Nasal immunization of mice with Lactobacillus casei expressing the Pneumococcal Surface Protein A: induction of antibodies, complement deposition and partial protection against Streptococcus pneumoniae challenge

Strategies for the development of new vaccines against Streptococcus pneumoniae infections try to overcome problems such as serotype coverage and high costs, present in currently available vaccines. Formulations based on protein candidates that can induce protection in animal models have been pointed as good alternatives. Among them, the Pneumococcal Surface Protein A (PspA) plays an important role during systemic infection at least in part through the inhibition of complement deposition on the pneumococcal surface, a mechanism of evasion from the immune system. Antigen delivery systems based on live recombinant lactic acid bacteria (LAB) represents a promising strategy for mucosal vaccination, since they are generally regarded as safe bacteria able to elicit both systemic and mucosal immune responses. In this work, the N-terminal region of clade I PspA was constitutively expressed in Lactobacillus casei and the recombinant bacteria was tested as a mucosal vaccine in mice. Nasal immunization with L. casei-PspA 1 induced anti-PspA antibodies that were able to bind to pneumococcal strains carrying both clade 1 and clade 2 PspAs and to induce complement deposition on the surface of the bacteria. In addition, an increase in survival of immunized mice after a systemic challenge with a virulent pneumococcal strain was observed. (C) 2008 Elsevier Masson SAS. All rights reserved.

Estudo da resistência do streptococcus pneumoniae à penicilina em pneumopatias infecciosas nas cidades de Porto Alegre e Caxias do Sul (RS - Brasil)

A resistência aos antibióticos dos patógenos mais comuns do trato respiratório está aumentando mundialmente. Recentemente, Streptococcus pneumoniae resistente à penicilina tem sido isolado em diversos países, e a freqüência dessas cepas tem elevado de modo alarmante. O aumento da resistência, com conseqüentes implicações terapêuticas, tem levado a uma reavaliação do uso dos antibióticos ß-lactâmicos para o tratamento de infecções pneumocócicas. No presente trabalho, um total de 107 amostras de Streptococcus pneumoniae, obtidas de materiais provenientes de pacientes adultos ambulatoriais e hospitalizados, em dois centros médicos de duas cidades do Rio Grande do Sul (Porto Alegre e Caxias do Sul), os quais apresentavam quadro clínico-radiológico de infecção pulmonar, foram analisadas com o objetivo de estudar-se a resistência do germe à penicilina. As amostras constituídas de escarro (80,4%), lavado brônquico (13,5%) e aspirado traqueal (6,6%) foram coletadas no período compreendido entre Julho de 1998 e Julho de 1999. O material foi semeado em meio de Agar sangue e as colônias suspeitas de Streptococcus pneumoniae foram transferidas para meio de Mueller-Hinton para teste de optoquina e de sensibilidade à penicilina com discos de oxacilina. Um halo de inibição da oxacilina menor do que 20 mm indicava a realização de teste para determinação da concentração inibitória mínima (MIC) com E-test. Um total de nove cepas foi identificado como tendo resistência intermediária à penicilina (MIC 0,1-1,0μg/ml) e nenhuma cepa resistente (resistência elevada: MIC > 2,0 μg/ml) foi identificada. Uma monitorização local das cepas quanto à resistência antimicrobiana é de grande importância para os clínicos no manejo de infecções pneumocócicas.

