Successful Use of Phage and Antibiotics Therapy for the Eradication of Two Bacterial Pathogens from the Respiratory Tract of an Infant.

Phage therapy can be a useful approach in a number of clinical cases associated with multidrug-resistant (MDR) bacterial infections. In this study, we describe a successful consecutive phage and antibiotic application to cure a 3-month-old girl suffering from severe bronchitis after tracheostomy. Bronchitis was associated with two bacterial agents, MDR Pseudomonas aeruginosa and a rare opportunistic pathogen Dolosigranulum pigrum. The phage cocktail "Pyobacteriophage" containing at least two different phages against isolated MDR P. aeruginosa strain was used via inhalation and nasal drops. Topical application of the phage cocktail removed most of P. aeruginosa cells and contributed to a change in the antimicrobial resistance profile of surviving P. aeruginosa cells. As a result, it became possible to choose and administer an appropriate antibiotic that was effective against both infectious agents. Complete recovery of the infant was recorded.

Native mitral valve infective endocarditis due to Dolosigranulum pigrum.

Dolosigranulum pigrum is an anaerobic, gram-positive coccus rarely identified as a pathogenic organism. However, case reports have suggested D. pigrum as a causative pathogen in bacteremia, nosocomial pneumonia, ventilator-associated pneumonia, synovitis, cholecystitis, and ophthalmologic infections. Herein, we present the first case of Dolosigranulum pigrum causing native mitral valve infective endocarditis. With the exception of erythromycin, the isolate displayed favorable minimum inhibitory concentrations (MIC) to all other antibiotics tested, including beta-lactams, levofloxacin, and vancomycin. The patient was successfully treated with a 6-week course of intravenous (IV) ceftriaxone followed by robotically assisted cardiac valve repair.

The role of respiratory microbiota in the protection against viral diseases: respiratory commensal bacteria as next-generation probiotics for COVID-19.

On March 11, 2020, the World Health Organization declared a pandemic of coronavirus infectious disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and imposed the biggest public health challenge for our civilization, with unforeseen impacts in the subsequent years. Similar to other respiratory infections, COVID-19 is associated with significant changes in the composition of the upper respiratory tract microbiome. Studies have pointed to a significant reduction of diversity and richness of the respiratory microbiota in COVID-19 patients. Furthermore, it has been suggested that Prevotella, Staphylococcus, and Streptococcus are associated with severe COVID-19 cases, while Dolosigranulum and Corynebacterium are significantly more abundant in asymptomatic subjects or with mild disease. These results have stimulated the search for new microorganisms from the respiratory microbiota with probiotic properties that could alleviate symptoms and even help in the fight against COVID-19. To date, the potential positive effects of probiotics in the context of SARS-CoV-2 infection and COVID-19 pandemics have been extrapolated from studies carried out with other viral pathogens, such as influenza virus and respiratory syncytial virus. However, scientific evidence has started to emerge demonstrating the capacity of immunomodulatory bacteria to beneficially influence the resistance against SARS-CoV-2 infection. Here we review the scientific knowledge regarding the role of the respiratory microbiota in viral infections in general and in the infection caused by SARS-CoV-2 in particular. In addition, the scientific work that supports the use of immunomodulatory probiotic microorganisms as beneficial tools to reduce the severity of respiratory viral infections is also reviewed. In particular, our recent studies that evaluated the role of immunomodulatory Dolosigranulum pigrum strains in the context of SARS-CoV-2 infection are highlighted.

Dolosigranulum pigrum en absceso corneal / Dolosigranulum pigrum in corneal abscess

Resumen Dolosigranulum pigrum es un coco gram positivo, anaerobio facultativo, que forma parte de la microbiota oral y del tracto respiratorio superior. Aunque los reportes de infecciones por este microorganismo son escasos, se lo ha asociado a un amplio espectro de enfermedades infecciosas. Se describe el caso de un hombre adulto con un absceso corneal del que se aisló D. pigrum. El microorganismo fue identificado por espectrometría de masas (MALDI-TOF MS) y secuenciación del gen 16S ARNr. A su vez, se logró la identificación presuntiva mediante pruebas fenotípicas claves, como la disposición en racimos en la coloración de Gram, la prueba negativa de la catalasa, la producción de pirrolidonil arilamidasa y leucina aminopeptidasa, el crecimiento en NaCl al 6,5% y la hidrólisis de esculina. Los datos de la literatura y el presente caso respaldan la asociación del microorganismo con infecciones oculares, a menudo de curso destructivo, principalmente en pacientes de edad avanzada.

