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.

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.

Chemotactic disruption as a method to control bacterial wilt caused by Ralstonia pseudosolanacearum.

We have demonstrated that chemotaxis to l-malate facilitated motility of Ralstonia pseudosolanacearum MAFF 106611, a causative agent of bacterial wilt, to plant roots. Here, we evaluated the assumption that the disruption of chemotaxis to l-malate leads to inhibition of plant infection by R. pseudosolanacearum MAFF 106611. Chemotactic assays revealed that chemotaxis to l-malate was completely or partially inhibited in the presence of l-, d-, and dl-malate, respectively. Moreover, l-malate served as a carbon and energy source for R. pseudosolanacearum MAFF 106611, while d-malate inhibited the growth of this bacterium. In the sand-soak inoculation virulence assay for tomato plants, the addition of l-, d-, and dl-malate to sand suppressed the plant infection. We concluded that supplementation of l- and dl-malate suppresses tomato plant infection with R. pseudosolanacearum MAFF 106611 by disrupting its chemotaxis to l-malate, while d-malate suppresses it by both the disruption of l-malate chemotaxis and inhibition of growth.

Elucidation of antibacterial effect of calcium chloride against Ralstonia pseudosolanacearum race 4 biovar 3 infecting ginger (Zingiber officinale Rosc.).

Bacterial wilt incited by Ralstonia pseudosolanacearum (Rps) race 4 biovar 3 is a serious threat to ginger (Zingiber officinale Rosc.) cultivation throughout the ginger growing tracts and warrants effective remedial measures since most of the strategies failed at field level implementation. After a series of experiments, calcium chloride was found to be effective against Rps both in vitro and in planta and its prophylactic effect has been successfully demonstrated under field conditions. CaCl2 at a concentration of > 2% significantly inhibited Rps under in vitro conditions. Calcium is an important nutritional element imparts a major role in plant disease resistance, and numerous studies have demonstrated the mitigating effect of calcium for disease management. CaCl2 being inhibitory to Rps, the mechanism of inhibition by CaCl2 against Rps was elucidated by a series of in vitro assays including swarming motility and biofilm formation. Direct inhibition was also studied using Scanning Electron Microscopy (SEM). The minimum bactericidal concentration and minimum inhibitory concentration were found to be around 3% while the EC 90 value was found to be 2.25%. The SEM analysis revealed the destruction of cell structure by making perforations on the cell surface. CaCl2 at the targeted concentrations inhibited biofilm formation as well as swarming motility of Rps. These findings suggest that CaCl2 exhibits strong antibacterial activity against Rps and has the potential to be used as an effective bactericide for Rps in managing bacterial wilt in ginger.

Potential Use of L-arabinose for the Control of Tomato Bacterial Wilt.

The present study aimed to investigate the potential of simple sugars for use as protection agents in the control of tomato bacterial wilt caused by Ralstonia pseudosolanacearum. Based on the sugar assimilation patterns of the pathogen, four unassimilable sugars (L-arabinose, maltose, D-raffinose, and D-ribose) were selected from 10 representative sugars present in tomato root exudates. These sugars were evaluated for their effects on bacterial wilt using a tomato seedling bioassay. The application of 0.25% L-arabinose significantly reduced disease severity and was, thus, selected as a candidate for further evaluations in a pot experiment under glasshouse conditions. The results obtained showed that the disease suppressive effects of L-arabinose slightly increased at higher concentrations; drench treatments at 0.1, 0.25, and 0.5% reduced disease severity by ca. 48, 70, and 87%, respectively. The drench treatment with 0.5% L-arabinose significantly reduced the pathogen population in the rhizosphere and stem tissues of tomato plants without any antibacterial activity. Real-time reverse-transcription PCR revealed that the expression of salicylic acid-dependent and ethylene-dependent defense genes was significantly enhanced in the stem tissues of L-arabinose-treated tomato plants following the pathogen inoculation. These results suggest that soil drenching with L-arabinose effectively suppresses tomato bacterial wilt by preventing pathogen proliferation in the rhizosphere and stem tissues of tomato plants. This is the first study to report the potential of L-arabinose as a safe, eco-friendly, and cost-effective plant protection agent for the control of tomato bacterial wilt.

