Detection of cultured and uncultured Burkholderia cepacia complex bacteria naturally occurring in the maize rhizosphere

The species composition of a Burkholderia cepacia complex population naturally occurring in the maize rhizosphere was investigated by using both culture-dependent and culture-independent methods. B. cepacia complex isolates were recovered from maize root slurry on the two selective media Pseudomonas cepacia azelaic acid tryptamine (PCAT) and trypan blue tetracycline (TB-T) and subjected to identification by a combination of restriction fragment length polymorphism (RFLP) analysis and species-specific polymerase chain reaction (PCR) tests of the recA gene. DNA extracted directly from root slurry was examined by means of nested PCR to amplify recA gene with species-specific B. cepacia complex primers and to obtain a library of PCR amplified recA genes. Using the culture-dependent method the species Burkholderia cepacia, Burkholderia cenocepacia, Burkholderia ambifaria and Burkholderia pyrrocinia were identified, whereas using the culture-independent method also the species Burkholderia vietnamiensis was detected. The latter method also allowed us to highlight a higher diversity within the B. cenocepacia species. In fact, by using the culture-independent method the species B. cenocepacia recA lineages IIIA and IIID besides B. cenocepacia recA lineage IIIB were detected. Moreover, higher heterogeneity of recA RFLP patterns was observed among clones assigned to the species B. cenocepacia than among B. cenocepacia isolates from selective media.     

Diversity and distribution of Burkholderia cepacia complex in the rhizosphere of rice and maize

A survey of Burkholderia cepacia complex (Bcc) species was conducted in agricultural fields within Hangzhou, China. Out of the 251 bacterial isolates recovered on the selective media from the rhizosphere of rice and maize, 112 of them were assigned to Bcc by PCR assays. The species composition of the Bcc isolates was analyzed by a combination of recA-restriction fragment length polymorphism assays, species-specific PCR tests and recA gene sequencing. The results revealed that the majority belong to B. cepacia, Burkholderia cenocepacia recA lineage IIIB, Burkholderia vietnamiensis and Burkholderia pyrrocinia. Burkholderia cenocepacia and B. vietnamiensis dominated the rhizosphere of maize and rice, respectively, indicating that species composition and abundance of Bcc may vary dramatically in different crop rhizospheres. In addition, one isolate (R456) formed a single discrete cluster within the phylogenetic analysis of the Bcc recA gene, and it may belong to a new genomovar.     

Development of a recA gene-based identification approach for the entire Burkholderia genus

Burkholderia is an important bacterial genus containing species of ecological, biotechnological, and pathogenic interest. With their taxonomy undergoing constant revision and the phenotypic similarity of several species, correct identification of Burkholderia is difficult. A genetic scheme based on the recA gene has greatly enhanced the identification of Burkholderia cepacia complex species. However, the PCR developed for the latter approach was limited by its specificity for the complex. By alignment of existing and novel Burkholderia recA sequences, we designed new PCR primers and evaluated their specificity by testing a representative panel of Burkholderia strains. PCR followed by restriction fragment length polymorphism analysis of an 869-bp portion of the Burkholderia recA gene was not sufficiently discriminatory. Nucleotide sequencing followed by phylogenetic analysis of this recA fragment differentiated both putative and known Burkholderia species and all members of the B. cepacia complex. In addition, it enabled the design of a Burkholderia genus-specific recA PCR that produced a 385-bp amplicon, the sequence of which was also able to discriminate all species examined. Phylogenetic analysis of 188 novel recA genes enabled clarification of the taxonomic position of several important Burkholderia strains and revealed the presence of four novel B. cepacia complex recA lineages. Although the recA phylogeny could not be used as a means to differentiate B. cepacia complex strains recovered from clinical infection versus the natural environment, it did facilitate the identification of clonal strain types of B. cepacia, B. stabilis, and B. ambifaria capable of residing in both niches.     

