Occurrence of Multiple Genomovars of Burkholderia cepacia in Cystic Fibrosis Patients and Proposal of Burkholderia multivorans sp. nov.

Burkholderia cepacia complex (Bcc) bacteria are opportunistic pathogens infecting hosts such as cystic fibrosis (CF) patients. Long-term Bcc infection of CF patients’ airways has been associated with emergence of phenotypic variation. Here we studied two Burkholderia multivorans clonal isolates displaying different morphotypes from a chronically infected CF patient to evaluate trait development during lung infection. Expression profiling of mucoid D2095 and non-mucoid D2214 isolates revealed decreased expression of genes encoding products related to virulence-associated traits and metabolism in D2214. Furthermore, D2214 showed no exopolysaccharide production, lower motility and chemotaxis, and more biofilm formation, particularly under microaerophilic conditions, than the clonal mucoid isolate D2095. When Galleria mellonella was used as acute infection model, D2214 at a cell number of approximately 7×106 c.f.u. caused a higher survival rate than D2095, although 6 days post-infection most of the larvae were dead. Infection with the same number of cells by mucoid D2095 caused larval death by day 4. The decreased expression of genes involved in carbon and nitrogen metabolism may reflect lower metabolic needs of D2214 caused by lack of exopolysaccharide, but also by the attenuation of pathways not required for survival. As a result, D2214 showed higher survival than D2095 in minimal medium for 28 days under aerobic conditions. Overall, adaptation during Bcc chronic lung infections gave rise to genotypic and phenotypic variation among isolates, contributing to their fitness while maintaining their capacity for survival in this opportunistic human niche.

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Characterization of the AmpC β-Lactamase fromBurkholderia multivorans

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01140-18 ◽

2018 ◽

Vol 62 (10) ◽

Cited By ~ 5

Author(s):

Scott A. Becka ◽

Elise T. Zeiser ◽

Melissa D. Barnes ◽

Magdalena A. Taracila ◽

Kevin Nguyen ◽

...

Keyword(s):

Cystic Fibrosis ◽

Active Site ◽

Burkholderia Cepacia ◽

Single Amino Acid ◽

Amino Acid Substitutions ◽

Enzyme Complex ◽

Active Site Residues ◽

Content Type ◽

Burkholderia Multivorans

ABSTRACTBurkholderia multivoransis a member of theBurkholderia cepaciacomplex, a group of >20 related species of nosocomial pathogens that commonly infect individuals suffering from cystic fibrosis. β-Lactam antibiotics are recommended as therapy for infections due toB.multivorans, which possesses two β-lactamase genes,blapenAandblaAmpC. PenA is a carbapenemase with a substrate profile similar to that of theKlebsiella pneumoniaecarbapenemase (KPC); in addition, expression of PenA is inducible by β-lactams inB.multivorans. Here, we characterize AmpC fromB.multivoransATCC 17616. AmpC possesses only 38 to 46% protein identity with non-BurkholderiaAmpC proteins (e.g., PDC-1 and CMY-2). Among 49 clinical isolates ofB.multivorans, we identified 27 different AmpC variants. Some variants possessed single amino acid substitutions within critical active-site motifs (Ω loop and R2 loop). Purified AmpC1 demonstrated minimal measurable catalytic activity toward β-lactams (i.e., nitrocefin and cephalothin). Moreover, avibactam was a poor inhibitor of AmpC1 (Kiapp> 600 μM), and acyl-enzyme complex formation with AmpC1 was slow, likely due to lack of productive interactions with active-site residues. Interestingly, immunoblotting using a polyclonal anti-AmpC antibody revealed that protein expression of AmpC1 was inducible inB.multivoransATCC 17616 after growth in subinhibitory concentrations of imipenem (1 μg/ml). AmpC is a unique inducible class C cephalosporinase that may play an ancillary role inB.multivoranscompared to PenA, which is the dominant β-lactamase inB.multivoransATCC 17616.

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A histone-like nucleoid structuring protein regulates several virulence traits in Burkholderia multivorans

Applied and Environmental Microbiology ◽

10.1128/aem.00369-21 ◽

2021 ◽

Author(s):

Sara C. Gomes ◽

Mirela R. Ferreira ◽

Andreia F. Tavares ◽

Inês N. Silva ◽

Jörg D. Becker ◽

...

