Biofilmes de Streptococcus pneumoniae: Genética, composición y terapia.

Tesis inédita de la Universidad Complutense de Madrid, Facultad de Ciencias Biológicas, Departamento de Microbiología III, leída el 23-03-2012Se ha estimado que los biofilmes están implicados en más de la mitad de las infecciones bacterianas humanas y, hasta en un 80%, en las infecciones crónicas. Además, las infecciones causadas por bacterias patógenas asociadas a la formación de biofilmes se muestran refractarias tanto a los agentes antimicrobianos como a los mecanismos de defensa del sistema inmunitario del hospedador. Todo ello nos ha llevado a plantear la necesidad de desarrollar nuevas vías de investigación en bacterias de importancia clínica relevante como es el caso de S. pneumoniae. Hasta el momento, el modo de vida sésil de S. pneumoniae apenas ha sido investigado y, sin embargo, la otitis media, y, posiblemente, la neumonía y la meningitis son enfermedades neumocócicas asociadas a la formación de biofilmes. Teniendo en cuenta lo expuesto anteriormente, los objetivos experimentales que nos planteamos investigar en la presente Tesis son los siguientes: 1. Búsqueda de nuevos genes implicados en la formación de biofilm. 2. Determinar el papel del polisacárido capsular en la formación de biofilm de S. pneumoniae. 3. Investigar la composición de la matriz extracelular de los biofilmes neumocócicos. 4. Estudiar los mecanismos de evasión del sistema inmune por parte de un biofilm. 5. Desarrollar y evaluar posibles medidas terapeúticas contra los biofilmes.La realización de esta Tesis ha sido posible gracias a la concesión de una beca incluida dentro del Programa de Becas Predoctorales de Formación de Investigadores del Ministerio de Ciencia e Innovación y al CIBER de Enfermedades Respiratorias (CIBERES).Peer reviewe

Biofilmes de "Streptococcus pneumoniae": genética, composición y terapia

Se ha estimado que los biofilmes están implicados en más de la mitad de las infecciones bacterianas humanas y, hasta en un 80%, en las infecciones crónicas. Además, las infecciones causadas por bacterias patógenas asociadas a la formación de biofilmes se muestran refractarias tanto a los agentes antimicrobianos como a los mecanismos de defensa del sistema inmunitario del hospedador. Todo ello nos ha llevado a plantear la necesidad de desarrollar nuevas vías de investigación en bacterias de importancia clínica relevante como es el caso de S. pneumoniae. Hasta el momento, el modo de vida sésil de S. pneumoniae apenas ha sido investigado y, sin embargo, la otitis media, y, posiblemente, la neumonía y la meningitis son enfermedades neumocócicas asociadas a la formación de biofilmes. Teniendo en cuenta lo expuesto anteriormente, los objetivos experimentales que nos planteamos investigar en la presente Tesis son los siguientes: 1. Búsqueda de nuevos genes implicados en la formación de biofilm. 2. Determinar el papel del polisacárido capsular en la formación de biofilm de S. pneumoniae. 3. Investigar la composición de la matriz extracelular de los biofilmes neumocócicos. 4. Estudiar los mecanismos de evasión del sistema inmune por parte de un biofilm. 5. Desarrollar y evaluar posibles medidas terapeúticas contra los biofilmes

Evidence of the presence of nucleic acids and β-glucan in the matrix of non-typeable Haemophilus influenzae in vitro biofilms

13 p.-9 fig.Non-typeable Haemophilus influenzae (NTHi) is a Gram-negative bacterium that frequently colonizes the human nasopharynx; it is a common cause of chronic and recurrent otitis media in children and of exacerbations of chronic obstructive pulmonary disease. To date, no exopolysaccharide clearly contributing to NTHi biofilms has been identified. Consequently, there is some debate as to whether NTHi forms biofilms during colonization and infection. The present work shows that NTHi can form biofilms in vitro, producing an extracellular matrix composed of proteins, nucleic acids, and a β-glucan. Extracellular DNA, visualized by immunostaining and using fluorochromes, is an important component of this matrix and appears to be essential in biofilm maintenance. Extracellular RNA appears to be required only in the first steps of biofilm formation. Evidence of a matrix polysaccharide was obtained by staining with Calcofluor white M2R and by disaggregating biofilms with cellulase. Using strain 54997, residues of Glcp(1→4) in the NTHi biofilm were confirmed by gas-liquid chromatography-mass spectrometry. Evidence that N-acetyl-L-cysteine shows notable killing activity towards in vitro NTHi biofilm-forming bacteria is also provided.This work was supported by a grant from the Ministerio de Economía y Competitividad (SAF2012-39444-C02-01). The CIBER de Enfermedades Respiratorias (CIBERES) is an initiative of the Instituto de Salud Carlos III.Peer reviewe

