The role of efflux and physiological adaptation in biofilm tolerance and resistance

Microbial biofilms demonstrate a decreased susceptibility to antimicrobial agents. Various mechanisms have been proposed to be involved in this recalcitrance. We focus on two of these factors. Firstly, the ability of sessile cells to actively mediate efflux of antimicrobial compounds has a profound impact on resistance and tolerance, and several studies point to the existence of biofilm-specific efflux systems. Secondly, biofilm-specific stress responses have a marked influence on cellular physiology, and contribute to the occurrence of persister cells. We provide an overview of the data that demonstrate that both processes are important for survival following exposure to antimicrobial agents

Molecular mechanisms of persistence in Burkholderia cenocepacia biofilms

Burkholderia cenocepacia J2315 is a member of the Burkholderia cepacia complex (Bcc), a group of 18 closely related metabolically versatile microorganisms. These opportunistic pathogens can cause severe lung infections in cystic fibrosis patients. Infections are often difficult to treat due to innate resistance of Bcc species to antimicrobials and because of their capacity to form biofilms. One of the mechanisms thought to be involved in biofilm resilience is the presence of persister cells. The main aim of this dissertation was to investigate whether persister cells are present in B. cenocepacia biofilms, what the molecular basis of antimicrobial tolerance is, and how persisters can be eradicated. We confirmed the presence of persister cells in B. cenocepacia biofilms and found that genes encoding proteins involved in ROS production are downregulated in persister cells, whereas the glyoxylate shunt is upregulated. Additionally, we found that toxin antitoxin (TA)-modules are involved in persistence. Searching for TA-modules we also identified an interesting operon (BCAM0257-8) located in the B. cepacia epidemic strain marker region. This module plays a role in persistence and in addition, BCAM0258 functions as a regulator influencing quorum sensing and activating cellular pathways involved in iron acquisition and biofilm formation. Overexpression of BCAM0257 was found to increase virulence in Galleria mellonella. Genes similar to BCAM0257-8 are found in all sequenced B. cenocepacia ET-12 genomes and its presence may help explaining why infections with strains of the B. cenocepacia ET-12 lineage are so difficult to treat. Furthermore, we evaluated in vitro the bacteriostatic and bactericidal activity of temocillin, a 6-α-methoxy-penicillin against planktonic and sessile Bcc bacteria. Our data indicate that, although temocillin has a good bacteriostatic activity against planktonic cultures and 4-h old biofilms, it is of limited use to eradicate mature biofilms

The role of reactive oxygen species in antibiotic-induced cell death in Burkholderia cepacia complex bacteria

It was recently proposed that bactericidal antibiotics, besides through specific drug-target interactions, kill bacteria by a common mechanism involving the production of reactive oxygen species (ROS). However, this mechanism involving the production of hydroxyl radicals has become the subject of a lot of debate. Since the contribution of ROS to antibiotic mediated killing most likely depends on the conditions, differences in experimental procedures are expected to be at the basis of the conflicting results. In the present study different methods (ROS specific stainings, gene-expression analyses, electron paramagnetic resonance, genetic and phenotypic experiments, detection of protein carbonylation and DNA oxidation) to measure the production of ROS upon antibiotic treatment in Burkholderia cepacia complex (Bcc) bacteria were compared. Different classes of antibiotics (tobramycin, ciprofloxacin, meropenem) were included, and both planktonic and biofilm cultures were studied. Our results indicate that some of the methods investigated were not sensitive enough to measure antibiotic induced production of ROS, including the spectrophotometric detection of protein carbonylation. Secondly, other methods were found to be useful only in specific conditions. For example, an increase in the expression of OxyR was measured in Burkholderia cenocepacia K56-2 after treatment with ciprofloxacin or meropenem (both in biofilms and planktonic cultures) but not after treatment with tobramycin. In addition results vary with the experimental conditions and the species tested. Nevertheless our data strongly suggest that ROS contribute to antibiotic mediated killing in Bcc species and that enhancing ROS production or interfering with the protection against ROS may form a novel strategy to improve antibiotic treatment

Genome-wide transcription start site profiling in biofilm-grown Burkholderia cenocepacia J2315

