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.     

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.     

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.     

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.     

Burkholderia cepacia complex: distribution of genomovars among isolates from the maize rhizosphere in Italy

Burkholderia cepacia is a 'complex' in which seven genomic species or genomovars have so far been identified. It appears that all seven B. cepacia genomovars are capable of causing infections in vulnerable persons; in particular, the importance of Burkholderia multivorans (genomovar II) and B. cepacia genomovar III among cystic fibrosis isolates, especially epidemic ones, has been emphasized. In order to acquire a better comprehension of the genomovar composition of environmental populations of B. cepacia, 120 strains were isolated from the rhizosphere of maize plants cultivated in fields located in northern, central and southern Italy. The identification of the different genomovars was accomplished by a combination of molecular polymerase chain reaction (PCR)-based techniques, such as restriction fragment length polymorphism (RFLP) analysis of 16S rDNA (ARDRA), genomovar-specific PCR tests and RFLP analyses based on polymorphisms in the recA gene whole-cell protein electrophoresis. ARDRA analysis allowed us to distinguish between all B. cepacia genomovars except B. cepacia genomovar I, B. cepacia genomovar III and Burkholderia ambifaria (genomovar VII). The latter genomovars were differentiated by means of recA PCR tests and RFLP analyses. Among the rhizospheric isolates of B. cepacia, we found only B. cepacia genomovar I, B. cepacia genomovar III, Burkholderia vietnamiensis (genomovar V) and B. ambifaria. B. cepacia genomovars I and III and B. ambifaria were recovered from all three fields, whereas B. vietnamiensis was detected only in the population isolated from the field located in central Italy. Among strains isolated from northern and southern Italy, the most abundant genomovars were B. ambifaria and B. cepacia genomovar III respectively; in contrast, the population isolated in central Italy showed an even distribution of strains among genomovars. These results indicate that it is not possible to differentiate clinical and environmental strains, or pathogenic and non-pathogenic strains, of the B. cepacia complex simply on the basis of genomovar status, and that the environment may serve as a reservoir for B. cepacia genomovar III infections in vulnerable humans.     

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.     

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.     

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.     

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.     

Species abundance and diversity of Burkholderia cepacia complex in the environment

Despite considerable interest in studying Burkholderia cepacia complex in the environment, we still do not have efficient methods to detect, isolate, and screen large numbers of B. cepacia isolates. To better describe the ecology and diversity of B. cepacia complex, a colony hybridization assay was developed to detect specifically all species of the complex based on polymorphism of the variable V3 region of the 16S rRNA sequence. The sensitivity of the assay was dramatically enhanced by using a probe consisting of three repeats of a B. cepacia complex-specific probe, each separated by a phosphoramidite spacer. In addition, a duplex PCR targeting B. cepacia complex-specific recA and 16S rRNA sequences was developed to enable a fast and reliable diagnostic assay for members of the complex. When applied to maize rhizosphere samples, colony hybridization results were in good agreement with those of most-probable-number duplex PCR, both indicating a >100-fold fluctuation of abundance between individual plants. Using restriction analysis of recA for a total of 285 confirmed isolates of the B. cepacia complex, up to seven B. cepacia complex species were identified; however, their diversity and abundance were not evenly distributed among individual plants, and several allelic variants were commonly found from the same rhizosphere sample. These results indicate that not only complex communities of B. cepacia complex species and closely related strains of the same species may coexist at high population levels but also species composition and abundance may dramatically vary between individual plants.     

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%.     

A novel strategy for the isolation and identification of environmental Burkholderia cepacia complex bacteria

The purpose of this study was to develop a novel strategy for the isolation and identification of Burkholderia cepacia complex bacteria from the home environment of cystic fibrosis (CF) patients. Water and soil samples were enriched in a broth containing 0.1% l-arabinose, 0.1% l-threonine, and a mixture of selective agents including 1 microgml(-1) C-390, 600U ml(-1) polymyxin B sulfate, 10 microgml(-1) gentamycin, 2 microgml(-1) vancomycin and 10 microgml(-1) cycloheximide. On selective media (consisting of the same components as above plus 1.8% agar), several dilutions of the enrichment broth were inoculated and incubated for 5 days at 28 degrees C. Isolates with different randomly amplified polymorphic DNA patterns were inoculated in Stewart's medium. Putative B. cepacia complex bacteria were confirmed by means of recA PCR and further identified by HaeIII-recA restriction fragment length polymorphism analysis. Our results suggest that these organisms may be more widespread in the home environment than previously assumed and that plant associated soil and pond water may be reservoirs of B. cepacia complex infection in CF patients.     

Detection and differentiation of Burkholderia pseudomallei, Burkholderia mallei and Burkholderia thailandensis by multiplex PCR

Burkholderia pseudomallei, a Gram-negative bacterium that causes melioidosis may be differentiated from closely related species of Burkholderia mallei that causes glanders and non-pathogenic species of Burkholderia thailandensis by multiplex PCR. The multiplex PCR consists of primers that flank a 10-bp repetitive element in B. pseudomallei and B. mallei amplifying PCR fragment of varying sizes between 400-700 bp, a unique sequence in B. thailandensis amplifying a PCR fragment of 308 bp and the metalloprotease gene amplifying a PCR fragment of 245 bp in B. pseudomallei and B. thailandensis. The multiplex PCR not only can differentiate the three Burkholderia species but can also be used for epidemiological typing of B. pseudomallei and B. mallei strains.     

