Burkholderia Isolates Research Articles

1252. A Challenging Burkholderia Outbreak Investigation Across Multiple Units at an Academic Medical Center From June 2017 to February 2018

BackgroundMost outbreak investigations involve short-term, geographically localized clusters. However, some organisms can form environmental reservoirs leading to more prolonged, widespread outbreaks. We describe a prolonged outbreak of Burkholderia at our institution.MethodsAn epidemiological investigation was conducted. Burkholderia isolates were genotyped using pulsed-field gel electrophoresis (PFGE) and recA gene sequencing. Initial isolates were sent to a national reference laboratory for multilocus sequence typing (MLST).Results32 patients on 12 units (see figure) had ≥1 positive culture for Burkholderia from June 2017 to February 2018. 21 had B. cenocepacia (PFGE pattern A, recA allele 365) and 11 had B. cepacia (PFGE pattern C, recA allele 53). MLST revealed that isolates with recA allele 365 were unique compared with previously identified B. cenocepacia strains. Of 32 patients, 28 (88%) had positive respiratory cultures. Of 32 patients, 3 (9%) had bacteremia. Thirty-day mortality was 4/29 (14%). A case–control study did not reveal a common point source. All surveillance cultures from asymptomatic patients were negative (n = 53). Two of nine sink drains in rooms of cases were positive for an unrelated strain of B. cepacia. Other environmental cultures were negative for Burkholderia (n = 49). Cases continued despite routine interventions (see figure), with some incident cases detected long after potential exposures. Ventilator/respiratory equipment (V/RE) cleaning was investigated. Multiple V/RE interventions were implemented: (1) ensuring a sterilization process for ventilator temperature probes (used in heated humidification) was occurring; (2) using disposable manometers on contact isolation patients; (3) reinforcing ventilator cleaning, including those in radiology suites after use.ConclusionNo definitive source of the outbreak was found. New cases continued after reinforcement of basic infection control practices, but subsided after focused attention on V/RE cleaning practices. Control of this outbreak was challenging due to the complexity of a prolonged “latency period” for Burkholderia, difficulty identifying reservoirs, and multiple possible modes of transmission, especially for organisms like Burkholderia that can persist on environmental surfaces and equipment. Disclosures All authors: No reported disclosures.

Multilocus sequence analysis and synergy tests of Burkholderia species isolated from cystic fibrosis and non-cystic fibrosis patients

Background: Burkholderia spp. are problematic infectious agents for cystic fibrosis (CF) or chronic granulomatous disease patients. Due to the failure of identification with conventional techniques and high intrinsic resistance to most antibiotics, these infections very difficult to treat and can cause a marked reduction in life expectancy. The aim of this study was to identify Burkholderia spp. strains isolated from CF and non-CF patients with multilocus sequence analysis (MLSA). Also, the antibiotic resistance and synergies of antibiotics were evaluated. Methods & Materials: A total of 38 Burkholderia spp. (25 CF, 13 non-CF) isolates were analyzed. All the Burkholderia spp. were identified by MLSA. Antibiotic susceptibilities of ceftazidime, meropenem, trimethoprim/sulfamethoxazole (TMP-SXT) and levofloxacin were determined by broth microdilution method according to CLSI guidelines. Synergy tests are performed by checkerboard method. Results: Multilocus sequence analysis identified clinical isolates as B. cenocepacia (n = 16), B. contaminans (n = 11), B. gladioli (n = 4), B. dolosa (n = 4), B. multivorans (n = 2) and B. seminalis (n = 1). Among the 38 Burkholderia spp. isolates, overall rates of resistance to ceftazidime, meropenem, TMP-SXT, and levofloxacin was 34.2%, 65.8%, 50% and 39.5%, respectively. No significant difference in antibiotic resistance between cystic fibrosis and non-cystic fibrosis patients were detected. Synergy has shown between meropenem and TMP-SXT at two isolates. Antagonism has detected at 15 isolates (between meropenem and ceftazidime (n = 12); between ceftazidime and TMP-SXT (n = 3)) Conclusion: A variety of molecular approaches have been developed for the identification of closely related species and MLSA has successfully provided a reliable advanced discrimination method for Burkholderia isolates. Expression of several resistance mechanisms in species may lead to higher resistance, whereas antagonism between meropenem and ceftazidime might be attributed to the expression of the beta-lactamases.

