic environments and soil. The bacteria can occasionally cause severe hospital-acquired bloodstream infections in immunocompromised patients and it has been recently recognized as an emerging opportunistic human pathogen. Furthermore, some R. mannitolilytica isolates are resistant to various antimicrobial agents, including ß-lactams and aminoglycosides, making antimicrobial therapy challenging and clinically problematic. However, clinical awareness of this pathogen is limited. To our knowledge, in Japan, there has been only one report of a carbapenem-resistant R. mannitolilytica clinical isolate from urine by Suzuki et al. in 2015. In this study, whole-genome sequencing analysis revealed the presence and genetic context of novel blaOXA-1176 and blaOXA-1177 genes on the 1.5 Mb megaplasmid from highly carbapenem-resistant R. mannitolilytica isolate and characterized the overall distribution of functional genes in the chromosome and megaplasmid. Our findings highlight the importance of further attention to R. mannitolilytica isolate in clinical settings.

Fermented botanical fertilizer controls bacterial wilt of tomatoes caused by Ralstonia pseudosolanacearum.

This study demonstrates the effect of fermented botanical product (FBP) on Ralstonia pseudosolanacearum-induced bacterial wilt disease and unravels its action mechanism. Soaking with diluted FBP solutions (0.1%-0.5%) significantly suppressed bacterial wilt in tomato plants, and FBP-treated tomato plants grew well against R. pseudosolanacearum infection. Growth assays showed that FBP had no antibacterial effect but promoted R. pseudosolanacearum growth. In contrast, few or no R. pseudosolanacearum cells were detected in aerial parts of tomato plants grown in FBP-soaked soil. Subsequent infection assays using the chemotaxis-deficient mutant (ΔcheA) or the root-dip inoculation method revealed that FBP does not affect pathogen migration to plant roots during infection. Moreover, FBP-pretreated tomato plants exhibited reduced bacterial wilt in the absence of FBP. These findings suggest that the plant, but not the pathogen, could be affected by FBP, resulting in an induced resistance against R. pseudosolanacearum, leading to a suppressive effect on bacterial wilt.

Comparative genomics and host range analysis of four Ralstonia pseudosolanacearum strains isolated from sunflower reveals genomic and phenotypic differences.

BACKGROUND: Bacterial wilt caused by Ralstonia solanacearum species complex (RSSC) is one of the devastating diseases in crop production, seriously reducing the yield of crops. R. pseudosolanacearum, is known for its broad infrasubspecific diversity and comprises 36 sequevars that are currently known. Previous studies found that R. pseudosolanacearum contained four sequevars (13, 14, 17 and 54) isolated from sunflowers sown in the same field. RESULTS: Here, we provided the complete genomes and the results of genome comparison of the four sequevars strains (RS639, RS642, RS647, and RS650). Four strains showed different pathogenicities to the same cultivars and different host ranges. Their genome sizes were about 5.84 ~ 5.94 Mb, encoding 5002 ~ 5079 genes and the average G + C content of 66.85% ~ 67%. Among the coding genes, 146 ~ 159 specific gene families (contained 150 ~ 160 genes) were found in the chromosomes and 34 ~ 77 specific gene families (contained 34 ~ 78 genes) in the megaplasmids from four strains. The average nucleotide identify (ANI) values between any two strains ranged from 99.05% ~ 99.71%, and the proportion of the total base length of collinear blocks accounts for the total gene length of corresponding genome was all more than 93.82%. Then, we performed a search for genomic islands, prophage sequences, the gene clusters macromolecular secretion systems, type III secreted effectors and other virulence factors in these strains, which provided detailed comparison results of their presence and distinctive features compared to the reference strain GMI1000. Among them, the number and types of T2SS gene clusters were different in the four strains, among which RS650 included all five types. T4SS gene cluster of RS639 and RS647 were missed. In the T6SS gene cluster, several genes were inserted in the RS639, RS647, and RS650, and gene deletion was also detected in the RS642. A total of 78 kinds of type III secreted effectors were found, which included 52 core and 9 specific effectors in four strains. CONCLUSION: This study not only provided the complete genomes of multiple R. pseudosolanacearum strains isolated from a new host, but also revealed the differences in their genomic levels through comparative genomics. Furthermore, these findings expand human knowledge about the range of hosts that Ralstonia can infect, and potentially contribute to exploring rules and factors of the genetic evolution and analyzing its pathogenic mechanism.

