Novel Ralstonia species from human infections: improved matrix-assisted laser desorption/ionization time-of-flight mass spectrometry-based identification and analysis of antimicrobial resistance patterns.

A collection of 161 Ralstonia isolates, including 90 isolates from persons with cystic fibrosis, 27 isolates from other human clinical samples, 8 isolates from the hospital environment, 7 isolates from industrial samples, and 19 environmental isolates, was subjected to matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) identification and yielded confident species level identification scores for only 62 (39%) of the isolates, including four that proved misidentified subsequently. Whole-genome sequence analysis of 32 representative isolates for which no confident MALDI-TOF MS species level identification was obtained revealed the presence of seven novel Ralstonia species, including three and four that were isolated from cystic fibrosis or other human clinical samples, respectively, and provided the basis for updating an in-house MALDI-TOF MS database. A reanalysis of all mass spectra with the updated MALDI-TOF MS database increased the percentage of isolates with confident species level identification up to 77%. The antimicrobial susceptibility of 30 isolates mainly representing novel human clinical and environmental Ralstonia species was tested toward 17 antimicrobial agents and demonstrated that the novel Ralstonia species were generally multi-resistant, yet susceptible to trimethoprim/sulfamethoxazole, ciprofloxacin, and tigecycline. An analysis of genomic antimicrobial resistance genes in 32 novel and publicly available genome sequences revealed broadly distributed beta-lactam resistance determinants.IMPORTANCEThe present study demonstrated that a commercial matrix-assisted laser desorption/ionization time-of-flight mass spectrometry identification database can be tailored to improve the identification of Ralstonia species. It also revealed the presence of seven novel Ralstonia species, including three and four that were isolated from cystic fibrosis or other human clinical samples, respectively. An analysis of minimum inhibitory concentration values demonstrated that the novel Ralstonia species were generally multi-resistant but susceptible to trimethoprim/sulfamethoxazole, ciprofloxacin, and tigecycline.

Contrasting genetic diversity and structure among Malagasy Ralstonia pseudosolanacearum phylotype I populations inferred from an optimized Multilocus Variable Number of Tandem Repeat Analysis scheme.

The Ralstonia solanacearum species complex (RSSC), composed of three species and four phylotypes, are globally distributed soil-borne bacteria with a very broad host range. In 2009, a devastating potato bacterial wilt outbreak was declared in the central highlands of Madagascar, which reduced the production of vegetable crops including potato, eggplant, tomato and pepper. A molecular epidemiology study of Malagasy RSSC strains carried out between 2013 and 2017 identified R. pseudosolanacearum (phylotypes I and III) and R. solanacearum (phylotype II). A previously published population biology analysis of phylotypes II and III using two MultiLocus Variable Number of Tandem Repeats Analysis (MLVA) schemes revealed an emergent epidemic phylotype II (sequevar 1) group and endemic phylotype III isolates. We developed an optimized MLVA scheme (RS1-MLVA14) to characterize phylotype I strains in Madagascar to understand their genetic diversity and structure. The collection included isolates from 16 fields of different Solanaceae species sampled in Analamanga and Itasy regions (highlands) in 2013 (123 strains) and in Atsinanana region (lowlands) in 2006 (25 strains). Thirty-one haplotypes were identified, two of them being particularly prevalent: MT007 (30.14%) and MT004 (16.44%) (sequevar 18). Genetic diversity analysis revealed a significant contrasting level of diversity according to elevation and sampling region. More diverse at low altitude than at high altitude, the Malagasy phylotype I isolates were structured in two clusters, probably resulting from different historical introductions. Interestingly, the most prevalent Malagasy phylotype I isolates were genetically distant from regional and worldwide isolates. In this work, we demonstrated that the RS1-MLVA14 scheme can resolve differences from regional to field scales and is thus suited for deciphering the epidemiology of phylotype I populations.

Nosocomial bloodstream infection and the emerging carbapenem-resistant pathogen Ralstonia insidiosa.

