Publications (57)210.74 Total impact
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Article: Biofilm Formation Avoids Complement Immunity and Phagocytosis of Streptococcus pneumoniae.
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ABSTRACT: 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 a reduced susceptibility to antimicrobial agents and hinder the recognition by the immune system. Host protection against biofilm-related pneumococcal disease has not been defined 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-growing bacteria demonstrating that pneumococcal biofilms avoid the activation of the classical complement pathway. Besides, recruitment of factor H, the down-regulator 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 way for host immune evasion both from the classical and the PspC-dependent alternative complement pathways.Infection and immunity 05/2013; · 4.21 Impact Factor -
Article: Macrolides and β-Lactam Antibiotics Enhance C3b Deposition on the Surface of Multidrug-Resistant Streptococcus pneumoniae Strains by a LytA Autolysin-Dependent Mechanism.
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ABSTRACT: The emergence of Streptococcus pneumoniae strains displaying high levels of multidrug resistance is of great concern worldwide and a serious threat for the outcome of the infection. Modifications of the bacterial envelope by antibiotics may assist the recognition and clearance of the pathogen by the host immune system. Recognition of S. pneumoniae resistant strains by the complement component C3b was increased in the presence of specific anti-pneumococcal antibodies and subinhibitory concentrations of different macrolides and β-lactam antibiotics for all the strains investigated. However, C3b levels were unchanged in the presence of serum containing specific antibodies and sub-MICs of levofloxacin. To investigate whether LytA, the main cell wall hydrolase of S. pneumoniae, might be involved in this process, lytA-deficient mutants were constructed. In the presence of antibiotics, loss of LytA was not associated with enhanced C3b deposition on the pneumococcal surface, which confirms the importance of LytA in this interaction. The results of this study offer new insights into the development of novel therapeutic strategies using certain antibiotics by increasing the efficacy of the host immune response to efficiently recognize pneumococcal resistant strains.Antimicrobial Agents and Chemotherapy 08/2012; 56(11):5534-40. · 4.84 Impact Factor -
Article: Insight into the composition of the intercellular matrix of Streptococcus pneumoniae biofilms.
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ABSTRACT: Biofilm matrices consist of a mixture of extracellular polymeric substances synthesized in large part by the biofilm-producing microorganisms themselves. These matrices are responsible for the cohesion and three-dimensional architecture of biofilms. The present study demonstrates the existence of a matrix composed of extracellular DNA, proteins and polysaccharides in the biofilm formed by the human pathogen Streptococcus pneumoniae. Extracellular DNA, visualized by fluorescent labelling, was an important component of this matrix. The existence of DNA-protein complexes associated with bacterial aggregates and other polymers was hypothesized based on the unexpected DNA binding activity of lysozyme LytC, a novel moonlighting protein. Actually, a 25-amino-acid-long peptide derived from LytC (positions 408 and 432 of the mature LytC) was also capable of efficiently binding to DNA. Moreover, the presence of intercellular DNA-LytC protein complexes in pneumococcal biofilms was demonstrated by confocal laser scanning microscopy. Evidence of extracellular polysaccharide different from the capsule was obtained by staining with Calcofluor dye and four types of lectin conjugated to Alexa fluorophores, and by incubation with glycoside hydrolases. The presence of residues of Glcp(1→4) and GlcNAc(1→4) (in its deacetylated form) in the pneumococcal biofilm was confirmed by GC-MS techniques.Environmental Microbiology 07/2012; · 5.84 Impact Factor -
Article: The galU gene expression in Streptococcus pneumoniae.
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ABSTRACT: The polysaccharide capsule of Streptococcus pneumoniae is the main virulence factor making the bacterium resistant to phagocytosis. The galU gene of S. pneumoniae encodes a UDP-glucose pyrophosphorylase absolutely required for capsule biosynthesis. In silico analyses indicated that the galU gene is co-transcribed with the gpdA gene, and four putative promoter regions located upstream of gpdA were predicted. One of them behaved as a functional promoter in a promoter reporter system. It is conceivable that the sequence responsible for initiating transcription of gpdA-galU operon is an extended -10 site TATGATA(T/G)AAT. Semi-quantitative real-time reverse transcription PCR experiments indicated that galU was expressed mainly in the exponential phase of growth.FEMS Microbiology Letters 04/2012; 332(1):47-53. · 2.04 Impact Factor -
Article: Cefditoren and Ceftriaxone Enhance Complement-Mediated Immunity in the Presence of Specific Antibodies against Antibiotic-Resistant Pneumococcal Strains.
