Steve P. Bernier

Concentration-dependent activity of antibiotics in natural environments

Bacterial responses to antibiotics are concentration-dependent. At high concentrations, antibiotics exhibit antimicrobial activities on susceptible cells, while subinhibitory concentrations induce diverse biological responses in bacteria. At non-lethal concentrations, bacteria may sense antibiotics as extracellular chemicals to trigger different cellular responses, which may include an altered antibiotic resistance/tolerance profile. In natural settings, microbes are typically in polymicrobial communities and antibiotic-mediated interactions between species may play a significant role in bacterial community structure and function. However, these aspects have not yet fully been explored at the community level. Here we discuss the different types of interactions mediated by antibiotics and non-antibiotic metabolites as a function of their concentrations and speculate on how these may amplify the overall antibiotic resistance/tolerance and the spread of antibiotic resistance determinants in a context of polymicrobial community.

Aminoglycoside resistance of Pseudomonas aeruginosa biofilms modulated by extracellular polysaccharide

Pseudomonas aeruginosa is an opportunistic pathogen that produces sessile communities known as biofilms that are highly resistant to antibiotic treatment. Limited information is available on the exact role of various components of the matrix in biofilm-associated antibiotic resistance. Here we show that the presence of extracellular polysaccharide reduced the extent of biofilm-associated antibiotic resistance for one class of antibiotics. Minimal bactericidal concentration (MBC) for planktonic and biofilm cells of P. aeruginosa PA14 was measured using a 96 well microtiter plate assay. The MBC of biofilm-grown ΔpelA mutant, which does not produce the Pel polysaccharide, was 4-fold higher for tobramycin and gentamicin, and unchanged for ΔbifA mutant, which overproduces Pel, when compared to the wild type. Biofilms of pelA mutants in two clinical isolates of P. aeruginosa showed 4- and 8-fold higher MBC for tobramycin as compared to wild type. There was no difference in the biofilm resistance of any of these strains when tested with fluoroquinolones. This work forms a basis for future studies revealing the mechanisms of biofilm-associated antibiotic resistance to aminoglycoside antibiotics by P. aeruginosa.

A LysR-Type Transcriptional Regulator in Burkholderia cenocepacia Influences Colony Morphology and Virulence

ABSTRACT Burkholderia cenocepacia strain K56-2 typically has rough colony morphology on agar medium; however, shiny colony variants (shv) can appear spontaneously. These shv all had a minimum of 50% reduction in biomass formation and were generally avirulent in an alfalfa seedling infection model. Three shv—K56-2 S15, K56-2 S76, and K56-2 S86—were analyzed for virulence in a chronic agar bead model of respiratory infection and, although all shv were able to establish chronic infection, they produced significantly less lung histopathology than the rough K56-2. Transmission electron microscopy revealed that an extracellular matrix surrounding bacterial cells was absent or reduced in the shv compared to the rough wild type. Transposon mutagenesis was performed on the rough wild-type strain and a mutant with an insertion upstream of ORF BCAS0225, coding for a putative LysR-type regulator, exhibited shiny colony morphology, reduced biofilm production, increased N-acyl homoserine lactone production, and avirulence in alfalfa. The rough parental colony morphotype, biofilm formation, and virulence in alfalfa were restored by providing BCAS0225 in trans in the BCAS0225::pGSVTp-luxCDABF mutant. Introduction of BCAS0225 restored the rough morphotype in several shv which were determined to have spontaneous mutations in this gene. In the present study, we show that the conversion from rough wild type to shv in B. cenocepacia correlates with reduced biofilm formation and virulence, and we determined that BCAS0225 is one gene involved in the regulation of these phenotypes.

Use of Suppression-Subtractive Hybridization To Identify Genes in the Burkholderia cepacia Complex That Are Unique to Burkholderia cenocepacia

We have previously shown differences in virulence between species of the Burkholderia cepacia complex using the alfalfa infection model and the rat agar bead chronic infection model. Burkholderia cenocepacia strains were more virulent in these two infection models than Burkholderia multivorans and Burkholderia stabilis strains. In order to identify genes that may account for the increased virulence of B. cenocepacia, suppression-subtractive hybridization was performed between B. cenocepacia K56-2 and B. multivorans C5393 and between B. cenocepacia K56-2 and B. stabilis LMG14294. Genes identified included DNA modification/phage-related/insertion sequences and genes involved in cell membrane/surface structures, resistance, transport, metabolism, regulation, secretion systems, as well as genes of unknown function. Several of these genes were present in the ET12 lineage of B. cenocepacia but not in other members of the B. cepacia complex. Virulence studies in a chronic lung infection model determined that the hypothetical YfjI protein, which is unique to the ET12 clone, contributes to lung pathology. Other genes specific to B. cenocepacia and/or the ET12 lineage were shown to play a role in biofilm formation and swarming or swimming motility.

Cyanide Toxicity to Burkholderia cenocepacia Is Modulated by Polymicrobial Communities and Environmental Factors

Microbes within polymicrobial communities can establish positive and negative interactions that have the potential to influence the overall behavior of the community. Pseudomonas aeruginosa and species of the Burkholderia cepacia complex (Bcc) can co-exist in the lower airways, however several studies have shown that P. aeruginosa can effectively kill the Bcc in vitro, for which hydrogen cyanide (HCN) was recently proposed to play a critical role. Here we show that modification of the environment (i.e., culture medium), long-term genetic adaptation of P. aeruginosa to the cystic fibrosis (CF) lung, or the addition of another bacterial species to the community can alter the sensitivity of Burkholderia cenocepacia to P. aeruginosa toxins. We specifically demonstrate that undefined rich media leads to higher susceptibility of B. cenocepacia to P. aeruginosa toxins like cyanide as compared to a synthetic medium (SCFM), that mimics the CF lung nutritional content. Overall, our study shows that the polymicrobial environment can have profound effects on negative interactions mediated by P. aeruginosa against B. cenocepacia. In fact, evolved P. aeruginosa or the presence of other species such as Staphylococcus aureus can directly abolish the direct competition mediated by cyanide and consequently maintaining a higher level of species diversity within the community.

