Social interactions in the Burkholderia cepacia complex : biofilms and quorum sensing

Burkholderia cepacia complex bacteria are opportunistic pathogens that cause respiratory tract infections in susceptible patients, mainly people with cystic fibrosis. There is convincing evidence that B. cepacia complex bacteria can form biofilms, not only on abiotic surfaces (e.g., glass and plastics), but also on biotic surfaces such as epithelial cells, leading to the suggestion that biofilm formation plays a key role in persistent infection of cystic fibrosis lungs. This article presents an overview of the molecular mechanisms involved in B. cepacla complex biofilm formation, the increased resistance of sessile B. cepacia complex cells and the role of quorum sensing in B. cepacia complex biofilm formation

Effect of β-lactamase inhibitors on in vitro activity of β-lactam antibiotics against Burkholderia cepacia complex species

Background: Bacteria belonging to the Burkholderia cepacia complex (Bcc) are an important cause of chronic respiratory tract infections in cystic fibrosis patients. Intrinsic resistance to a wide range of antimicrobial agents, including a variety of beta-lactam antibiotics, is frequently observed in Bcc strains. Resistance to beta-lactams is most commonly mediated by efflux pumps, alterations in penicillin-binding proteins or the expression of beta-lactamases. beta-lactamase inhibitors are able to restore the in vitro activity of beta-lactam molecules against a variety of Gram-negative species, but the effect of these inhibitors on the activity of beta-lactam treatment against Bcc species is still poorly investigated. Methods: In the present study, the susceptibility of a panel of Bcc strains was determined towards the beta-lactam antibiotics ceftazidime, meropenem, amoxicillin, cefoxitin, cefepime and aztreonam; alone or in combination with a beta-lactamase inhibitor (clavulanic acid, sulbactam, tazobactam and avibactam). Consequently, beta-lactamase activity was determined for active beta-lactam/beta-lactamase inhibitor combinations. Results: Clavulanic acid had no effect on minimum inhibitory concentrations, but addition of sulbactam, tazobactam or avibactam to ceftazidime, amoxicillin, cefoxitin, cefepime or aztreonam leads to increased susceptibility (at least 4-fold MIC-decrease) in some Bcc strains. The effect of beta-lactamase inhibitors on beta-lactamase activity is both strain-and/or antibiotic-dependent, and other mechanisms of beta-lactam resistance (besides production of beta-lactamases) appear to be important. Conclusions: Considerable differences in susceptibility of Bcc strains to beta-lactam antibiotics were observed. Results obtained in the present study suggest that resistance of Bcc strains against beta-lactam antibiotics is mediated by both beta-lactamases and non-beta-lactamase-mediated resistance mechanisms

Developing selective media for quantification of multispecies biofilms following antibiotic treatment

The lungs of cystic fibrosis (CF) patients are chronically colonized by a polymicrobial biofilm community, leading to difficult-to-treat infections. To combat these infections, CF patients are commonly treated with a variety of antibiotics. Understanding the dynamics of polymicrobial community composition in response to antibiotic therapy is essential in the search for novel therapies. Culture-dependent quantification of individual bacteria from defined multi-species biofilms is frequently carried out by plating on selective media. However, the influence of the selective agents in these media on quantitative recovery before or after antibiotic treatment is often unknown. In the present study we developed selective media for six bacterial species that are frequently co-isolated from the CF lung, i.e. Pseudomonas aeruginosa, Staphylococcus aureus, Streptococcus anginosus, Achromobacter xylosoxidans, Rothia mucilaginosa, and Gemella haemolysans. We show that certain supplementations to selective media strongly influence quantitative recovery of (un) treated biofilms. Hence, the developed media were optimized for selectivity and quantitative recovery before or after treatment with antibiotics of four major classes, i.e. ceftazidime, ciprofloxacin, colistin, or tobramycin. Finally, in a proof of concept experiment the novel selective media were applied to determine the community composition of multispecies biofilms before and after treatment with tobramycin

In vitro activity of ceftazidime, ciprofloxacin, meropenem, minocycline, tobramycin and trimethoprim/sulfamethoxazole against planktonic and sessile Burkholderia cepacia complex bacteria