Development of antibody to virulence factor flagellin and its evaluation in screening Ralstonia pseudosolanacearum.

The bacterial wilt disease caused by Ralstonia pseudosolanacearum presents a notable economic risk to a variety of crucial crops worldwide. During preliminary isolation of this phytopathogen, several colonies of other saprophytic bacteria may be mistaken with it. So, the present study aims to address this issue by proposing the application of immunogenic proteins, particularly flagellin (FliC), to enable a rapid and early identification of bacterial wilt. In this study, a novel approach is unveiled for the early detection of R. pseudosolanacearum. The study exploits the immunogenic attributes of flagellin (FliC), by generating polyclonal antibodies against recombinant FliC within model organisms-rabbits and mice. The efficacy of these antibodies is meticulously assessed through discerning techniques, including DAS-ELISA and Western blot analyses, which elucidate their remarkable specificity in identifying various R. pseudosolanacearum strains. Furthermore, the introduction of antibody-coated latex agglutinating reagents offers an additional layer of confirmation, substantiating the feasibility of establishing a laboratory-based toolkit for swift screening and unambiguous identification of the bacterial wilt pathogen. This study presents a significant stride toward enhancing early diagnostic capabilities, potentially revolutionizing agricultural practices by safeguarding crop yield and quality through proactive pathogen detection and mitigation strategies.

Molecular basis for the interference of the Arabidopsis WRKY54-mediated immune response by two sequence-unrelated bacterial effectors.

Arabidopsis thaliana WRKY proteins are potential targets of pathogen-secreted effectors. RESISTANT TO RALSTONIA SOLANACEARUM 1 (RRS1; AtWRKY52) is a well-studied Arabidopsis nucleotide-binding and leucine-rich repeat (NLR) immune receptor carrying a C-terminal WRKY domain that functions as an integrated decoy. RRS1-R recognizes the effectors AvrRps4 from Pseudomonas syringae pv. pisi and PopP2 from Ralstonia pseudosolanacearum by direct interaction through its WRKY domain. AvrRps4 and PopP2 were previously shown to interact with several AtWRKYs. However, how these effectors selectively interact with their virulence targets remains unknown. Here, we show that several members of subgroup IIIb of the AtWRKY family are targeted by AvrRps4 and PopP2. We demonstrate that several AtWRKYs induce cell death when transiently expressed in Nicotiana benthamiana, indicating the activation of immune responses. AtWRKY54 was the only cell death-inducing AtWRKY that interacted with both AvrRps4 and PopP2. We found that AvrRps4 and PopP2 specifically suppress AtWRKY54-induced cell death. We also demonstrate that the amino acid residues required for the avirulence function of AvrRps4 and PopP2 are critical for suppressing AtWRKY54-induced cell death. AtWRKY54 residues predicted to form a binding interface with AvrRps4 were predominantly located in the DNA binding domain and necessary for inducing cell death. Notably, one AtWRKY54 residue, E164, contributes to affinity with AvrRps4 and is exclusively present among subgroup IIIb AtWRKYs, yet is located outside of the DNA-binding domain. Surprisingly, AtWRKY54 mutated at E164 evaded AvrRps4-mediated cell death suppression. Taking our observations together, we propose that AvrRp4 and PopP2 specifically target AtWRKY54 to suppress plant immune responses.

Transcriptomic profiling reveals host-specific evolutionary pathways promoting enhanced fitness in the plant pathogen Ralstonia pseudosolanacearum.

