Characterization of Burkholderia cepacia complex from cystic fibrosis patients in China and their chitosan susceptibility

A survey of Burkholderia cepacia complex (Bcc) species was conducted in sputum from cystic fibrosis (CF) patients in China. One hundred and four bacterial isolates were recovered on B. cepacia selective agar and 42 of them were assigned to Bcc by PCR assays. The species composition of the Bcc isolates from CF sputum 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 42 Bcc isolates belong to B. cepacia, B. cenocepacia and B. contaminans while predominant Bcc species was B. cenocepacia. This is the first report of B. contaminans from CF sputum in China. In addition, results from this study showed that chitosan solution at 10, 25, 50 and 100 μg/ml markedly inhibited the growth of the 16 representative isolates from the three different Bcc species, which indicated that chitosan was a potential bactericide against Bcc bacteria.     

Diversity analysis of Burkholderia cepacia complex in the water bodies of West Lake, Hangzhou, China

A survey of Burkholderia cepacia complex (Bcc) species was conducted in water bodies of West Lake in China. A total of 670 bacterial isolates were recovered on selective media. Out of them, 39.6% (265 isolates) were assigned to the following species: Burkholderia multivorans, Burkholderia cenocepacia recA lineage IIIA, IIIB, Burkholderia stabilis, Burkholderia vietnamiensis, and Burkholderia seminalis while B. cenocepacia is documented as a dominant Bcc species in water of West Lake. In addition, all Bcc isolates tested were PCR negative for the cblA and esmR transmissibility marker genes except B. cenocepacia IIIB A8 which was positive for esmR genelater. The present study raises great concerns on the role of West Lake as a “reservoir” for potential Bcc pathogenic strains.     

Burkholderia pseudomultivorans sp. nov., a novel Burkholderia cepacia complex species from human respiratory samples and the rhizosphere

Eleven Burkholderia cepacia-like isolates of human clinical and environmental origin were examined by a polyphasic approach including recA and 16S rRNA sequence analysis, multilocus sequence analysis (MLSA), DNA base content determination, fatty acid methyl ester analysis, and biochemical characterization. The results of this study demonstrate that these isolates represent a novel species within the B. cepacia complex (Bcc) for which we propose the name Burkholderia pseudomultivorans. The type strain is strain LMG 26883^T (=CCUG 62895^T). B. pseudomultivorans can be differentiated from other Bcc species by recA gene sequence analysis, MLSA, and several biochemical tests including growth at 42 °C, acidification of sucrose and adonitol, lysine decarboxylase and β-galactosidase activity, and esculin hydrolysis.     

The Contribution of Antibiotic Resistance Mechanisms in Clinical Burkholderia cepacia Complex Isolates: An Emphasis on Efflux Pump Activity

Due to the limited information of the contribution of various antibiotic resistance mechanisms in clinical Burkholderia cepacia complex isolates, Antibiotic resistance mechanisms, including integron analysis, identification of quinolone resistance-determining region mutations, measurement of efflux pump activity, and sequence analysis of efflux pump regulators, were investigated in 66 clinical B. cepacia complex isolates. Species were identified via recA-RFLP and MALDI-TOF. Four genomovars were identified by recA-RFLP. B. cenocepacia (genomovar III) was the most prevalent genomovar (90.1%). Most isolates (60/66, 90.9%) were correctly identified by MALDI-TOF analysis. Clonal relatedness determined by PFGE analysis revealed 30 pulsotypes, including two major pulsotypes that comprised 22.7% and 18.2% of the isolates, respectively. Seventeen (25.8%) isolates harboured class 1 integron with various combinations of resistance genes. Among six levofloxacin-resistant isolates, five had single-base substitutions in the gyrA gene and three demonstrated efflux pump activities. Among the 42 isolates exhibiting resistance to at least one antimicrobial agent, 94.4% ceftazidime-resistant isolates (17/18) and 72.7% chloramphenicol-resistant isolates (16/22) demonstrated efflux pump activity. Quantitation of efflux pump RNA level and sequence analysis revealed that over-expression of the RND-3 efflux pump was attributable to specific mutations in the RND-3 efflux pump regulator gene. In conclusion, high-level expression of efflux pumps is prevalent in B. cepacia complex isolates. Mutations in the RND-3 efflux pump regulator gene are the major cause of efflux pump activity, resulting in the resistance to antibiotics in clinical B. cepacia complex isolates.     

