RESUMEN

In previous studies, it was demonstrated that Corynebacterium pseudodiphtheriticum 090104, isolated from the human nasopharynx, modulates respiratory immunity, improving protection against infections. Here, the antagonistic effect of the 090104 strain on respiratory pathogens, including Streptococcus pneumoniae, Staphylococcus aureus, Klebsiella pneumoniae, Pseudomonas aeruginosa, and Acinetobacter baumannii, was explored. In a series of in vitro studies, the capacity of C. pseudodiphtheriticum 090104, its bacterium-like particles, and its culture supernatants to coaggregate, inhibit the growth, and change the virulent phenotype of pathogenic bacteria was evaluated. The results showed that the 090104 strain was able to exert a bacteriostatic effect on K. pneumoniae and S. pneumoniae growth. In addition, C. pseudodiphtheriticum 090104 coaggregated, inhibited biofilm formation, and induced phenotypic changes in all the respiratory pathogens evaluated. In conclusion, this work demonstrated that, in addition to its beneficial effects exerted by host-microbe interactions, C. pseudodiphtheriticum 090104 can enhance protection against respiratory pathogens through its microbe-microbe interactions. The mechanisms involved in such interactions should be evaluated in future research.

RESUMEN

Previously, it was shown that intranasally (i.n.) administered Corynebacterium pseudodiphtheriticum 090104 (Cp) or CP-derived bacterium-like particles (BLPs) improve the immunogenicity of the pneumococcal conjugate vaccine (PCV). This work aimed to deepen the characterization of the adjuvant properties of Cp and CP-derived BLPs for their use in the development of pneumococcal vaccines. The ability of Cp and CP-derived BLPs to improve both the humoral and cellular specific immune responses induced by i.n. administered polysaccharide-based commercial pneumococcal vaccine (Pneumovax 23®) and the chimeric recombinant PSPF (PsaA-Spr1875-PspA-FliC) protein was evaluated, as well as the protection against Streptococcus pneumoniae infection in infant mice. Additionally, whether the immunization protocols, including Cp and CP-derived BLPs, together with the pneumococcal vaccines can enhance the resistance to secondary pneumococcal pneumonia induced after inflammatory lung damage mediated by the activation of Toll-like receptor 3 (TLR3) was assessed. The results showed that both Cp and CP-derived BLPs increased the immunogenicity and protection induced by two pneumococcal vaccines administered through the nasal route. Of note, the nasal priming with the PSPF T-dependent antigen co-administered with Cp or CP-derived BLPs efficiently stimulated humoral and cellular immunity and increased the resistance to primary and secondary pneumococcal infections. The CP-derived BLPs presented a stronger effect than live bacteria. Given safety concerns associated with live bacterium administration, especially in high-risk populations, such as infants, the elderly, and immunocompromised patients, BLPs emerge as an attractive mucosal adjuvant to improve the host response to pneumococcal infections and to enhance the vaccines already in the market or in development.

RESUMEN

Previously, we demonstrated that nasally administered Corynebacterium pseudodiphtheriticum 090104 (Cp) or its bacterium-like particles (BLPs) increase the resistance of mice against bacterial and viral respiratory pathogens by modulating the innate immunity. In this work, we evaluated the ability of Cp and BLPs to stimulate alveolar macrophages, and to enhance the humoral immune response induced by a commercial vaccine against Streptococcus pneumoniae. In the first set of experiments, Cp or the BLPs were incubated with primary cultures of murine alveolar macrophages and the phagocytic activity, and the production of cytokines was evaluated. The results revealed that Cp and BLPs were efficiently phagocyted by respiratory macrophages and that both treatments triggered the production of TNF-α, IFN-γ, IL-6, and IL-1ß. In the second set of experiments, 3-week-old Swiss mice were intranasally immunized at days 0, 14, and 28 with the pneumococcal vaccine Prevenar®13 (PCV), Cp + PCV, or BLPs + PCV. On day 33, samples of bronco-alveolar lavages (BAL) and serum were collected for the study of specific antibodies. In addition, immunized mice were challenged with S. pneumoniae serotypes 6B or 19F on day 33 and sacrificed on day 35 (day 2 post-infection) to evaluate the resistance to the infection. Both Cp + PCV and BLPs + PCV groups had higher specific serum IgG and BAL IgA antibodies than the PCV control mice. In addition, the mice that were immunized with Cp + PCV or BLPs + PCV had lower lung and blood pneumococcal cell counts as well as lower levels of BAL albumin and LDH, indicating a reduced lung damage compared to the control mice. Improved levels of anti-pneumococcal antibodies were also detected in the serum and BAL samples after the challenges with the pathogens. The results demonstrated that C. pseudodiphtheriticum 090104 and its bacterium-like particles are capable of stimulating the respiratory innate immune system serving as adjuvants to potentiate the adaptive humoral immune response. Our study is a step forward in the positioning of this respiratory commensal bacterium as a promising mucosal adjuvant for vaccine formulations aimed at combating respiratory infectious diseases.