Molecular characterization of Burkholderia strains isolated from rice cultivars (Oryza sativa L.) for species identification and phylogenetic grouping

The genus Burkholderia consists of extremely versatile bacteria that occupy diverse niches and are commonly encountered in the rhizosphere of crop plants. In this study, we characterized three plant growth promoting strains assigned as Burkholderia sp. using biochemical and molecular characterization. The Burkholderia spp. strains CBMB40, CBPB-HIM, and CBPB-HOD were characterized using biochemical tests, BIOLOG carbon substrate utilization, fatty acid methyl ester analysis, analysis of recA gene sequences, and DNA-DNA hybridization. The results from these studies indicated that the strains CBMB40, CBPB-HIM, and CBPBHOD can be assigned under Burkholderia vietnamiensis, Burkholderia ubonensis, and Burkholderia pyrrocinia, respectively.     

Identification and onion pathogenicity of Burkholderia cepacia complex isolates from the onion rhizosphere and onion field soil

Burkholderia cepacia complex strains are genetically related but phenotypically diverse organisms that are important opportunistic pathogens in patients with cystic fibrosis (CF,) as well as pathogens of onion and banana, colonizers of the rhizospheres of many plant species, and common inhabitants of bulk soil. Genotypic identification and pathogenicity characterization were performed on B. cepacia complex isolates from the rhizosphere of onion and organic soils in Michigan. A total of 3,798 putative B. cepacia complex isolates were recovered on Pseudomonas cepacia azelaic acid tryptamine and trypan blue tetracycline semiselective media during the 2004 growing season from six commercial onion fields located in two counties in Michigan. Putative B. cepacia complex isolates were identified by hybridization to a 16S rRNA gene probe, followed by duplex PCR using primers targeted to the 16S rRNA gene and recA sequences and restriction fragment length polymorphism analysis of the recA sequence. A total of 1,290 isolates, 980 rhizosphere and 310 soil isolates, were assigned to the species B. cepacia (160), B. cenocepacia (480), B. ambifaria (623), and B. pyrrocinia (27). The majority of isolates identified as B. cepacia (85%), B. cenocepacia (90%), and B. ambifaria (76%) were pathogenic in a detached onion bulb scale assay and caused symptoms of water soaking, maceration, and/or necrosis. A phylogenetic analysis of recA sequences from representative B. cepacia complex type and panel strains, along with isolates collected in this study, revealed that the B. cenocepacia isolates associated with onion grouped within the III-B lineage and that some strains were closely related to strain AU1054, which was isolated from a CF patient. This study revealed that multiple B. cepacia complex species colonize the onion rhizosphere and have the potential to cause sour skin rot disease of onion. In addition, the onion rhizosphere is a natural habitat and a potential environmental source of B. cenocepacia.     

RecA gene sequence and Multilocus Sequence Typing for species-level resolution of Burkholderia cepacia complex isolates

Aim:  To identify, by means of recA sequencing and multilocus sequence typing (MLST), Burkholderia cepacia complex (BCC) isolates of environmental and clinical origin, which failed to be identified by recA RFLP and species-specific PCR.
Methods and Results:  By using recA sequence-based identification, 17 out of 26 BCC isolates were resolved at the level of species and lineage (ten Burkholderia cenocepacia IIIB, two Burkholderia arboris and five Burkholderia lata ). By using MLST method, 24 BCC isolates were identified. MLST confirmed recA sequence results, and, furthermore, enabled to identify isolates of the BCC5 group, and showed relatedness with Burkholderia contaminans for one of the two isolates not identified.
Conclusions:  recA sequence-based identification allowed to resolve, at the level of species and lineage, 65·4%, of the BCC isolates examined, whilst MLST increased this percentage to 88·5%.
Significance and Impact of the Study:  BCC isolates previously not resolved by recA RFLP and species-specific PCR were successfully identified by means of recA sequencing and MLST, which represent the most appropriate methods to identify difficult strains for epidemiological purposes and cystic fibrosis patients management.     