The impact of host diversity on the genotypic and phenotypic evolution of broad-spectrum pathogens is an open issue. Here, we used populations of the plant pathogen Ralstonia pseudosolanacearum that were experimentally evolved on five types of host plants, either belonging to different botanical families or differing in their susceptibility or resistance to the pathogen. We investigated whether changes in transcriptomic profiles, associated with or independent of genetic changes, could occur during the process of host adaptation, and whether transcriptomic reprogramming was dependent on host type. Genomic and transcriptomic variations were established for 31 evolved clones that showed better fitness in their experimental host than the ancestral clone. Few genomic polymorphisms were detected in these clones, but significant transcriptomic variations were observed, with a large number of differentially expressed genes (DEGs). In a very clear way, a group of genes belonging to the network of regulation of the bacterial virulence such as efpR, efpH or hrpB, among others, were deregulated in several independent evolutionary lineages and appeared to play a key role in the transcriptomic rewiring observed in evolved clones. A double hierarchical clustering based on the 400 top DEGs for each clone revealed 2 major patterns of gene deregulation that depend on host genotype, but not on host susceptibility or resistance to the pathogen. This work therefore highlights the existence of two major evolutionary paths that result in a significant reorganization of gene expression during adaptive evolution and underscore clusters of co-regulated genes associated with bacterial adaptation on different host lines.

A case of meningitis caused by Ralstonia insidiosa, a rare opportunistic pathogen.

BACKGROUND: Ralstonia is a genus of Gram-negative opportunistic bacteria that can survive in many kinds of solutions and cause a variety of infections. Ralstonia spp. have increasingly been isolated and reported to cause infections in recent years, thanks to the development of identification methods such as matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) and gene sequencing. However, infections caused by Ralstonia insidiosa are still rare. Only a few cases of respiratory infections and bloodstream infections have been reported, none of which involved meningitis. To the best of our knowledge, this is the first reported case of meningitis caused by R. insidiosa worldwide. It is necessary to report and review this case. CASE PRESENTATION: We report a case of meningitis caused by R. insidiosa following lumbar surgery in China. The patient exhibited symptoms of headache, dizziness, and recurrent fever. The fever remained unresolved after empiric antibiotic therapy with intravenous cefotaxime and vancomycin in the initial days. Cerebrospinal fluid (CSF) culture yielded Gram-negative non-fermentative bacteria, which were identified as R. insidiosa. As there was a lack of antibiotic susceptibility testing results, clinical pharmacists conducted a literature review to select appropriate antibiotics. The patient's condition improved after receiving effective treatment with intravenous cefepime and levofloxacin. CONCLUSIONS: Uncommon pathogens, such as R. insidiosa, should be considered in postoperative central nervous system (CNS) infections, particularly in cases with unsatisfactory results of empiric anti-infective therapy. This is the first reported case of meningitis caused by R. insidiosa worldwide. MALDI-TOF MS provides rapid and accurate identification of this pathogen. The antibiotic susceptibility testing results of R. indiosa may be interpreted based on the breakpoints for Pseudomonas spp., Burkholderia cepacia spp., and Acinetobacter spp. Our case presents a potential option for empiric therapy against this pathogen, at least in the local area. This is crucial to minimize the severity and mortality rates associated with meningitis. Standardized antibiotic susceptibility testing and breakpoints for the Ralstonia genus should be established in the future as cases accumulate. Cefepime and levofloxacin may be potential antibiotics for infections caused by R. indiosa.

Antibacterial activity and action mechanism of curcusionol from Carex siderosticta Hance against Ralstonia nicotianae.