Objectives: The goal of the present study was to obtain a comprehensive overview of the bacteriostatic and bactericidal effects of six commonly used antibiotics on planktonic as well as on sessile Burkholderia cepacia complex cells. Methods: The bacteriostatic and bactericidal activities of ceftazidime, ciprofloxacin, meropenem, minocycline, tobramycin and trimethoprim/sulfamethoxazole were determined against 38 B. cepacia complex strains. MICs and minimal biofilm inhibitory concentrations (MBICs) were determined using a traditional broth microdilution method and a novel resazurin-based viability staining, respectively. The bactericidal effects of the investigated antibiotics (using antibiotic concentrations corresponding to 10 x MIC; except for tobramycin, for which a final concentration of 4 x MIC was tested) on stationary phase planktonic cultures and on 24-h-old biofilms were evaluated using conventional plate count methods. Results: Our results confirm the innate resistance of B. cepacia complex organisms to six first-line antibiotics used to treat infected cystic fibrosis patients. All antibiotics showed similar bacteriostatic activities against exponentially growing B. cepacia complex planktonic cells and freshly adhered sessile cells (4 h). In addition, most of the antibiotics showed similar bactericidal effects on stationary phase planktonic cultures and on young and older biofilms. Conclusions: Despite the general assumption that sessile cells show a decreased susceptibility to antibiotics, our data indicate similar bacteriostatic and bactericidal activity of six selected antibiotics against planktonic and sessile B. cepacia complex bacteria

Identification of small RNAs abundant in Burkholderia cenocepacia biofilms reveal putative regulators with a potential role in carbon and iron metabolism

Small RNAs play a regulatory role in many central metabolic processes of bacteria, as well as in developmental processes such as biofilm formation. Small RNAs of Burkholderia cenocepacia, an opportunistic pathogenic beta-proteobacterium, are to date not well characterised. To address that, we performed genome-wide transcriptome structure analysis of biofilm grown B. cenocepacia J2315. 41 unannotated short transcripts were identified in intergenic regions of the B. cenocepacia genome. 15 of these short transcripts, highly abundant in biofilms, widely conserved in Burkholderia sp. and without known function, were selected for in-depth analysis. Expression profiling showed that most of these sRNAs are more abundant in biofilms than in planktonic cultures. Many are also highly abundant in cells grown in minimal media, suggesting they are involved in adaptation to nutrient limitation and growth arrest. Their computationally predicted targets include a high proportion of genes involved in carbon metabolism. Expression and target genes of one sRNA suggest a potential role in regulating iron homoeostasis. The strategy used for this study to detect sRNAs expressed in B. cenocepacia biofilms has successfully identified sRNAs with a regulatory function

Detection and quantification of viable airborne bacteria and fungi using solid-phase cytometry

This protocol describes the use of solid-phase cytometry for the enumeration of airborne bacteria and fungi. In contrast with conventional methods, accurate results can be obtained in real time, especially for air samples with low numbers of microorganisms. Air samples are collected by impaction on a water-soluble polymer that is subsequently dissolved. Part of the sample can be filtered over two membrane filters with different pore sizes. One filter is used to obtain a total count of all viable microorganisms, and a second filter is used to determine the number of airborne fungi. Microorganisms present on the filter are labeled with a viability substrate and subsequently detected and quantified using a solid-phase cytometer. The detected spots are microscopically validated using an epifluorescence microscope to discriminate between bacteria, fungi and fluorescent particles. The whole procedure takes 5 h to complete and results in the accurate quantification of airborne bacteria and fungi for samples with a low or high microbial load

Response of sessile cells to stress : from changes in gene expression to phenotypic adaptation

A better understanding of the genotypic and phenotypic adaptation of sessile (biofilm-associated) microorganisms to various forms of stress is required in order to develop more effective antibiofilm strategies. This review presents an overview of what high-throughput transcriptomic analyses have taught us concerning the response of various clinically relevant microorganisms (including Pseudomonas aeruginosa, Burkholderia cenocepacia and Candida albicans) to treatment with antibiotics or disinfectants. In addition, several problems associated with identifying gene expression patterns in biofilms in general and their implications for identifying the response to stress are discussed (with a focus on heterogeneity in microbial biofilms and the role of small RNAs in microbial group behavior)

Influence of the lung microbiome on antibiotic susceptibility of cystic fibrosis pathogens

The lungs of patients with cystic fibrosis (CF) are colonised by a microbial community comprised of pathogenic species, such as Pseudomonas aeruginosa and Staphylococcus aureus, and microorganisms that are typically not associated with worse clinical outcomes (considered as commensals). Antibiotics directed at CF pathogens are often not effective and a discrepancy is observed between activity of these agents in vitro and in the patient. This review describes how interspecies interactions within the lung microbiome might influence the outcome of antibiotic treatment targeted at common CF pathogens. Protective mechanisms by members of the microbiome such as antibiotic degradation (indirect pathogenicity), alterations of the cell wall, production of matrix components decreasing antibiotic penetration, and changes in metabolism are discussed. Interspecies interactions that increase bacterial susceptibility are also addressed. Furthermore, we discuss how experimental conditions, such as culture media, oxygen levels, incorporation of host-pathogen interactions, and microbial community composition may influence the outcome of microbial interaction studies related to antibiotic activity. Hereby, the importance to create in vitro conditions reflective of the CF lung microenvironment is highlighted. Understanding the role of the CF lung microbiome in antibiotic efficacy may help find novel therapeutic and diagnostic approaches to better tackle chronic lung infections in this patient population