Combination of Antibodies and Antibiotics as a Promising Strategy Against Multidrug-Resistant Pathogens of the Respiratory Tract

The emergence of clinical isolates associated to multidrug resistance is a serious threat worldwide in terms of public health since complicates the success of the antibiotic treatment and the resolution of the infectious process. This is of great concern in pathogens affecting the lower respiratory tract as these infections are one of the major causes of mortality in children and adults. In most cases where the respiratory pathogen is associated to multidrug-resistance, antimicrobial concentrations both in serum and at the site of infection may be insufficient and the resolution of the infection depends on the interaction of the invading pathogen with the host immune response. The outcome of these infections largely depends on the susceptibility of the pathogen to the antibiotic treatment, although the humoral and cellular immune responses also play an important role in this process. Hence, prophylactic measures or even immunotherapy are alternatives against these multi-resistant pathogens. In this sense, specific antibodies and antibiotics may act concomitantly against the respiratory pathogen. Alteration of cell surface structures by antimicrobial drugs even at sub-inhibitory concentrations might result in greater exposure of microbial ligands that are normally hidden or hardly exposed. This alteration of the bacterial envelope may stimulate opsonization by natural and/or specific antibodies or even by host defense components, increasing the recognition of the microbial pathogen by circulating phagocytes. In this review we will explain the most relevant studies, where vaccination or the use of monoclonal antibodies in combination with antimicrobial treatment has demonstrated to be an alternative strategy to overcome the impact of multidrug resistance in respiratory pathogens

Biofilm formation avoids complement immunity and phagocytosis of Streptococcus pneumoniae

Streptococcus pneumoniae is a frequent member of the microbiota of the human nasopharynx. Colonization of the nasopharyngeal tract is a first and necessary step in the infectious process and often involves the formation of sessile microbial communities by this human pathogen. The ability to grow and persist as biofilms is an advantage for many microorganisms, because biofilm-grown bacteria show reduced susceptibility to antimicrobial agents and hinder recognition by the immune system. The extent of host protection against biofilm-related pneumococcal disease has not been determined yet. Using pneumococcal strains growing as planktonic cultures or as biofilms, we have investigated the recognition of S. pneumoniae by the complement system and its interactions with human neutrophils. Deposition of C3b, the key complement component, was impaired on S. pneumoniae biofilms. In addition, binding of C-reactive protein and the complement component C1q to the pneumococcal surface was reduced in biofilm bacteria, demonstrating that pneumococcal biofilms avoid the activation of the classical complement pathway. In addition, recruitment of factor H, the downregulator of the alternative pathway, was enhanced by S. pneumoniae growing as biofilms. Our results also show that biofilm formation diverts the alternative complement pathway activation by a PspC-mediated mechanism. Furthermore, phagocytosis of pneumococcal biofilms was also impaired. The present study confirms that biofilm formation in S. pneumoniae is an efficient means of evading both the classical and the PspC-dependent alternative complement pathways the host immune system.This work was supported by grants SAF2009-10824; and SAF2012-39444-C02-01/02 from Dirección General de Investigación Científica y Técnica and MINECO, respectively. Centro de Investigación Biomédica en Red de Enfermedades Respiratorias (CIBERES) is an initiative of the ISCIII. M.D. and E.R-S. were supported by FPI and FPU fellowships, respectively, from Ministerio de Economía y Competitividad.S

Clinical Relevance and Molecular Pathogenesis of the Emerging Serotypes 22F and 33F of Streptococcus pneumoniae in Spain