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In a previous work, we demonstrated that nasally administered Corynebacterium pseudodiphtheriticum 090104 beneficially modulated the respiratory innate immune response and improved the protection against Respiratory Syncytial Virus and Streptococcus pneumoniae in mice. In this work, we aimed to evaluate whether the immunomodulatory 090104 strain was able to enhance the resistance against the respiratory infection induced by hypermucoviscous carbapenemase-producing (KPC-2) Klebsiella pneumoniae strains belonging to the sequence type (ST) 25. The nasal treatment of mice with C. pseudodiphtheriticum 090104 before the challenge with multiresistant K. pneumoniae ST25 strains significantly reduced lung bacterial cell counts and lung tissue damage. The protective effect of the 090104 strain was related to its ability to regulate the respiratory innate immune response triggered by K. pneumoniae challenge. C. pseudifteriticum 090104 differentially modulated the recruitment of leukocytes into the lung and the production of TNF-α, IFN-γ and IL-10 levels in the respiratory tract and serum. Our results make an advance in the positioning of C. pseudodiphtheriticum 090104 as a next-generation probiotic for the respiratory tract and encourage further research of this bacterium as a promising alternative to develop non-antibiotic therapeutical approaches to enhance the prevention of infections produced by microorganisms with multiple resistance to antimicrobials such as KPC-2-producing hypermucoviscous K. pneumoniae strains belonging to ST25.

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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.

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Corynebacterium pseudodiphtheriticum forma parte de la microbiota normal de orofaringe y de piel. Sin embargo, en las últimas décadas está emergiendo como oportunista causante de infecciones clínicamente significativas en pacientes con algún compromiso previo. Se refiere el caso clínico de una paciente de 76 años con antecedentes de hipertensión arterial, hipotiroidismo, diabetes tipo 2 e insuficiencia renal crónica, que presentó neumonía durante su estadía en terapia intensiva. El examen directo del esputo inducido (coloración de Gram) reveló una muestra representativa con abundante microbiota monomicrobiana constituida por bacilos gram positivos pleomórficos corineformes y el cultivo presencia de C. pseudodiphtheriticum. La paciente recibió medicación empírica con cefalosporina de tercera generación con evolución favorable.

Microorganisms of the genera Corynebacterium, specie pseudodiphtheriticum are a part of the indigenous microbiota of human skin and oropharinx. Nevertheless in recent decades these bacilli are emerging as opportunistic pathogens causing clinically significant infections in patients with previous compromise. We report the case of a 76 years old female patient, with a history of hypertension, hypothyroidism, type 2 diabetes and chronic renal failure, who presented pneumonia during their stay at the intensive care unit. The induced sputum revealed a representative sample with monomicrobial gram positive pleomorphic coryneform rods (Gram stain) and cultures demonstrated the presence of C. pseudodiphtheriticum as the only bacteria recovered. The pacient received an empirical third generation cephalosporin medication with a succesfull recovery.

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RESUMEN

Microorganisms of the genera Corynebacterium, specie pseudodiphtheriticum are a part of the indigenous microbiota of human skin and oropharinx. Nevertheless in recent decades these bacilli are emerging as opportunistic pathogens causing clinically significant infections in patients with previous compromise. We report the case of a 76 years old female patient, with a history of hypertension, hypothyroidism, type 2 diabetes and chronic renal failure, who presented pneumonia during their stay at the intensive care unit. The induced sputum revealed a representative sample with monomicrobial gram positive pleomorphic coryneform rods (Gram stain) and cultures demonstrated the presence of C. pseudodiphtheriticum as the only bacteria recovered. The pacient received an empirical third generation cephalosporin medication with a succesfull recovery.

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A ocorrência de fenótipos multirresistentes de Corynebacterium pseudodiphtheriticum e sua associação a infecções graves, com elevada mortalidade em pacientes imunocomprometidos, aliados ao escasso conhecimento da virulência e patogenia destas infecções, motivou esta pesquisa, que teve como objetivo investigar mecanismos de virulência e resistência microbiana deste agente entre pacientes de um hospital universitário brasileiro. Um total de 113 amostras de C. pseudodiphtheriticum identificadas por métodos bioquímicos convencionais e sistema API-Coryne isoladas de pacientes de diferentes grupos etários. Os micro-organismos eram, em sua maioria, relacionados a infecções no trato respiratório (27,45%), urinário (29,20%) e sitios intravenosos (18,60%) e cerca de 32,70% das amostras foram provenientes de pacientes com pelo menos uma das condições predisponentes: insuficiência renal; transplante renal, tuberculose em paciente HIV+, câncer, cirrose hepática, hemodiálise e uso de cateter. As amostras testadas revelaram-se multirresistentes sendo a maioria resistente à oxacilina, eritromicina e clindamicina. A adesão das cepas ao poliestireno e ao poliuretano indicou o envolvimento de hidrofobicidade da superfície celular na fase inicial da formação de biofilmes. O crescimento subsequente conduziu à formação de microcolônias, agregados bacterianos densos incorporados na matriz exopolimérica rodeada por espaços vazios, típica de biofilmes maduros...

