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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. 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 tra, Depto. de Genética, Fisiología y Microbiología, Fac. de Ciencias Biológicas, TRUE, pub

Background: Introduction of pneumococcal conjugate vaccines (PCVs) has reduced the disease caused by vaccine serotypes in children, providing herd protection to adults. However, the emergence of nonvaccine serotypes is of great concern worldwide. Methods: This study includes national laboratory data from invasive pneumococcal disease (IPD) cases that affected pediatric and adult populations during 2009–2019. The impact of implementing different vaccine strategies for immunocompetent adults by comparing Spanish regions that used the 13-valent PCV (PCV13) vs regions that used the 23-valent pneumococcal polysaccharide vaccine (PPV23) was also analyzed for 2017−2019. Results: The overall reductions in IPD cases by PCV13 serotypes in children and adults were 88% and 59%, respectively, during 2009–2019, with a constant increase in 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 serotypes 3 and 8 accounted for 36% of IPD cases in adults. Introduction of PCV13 or PPV23 in the adult calendar of certain Spanish regions reduced the IPD cases by PCV13 serotypes by up to 25% and 11%, respectively, showing a decrease of serotype 3 when PCV13 was used. Conclusions: Use of PCV13 in children has affected the epidemiology, reducing the burden of IPD in children but also in adults by herd protection; however, the increase in serotype 8 in adults is worrisome. Vaccination with PCV13 in adults seems to control IPD cases by PCV13 serotypes including serotype 3., Ministerio de Economía, Industria y Competitividad, Instituto de Salud Carlos III, Depto. de Genética, Fisiología y Microbiología, Fac. de Ciencias Biológicas, TRUE, pub

The cell wall glucosaminidase LytB of Streptococcus pneumoniae is a surface exposed protein involved in daughter cell separation, biofilm formation and contributes to different aspects of the pathogenesis process. In this study we have characterized the antibody responses after immunization of mice with LytB in the presence of alhydrogel as an adjuvant. Enzyme-linked immunosorbent assays measuring different subclasses of immunoglobulin G, demonstrated that the antibody responses to LytB were predominantly IgG1 and IgG2b, followed by IgG3 and IgG2a subclasses. Complement-mediated immunity against two different pneumococcal serotypes was investigated using sera from immunized mice. Immunization with LytB increased the recognition of S. pneumoniae by complement components C1q and C3b demonstrating that anti-LytB antibodies trigger activation of the classical pathway. Phagocytosis assays showed that serum containing antibodies to LytB stimulates neutrophil-mediated phagocytosis against S. pneumoniae. Animal models of infection including invasive pneumonia and sepsis were performed with two different clinical isolates. Vaccination with LytB increased bacterial clearance and induced protection demonstrating that LytB might be a good candidate to be considered in a future protein-based vaccine against S. pneumoniae.

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.

Capsular switching allows pre-existing clones of Streptococcus pneumoniae expressing vaccine serotypes to escape the vaccine-induced immunity by acquisition of capsular genes from pneumococci of a non-vaccine serotype. Here, we have analysed the clonal composition of 492 clinical isolates of serotype 11A causing invasive disease in Spain (2000–2012), and their ability to evade the host immune response. Antibiograms, serotyping and molecular typing were performed. The restriction profiles of pbp2x, pbp1a and pbp2b genes were also analysed. Interaction with the complement components C1q, C3b, C4BP, and factor H was explored whereas opsonophagocytosis assays were performed using a human cell line differentiated to neutrophils. Biofilm formation and the polymorphisms of the major autolysin LytA were evaluated. The main genotypes of the 11A pneumococci were: ST62 (447 isolates, 90.6%), followed by ST6521 (35 isolates, 7.3%) and ST838 (10 isolates, 2.1%). Beta lactam resistant serotype 11A variants of genotypes ST838 and ST6521 closely related to the Spain9V-ST156 clone were first detected in 2005. A different pattern of evasion of complement immunity and phagocytosis was observed between genotypes. The emergence of one vaccine escape variant of Spain9V-ST156 (ST652111A), showing a high potential to avoid the host immune response, was observed.In addition, isolates of ST652111A showed higher ability to produce biofilms than ST83811A or ST6211A, which may have contributed to the emergence of this PEN-resistant ST652111A genotype in the last few years. The emergence of penicillin-resistant 11A invasive variants of the highly successful ST156 clonal complex merits close monitoring.