Plant growth promoting rhizobacteria- Bacillus amyloliquefaciens improves plant growth and induces resistance in chilli against anthracnose disease

The present study was aimed to screen plant growth promoting rhizobacteria (PGPR) for their effect on induction of resistance in chilli (Capsicum annum L.) against anthracnose disease. A total of 59 PGPR were evaluated for their antagonistic activity against Colletotrichum truncatum and only fourteen PGPR were able to inhibit the mycelial growth of the pathogen. The pathogenicity and root colonization of the selected PGPR showed that out of fourteen isolates only eight isolates of Achromobacter, Bacillus, Burkholderia, Enterobacter, Ochrobactrum and Providencia genera were able to be both non-pathogenic and possessed the ability to colonize the host plant. Among the PGPR isolates, seed treatment with Bacillus amyloliquefaciens resulted in maximum enhancement of seed germination (84.75%), seedling vigor (1423.8) along with an increase in vegetative growth parameters. Significant disease protection of 71% against anthracnose disease was observed in plants pretreated with B. amyloliquefaciens followed by B. cepacia and P. rettgeri, under greenhouse conditions. The induced resistance obtained upon PGPR treatment was in line with higher activity of defense enzymes [phenylalanine ammonia lyase (PAL), peroxidase (POX), polyphenol oxidase (PPO) and β-1,3-glucanase]. It was observed that, almost one to two-fold increase in enzyme activities was observed in PGPR treated challenge inoculated seedlings compared to control inoculated seedlings. The results evidenced that the PGPR- B. amyloliquefaciens possesses immense potential to increase the plant growth apart from suppressing anthracnose disease in chilli.

Effect of Microbial inoculation in combating the aluminium toxicity effect on growth of Zea mays.

The present study is aimed at improving the aluminium tolerance in maize crop employing the potential of microbial inoculants in conferring resistance to these toxicities via production of certain chelating compounds like siderophores, exopolysachharides and organic acids. Acid soils have now-a-days become one of the key factors for limiting growth of many agriculturally important crops. Aluminium is one of the major elements present in acid soils and is mainly responsible for toxicity in the soil. This aluminium is rapidly soluble in soil water and hence absorbed by plant roots under conditions where soil pH is below 5. This toxicity leads to severe root growth inhibition, thereby limiting the production of maize crops. It was observed that use of microbial inoculums can be helpful in elimination of these toxic compounds and prevent the inhibition of root growth . It was found that the soils contaminated with aluminium toxicity decreased the root length of maize plant significantly by 65% but Bacillus and Burkholderia inoculation increased this root length significantly by 1.4- folds and 2- folds respectively thereby combating the effect of aluminium toxicity. Aluminium concentration was found maximum in roots of plants which were grown under aluminium stress condition. But this aluminium accumulation decreased ̴ 2-folds when Burkholderia was used as seed inoculants under aluminium stress conditions. Also, at 60mM aluminium accumulation, phosphorus solubilisation in roots was found to be increased upto 30% on Burkholderia inoculation. However, Bacillus inoculation didn't show any significant difference in either of the case. Thus, the inoculation of seeds with Burkholderia isolates could prove to be a boon in sequestering aluminium toxicity in Zea mays.

Burkholderia isolates from a sand dune leaf litter display biocontrol activity against the bole rot disease of Agave sisalana

Sisal (Agave sisalana) is an important source of biodegradable hard fiber for diverse industrial uses. The disease known as sisal bole rot leads to plant death and is caused by black aspergilli, including Aspergillus niger, A. brasiliensis and A. tubingensis. Bole rot is among the factors responsible for the decline of sisal production in Brazil. The potential of 16 Burkholderia sp. isolates obtained from sand dune leaf litter to control the disease was evaluated as part of a biocontrol program. The isolates of Burkholderia clustered in a new phylogenetic clade related to, but different from B. gladioli, B. plantari and B. glumae, its closest known relatives. All isolates secreted several enzymes, diffusible compounds, siderophores, volatile organic compounds (VOCs) and were able to solubilize phosphate, and inhibit mycelial growth and spore germination of Aspergillus niger in vitro. The Burkholderia isolates varied in their activity in vitro and in a greenhouse experiment, but consistently reduced disease severity by 54–77% in two independent field experiments. Because species of Burkholderia are opportunistic and might be pathogenic to plants and animals, the development of biocontrol products containing living cells of these bacteria has been viewed with care by regulatory agencies and scientists around the world. Future studies will focus on the development of products containing metabolites and heat-killed cells of these isolates to control sisal bole rot disease.