Streptococcus pneumoniae is the main bacterial cause of respiratory infections in children and the elderly worldwide. Serotype replacement is a frequent phenomenon after the introduction of conjugated vaccines, with emerging serotypes 22F and 33F as frequent non-PCV13 serotypes in children and adults in North America and other countries. Characterization of mechanisms involved in evasion of the host immune response by these serotypes is of great importance in public health because they are included in the future conjugated vaccines PCV15 and PCV20. One of the main strategies of S. pneumoniae to persistently colonize and causes infection is biofilm formation. In this study, we have evaluated the influence of capsule polysaccharide in biofilm formation and immune evasion by using clinical isolates from different sources and isogenic strains with capsules from prevalent serotypes. Since the introduction of PCV13 in Spain in the year 2010, isolates of serotypes 22F and 33F are rising among risk populations. The predominant circulating genotypes are ST43322F and ST71733F , being CC433 in 22F and CC717 in 33F the main clonal complexes in Spain. The use of clinical isolates of different origin, demonstrated that pediatric isolates of serotypes 22F and 33F formed better biofilms than adult isolates and this was statistically significant. This phenotype was greater in clinical isolates from blood origin compared to those from cerebrospinal fluid, pleural fluid and otitis. Opsonophagocytosis assays showed that serotype 22F and 33F were recognized by the PSGL-1 receptor on leukocytes, although serotype 22F, was more resistant than serotype 33F to phagocytosis killing and more lethal in a mouse sepsis model. Overall, the emergence of additional PCV15 serotypes, especially 22F, could be associated to an enhanced ability to divert the host immune response that markedly increased in a biofilm state. Our findings demonstrate that pediatric isolates of 22F and 33F, that form better biofilm than isolates from adults, could have an advantage to colonize the nasopharynx of children and therefore, be important in carriage and subsequent dissemination to the elderly. The increased ability of serotype 22F to avoid the host immune response, might explain the emergence of this serotype in the last years.This work was partially supported by a grant from the MSD-USA (MISP Call, Reference: 57320), Ministerio de Economía, Industria y Competitividad (MINECO) (SAF2017-83388-R), and CIBER de Enfermedades Respiratorias (CIBERES) is an initiative of the Instituto de Salud Carlos III

Combination of Cefditoren and N-acetyl-l-Cysteine Shows a Synergistic Effect against Multidrug-Resistant Streptococcus pneumoniae Biofilms

Biofilm formation by Streptococcus pneumoniae is associated with colonization of the upper respiratory tract, including the carrier state, and with chronic respiratory infections in patients suffering from chronic obstructive pulmonary disease (COPD). The use of antibiotics alone to treat recalcitrant infections caused by biofilms is insufficient in many cases, requiring novel strategies based on a combination of antibiotics with other agents, including antibodies, enzybiotics, and antioxidants. In this work, we demonstrate that the third-generation oral cephalosporin cefditoren (CDN) and the antioxidant N-acetyl-l-cysteine (NAC) are synergistic against pneumococcal biofilms. Additionally, the combination of CDN and NAC resulted in the inhibition of bacterial growth (planktonic and biofilm cells) and destruction of the biofilm biomass. This marked antimicrobial effect was also observed in terms of viability in both inhibition (prevention) and disaggregation (treatment) assays. Moreover, the use of CDN and NAC reduced bacterial adhesion to human lung epithelial cells, confirming that this strategy of combining these two compounds is effective against resistant pneumococcal strains colonizing the lung epithelium. Finally, administration of CDN and NAC in mice suffering acute pneumococcal pneumonia caused by a multidrug-resistant strain was effective in clearing the bacteria from the respiratory tract in comparison to treatment with either compound alone. Overall, these results demonstrate that the combination of oral cephalosporins and antioxidants, such as CDN and NAC, respectively, is a promising strategy against respiratory biofilms caused by S. pneumoniae. IMPORTANCE Streptococcus pneumoniae is one of the deadliest bacterial pathogens, accounting for up to 2 million deaths annually prior to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Vaccines have decreased the burden of diseases produced by S. pneumoniae, but the rise of antibiotic-resistant strains and nonvaccine serotypes is worrisome. Pneumococcal biofilms are associated with chronic respiratory infections, and treatment is challenging, making the search for new antibiofilm therapies a priority as biofilms become resistant to traditional antibiotics. In this work, we used the combination of an antibiotic (CDN) and an antioxidant (NAC) to treat the pneumococcal biofilms of relevant clinical isolates. We demonstrated a synergy between CDN and NAC that inhibited and treated pneumococcal biofilms, impaired pneumococcal adherence to the lung epithelium, and treated pneumonia in a mouse pneumonia model. We propose the widely used cephalosporin CDN and the repurposed drug NAC as a new antibiofilm therapy against S. pneumoniae biofilms, including those formed by antibiotic-resistant clinical isolates.This work was supported by Ministerio de Ciencia e Innovación (MICINN) (grant PID2020-119298RB-I00) and by Meiji Pharma Spain (grant MVP 119/20). J.Y. has received grants from MSD-USA (MISP Call) and Pfizer that are not related to this work. J.Y. has participated in advisory boards organized by MSD and Pfizer. M.G. and P.C. are members of the Scientific Department of Meiji Pharma Spain. The other authors declare no competing interests.S