Draft Genome Sequence of Burkholderia dolosa PC543 Isolated from Cystic Fibrosis Airways

ABSTRACT Burkholderia dolosa is a member of the Burkholderia cepacia complex, a group of opportunistic bacterial pathogens often associated with fatal chronic infections in the lungs of patients suffering from cystic fibrosis (CF). Here, we announce the draft genome sequence of B. dolosa PC543 (LMG 19468), a CF airway isolate.

The attenuated virulence of a Burkholderia cenocepacia paaABCDE mutant is due to inhibition of quorum sensing by release of phenylacetic acid.

The phenylacetic acid degradation pathway of Burkholderia cenocepacia is active during cystic fibrosis‐like conditions and is necessary for full pathogenicity of B. cenocepacia in nematode and rat infection models; however, the reasons for such requirements are unknown. Here, we show that the attenuated virulence of a phenylacetic acid catabolism mutant is due to quorum sensing inhibition. Unlike wild‐type B. cenocepacia, a deletion mutant of the phenylacetyl‐CoA monooxygenase complex (ΔpaaABCDE) released phenylacetic acid in the medium that favours infection in Caenorhabditis elegans. Addition of phenylacetic acid further decreased the pathogenicity of the ΔpaaABCDE, which cannot metabolize phenylacetic acid, but did not affect the wild‐type, due to phenylacetic acid consumption. In line with reduced detection of acyl‐homoserine lactones in spent medium, the ΔpaaABCDE exhibited transcriptional inhibition of the quorum sensing system cepIR. Phenotypes repressed in ΔpaaABCDE, protease activity and pathogenicity against C. elegans, increased with exogenous N‐octanoyl‐L‐homoserine lactone. Thus, we demonstrate that the attenuated phenotype of B. cenocepacia ΔpaaABCDE is due to quorum sensing inhibition by release of phenylacetic acid, affecting N‐octanoyl‐L‐homoserine lactone signalling. Further, we propose that active degradation of phenylacetic acid by B. cenocepacia during growth in cystic fibrosis‐like conditions prevents accumulation of a quorum sensing inhibiting compound.

Genetic signature of bacterial pathogen adaptation during chronic pulmonary infections

To establish and maintain chronic infections, many pathogens adapt in response to selective pressures within the host, leaving unique genetic signatures. A new study uses whole-genome and population sequencing approaches to identify evidence of adaptive evolution in Burkholderia dolosa genomes isolated from chronic infections in patients with cystic fibrosis.

The Mla Pathway Plays an Essential Role in the Intrinsic Resistance of Burkholderia cepacia Complex Species to Antimicrobials and Host Innate Components

Antibiotic resistance is a threat to our modern society, and new strategies leading to the identification of new molecules or targets to combat multidrug-resistant pathogens are needed. Species of the genus Burkholderia, including the Burkholderia cepacia complex (Bcc), Burkholderia pseudomallei, and Burkholderia mallei, can be highly pathogenic and are intrinsically resistant to multiple classes of antibiotics. Bcc species are nonetheless sensitive to extracellular products released by Pseudomonas aeruginosa in interspecies competition. We screened for Burkholderia transposon mutants with increased sensitivity to P. aeruginosa spent medium and identified multiple mutants in genes sharing homology with the Mla pathway. Insertional mutants in representative genes of the Bcc Mla pathway had a compromised cell membrane and were more sensitive to various extracellular stresses, including antibiotics and human serum. More precisely, mla mutants in the Bcc species Burkholderia cenocepacia and Burkholderia dolosa were more susceptible to Gram-positive antibiotics (i.e., macrolides and rifampin), fluoroquinolones, tetracyclines, and chloramphenicol. Genetic complementation of mlaC insertional mutants restored cell permeability and resistance to Gram-positive antibiotics. Importantly, Bcc mla mutants were not universally weaker strains since their susceptibilities to other classes of antibiotics were unaffected. Although cell permeability of homologous mla mutants in Escherichia coli or P. aeruginosa was also impaired, they were not more sensitive to Gram-positive antibiotics or other antimicrobials as was observed in Bcc mla mutants. Together, the data suggest that the Mla pathway in Burkholderia may play a different biological role, which could potentially represent a Burkholderia-specific drug target in combination therapy with antibiotic adjuvants.IMPORTANCE The outer membrane of Gram-negative bacteria acts as an effective barrier against toxic compounds, and therefore compromising this structure could increase sensitivity to currently available antibiotics. In this study, we show that the Mla pathway, a system involved in maintaining the integrity of the outer membrane, is genetically and functionally different in Burkholderia cepacia complex species compared to that in other proteobacteria. Mutants in mla genes of Burkholderia cenocepacia or Burkholderia dolosa were sensitive to Gram-positive antibiotics, while this effect was not observed in Escherichia coli or Pseudomonas aeruginosa The Mla pathway in Burkholderia species may represent an ideal genus-specific target to address their intrinsic antimicrobial resistances.