Antimicrobial resistance of isolated Streptococcus pneumoniae in a hospital of the Brazilian public system

Streptococcus pneumoniae is the predominant bacterial agent that affects the human population with pneumonia. This disease is an important cause of death in the elderly and the children under five years old. In this study, 29 strains of invasive S. pneumoniae were isolated from 29 patients of pneumonia, bacteremia and meningitis in the laboratory of the Municipal Hospital in Paulinia, Brazil, from May 2006 to October 2007. Patients' age ranged from 8 months old to 60 years old. These strains of S. pneumoniae were isolated from blood, pleural fluid and cerebrospinal fluid (CSF) of patients. After typing of encapsulated strains of S. pneumoniae through quellung reaction, their resistance to antimicrobial agents was gauged through Disc Diffusion Technique followed by determination of minimum inhibitory concentration (MIC). Among the 29 strains analyzed, 23 were methicillin-sensitive and six were methicillin-resistant and penicillin intermediate resistant. No strain presented full resistance to penicillin. Serotyping was performed only in two samples, which belonged to serotype 18. Our data may alert ambulatory regarding the incidence of pneumococcal strains resistant to the most common drugs due to inappropriate use of antimicrobials and also collaborate to the elaboration of pneumococcal conjugate vaccines specific to each region.

Streptococcus pneumoniae: susceptibility to penicillin and moxifloxacin

Objective: To determine the minimum inhibitory concentrations (MICs) of parenteral penicillin and moxifloxacin against Streptococcus pneumoniae strains isolated at a hospital center. Methods: In-vitro, prospective study involving 100 S. pneumoniae isolates collected from patients who had been treated, between October of 2008 and July of 2010, at the Hospital das Clinicas complex of the University of Sao Paulo School of Medicine, located in the city of Sao Paulo, Brazil. The isolates were obtained from respiratory tract cultures or blood samples unrelated to meningeal infections, and they were tested for penicillin and moxifloxacin susceptibility by E-test. The MIC category interpretations were based on updated standards. Results: All isolates were fully susceptible to parenteral penicillin (MIC <= 2 mu g/mL), and, consequently, they were also susceptible to amoxicillin, ampicillin, third/fourth generation cephalosporins, and ertapenem. Of the S. pneumoniae strains, 99% were also susceptible to moxifloxacin, and only one strain showed an MIC = 1.5 mu g/mL (intermediate). Conclusions: Our results showed high susceptibility rates to parenteral penicillin and moxifloxacin among S. pneumoniae isolates unrelated to meningitis, which differs from international reports. Reports on penicillin resistance should be based on updated breakpoints for non-meningitis isolates in order to guide the selection of an antimicrobial therapy and to improve the prediction of the clinical outcomes.

Custos hospitalares da meningite causada por Streptococcus pneumoniae na cidade de São José dos Campos, São Paulo, Brasil

O conhecimento dos custos hospitalares é de grande importância para os processos de tomada de decisão em saúde pública. O objetivo deste estudo foi estimar os custos hospitalares diretos relacionados à meningite pneumocócica em crianças com até 13 anos (inclusive), na cidade de São José dos Campos, São Paulo, Brasil, de janeiro de 1999 a dezembro de 2008. Foram obtidos dados de prontuários médicos. O cálculo foi realizado pelo método misto de mensuração das quantidades dos itens de custos e atribuição de valor aos itens consumidos (micro-costing e gross-costing). Os valores monetários referem-se a novembro de 2009, sendo expressos em reais. A análise das frequências e médias foi realizada pelo programa Epi Info versão 3.5.1. Foram notificados 41 casos. Os custos hospitalares diretos variaram de R$ 1.277,90 a R$ 19.887,56 (média = R$ 5.666,43), ou seja, 10 a 20 vezes maiores que o custo médio de internações pago pelo SUS. Os custos dos honorários profissionais foram os mais relevantes, seguidos pelos custos dos medicamentos, procedimentos, materiais e exames laboratoriais.