A Unique Combination of Two Different Quorum Sensing Systems in the ß-Rhizobium Cupriavidus taiwanensis.

Cupriavidus taiwanensis LMG19424, a ß-rhizobial symbiont of Mimosa pudica, harbors phc and tqs quorum sensing (QS), which are the homologous cell-cell communication systems previously identified from the plant pathogen Ralstonia solanacearum and the human pathogen Vibrio cholerae, respectively. However, there has been no experimental evidence reported that these QS systems function in C. taiwanensis LMG19424. We identified (R)-methyl 3-hydroxymyristate (3-OH MAME) and (S)-3-hydroxypentadecan-4-one (C15-AHK) as phc and tqs QS signals, respectively, and characterized these QS systems. The expression of the signal synthase gene phcB and tqsA in E. coli BL21(DE3) resulted in the high production of 3-OH MAME and C15-AHK, respectively. Their structures were elucidated by comparison of EI-MS data and GC/chiral LC retention times with synthetic standards. The deletion of phcB reduced cell motility and increased biofilm formation, and the double deletion of phcB/tqsA caused the accumulation of the metal chelator coproporphyrin III in its mutant culture. Although the deletion of phcB and tqsA slightly reduced its ability to nodulate on aseptically grown seedlings of M. pudica, there was no significant difference in nodule formation between LMG19424 and its QS mutants when commercial soils were used. Taken together, this is the first example of the simultaneous production of 3-OH MAME/C15-AHK as QS signals in a bacterial species, and the importance of the phc/tqs QS systems in the saprophytic stage of C. taiwanensis LMG19424 is suggested.

Synthesis of Thiazolium-Labeled 1,3,4-Oxadiazole Thioethers as Prospective Antimicrobials: In Vitro and in Vivo Bioactivity and Mechanism of Action.

In this study, a type of thiazolium-labeled 1,3,4-oxadiazole thioether bridged by diverse alkyl chain lengths was constructed. The antimicrobial activity of the fabricated thioether toward plant pathogenic bacteria and fungi was then screened. Antibacterial evaluation indicated that title compounds possess specific characteristics that enable them to severely attack three phytopathogens, namely, Xanthomonas oryzae pv. oryzae, Ralstonia solanacearum, and Xanthomonas axonopodis pv. citri with minimal EC50 values of 0.10, 3.27, and 3.50 µg/mL, respectively. Three-dimensional quantitative structure-activity relationship models were established to direct the following excogitation for exploring higher active drugs. The in vivo study against plant bacterial diseases further identified the prospective application of title compounds as alternative antibacterial agents. The proteomic technique, scanning electron microscopy patterns, and fluorescence spectrometry were exploited to investigate the antibacterial mechanism. Additionally, some target compounds performed superior inhibitory actions against three tested fungal strains. In view of their simple molecular architecture and highly efficient bioactivity, these substrates could be further explored as promising surrogates for fighting against plant microbial infections.

Labrenzbactin from a coral-associated bacterium Labrenzia sp.

A new catecholate-containing siderophore, labrenzbactin (1), was isolated from the fermentation broth of a coral-associated bacterium Labrenzia sp. The structure and absolute configuration of 1 was determined by spectroscopic methods and Marfey's analysis. Overall, 1 showed antimicrobial activity against Ralstonia solanacearum SUPP1541 and Micrococcus luteus ATCC9341 with MIC values of 25 and 50 µg ml-1, respectively, and cytotoxicity against P388 murine leukemia cells with an IC50 of 13 µM.

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.

Green synthesis of silver nanoparticles using tomato leaf extract and their entrapment in chitosan nanoparticles to control bacterial wilt.