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ABSTRACT: Specific antibodies mediate humoral and cellular protection against invading pathogens such as Streptococcus pneumoniae by activating complement mediated immunity, promoting phagocytosis and stimulating bacterial clearance. The emergence of pneumococcal strains with high levels of antibiotic resistance is of great concern worldwide and a serious threat for public health. Flow cytometry was used to determine whether complement-mediated immunity against three antibiotic-resistant S. pneumoniae clinical isolates is enhanced in the presence of sub-inhibitory concentrations of cefditoren and ceftriaxone. The binding of acute phase proteins such as C-reactive protein and serum amyloid P component, and of complement component C1q, to pneumococci was enhanced in the presence of serum plus either of these antibiotics. Both antibiotics therefore trigger the activation of the classical complement pathway against S. pneumoniae. C3b deposition was also increased in the presence of specific anti-pneumococcal antibodies and sub-inhibitory concentrations of cefditoren and ceftriaxone confirming that the presence of these antibiotics enhances complement-mediated immunity to S. pneumoniae. Using cefditoren and ceftriaxone to promote the binding of acute phase proteins and C1q to pneumococci, and to increase C3b deposition, when anti-pneumococcal antibodies are present, might help reduce the impact of antibiotic resistance in S. pneumoniae infections.PLoS ONE 01/2012; 7(9):e44135. · 4.09 Impact Factor -
Article: Thermal Stability of Cpl-7 Endolysin from the Streptococcus pneumoniae Bacteriophage Cp-7; Cell Wall-Targeting of Its CW_7 Motifs.
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ABSTRACT: Endolysins comprise a novel class of selective antibacterials refractory to develop resistances. The Cpl-7 endolysin, encoded by the Streptococcus pneumoniae bacteriophage Cp-7, consists of a catalytic module (CM) with muramidase activity and a cell wall-binding module (CWBM) made of three fully conserved CW_7 repeats essential for activity. Firstly identified in the Cpl-7 endolysin, CW_7 motifs are also present in a great variety of cell wall hydrolases encoded, among others, by human and live-stock pathogens. However, the nature of CW_7 receptors on the bacterial envelope remains unknown. In the present study, the structural stability of Cpl-7 and the target recognized by CW_7 repeats, relevant for exploitation of Cpl-7 as antimicrobial, have been analyzed, and transitions from the CM and the CWBM assigned, using circular dichroism and differential scanning calorimetry. Cpl-7 stability is maximum around 6.0-6.5, near the optimal pH for activity. Above pH 8.0 the CM becomes extremely unstable, probably due to deprotonation of the N-terminal amino-group, whereas the CWBM is rather insensitive to pH variation and its structural stabilization by GlcNAc-MurNAc-l-Ala-d-isoGln points to the cell wall muropeptide as the cell wall target recognized by the CW_7 repeats. Denaturation data also revealed that Cpl-7 is organized into two essentially independent folding units, which will facilitate the recombination of the CM and the CWBM with other catalytic domains and/or cell wall-binding motifs to yield new tailored chimeric lysins with higher bactericidal activities or new pathogen specificities.PLoS ONE 01/2012; 7(10):e46654. · 4.09 Impact Factor -
Article: Biofilm formation in Streptococcus pneumoniae.
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ABSTRACT: Biofilm-grown bacteria are refractory to antimicrobial agents and show an increased capacity to evade the host immune system. In recent years, studies have begun on biofilm formation by Streptococcus pneumoniae, an important human pathogen, using a variety of in vitro model systems. The bacterial cells in these biofilms are held together by an extracellular matrix composed of DNA, proteins and, possibly, polysaccharide(s). Although neither the precise nature of these proteins nor the composition of the putative polysaccharide(s) is clear, it is known that choline-binding proteins are required for successful biofilm formation. Further, many genes appear to be involved, although the role of each appears to vary when biofilms are produced in batch or continuous culture. Prophylactic and therapeutic measures need to be developed to fight S. pneumoniae biofilm formation. However, much care needs to be taken when choosing strains for such studies because different S. pneumoniae isolates can show remarkable genomic differences. Multispecies and in vivo biofilm models must also be developed to provide a more complete understanding of biofilm formation and maintenance.Microbial Biotechnology 09/2011; 5(4):455-65. · 2.53 Impact Factor -
Article: In vitro destruction of Streptococcus pneumoniae biofilms with bacterial and phage peptidoglycan hydrolases.