Diversity of cultivated endophytic bacteria from sugarcane: genetic and biochemical characterization of Burkholderia cepacia complex isolates

Bacteria were isolated from the rhizosphere and from inside the roots and stems of sugarcane plants grown in the field in Brazil. Endophytic bacteria were found in both the roots and the stems of sugarcane plants, with a significantly higher density in the roots. Many of the cultivated endophytic bacteria were shown to produce the plant growth hormone indoleacetic acid, and this trait was more frequently found among bacteria from the stem. 16S rRNA gene sequence analysis revealed that the selected isolates of the endophytic bacterial community of sugarcane belong to the genera of Burkholderia, Pantoea, Pseudomonas, and Microbacterium. Bacterial isolates belonging to the genus Burkholderia were the most predominant among the endophytic bacteria. Many of the Burkholderia isolates produced the antifungal metabolite pyrrolnitrin, and all were able to grow at 37 degrees C. Phylogenetic analyses of the 16S rRNA gene and recA gene sequences indicated that the endophytic Burkholderia isolates from sugarcane are closely related to clinical isolates of the Burkholderia cepacia complex and clustered with B. cenocepacia (gv. III) isolates from cystic fibrosis patients. These results suggest that isolates of the B. cepacia complex are an integral part of the endophytic bacterial community of sugarcane in Brazil and reinforce the hypothesis that plant-associated environments may act as a niche for putative opportunistic human pathogenic bacteria.     

PCR-based identification and characterization of Burkholderia cepacia complex bacteria from clinical and environmental sources

AIMS: To study the genotypic identification and characterization of the 119 Burkholderia cepacia complex (Bcc) strains recovered from clinical and environmental sources in Japan and Thailand. METHODS AND RESULTS: Based on the results of analysis by 16S rDNA RFLP generated after digestion with DdeI, the Bcc strains were differentiated into two patterns: pattern 1 (including Burkholderia vietnamiensis) and pattern 2 (including B. cepacia genomovar I, Burkholderia cenocepacia and Burkholderia stabilis). All strains belonged to pattern 2 except for one strain. In the RFLP analysis of the recA gene using HaeIII, strains were separated into eight patterns designated as A, D, E, G, H, I, J and K, of which pattern K was new. Burkholderia cepacia epidemic strain marker (BCESM) encoded by esmR [corrected] and the pyrrolnitrin biosynthetic locus encoded by prnC were present in 22 strains (18%) and 88 strains (74%) from all sources, respectively. All esmR-positive [corrected] strains belonged to B. cenocepacia, whereas most prnC-positive strains belonged to B. cepacia genomovar I. CONCLUSIONS: Strains derived from clinical sources were assigned to B. cepacia genomovar I, B. cenocepacia, B. stabilis and B. vietnamiensis. The majority of Bcc strains from environmental sources (77 of a total 95 strains) belonged to B. cepacia genomovar I, whereas the rest belonged to B. cenocepacia. On the basis of genomovar-specific PCR and prnC RFLP analysis, strains belonging to recA pattern K were identified as B. cepacia genomovar I. SIGNIFICANCE AND IMPACT OF THE STUDY: This work provides the genotypic identification of a collection of the Bcc strains from Japan and Thailand. RFLP analysis of the prnC gene promises to be a useful method for differentiating Burkholderia pyrrocinia from B. cepacia genomovar I strains.     

Investigating Burkholderia cepacia complex populations recovered from Italian maize rhizosphere by multilocus sequence typing