Objectives: To phenotypically and genotypically characterize 11 strains (isolated in four different centres) exhibiting penicillin MIC of 8-32 mg/L among isolates of the SPICE project. Nine isolates were from Romania (9/162; 5.56%) and two from Poland (2/305; 0.66%). Methods: In vitro susceptibility was determined in triplicate by microdilution (CLSI guidelines), and additionally, MICs of penicillin, cefotaxime and amoxicillin were confirmed in triplicate by agar dilution. Multilocus sequence typing (MLST), PFGE and gene amplification and sequencing were performed. Results: For the nine Romanian isolates, MICs were >/=16 mg/L for penicillin, cefotaxime and amoxicillin, >/=32 mg/L for cefuroxime and cefpodoxime, 4-8 mg/L for cefditoren and >/=128 mg/L for erythromycin and gentamicin. All isolates were non-susceptible to imipenem (MIC = 0.5-1 mg/L) and susceptible to levofloxacin (MIC = 0.5-1 mg/L) and vancomycin (MIC = 0.25-0.5 mg/L). These Romanian strains presented a new cluster in the 595-600 region of PBP2X (YSGIQL-->LSTPWF) conferring 98% homology with Streptococcus mitis PBP2X, with a new MurM allele (seven strains) with eight amino acid changes versus R6. PBP nucleotide sequences were highly conserved suggesting a common origin. Allelic profiles of two strains gave sequence type 321, three strains exhibited a single- and four a double-locus variance. MLST-predicted serotype was 23F in all but one strain (19F), but three strains were 19A by Quellung. Conclusions: The multidrug high resistance (precluding adequate oral therapy in children), its origin, the prevalence found in Romania and the presence of non-vaccine (7-valent) serotypes should worry the medical community because of a possible clonal diffusion that would limit therapeutic alternatives., Tedec-Meiji Farma S.A., Depto. de Medicina, Fac. de Medicina, TRUE, pub

Background: Although protein binding is a reversible phenomenon, it is assumed that antibacterial activity is exclusively exerted by the free (unbound) fraction of antibiotics. Methodology/principal findings: Activity of cefditoren, a highly protein bound 3(rd) generation cephalosporin, over 24h after an oral 400 mg cefditoren-pivoxil bid regimen was studied against six S. pneumoniae strains (penicillin/cefditoren MICs; microg/ml): S1 (0.12/0.25), S2 (0.25/0.25), S3 and S4 (0.5/0.5), S5 (1/0.5) and S6 (4/0.5). A computerized pharmacodynamic simulation with media consisting in 75% human serum and 25% broth (mean albumin concentrations = 4.85+/-0.12 g/dL) was performed. Protein binding was measured. The cumulative percentage of a 24h-period that drug concentrations exceeded the MIC for total (T > MIC) and unbound concentrations (fT > MIC), expressed as percentage of the dosing interval, were determined. Protein binding was 87.1%. Bactericidal activity (> or = 99.9% initial inocula reduction) was obtained against strains S1 and S2 at 24h (T > MIC = 77.6%, fT > MIC = 23.7%). With T > MIC of 61.6% (fT > MIC = 1.7%), reductions against S3 and S4 ranged from 90% to 97% at 12h and 24h; against S5, reduction was 45.1% at 12h and up to 85.0% at 24h; and against S6, reduction was 91.8% at 12h, but due to regrowth of 52.9% at 24h. Cefditoren physiological concentrations exerted antibacterial activity against strains exhibiting MICs of 0.25 and 0.5 microg/ml under protein binding conditions similar to those in humans. Conclusions/significance: The results of this study suggest that, from the pharmacodynamic perspective, the presence of physiological albumin concentrations may not preclude antipneumococcal activity of highly bound cephalosporins as cefditoren., Tedec-Meiji Farma S.A., Depto. de Medicina, Fac. de Medicina, TRUE, pub