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ABSTRACT: Host- and phage-coded cell wall hydrolases have been used to fight Streptococcus pneumoniae growing as planktonic cells in vitro as well as in animal models. Until now, however, the usefulness of these enzymes in biofilm-grown pneumococci has gone untested. The antipneumococcal activity of different cell wall hydrolases produced by S. pneumoniae and a number of its phages was examined in an in vitro biofilm model. The major pneumococcal autolysin LytA, an N-acetylmuramoyl-l-alanine amidase, showed the greatest efficiency in disintegrating S. pneumoniae biofilms. The phage-encoded lysozymes Cpl-1 and Cpl-7 were also very efficient. Biofilms formed by the close pneumococcal relatives Streptococcus pseudopneumoniae and Streptococcus oralis were also destroyed by the phage endolysins but not by the S. pneumoniae autolysin LytA. A cooperative effect of LytA and Cpl-1 in the disintegration of S. pneumoniae biofilms was recorded.Antimicrobial Agents and Chemotherapy 09/2011; 55(9):4144-8. · 4.84 Impact Factor -
Article: Characterization of invasive Pneumococci of serogroup 6 from adults in Barcelona, Spain, in 1994 to 2008.
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ABSTRACT: A total of 91 of 1,480 invasive isolates (6.1%) collected from adults in Barcelona, Spain, in the period of 1994 to 2008 were of serogroup 6 (6B, 47 isolates; 6A, 28; and 6C, 16). Throughout this period, serotype 6B (Spain(6B)-ST90) decreased, and serotype 6A remained stable. An increase in serotype 6C (ST224) in the 2004-2008 period was observed.Journal of clinical microbiology 03/2011; 49(6):2328-30. · 4.16 Impact Factor -
Article: Development of amperometric magnetogenosensors coupled to asymmetric PCR for the specific detection of Streptococcus pneumoniae.
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ABSTRACT: A disposable magnetogenosensor for the rapid, specific and sensitive detection of Streptococcus pneumoniae is reported. The developed procedure involves the use of streptavidin-modified magnetic beads, a specific biotinylated capture probe that hybridizes with a specific region of lytA, the gene encoding the pneumococcal major autolysin, and appropriate primers for asymmetric polymerase chain reaction (PCR) amplification. Capture probes and amplicons specific for S. pneumoniae were selected by a careful analysis of all lytA alleles available. The selected primers amplify a 235-bp fragment of pneumococcal lytA. A detection limit (LOD) of 5.1 nM was obtained for a 20-mer synthetic target DNA without any amplification protocol, while the LOD for the asymmetric PCR amplicon was 1.1 nM. A RSD value of 6.9% was obtained for measurements carried out with seven different genosensors for 1.1-nM aPCR product. The strict specificity of the designed primers was demonstrated by aPCR amplification of genomic DNA prepared from different bacteria, including some closely related streptococci. Direct asymmetric PCR (daPCR), using cells directly from broth cultures of S. pneumoniae, showed that daPCR products could be prepared with as few as 2 colony-forming units (CFU). Furthermore, this methodology did not show any cross-reaction with closely related streptococci such as Streptococcus mitis (or Streptococcus pseudopneumoniae) even when present in the culture at concentrations up to 10(5) times higher than that of S. pneumoniae. Preliminary data for rapid detection of pneumococcus directly in clinical samples has shown that it is possible to discriminate between non-inoculated blood and urine samples and samples inoculated with only 10(3) CFU mL(-1) S. pneumoniae.Analytical and Bioanalytical Chemistry 03/2011; 399(7):2413-20. · 3.78 Impact Factor -
Article: Nasopharyngeal colonization and invasive disease are enhanced by the cell wall hydrolases LytB and LytC of Streptococcus pneumoniae.