Abstract Dolosigranulum pigrum is a gram-positive, facultatively anaerobic coccus, which is part of the oral and upper respiratory tract microbiota. Although reports of infections by this microorganism are scarce, it has been associated with a wide spectrum of infectious diseases. The case of an elderly man with a lower corneal abscess, in which Dolosigranulum pigrum was isolated, is described. The microorganism was identified by mass spectrometry (MALDI-TOF MS) and by the sequencingof the 16S rRNAgene. Furthermore, the presumptive identification of the causative agent was achieved by using key phenotypic tests such as the cluster arrangement in Gram stain, the negative catalase test, the production of pyrrolidonyl arylamidase and leucine aminopeptidase activity, the growth in 6.5% NaCl and esculin hydrolysis. The data from the literature (and the present case) support the association of the microorganism with ocular infections, which often take a destructive course, mainly in elderly patients.

[Dolosigranulum pigrum in corneal abscess]. / Dolosigranulum pigrum en absceso corneal.

Dolosigranulum pigrum is a gram-positive, facultatively anaerobic coccus, which is part of the oral and upper respiratory tract microbiota. Although reports of infections by this microorganism are scarce, it has been associated with a wide spectrum of infectious diseases. The case of an elderly man with a lower corneal abscess, in which Dolosigranulum pigrum was isolated, is described. The microorganism was identified by mass spectrometry (MALDI-TOF MS) and by the sequencing of the 16S rRNA gene. Furthermore, the presumptive identification of the causative agent was achieved by using key phenotypic tests such as the cluster arrangement in Gram stain, the negative catalase test, the production of pyrrolidonyl arylamidase and leucine aminopeptidase activity, the growth in 6.5% NaCl and esculin hydrolysis. The data from the literature (and the present case) support the association of the microorganism with ocular infections, which often take a destructive course, mainly in elderly patients.

Towards the human nasal microbiome: Simulating D. pigrum and S. aureus.

The human nose harbors various microbes that decisively influence the wellbeing and health of their host. Among the most threatening pathogens in this habitat is Staphylococcus aureus. Multiple epidemiological studies identify Dolosigranulum pigrum as a likely beneficial bacterium based on its positive association with health, including negative associations with S. aureus. Carefully curated GEMs are available for both bacterial species that reliably simulate their growth behavior in isolation. To unravel the mutual effects among bacteria, building community models for simulating co-culture growth is necessary. However, modeling microbial communities remains challenging. This article illustrates how applying the NCMW fosters our understanding of two microbes' joint growth conditions in the nasal habitat and their intricate interplay from a metabolic modeling perspective. The resulting community model combines the latest available curated GEMs of D. pigrum and S. aureus. This uses case illustrates how to incorporate genuine GEM of participating microorganisms and creates a basic community model mimicking the human nasal environment. Our analysis supports the role of negative microbe-microbe interactions involving D. pigrum examined experimentally in the lab. By this, we identify and characterize metabolic exchange factors involved in a specific interaction between D. pigrum and S. aureus as an in silico candidate factor for a deep insight into the associated species. This method may serve as a blueprint for developing more complex microbial interaction models. Its direct application suggests new ways to prevent disease-causing infections by inhibiting the growth of pathogens such as S. aureus through microbe-microbe interactions.

Genomic Stability and Genetic Defense Systems in Dolosigranulum pigrum, a Candidate Beneficial Bacterium from the Human Microbiome.