Background: Burkholderia cenocepacia is a soil-dwelling Gram-negative Betaproteobacterium with an important role as opportunistic pathogen in humans. Infections with B. cenocepacia are very difficult to treat due to their high intrinsic resistance to most antibiotics. Biofilm formation further adds to their antibiotic resistance. B. cenocepacia harbours a large, multi-replicon genome with a high GC-content, the reference genome of strain J2315 includes 7374 annotated genes. This study aims to annotate transcription start sites and identify novel transcripts on a whole genome scale. Methods: RNA extracted from B. cenocepacia J2315 biofilms was analysed by differential RNA-sequencing and the resulting dataset compared to data derived from conventional, global RNA-sequencing. Transcription start sites were annotated and further analysed according to their position relative to annotated genes. Results: Four thousand ten transcription start sites were mapped over the whole B. cenocepacia genome and the primary transcription start site of 2089 genes expressed in B. cenocepacia biofilms were defined. For 64 genes a start codon alternative to the annotated one was proposed. Substantial antisense transcription for 105 genes and two novel protein coding sequences were identified. The distribution of internal transcription start sites can be used to identify genomic islands in B. cenocepacia. A potassium pump strongly induced only under biofilm conditions was found and 15 non-coding small RNAs highly expressed in biofilms were discovered. Conclusions: Mapping transcription start sites across the B. cenocepacia genome added relevant information to the J2315 annotation. Genes and novel regulatory RNAs putatively involved in B. cenocepacia biofilm formation were identified. These findings will help in understanding regulation of B. cenocepacia biofilm formation

Targeting the nonmevalonate pathway in Burkholderia cenocepacia increases susceptibility to certain β-lactam antibiotics

The nonmevalonate pathway is the sole pathway for isoprenoid biosynthesis in Burkholderia cenocepacia and is possibly a novel target for the development of antibacterial chemotherapy. The goals of the present study were to evaluate the essentiality of dxr, the second gene of the nonmevalonate pathway, in B. cenocepacia and to determine whether interfering with the nonmevalonate pathway increases susceptibility toward antibiotics. To this end, a rhamnose-inducible conditional dxr knockdown mutant of B. cenocepacia strain K56-2 (B. cenocepacia K56-2dxr) was constructed, using a plasmid which enables the delivery of a rhamnose-inducible promoter in the chromosome. Expression of dxr is essential for bacterial growth; the growth defect observed in the dxr mutant could be complemented by expressing dxr in trans under the control of a constitutive promoter, but not by providing 2C-methyl-D-erythritol-4-phosphate, the reaction product of DXR (1-deoxy-D-xylulose 5-phosphate reductoisomerase). B. cenocepacia K56-2dxr showed markedly increased susceptibility to the beta-lactam antibiotics aztreonam, ceftazidime, and cefotaxime, while susceptibility to other antibiotics was not (or was much less) affected; this increased susceptibility could also be complemented by in trans expression of dxr. A similarly increased susceptibility was observed when antibiotics were combined with FR900098, a known DXR inhibitor. Our data confirm that the nonmevalonate pathway is essential in B. cenocepacia and suggest that combining potent DXR inhibitors with selected beta-lactam antibiotics is a useful strategy to combat B. cenocepacia infections

Host metabolites stimulate the bacterial proton motive force to enhance the activity of aminoglycoside antibiotics