The Burkholderia cepacia complex (BCC) comprises at least nine closely related species of abundant environmental microorganisms. Some of these species are highly spread in the rhizosphere of several crop plants, particularly of maize; additionally, as opportunistic pathogens, strains of the BCC are capable of colonizing humans. We have developed and validated a multilocus sequence typing (MLST) scheme for the BCC. Although widely applied to understand the epidemiology of bacterial pathogens, MLST has seen limited application to the population analysis of species residing in the natural environment; we describe its novel application to BCC populations within maize rhizospheres. 115 BCC isolates were recovered from the roots of different maize cultivars from three different Italian regions over a 9-year period (1994-2002). A total of 44 sequence types (STs) were found of which 41 were novel when compared with existing MLST data which encompassed a global database of 1000 clinical and environmental strains representing nearly 400 STs. In this study of rhizosphere isolates approximately 2.5 isolates per ST was found, comparable to that found for the whole BCC population. Multilocus sequence typing also resolved inaccuracies associated with previous identification of the maize isolates based on recA gene restriction fragment length polymorphims and species-specific polymerase chain reaction. The 115 maize isolates comprised the following BCC species groups, B. ambifaria (39%), BCC6 (29%), BCC5 (10%), B. pyrrocinia (8%), B. cenocepacia IIIB (7%) and B. cepacia (6%), with BCC5 and BCC6 potentially constituting novel species groups within the complex. Closely related clonal complexes of strains were identified within B. cepacia, B. cenocepacia IIIB, BCC5 and BCC6, with one of the BCC5 clonal complexes being distributed across all three sampling sites. Overall, our analysis demonstrates that the maize rhizosphere harbours a massive diversity of novel BCC STs, so that their addition to our global MLST database increased the ST diversity by 10%.     

Application of a recA gene-based identification approach to the maize rhizosphere reveals novel diversity in Burkholderia species

Burkholderia species are widely distributed in the natural environment. We evaluated the use of the recA gene in a cultivation-independent approach to examine the Burkholderia diversity associated with the maize rhizosphere. Two types of recA gene library were constructed, one with broad-specificity recA primers (BUR1 and BUR2) and a second from the products of nested PCRs using Burkholderia-specific primers (BUR3 and BUR4). The broad-specificity primer set provided near full-length recA sequences (869 bp) suitable for the creation of robust environmental sequence data sets; however, the nested PCR approach demonstrated the greatest specificity (84%) for detection of Burkholderia species recA genes. In addition, the screening approach was able to identify recA phylotypes matching Burkholderia cepacia complex species previously cultivated from the maize samples and discriminate these from other Burkholderia. The ecological benefit of Burkholderia species cultivated from maize rhizosphere is well documented, however, the fact that the majority of Burkholderia recA genes detected in this study (90%) were suggestive of novel taxa indicates that a wealth of potentially important interactions with uncultivated Burkholderia species remain unstudied in this habitat.     

Burkholderia anthina sp. nov. and Burkholderia pyrrocinia,two additional Burkholderia cepacia complex bacteria,may confound results of new molecular diagnostic tools

Nineteen Burkholderia cepacia-like isolates of human and environmental origin could not be assigned to one of the seven currently established genomovars using recently developed molecular diagnostic tools for B. cepacia complex bacteria. Various genotypic and phenotypic characteristics were examined. The results of this polyphasic study allowed classification of the 19 isolates as an eighth B. cepacia complex genomovar (Burkholderia anthina sp. nov.) and to design tools for its identification in the diagnostic laboratory. In addition, new and published data for Burkholderia pyrrocinia indicated that this soil bacterium is also a member of the B. cepacia complex. This highlights another potential source for diagnostic problems with B. cepacia-like bacteria.     

Use of the gyrB gene to discriminate among species of the Burkholderia cepacia complex

Bacteria of the Burkholderia cepacia complex (Bcc) are opportunistic pathogens that can cause serious infections in lungs of cystic fibrosis patients. The Bcc comprises at least nine species that have been discriminated by a polyphasic taxonomic approach. In this study, we focused on the gyrB gene, universally distributed among bacteria, as a new target gene to discriminate among the Bcc species. New PCR primers were designed to amplify a gyrB DNA fragment of about 1900 bp from 76 strains representative of all Bcc species. Nucleotide sequences of PCR products were determined and showed more than 400 polymorphic sites with high sequence similarity values from most isolates of the same species. Phylogenetic tree analysis revealed that most of the 76 gyrB sequences grouped, forming clusters, each corresponding to a given Bcc species.     