BACKGROUND: Silver nanoparticles (AgNPs), particularly those entrapped in polymeric nanosystems, have arisen as options for managing plant bacterial diseases. Among the biopolymers useful for the entrapment of AgNPs, chitosan is promising because of its low cost, good biocompatibility, antimicrobial properties and biodegradability. The present study aimed: (i) to greenly-synthesize AgNPs using different concentrations of aqueous extract of tomato leaves followed by entrapment of AgNPs with chitosan (CH-AgNPs); (ii) to characterize the optical, structural and biological properties of the nanosystems produced; (iii) to evaluate the antimicrobial activities of AgNPs and nanomaterials; and (iv) to assess the effectiveness of AgNPs and nanomaterials for controlling tomato bacterial wilt caused by Ralstonia solanacearum. RESULTS: Spherical and oval AgNPs had incipient colloidal instability, although the concentration of the tomato leaf extract influenced both size (< 87 nm) and the polydispersity index. Nanomaterials (< 271 nm in size) were characterized by a highly stable matrix of chitosan containing polydisperse AgNPs. Free AgNPs and CH-AgNPs were stable for up to 30 days, with no significant alteration in physicochemical parameters. The AgNPs and nanomaterials had antibacterial activity and decreased bacterial growth at micromolar concentrations after 48 h. Morphological changes in R. solanacearum cells were observed after treatment with CH-AgNPs. The application of CH-AgNPs at 256 µmol L-1 reduced the incidence of bacterial wilt in a partially resistant tomato genotype but not in the susceptible line. CONCLUSION: Greenly-synthesized chitosan-derived nanomaterials containing AgNPs produced with leaf extracts from their own species appear to comprise a promising and sustainable alternative in an integrated management approach aiming to reduce the yield losses caused by bacterial wilt. © 2019 Society of Chemical Industry.

Association between gentamicin resistance and stress tolerance in water isolates of Ralstonia pickettii and R. mannitolilytica.

Members of the species Ralstonia pickettii and R. mannitolilytica, although ubiquitous and lacking major virulence factors, have been associated with nosocomial outbreaks. Tolerance to metals, antibiotics, and disinfectants may represent an advantage for their ubiquity and opportunistic pathogenic potential. In this study, we compared five strains that differed on the origin (hospital effluent, tap water, mineral water) and in the susceptibility to aminoglycosides, regarding their tolerance to metals and disinfection. The growth kinetics and biofilm formation capacity were tested in four R. pickettii strains and one R. mannitolilytica at sub-inhibitory concentrations of aminoglycosides or arsenite. The survival to UV radiation, chlorine, or hydrogen peroxide was also compared in aminoglycoside resistant and susceptible strains. Aminoglycoside-resistant strains presented a higher tolerance to arsenite than the susceptible ones and either aminoglycosides or arsenite was observed to stimulate the biofilm formation. Sub-inhibitory concentrations of the aminoglycoside gentamicin or arsenite significantly decreased the growth rate and yield, but only arsenite caused a significant increase of the lag phase. Hydrogen peroxide presented higher disinfection effectiveness against aminoglycoside susceptible than against resistant strains, an effect that was not observed for UV or chlorine. Although this conclusion needs validation based on a larger number of isolates, including clinical, the results suggest that aminoglycoside resistance may be associated with traits that influence Ralstonia spp. fitness in the environment.

Hydroxycoumarins: New, effective plant-derived compounds reduce Ralstonia pseudosolanacearum populations and control tobacco bacterial wilt.