Dolosigranulum pigrum is positively associated with indicators of health in multiple epidemiological studies of human nasal microbiota. Knowledge of the basic biology of D. pigrum is a prerequisite for evaluating its potential for future therapeutic use; however, such data are very limited. To gain insight into D. pigrum's chromosomal structure, pangenome, and genomic stability, we compared the genomes of 28 D. pigrum strains that were collected across 20 years. Phylogenomic analysis showed closely related strains circulating over this period and closure of 19 genomes revealed highly conserved chromosomal synteny. Gene clusters involved in the mobilome and in defense against mobile genetic elements (MGEs) were enriched in the accessory genome versus the core genome. A systematic analysis for MGEs identified the first candidate D. pigrum prophage and insertion sequence. A systematic analysis for genetic elements that limit the spread of MGEs, including restriction modification (RM), CRISPR-Cas, and deity-named defense systems, revealed strain-level diversity in host defense systems that localized to specific genomic sites, including one RM system hot spot. Analysis of CRISPR spacers pointed to a wealth of MGEs against which D. pigrum defends itself. These results reveal a role for horizontal gene transfer and mobile genetic elements in strain diversification while highlighting that in D. pigrum this occurs within the context of a highly stable chromosomal organization protected by a variety of defense mechanisms. IMPORTANCE Dolosigranulum pigrum is a candidate beneficial bacterium with potential for future therapeutic use. This is based on its positive associations with characteristics of health in multiple studies of human nasal microbiota across the span of human life. For example, high levels of D. pigrum nasal colonization in adults predicts the absence of Staphylococcus aureus nasal colonization. Also, D. pigrum nasal colonization in young children is associated with healthy control groups in studies of middle ear infections. Our analysis of 28 genomes revealed a remarkable stability of D. pigrum strains colonizing people in the United States across a 20-year span. We subsequently identified factors that can influence this stability, including genomic stability, phage predators, the role of MGEs in strain-level variation, and defenses against MGEs. Finally, these D. pigrum strains also lacked predicted virulence factors. Overall, these findings add additional support to the potential for D. pigrum as a therapeutic bacterium.

The Respiratory Commensal Bacterium Dolosigranulum pigrum 040417 Improves the Innate Immune Response to Streptococcus pneumoniae.

Previously, we demonstrated that the nasal administration of Dolosigranulum pigrum 040417 differentially modulated the respiratory innate immune response triggered by the activation of Toll-like receptor 2 in infant mice. In this work, we aimed to evaluate the beneficial effects of D. pigrum 040417 in the context of Streptococcus pneumoniae infection and characterize the role of alveolar macrophages (AMs) in the immunomodulatory properties of this respiratory commensal bacterium. The nasal administration of D. pigrum 040417 to infant mice significantly increased their resistance to pneumococcal infection, differentially modulated respiratory cytokines production, and reduced lung injuries. These effects were associated to the ability of the 040417 strain to modulate AMs function. Depletion of AMs significantly reduced the capacity of the 040417 strain to improve both the reduction of pathogen loads and the protection against lung tissue damage. We also demonstrated that the immunomodulatory properties of D. pigrum are strain-specific, as D. pigrum 030918 was not able to modulate respiratory immunity or to increase the resistance of mice to an S. pneumoniae infection. These findings enhanced our knowledge regarding the immunological mechanisms involved in modulation of respiratory immunity induced by beneficial respiratory commensal bacteria and suggested that particular strains could be used as next-generation probiotics.

Dolosigranulum pigrum Modulates Immunity against SARS-CoV-2 in Respiratory Epithelial Cells.

In a previous work, we demonstrated that nasally administered Dolosigranulum pigrum 040417 beneficially modulated the respiratory innate immune response triggered by the activation of Toll-like receptor 3 (TLR3) and improved protection against Respiratory Syncytial Virus (RSV) in mice. In this work, we aimed to evaluate the immunomodulatory effects of D. pigrum 040417 in human respiratory epithelial cells and the potential ability of this immunobiotic bacterium to increase the protection against Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). The respiratory commensal bacterium D. pigrum 040417 differentially modulated the production of IFN-ß, IL-6, CXCL8, CCL5 and CXCL10 in the culture supernatants of Calu-3 cells stimulated with poly(I:C) or challenged with SARS-CoV-2. The differential cytokine profile induced by the 040417 strain was associated with a significant reduction in viral replication and cellular damage after coronavirus infection. Of note, D. pigrum 030918 was not able to modify the resistance of Calu-3 cells to SARS-CoV-2 infection, indicating a strain-specific immunomodulatory effect for respiratory commensal bacteria. The findings of this work improve our understanding of the immunological mechanisms involved in the modulation of respiratory immunity induced by respiratory commensal bacteria, by demonstrating their specific effect on respiratory epithelial cells. In addition, the results suggest that particular strains such as D. pigrum 040417 could be used as a promising alternative for combating SARS-CoV-2 and reducing the severity of COVID-19.

First Genome-Scale Metabolic Model of Dolosigranulum pigrum Confirms Multiple Auxotrophies.

Dolosigranulum pigrum is a quite recently discovered Gram-positive coccus. It has gained increasing attention due to its negative correlation with Staphylococcus aureus, which is one of the most successful modern pathogens causing severe infections with tremendous morbidity and mortality due to its multiple resistances. As the possible mechanisms behind its inhibition of S. aureus remain unclear, a genome-scale metabolic model (GEM) is of enormous interest and high importance to better study its role in this fight. This article presents the first GEM of D. pigrum, which was curated using automated reconstruction tools and extensive manual curation steps to yield a high-quality GEM. It was evaluated and validated using all currently available experimental data of D. pigrum. With this model, already predicted auxotrophies and biosynthetic pathways could be verified. The model was used to define a minimal medium for further laboratory experiments and to predict various carbon sources' growth capacities. This model will pave the way to better understand D. pigrum's role in the fight against S. aureus.