Keyword(s):

Gene Expression ◽

Cystic Fibrosis ◽

Burkholderia Cepacia ◽

Gc Content ◽

Mobile Element ◽

Surface Hydrophobicity ◽

Upstream Region ◽

Cell Physiology ◽

Burkholderia Multivorans ◽

Virulence Traits

Burkholderia cepacia complex bacteria comprises opportunistic pathogens causing chronic respiratory infections in cystic fibrosis (CF) patients. These microorganisms produce an exopolysaccharide named cepacian, which is considered a virulence determinant. To find genes implicated in the regulation of cepacian biosynthesis, we characterized an evolved nonmucoid variant (17616nmv) derived from the ancestor, Burkholderia multivorans ATCC 17616, after prolonged stationary phase. Lack of cepacian biosynthesis was correlated with downregulation of the expression of bce genes implicated in its biosynthesis. Furthermore, genome sequencing of the variant identified the transposition of the mobile element IS406 upstream the coding sequence of an hns-like gene (Bmul_0158) encoding a histone-like nucleoid structuring protein, a known global transcriptional repressor. This IS element upregulated the expression of Bmul_0158 by 4-fold. Transcriptome analysis identified the global effects of this mutation on gene expression, with major changes in genes implicated in motility, pili synthesis, type VI secretion, and chromosome associated functions. Concomitant with these differences, the nonmucoid variant displays reduced adherence to a CF lung bronchial cell line, reduced surface hydrophobicity, forms smaller cellular aggregates, but has an increase in swimming and swarming motilities. Finally, analysis of the GC content of the upstream region of differentially expressed genes led to the identification of various genomic regions, possibly acquired by horizontal gene transfer, which were transcriptionally repressed by the increased expression of Bmul_0158 gene in the 17616nmv strain. Taken together, the results revealed a significant role for this H-NS protein in the regulation of B. multivorans persistence- and virulence-associated genes. IMPORTANCE Members of the histone-like nucleoid-structuring (H-NS) family of proteins, present in many bacteria, are important global regulators of gene expression. Many of the regulated genes were acquired horizontally and include pathogenicity islands and prophages, among others. Additionally, H-NS can play a structural role by bridging and compacting DNA, presenting a crucial role in cell physiology. Several virulence phenotypes have been frequently identified in several bacteria as dependent on H-NS activity. Here, we describe an H-NS-like protein of the opportunistic pathogen Burkholderia multivorans, a species commonly infecting the respiratory tract of cystic fibrosis patients. Our results indicate that this protein is involved in regulating virulence traits such as exopolysaccharide biosynthesis, adhesion to biotic surfaces, cellular aggregation, and motility. Furthermore, this H-NS-like protein, is one out of eight orthologs present in B. multivorans ATCC 17616 genome, posing relevant questions to be investigated on how these proteins coordinate the expression of virulence traits.

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Discrimination of Burkholderia multivorans and Burkholderia vietnamiensis fromBurkholderia cepacia Genomovars I, III, and IV by PCR

Journal of Clinical Microbiology ◽

10.1128/jcm.37.5.1335-1339.1999 ◽

1999 ◽

Vol 37 (5) ◽

pp. 1335-1339 ◽

Cited By ~ 38

Author(s):

Adolf Bauernfeind ◽

Ines Schneider ◽

Renate Jungwirth ◽

Carsten Roller

Keyword(s):

Cystic Fibrosis ◽

Burkholderia Cepacia ◽

General Sense ◽

Burkholderia Multivorans ◽

Burkholderia Vietnamiensis ◽

Definition Of ◽

Species Specific ◽

Optimized Conditions ◽

Ribosomal Dnas ◽

Sense Primer

We present a PCR procedure for identification of Burkholderia cepacia, Burkholderia multivorans, andBurkholderia vietnamiensis. 16S and 23S ribosomal DNAs (rDNAs) of B. multivorans and B. vietnamiensiswere sequenced and aligned with published sequences for definition of species-specific 18-mer oligonucleotide primers. Specific antisense 16S rDNA primers (for B. cepacia, 5′-AGC ACT CCC RCC TCT CAG-3′; for B. multivorans, 5′-AGC ACT CCC GAA TCT CTT-3′) and 23S rDNA primers (for B. vietnamiensis, 5′-TCC TAC CAT GCG TGC AA-3′) were paired with a general sense primer of 16S rDNAs (5′-AGR GTT YGA TYM TGG CTC AG-3′) or with a sense primer of 23S rDNA (5′-CCT TTG GGT CAT CCT GGA-3′). PCR with these primers under optimized conditions is appropriate to specifically and rapidly identify B. multivorans, B. vietnamiensis, and B. cepacia (genomovars I, III, and IV are not discriminated). In comparison with the polyphasic taxonomic analyses presently necessary for species and genomovar identification within the B. cepacia complex, our procedure is more rapid and easier to perform and may contribute to clarifying the clinical significance of individual members of the complex in cystic fibrosis.