Prevalence of Burkholderia species, including members of Burkholderia cepacia complex, among UK cystic and non-cystic fibrosis patients.

We aimed to establish the prevalence of different Burkholderia species among UK cystic fibrosis (CF) and non-CF patients over a 2 year period. Matrix-assisted laser desorption/ionization-time of flight mass spectrometry was used to identify isolates to genus level, followed by recA/gyrB sequence clustering or species-specific PCR. In all, 1047 Burkholderia isolates were submitted for identification from 361 CF patients and 112 non-CF patients, 25 from the hospital environment and three from a commercial company. Potential cross-infection was assessed by pulsed-field gel electrophoresis (PFGE) and multi- locus-sequence typing (MLST). MICs were determined for 161 Burkholderia cepacia complex (Bcc) isolates. CF Trust registry data were sought to examine clinical parameters relating to Bcc infection. Burkholderia multivorans was the most prevalent species among CF patients affecting 56 % (192) patients, followed by Burkholderia cenocepacia IIIA (15 %; 52 patients). Five novel recA clusters were found. Among non-CF patients, Burkholderia cepacia was the most prevalent species (37/112; 34 %), with 18 of 40 isolates part of a UK-wide B. cepacia 'cluster'. This and three other clusters were investigated by PFGE and MLST. Cable-pili positive isolates included two novel sequence types and representatives of ET12. Antibiotic susceptibility varied between and within species and CF/non- CF isolates. CF Trust registry data suggested no significant difference in lung function between patients harbouring B. cenocepacia, B. multivorans and other Bcc species (P=0.81). The dominance of B. multivorans in CF, the presence of a B. cepacia cluster among non-CF patients and the existence of putative novel species all highlighted the continuing role of Burkholderia species as opportunistic pathogens.

Prevalence and Identification of Burkholderia pseudomallei and Near-Neighbor Species in the Malabar Coastal Region of India.

Accurate identification of pathogens with biowarfare importance requires detection tools that specifically differentiate them from near-neighbor species. Burkholderia pseudomallei, the causative agent of a fatal disease melioidosis, is one such biothreat agent whose differentiation from its near-neighbor species is always a challenge. This is because of its phenotypic similarity with other Burkholderia species which have a wide spread geographical distribution with shared environmental niches. Melioidosis is a major public health concern in endemic regions including Southeast Asia and northern Australia. In India, the disease is still considered to be emerging. Prevalence surveys of this saprophytic bacterium in environment are under-reported in the country. A major challenge in this case is the specific identification and differentiation of B. pseudomallei from the growing list of species of Burkholderia genus. The objectives of this study included examining the prevalence of B. pseudomallei and near-neighbor species in coastal region of South India and development of a novel detection tool for specific identification and differentiation of Burkholderia species. Briefly, we analyzed soil and water samples collected from Malabar coastal region of Kerala, South India for prevalence of B. pseudomallei. The presumptive Burkholderia isolates were identified using recA PCR assay. The recA PCR assay identified 22 of the total 40 presumptive isolates as Burkholderia strains (22.72% and 77.27% B. pseudomallei and non-pseudomallei Burkholderia respectively). In order to identify each isolate screened, we performed recA and 16S rDNA sequencing. This two genes sequencing revealed that the presumptive isolates included B. pseudomallei, non-pseudomallei Burkholderia as well as non-Burkholderia strains. Furthermore, a gene termed D-beta hydroxybutyrate dehydrogenase (bdha) was studied both in silico and in vitro for accurate detection of Burkholderia genus. The optimized bdha based PCR assay when evaluated on the Burkholderia isolates of this study, it was found to be highly specific (100%) in its detection feature and a clear detection sensitivity of 10 pg/μl of purified gDNA was recorded. Nucleotide sequence variations of bdha among interspecies, as per in silico analysis, ranged from 8 to 29% within the target stretch of 730 bp highlighting the potential utility of bdha sequencing method in specific detection of Burkholderia species. Further, sequencing of the 730 bp bdha PCR amplicon of each Burkholderia strain isolated could differentiate the species and the data was comparable with recA sequence data of the strains. All sequencing results obtained were submitted to NCBI database. Bayesian phylogenetic analysis of bdha in comparison with recA and 16S rDNA showed that the bdha gene provided comparable identification of Burkholderia species.