Study of two novel streptococcus pneumoniae operons: pilus islet 2 and the lantibiotic operon

Analysis of publicly available genomes of Streptococcus pneumoniae has led to the identification of a new genomic element resembling gram-positive pilus islets (PIs). Here, we demonstrate that this genomic region, herein referred to as PI-2 (containing the genes pitA, sipA, pitB, srtG1, and srtG2) codes for a novel functional pilus in pneumococcus. Therefore, there are two pilus islets identified so far in this pathogen (PI-1 and PI-2). Polymerization of the PI-2 pilus requires the backbone protein PitB as well as the sortase SrtG1 and the signal peptidase-like protein SipA. PI-2 is associated with serotypes 1, 2, 7F, 19A, and 19F, considered to be emerging in both industrialized and developing countries. Interestingly, strains belonging to clonal complex 271 (CC271) contain both PI-1 and PI-2, as revealed by genome analyses. In these strains both pili are surface exposed and independently assembled. Furthermore, in vitro experiments provide evidence that the pilus encoded by PI-2 of S. pneumoniae is involved in adherence. Thus, pneumococci encode at least two types of pili that may play a role in the initial host cell contact to the respiratory tract. In addition, the pilus proteins are potential antigens for inclusion in a new generation of pneumococcal vaccines. Adherence by pili could represent important factor in bacterial community formation, since it has been demonstrated that bacterial community formation plays an important role in pneumococcal otitis media. In vitro quantification of bacterial community formation by S. pneumoniae was performed in order to investigate the possible role of pneumococcal pili to form communities. By using different growth media we were not able to see clear association between pili and community formation. But our findings revealed that strains belonging to MLST clonal complex CC15 efficiently form bacterial communities in vitro in a glucose dependent manner. We compared the genome of forty-four pneumococcal isolates discovering four open reading frames specifically associated with CC15. These four genes are annotated as members of an operon responsible for the biosynthesis of a putative lanctibiotic peptide, described to be involved in bacterial community formation. Our experiments show that the lanctibiotic operon deletion affects glucose mediated community formation in CC 15 strain INV200. Moreover, since glucose consumption during bacterial growth produce an acidic environment, we tested bacterial community formation at different pH and we showed that the lanctibiotic operon deletion affected pH mediated community formation in CC 15 strain INV200. In conclusion, these data demonstrate that the putative lanctibiotic operon is associated with pneumococcal CC 15 strains in vitro bacterial community formation.

Epidemiological and functional characterization of Streptococcus pneumoniae pili

Streptococcus pneumoniae is an important life threatening human pathogen causing agent of invasive diseases such as otitis media, pneumonia, sepsis and meningitis, but is also a common inhabitant of the respiratory tract of children and healthy adults. Likewise most streptococci, S. pneumoniae decorates its surface with adhesive pili, composed of covalently linked subunits and involved in the attachment to epithelial cells and virulence. The pneumococcal pili are encoded by two genomic regions, pilus islet 1 (PI-1), and pilus islet-2 (PI-2), which are present in about 30% and 16% of the pneumococcal strains, respectively. PI-1 exists in three clonally related variants, whereas PI-2 is highly conserved. The presence of the islets does not correlate with the serotype of the strains, but with the genotype (as determined by Multi Locus Sequence Typing). The prevalence of PI-1 and PI-2 positive strains is similar in isolates from invasive disease and carriage. To better dissect a possible association between PIs presence and disease we evaluated the distribution of the two PIs in a panel of 113 acute otitis media (AOM) clinical isolates from Israel. PI-1 was present in 30.1% (N=34) of the isolates tested, and PI-2 in 7% (N=8). We found that 50% of the PI-1 positive isolates belonged to the international clones Spain9V-3 (ST156) and Taiwan19F-14 (ST236), and that PI-2 was not present in the absence of Pl-1. In conclusion, there was no correlation between PIs presence and AOM, and, in general, the observed differences in PIs prevalence are strictly dependent upon regional differences in the distribution of the clones. Finally, in the AOM collection the prevalence of PI-1 was higher among antibiotic resistant isolates, confirming previous indications obtained by the in silico analysis of the MLST database collection. Since the pilus-1 subunits were shown to confer protection in mouse models of infection both in active and passive immunization studies, and were regarded as potential candidates for a new generation of protein-based vaccines, the functional characterization was mainly focused on S. pneumoniae pilus -1 components. The pneumococcal pilus-1 is composed of three subunits, RrgA, RrgB and RrgC, each stabilized by intra-molecular isopeptide bonds and covalently polymerized by means of inter-molecular isopeptide bonds to form an extended fibre. The pilus shaft is a multimeric structure mainly composed by the RrgB backbone subunit. The minor ancillary proteins are located at the tip and at the base of the pilus, where they have been proposed to act as the major adhesin (RrgA) and as the pilus anchor (RrgC), respectively. RrgA is protective in in vivo mouse models, and exists in two variants (clades I and II). Mapping of the sequence variability onto the RrgA structure predicted from X-ray data showed that the diversity was restricted to the “head” of the protein, which contains the putative binding domains, whereas the elongated “stalk” was mostly conserved. To investigate whether this variability could influence the adhesive capacity of RrgA and to map the regions important for binding, two full-length protein variants and three recombinant RrgA portions were tested for adhesion to lung epithelial cells and to purified extracellular matrix (ECM) components. The two RrgA variants displayed similar binding abilities, whereas none of the recombinant fragments adhered at levels comparable to those of the full-length protein, suggesting that proper folding and structural arrangement are crucial to retain protein functionality. Furthermore, the two RrgA variants were shown to be cross-reactive in vitro and cross-protective in vivo in a murine model of passive immunization. Taken together, these data indicate that the region implicated in adhesion and the functional epitopes responsible for the protective ability of RrgA may be conserved and that the considerable level of variation found within the “head” domain of RrgA may have been generated by immunologic pressure without impairing the functional integrity of the pilus.