BACKGROUND: Ralstonia picketti, Ralstonia mannitolilytica, and Ralstonia insidiosa have recently been regarded as emerging pathogens of infectious diseases, in particular as the pathogens responsible for nosocomial infection in immunocompromised patients. R. insidiosa differs from R. picketti and R. mannitolilytica, and its related infections are rarely reported. METHODS: Clinical data from two nosocomial bloodstream infection cases were extracted and analyzed. The causable isolates were identified by the VITEK 2 Compact system, matrix assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS), and molecular identification methods using PCR with universal and species-specific primers. Antimicrobial susceptibility testing was performed using the broth microdilution method. Both of the isolates were subjected to whole genome sequencing using a HiSeq X10 Sequencer. Antimicrobial resistance genes, virulence factors, and plasmid replicons were identified from assembled genomes. A real-time RT-PCR experiment and a cloning experiment were conducted to explore the related class D ß-lactamase-encoding genes. RESULTS: Both patients recovered under therapy with antibiotics. Isolates were initially misidentified as R. mannitolilytica by the VITEK 2 Compact system rather than R. insidiosa, as identified by both MALDI-TOF MS and 16S rRNA gene sequencing. Both isolates were resistant to aminoglycosides, ß-lactams, and polymyxin B. One isolate harboring blaOXA-570 was resistant to carbapenems. The whole genome sequencing data confirmed species identification based on average nucleotide identity (ANI) and revealed two variants of class D ß-lactamase-encoding gene blaOXA (blaOXA-573 and blaOXA-574). The real-time RT-PCR experiment showed no difference in gene expression between blaOXA-570 and blaOXA-573 in our strains. The cloning experiment showed that variant OXA-573 had no carbapenem hydrolase activity. CONCLUSIONS: We described two cases of nosocomial bloodstream infection caused by R. insidiosa strains. MALDI-TOF MS was cost-effective for rapid species identification. Clinicians should be aware that R. insidiosa can be resistant to commonly used antibiotics, even carbapenems.

The Ptr1 Locus of Solanum lycopersicoides Confers Resistance to Race 1 Strains of Pseudomonas syringae pv. tomato and to Ralstonia pseudosolanacearum by Recognizing the Type III Effectors AvrRpt2 and RipBN.

Race 1 strains of Pseudomonas syringae pv. tomato, which cause bacterial speck disease of tomato, are becoming increasingly common and no simply inherited genetic resistance to such strains is known. We discovered that a locus in Solanum lycopersicoides, termed Pseudomonas tomato race 1 (Ptr1), confers resistance to race 1 P. syringae pv. tomato strains by detecting the activity of type III effector AvrRpt2. In Arabidopsis, AvrRpt2 degrades the RIN4 protein, thereby activating RPS2-mediated immunity. Using site-directed mutagenesis of AvrRpt2, we found that, like RPS2, activation of Ptr1 requires AvrRpt2 proteolytic activity. Ptr1 also detected the activity of AvrRpt2 homologs from diverse bacteria, including one in Ralstonia pseudosolanacearum. The genome sequence of S. lycopersicoides revealed no RPS2 homolog in the Ptr1 region. Ptr1 could play an important role in controlling bacterial speck disease and its future cloning may shed light on an example of convergent evolution for recognition of a widespread type III effector.

Genomic and biological characterization of a new member of the genus Phikmvvirus infecting phytopathogenic Ralstonia bacteria.

Bacterial wilt caused by Ralstonia spp., soil-borne Gram-negative bacteria, is considered one of the most important plant diseases in tropical and subtropical regions of the world. A large number of bacteriophages capable of lysing or physiologically reprogramming cells of Ralstonia spp. have been reported in Asia. Despite the potential use of these organisms in the management of bacterial wilt, information on viruses that infect Ralstonia spp. is nonexistent in the Americas. We isolated a virus that infects Ralstonia spp. from a soil sample in the state of Minas Gerais, Brazil. Microscopy and genomic and phylogenetic analysis allowed us to classify the virus as a member of the family Podoviridae, genus Phikmvvirus. In spite of its relationship to Ralstonia virus RSB3, an Asian isolate, genomic and biological characteristics showed that the virus isolated in Brazil, tentatively named "Ralstonia virus phiAP1" (phiAP1), belongs to a new species. phiAP1 has EPS depolymerase activity and contains two putative virion-associated peptidoglycan hydrolases (VAPGHs), which reveals a robust mechanism of pathogenesis. Furthermore, phiAP1 specifically infects Ralstonia solanacearum, R. pseudosolanacearum and R. syzygii, causing cell lysis, but it was not able to infect thirteen other bacteria that were tested. Together, these characteristics highlight the biotechnological potential of this virus for the management of bacterial wilt.