Distance‐dependent effects of pathogenic fungi on seedlings of a legume tree: impaired nodule formation and identification of antagonistic rhizosphere bacteria

Summary The Janzen‐Connell (JC) hypothesis proposes that diversity of tree communities is promoted by species‐specific enemies that suppress seedling recruitment. Pathogenic soil‐borne fungi are often responsible for JC effects. However, previous ecological studies have not placed JC effects in the context of beneficial soil bacteria. Using the JC effect surrounding a subtropical legume tree (Ormosia glaberrima) as a model, we characterized tripartite interactions between seedlings, soil fungi and plant‐associated bacteria. Survival of seedlings grown in soil inocula collected at close distances to focal adult trees was reduced. Half of the experimental units were treated with fungicides to confirm the presence of pathogenic fungi. In a parallel experiment, nitrogen‐fixing rhizobia induced fewer nodules on O. glaberrima seedlings when soil inocula were collected closer to focal adult trees. An inoculation experiment with rhizobial isolates promoted nodulation of seedlings grown in soil taken at close distances to adult trees, suggesting that rhizobial densities are suboptimal at these locations. Fusarium oxysporum, a pathogen of O. glaberrima seedlings, produced exudates that inhibited growth of most bacteria isolated from nodules. Accordingly, F. oxysporum negatively affected rhizosphere colonization of rhizobia in a co‐inoculation experiment. Contrariwise, certain Burkholderia isolates (from nodules and the rhizosphere) were identified that possess the ability to inhibit growth of F. oxysporum. The majority of the antagonistic bacteria were isolated from locations with fungal pathogens, suggesting competition between fungi and rhizosphere bacteria. Exclusion of pathogenic fungi by application of fungicides promoted nodulation of O. glaberrima seedlings under growth‐room and field conditions. Synthesis. Our findings indicate that soil‐borne pathogens surrounding adult trees are microbial keystone species that locally influence interactions between seedlings and plant‐associated bacteria.

Food preparation with mucoralean fungi: A potential biosafety issue?

Mucorales have been used for production of fermented food in Asia and Africa since time immemorial. Particularly Rhizopus species are rapidly growing, active producers of lipases and proteases and occur naturally during the first stages of soybean fermentation. Two biosafety issues have been raised in recent literature: (1) pathogenicity, Rhizopus species being prevalent opportunists causing erosive infections in severely compromised patients, and (2) toxicity, strains harbouring endosymbiotic Burkholderia producing toxic secondary metabolites. At the molecular level, based on different gene markers, species identity was found between strains used for food processing and clinical strains. In this study, we screened for bacterial symbionts in 64 Rhizopus strains by light microscopy, 16S rRNA sequencing, and HPLC. Seven strains (11 %) carried bacteria identified as Burkholderia rhizoxinica and Burkholderia endofungorum, and an unknown Burkholderia species. The Burkholderia isolates proved to be able to produce toxic rhizoxins. Strains with endosymbionts originated from food, soil, and a clinical source, and thus their presence could not be linked to particular habitats. The presence of Burkholderia in Rhizopus producing toxins could not be excluded as a potential risk for human health. In contrast, given the type of diseases caused by Rhizopus species, we regard the practical risk of infection via the food industry as negligible.

Diverse Burkholderia Species Isolated from Soils in the Southern United States with No Evidence of B. pseudomallei.