Burkholderia tuberum sp. nov. and Burkholderia phymatum sp. nov., nodulate the roots of tropical legumes

The taxonomic status of five root nodule isolates from tropical legumes was determined using a polyphasic taxonomic approach. Two isolates were identified as B. caribensis, an organism originally isolated from soil in Martinique (the French West Indies). One isolate was identified as Burkholderia cepacia genomovar VI, a B. cepacia complex genomovar thus far only isolated from sputum of cystic fibrosis patients. The remaining two isolates were identified as novel Burkholderia species for which we propose the names Burkholderia tuberum sp. nov. and Burkholderia phymatum sp. nov. The type strains are LMG 21444T and LMG 21445T, respectively.     

Culture-based and non-growth-dependent detection of the Burkholderia cepacia complex in soil environments

Burkholderia cepacia complex (Bcc) bacteria reside in soil, plant rhizospheres, and water, but their prevalence and distribution in outdoor environments is not clear. We sampled a variety of soil and rhizosphere environments with which people may have contact: playgrounds, athletic fields, parks, hiking trails, residential yards, and gardens. A total of 91 sites was sampled in three large U.S. cities. In the first phase of the study, putative Bcc isolates were recovered on Burkholderia cepacia selective agar and trypan blue tetracycline medium and subsequently examined for biochemical reactivity and growth at 32 and 22 degrees C. Isolates were further examined by PCR assays targeting Bcc-specific ribosomal DNA and recA gene sequences. Among the 1,013 bacterial isolates examined, 68 were identified as Bcc; 14 (15%) of 91 sampled sites yielded Bcc isolates. In the second phase, DNA was extracted directly from soil samples and examined with PCR assays targeting Bcc 16S rRNA gene sequences. Either 82 or 93% of the soil samples were positive for at least one Bcc genomovar, depending on the PCR assay system used. Cloning and sequencing were performed to check the specificity of the PCR assays. Sequence analysis of the 463-bp 16S rRNA inserts from eight clones indicated that all were from members of the Bcc. The four soil samples from which these clones were generated did not yield isolates identified as Bcc. Based on PCR detection, Bcc appears to be prevalent in soil from urban and suburban environments. Culture-based recovery of Bcc may underestimate environmental populations.     

Burkholderia fungorum DBT1: a promising bacterial strain for bioremediation of PAHs-contaminated soils

An extensive taxonomic analysis of the bacterial strain Burkholderia sp. DBT1, previously isolated from an oil refinery wastewater drainage, is discussed here. This strain is capable of transforming dibenzothiophene through the 'destructive' oxidative pathway referred to as the Kodama pathway. Burkholderia DBT1 has also been proved to use fluorene, naphthalene and phenanthrene as carbon and energy sources, although growth on the first two compounds requires a preinduction step. This evidence suggests that the strain DBT1 exerts a versatile metabolism towards polycyclic aromatic hydrocarbons other than condensed thiophenes. Phylogenetic characterization using a polyphasic approach was carried out to clarify the actual taxonomic position of this strain, potentially exploitable in bioremediation. In particular, investigations were focused on the possible exclusion of Burkholderia sp. DBT1 from the Burkholderia cepacia complex. Analysis of the sequences of 16S, recA and gyrB genes along with the DNA-DNA hybridization procedure indicated that the strain DBT1 belongs to the species Burkholderia fungorum, suggesting the proposal of the taxonomic denomination B. fungorum DBT1.     

A novel recombinant ethyl ferulate esterase from Burkholderia multivorans

AIMS: Isolation and identification of bacterial isolates with specific ferulic acid (FA) esterase activity and cloning of a gene encoding activity. METHODS AND RESULTS: A micro-organism with ethyl ferulate hydrolysing (EFH) activity was isolated by culture enrichment techniques. Detailed molecular identification based on species-specific primers and two conserved genes (16S rRNA and recA) led to the identification of the isolate as Burkholderia multivorans UWC10. A gene (designated estEFH5) encoding an EFH enzyme was cloned and its nucleotide sequence determined. Translational analysis revealed that estEFH5 encoded a polypeptide of 326 amino acids with an estimated molecular weight of 34.83 kDa. The EstEFH5 primary structure showed a typical serine hydrolase motif (G-H-S-L-G). The estEFH5 gene was over-expressed in Escherichia coli in an insoluble form. Following urea denaturation and in vitro refolding, the enzyme was purified using one-step His Select Nickel chromatographic column. CONCLUSION: Purified EstEFH5 showed a preference for short-chain rho-nitrophenyl esters (C2 and C3) a typical feature for carboxylesterase. Furthermore, the recombinant enzyme also retained the activity against ethyl ferulate (EF). SIGNIFICANCE AND IMPACT OF THE STUDY: A biocatalytic process for the production of FA from EF as a model substrate was demonstrated. This is the first report that describes the cloning and expression of a gene encoding FA esterase activity from the genus Burkholderia.     

