R othia mucilaginosa is an anti-inflammatory bacterium in the respiratory tract of patients with chronic lung disease.

BACKGROUND: Chronic airway inflammation is the main driver of pathogenesis in respiratory diseases such as severe asthma, chronic obstructive pulmonary disease, cystic fibrosis (CF) and bronchiectasis. While the role of common pathogens in airway inflammation is widely recognised, the influence of other microbiota members is still poorly understood. METHODS: We hypothesised that the lung microbiota contains bacteria with immunomodulatory activity which modulate net levels of immune activation by key respiratory pathogens. Therefore, we assessed the immunomodulatory effect of several members of the lung microbiota frequently reported as present in CF lower respiratory tract samples. RESULTS: We show that Rothia mucilaginosa, a common resident of the oral cavity that is also often detectable in the lower airways in chronic disease, has an inhibitory effect on pathogen- or lipopolysaccharide-induced pro-inflammatory responses, in vitro (three-dimensional cell culture model) and in vivo (mouse model). Furthermore, in a cohort of adults with bronchiectasis, the abundance of Rothia species was negatively correlated with pro-inflammatory markers (interleukin (IL)-8 and IL-1ß) and matrix metalloproteinase (MMP)-1, MMP-8 and MMP-9 in sputum. Mechanistic studies revealed that R. mucilaginosa inhibits NF-κB pathway activation by reducing the phosphorylation of IκBα and consequently the expression of NF-κB target genes. CONCLUSIONS: These findings indicate that the presence of R. mucilaginosa in the lower airways potentially mitigates inflammation, which could in turn influence the severity and progression of chronic respiratory disorders.

Bacterial Interference With Lactate Dehydrogenase Assay Leads to an Underestimation of Cytotoxicity.

Models to study host-pathogen interactions in vitro are 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 by Pseudomonas aeruginosa, Klebsiella pneumoniae, Stenotrophomonas maltophilia, and Streptococcus 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 (by K. pneumoniae and S. pneumoniae) and protease production (by P. aeruginosa and S. 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.

Antibiotic susceptibility of cystic fibrosis lung microbiome members in a multispecies biofilm.

The lungs of cystic fibrosis (CF) patients are often chronically colonized by multiple microbial species that can form biofilms, including the major CF pathogen Pseudomonas aeruginosa. Herewith, lower microbial diversity in CF airways is typically associated with worse health outcomes. In an attempt to treat CF lung infections patients are frequently exposed to antibiotics, which may affect microbial diversity. This study aimed at understanding if common antibiotics that target P. aeruginosa influence microbial diversity. To this end, a microaerophilic multispecies biofilm model of frequently co-isolated members of the CF lung microbiome (Pseudomonas aeruginosa, Staphylococcus aureus, Streptococcus anginosus, Achromobacter xylosoxidans, Rothia mucilaginosa, and Gemella haemolysans) was exposed to antipseudomonal antibiotics. We found that antibiotics that affected several dominant species (i.e. ceftazidime, tobramycin) resulted in higher species evenness compared to colistin, which is only active against P. aeruginosa. Furthermore, susceptibility of individual species in the multispecies biofilm following antibiotic treatment was compared to that of the respective single-species biofilms, showing no differences. Adding three anaerobic species (Prevotella melaninogenica, Veillonella parvula, and Fusobacterium nucleatum) to the multispecies biofilm did not influence antibiotic susceptibility. In conclusion, our study demonstrates antibiotic-dependent effects on microbial community diversity of multispecies biofilms comprised of CF microbiome members.

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-C12-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 ß-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.

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.

Influence of three-dimensional lung epithelial cells and interspecies interactions on antibiotic efficacy against Mycobacterium abscessus and Pseudomonas aeruginosa.

Mycobacterium abscessus lung infection is a major health problem for cystic fibrosis (CF) patients. Understanding the in vivo factors that influence the outcome of therapy may help addressing the poor correlation between in vitro and in vivo antibiotic efficacy. We evaluated the influence of interspecies interactions and lung epithelial cells on antibiotic efficacy. Therefore, single and dual-species biofilms of M. abscessus and a major CF pathogen (Pseudomonas aeruginosa) were cultured on a plastic surface or on in vivo-like three-dimensional (3-D) lung epithelial cells, and the activity of antibiotics (colistin, amikacin, clarithromycin, ceftazidime) in inhibiting biofilm formation was evaluated. Using the most physiologically relevant model (dual-species biofilms on 3-D cells), we observed that treatment with antibiotics during biofilm development inhibited P. aeruginosa but not M. abscessus biofilms, resulting in a competitive advantage for the latter. Clarithromycin efficacy against P. aeruginosa was inhibited by 3-D lung cells. In addition, biofilm induction of M. abscessus was observed by certain antibiotics on plastic but not on 3-D cells. Pseudomonas aeruginosa influenced the efficacy of certain antibiotics against M. abscessus, but not vice versa. In conclusion, these results suggest a role of host cells and interspecies interactions in bacterial responses to antimicrobials.

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 multispecies 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.