A national longitudinal study evaluating the activity of cefditoren and other antibiotics against non-susceptible Streptococcus pneumoniae strains during the period 2004-20 in Spain.

BACKGROUND: Surveillance studies including antibiotic resistance and evolution of pneumococcal serotypes are critical to evaluate the susceptibility of commonly used antibiotics and the contribution of conjugate vaccines against resistant strains. OBJECTIVES: To determine the susceptibility of clinical isolates of Streptococcus pneumoniae with reduced susceptibility to penicillin to a panel of antibiotics during the period 2004-20 and characterize the impact of pneumococcal conjugate vaccines in the evolution of resistant serotypes. METHODS: We selected 3017 clinical isolates in order to determine the minimal inhibitory concentration to penicillin, amoxicillin, cefotaxime, erythromycin, levofloxacin and oral cephalosporins, including cefditoren, cefixime and cefpodoxime. RESULTS: The antibiotics with the lowest proportion of resistant strains from 2004 to 2020 were cefditoren (<0.4%), followed by cefotaxime (<5%), penicillin (<6.5%) and levofloxacin (<7%). Among oral cephalosporins, cefixime was the cephalosporin with the highest MIC90 (32 mg/L) and MIC50 (8-16 mg/L) throughout the study, followed by cefpodoxime with highest values of MIC90 (4 mg/L) and MIC50 (2 mg/L) for the majority of the study period. In contrast, cefditoren was the cephalosporin with the lowest MIC90 (1 mg/L) and MIC50 (0.25-0.5 mg/L). CONCLUSIONS: Cefditoren was the antibiotic with the highest proportion of susceptible strains. Hence, more than 80% of the clinical strains were susceptible to cefditoren throughout the period 2004-20. The proportion of resistant isolates to cefditoren and cefotaxime was scarce, being less than 0.4% for cefditoren and lower than 5% for cefotaxime, despite the increased rates of serotypes not covered by the 13-valent pneumococcal conjugate vaccine.

New live attenuated tuberculosis vaccine MTBVAC induces trained immunity and confers protection against experimental lethal pneumonia.

Among infectious diseases, tuberculosis is the leading cause of death worldwide, and represents a serious threat, especially in developing countries. The protective effects of Bacillus Calmette-Guerin (BCG), the current vaccine against tuberculosis, have been related not only to specific induction of T-cell immunity, but also with the long-term epigenetic and metabolic reprogramming of the cells from the innate immune system through a process termed trained immunity. Here we show that MTBVAC, a live attenuated strain of Mycobacterium tuberculosis, safe and immunogenic against tuberculosis antigens in adults and newborns, is also able to generate trained immunity through the induction of glycolysis and glutaminolysis and the accumulation of histone methylation marks at the promoters of proinflammatory genes, facilitating an enhanced response after secondary challenge with non-related bacterial stimuli. Importantly, these findings in human primary myeloid cells are complemented by a strong MTBVAC-induced heterologous protection against a lethal challenge with Streptococcus pneumoniae in an experimental murine model of pneumonia.

Nationwide Trends of Invasive Pneumococcal Disease in Spain From 2009 Through 2019 in Children and Adults During the Pneumococcal Conjugate Vaccine Era.

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.

The N-Acetylglucosaminidase LytB of Streptococcus pneumoniae Is Involved in the Structure and Formation of Biofilms.