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ABSTRACT: Streptococcus pneumoniae is a common colonizer of the human nasopharynx and one of the major pathogens causing invasive disease worldwide. Dissection of the molecular pathways responsible for colonization, invasion, and evasion of the immune system will provide new targets for antimicrobial or vaccine therapies for this common pathogen. We have constructed mutants lacking the pneumococcal cell wall hydrolases (CWHs) LytB and LytC to investigate the role of these proteins in different phases of the pneumococcal pathogenesis. Our results show that LytB and LytC are involved in the attachment of S. pneumoniae to human nasopharyngeal cells both in vitro and in vivo. The interaction of both proteins with phagocytic cells demonstrated that LytB and LytC act in concert avoiding pneumococcal phagocytosis mediated by neutrophils and alveolar macrophages. Furthermore, C3b deposition was increased on the lytC mutant confirming that LytC is involved in complement evasion. As a result, the lytC mutant showed a reduced ability to successfully cause pneumococcal pneumonia and sepsis. Bacterial mutants lacking both LytB and LytC showed a dramatically impaired attachment to nasopharyngeal cells as well as a marked degree of attenuation in a mouse model of colonization. In addition, C3b deposition and phagocytosis was more efficient for the double lytB lytC mutant and its virulence was greatly impaired in both systemic and pulmonary models of infection. This study confirms that the CWHs LytB and LytC of S. pneumoniae are essential virulence factors involved in the colonization of the nasopharynx and in the progress of invasive disease by avoiding host immunity.PLoS ONE 01/2011; 6(8):e23626. · 4.09 Impact Factor -
Article: Disposable amperometric magnetoimmunosensors for the specific detection of Streptococcus pneumoniae.
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ABSTRACT: Disposable amperometric magnetoimmunosensors, based on the use of functionalized magnetic beads and gold screen-printed electrodes, have been developed for the selective detection and quantification of Streptococcus pneumoniae. A specific antibody prepared against a serotype 37 S. pneumoniae strain, selected by flow cytometry among seven anticapsular or antisomatic antibodies, was linked to Protein A-modified magnetic beads and incubated with bacteria. The same antibody, conjugated with horseradish peroxidase, was attached to the bacteria and the resulting modified magnetic beads were captured by a magnetic field on the surface of tetrathiafulvalene-modified gold screen-printed electrodes. The amperometric response obtained at -0.15 V vs. the silver pseudoreference electrode of the Au/SPE after the addition of H(2)O(2) was used as transduction signal. Different assay formats were examined and the experimental variables optimized. The limits of detection achieved, without pre-concentration or pre-enrichment steps, were 1.5×10(4) cfu mL(-1) (colony forming unit) and 6.3×10(5) cfu mL(-1) for S. pneumoniae strains Dawn (serotype 37) and R6 (non-encapsulated), respectively. The developed methodology shows a good selectivity against closely related streptococci and its usefulness for the analysis of inoculated urine samples has been demonstrated. The total analysis time of 3.5 h from sampling to measurement, the possibility to prepare up to 30 sensors per day and the use of small amounts of test solution for S. pneumoniae identification, constitute important advantages that make the developed methodology a promising alternative for clinical diagnosis.Biosensors & bioelectronics 12/2010; 26(4):1225-30. · 5.43 Impact Factor -
Article: Dissemination of an erythromycin-resistant penicillin-nonsusceptible Streptococcus pneumoniae Poland(6B)-20 clone in Argentina.
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ABSTRACT: Prevalence of serotype 6B penicillin (PEN)-nonsusceptible Streptococcus pneumoniae significantly increased from 15.8% (1993-1997) to 67.3% (1998-2002) (p<0.001) in Argentina. Serogroup 6 ranks fourth among different capsular types within invasive isolates from Argentinean patients <6 years of age. To evaluate whether the increase in PEN resistance in serotype 6B pneumococci was due to the dissemination of one or more clones, the genetic diversity of 93 S. pneumoniae serotype 6B isolates was analyzed. Five BOX-polymerase chain reaction types were obtained (65.5% isolates) and a group of 15 isolates, representing 41.6% of those having a decreased susceptibility to PEN, were further characterized. The antibiotype of these isolates showed their multiresistance, with 100% of the isolates being resistant to erythromycin, 80% to tetracycline, and 73.3% to trimethoprim-sulfamethoxazole. Of the 15 isolates, 13 belonged to the same pulsed-field gel electrophoresis type and galU cluster and were members of the same clone. The identity of the clone was confirmed in four isolates by multilocus sequence typing. The sequence type found (ST315) corresponds to the Poland(6B)-20 clone. In summary, BOX-polymerase chain reaction, pulsed-field gel electrophoresis, and galU polymorphism were useful tools to detect the presence of a clone whose identity was confirmed by multilocus sequence typing. The isolates belonging to Poland(6B)-20 found in this work are described for the first time in Latin America.Microbial drug resistance (Larchmont, N.Y.) 12/2010; 17(1):75-81. · 1.99 Impact Factor -
Article: Cpl-7, a lysozyme encoded by a pneumococcal bacteriophage with a novel cell wall-binding motif.