Immunoproteomic Analysis of Proteins Expressed by Two Related Pathogens,Burkholderia multivorans and Burkholderia cenocepacia,during Human Infection

Burkholderia cepacia complex (Bcc) is an opportunistic bacterial pathogen that causes chronic infections in people with cystic fibrosis (CF). It is a highly antibiotic resistant organism and Bcc infections are rarely cleared from patients, once they are colonized. The two most clinically relevant species within Bcc are Burkholderia cenocepacia and Burkholderia multivorans. The virulence of these pathogens has not been fully elucidated and the virulence proteins expressed during human infection have not been identified to date. Furthermore, given its antibiotic resistance, prevention of infection with a prophylactic vaccine may represent a better alternative than eradication of an existing infection. We have compared the immunoproteome of two strains each from these two species of Bcc, with the aim of identifying immunogenic proteins which are common to both species. Fourteen immunoreactive proteins were exclusive to both B. cenocepacia strains, while 15 were exclusive to B. multivorans. A total of 15 proteins were immunogenic across both species. DNA-directed RNA polymerase, GroEL, 38kDa porin and elongation factor-Tu were immunoreactive proteins expressed by all four strains examined. Many proteins which were immunoreactive in both species, warrant further investigations in order to aid in the elucidation of the mechanisms of pathogenesis of this difficult organism. In addition, identification of some of these could also allow the development of protective vaccines which may prevent colonisation.     

Diversity of <Emphasis Type="Italic">Burkholderia cepacia</Emphasis> Complex from the Moso Bamboo (<Emphasis Type="Italic">Phyllostachys edulis</Emphasis>) Rhizhosphere Soil

The purpose of this study was to determine the existence of Burkholderia cepacia complex (Bcc) at species level and the predominant species in the environment of moso bamboo plantations in Hangzhou, China. A total of 423 isolates were recovered from moso bamboo rhizhosphere soil samples of three sites on the selective medium during 2007–2008. Isolates were identified by Bcc-specific PCR assays, followed by recA-restriction fragment length polymorphism assays, species-specific PCR analysis, recA gene sequencing, multilocus sequence typing (MLST) scheme, and BOX-PCR fingerprinting for genomic diversity. Out of 423 isolates, 278 isolates were assigned to the following Bcc species, eight B. stabilis, 26 B. anthina, 193 B. pyrrocinia, and 51 B. arboris, which indicated B. pyrrocinia as the most dominant species followed by B. arboris. Moreover, false positives were observed in certain isolates of B. arboris while performing species-specific PCR test. Furthermore, the results of recA gene sequence similarity and MLST data demonstrated that nine isolates formed a single discrete cluster but were PCR negative to species-specific primers representing novel species may exist within the Bcc. In addition, BOX-PCR fingerprinting for all the Bcc isolates also showed the strain diversity. It is the first report of the existence of B. arboris and predominance of B. pyrrocinia in the moso bamboo environment.     

Metabolic Profiling of Burkholderia cenocepacia, Burkholderia ambifaria, and Burkholderia pyrrocinia Isolates from Maize Rhizosphere

Burkholderia cenocepacia, Burkholderia ambifaria, and Burkholderia pyrrocinia are the Burkholderia cepacia complex (Bcc) species most frequently associated with roots of crop plants. To investigate the ecophysiological diversity of these species, metabolic profiling of maize rhizosphere isolates was carried out by means of the Biolog system, using GN2 and SFN2 plates and different parameters related to optical density (OD). The metabolic profiles produced by the SFN2 and GN2 plates were identical, but the SFN2's narrower range of OD values and significantly longer reaction times made these plates less suitable for differentiation of isolates. Principal component analysis of maximum OD (OD_M) and maximum substrate oxidation rate (μ_M) data generated by GN2 plates allowed the selection of a reduced number of carbon sources. Statistical analysis of OD_M values highlighted marked differences between the metabolic profiles of B. cenocepacia and B. ambifaria, whereas metabolic profiles of B. pyrrocinia clustered very often with those of B. cenocepacia. Analysis of the μ_M parameter resulted in a slightly lower differentiation among the three Bcc species and a higher metabolic diversity within the single species, in particular within B. cenocepacia. Finally, B. cenocepacia and B. pyrrocinia showed generally higher oxidation rates than B. ambifaria on those GN2 substrates that commonly occur in maize root exudates.     

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°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.     

Regulation of phenylacetic acid degradation genes of Burkholderia cenocepacia K56-2

Background  

Metabolically versatile soil bacteria Burkholderia cepacia complex (Bcc) have emerged as opportunistic pathogens, especially of cystic fibrosis (CF). Previously, we initiated the characterization of the phenylacetic acid (PA) degradation pathway in B. cenocepacia, a member of the Bcc, and demonstrated the necessity of a functional PA catabolic pathway for full virulence in Caenorhabditis elegans. In this study, we aimed to characterize regulatory elements and nutritional requirements that control the PA catabolic genes in B. cenocepacia K56-2.     