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.     

Evidence of transmission of Burkholderia cepacia, Burkholderia multivorans and Burkholderia dolosa among persons with cystic fibrosis

Previous studies have identified specific Burkholderia cepacia complex strains that are common to multiple persons with cystic fibrosis (CF). Such so-called epidemic strains have an apparent enhanced capacity for inter-patient spread and reside primarily in Burkholderia cenocepacia (formerly B. cepacia complex genomovar III). We sought to identify strains from B. cepacia complex species other than B. cenocepacia that are similarly shared by multiple CF patients. We performed genotype analysis of 360 recent sputum culture isolates from 360 persons residing in 29 cities by using repetitive extragenic palendromic polymerase chain reaction (rep-PCR) and pulsed field gel electrophoresis. The results indicate that sharing of a common Burkholderia multivorans strain occurs relatively infrequently; however, several small clusters of patients infected with the same strain were identified. A cluster of seven patients infected with the same B. cepacia (genomovar I) strain was found. We also identified a large group of 28 patients receiving care in the same treatment center and infected with the same Burkholderia dolosa strain. These observations suggest that B. cepacia complex strains in species other than B. cenocepacia may be spread among CF patients.     

Burkholderia cepacia complex bacteria: opportunistic pathogens with important natural biology

Interaction with plants around their roots and foliage forms the natural habitat for a wide range of gram-negative bacteria such as Burkholderia, Pseudomonas and Ralstonia. During these interactions many of these bacteria facilitate highly beneficial processes such as the breakdown of pollutants or enhancement of crop growth. All these bacterial species are also capable of causing opportunistic infections in vulnerable individuals, especially people with cystic fibrosis (CF). Here we will review the current understanding of the Burkholderia cepacia complex (Bcc) as a group of model opportunistic pathogens, contrasting their clinical epidemiology with their ecological importance. Currently, the B. cepacia complex is composed of nine formally named species groups which are all difficult to identify using phenotypic methods. Genetic methods such as 16S rRNA and recA gene sequence analysis have proven useful for Bcc species identification. Multilocus sequence typing (MLST) is also emerging as a very useful tool for both Bcc strain and species identification. Historically, Burkholderia cenocepacia was the most dominant Bcc pathogen in CF, however, probably as a result of strict infection control practices introduced to control the spread of this species, its prevalence has been reduced. Burkholderia multivorans is the now the most dominant Bcc infection encountered in the UK CF population, a changing epidemiology that also appears to be occurring in the US CF population. The distribution of Bcc species residing in the natural environment may vary considerably with the type of environment examined. Clonally identical Bcc strains have been found to occur in the natural environment and cause infection. The contamination of medical devices, disinfectants and pharmaceutical formulations has also been directly linked to several outbreaks of infection. In the last 10 years considerable progress has been made in understanding the natural biology and clinical infections caused by this fascinating group of bacteria.     

从植物根际定向批量筛选广谱有机溶剂耐受性脂肪酶产生菌

洋葱伯克霍尔德菌脂肪酶是一类具有重要工业应用价值的优良脂肪酶之一。根据已公布的洋葱伯克霍尔德菌基因组信息, 在传统的洋葱伯克霍尔德菌选择性培养基中添加适量的氨苄青霉素和卡那霉素, 从植物根际的土壤中筛选洋葱伯克霍尔德菌。对获得的单菌落再用含罗丹明B指示剂的产脂肪酶定性检测平板检测, 从4个根际土壤中筛选到35株产脂肪酶的洋葱伯克霍尔德菌, 阳性率达到65%。其中15株对体积浓度为10%的苯、己烷和正庚烷同时具有耐受性。用recA基因分子鉴定上述15株菌种, 全部属于洋葱伯克霍尔德菌菌群。     

Burkholderia spp. alter Pseudomonas aeruginosa physiology through iron sequestration

Pseudomonas aeruginosa and members of the Burkholderia cepacia complex often coexist in both the soil and the lungs of cystic fibrosis patients. To gain an understanding of how these different species affect each other's physiology when coexisting, we performed a screen to identify P. aeruginosa genes that are induced in the presence of Burkholderia: A random gene fusion library was constructed in P. aeruginosa PA14 by using a transposon containing a promoterless lacZ gene. Fusion strains were screened for their ability to be induced in the presence of Burkholderia strains in a cross-streak assay. Three fusion strains were induced specifically by Burkholderia species; all three had transposon insertions in genes known to be iron regulated. One of these fusion strains, containing a transposon insertion in gene PA4467, was used to characterize the inducing activity from Burkholderia: Biochemical and genetic evidence demonstrate that ornibactin, a siderophore produced by nearly all B. cepacia strains, can induce P. aeruginosa PA4467. Significantly, PA4467 is induced early in coculture with an ornibactin-producing but not an ornibactin-deficient B. cepacia strain, indicating that ornibactin can be produced by B. cepacia and detected by P. aeruginosa when the two species coexist.     

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.