BACKGROUND: Tobacco bacterial wilt is a typical soil-borne disease caused by Ralstonia nicotianae, which causes huge losses in tobacco production every year. The crude extract of Carex siderosticta Hance was shown to have antibacterial activity against R. nicotianae during our search, and the natural antibacterial components were sought after using bioassay-guided fractionation of the compounds. RESULT: Ethanol extract of Carex siderosticta Hance with the minimum inhibitory concentration (MIC) value of 100 µg/mL against R. nicotianae in vitro. The potential of these compounds as antibactericides against R. nicotianae were assessed. Curcusionol (1), showed the highest antibacterial activity against R. nicotianae with MIC value of 12.5 µg/mL in vitro. In the protective effect tests, the control effect of curcusionol (1) was 92.31 and 72.60%, respectively, after application of 7 and 14 days, at a concentration of 1500 µg/mL, being comparable to that of streptomycin sulfate at a concentration of 500 µg/mL, confirming that curcusionol (1) showed the potential for the development of new antibacterial drugs. RNA-sequencing, scanning electron microscopy (SEM) and transmission electron microscopy (TEM) analysis confirmed that curcusionol mainly destroys R. nicotianae cell membrane structure and affects quorum sensing (QS) to inhibit pathogenic bacteria. CONCLUSION: This study revealed that the antibacterial activity of Carex siderosticta Hance makes it a botanical bactericide against R. nicotianae, while curcusionol as lead structures for antibacterial development is obvious by its potent antibacterial activity. © 2023 Society of Chemical Industry.

Virulence of Novel Ralstonia pseudosolanacearum (Phylotype I) Strains from Rose, Blueberry, and Mandevilla on Seed Potato.

Potato (Solanum tuberosum L.) ranks fourth among the most important staple food in the world. Ralstonia solanacearum (phylotype [phy] IIB, sequevar [seq] 1 and 2), also known as R3B2, the causal agent of brown rot disease on potato, is extremely damaging, causing great economical losses to potato in temperate regions. It is thought that members of Ralstonia pseudosolanacearum (phy I) are not pathogenic at low temperatures and are usually found in warmer climates. R. pseudosolanacearum strain PD 7123 (seq 33) isolated from roses in the Netherlands, strain P824 (seq 13) isolated from blueberry, and strain P781 (seq 14) from mandevilla in Florida are phylogenetically closely related and could share the same host. The virulence and ability of these novel strains to multiply latently in potato in temperate regions is unknown. The objective of this work was to assess the virulence and presence of latent infections of the mentioned R. pseudosolanacearum strains on three commercial seed potato cultivars under warmer (28°C) and temperate (20°C) temperatures. At 28°C, all three R. pseudosolanacearum strains caused severe symptoms on all potato cultivars. Overall disease severity on potato was lower at 20°C than 28°C, but major differences in virulence of the three strains were observed at 42 days postinoculation (dpi) among potato cultivars. All asymptomatic potato plants and most of their daughter tubers had latent infections at 20°C. Altogether, these results show that the phy I strains from rose, blueberry, and mandevilla may pose a threat to potato production in temperate climates and the worldwide movement of seed potatoes.[Formula: see text] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY 4.0 International license.

Plant-Pathogenic Ralstonia Phylotypes Evolved Divergent Respiratory Strategies and Behaviors To Thrive in Xylem.

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.

Identification, genetic diversity, and pathogenicity of Ralstonia pseudosolanacearum causing cigar tobacco bacterial wilt in China.

Ralstonia pseudosolanacearum, previously known as R. solanacearum species complex (RSSC) phylotypes I and III, is a plant pathogenic bacterium causing significant yield losses in economical crops. In the May of 2020 and 2021, cigar tobacco bacterial wilt was first observed in fields in Danzhou, Hainan Province, China. A total of eight bacterial isolates were isolated and identified as R. pseudosolanacearum with race 1, biovar III by 16S rRNA gene sequencing, Biolog, and host identification. The amino acid sequence showed that Hainan strains and 15 R. pseudosolanacearum reference strains from flue-cured tobacco in Shandong and Guizhou Provinces, all belonged to RS1000 type containing the avrA gene, only Guizhou strains also had the popP1 gene. On the basis of phylotype-specific multiplex PCR amplification, mismatch repair gene and endoglucanase gene-base tree, Hainan strains were identified as phylotype I sequevar 70, and showed stronger pathogenic capabilities on three different varieties than those reference strains. This is the first report of cigar tobacco bacterial wilt caused by R. pseudosolanacearum sequevar 70. The results revealed the diversity of RSSC in Nicotiana tabacum in China and provided useful information regarding the epidemiology of cigar tobacco wilt disease, as well as the breeding for disease resistance in local cigar tobacco.