Ralstonia infection in cystic fibrosis.

This study aimed to determine prevalence of Ralstonia spp. in cystic fibrosis patients, look for any evidence of cross infection and to describe clinical outcomes for patients infected by Ralstonia spp. Prevalence of Ralstonia spp. was calculated annually from 2008 to 2016. Pulsed-field gel electrophoresis was performed on ⩾1 sample from patients with an isolation of Ralstonia spp. between 2008 and 2016. A prospective, longitudinal observational study of adult patients was performed with 12 months follow-up from recruitment. Prevalence of Ralstonia spp. rose from 0·6% in 2008 to 2·4% in 2016. In total 12 out of 14 (86%) patients with ⩾1 isolation of Ralstonia spp. developed chronic infection. A pair and a group of three unrelated patients with epidemiological connections shared strains of Ralstonia mannitolilytica. Lung function of Ralstonia spp. infected patients was moderately to severely impaired. Prevalence of Ralstonia spp. is low but increasing. The risk of a patient developing chronic Ralstonia spp. infection following first acquisition is high and cross-infection may be possible. Whether Ralstonia spp. infection causes increased pulmonary exacerbation frequency and lung function decline needs to be evaluated in larger prospective studies.

Temperature drives the assembly of endophytic communities' seasonal succession.

Endophytic microorganisms asymptomatically colonise plant tissues. Exploring the assembly dynamics of bacterial endophytic communities is essential to understand the functioning of the plant holobiont and to optimise their possible use as biopesticides or plant biostimulants. The variation in endophytic communities in above and below-ground organs in Vitis vinifera in the field were studied. To understand the specific effect of temperature on endophytic communities, a separate experiment was set up where grapevine cuttings were grown under controlled conditions at three different temperatures. The findings revealed the succession of endophytic communities over the year. Endophytic communities of roots and stems differ in terms of composition and dynamic response to temperature. Noticeably, compositional differences during the seasons affected bacterial taxa more in stems than in roots, suggesting that roots offer a more stable and less easily perturbed environment. Correlation abundance networks showed that the presence of several taxa (including Bradyrhizobium, Burkholderia, Dyella, Mesorhizobium, Propionibacterium and Ralstonia) is linked in both the field and the greenhouse.

Ralstonia picketti neonatal sepsis: a case report.

BACKGROUND: Ralstonia genus are gram negative bacillus and includes four bacteria namely Ralstonia picketti, Ralstonia Solanacearum, Ralstonia insidiosa and Ralstonia mannitolilytica. These are opportunistic pathogens and cause infections in immunocompromised host. The sources of infection are usually contaminated solutions and water. The majority of the reported cases are caused by R. picketti. It is very rare cause of neonatal sepsis with less than twenty cases reported in literature till date. CASE PRESENTATION: A late preterm male infant, Indian race was admitted to the neonatal intensive care unit for respiratory distress developing soon after birth. The infant was managed with respiratory support and gradually infant improved and diagnosis of transient tachypnea of newborn was made. At age of 84 h of postnatal life, the infant developed features of neonatal sepsis and investigations were suggestive of sepsis. The infant was started on intravenous antibiotic, multiple vasopressors and steroids. The blood culture showed growth of multi-drug resistant R. picketti. The antibiotics were changed as per sensitivity pattern and infant was discharged in good condition and was accepting breast feeding at the time of discharge. There was also no other case of R. picketti in the nursery during the same time period. CONCLUSION: Ralstonia picketti is an uncommon cause of neonatal sepsis and usually source of infection are contaminated solutions and medical products. The management involves early detection, treatment with appropriate antibiotics and doing surveillance culture to identify the possible source of infection.

Identification and characterization of chemosensors for d-malate, unnatural enantiomer of malate, in Ralstonia pseudosolanacearum.