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ABSTRACT: Bacteriophage endolysins include a group of new antibacterials reluctant to development of resistance. We present here the first structural study of the Cpl-7 endolysin, encoded by pneumococcal bacteriophage Cp-7. It contains an N-terminal catalytic module (CM) belonging to the GH25 family of glycosyl hydrolases and a C-terminal region encompassing three identical repeats of 42 amino acids (CW_7 repeats). These repeats are unrelated to choline-targeting motifs present in other cell wall hydrolases produced by Streptococcus pneumoniae and its bacteriophages, and are responsible for the protein attachment to the cell wall. By combining different biophysical techniques and molecular modeling, a three-dimensional model of the overall protein structure is proposed, consistent with circular dichroism and sequence-based secondary structure prediction, small angle x-ray scattering data, and Cpl-7 hydrodynamic behavior. Cpl-7 is an ∼115-Å long molecule with two well differentiated regions, corresponding to the CM and the cell wall binding region (CWBR), arranged in a lateral disposition. The CM displays the (βα)(5)β(3) barrel topology characteristic of the GH25 family, and the impact of sequence differences with the CM of the Cpl-1 lysozyme in substrate binding is discussed. The CWBR is organized in three tandemly assembled three-helical bundles whose dispositions remind us of a super-helical structure. Its approximate dimensions are 60 × 20 × 20 Å and presents a concave face that might constitute the functional region involved in bacterial surface recognition. The distribution of CW_7 repeats in the sequences deposited in the Entrez Database have been examined, and the results drastically expanded the antimicrobial potential of the Cpl-7 endolysin.Journal of Biological Chemistry 10/2010; 285(43):33184-96. · 4.77 Impact Factor -
Article: Vancomycin tolerance in clinical and laboratory Streptococcus pneumoniae isolates depends on reduced enzyme activity of the major LytA autolysin or cooperation between CiaH histidine kinase and capsular polysaccharide.
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ABSTRACT: Summary Vancomycin is frequently added to standard therapy for pneumococcal meningitis. Although vancomycin-resistant Streptococcus pneumoniae strains have not been isolated, reports on the emergence of vancomycin-tolerant pneumococci are a cause of concern. To date, the molecular basis of vancomycin tolerance in S. pneumoniae is essentially unknown. We examined two vancomycin-tolerant clinical isolates, i.e. a purported autolysin negative (LytA(-)), serotype 23F isolate (strain S3) and the serotype 14 strain 'Tupelo', which is considered a paradigm of vancomycin tolerance. S3 was characterized here as carrying a frameshift mutation in the lytA gene encoding the main pneumococcal autolysin. The vancomycin tolerance of strain S3 was abolished by transformation to the autolysin-proficient phenotype. The original Tupelo strain was discovered to be a mixture: a strain showing a vancomycin-tolerant phenotype (Tupelo_VT) and a vancomycin-nontolerant strain (Tupelo_VNT). The two strains differed only in terms of a single mutation in the ciaH gene present in the VT strain. Most interestingly, although the vancomycin tolerance of Tupelo_VT could be overcome by increasing the LytA dosage upon transformation by a multicopy plasmid or by externally adding the autolysin, we show that vancomycin tolerance in S. pneumoniae requires the simultaneous presence of a mutated CiaH histidine kinase and capsular polysaccharide.Molecular Microbiology 06/2010; · 5.01 Impact Factor -
Article: Evidence of localized prophage-host recombination in the lytA gene, encoding the major pneumococcal autolysin.