The occurrence of multiresistant phenotypes and associated with severe infections, with high mortality in immunocompromised hosts due to Corynebacterium pseudodiphtheriticum, allied to little known about virulence and pathogenesis these infections, led to present investigation. The investigation aims to examine the virulence mechanisms and resistance to antimicrobial agents of C. pseudodiphtheriticum among patients with bacterial infections at a Brazilian teaching hospital. A total of 113 C. pseudodiphtheriticum strains identified by conventional biochemical methods and API-Coryne System were recovered from patients from different age groups. Micro-organisms were mostly related to infections in the respiratory tracts (27.45%), urinary (29.20%) and intravenous sites (18.60%) and approximately 32.70% samples were obtained of patients presenting at least one of the pre-disposing conditions: end-stage renal disease; renal transplant; AIDS and Mycobacterium tuberculosis infection; cancer, hepatic cirrhosis; haemodialysis and catheter use. Antimicrobial susceptibility tests identified multiresistant phenotypes. Most strains were resistant to oxacillin, erythromycin and clindamycin. Adherence to polystyrene and polyurethane indicated the involvement of cell surface hydrophobicity in the initial stage of biofilm formation. Further growth led to the formation of dense bacterial aggregates embedded in the exopolymeric matrix surrounded by voids, typical of mature biofilms...

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Corynebacterium pseudodiphtheriticum is a well-known human pathogen that mainly causes respiratory disease and is associated with high mortality in compromised hosts. Little is known about the virulence factors and pathogenesis of C. pseudodiphtheriticum. In this study, cultured human epithelial (HEp-2) cells were used to analyse the adherence pattern, internalisation and intracellular survival of the ATCC 10700 type strain and two additional clinical isolates. These microorganisms exhibited an aggregative adherence-like pattern to HEp-2 cells characterised by clumps of bacteria with a "stacked-brick" appearance. The differences in the ability of these microorganisms to invade and survive within HEp-2 cells and replicate in the extracellular environment up to 24 h post infection were evaluated. The fluorescent actin staining test demonstrated that actin polymerisation is involved in the internalisation of the C. pseudodiphtheriticum strains. The depolymerisation of microfilaments by cytochalasin E significantly reduced the internalisation of C. pseudodiphtheriticum by HEp-2 cells. Bacterial internalisation and cytoskeletal rearrangement seemed to be partially triggered by the activation of tyrosine kinase activity. Although C. pseudodiphtheriticum strains did not demonstrate an ability to replicate intracellularly, HEp-2 cells were unable to fully clear the pathogen within 24 h. These characteristics may explain how some C. pseudodiphtheriticum strains cause severe infection in human patients.

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We report here a rare case of cutaneous infection due to Corynebacterium pseudodiphtheriticum. The patient presented to the clinical laboratory with a skin ulcer on his left leg. Gram-stained preparation of the purulent secretion revealed the presence of numerous rod-shaped Gram-positive organisms in the absence of any other species. The organism was grown in pure culture on sheep blood agar and was further identified as C. pseudodiphtheriticum using a commercial identification system (API-Coryne, BioMérieux, France). The infection was successfully treated with ciprofloxacin. This case emphasizes the importance of the clinical microbiology laboratory in correctly identifying Gram-positive organisms obtained in pure culture from skin ulcers.

Reportamos o isolamento de Corynebacterium pseudodiphtheriticum de um caso de infecção cutânea. O paciente apresentou-se ao laboratório clínico com uma úlcera na perna esquerda. A coloração de Gram do material revelou a presença de bacilos Gram-positivos e ausência de outras espécies bacterianas. O organismo foi isolado em cultura pura no ágar sangue de carneiro e foi identificado como C. pseudodiphtheriticum através de um sistema de identificação comercial (API-Coryne, BioMérieux, França). A infecção foi tratada com sucesso através do uso de ciprofloxacina. Este caso reforça a importância do laboratório de microbiologia clínica na identificação de organismos Gram-positivos isolados de cultura pura de amostras de úlceras cutâneas.

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