Spherical pneumonia caused by Ralstonia mannitolilytica: a case report and literature review.

BACKGROUND: Spherical pneumonia is an extremely rare condition that is difficult to diagnose. It is a specific type of lung infection that often manifests as a round or round-like mass on chest imaging. Spherical pneumonia is easily misdiagnosed as a pulmonary tumor; therefore, awareness of this disease must be strengthened. CASE PRESENTATION: The patient was a 29-year-old female who had persistent cough and sputum for approximately 1 month and fever for 5 days. Chest computed tomography (CT) at our hospital revealed a mass in the lower lobe of the right lung near the hilar region, with obstructive pulmonary atelectasis and obstructive pneumonia. Although lung cancer was suspected, Ralstonia mannitolilytica was detected by metagenomic next-generation sequencing (mNGS) of bronchoalveolar lavage fluid, and no cancer cells or Mycobacterium tuberculosis were detected. Finally, the patient was diagnosed with spherical pneumonia caused by R. mannitolilytica. Anti-infective treatment, symptomatic treatment, and administration of a traditional Chinese medicine decoction were performed based on the syndrome differentiation. After 10 days of treatment, chest CT revealed few lesions in the lower lobe of the right lung, which were significantly reduced compared with those in the past. CONCLUSIONS: Spherical pneumonia caused by R. mannitolilytica has not yet been reported and differential diagnosis is key in clinical diagnosis. When spherical pneumonia is difficult to diagnose, mNGS may be a better alternative.

Presence of Ralstonia pseudosolanacearum (Phylotype I) in Aquatic Environments in the Netherlands.

Ralstonia pseudosolanacearum, a European Union quarantine organism, was until recently absent in the aquatic environments and outdoor cultivation systems of the region. This bacterium was only sporadically reported in restricted greenhouse cultivation systems in some EU countries. In this paper, we report the first findings of R. pseudosolanacearum (phylotype I) in surface water in two distinct geographic locations in the Netherlands in 2020. In 2021, the population of R. pseudosolanacearum in surface water ranged from 104 to 106 CFU/liter. An inoculum reservoir for R. pseudosolanacearum in these aquatic environments was the wild bittersweet plant where population densities ranged from 105 to 107 CFU/ml concentrated bittersweet extract. The virulence of the R. pseudosolanacearum isolates from surface water and bittersweet was confirmed by a pathogenicity test on Solanum lycopersicum cv. Moneymaker plants, resulting in wilting and necrosis of the plants. Sequence analysis of the egl locus of R. pseudosolanacearum isolates from surface water and bittersweet revealed that these isolates are closely related to R. pseudosolanacearum (phylotype I) isolates found previously in the Netherlands on rose. R. pseudosolanacearum (phylotype I) has a very broad host plant range, including potato, many ornamentals, and other economically important crops. This highlights the risk for various host plants grown in the vicinity of the geographic locations where R. pseudosolanacearum has been found and shows the importance of unraveling the epidemiological parameters of the survival, establishment, and spread of R. pseudosolanacearum in temperate climates.

The genus Ralstonia: The new kid on the block.