Phenotypic and genotypic identification of bacteria from the Burkholderia cepacia complex

AIM: Evaluation of the diagnostic value of pheno- and genotypic characteristics of B. cepacia strains collection. MATERIALS AND METHODS: Phenotypic and genetic methods of identification and differentiation of 25 strains of the B. cepacia complex. RESULTS: Polyphasic taxonomic approach utilizing multiple diagnostic tests was used for accurate identification of Burkholderia species. Algorithm for identification of microorganisms from the B. cepacia complex was developed. CONCLUSION: Combined use of phenotypic and molecular genetic tests, such as recA gene PCR, is recommended for differentiation of the B. cepacia complex genomovars.     

Diversity of Burkholderia isolates from woodland rhizosphere environments

AIMS: Determination of genetic diversity among UK Burkholderia cepacia isolates from various environmental niches, principally woodland tree rhizospheres and onions. METHODS AND RESULTS: Genus determination was made using polymerase chain reaction (PCR) amplification and fatty acid methyl ester profiling. Genetic diversity was investigated by repetitive sequence genetic PCR fingerprinting. Several onion isolates were similar to clinical isolates but others were diverse. Some environmental isolates were possibly synonymous with B. cepacia and B. gladioli but most from woodland rhizospheres were distinct and clustered together. The 16S rRNA genes of representatives from these clusters were PCR amplified, sequenced and phylogenetically compared with all known Burkholderia and related species. This revealed that the rhizospheric isolates had closest affinity with Burkholderia spp. with known bioremediative and biocontrol capabilities and were unrelated to taxa comprising plant or human pathogenic strains. CONCLUSIONS: All of the analyses investigated revealed that environmental and onion isolates of B. cepacia complex bacteria are genetically diverse but that woodland rhizospheric isolates are related to each other and unrelated to plant or human pathogenic strains. SIGNIFICANCE AND IMPACT OF THE STUDY: Woodland rhizospheric isolates of B. cepacia are potentially good candidates for use in bioremediation and biocontrol, as they appear distinct from plant or human pathogenic strains.     

Burkholderia cepacia genomovar III Is a common plant-associated bacterium

A polyphasic taxonomic study involving DNA-DNA hybridization, whole-cell protein electrophoresis, and 16S ribosomal DNA sequence analysis revealed that a group of Burkholderia cepacia-like organisms isolated from the rhizosphere or tissues of maize, wheat, and lupine belong to B. cepacia genomovar III, a genomic species associated with "cepacia syndrome" in cystic fibrosis patients. The present study also revealed considerable protein electrophoretic heterogeneity within this species and demonstrated that the B. cepacia complex consists of two independent phylogenetic lineages.     

Development of a species-specific recA-based PCR test for Burkholderia fungorum

The genus Burkholderia comprises over 28 species and species-specific, recA-based polymerase chain reaction (PCR) tests are available for several species, but not for some soil-inhabiting species including B. fungorum. Previous analysis of several novel rhizospheric, environmental isolates belonging to the B. cepacia complex suggested they may be closely related to B. fungorum. To discover any relationship between these isolates and B. fungorum we set out to clone and sequence a portion of the B. fungorum recA gene in order to design species-specific primer pairs for use in a recA-based PCR assay. Using a similar procedure we extended the recA-based PCR assay to identify B. sacchari and B. caledonica, two additional soil-inhabiting Burkholderia spp.