Plant wilt disease caused by the soilborne bacterial pathogen Ralstonia pseudosolanacearum is one of the most devastating plant diseases; however, no effective protection against this disease has been developed. Coumarins are important natural plant-derived compounds with a wide range of bioactivities and extensive applications in medicine and agriculture. In the present study, three hydroxycoumarins (Hycs), umbelliferone (UM), esculetin (ES) and daphnetin (DA) significantly inhibited the growth of R. pseudosolanacearum on solid medium in a concentration-dependent manner, and the minimum inhibitory concentration (MICs) of these compounds was 325  mg L-1, 125 mg L-1 and 75 mg L-1, respectively. The percentage of live cells of R. pseudosolanacearum when supplemented with UM, ES, and DA was 63.61%, 17.81% and 7.23%, respectively, which were significantly lower than the DMSO treatment with 92%. Furthermore, irrigating roots with hydroxycoumarins (Hycs) 24 h before inoculation with R. pseudosolanacearum significantly delayed the occurrence of tobacco bacterial wilt, with the control efficiency of the DA treatment (the most efficient of Hycs treatment) 80.03%, 69.83%, 59.19%, 45.49%, 44.12%, 38.27% at 6, 8, 10, 12, 14, and 16 days after inoculation, respectively. Compared with the DMSO treatment, the pathogen populations of tobacco stems supplemented with 100 mg L-1 DA were the lowest, with population significantly reduced by 22.46%, 27.34%, and 18.06% at 4, 7, and 10 days after inoculation, respectively. Based on this study, these Hycs could be applied as potential protective agents in the management of tobacco bacterial wilt.

[Drug resistance and protoporphyrin ferrochelatase of Ralstonia mannitolilytica].

OBJECTIVE: To investigate the drug resistance, ß-lactamase-encoding genes and protoporphyrin ferrochelatase-encoding genes of Ralstonia mannitolilytica, and to explore its structure and pathogenic function. METHODS: The strain was isolated by plate streaking method and identified by automatic bacteria detection system and 16S RNA gene PCR. Microdilution method was applied for drug susceptibility test. ß-lactamases, extended spectrum ß-lactamases (ESBL) and carbapenemases were detected using nitrocefin-disk, Kirby-Bauer disk, and Hodge test, respectively. Five ß-lactamase-encoding genes and protoporphyrin ferrochelatase-encoding gene of the isolate were amplified by PCR for sequencing. Bioinformatic softwares were used to analyze the structure and function of the product of protoporphyrin ferrochelatase-encoding gene. RESULTS: A strain belonging to Ralstonia mannitolilytica was isolated. This isolate was sensitive to cefepime, ciprofloxacin, ofloxacin and tigecycline, but resistant to five penicillins, four cephalosporins and two carbapenems antibiotics. The isolate produced ß-lactamases but did not produce ESBL and carbapenemases. The isolate had five distinct ß-lactamase-encoding genes and protoporphyrin ferrochelatase-encoding gene. The product of protoporphyrin ferrochelatase-encoding gene contained two functional domains of protoporphyrin ferrochelatase belonging to type Ⅱ chelatase superfamily that presented the most closely genetic relationship with the protoporphyrin ferrochelatase of Neisseria meningidis. CONCLUSIONS: The isolate of Ralstonia mannitolilytica has a higher resistance to ß-lactam antibiotics and its ß-lactamase-encoding genes are different with the common bacterial ß-lactamase-encoding genes. Protoporphyrin ferrochelatase may act as an important virulence factor of Ralstonia mannitolilytica.

Negative chemotaxis of Ralstonia pseudosolanacearum to maleate and identification of the maleate chemosensory protein.

Ralstonia pseudosolanacearum Ps29 was repelled by maleate. Screening of a complete collection of Ps29 single-methyl-accepting chemotaxis protein (mcp) gene mutants identified the RSp0303 homolog (McpP) as a chemotaxis sensor mediating negative chemotaxis to maleate. Interestingly, the mcpP-deletion mutant was attracted to maleate, indicating that this bacterium expresses a MCP(s) for both positive and negative chemotaxis to maleate. We constructed a Ps29 derivative (designated POC14) harboring deletions in 14 individual mcp genes, including mcpP, to characterize McpP. Introduction of a plasmid harboring the mcpP gene (pPS16) restored the ability to negatively respond to maleate, confirming that McpP is a MCP for negative chemotaxis to maleate. We thought that maleate might be applied to controlling plant infection by R. pseudosolanacearum. To evaluate this possibility, we measured chemotactic responses of seven other virulent R. pseudosolanacearum strains to maleate. We confirmed that they harbored functional mcpP orthologues, but they showed no chemotactic responses to maleate. Quantitative RT-PCR analysis revealed that these seven R. pseudosolanacearum strains did not show negative chemotaxis to maleate because of negligible transcription of the mcpP genes. We compared the chemotactic responses of POC14 and POC14[pPS16] toward various chemicals and found that McpP senses inorganic phosphate as a chemoattractant.