Ralstonia pseudosolanacearum Ps29 is attracted by nonmetabolizable d-malate, an unnatural enantiomer. Screening of a complete collection of single-mcp-gene deletion mutants of Ps29 revealed that the RSc1156 homologue is a chemosensor for d-malate. An RSc1156 homologue deletion mutant of Ps29 showed decreased but significant responses to d-malate, suggesting the existence of another d-malate chemosensor. McpM previously had been identified as a chemosensor for l-malate. We constructed an RSc1156 homologue mcpM double deletion mutant and noted that this mutant failed to respond to d-malate; thus, the RSc1156 homologue and McpM are the major chemosensors for d-malate in this organism. To further characterize the ligand specificities of the RSc1156 homologue and McpM, we constructed a Ps29 derivative (designated K18) harbouring deletions in 18 individual mcp genes, including mcpM and RSc1156. K18 harbouring the RSc1156 homologue responded strongly to l-tartrate and d-malate and moderately to d-tartrate, but not to l-malate or succinate. K18 harbouring mcpM responded strongly to l-malate and d-tartrate and moderately to succinate, fumarate and d-malate. Ps29 utilizes l-malate and l-tartrate, but not d-malate. We therefore concluded that l-tartrate and l-malate are natural ligands of the RSc1156 homologue and McpM, respectively, and that chemotaxis toward d-malate is a fortuitous response by the RSc1156 homologue and McpM in Ps29. We propose re-designation of the RSc1156 homologue as McpT. In tomato plant infection assays, the mcpT deletion mutant of highly virulent R. pseudosolanacearum MAFF106611 was as infectious as wild-type MAFF106611, suggesting that McpT-mediated chemotaxis does not play an important role in tomato plant infection.

Phylogenomic analysis of the genus Ralstonia based on 686 single-copy genes.

The genus Ralstonia contains species that are devastating plant pathogens, opportunistic human pathogens, and/or important degraders of xenobiotic and recalcitrant compounds. However, significant nomenclature problems exist, especially for the Ralstonia solanacearum species complex which consists of four phylotypes. Phylogenomics of the Ralstonia genus was investigated via a comprehensive analysis of 39 Ralstonia genomes as well as four genomes of Cupriavidus necator (more commonly known by its previous name Ralstonia eutropha). These data revealed 686 single-copy orthologs that could be extracted from the Ralstonia core-genome and used to reconstruct the phylogeny of the genus Ralstonia. The generated tree has strong bootstrap support for almost all branches. We also estimated the in silico DNA-DNA hybridization (isDDH) and the average nucleotide identity (ANI) values between each genome. Our data confirmed that whole genome sequence data provides a powerful tool to resolve the complex taxonomic questions of the genus Ralstonia, e.g. strains of Ralstonia solanacearum phylotype IIA and IIB may represent two subspecies of R. solanacearum, and strains of R. solanacearum phylotype I and III may be classified into two subspecies of Ralstonia pseudosolanacearum. Recently, strains of R. solanacearum phylotype IV were proposed to be reclassified into different subspecies of Ralstonia syzygii; our study, however, showed that phylotype IV strains had high isDDH values (83.8-96.1 %), indicating it may be not appropriate to classify these closely related strains into different subspecies. We also evaluated the performance of six chromosomal housekeeping genes (gdhA, mutS, adk, leuS, rplB and gyrB) used in Ralstonia phylogenetic inference. The multilocus sequence analysis of these six marker genes was able to reliably infer the phylogenetic relationships of the genus Ralstonia.

The microbiome and emerging pathogens in cystic fibrosis and non-cystic fibrosis bronchiectasis.

Chronic pulmonary sepsis is the predominant cause of morbidity for patients with cystic fibrosis (CF) and non-CF bronchiectasis. Previously it was thought that respiratory infection in these patients was mostly limited to a very small number of typical pathogens; however, in recent years there have been increasing reports of infection with other emerging potential pathogens including Burkholderia, Stenotrophomonas, Achromobacter, Ralstonia, Pandoraea, nontuberculous mycobacteria, and fungal species. Furthermore, culture-independent methodologies have established that the lungs of patients with CF and non-CF bronchiectasis comprise mixed microbiological communities of aerobic and anaerobic bacteria, fungal and viral species, collectively referred to as the lung microbiome. This article addresses the clinical relevance of emerging pathogens and the lung microbiome in CF and non-CF bronchiectasis.