Capsule type of Streptococcus pneumoniae determines growth phenotype

The polysaccharide capsule of Streptococcus pneumoniae defines over ninety serotypes, which differ in their carriage prevalence and invasiveness for poorly understood reasons. Recently, an inverse correlation between carriage prevalence and oligosaccharide structure of a given capsule has been described. Our previous work suggested a link between serotype and growth in vitro. Here we investigate whether capsule production interferes with growth in vitro and whether this predicts carriage prevalence in vivo. Eighty-one capsule switch mutants were constructed representing nine different serotypes, five of low (4, 7F, 14, 15, 18C) and four of high carriage prevalence (6B, 9V, 19F, 23F). Growth (length of lag phase, maximum optical density) of wildtype strains, nontypeable mutants and capsule switch mutants was studied in nutrient-restricted Lacks medium (MLM) and in rich undefined brain heart infusion broth supplemented with 5% foetal calf serum (BHI+FCS). In MLM growth phenotype depended on, and was transferred with, capsule operon type. Colonization efficiency of mouse nasopharynx also depended on, and was transferred with, capsule operon type. Capsule production interfered with growth, which correlated inversely with serotype-specific carriage prevalence. Serotypes with better growth and higher carriage prevalence produced thicker capsules (by electron microscopy, FITC-dextran exclusion assays and HPLC) than serotypes with delayed growth and low carriage prevalence. However, expression of cpsA, the first capsule gene, (by quantitative RT-PCR) correlated inversely with capsule thickness. Energy spent for capsule production (incorporation of H3-glucose) relative to amount of capsule produced was higher for serotypes with low carriage prevalence. Experiments in BHI+FCS showed overall better bacterial growth and more capsule production than growth in MLM and differences between serotypes were no longer apparent. Production of polysaccharide capsule in S. pneumoniae interferes with growth in nutrient-limiting conditions probably by competition for energy against the central metabolism. Serotype-specific nasopharyngeal carriage prevalence in vivo is predicted by the growth phenotype.