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ABSTRACT: According to a highly polymorphic region in the lytA gene, encoding the major autolysin of Streptococcus pneumoniae, two different families of alleles can be differentiated by PCR and restriction digestion. Here, we provide evidence that this polymorphic region arose from recombination events with homologous genes of pneumococcal temperate phages.Journal of bacteriology 03/2010; 192(10):2624-32. · 3.94 Impact Factor -
Article: Comparative efficacy of novobiocin and amoxicillin in experimental sepsis caused by beta-lactam-susceptible and highly resistant pneumococci.
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ABSTRACT: Therapeutic alternatives are needed against infections caused by highly multidrug-resistant Streptococcus pneumoniae. Novobiocin, an old antibiotic, was tested in vitro and in a murine sepsis model against one amoxicillin-susceptible and three amoxicillin-resistant strains [minimum inhibitory concentrations (MICs) 8-64 mg/L]. Novobiocin MICs for all strains were 0.25-0.5 mg/L. In sepsis, novobiocin and amoxicillin were evaluated at 25, 50, 100 and 200 mg/kg given at 1, 5, 24 and 48 h post bacterial challenge. The most effective regimens in animals infected with the amoxicillin-susceptible strain were 200 mg/kg novobiocin and 25 mg/kg amoxicillin, achieving 100% survival and undetectable organisms in the peritoneum. Among mice infected with amoxicillin-resistant S. pneumoniae, 200 mg/kg novobiocin gave the highest protection (90-100% survivors), followed by 200mg/kg amoxicillin (60-100%), 100 mg/kg novobiocin (50-87.5%) and 50 mg/kg amoxicillin (14.3-25%). The killing effect of antibiotics in the peritoneum (mean Deltalog(10) colony-forming units/mL between treated and control mice) was as follows: 200 mg/kg novobiocin (-6.6)>200 mg/kg amoxicillin (-5.6)>100 mg/kg novobiocin (-3.7) > 50 mg/kg amoxicillin (-0.7). Total plasma and ultrafiltrate pharmacokinetics of novobiocin (200 mg/kg, single dose) in non-infected mice showed, respectively, half-lives of 151 min and 215 min, area under the concentration-time curves (AUCs) of 945.0 mgh/L and 136.6 mgh/L and maximal concentrations of 147 mg/L and 18 mg/L. Novobiocin may be a promising agent for therapy of highly beta-lactam-resistant pneumococcal infections.International journal of antimicrobial agents 03/2010; 35(6):544-9. · 3.03 Impact Factor -
Article: Enhanced in vivo activity of cefditoren in pre-immunized mice against penicillin-resistant S. pneumoniae (serotypes 6B, 19F and 23F) in a sepsis model.
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ABSTRACT: Specific antibodies are likely to be present before S. pneumoniae infection. We explored cefditoren (CDN) total and free values of serum concentrations exceeding the MIC (t>MIC) related to efficacy in a mice sepsis model, and the effect of specific gammaglobulins on in-vitro phagocytosis and in-vivo efficacy. We used three pneumococcal isolates (serotype, MIC OF CDN): Strain 1 (6B, 1 microg/ml), Strain 2 (19F, 2 microg/ml) and Strain 3 (23F, 4 microg/ml). Hyperimmune serum (HS) was obtained from mice immunized with heat-inactivated strains. In-vitro, phagocytosis by HS diluted 1/10 in presence/absence of sub-inhibitory concentrations was measured by flow cytometry including fluorescent bacteria and a neutrophil cell line. In-vivo dose-ranging experiments with HS (dilutions 1/2-1/16) and CDN (6.25 mg/kg-100 mg/kg tid for 48 h) were performed to determine the minimal protective dilution/dose (highest survival) and the non-protective highest dilution/dose (highest mortality: HS-np dilution and CDN-np dose) over 7 days. Efficacy of CDN-np in animals pre-immunized with HS-np (combined strategy) was explored and blood bacterial clearance determined. The CDN measured protein binding was 86.9%. In-vitro, CDN significantly increased phagocytosis (vs. HS 1/10). In non pre-immunized animals, t>MIC values for CDN of approximately 35% (total) and approximately 19% (free) were associated with 100% survival. Significant differences in survival were found between HS-np alone (< or = 20%) or CDN-np alone (< or = 20%) vs. the combined strategy (90%, 60% and 60% for Stains 1, 2 and 3), with t>MIC (total/free) of 22.8%/14.3%, 26.8%/16.0%, and 22.4%/12.7% for Strains 1, 2 and 3, respectively. Prior to the second dose (8 h), median bacterial counts were significantly lower in animals surviving vs. dead at day 7. In mice (CDN protein binding similar to humans) total t>MIC values of approximately 35% (approximately 19% free) were efficacious, with a decrease in the required values in pre-immunized animals. This reinforces that immunoprotection to overcome resistance may provide lifesaving strategies.PLoS ONE 01/2010; 5(8):e12041. · 4.09 Impact Factor -
Article: Versatility of the capsular genes during biofilm formation by Streptococcus pneumoniae.