<div><p>Antibiotic susceptibility of bacterial pathogens is typically evaluated using <i>in vitro</i> assays that do not consider the complex host microenvironment. This may help explaining a significant discrepancy between antibiotic efficacy <i>in vitro</i> and <i>in vivo</i>, with some antibiotics being effective <i>in vitro</i> but not <i>in vivo</i> or vice versa. Nevertheless, it is well-known that antibiotic susceptibility of bacteria is driven by environmental factors. Lung epithelial cells enhance the activity of aminoglycoside antibiotics against the opportunistic pathogen <i>Pseudomonas aeruginosa</i>, yet the mechanism behind is unknown. The present study addresses this gap and provides mechanistic understanding on how lung epithelial cells stimulate aminoglycoside activity. To investigate the influence of the local host microenvironment on antibiotic activity, an <i>in vivo</i>-like three-dimensional (3-D) lung epithelial cell model was used. We report that conditioned medium of 3-D lung cells, containing secreted but not cellular components, potentiated the bactericidal activity of aminoglycosides against <i>P</i>. <i>aeruginosa</i>, including resistant clinical isolates, and several other pathogens. In contrast, conditioned medium obtained from the same cell type, but grown as conventional (2-D) monolayers did not influence antibiotic efficacy. We found that 3-D lung cells secreted endogenous metabolites (including succinate and glutamate) that enhanced aminoglycoside activity, and provide evidence that bacterial pyruvate metabolism is linked to the observed potentiation of antimicrobial activity. Biochemical and phenotypic assays indicated that 3-D cell conditioned medium stimulated the proton motive force (PMF), resulting in increased bacterial intracellular pH. The latter stimulated antibiotic uptake, as determined using fluorescently labelled tobramycin in combination with flow cytometry analysis. Our findings reveal a cross-talk between host and bacterial metabolic pathways, that influence downstream activity of antibiotics. Understanding the underlying basis of the discrepancy between the activity of antibiotics <i>in vitro</i> and <i>in vivo</i> may lead to improved diagnostic approaches and pave the way towards novel means to stimulate antibiotic activity.</p></div

The role of small proteins in Burkholderia cenocepacia J2315 biofilm formation, persistence and intracellular growth

Burkholderia cenocepacia infections are difficult to treat due to resistance, biofilm formation and persistence. B. cenocepacia strain J2315 has a large multi-replicon genome (8.06 Mb) and the function of a large fraction of (conserved) hypothetical genes remains elusive. The goal of the present study is to elucidate the role of small proteins in B. cenocepacia, focusing on genes smaller than 300 base pairs of which the function is unknown. Almost 10% (572) of the B. cenocepacia J2315 genes are smaller than 300 base pairs and more than half of these are annotated as coding for hypothetical proteins. For 234 of them no similarity could be found with non-hypothetical genes in other bacteria using BLAST. Using available RNA sequencing data obtained from biofilms, a list of 27 highly expressed B. cenocepacia J2315 genes coding for small proteins was compiled. For nine of them expression in biofilms was also confirmed using LC-MS based proteomics and/or expression was confirmed using eGFP translational fusions. Overexpression of two of these genes negatively impacted growth, whereas for four others overexpression led to an increase in biofilm biomass. Overexpression did not have an influence on the MIC for tobramycin, ciprofloxacin or meropenem but for five small protein encoding genes, overexpression had an effect on the number of persister cells in biofilms. While there were no significant differences in adherence to and invasion of A549 epithelial cells between the overexpression mutants and the WT, significant differences were observed in intracellular growth/survival. Finally, the small protein BCAM0271 was identified as an antitoxin belonging to a toxin-antitoxin module. The toxin was found to encode a tRNA acetylase that inhibits translation. In conclusion, our results confirm that small proteins are present in the genome of B. cenocepacia J2315 and indicate that they are involved in various biological processes, including biofilm formation, persistence and intracellular growth.SCOPUS: ar.jinfo:eu-repo/semantics/publishe

Coumarin reduces virulence and biofilm formation in Pseudomonas aeruginosa by affecting quorum sensing, type III secretion and C-di-GMP levels

As one of the major pathogens in wound infections, Pseudomonas aeruginosa produces several virulence factors and forms biofilms; these processes are under the regulation of various quorum sensing (QS) systems. Therefore, QS has been regarded as a promising target to treat P. aeruginosa infections. In the present study, we evaluated the effect of the plant-derived QS inhibitor coumarin on P. aeruginosa biofilms and virulence. Coumarin inhibited QS in the P. aeruginosa QSIS2 biosensor strain, reduced protease and pyocyanin production, and inhibited biofilm formation in microtiter plates in different P. aeruginosa strains. The effects of coumarin in inhibiting biofilm formation in an in vitro wound model and reducing P. aeruginosa virulence in the Lucilia sericata infection model were strain-dependent. Transcriptome analysis revealed that several key genes involved in the las, rhl, Pseudomonas quinolone signal (PQS), and integrated QS (IQS) systems were downregulated in coumarin-treated biofilms of P. aeruginosa PAO1. Coumarin also changed the expression of genes related to type III secretion and cyclic diguanylate (c-di-GMP) metabolism. The cellular c-di-GMP level of P. aeruginosa PAO1 and recent clinical P. aeruginosa strains was significantly reduced by coumarin. These results provide new evidence for the possible application of coumarin as an anti-biofilm and anti-virulence agent against P. aeruginosa in wound infections