The global distribution of the soil-dwelling bacterium Burkholderia pseudomallei, causative agent of melioidosis, is poorly understood. We used established culturing methods developed for B. pseudomallei to isolate Burkholderia species from soil collected at 18 sampling sites in three states in the southern United States (Arizona (n = 4), Florida (n = 7), and Louisiana (n = 7)). Using multi-locus sequence typing (MLST) of seven genes, we identified 35 Burkholderia isolates from these soil samples. All species belonged to the B. cepacia complex (Bcc), including B. cenocepacia, B. cepacia, B. contaminans, B. diffusa, B. metallica, B. seminalis, B. vietnamiensis and two unnamed members of the Bcc. The MLST analysis provided a high level of resolution among and within these species. Despite previous clinical cases within the U.S. involving B. pseudomallei and its close phylogenetic relatives, we did not isolate any of these taxa. The Bcc contains a number of opportunistic pathogens that cause infections in cystic fibrosis patients. Interestingly, we found that B. vietnamiensis was present in soil from all three states, suggesting it may be a common component in southern U.S. soils. Most of the Burkholderia isolates collected in this study were from Florida (30/35; 86%), which may be due to the combination of relatively moist, sandy, and acidic soils found there compared to the other two states. We also investigated one MLST gene, recA, for its ability to identify species within Burkholderia. A 365bp fragment of recA recovered nearly the same species-level identification as MLST, thus demonstrating its cost effective utility when conducting environmental surveys for Burkholderia. Although we did not find B. pseudomallei, our findings document that other diverse Burkholderia species are present in soils in the southern United States.

Burkholderia kirstenboschensis sp. nov. nodulates papilionoid legumes indigenous to South Africa

Despite the diversity of Burkholderia species known to nodulate legumes in introduced and native regions, relatively few taxa have been formally described. For example, the Cape Floristic Region of South Africa is thought to represent one of the major centres of diversity for the rhizobial members of Burkholderia, yet only five species have been described from legumes occurring in this region and numerous are still awaiting taxonomic treatment. Here, we investigated the taxonomic status of 12 South African root-nodulating Burkholderia isolates from native papilionoid legumes (Hypocalyptus coluteoides, H. oxalidifolius, H. sophoroides and Virgilia oroboides). Analysis of four gene regions (16S rRNA, recA, atpD and rpoB) revealed that the isolates represent a genealogically unique and exclusive assemblage within the genus. Its distinctness was supported by all other aspects of the polyphasic approach utilized, including the genome-based criteria DNA−DNA hybridization (≥70.9%) and average nucleotide identities (≥96%). We accordingly propose the name B. kirstenboschensis sp. nov. for this taxon with isolate Kb15T (=LMG 28727T; =SARC 695T) as its type strain. Our data showed that intraspecific genome size differences (≥0.81 Mb) and the occurrence of large DNA regions that are apparently unique to single individuals (16−23% of an isolate's genome) can significantly limit the value of data obtained from DNA−DNA hybridization experiments. Substitution of DNA−DNA hybridization with whole genome sequencing as a prerequisite for the description of Burkholderia species will undoubtedly speed up the pace at which their diversity are documented, especially in hyperdiverse regions such as the Cape Floristic Region.

Burkholderia bacteria infectiously induce the proto-farming symbiosis of Dictyostelium amoebae and food bacteria

Symbiotic associations can allow an organism to acquire novel traits by accessing the genetic repertoire of its partner. In the Dictyostelium discoideum farming symbiosis, certain amoebas (termed "farmers") stably associate with bacterial partners. Farmers can suffer a reproductive cost but also gain beneficial capabilities, such as carriage of bacterial food (proto-farming) and defense against competitors. Farming status previously has been attributed to amoeba genotype, but the role of bacterial partners in its induction has not been examined. Here, we explore the role of bacterial associates in the initiation, maintenance, and phenotypic effects of the farming symbiosis. We demonstrate that two clades of farmer-associated Burkholderia isolates colonize D. discoideum nonfarmers and infectiously endow them with farmer-like characteristics, indicating that Burkholderia symbionts are a major driver of the farming phenomenon. Under food-rich conditions, Burkholderia-colonized amoebas produce fewer spores than uncolonized counterparts, with the severity of this reduction being dependent on the Burkholderia colonizer. However, the induction of food carriage by Burkholderia colonization may be considered a conditionally adaptive trait because it can confer an advantage to the amoeba host when grown in food-limiting conditions. We observed Burkholderia inside and outside colonized D. discoideum spores after fruiting body formation; this observation, together with the ability of Burkholderia to colonize new amoebas, suggests a mixed mode of symbiont transmission. These results change our understanding of the D. discoideum farming symbiosis by establishing that the bacterial partner, Burkholderia, is an important causative agent of the farming phenomenon.