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ABSTRACT: Streptococcus pneumoniae forms part of the natural microbiota of the nasopharynx. For the pneumococcus to cause infection, colonization needs to occur and this process is mediated by adherence of bacteria to the respiratory epithelium. Although the capsular polysaccharide (CPS) of S. pneumoniae is known to be important for infection to occur, its role in colonization is controversial. Biofilm models are starting to emerge as a promising tool to investigate the role of CPS during nasopharyngeal carriage, which is the first step in the dissemination and initiation of a pneumococcal infection. Using a well-defined model system to analyse in vitro biofilm formation in pneumococcus, here we explore the molecular changes underlying the appearance of capsular mutants using type 3 S. pneumoniae cells. Spontaneous colony phase variants show promoter mutations, as well as duplications, deletions and point mutations in the cap3A gene, which codes for a UDP-glucose dehydrogenase (UDP-GlcDH). Increased biofilm-forming capacity could usually be correlated with a reduction both in colony size and in the relative amount of CPS present on the cell surface of each colony variant. However, a mutation in Cap3A Thr83Ile (a strictly conserved residue in bacterial UDP-GlcDHs) that resulted in very low CPS production also led to impaired biofilm formation. We propose that non-encapsulated mutants of pneumococcal type 3 strains are essentially involved in the initial stages (the attachment stage) of biofilm formation during colonization/pathogenesis.Environmental Microbiology 07/2009; 11(10):2542-55. · 5.84 Impact Factor -
Article: Comparative genomic analysis of ten Streptococcus pneumoniae temperate bacteriophages.
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ABSTRACT: Streptococcus pneumoniae is an important human pathogen that often carries temperate bacteriophages. As part of a program to characterize the genetic makeup of prophages associated with clinical strains and to assess the potential roles that they play in the biology and pathogenesis in their host, we performed comparative genomic analysis of 10 temperate pneumococcal phages. All of the genomes are organized into five major gene clusters: lysogeny, replication, packaging, morphogenesis, and lysis clusters. All of the phage particles observed showed a Siphoviridae morphology. The only genes that are well conserved in all the genomes studied are those involved in the integration and the lysis of the host in addition to two genes, of unknown function, within the replication module. We observed that a high percentage of the open reading frames contained no similarities to any sequences catalogued in public databases; however, genes that were homologous to known phage virulence genes, including the pblB gene of Streptococcus mitis and the vapE gene of Dichelobacter nodosus, were also identified. Interestingly, bioinformatic tools showed the presence of a toxin-antitoxin system in the phage phiSpn_6, and this represents the first time that an addition system in a pneumophage has been identified. Collectively, the temperate pneumophages contain a diverse set of genes with various levels of similarity among them.Journal of bacteriology 07/2009; 191(15):4854-62. · 3.94 Impact Factor
Top co-authors
Top Journals
Institutions
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2005–2012
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Centro de Investigaciones Biológicas
Madrid, Madrid, Spain -
University of Buenos Aires
- Facultad de Farmacia y Bioquímica
Buenos Aires, Buenos Aires F.D., Argentina
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1998–2011
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Spanish National Research Council
- Biological Research Centre
Madrid, Madrid, Spain
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2010
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Centro de Investigación Biomédica en Red de Enfermedades Raras
Valencia, Valencia, Spain
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2007
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Fundación Jiménez Díaz
Madrid, Madrid, Spain
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2003
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Instituto de Salud Carlos III
Madrid, Madrid, Spain
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