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Burkholderia cepacia Complex Contact-Dependent Growth Inhibition Systems Mediate Interbacterial Competition

Journal of Bacteriology ◽

10.1128/jb.00012-19 ◽

2019 ◽

Vol 201 (12) ◽

Cited By ~ 1

Author(s):

Tanya Myers-Morales ◽

A. Elizabeth Oates ◽

Matthew S. Byrd ◽

Erin C. Garcia

Keyword(s):

Cystic Fibrosis ◽

Growth Inhibition ◽

Burkholderia Cepacia ◽

Gram Negative Bacteria ◽

Immunity Protein ◽

Gram Negative ◽

Content Type ◽

Burkholderia Multivorans ◽

Dependent Growth ◽

Contact Dependent Growth Inhibition

ABSTRACT Burkholderia species, including opportunistic pathogens in the Burkholderia cepacia complex (Bcc), have genes to produce contact-dependent growth inhibition (CDI) system proteins. CDI is a phenomenon in which Gram-negative bacteria use the toxic C terminus of a polymorphic surface-exposed exoprotein, BcpA, to inhibit the growth of susceptible bacteria upon direct cell-cell contact. Production of a small immunity protein, BcpI, prevents autoinhibition. Although CDI systems appear widespread in Gram-negative bacteria, their function has been primarily examined in several model species. Here we demonstrate that genes encoding predicted CDI systems in Bcc species exhibit considerable diversity. We also show that Burkholderia multivorans, which causes pulmonary infections in patients with cystic fibrosis, expresses genes that encode two CDI systems, both of which appear distinct from the typical Burkholderia-type CDI system. Each system can mediate intrastrain interbacterial competition and contributes to bacterial adherence. Surprisingly, the immunity-protein-encoding bcpI gene of CDI system 1 could be mutated without obvious deleterious effects. We also show that nonpathogenic Burkholderia thailandensis uses CDI to control B. multivorans growth during coculture, providing one of the first examples of interspecies CDI and suggesting that CDI systems could be manipulated to develop therapeutic strategies targeting Bcc pathogens. IMPORTANCE Competition among bacteria affects microbial colonization of environmental niches and host organisms, particularly during polymicrobial infections. The Bcc is a group of environmental bacteria that can cause life-threatening opportunistic infections in patients who have cystic fibrosis or are immunocompromised. Understanding the mechanisms used by these bacterial pathogens to compete with one another may lead to the development of more effective therapies. Findings presented here demonstrate that a Bcc species, Burkholderia multivorans, produces functional CDI system proteins and that growth of this pathogen can be controlled by CDI system proteins produced by neighboring Burkholderia cells.

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Inhibition of co-colonizing cystic fibrosis-associated pathogens by Pseudomonas aeruginosa and Burkholderia multivorans

Microbiology ◽

10.1099/mic.0.074203-0 ◽

2014 ◽

Vol 160 (7) ◽

pp. 1474-1487 ◽

Cited By ~ 24

Author(s):

Anne Costello ◽

F. Jerry Reen ◽

Fergal O’Gara ◽

Máire Callaghan ◽

Siobhán McClean

Keyword(s):