Interrogation of the Burkholderia pseudomallei genome to address differential virulence among isolates.

Infection by the Gram-negative pathogen Burkholderia pseudomallei results in the disease melioidosis, acquired from the environment in parts of southeast Asia and northern Australia. Clinical symptoms of melioidosis range from acute (fever, pneumonia, septicemia, and localized infection) to chronic (abscesses in various organs and tissues, most commonly occurring in the lungs, liver, spleen, kidney, prostate and skeletal muscle), and persistent infections in humans are difficult to cure. Understanding the basic biology and genomics of B. pseudomallei is imperative for the development of new vaccines and therapeutic interventions. This formidable task is becoming more tractable due to the increasing number of B. pseudomallei genomes that are being sequenced and compared.Here, we compared three B. pseudomallei genomes, from strains MSHR668, K96243 and 1106a, to identify features that might explain why MSHR668 is more virulent than K96243 and 1106a in a mouse model of B. pseudomallei infection. Our analyses focused on metabolic, virulence and regulatory genes that were present in MSHR668 but absent from both K96243 and 1106a. We also noted features present in K96243 and 1106a but absent from MSHR668, and identified genomic differences that may contribute to variations in virulence noted among the three B. pseudomallei isolates. While this work contributes to our understanding of B. pseudomallei genomics, more detailed experiments are necessary to characterize the relevance of specific genomic features to B. pseudomallei metabolism and virulence. Functional analyses of metabolic networks, virulence and regulation shows promise for examining the effects of B. pseudomallei on host cell metabolism and will lay a foundation for future prediction of the virulence of emerging strains. Continued emphasis in this area will be critical for protection against melioidosis, as a better understanding of what constitutes a fully virulent Burkholderia isolate may provide for better diagnostic and medical countermeasure strategies.

Biodiversity and physiological characteristics of Antarctic and Arctic lichens-associated bacteria

The diversity and physiological characteristics of culturable bacteria associated with lichens from different habitats of the Arctic and Antarctica were investigated. The 68 retrieved isolates could be grouped on the basis of their 16S rRNA gene sequences into 26 phylotypes affiliated with the phyla Actinobacteria, Bacteroidetes, Deinococcus-Thermus, and Firmicutes and with the classes Alphaproteobacteria, Betaproteobacteria, and Gammaproteobacteria. Isolates belonging to the Alphaproteobacteria were the most abundant, followed by those belonging to Actinobacteria, Betaproteobacteria, Gammaproteobacteria, Bacteroidetes, Firmicutes, and Deinococcus-Thermus. Phylogenetic analysis showed that approximately 21 % of the total isolates represented a potentially novel species or genus (≤97 % sequence similarity). Strains belonging to the genera Sphingomonas, Frondihabitans, Hymenobacter, and Burkholderia were recovered from lichen samples from both geographic locations, implying common and important bacterial functions within lichens. Extracellular protease activities were detected in six isolates, affiliated with Burkholderia, Frondihabitans, Hymenobacter, Pseudomonas, and Rhodanobacter. Extracellular lipase activities were detected in 37 isolates of the genera Burkholderia, Deinococcus, Frondihabitans, Pseudomonas, Rhodanobacter, Sphingomonas, and Subtercola. This is the first report on the culturable bacterial diversity present within lichens from Arctic and Antarctica and the isolates described herein are valuable resources to decode the functional and ecological roles of bacteria within lichens. In addition, the low similarity (≤97 %) of the recovered isolates to known species and their production of cold-active enzymes together suggest that lichens are noteworthy sources of novel bacterial strains for use in biotechnological applications.