Dolosigranulum pigrum Cooperation and Competition in Human Nasal Microbiota.

Multiple epidemiological studies identify Dolosigranulum pigrum as a candidate beneficial bacterium based on its positive association with health, including negative associations with nasal/nasopharyngeal colonization by the pathogenic species Staphylococcus aureus and Streptococcus pneumoniae Using a multipronged approach to gain new insights into D. pigrum function, we observed phenotypic interactions and predictions of genomic capacity that support the idea of a role for microbe-microbe interactions involving D. pigrum in shaping the composition of human nasal microbiota. We identified in vivo community-level and in vitro phenotypic cooperation by specific nasal Corynebacterium species. Also, D. pigrum inhibited S. aureus growth in vitro, whereas robust inhibition of S. pneumoniae required both D. pigrum and a nasal Corynebacterium together. D. pigrum l-lactic acid production was insufficient to account for these inhibitions. Genomic analysis of 11 strains revealed that D. pigrum has a small genome (average 1.86 Mb) and multiple predicted auxotrophies consistent with D. pigrum relying on its human host and on cocolonizing bacteria for key nutrients. Further, the accessory genome of D. pigrum harbored a diverse repertoire of biosynthetic gene clusters, some of which may have a role in microbe-microbe interactions. These new insights into D. pigrum's functions advance the field from compositional analysis to genomic and phenotypic experimentation on a potentially beneficial bacterial resident of the human upper respiratory tract and lay the foundation for future animal and clinical experiments.IMPORTANCEStaphylococcus aureus and Streptococcus pneumoniae infections cause significant morbidity and mortality in humans. For both, nasal colonization is a risk factor for infection. Studies of nasal microbiota identify Dolosigranulum pigrum as a benign bacterium present when adults are free of S. aureus or when children are free of S. pneumoniae Here, we validated these in vivo associations with functional assays. We found that D. pigrum inhibited S. aureusin vitro and, together with a specific nasal Corynebacterium species, also inhibited S. pneumoniae Furthermore, genomic analysis of D. pigrum indicated that it must obtain key nutrients from other nasal bacteria or from humans. These phenotypic interactions support the idea of a role for microbe-microbe interactions in shaping the composition of human nasal microbiota and implicate D. pigrum as a mutualist of humans. These findings support the feasibility of future development of microbe-targeted interventions to reshape nasal microbiota composition to exclude S. aureus and/or S. pneumoniae.

Dolosigranulum pigrum: Predicting Severity of Infection.

In this report, we describe a case of a 61-year-old male patient who had the bacterium Dolosigranulum pigrum growing in a blood culture. It was susceptible to ampicillin, ceftriaxone, levofloxacin, and vancomycin but was intermediately resistant to erythromycin. The patient did not have a negative outcome as a consequence of this bacterium, which retrospectively could have been predicted based on the epidemiological data within the patient's profile.

The Ability of Respiratory Commensal Bacteria to Beneficially Modulate the Lung Innate Immune Response Is a Strain Dependent Characteristic.

We investigated whether the ability of commensal respiratory bacteria to modulate the innate immune response against bacterial and viral pathogens was a shared or strain-specific characteristic. Bacterial strains belonging to the Corynebacterium pseudodiphtheriticum and Dolosigranulum pigrum species were compared by studying their influence in the Toll-like receptor (TLR)-2- and TLR3-triggered immune responses in the respiratory tract, as well as in the resistance to Respiratory Syncytial Virus (RSV) and Streptococcus pneumoniae infections. We demonstrated that nasally administered C. pseudodiphteriticum 090104 or D. pigrum 040417 were able to modulate respiratory immunity and increase the resistance against pathogens, while other strains of the same species did not influence the respiratory immune responses, demonstrating a clear strain-dependent immunomodulatory effect of respiratory commensal bacteria. We also reported here that bacterium-like particles (BLP) and cell walls derived from immunomodulatory respiratory commensal bacteria are an interesting alternative for the modulation of the respiratory immune system. Our study is a step forward in the positioning of certain strains of respiratory commensal bacteria as next-generation probiotics for the respiratory tract.