Tobramycin-Treated Pseudomonas aeruginosa PA14 Enhances Streptococcus constellatus 7155 Biofilm Formation in a Cystic Fibrosis Model System.

UNLABELLED: Cystic fibrosis (CF) is a human genetic disorder which results in a lung environment that is highly conducive to chronic microbial infection. Over the past decade, deep-sequencing studies have demonstrated that the CF lung can harbor a highly diverse polymicrobial community. We expanded our existing in vitro model of Pseudomonas aeruginosa biofilm formation on CF-derived airway cells to include this broader set of CF airway colonizers to investigate their contributions to CF lung disease, particularly as they relate to the antibiotic response of the population. Using this system, we identified an interspecies interaction between P. aeruginosa, a bacterium associated with declining lung function and worsening disease, and Streptococcus constellatus, a bacterium correlated with the onset of pulmonary exacerbations in CF patients. The growth rate and cytotoxicity of S. constellatus 7155 and P. aeruginosa PA14 were unchanged when grown together as mixed biofilms in the absence of antibiotics. However, the addition of tobramycin, the frontline maintenance therapy antibiotic for individuals with CF, to a mixed biofilm of S. constellatus 7155 and P. aeruginosa PA14 resulted in enhanced S. constellatus biofilm formation. Through a candidate genetic approach, we showed that P. aeruginosa rhamnolipids were reduced upon tobramycin exposure, allowing for S. constellatus 7155 biofilm enhancement, and monorhamnolipids were sufficient to reduce S. constellatus 7155 biofilm viability in the absence of tobramycin. While the findings presented here are specific to a biofilm of S. constellatus 7155 and P. aeruginosa PA14, they highlight the potential of polymicrobial interactions to impact antibiotic tolerance in unanticipated ways. IMPORTANCE: Deep-sequencing studies have demonstrated that the CF lung can harbor a diverse polymicrobial community. By recapitulating the polymicrobial communities observed in the CF lung and identifying mechanisms of interspecies interactions, we have the potential to select the best therapy for a given bacterial community and reveal potential opportunities for novel therapeutic interventions. Using an in vitro model of bacterial infection on CF airway cells, we tested how a particular polymicrobial community grows, damages human cells, and responds to antibiotics in single and mixed infections. We describe here the mechanism of an interspecies interaction between two pathogens in the CF lung, P. aeruginosa and S. constellatus, which is potentiated by a commonly prescribed antibiotic, tobramycin.

Analysis of the Relationship Between Micrognathia and Cleft Palate: A Systematic Review.

Objective To gather data from relevant experimental and observational studies to determine the relationship between micrognathia and cleft palate. The goal is to raise awareness and motivate clinicians to consider the cause and effect relationship when confronted with patients with cleft palate, even if there is no clearly noticeable mandibular abnormality. Design Several electronic databases were systematically examined to find articles for this review, using search terms including "cleft palate," "micrognathia," "tongue," and "airway obstruction." PubMed was the source of all the articles chosen to be included. Exclusion criteria included case reports, articles focused on treatment options, and articles only tangentially related to cleft palate and/or micrognathia. Results A total of 930 articles were screened for relevance, and 82 articles were chosen for further analysis. Evidence gathered in this review includes a variety of etiological factors that are causative or associated with both micrognathia and cleft palate. Observational studies relating the two abnormalities are also included. Much of the included literature recognizes a cause-and-effect relationship between micrognathia and cleft palate. Conclusion On the basis of the published data, we suggest that micrognathia does induce cleft palate in humans and animals. With knowledge of this causative relationship, clinicians should consider the importance of gathering cephalometric data on the mandibles and tongues of patients presenting with isolated cleft palate to determine whether they have micrognathia as well. With more data, patterns may emerge that could give insight into the complex etiology of nonsyndromic cleft palate.

Mannitol Does Not Enhance Tobramycin Killing of Pseudomonas aeruginosa in a Cystic Fibrosis Model System of Biofilm Formation.