Preparative separation of TL1-1 from Daldinia eschscholzii extract by macroporous resin and evaluation of its antimicrobial activities.

2,3-Dihydro-5-hydroxy-2-methylchromen-4-one (TL1-1) has already been reported to exhibit significant activities such as cytotoxicity, antifungal activity and growth inhibitory activity. In order to simply and efficiently separate TL1-1 from crude extracts of Daldinia eschscholzii on a large-preparative scale, XAD-16 resin was selected from ten types of resin based on its superior adsorption and desorption performance. Adsorption equilibrium data for this resin fitted well with pseudo-first order kinetics and the Freundlich model, which were elucidated from kinetic experiments and adsorption isotherms. Under optimized conditions, the purity of TL1-1 increased from 19.21% (w/w) in the crude extract, to 84.64% (w/w) in the final product, with a recovery yield of 75.06% (w/w) by a one-step treatment. Moreover, in a large-scale separation, the purity and recovery of TL1-1 was 80.33% and 72.02% (w/w), respectively. These results demonstrated that a simple adsorption-desorption strategy, using XAD-16 resin, was efficient, which also highlighted its potential for the future large-scale purification and preparation of TL1-1. In addition, studies showed that the purified TL1-1 exhibited moderate antibacterial activity against Ralstonia solanacearum.

Diverse microbial communities in non-aerated compost teas suppress bacterial wilt.

Non-aerated compost teas (NCTs) are water extracts of composted organic materials and are used to suppress soil borne and foliar disease in many pathosystems. Greenhouse trials were used to test the effectiveness of NCTs to suppress potato bacterial wilt caused by Ralstonia solanacearum on plants grown in soils inoculated with a virulent isolate of the pathogen (biovar II). NCTs prepared from matured compost sources: agricultural waste (AWCT), vermicompost (VCT) and solid municipal waste (SMWCT) were evaluated at three initial application times (7 days before inoculation, at time of inoculation and 7 days after inoculation) prior to weekly applications, in a randomized complete-block design. AWCT applied initially at the time of inoculation resulted in the greatest disease suppression, with the disease severity index 2.5-fold less than the non-treated plants and the "area under the disease progress curve" (AUDPC) 3.2-fold less. VCT and SMWCT were less suppressive than AWCT regardless of initial application time. Next generation sequencing of the v4 region of 16S rRNA gene and the internal transcribed spacer region (ITS1) revealed that diversity and composition of the bacterial and fungal communities across the NCTs varied significantly. Dominant bacterial phyla such as Actinobacteria, Bacteroidetes, Firmicutes, Proteobacteria, Verrucomicrobia, Chloroflexi, Planctomycetes, Acidobacteria, and a fungal phylum Ascomycota were detected in all NCTs. AWCT had optimum physico-chemical measurements with higher bacterial Shannon diversity indices (H) and fungal richness (S) than the other treatments. We conclude that bacterial wilt of potatoes grown in controlled conditions can be suppressed by a non-aerated compost tea with a high microbial diversity when applied at planting and weekly thereafter.

Ralstonia species - do these bacteria matter in cystic fibrosis?

