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

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

Effect of shear stress on Pseudomonas aeruginosa isolated from the cystic fibrosis lung

Chronic colonization of the lungs by Pseudomonas aeruginosa is one of the major causes of morbidity and mortality in cystic fibrosis (CF) patients. To gain insights into the characteristic biofilm phenotype of P. aeruginosa in the CF lungs, mimicking the CF lung environment is critical. We previously showed that growth of the non-CF-adapted P. aeruginosa PAO1 strain in a rotating wall vessel, a device that simulates the low fluid shear (LS) conditions present in the CF lung, leads to the formation of in-suspension, self-aggregating biofilms. In the present study, we determined the phenotypic and transcriptomic changes associated with the growth of a highly adapted, transmissible P. aeruginosa CF strain in artificial sputum medium under LS conditions. Robust self-aggregating biofilms were observed only under LS conditions. Growth under LS conditions resulted in the upregulation of genes involved in stress response, alginate biosynthesis, denitrification, glycine betaine biosynthesis, glycerol metabolism, and cell shape maintenance, while genes involved in phenazine biosynthesis, type VI secretion, and multidrug efflux were downregulated. In addition, a number of small RNAs appeared to be involved in the response to shear stress. Finally, quorum sensing was found to be slightly but significantly affected by shear stress, resulting in higher production of autoinducer molecules during growth under high fluid shear (HS) conditions. In summary, our study revealed a way to modulate the behavior of a highly adapted P. aeruginosa CF strain by means of introducing shear stress, driving it from a biofilm lifestyle to a more planktonic lifestyle. IMPORTANCE: Biofilm formation by Pseudomonas aeruginosa is one of the hallmarks of chronic cystic fibrosis (CF) lung infections. The biofilm matrix protects this bacterium from antibiotics as well as from the immune system. Hence, the prevention or reversion of biofilm formation is believed to have a great impact on treatment of chronic P. aeruginosa CF lung infections. In the present study, we showed that it is possible to modulate the behavior of a highly adapted transmissible P. aeruginosa CF isolate at both the transcriptomic and phenotypic levels by introducing shear stress in a CF-like environment, driving it from a biofilm to a planktonic lifestyle. Consequently, the results obtained in this study are of great importance with regard to therapeutic applications that introduce shear stress in the lungs of CF patients

Spaceflight modulates gene expression in the whole blood of astronauts

Astronauts are exposed to a unique combination of stressors during spaceflight, which leads to alterations in their physiology and potentially increases their susceptibility to disease, including infectious diseases. To evaluate the potential impact of the spaceflight environment on the regulation of molecular pathways mediating cellular stress responses, we performed a first-of-its-kind pilot study to assess spaceflight-related gene-expression changes in the whole blood of astronauts. Using an array comprised of 234 well-characterized stress-response genes, we profiled transcriptomic changes in six astronauts (four men and two women) from blood preserved before and immediately following the spaceflight. Differentially regulated transcripts included those important for DNA repair, oxidative stress, and protein folding/degradation, including HSP90AB1, HSP27, GPX1, XRCC1, BAG-1, HHR23A, FAP48, and C-FOS. No gender-specific differences or relationship to number of missions flown was observed. This study provides a first assessment of transcriptomic changes occurring in the whole blood of astronauts in response to spaceflight

Cutibacterium acnes phylotype I and II strains interact differently with human skin cells

Acne vulgaris is one of the most common skin disorders and affects the pilosebaceous units. Although the exact pathogenesis of acne is still unknown, Cutibacterium acnes (formerly known as Propionibacterium acnes) is considered one of the key contributing factors. In fact, a significant association exists between C. acnes strains belonging to phylotype I and acne. However, there is still heavy debate on the exact role of C. acnes in acne and its behavior in the pilosebaceous unit, and more specifically its interactions with the human skin cells. In this study, key elements of the host-pathogen interaction were studied for a collection of C. acnes strains, belonging to phylotype I and II, including association with HaCaT keratinocytes and SZ95 sebocytes, the effect of C. acnes on keratinocyte tight junctions in a HaCaT monoculture and in an additional keratinocyte-sebocyte co-culture model, and C. acnes invasion through the keratinocyte cell layer. Our data showed association of all C. acnes strains to both skin cell lines, with a significantly higher association of type I strains compared to type II strains. Microscopic imaging and western blot analysis of the tight junction protein ZO-1, together with transepithelial electrical resistance (TEER) measurements revealed an initial induction of keratinocyte tight junctions after 24 h infection but a degradation after 48 h, demonstrating a decline in cell lining integrity during infection. Subsequently, C. acnes was able to invade after 48 h of infection, although invasion frequency was significantly higher for type II strains compared to type I strains

Bacterial interference with lactate dehydrogenase assay leads to an underestimation of cytotoxicity