The genus Ralstonia comprises of aerobic, gram-negative, oxidase positive, nonfermentative, largely environmental organisms. They are an emerging pathogen in the hospital setting and are increasingly associated with opportunistic infections and outbreaks. We hereby present a case series of six patients diagnosed with bacteraemia caused by Ralstonia spp. and a brief review of literature. These cases highlight that isolation of a nonfermenting gram-negative bacillus from blood culture of a patient admitted in critical care setting should not be ignored as mere contaminant. Clinicians and microbiologists need to work as a team to combat this novel bug.

Getting to the root of Ralstonia invasion.

Plant diseases caused by soilborne pathogens are a major limiting factor in crop production. Bacterial wilt disease, caused by soilborne bacteria in the Ralstonia solanacearum Species Complex (Ralstonia), results in significant crop loss throughout the world. Ralstonia invades root systems and colonizes plant xylem, changing plant physiology and ultimately causing plant wilting in susceptible varieties. Elucidating how Ralstonia invades and colonizes plants is central to developing strategies for crop protection. Here we review Ralstonia pathogenesis from root detection and attachment, early root colonization, xylem invasion and subsequent wilting. We focus primarily on studies in tomato from the last 5-10 years. Recent work has identified elegant mechanisms Ralstonia uses to adapt to the plant xylem, and has discovered new genes that function in Ralstonia fitness in planta. A picture is emerging of an amazingly versatile pathogen that uses multiple strategies to make its surrounding environment more hospitable and can adapt to new environments.

Validating Methods To Eradicate Plant-Pathogenic Ralstonia Strains Reveals that Growth In Planta Increases Bacterial Stress Tolerance.

Plant-pathogenic bacteria in the Ralstonia solanacearum species complex (RSSC) cause highly destructive bacterial wilt disease of diverse crops. Wilt disease prevention and management is difficult because RSSC persists in soil, water, and plant material. Growers need practical methods to kill these pathogens in irrigation water, a common source of disease outbreaks. Additionally, the R. solanacearum race 3 biovar 2 (R3bv2) subgroup is a quarantine pest in many countries and a highly regulated select agent pathogen in the United States. Plant protection officials and researchers need validated protocols to eradicate R3bv2 for regulatory compliance. To meet these needs, we measured the survival of four R3bv2 and three phylotype I RSSC strains following treatment with hydrogen peroxide, stabilized hydrogen peroxide (Huwa-San), active chlorine, heat, UV radiation, and desiccation. No surviving RSSC cells were detected after cultured bacteria were exposed for 10 min to 400 ppm hydrogen peroxide, 50 ppm Huwa-San, 50 ppm active chlorine, or temperatures above 50°C. RSSC cells on agar plates were eradicated by 30 s of UV irradiation and killed by desiccation on most biotic and all abiotic surfaces tested. RSSC bacteria did not survive the cell lysis steps of four nucleic acid extraction protocols. However, bacteria in planta were more difficult to kill. Stems of infected tomato plants contained a subpopulation of bacteria with increased tolerance of heat and UV light, but not oxidative stress. This result has significant management implications. We demonstrate the utility of these protocols for compliance with select agent research regulations and for management of a bacterial wilt outbreak in the field. IMPORTANCE Bacteria in the Ralstonia solanacearum species complex (RSSC) are globally distributed and cause destructive vascular wilt diseases of many high-value crops. These aggressive pathogens spread in diseased plant material and via contaminated soil, tools, and irrigation water. A subgroup of the RSSC, race 3 biovar 2, is a European and Canadian quarantine pathogen and a U.S. select agent subject to stringent and constantly evolving regulations intended to prevent pathogen introduction or release. We validated eradication and inactivation methods that can be used by (i) growers seeking to disinfest water and manage bacterial wilt disease outbreaks, (ii) researchers who must remain in compliance with regulations, and (iii) regulators who are expected to define containment practices. Relevant to all these stakeholders, we show that while cultured RSSC cells are sensitive to relatively low levels of oxidative chemicals, desiccation, and heat, more aggressive treatment, such as autoclaving or incineration, is required to eradicate plant-pathogenic Ralstonia growing inside plant material.