Emerging, non-PCV13 serotypes 11A and 35B of Streptococcus pneumoniae show high potential for biofilm formation in vitro

Background: Since the use of pneumococcal conjugate vaccines PCV7 and PCV13 in children became widespread, invasive pneumococcal disease (IPD) has dramatically decreased. Nevertheless, there has been a rise in incidence of Streptococcus pneumoniae non-vaccine serotypes (NVT) colonising the human nasopharynx. Nasopharyngeal colonisation, an essential step in the development of S. pneumoniae-induced IPD, is associated with biofilm formation. Although the capsule is the main pneumococcal virulence factor, the formation of pneumococcal biofilms might, in fact, be limited by the presence of capsular polysaccharide (CPS). Methodology/Principal Findings: We used clinical isolates of 16 emerging, non-PCV13 serotypes as well as isogenic transformants of the same serotypes. The biofilm formation capacity of isogenic transformants expressing CPSs from NVT was evaluated in vitro to ascertain whether this trait can be used to predict the emergence of NVT. Fourteen out of 16 NVT analysed were not good biofilm formers, presumably because of the presence of CPS. In contrast, serotypes 11A and 35B formed >45% of the biofilm produced by the non-encapsulated M11 strain. Conclusions/Significance This study suggest that emerging, NVT serotypes 11A and 35B deserve a close surveillance

Auranofin-loaded nanoparticles as a new therapeutic tool to fight streptococcal infections

Drug-loaded nanoparticles (NPs) can improve infection treatment by ensuring drug concentration at the right place within the therapeutic window. Poly(lactic-co-glycolic acid) (PLGA) NPs are able to enhance drug localization in target site and to sustainably release the entrapped molecule, reducing the secondary effects caused by systemic antibiotic administration. We have loaded auranofin, a gold compound traditionally used for treatment of rheumatoid arthritis,into PLGA NPs and their efficiency as antibacterial agent against two Gram-positive pathogens, Streptococcus pneumoniae and Streptococcus pyogenes was evaluated. Auranofin-PLGA NPs showed a strong bactericidal effect as cultures of multiresistant pneumococcal strains were practically sterilized after 6 h of treatment with such auranofin-NPs at 0.25 μM. Moreover, this potent bactericidal effect was also observed in S. pneumoniae and S. pyogenes biofilms, where the same concentration of auranofin-NPs was capable of decreasing the bacterial population about 4 logs more than free auranofin. These results were validated using zebrafish embryo model demonstrating that treatment with auranofin loaded into NPs achieved a noticeable survival against pneumococcal infections. All these approaches displayed a clear superiority of loaded auranofin PLGA nanocarriers compared to free administration of the drug, which supports their potential application for the treatment of streptococcal infections

Nationwide trends of invasive pneumococcal disease in Spain (2009-2019) in children and adults during the pneumococcal conjugate vaccine era.

Introduction of pneumococcal conjugate vaccines (PCVs) has shown a marked reduction in the disease caused by vaccine serotypes in children providing herd protection to the elderly group. However, the emergence of non-vaccine serotypes is of great concern worldwide. This study includes national laboratory data from invasive pneumococcal disease (IPD) cases affecting pediatric and adult population during 2009-2019. The impact of implementing different vaccine strategies for immunocompetent adults comparing Spanish regions using PCV13 vs regions using PPV23 vaccine was also analyzed for 2017-2019. The overall reductions of IPD cases by PCV13 serotypes in children and adults were 88% and 59% respectively during 2009-2019 with a constant increase of serotype 8 in adults since 2015. IPD cases by additional serotypes covered by PPV23 increased from 20% in 2009 to 52% in 2019. In children, serotype 24F was the most frequent in 2019 whereas in adults, serotypes 3 and 8 accounted for 36% of IPD cases. Introduction of PCV13 or PPV23 in the adult calendar of certain Spanish regions reduced up to 25% and 11% respectively the IPD cases by PCV13 serotypes, showing a decrease of serotype 3 when PCV13 was used. Use of PCV13 in children has shown a clear impact in pneumococcal epidemiology reducing the burden of IPD in children but also in adults by herd protection although the increase of serotype 8 in adults is worrisome. Vaccination with PCV13 in immunocompetent adults seems to control IPD cases by PCV13 serotypes including serotype 3.This work was supported by Ministerio de Economía, Industria y Competitividad (MINECO) [grant SAF2017-83388] and internal funding fromS