Polyphasic taxonomic revision of the Ralstonia solanacearum species complex: proposal to emend the descriptions of Ralstonia solanacearum and Ralstonia syzygii and reclassify current R. syzygii strains as Ralstonia syzygii subsp. syzygii subsp. nov., R. solanacearum phylotype IV strains as Ralstonia syzygii subsp. indonesiensis subsp. nov., banana blood disease bacterium strains as Ralstonia syzygii subsp. celebesensis subsp. nov. and R. solanacearum phylotype I and III strains as Ralstonia pseudosolanacearum sp. nov.

The Ralstonia solanacearum species complex has long been recognized as a group of phenotypically diverse strains that can be subdivided into four phylotypes. Using a polyphasic taxonomic approach on an extensive set of strains, this study provides evidence for a taxonomic and nomenclatural revision of members of this complex. Data obtained from phylogenetic analysis of 16S-23S rRNA ITS gene sequences, 16S-23S rRNA intergenic spacer (ITS) region sequences and partial endoglucanase (egl) gene sequences and DNA-DNA hybridizations demonstrate that the R. solanacearum species complex comprises three genospecies. One of these includes the type strain of Ralstonia solanacearum and consists of strains of R. solanacearum phylotype II only. The second genospecies includes the type strain of Ralstonia syzygii and contains only phylotype IV strains. This genospecies is subdivided into three distinct groups, namely R. syzygii, the causal agent of Sumatra disease on clove trees in Indonesia, R. solanacearum phylotype IV strains isolated from different host plants mostly from Indonesia, and strains of the blood disease bacterium (BDB), the causal agent of the banana blood disease, a bacterial wilt disease in Indonesia that affects bananas and plantains. The last genospecies is composed of R. solanacearum strains that belong to phylotypes I and III. As these genospecies are also supported by phenotypic data that allow the differentiation of the three genospecies, the following taxonomic proposals are made: emendation of the descriptions of Ralstonia solanacearum and Ralstonia syzygii and descriptions of Ralstonia syzygii subsp. nov. (type strain R 001(T) = LMG 10661(T) = DSM 7385(T)) for the current R. syzygii strains, Ralstonia syzygii subsp. indonesiensis subsp. nov. (type strain UQRS 464(T) = LMG 27703(T) = DSM 27478(T)) for the current R. solanacearum phylotype IV strains, Ralstonia syzygii subsp. celebesensis subsp. nov. (type strain UQRS 627(T) = LMG 27706(T) = DSM 27477(T)) for the BDB strains and Ralstonia pseudosolanacearum sp. nov. (type strain UQRS 461(T) = LMG 9673(T) = NCPPB 1029(T)) for the strains of R. solanacearum phylotypes I and III.

Brote de Ralstonia mannitolilytica a partir de bacteriemias asociadas a catéteres implantables y semiimplantables en un hospital pediátrico / Outbreak of Raistonia mannitolilytica by implantable and semi-implantable catheter-related bacteremia in a pediatric hospital

Ralstonia mannitolilytica junto con Ralstonia pickettii han sido asociadas con brotes hospitalarios relacionados con la contaminación de algún dispositivo o fluido. El objetivo de este trabajo fue describir un brote por R. mannitolilytica a partir de bacteriemias asociadas a catéteres implantables y semiimplantables ocurrido en un hospital pediátrico de alta complejidad y evaluar la utilidad del empleo de métodos moleculares para su investigación.Se detectó la presencia de bacilos gram negativos no fermentadores, con igual antibiotipo, en hemocultivos y retrocultivos a partir de dos pacientes que tenían catéteres implantables y estaban atendidos en una misma área del hospital. Se realizaron estudios microbiológicos de muestras de frascos de heparina, soluciones de dextrosa y soluciones antisépticas con resultado negativo. Algunos pacientes tuvieron signos y/o síntomas clínicos de bacteriemia al habilitar los catéteres para su uso. Se citaron para su estudio a todos los pacientes que habían tenido un procedimiento de apertura y cierre de catéter durante las fechas cercanas a los hallazgos en hemocultivos (N expuestos = 45). Ocurrieron 17 casos (infectados), a partir de los cuales se analizaron 23 aislamientos, en los que se pudo documentar la presencia de R. mannitolilytica (23 aislamientos). Por métodos moleculares se determinó que los aislamientos provenientes de muestras de pacientes involucrados en el brote se encontraban estrechamente relacionados y podrían representar una misma cepa o clon. Por evidencia circunstancial se consideró a la "solución heparínica de cierre" como fuente posible del brote (AU)