Cystic Fibrosis ◽

Pseudomonas Aeruginosa ◽

Burkholderia Cepacia ◽

Inflammatory Responses ◽

Respiratory Infections ◽

Lung Damage ◽

Burkholderia Cenocepacia ◽

Antibiotic Resistant ◽

Bacterial Dna ◽

Burkholderia Multivorans

Cystic fibrosis (CF) is a recessive genetic disease characterized by chronic respiratory infections and inflammation causing permanent lung damage. Recurrent infections are caused by Gram-negative antibiotic-resistant bacterial pathogens such as Pseudomonas aeruginosa, Burkholderia cepacia complex (Bcc) and the emerging pathogen genus Pandoraea. In this study, the interactions between co-colonizing CF pathogens were investigated. Both Pandoraea and Bcc elicited potent pro-inflammatory responses that were significantly greater than Ps. aeruginosa. The original aim was to examine whether combinations of pro-inflammatory pathogens would further exacerbate inflammation. In contrast, when these pathogens were colonized in the presence of Ps. aeruginosa the pro-inflammatory response was significantly decreased. Real-time PCR quantification of bacterial DNA from mixed cultures indicated that Ps. aeruginosa significantly inhibited the growth of Burkholderia multivorans, Burkholderia cenocepacia, Pandoraea pulmonicola and Pandoraea apista, which may be a factor in its dominance as a colonizer of CF patients. Ps. aeruginosa cell-free supernatant also suppressed growth of these pathogens, indicating that inhibition was innate rather than a response to the presence of a competitor. Screening of a Ps. aeruginosa mutant library highlighted a role for quorum sensing and pyoverdine biosynthesis genes in the inhibition of B. cenocepacia. Pyoverdine was confirmed to contribute to the inhibition of B. cenocepacia strain J2315. B. multivorans was the only species that could significantly inhibit Ps. aeruginosa growth. B. multivorans also inhibited B. cenocepacia and Pa. apista. In conclusion, both Ps. aeruginosa and B. multivorans are capable of suppressing growth and virulence of co-colonizing CF pathogens.

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Evaluation of antimicrobial susceptibility testing methods for Burkholderia cenocepacia and Burkholderia multivorans isolates from cystic fibrosis patients

Journal of Clinical Microbiology ◽

10.1128/jcm.01447-21 ◽

2021 ◽

Author(s):

Holly K. Huse ◽

Mark J. Lee ◽

Mandy Wootton ◽

Susan E. Sharp ◽

Maria Traczewski ◽

...

Keyword(s):

Cystic Fibrosis ◽

Antimicrobial Susceptibility ◽

Burkholderia Cepacia ◽

Susceptibility Testing ◽

Antimicrobial Susceptibility Testing ◽

Burkholderia Cenocepacia ◽

Acceptance Criteria ◽

Method Performance ◽

Burkholderia Multivorans ◽

Microscan Walkaway

The Burkholderia cepacia complex (BCC) is known for causing serious lung infections in people with cystic fibrosis (CF). These infections can require lung transplantation, eligibility for which may be guided by antimicrobial susceptibility testing (AST). While the Clinical and Laboratory Standards Institute recommends AST for BCC, the European Committee on Antimicrobial Susceptibility Testing (EUCAST) does not due to poor method performance and correlation with clinical outcomes. Furthermore, limited data exists on the performance of automated AST methods for BCC. To address these issues, reproducibility and accuracy were evaluated for disk diffusion (DD), broth microdilution (BMD), and MicroScan WalkAway using 50 B. cenocepacia and 50 B. multivorans isolates collected from people with CF. The following drugs were evaluated in triplicate: chloramphenicol (CAM), ceftazidime (CAZ), meropenem (MEM), trimethoprim-sulfamethoxazole (TMP-SMX), minocycline (MIN), levofloxacin (LVX), ciprofloxacin (CIP) and piperacillin-tazobactam (PIP-TAZ). BMD reproducibility was ≥ 95% for MEM and MIN only, and MicroScan WalkAway reproducibility was similar to BMD. DD reproducibility was < 90% for all drugs tested when a 3 mm cut-off was applied. When comparing the accuracy of DD to BMD, only MEM met all acceptance criteria. TMP-SMX and LVX had high minor errors, CAZ had unacceptable very major errors (VME), and MIN, PIP-TAZ, and CIP had both unacceptable minor errors and VMEs. For MicroScan WalkAway, no drugs met acceptance criteria. Analyses also showed that errors were not attributed to one species. In general, our data agree with EUCAST recommendations that routine AST should not be performed for BCC CF isolates.

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Epidemiology of Burkholderia cepacia complex species recovered from cystic fibrosis patients: issues related to patient segregation

Journal of Medical Microbiology ◽

10.1099/jmm.0.45557-0 ◽

2004 ◽

Vol 53 (7) ◽

pp. 663-668 ◽

Cited By ~ 42

Author(s):

Andrew McDowell ◽

Eshwar Mahenthiralingam ◽

Kerstin E.A. Dunbar ◽

John E. Moore ◽

Mary Crowe ◽

...