Phenotypic and genotypic characterisation of Burkholderia cenocepacia J2315 mutants affected in homoserine lactone and diffusible signal factor-based quorum sensing systems suggests interplay between both types of systems

Many putative virulence factors of Burkholderia cenocepacia are controlled by various quorum sensing (QS) circuits. These QS systems either use N-acyl homoserine lactones (AHL) or cis-2-dodecenoic acid ("Burkholderia diffusible signal factor'', BDSF) as signalling molecules. Previous work suggested that there is little cross-talk between both types of systems. We constructed mutants in B. cenocepacia strain J2315, in which genes encoding CepI (BCAM1870), CciI (BCAM0239a) and the BDSF synthase (BCAM0581) were inactivated, and also constructed double (Delta cepI Delta BCAM0581, Delta cciI Delta BCAM0581 and Delta cepI Delta cciI) mutants and a triple (Delta cepI Delta cciI Delta BCAM0581) mutant. Subsequently we investigated phenotypic properties (antibiotic susceptibility, biofilm formation, production of AHL and BDSF, protease activity and virulence in Caenorhabditis elegans) and measured gene expression in these mutants, and this in the presence and absence of added BDSF, AHL or both. The triple mutant was significantly more affected in biofilm formation, antimicrobial susceptibility, virulence in C. elegans, and protease production than either the single or double mutants. The Delta BCAM0581 mutant and the Delta cepI Delta BCAM0581 and Delta cciI Delta BCAM0581 double mutants produced significantly less AHL compared to the WT strain and the Delta cepI and Delta cciI single mutant, respectively. The expression of cepI and cciI in Delta BCAM0581, was approximately 3-fold and 7-fold (p < 0.05) lower than in the WT, respectively. The observed differences in AHL production, expression of cepI and cciI and QS-controlled phenotypes in the Delta BCAM0581 mutant could (at least partially) be restored by addition of BDSF. Our data suggest that, in B. cenocepacia J2315, AHL and BDSF-based QS systems co-regulate the same set of genes, regulate different sets of genes that are involved in the same phenotypes and/or that the BDSF system controls the AHL-based QS system. As the expression of the gene encoding the C6-HSL synthase CciI (and to a lesser extent the C8-HSL synthase CepI) is partially controlled by BDSF, it seems likely that the BDSF QS systems controls AHL production through this system

Various Evolutionary Trajectories Lead to Loss of the Tobramycin-Potentiating Activity of the Quorum-Sensing Inhibitor Baicalin Hydrate in Burkholderia cenocepacia Biofilms

Combining antibiotics with potentiators that increase their activity is a promising strategy to tackle infections caused by antibiotic-resistant bacteria. As potentiators do not interfere with essential processes, it has been hypothesized that they are less likely to induce resistance. However, evidence supporting this hypothesis is lacking. In the present study, we investigated whether Burkholderia cenocepacia J2315 biofilms develop reduced susceptibility toward one such adjuvant, baicalin hydrate (BH). Biofilms were repeatedly and intermittently treated with tobramycin (TOB) alone or in combination with BH for 24 h. After treatment, the remaining cells were quantified using plate counting. After 15 cycles, biofilm cells were less susceptible to TOB and TOB + BH compared to the start population, and the potentiating effect of BH toward TOB was lost. Whole-genome sequencing was performed to probe which changes were involved in the reduced effect of BH, and mutations in 14 protein-coding genes were identified (including mutations in genes involved in central metabolism and in BCAL0296, encoding an ABC transporter). No changes in the MIC or MBC of TOB or changes in the number of persister cells were observed. However, basal intracellular levels of reactive oxygen species (ROS) and ROS levels found after treatment with TOB were markedly decreased in the evolved populations. In addition, in evolved cultures with mutations in BCAL0296, a significantly reduced uptake of TOB was observed. Our results indicate that B. cenocepacia J2315 biofilms rapidly lose susceptibility toward the antibiotic-potentiating activity of BH and point to changes in central metabolism, reduced ROS production, and reduced TOB uptake as mechanisms