Membrane-active mechanism of LFchimera against Burkholderia pseudomallei and Burkholderia thailandensis.

LFchimera, a construct combining two antimicrobial domains of bovine lactoferrin, lactoferrampin265-284 and lactoferricin17-30, possesses strong bactericidal activity. As yet, no experimental evidence was presented to evaluate the mechanisms of LFchimera against Burkholderia isolates. In this study we analyzed the killing activity of LFchimera on the category B pathogen Burkholderia pseudomallei in comparison to the lesser virulent Burkholderia thailandensis often used as a model for the highly virulent B. pseudomallei. Killing kinetics showed that B. thailandensis E264 was more susceptible for LFchimera than B. pseudomallei 1026b. Interestingly the bactericidal activity of LFchimera appeared highly pH dependent; B. thailandensis killing was completely abolished at and below pH 6.4. FITC-labeled LFchimera caused a rapid accumulation within 15min in the cytoplasm of both bacterial species. Moreover, freeze-fracture electron microscopy demonstrated extreme effects on the membrane morphology of both bacterial species within 1h of incubation, accompanied by altered membrane permeability monitored as leakage of nucleotides. These data indicate that the mechanism of action of LFchimera is similar for both species and encompasses disruption of the plasma membrane and subsequently leakage of intracellular nucleotides leading to cell dead.

Burkholderia sp. induces functional nodules on the South African invasive legume Dipogon lignosus (Phaseoleae) in New Zealand soils.

The South African invasive legume Dipogon lignosus (Phaseoleae) produces nodules with both determinate and indeterminate characteristics in New Zealand (NZ) soils. Ten bacterial isolates produced functional nodules on D. lignosus. The 16S ribosomal RNA (rRNA) gene sequences identified one isolate as Bradyrhizobium sp., one isolate as Rhizobium sp. and eight isolates as Burkholderia sp. The Bradyrhizobium sp. and Rhizobium sp. 16S rRNA sequences were identical to those of strains previously isolated from crop plants and may have originated from inocula used on crops. Both 16S rRNA and DNA recombinase A (recA) gene sequences placed the eight Burkholderia isolates separate from previously described Burkholderia rhizobial species. However, the isolates showed a very close relationship to Burkholderia rhizobial strains isolated from South African plants with respect to their nitrogenase iron protein (nifH), N-acyltransferase nodulation protein A (nodA) and N-acetylglucosaminyl transferase nodulation protein C (nodC) gene sequences. Gene sequences and enterobacterial repetitive intergenic consensus (ERIC) PCR and repetitive element palindromic PCR (rep-PCR) banding patterns indicated that the eight Burkholderia isolates separated into five clones of one strain and three of another. One strain was tested and shown to produce functional nodules on a range of South African plants previously reported to be nodulated by Burkholderia tuberum STM678(T) which was isolated from the Cape Region. Thus, evidence is strong that the Burkholderia strains isolated here originated in South Africa and were somehow transported with the plants from their native habitat to NZ. It is possible that the strains are of a new species capable of nodulating legumes.

English

  Six isolates of Burkholderia were obtained from rhizosphere soils of rice grown in tropical lowlands of South Assam, India. Among the identified Burkholderia isolates, SDSA-I10/1 has shown higher nitrogen fixing potential and it was selected for 16S rDNA sequencing. The isolate SDSA-I10/1 showed highest resemblance to Burkholderia caribensis MWAP84 (Y17011) and hence, identified as Burkholderia caribensis strain SDSA-I10/1 (GU372342). Inoculation of this strain improved the growth and yield parameters of rice significantly over uninoculated control plants, thus it may be used as indigenous microbial inoculant for intensive rice cropping in tropical lowlands.   Key words: Burkholderia caribensis strain SDSA-I10/1, 16S rDNA sequence, biofertilizer, Diazotroph, nitrogenase activity, tropical lowland.