Garlic Revisited: Antimicrobial Activity of Allicin-Containing Garlic Extracts against Burkholderia cepacia Complex

The antimicrobial activities of garlic and other plant alliums are primarily based on allicin, a thiosulphinate present in crushed garlic bulbs. We set out to determine if pure allicin and aqueous garlic extracts (AGE) exhibit antimicrobial properties against the Burkholderia cepacia complex (Bcc), the major bacterial phytopathogen for alliums and an intrinsically multiresistant and life-threatening human pathogen. We prepared an AGE from commercial garlic bulbs and used HPLC to quantify the amount of allicin therein using an aqueous allicin standard (AAS). Initially we determined the minimum inhibitory concentrations (MICs) of the AGE against 38 Bcc isolates; these MICs ranged from 0.5 to 3% (v/v). The antimicrobial activity of pure allicin (AAS) was confirmed by MIC and minimum bactericidal concentration (MBC) assays against a smaller panel of five Bcc isolates; these included three representative strains of the most clinically important species, B. cenocepacia. Time kill assays, in the presence of ten times MIC, showed that the bactericidal activity of AGE and AAS against B. cenocepacia C6433 correlated with the concentration of allicin. We also used protein mass spectrometry analysis to begin to investigate the possible molecular mechanisms of allicin with a recombinant form of a thiol-dependent peroxiredoxin (BCP, Prx) from B. cenocepacia. This revealed that AAS and AGE modifies an essential BCP catalytic cysteine residue and suggests a role for allicin as a general electrophilic reagent that targets protein thiols. To our knowledge, we report the first evidence that allicin and allicin-containing garlic extracts possess inhibitory and bactericidal activities against the Bcc. Present therapeutic options against these life-threatening pathogens are limited; thus, allicin-containing compounds merit investigation as adjuncts to existing antibiotics.     

日化产品中洋葱伯克霍尔德氏菌复合群(Bcc)的分类和神秘伯克霍尔德氏菌的耐药性研究

【目的】对从2020–2022年不同日化产品中分离的29株洋葱伯克霍尔德氏菌复合群(Burkholderia cepacia complex,Bcc)进行分类和分型,另将2020年前来源于日化产品中6株被鉴定为Burkholderia lata的菌株进行分类更正。探究神秘伯克霍尔德氏菌(Burkholderia aenigmatica)的耐药性。【方法】本文主要应用多位点分型研究方法(multilocus sequence typing,MLST),PCR扩增atpD、gltB、gyrB、recA、lepA、phaC和trp B 7个管家基因片段,将测序结果与MLST数据库中的数据比对分析,获得菌株各管家基因的编号和ST型(sequence type),对本检测中心分离自日化产品的Bcc进行分型;利用多位点序列分析(multilocus sequence analysis,MLSA),结合MLST中等位基因的核苷酸序列构建进化树,从而对Bcc进行系统发育分析和鉴定。利用最小抑菌浓度法(minimum inhibitory concentration,MIC)测定Bcc对常见防腐剂(1,...     

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.     

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.     

Differences in strategies to combat osmotic stress in Burkholderia cenocepacia elucidated by NMR-based metabolic profiling

Aims: To investigate mechanisms of osmotic tolerance in Burkholderia cenocepacia, a member of the B. cepacia complex (Bcc) of closely related strains, which is of clinical as well as environmental importance. Methods and Results: We employed NMR‐based metabolic profiling (metabolomics) to elucidate the metabolic consequences of high osmotic stress for five isolates of B. cenocepacia. The strains differed significantly in their levels of osmotic stress tolerance, and we identified three different sets of metabolic responses with the strains least impacted by osmotic stress exhibiting higher levels of the osmo‐protective metabolites glycine‐betaine and/or trehalose. Strains either increased concentrations or had constitutively high levels of these metabolites. Conclusions: Even within the small set of B. cenocepacia isolates, there was a surprising degree of variability in the metabolic responses to osmotic stress. Significance and impact of the study: The metabolic responses, and hence osmotic stress tolerance, vary between different B. cenocepacia isolates. This study provides a first look into the potentially highly diverse physiology of closely related strains of one species of the Bcc and illustrates that physiological or clinically relevant phenotypes are unlikely to be inferable from genetic relatedness within this species group.     