Objectives: To investigate antimicrobial effects on a mixed culture of five Streptococcus pneumoniae serotypes (S) as an approach to ecology of population dynamics. Methods: A computerized pharmacodynamic model simulating concentrations obtained after levofloxacin, ciprofloxacin and azithromycin doses was used. Resistance patterns were S12, susceptible to study drugs; S31, low-level macrolide-resistant (efflux phenotype); S11, high-level macrolide-resistant (erm genotype); S9V, low-level quinolone-resistant; and S3, high-level quinolone-resistant. Initial mixed inocula (time 0) included similar percentages of each serotype. Results: At 24 h of control drug-free experiments, dominant strains were S9V (57.4%) and S12 (41.8%) with marginal populations of S31, S3 and S11. Azithromycin selected to a much higher extent the strain with low-level resistance to macrolides (S31) rather than the strain with high-level resistance (S11) (accounting for 99.9% versus 0.1% of the total population at 24 h). Ciprofloxacin selected to a higher extent low-level (S9V) rather than high-level (S3) quinolone resistance (72.4% versus 27.6%). Levofloxacin decreased the proportion of the predominant S9V in controls to 22.2% (an intermediate-resistant strain with MIC = 4 mg/L) and unmasked the high-level resistant strain (MIC = 32 mg/L) up to 77.8%. Conclusions: Strain distribution in an antibiotic-free environment depends on bacterial fitness in mono- and multi-strain niches. Selective pressure of antimicrobial regimens eradicate some populations and unmask minor populations, thus redistributing the whole population. Selective potential only for resistance phenotypes with very low prevalence (such as high-level quinolone resistance) in the community should be preferred to that selecting more prevalent resistance phenotypes., GlaxoSmithKline S.A., Sanofi-Aventis S.A., Depto. de Medicina, Fac. de Medicina, TRUE, pub

Objectives: High-level penicillin resistance in Streptococcus pneumoniae requires extensive re-modulation of the penicillin-binding proteins (PBPs), and murM gene function is also required for the expression of resistance. In this work, we determined whether specific changes in PBPs were associated with specific MurM variants. Methods: Two collections of highly penicillin-resistant (MIC 2-8 mg/L) isolates, including 10 early (1997-1998) and 23 contemporary (2002-2004) isolates, were studied. Results: Most of the isolates belonged to clones Spain(6B)-2 (13 strains), Spain(23F)-1 (10 isolates) and Spain(14)-5 (20 isolates). Different protein variants of MurM (MA, MB5, MB6, MB9 and MB10), PBP1A (A-C), PBP2B (A-D) and PBP2X (A-C) were recognized, including two murM alleles not previously described. Particular [MurM-PBP1A-2B-2X] allelic combinations were predominant among the different clones, including [MA-B-B-B] for old (MIC 2 mg/L) and [MB10-C-A-B] for recent (MIC 4-8 mg/L) Spain(6B)-2 isolates, [MA-A-C-A] for Spain(23F)-1 and [MB5-A-A-A] in Spain(14)-5 isolates. Conclusions: Although S. pneumoniae has a basic recombinational population structure, our results indicate remarkable conservation of PBPs and MurM protein types within each clone. This suggests that particular PBPs-MurM combinations tend to be preserved and may have an independent evolutionary history in particular clones., Depto. de Medicina, Fac. de Medicina, TRUE, pub

Objectives: Phage-coded lysins, i.e. murein hydrolases, are enzymes that destroy the cell wall of bacteria. A rapid killing of Streptococcus pneumoniae in the nasopharynx of mice has been described recently using a phage-coded murein hydrolase (enzybiotic). The in vivo effects of a dose-ranging treatment, using either of the phage-coded lytic enzymes Cpl-1 lysozyme or the Pal amidase, have been investigated here in a murine sepsis model., Methods: Purified Pal amidase and/or Cpl-1 lysozyme were used alone or in combination. These enzymes were injected intraperitoneally at different times after challenge with 5 × 107 cfu of a type 6B, antibiotic-resistant S. pneumoniae clinical isolate., Results: Animals challenged with 5 × 107 cfu of this strain alone died within 72 h, whereas a single intraperitoneal injection of Cpl-1 or Pal (200 µg; 1100 U) administered 1 h after the bacterial challenge was sufficient to effectively protect the mice, according to unpaired t-test (P < 0.0001). Bacteraemia in unprotected mice reached colony counts >107 cfu/mL, whereas the mean colony count in lysin-protected animals was <106 cfu/mL over time and ultimately became undetectable. Interestingly, a synergic effect in vivo was observed with the combined use of 2.5 µg each of Cpl-1 and Pal., Conclusions: Our findings suggest strongly that phage lysins protect animals from bacteraemia and death. Moreover, the simultaneous attack of the pneumococcal peptidoglycan by a lysozyme and an amidase leads to a remarkable effect through enhanced destruction of the bacterial cell wall. The benefits of therapy with enzybiotics against pneumococcus reported here might warrant the examination of alternative strategies for the treatment of diseases caused by clinically relevant pathogens.