Both Ralstonia mannitolilytica and Ralstonia pickettii have been associated with hospital outbreaks due to device or fluid contamination. The aim of this study was to describe an implantable- or semi-implantable-catheter-related bacteremia outbreak by R. mannitolilytica in a tertiary-care hospital and to assess the usefulness of molecular analysis for the identification of the organism. Non-fermenting gram-negative bacilli, with identical antibiotypes, were detected in hemocultures of two patients with implantable catheters in the same hospital area. Microbiological studies of heparin and dextrose and antiseptic solution vials were negative. Some of the patients had clinical signs and/or symptoms of bacteremia when the catheter was prepared for use. All patients who underwent a procedure of accessing or locking the port of the catheter around the time of the positive hemoculture findings were contacted (N exposed = 45). Seventeen infections were detected, of which 23 isolates were analyzed. The presence of R. mannitolilytica was recorded in 23 isolates. Molecular analysis showed that the isolates from the samples of the patients involved in the outbreak were closely related and might represent the same strain or clone. Circumstantial evidence suggested that the heparin-lock solution may have been the source of the outbreak (AU)

The antibiotic susceptibility of water-based bacteria Ralstonia pickettii and Ralstonia insidiosa.

Ralstonia pickettii and Ralstonia insidiosa are waterborne bacteria that can survive and grow in various water sources, and that are emerging pathogens in hospital settings. Not much is known about the antibiotic resistance of these bacteria. Previous reports of antimicrobial susceptibility have been largely limited to a few clinical strains with no accounting for genotypic or phenotypic diversity or that these species could vary from the set breakpoints. Etests and disc diffusion tests were carried out to compare the antimicrobial susceptibilities to 12 different antibiotics of 68 different isolates of R. pickettii (53) and R. insidiosa (15) from varying environments, which have previously been well characterized both phenotypically and genetically. The majority of the R. pickettii and R. insidiosa isolates showed susceptibility to most of the antibiotics tested in this study. The most effective were found to be the quinolones and sulfamethoxazole/trimethoprim. Antibiotic susceptibility was also found not to vary between environmental niches for R. pickettii and R. insidiosa isolates.

Isolation and characterization of a soluble and thermostable phosphite dehydrogenase from Ralstonia sp. strain 4506.

Phosphite dehydrogenase (PtxD), which catalyzes the nearly irreversible oxidation of phosphite to phosphate with the concomitant reduction of NAD(+) to NADH, has great potential for NADH regeneration in industrial biocatalysts. Here, we isolated a soil bacterium, Ralstonia sp. strain 4506, that grew at 45°C on a minimal medium containing phosphite as the sole source of phosphorus. A recombinant PtxD of Ralstonia sp. (PtxD(R4506)) appeared in the soluble fraction in Escherichia coli. The purified PtxD(R4506) showed 6.7-fold greater catalytic efficiency (V(max)/K(m)) than the first characterized PtxD of Pseudomonas stutzeri (PtxD(PS)). Moreover, the purified PtxD(R4506) showed maximum activity at 50°C, and its half-life of thermal inactivation at 45°C was 80.5h, which is approximately 3,450-fold greater than that of PtxD(PS). Therefore, we concluded that PtxD(R4506), which shows high catalytic efficiency, solubility, and thermostability, would be useful for NADH regeneration applications.

Ralstonia syzygii, the Blood Disease Bacterium and some Asian R. solanacearum strains form a single genomic species despite divergent lifestyles.