Ralstonia species, often regarded as an environmental organism of low pathogenicity, can cause significant disease in certain at-risk patient groups, including those with cystic fibrosis. Difficulties with its identification in the clinical laboratory mean that it may be misidentified and therefore under recognised as a cause of disease. A number of outbreaks have been associated with the use of devices for inhaled respiratory therapy, putting those with chronic respiratory conditions at risk. Antimicrobial treatment of infection is challenging and limited due to frequent antimicrobial resistance. This review highlights issues regarding the identification, treatment and prevention of infection due to Ralstonia spp. in children with cystic fibrosis.

The long-term effect of uranium and pH on the community composition of an artificial consortium.

In the environment, microorganisms are living in diverse communities, which are impacted by the prevailing environmental conditions. Here, we present a study investigating the effect of low pH and elevated uranium concentration on the dynamics of an artificial microbial consortium. The members (Caulobacter sp. OR37, Asinibacterium sp. OR53, Ralstonia sp. OR214 and Rhodanobacter sp. OR444) were isolated from a uranium contaminated and acidic subsurface sediment. In pure culture, Ralstonia sp. OR214 had the highest growth rate at neutral and low pH and only Caulobacter sp. OR37 and Asinibacterium sp. OR53 grew in the presence uranium. The four strains were mixed in equal ratios, incubated at neutral and low pH and in the presence uranium and transferred to fresh medium once per week for 30 weeks. After 30 weeks, Ralstonia sp. OR214 was dominant at low and neutral pH and Caulobacter sp. OR37 and Asinibacterium sp. OR53 were dominant in the presence of uranium. After 12 weeks, the cultures were also transferred to new conditions to access the response of the consortia to changing conditions. The transfers showed an irreversible effect of uranium, but not of low pH on the consortia. Overall, the strains initially tolerant to the respective conditions persisted over time in high abundances in the consortia.

Antibacterial activity of cyclo(L-Pro-L-Tyr) and cyclo(D-Pro-L-Tyr) from Streptomyces sp. strain 22-4 against phytopathogenic bacteria.

Two bioactive cyclic dipeptides, cyclo(L-Pro-L-Tyr) and cyclo(D-Pro-L-Tyr), were isolated from the culture broth of Streptomyces sp. strain 22-4 and tested against three economically important plant pathogens, Xanthomonas axonopodis pv. citri, Ralstonia solanacearum and Clavibacter michiganensis. Both cyclic dipeptides were active against X. axonopodis pv. citri and R. Solanacearum with MIC of 31.25 µg/mL. No activity could be observed against C. michiganensis.

Effects of acoustic streaming from moderate-intensity pulsed ultrasound for enhancing biofilm mitigation effectiveness of drug-loaded liposomes.

Because biofilms have resistance to antibiotics, their control using minimum amounts of chemicals and energy becomes a critical issue particularly for resource-constrained long-term space and deep-sea explorations. This preliminary study investigates how ultrasound promoting penetration of antibiotic-loaded liposomes into alginate-based bacterial biofilms, resulting in enhanced bacterial (Ralstonia insidiosa) killing. Nano-sized liposomes are used as a delivery vehicle for the antibiotic gentamicin. Alginate-based synthetic biofilms, which are widely acknowledged as biofilm phantoms, filled with liposome solution are formed at the bottoms of six-well Petri dishes and exposed to ultrasound (frequency = 2.25 MHz, 10% duty cycle, and spatially and temporally averaged intensity ISAPA = 4.4 W/cm(2)). Gentamicin is released from liposomes after they are lysed using detergent solution (0.05% sodium dodecyl sulfate, 1.0% Triton X-100) and incubated for 20 min. The alginate biofilm is dissolved and diluted, counting of colony-forming units shows about 80% of the bacteria are killed. It has also been shown the liposome-capture density by the alginate film increases linearly with the ultrasound intensity up to ISAPA = 6.2 W/cm(2) reaching approximately threefold that without ultrasound. Measurement by using particle-image velocimetry has demonstrated the acoustic streaming with modification by thermal convection controls the enhancement of the liposome capture rate.