Inflammasome-dependent IFN-γ drives pathogenesis in Streptococcus pneumoniae meningitis

The pathology associated with Streptococcus pneumoniae meningitis results largely from activation of immune-associated pathways. We systematically investigated the production of IFN subtypes, as well as their influence on pathology, in a mouse model of S. pneumoniae meningitis. Despite the occurrence of a mixed IFN type I/II gene signature, no evidence for production or involvement of type I IFNs in disease progression was found. In contrast, type II IFN (IFN-γ) was strongly induced, and IFN-γ(-/-) mice were significantly protected from severe disease. Using intracellular cytokine staining and targeted cell-depletion approaches, NK cells were found to be the dominant source of IFN-γ. Furthermore, production of IFN-γ was found to be dependent upon ASC and IL-18, indicating that an ASC-dependent inflammasome pathway was responsible for mediating IFN-γ induction. The influence of IFN-γ gene deletion on a range of processes known to be involved in bacterial meningitis pathogenesis was examined. Although neutrophil numbers in the brain were similar in infected wild-type and IFN-γ(-/-) mice, both monocyte recruitment and CCL2 production were less in infected IFN-γ(-/-) mice compared with infected wild-type controls. Additionally, gene expression of NO synthase was strongly diminished in infected IFN-γ(-/-) mice compared with infected controls. Finally, bacterial clearance was enhanced in IFN-γ(-/-) mice, although the underlying mechanism remains unclear. Together, these data suggest that inflammasome-dependent IFN-γ contributes via multiple pathways to pathology during S. pneumoniae meningitis.

Heteroresistance to Fosfomycin Is Predominant in Streptococcus pneumoniae and Depends on the murA1 Gene

Fosfomycin targets the first step of peptidoglycan biosynthesis in Streptococcus pneumoniae catalyzed by UDP-N-acetylglucosamine enolpyruvyltransferase (MurA1). We investigated whether heteroresistance to fosfomycin occurs in S. pneumoniae. We found that of 11 strains tested, all but 1 (Hungary(19A)) displayed heteroresistance and that deletion of murA1 abolished heteroresistance. Hungary(19A) differs from the other strains by a single amino acid substitution in MurA1 (Ala364Thr). To test whether this substitution is responsible for the lack of heteroresistance, it was introduced into strain D39. The heteroresistance phenotype of strain D39 was not changed. Furthermore, no relevant structural differences between the MurA1 crystal structures of heteroresistant strain D39 and nonheteroresistant strain Hungary(19A) were found. Our results reveal that heteroresistance to fosfomycin is the predominant phenotype of S. pneumoniae and that MurA1 is required for heteroresistance to fosfomycin but is not the only factor involved. The findings provide a caveat for any future use of fosfomycin in the treatment of pneumococcal infections.

Serotype-specific invasiveness and colonization prevalence in Streptococcus pneumoniae correlate with the lag phase during in vitro growth

Serotypes of Streptococcus pneumoniae differ in colonization prevalence and the likelihood of causing disease. In vitro growth in brain heart infusion broth with or without 5% fetal calf serum (FCS) was compared for 47 clinical isolates representing 15 pneumococcal serotypes. Serotype-specific colonization prevalence and odds ratios for the invasive potential were obtained from an international and a local epidemiological study. The duration of the lag phase increased with the invasiveness and was inversely associated with the colonization prevalence of a serotype. Supplementation with FCS shortened the lag phase preferentially in serotypes associated with invasive disease (P=0.007). Reduction of oxidative stress by addition of manganese (Mn(2+)), Tiron, mannitol or catalase did not influence the duration of the lag phase significantly. Serotype specific invasiveness and colonization prevalence of S. pneumoniae are associated with the length of the lag phase during in vitro growth. This may correlate with serotype specific selection in vivo.