The Ralstonia solanacearum species complex includes R. solanacearum, R. syzygii, and the Blood Disease Bacterium (BDB). All colonize plant xylem vessels and cause wilt diseases, but with significant biological differences. R. solanacearum is a soilborne bacterium that infects the roots of a broad range of plants. R. syzygii causes Sumatra disease of clove trees and is actively transmitted by cercopoid insects. BDB is also pathogenic to a single host, banana, and is transmitted by pollinating insects. Sequencing and DNA-DNA hybridization studies indicated that despite their phenotypic differences, these three plant pathogens are actually very closely related, falling into the Phylotype IV subgroup of the R. solanacearum species complex. To better understand the relationships among these bacteria, we sequenced and annotated the genomes of R. syzygii strain R24 and BDB strain R229. These genomes were compared to strain PSI07, a closely related Phylotype IV tomato isolate of R. solanacearum, and to five additional R. solanacearum genomes. Whole-genome comparisons confirmed previous phylogenetic results: the three phylotype IV strains share more and larger syntenic regions with each other than with other R. solanacearum strains. Furthermore, the genetic distances between strains, assessed by an in-silico equivalent of DNA-DNA hybridization, unambiguously showed that phylotype IV strains of BDB, R. syzygii and R. solanacearum form one genomic species. Based on these comprehensive data we propose a revision of the taxonomy of the R. solanacearum species complex. The BDB and R. syzygii genomes encoded no obvious unique metabolic capacities and contained no evidence of horizontal gene transfer from bacteria occupying similar niches. Genes specific to R. syzygii and BDB were almost all of unknown function or extrachromosomal origin. Thus, the pathogenic life-styles of these organisms are more probably due to ecological adaptation and genomic convergence during vertical evolution than to the acquisition of DNA by horizontal transfer.

Genotypic and phenotypic diversity of Ralstonia pickettii and Ralstonia insidiosa isolates from clinical and environmental sources including High-purity Water. Diversity in Ralstonia pickettii.

BACKGROUND: Ralstonia pickettii is a nosocomial infectious agent and a significant industrial contaminant. It has been found in many different environments including clinical situations, soil and industrial High Purity Water. This study compares the phenotypic and genotypic diversity of a selection of strains of Ralstonia collected from a variety of sources. RESULTS: Ralstonia isolates (fifty-nine) from clinical, industrial and environmental origins were compared genotypically using i) Species-specific-PCR, ii) PCR and sequencing of the 16S-23S rRNA Interspatial region (ISR) iii) the fliC gene genes, iv) RAPD and BOX-PCR and v) phenotypically using biochemical testing. The species specific-PCR identified fifteen out of fifty-nine designated R. pickettii isolates as actually being the closely related species R. insidiosa. PCR-ribotyping of the 16S-23S rRNA ISR indicated few major differences between the isolates. Analysis of all isolates demonstrated different banding patterns for both the RAPD and BOX primers however these were found not to vary significantly. CONCLUSIONS: R. pickettii species isolated from wide geographic and environmental sources appear to be reasonably homogenous based on genotypic and phenotypic characteristics. R. insidiosa can at present only be distinguished from R. pickettii using species specific PCR. R. pickettii and R. insidiosa isolates do not differ significantly phenotypically or genotypically based on environmental or geographical origin.

Differentiating the growing nosocomial infectious threats Ralstonia pickettii and Ralstonia insidiosa.

Differentiation of the growing nosocomial infectious threats, Ralstonia pickettii and Ralstonia insidiosa, based on nitrate reduction, desferrioxamine susceptibility, arabinose, N-acetyl-glucosamine and phenylacetate assimilation is described. These tests can be used for preliminary identification of Ralstonia pickettii and Ralstonia insidiosa resulting in more accurate identification of these species.

A novel alkaline lipase from Ralstonia with potential application in biodiesel production.

With the aim of isolating a biocatalyst able to catalyze biodiesel production from microbial source, Ralstonia sp. CS274 was isolated and a lipase from the strain (RL74) was purified. Molecular weight of RL74 was estimated to be 28,000 Da by SDS-PAGE. The activity was highest at 50-55°C and pH 8.0-9.5 and was stable at pH 7.0-12.0 and up to 45°C. It was resistant to oxidizing and reducing agents and the activity was enhanced by detergents. RL74 was 1,3 specific and K(m) and V(max) for p-nitrophenyl palmitate were 2.73 ± 0.6mM and 101.4 ± 1.9 mM/min mg, respectively. N-terminal amino acid sequence showed partial homology with that of Penicillium lipases. RL74 produced biodiesel more efficiently in palm oil than in soybean oil; and the production was highest at pH 8.0, at 5% methanol and at 20% water content.