Cystic Fibrosis (CF) is a human genetic disease that results in the accumulation of thick, sticky mucus in the airways, which results in chronic, life-long bacterial biofilm infections that are difficult to clear with antibiotics. Pseudomonas aeruginosa lung infection is correlated with worsening lung disease and P. aeruginosa transitions to an antibiotic tolerant state during chronic infections. Tobramycin is an aminoglycoside currently used to combat lung infections in individuals with CF. While tobramycin is effective at eradicating P. aeruginosa in the airways of young patients, it is unable to completely clear the chronic P. aeruginosa infections in older patients. A recent report showed that co-addition of tobramycin and mannitol enhanced killing of P. aeruginosa grown in vitro as a biofilm on an abiotic surface. Here we employed a model system of bacterial biofilms formed on the surface of CF-derived airway cells to determine if mannitol would enhance the antibacterial activity of tobramycin against P. aeruginosa grown on a more clinically relevant surface. Using this model system, which allows the growth of robust biofilms with high-level antibiotic tolerance analogous to in vivo biofilms, we were unable to find evidence for enhanced antibacterial activity of tobramycin with the addition of mannitol, supporting the observation that this type of co-treatment failed to reduce the P. aeruginosa bacterial load in a clinical setting.

Iron supplementation does not worsen respiratory health or alter the sputum microbiome in cystic fibrosis.

BACKGROUND: Iron supplementation for hypoferremic anemia could potentiate bacterial growth in the cystic fibrosis (CF) lung, but clinical trials testing this hypothesis are lacking. METHODS: Twenty-two adults with CF and hypoferremic anemia participated in a randomized, double-blind, placebo-controlled, crossover trial of ferrous sulfate 325mg daily for 6weeks. Iron-related hematologic parameters, anthropometric data, sputum iron, Akron Pulmonary Exacerbation Score (PES), and the sputum microbiome were serially assessed. Fixed-effect models were used to describe how ferrous sulfate affected these variables. RESULTS: Ferrous sulfate increased serum iron by 22.3% and transferrin saturation (TSAT) by 26.8% from baseline (p<0.05) but did not affect hemoglobin, sputum iron, Akron PES, and the sputum microbiome. CONCLUSIONS: Low-dose ferrous sulfate improved hypoferremia without correcting anemia after 6weeks. We did not observe significant effects on sputum iron, Akron PES, and the sputum microbiome. Although we did not identify untoward health effects of iron supplementation, a larger blinded randomized controlled trial would be needed to fully demonstrate safety.

Predator odor-evoked BOLD activation in the awake rat: modulation by oxytocin and V1a vasopressin receptor antagonists.

Modulators of unconditioned fear are potential targets for developing treatments for anxiety disorders. We used blood oxygen level dependent (BOLD) MRI to investigate the pattern of brain activity during the presentation of a predator odor (cat fur) and a repulsive novel odor, butyric acid (BA), to awake rats. We further tested whether odor-evoked BOLD activation involved oxytocin (OT) and vasopressin V(1a) receptors. Animals were subdivided into groups either administered an intracerebroventricular injection of artificial cerebrospinal fluid (CSF), an OT receptor antagonist or a V(1a) antagonist (125 ng/10 µL each) 90 min before studies. BA odor evoked robust brain activation across olfactory, sensory, memory and limbic regions. The magnitude of BOLD activation across these regions was greater for BA than with cat fur. However, blockade of OT and V(1a) receptors differentially modulated odor evoked neural activity, particularly in the amygdala. OT and V(1a) antagonism preferentially modulated BOLD responding to BA in the cortical amygdala. While, OT and V(1a) antagonisms preferentially modulated BOLD responding to cat fur in the central amygdala. The data suggest that although OT receptors modulate BOLD activation in response to a novel and repulsive odor such as BA, vasopressin V(1a) receptors exert a modulatory influence on the neural response to a predator odor.