Tangled bank of experimentally evolvedBurkholderiabiofilms reflects selection during chronic infections

How diversity evolves and persists in biofilms is essential for understanding much of microbial life, including the uncertain dynamics of chronic infections. We developed a biofilm model enabling long-term selection for daily adherence to and dispersal from a plastic bead in a test tube. Focusing on a pathogen of the cystic fibrosis lung, Burkholderia cenocepacia, we sequenced clones and metagenomes to unravel the mutations and evolutionary forces responsible for adaptation and diversification of a single biofilm community during 1,050 generations of selection. The mutational patterns revealed recurrent evolution of biofilm specialists from generalist types and multiple adaptive alleles at relatively few loci. Fitness assays also demonstrated strong interference competition among contending mutants that preserved genetic diversity. Metagenomes from five other independently evolved biofilm lineages revealed extraordinary mutational parallelism that outlined common routes of adaptation, a subset of which was found, surprisingly, in a planktonic population. These mutations in turn were surprisingly well represented among mutations that evolved in cystic fibrosis isolates of both Burkholderia and Pseudomonas. These convergent pathways included altered metabolism of cyclic diguanosine monophosphate, polysaccharide production, tricarboxylic acid cycle enzymes, global transcription, and iron scavenging. Evolution in chronic infections therefore may be driven by mutations in relatively few pathways also favored during laboratory selection, creating hope that experimental evolution may illuminate the ecology and selective dynamics of chronic infections and improve treatment strategies.

Burkholdines from Burkholderia ambifaria: Antifungal Agents and Possible Virulence Factors

Burkholdines are cyclic lipopeptides with unusual antifungal potency, making them promising leads as a new class of antifungal agents. However, a recent report using knockout mutagenesis indicates that these and related compounds, such as occidiofungins, xylocandins, and cepacidines, may also be synonymous with the long-known hemolytic virulence factors found in diverse Burkholderia isolates. Because of their possible roles in causing Burkholderia infections or curing fungal infections, it is important to fully define their structures and biological activities using pure compounds. Here, we report the structures of three further burkholdines, Bk-1119, Bk-1213, and Bk-1215, which were elucidated using spectroscopic methods. The absolute configuration of this compound class was determined for the first time using a combination of spectroscopy and chemical degradation techniques. Antifungal and hemolytic activities were assessed for five pure burkholdines, representative of the structural diversity of this lipopeptide class. All of the burkholdines were potent antifungal and hemolytic agents, validating their probable role in virulence. However, one of the burkholdines (Bk-1119) exhibited a >30-fold selectivity for fungi versus sheep erythrocytes and was more than 25-fold more potent than amphotericin against some fungal strains. Therefore, burkholdines have potential to selectively target fungal infections.

Diazotrophic Burkholderia species isolated from the Amazon region exhibit phenotypical, functional and genetic diversity

Forty-eight Burkholderia isolates from different land use systems in the Amazon region were compared to type strains of Burkholderia species for phenotypic and functional characteristics that can be used to promote plant growth. Most of these isolates (n=46) were obtained by using siratro (Macroptilium atropurpureum – 44) and common bean (Phaseolus vulgaris – 2) as the trap plant species; two isolates were obtained from nodules collected in the field from Indigofera suffruticosa and Pithecellobium sp. The evaluated characteristics were the following: colony characterisation on “79” medium, assimilation of different carbon sources, enzymatic activities, solubilisation of phosphates, nitrogenase activity and antifungal activity against Fusarium oxysporium f. sp. phaseoli. Whole cell protein profiles, 16S rRNA, gyrB, and recA gene sequencing and multilocus sequence typing were used to identify the isolates. The isolates showed different cultural and biochemical characteristics depending on the legume species from which they were obtained. Except for one isolate from I. suffruticosa, all isolates were able to solubilise calcium phosphate and present nitrogenase activity under free-living conditions. Only one isolate from common beans, showed antifungal activity. The forty four isolates from siratro nodules were identified as B. fungorum; isolates UFLA02-27 and UFLA02-28, obtained from common bean plants, were identified as B. contaminans; isolate INPA89A, isolated from Indigofera suffruticosa, was a close relative of B. caribensis but could not be assigned to an established species; isolate INPA42B, isolated from Pithecellobium sp., was identified as B. lata. This is the first report of nitrogenase activity in B. fungorum, B. lata and B. contaminans.