The prevalence of penicillin-non-susceptible Streptococcus pneumoniae among children aged < 5 years correlates with the biannual epidemic activity of respiratory syncytial virus

This study investigated whether the epidemiology of penicillin-non-susceptible pneumococci (PNSP) colonising small children correlated with the biannual epidemic activity of respiratory syncytial virus (RSV). Colonisation rates and the prevalence of PNSP among paediatric outpatients aged < 5 years was analysed between January 1998 and September 2003 using an established national surveillance network. Resistance trends were investigated using time-series analysis to assess the correlation with the biannual pattern of RSV infections and national sales of oral paediatric formulations of antibiotics and antibiotic prescriptions to children aged < 5 years for acute respiratory tract infections. PNSP rates exhibited a biannual cycle in phase with the biannual seasonal RSV epidemics (p < 0.05). Resistance rates were higher during the winter seasons of 1998-1999 (20.1%), 2000-2001 (16.0%) and 2002-2003 (19.1%), compared with the winter seasons of 1997-1998 (8.2%), 1999-2000 (11.6%) and 2001-2002 (9.5%). Antibiotic sales and prescriptions showed regular peaks during each winter, with no significant correlation with the biannual pattern of RSV activity and seasonal trends of PNSP. RSV is an important determinant of the spread of PNSP and must be considered in strategies aimed at antimicrobial resistance control.

Distribution and invasiveness of Streptococcus pneumoniae serotypes in Switzerland, a country with low antibiotic selection pressure, from 2001 to 2004

To describe the serotype-specific epidemiology of colonizing and invasive Streptococcus pneumoniae isolates, which is important for vaccination strategies, we analyzed a total of 2,388 invasive and 1,540 colonizing S. pneumoniae isolates collected between January 2001 and December 2004 within two nationwide surveillance programs. We found that the relative rank orders of the most frequent serotypes (serotypes 1, 3, 4, 6B, 7F, 14, 19F, and 23F) differed among invasive and colonizing isolates. Serotypes 1, 4, 5, 7F, 8, 9V, and 14 had increased invasive potential, and serotypes/serogroups 3, 6A, 7, 10, 11, 19F, and 23F were associated with colonization. The proportion of pediatric serotypes was higher among children < 5 years old (48.5%) and persons > 64 years old (34.1%) than among other age groups (29.1%); it was also higher in West Switzerland (40.2%) than in other geographic regions (34.7%). Likewise, serotype-specific proportions of penicillin-resistant isolates for types 6B, 9V, 14, and 19F were significantly higher in West Switzerland. The relative frequency of pediatric serotypes corresponded with antibiotic consumption patterns. We conclude that the epidemiology of invasive and colonizing S. pneumoniae isolates is influenced by the serotype-specific potential for invasiveness, and therefore, surveillance programs should include colonizing and invasive S. pneumoniae isolates. Antibiotic selection pressure determines the serotype distribution in different age groups and geographic regions and therefore the expected direct and indirect effects of the 7-valent conjugate vaccine.

Clindamycin is neuroprotective in experimental Streptococcus pneumoniae meningitis compared with ceftriaxone

In animal models of Streptococcus pneumoniae meningitis, rifampin is neuroprotective in comparison to ceftriaxone. So far it is not clear whether this can be generalized for other protein synthesis-inhibiting antimicrobial agents. We examined the effects of the bactericidal protein synthesis-inhibiting clindamycin (n = 12) on the release of proinflammatory bacterial components, the formation of neurotoxic compounds and neuronal injury compared with the standard therapy with ceftriaxone (n = 12) in a rabbit model of pneumococcal meningitis. Analysis of the CSF and histological evaluation were combined with microdialysis from the hippocampal formation and the neocortex. Compared with ceftriaxone, clindamycin reduced the release of lipoteichoic acids from the bacteria (p = 0.004) into the CSF and the CSF leucocyte count (p = 0.011). This led to lower extracellular concentrations of hydroxyl radicals (p = 0.034) and glutamate (p = 0.016) in the hippocampal formation and a subsequent reduction of extracellular glycerol levels (p = 0.018) and neuronal apoptosis in the dentate gyrus (p = 0.008). The present data document beneficial effects of clindamycin compared with ceftriaxone on various parameters linked with the pathophysiology of pneumococcal meningitis and development of neuronal injury. This study suggests neuroprotection to be a group effect of bactericidal protein synthesis-inhibiting antimicrobial agents compared with the standard therapy with beta-lactam antibiotics in meningitis.