Burkholderia cepacia Complex Phage-Antibiotic Synergy (PAS): Antibiotics Stimulate Lytic Phage Activity

The Burkholderia cepacia complex (Bcc) is a group of at least 18 species of Gram-negative opportunistic pathogens that can cause chronic lung infection in cystic fibrosis (CF) patients. Bcc organisms possess high levels of innate antimicrobial resistance, and alternative therapeutic strategies are urgently needed. One proposed alternative treatment is phage therapy, the therapeutic application of bacterial viruses (or bacteriophages). Recently, some phages have been observed to form larger plaques in the presence of sublethal concentrations of certain antibiotics; this effect has been termed phage-antibiotic synergy (PAS). Those reports suggest that some antibiotics stimulate increased production of phages under certain conditions. The aim of this study is to examine PAS in phages that infect Burkholderia cenocepacia strains C6433 and K56-2. Bcc phages KS12 and KS14 were tested for PAS, using 6 antibiotics representing 4 different drug classes. Of the antibiotics tested, the most pronounced effects were observed for meropenem, ciprofloxacin, and tetracycline. When grown with subinhibitory concentrations of these three antibiotics, cells developed a chain-like arrangement, an elongated morphology, and a clustered arrangement, respectively. When treated with progressively higher antibiotic concentrations, both the sizes of plaques and phage titers increased, up to a maximum. B. cenocepacia K56-2-infected Galleria mellonella larvae treated with phage KS12 and low-dose meropenem demonstrated increased survival over controls treated with KS12 or antibiotic alone. These results suggest that antibiotics can be combined with phages to stimulate increased phage production and/or activity and thus improve the efficacy of bacterial killing.     

Proteomic Profiling of Burkholderia cenocepacia Clonal Isolates with Different Virulence Potential Retrieved from a Cystic Fibrosis Patient during Chronic Lung Infection

Respiratory infections with Burkholderia cepacia complex (Bcc) bacteria in cystic fibrosis (CF) are associated with a worse prognosis and increased risk of death. In this work, we assessed the virulence potential of three B. cenocepacia clonal isolates obtained from a CF patient between the onset of infection (isolate IST439) and before death with cepacia syndrome 3.5 years later (isolate IST4113 followed by IST4134), based on their ability to invade epithelial cells and compromise epithelial monolayer integrity. The two clonal isolates retrieved during late-stage disease were significantly more virulent than IST439. Proteomic profiling by 2-D DIGE of the last isolate recovered before the patient’s death, IST4134, and clonal isolate IST439, was performed and compared with a prior analysis of IST4113 vs. IST439. The cytoplasmic and membrane-associated enriched fractions were examined and 52 proteins were found to be similarly altered in the two last isolates compared with IST439. These proteins are involved in metabolic functions, nucleotide synthesis, translation and protein folding, cell envelope biogenesis and iron homeostasis. Results are suggestive of the important role played by metabolic reprogramming in the virulence potential and persistence of B. cenocepacia, in particular regarding bacterial adaptation to microaerophilic conditions. Also, the content of the virulence determinant AidA was higher in the last 2 isolates. Significant levels of siderophores were found to be secreted by the three clonal isolates in an iron-depleted environment, but the two late isolates were more tolerant to low iron concentrations than IST439, consistent with the relative abundance of proteins involved in iron uptake.     

Identification of Burkholderia pseudomallei Near-Neighbor Species in the Northern Territory of Australia

Identification and characterization of near-neighbor species are critical to the development of robust molecular diagnostic tools for biothreat agents. One such agent, Burkholderia pseudomallei, a soil bacterium and the causative agent of melioidosis, is lacking in this area because of its genomic diversity and widespread geographic distribution. The Burkholderia genus contains over 60 species and occupies a large range of environments including soil, plants, rhizospheres, water, animals and humans. The identification of novel species in new locations necessitates the need to identify the true global distribution of Burkholderia species, especially the members that are closely related to B. pseudomallei. In our current study, we used the Burkholderia-specific recA sequencing assay to analyze environmental samples from the Darwin region in the Northern Territory of Australia where melioidosis is endemic. Burkholderia recA PCR negative samples were further characterized using 16s rRNA sequencing for species identification. Phylogenetic analysis demonstrated that over 70% of the bacterial isolates were identified as B. ubonensis indicating that this species is common in the soil where B. pseudomallei is endemic. Bayesian phylogenetic analysis reveals many novel branches within the B. cepacia complex, one novel B. oklahomensis-like species, and one novel branch containing one isolate that is distinct from all other samples on the phylogenetic tree. During the analysis with recA sequencing, we discovered 2 single nucleotide polymorphisms in the reverse priming region of B. oklahomensis. A degenerate primer was developed and is proposed for future use. We conclude that the recA sequencing technique is an effective tool to classify Burkholderia and identify soil organisms in a melioidosis endemic area.     

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°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.