Unique microbial communities persist in individual cystic fibrosis patients throughout a clinical exacerbation.

BACKGROUND: Cystic fibrosis (CF) is caused by inherited mutations in the cystic fibrosis transmembrane conductance regulator gene and results in a lung environment that is highly conducive to polymicrobial infection. Over a lifetime, decreasing bacterial diversity and the presence of Pseudomonas aeruginosa in the lung are correlated with worsening lung disease. However, to date, no change in community diversity, overall microbial load or individual microbes has been shown to correlate with the onset of an acute exacerbation in CF patients. We followed 17 adult CF patients throughout the course of clinical exacerbation, treatment and recovery, using deep sequencing and quantitative PCR to characterize spontaneously expectorated sputum samples RESULTS: We identified approximately 170 bacterial genera, 12 of which accounted for over 90% of the total bacterial load across all patient samples. Genera abundant in any single patient sample tended to be detectable in most samples. We found that clinical stages could not be distinguished by absolute Pseudomonas aeruginosa load, absolute total bacterial load or the relative abundance of any individual genus detected, or community diversity. Instead, we found that the microbial structure of each patient's sputum microbiome was distinct and resilient to exacerbation and antibiotic treatment. CONCLUSION: Consistent with previously reported sputum microbiome studies we found that total and relative abundance of genera at the population level were remarkably stable for individual patients regardless of clinical status. Patient-by-patient analysis of diversity and relative abundance of each individual genus revealed a complex microbial landscape and highlighted the difficulty of identifying a universal microbial signature of exacerbation. Overall, at the genus level, we find no evidence of a microbial signature of clinical stage.

Export requirements of pneumolysin in Streptococcus pneumoniae.

Streptococcus pneumoniae is a major causative agent of otitis media, pneumonia, bacteremia, and meningitis. Pneumolysin (Ply), a member of the cholesterol-dependent cytolysins (CDCs), is produced by virtually all clinical isolates of S. pneumoniae, and ply mutant strains are severely attenuated in mouse models of colonization and infection. In contrast to all other known members of the CDC family, Ply lacks a signal peptide for export outside the cell. Instead, Ply has been hypothesized to be released upon autolysis or, alternatively, via a nonautolytic mechanism that remains undefined. We show that an exogenously added signal sequence is not sufficient for Sec-dependent Ply secretion in S. pneumoniae but is sufficient in the surrogate host Bacillus subtilis. Previously, we showed that Ply is localized primarily to the cell wall compartment in the absence of detectable cell lysis. Here we show that Ply released by autolysis cannot reassociate with intact cells, suggesting that there is a Ply export mechanism that is coupled to cell wall localization of the protein. This putative export mechanism is capable of secreting a related CDC without its signal sequence. We show that B. subtilis can export Ply, suggesting that the export pathway is conserved. Finally, through truncation and domain swapping analyses, we show that export is dependent on domain 2 of Ply.

Pneumolysin localizes to the cell wall of Streptococcus pneumoniae.

Streptococcus pneumoniae is the causative agent of multiple diseases, including otitis media, pneumonia, bacteremia, and meningitis. Pneumolysin (Ply), a member of the cholesterol-dependent cytolytic pore-forming toxins, is produced by virtually all clinical isolates of S. pneumoniae, and strains in which the Ply gene has been deleted are severely attenuated in mouse models of infection. In contrast to all other members of the cholesterol-dependent cytolysin family, Ply lacks a signal peptide for export. Instead, Ply has been hypothesized to be released upon autolysis or, alternatively, via a nonautolytic mechanism that remains ill defined. We determined by use of cell fractionation and Western blotting that, during in vitro growth, exported Ply is localized primarily to the cell wall compartment in 18 different serotypes in the absence of detectable cell lysis. Hemolytic assays revealed that this cell wall-localized Ply is active. Additionally, cell wall-localized Ply is accessible to extracellular protease and is detergent releasable.