Keyword(s):

Cystic Fibrosis ◽

Burkholderia Cepacia ◽

Cross Infection ◽

Burkholderia Cenocepacia ◽

Burkholderia Cepacia Complex ◽

Complex Species ◽

Burkholderia Multivorans ◽

Burkholderia Vietnamiensis ◽

Random Amplification ◽

Geographical Regions

Studies of the prevalence of Burkholderia cepacia complex species amongst cystic fibrosis (CF) patients in different geographical regions, and the association between cross-infection and putative transmissibility markers, will further our understanding of these organisms and help to address infection-control issues. In this study, B. cepacia complex isolates from CF patients in different regions of Europe were analysed. Isolates were examined for B. cepacia complex species and putative transmissibility markers [cable pilin subunit gene (cblA) and the B. cepacia epidemic strain marker (BCESM)]. Sporadic and cross-infective strains were identified by random amplification of polymorphic DNA (RAPD). In total, 79 % of patients were infected with Burkholderia cenocepacia (genomovar III), 18 % with Burkholderia multivorans (genomovar II) and less than 5 % of patients with B. cepacia (genomovar I), Burkholderia stabilis (genomovar IV) or Burkholderia vietnamiensis (genomovar V). The cblA and BCESM transmissibility markers were only detected in strains of B. cenocepacia. The BCESM was a more sensitive marker for transmissible B. cenocepacia strains than cblA, although sporadic B. cenocepacia strains containing the BCESM, but lacking cblA, were also observed. Furthermore, clusters of cross-infection with transmissibility marker-negative strains of B. multivorans were identified. In conclusion, B. cenocepacia was the greatest cause of cross-infection, and the most widely distributed B. cepacia complex species, within these CF populations. However, cross-infection was not exclusive to B. cenocepacia and cblA and the BCESM were not absolute markers for transmissible B. cenocepacia, or other B. cepacia complex strains. It is therefore suggested that CF centres cohort patients based on the presence or absence of B. cepacia complex infection and not on the basis of transmissibility marker-positive B. cenocepacia as previously suggested.

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Genomics of an endemic cystic fibrosis Burkholderia multivorans strain reveals low within-patient evolution but high between-patient diversity

PLoS Pathogens ◽

10.1371/journal.ppat.1009418 ◽

2021 ◽

Vol 17 (3) ◽

pp. e1009418

Author(s):

Cédric Lood ◽

Charlotte Peeters ◽

Quentin Lamy-Besnier ◽

Jeroen Wagemans ◽

Daniel De Vos ◽

...

Keyword(s):

Cystic Fibrosis ◽

Burkholderia Cepacia ◽

Genomic Analysis ◽

Genomic Diversity ◽

Epidemiological Surveillance ◽

Nucleotide Polymorphisms ◽

Whole Genome Analysis ◽

Structural Genomic ◽

Burkholderia Multivorans ◽

Sequencing Technologies

Burkholderia multivorans is a member of the Burkholderia cepacia complex (Bcc), notorious for its pathogenicity in persons with cystic fibrosis. Epidemiological surveillance suggests that patients predominantly acquire B. multivorans from environmental sources, with rare cases of patient-to-patient transmission. Here we report on the genomic analysis of thirteen isolates from an endemic B. multivorans strain infecting four cystic fibrosis patients treated in different pediatric cystic fibrosis centers in Belgium, with no evidence of cross-infection. All isolates share an identical sequence type (ST-742) but whole genome analysis shows that they exhibit peculiar patterns of genomic diversity between patients. By combining short and long reads sequencing technologies, we highlight key differences in terms of small nucleotide polymorphisms indicative of low rates of adaptive evolution within patient, and well-defined, hundred Kbps-long segments of high enrichment in mutations between patients. In addition, we observed large structural genomic variations amongst the isolates which revealed different plasmid contents, active roles for transposase IS3 and IS5 in the deactivation of genes, and mobile prophage elements. Our study shows limited within-patient B. multivorans evolution and high between-patient strain diversity, indicating that an environmental microdiverse reservoir must be present for this endemic strain, in which active diversification is taking place. Furthermore, our analysis also reveals a set of 30 parallel adaptations across multiple patients, indicating that the specific genomic background of a given strain may dictate the route of adaptation within the cystic fibrosis lung.

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