Models to study host-pathogen interactionsin vitroare an important tool for investigating the infectious disease process and evaluating the efficacy of antimicrobial compounds. In these models, the viability of mammalian cells is often determined using the lactate dehydrogenase (LDH) cytotoxicity assay. In the present study we evaluated whether bacteria could interfere with the LDH assay. As a model for host-pathogen interactions, we co-cultured lung epithelial cells with eight bacteria encountered in the lower respiratory tract. We show that LDH activity is affected byPseudomonas aeruginosa, Klebsiella pneumoniae, Stenotrophomonas maltophilia, andStreptococcus pneumoniae, and that this depends on the density of the start inoculum and the duration of infection. Two different mechanisms were discovered through which bacteria interfered with LDH activity, i.e., acidification of the cell culture medium (byK. pneumoniaeandS. pneumoniae) and protease production (byP. aeruginosaandS. maltophilia). In addition, we developed and validated a modified protocol to evaluate cytotoxicity using the LDH assay, where bacterial interference with LDH quantification is avoided

In vitro evolution of Pseudomonas aeruginosa AA2 biofilms in the presence of cystic fibrosis lung microbiome members

In cystic fibrosis (CF) airways, the opportunistic pathogen Pseudomonas aeruginosa evolves from an acute to a chronic infection phenotype. Yet, the in vivo factors influencing the evolutionary trajectory of P. aeruginosa are poorly understood. This study aimed at understanding the role of the CF lung microbiome in P. aeruginosa evolution. Therefore, we investigated the in vitro biofilm evolution of an early CF P. aeruginosa isolate, AA2, in the presence or absence of a synthetic CF lung microbiome. Whole genome sequencing of evolved populations revealed mutations in quorum sensing (QS) genes (lasR, pqsR) with and without the microbiome. Phenotypic assays confirmed decreased production of the QS molecule 3-O-C-12-homoserine lactone, and QS-regulated virulence factors pyocyanin and protease. Furthermore, a mixture of lasR and lasR pqsR mutants was found, in which double mutants showed less pyocyanin and protease production than lasR mutants. While the microbial community did not influence the production of the tested P. aeruginosa virulence factors, we observed a trend towards more mutations in the transcriptional regulators gntR and mexL when P. aeruginosa was grown alone. P. aeruginosa developed resistance to beta-lactam antibiotics during evolution, when grown with and without the microbiome. In conclusion, in an experimental biofilm environment, the early P. aeruginosa CF isolate AA2 evolves towards a CF-like genotype and phenotype, and most studied evolutionary adaptations are not impacted by CF microbiome members

Evaluation of combination therapy for Burkholderia cenocepacia lung infection in different in vitro and in vivo models

Burkholderia cenocepacia is an opportunistic pathogen responsible for life-threatening infections in cystic fibrosis patients. B. cenocepacia is extremely resistant towards antibiotics and therapy is complicated by its ability to form biofilms. We investigated the efficacy of an alternative antimicrobial strategy for B. cenocepacia lung infections using in vitro and in vivo models. A screening of the NIH Clinical Collection 1&2 was performed against B. cenocepacia biofilms formed in 96-well microtiter plates in the presence of tobramycin to identify repurposing candidates with potentiator activity. The efficacy of selected hits was evaluated in a three-dimensional (3D) organotypic human lung epithelial cell culture model. The in vivo effect was evaluated in the invertebrate Galleria mellonella and in a murine B. cenocepacia lung infection model. The screening resulted in 60 hits that potentiated the activity of tobramycin against B. cenocepacia biofilms, including four imidazoles of which econazole and miconazole were selected for further investigation. However, a potentiator effect was not observed in the 3D organotypic human lung epithelial cell culture model. Combination treatment was also not able to increase survival of infected G. mellonella. Also in mice, there was no added value for the combination treatment. Although potentiators of tobramycin with activity against biofilms of B. cenocepacia were identified in a repurposing screen, the in vitro activity could not be confirmed nor in a more sophisticated in vitro model, neither in vivo. This stresses the importance of validating hits resulting from in vitro studies in physiologically relevant model systems

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

Efficacy against dual-species biofilms using phage-antibiotics combinations is independent of the biofilm model

Pseudomonas aeruginosa and Staphylococcus aureus are opportunistic pathogens commonly found in polymicrobial infections, namely in wounds and in respiratory tract infections. Both organisms frequently cause chronic biofilm infections and due to their antibiotic tolerance are very challenging to control. We have previously shown that the combined treatments of Pseudomonas phage EPA1 and gentamicin have increased anti-biofilm activity against mono and dual-species biofilms formed in microtiter plates. Here, we developed an innovative approach to study the efficacy of phage-antibiotic combinations in two in vivo-like models: a three-dimensional lung epithelial model that mimics aspects of the parental tissue and an artificial wound model. The efficacy of single, simultaneous and sequential treatments were compared. In the lung model, the sequential treatment of phages and gentamicin resulted in P. aeruginosa biofilm eradication. In artificial dermis, sequential treatment was also the treatment where higher reductions of culturable cells was observed in dual-species biofilms. Globally, our data suggests that the sequential phage treatment causes an adjuvant effect by lowering the MIC value of the phage-surviving population. LDH test showed that this sequential application of phages and antibiotics is not cytotoxic to lung cells. In addition, we observed that on the lung model the 3-D cell integrity was not affected by sequential treatments. We also demonstrated that the order in which phages and antibiotics are applied lead to different efficacy outcomes, showing that in clinical practice the timing to apply antibiotics will be very crucial for the success of treatment. The sequential application of phages and ciprofloxacin was shown to be safe and very efficient against dual-species biofilms formed in different models simulating different types of infection and opening new perspectives for their clinical application.info:eu-repo/semantics/publishedVersio