The N-acetylglucosaminidase LytB of Streptococcus pneumoniae is involved in nasopharyngeal colonization and is responsible for cell separation at the end of cell division; thus, ΔlytB mutants form long chains of cells. This paper reports the construction and properties of a defective pneumococcal mutant producing an inactive LytB protein (LytBE585A). It is shown that an enzymatically active LytB is required for in vitro biofilm formation, as lytB mutants (either ΔlytB or producing the inactive LytBE585A) are incapable of forming substantial biofilms, despite that extracellular DNA is present in the biofilm matrix. Adding small amounts (0.5 to 2.0 µg/ml) of exogenous LytB or some LytB constructs restored the biofilm-forming capacity of lytB mutants to wild-type levels. The LytBE585A mutant formed biofilm more rapidly than ΔlytB mutants in the presence of LytB. This suggests that the mutant protein acted in a structural role, likely through the formation of complexes with extracellular DNA. The chain-dispersing capacity of LytB allowed the separation of daughter cells, presumably facilitating the formation of microcolonies and, finally, of biofilms. A role for the possible involvement of LytB in the synthesis of the extracellular polysaccharide component of the biofilm matrix is also discussed.IMPORTANCE It has been previously accepted that biofilm formation in S. pneumoniae must be a multigenic trait because the mutation of a single gene has led to only to partial inhibition of biofilm production. In the present study, however, evidence that the N-acetylglucosaminidase LytB is crucial in biofilm formation is provided. Despite the presence of extracellular DNA, strains either deficient in LytB or producing a defective LytB enzyme formed only shallow biofilms.

PSGL-1 on Leukocytes is a Critical Component of the Host Immune Response against Invasive Pneumococcal Disease.

Bacterial uptake by phagocytic cells is a vital event in the clearance of invading pathogens such as Streptococcus pneumoniae. A major role of the P-selectin glycoprotein ligand-1 (PSGL-1) on leukocytes against invasive pneumococcal disease is described in this study. Phagocytosis experiments using different serotypes demonstrated that PSGL-1 is involved in the recognition, uptake and killing of S. pneumoniae. Co-localization of several clinical isolates of S. pneumoniae with PSGL-1 was demonstrated, observing a rapid and active phagocytosis in the presence of PSGL-1. Furthermore, the pneumococcal capsular polysaccharide and the main autolysin of the bacterium--the amidase LytA--were identified as bacterial ligands for PSGL-1. Experimental models of pneumococcal disease including invasive pneumonia and systemic infection showed that bacterial levels were markedly increased in the blood of PSGL-1-/- mice. During pneumonia, PSGL-1 controls the severity of pneumococcal dissemination from the lung to the bloodstream. In systemic infection, a major role of PSGL-1 in host defense is to clear the bacteria in the systemic circulation controlling bacterial replication. These results confirmed the importance of this receptor in the recognition and clearance of S. pneumoniae during invasive pneumococcal disease. Histological and cellular analysis demonstrated that PSGL-1-/- mice have increased levels of T cells migrating to the lung than the corresponding wild-type mice. In contrast, during systemic infection, PSGL-1-/- mice had increased numbers of neutrophils and macrophages in blood, but were less effective controlling the infection process due to the lack of this functional receptor. Overall, this study demonstrates that PSGL-1 is a novel receptor for S. pneumoniae that contributes to protection against invasive pneumococcal disease.

N-Acetyl-l-Cysteine and Cysteamine as New Strategies against Mixed Biofilms of Nonencapsulated Streptococcus pneumoniae and Nontypeable Haemophilus influenzae.

Acute otitis media, a polymicrobial disease of the middle ear cavity of children, is a significant public health problem worldwide. It is most frequently caused by encapsulated Streptococcus pneumoniae and nontypeable Haemophilus influenzae, although the widespread use of pneumococcal conjugate vaccines is apparently producing an increase in the carriage of nonencapsulated S. pneumoniae Frequently, pneumococci and H. influenzae live together in the human nasopharynx, forming a self-produced biofilm. Biofilms present a global medical challenge since the inherent antibiotic resistance of their producers demands the use of large doses of antibiotics over prolonged periods. Frequently, these therapeutic measures fail, contributing to bacterial persistence. Here, we describe the development of an in vitro nonencapsulated S. pneumoniae-nontypeable H. influenzae biofilm system with polystyrene or glass-bottom plates. Confocal laser scanning microscopy and specific fluorescent labeling of pneumococcal cells with Helix pomatia agglutinin revealed an even distribution of both species within the biofilm. This simple and robust protocol of mixed biofilms was used to test the antimicrobial properties of two well-known antioxidants that are widely used in the clinical setting, i.e., N-acetyl-l-cysteine and cysteamine. This repurposing approach showed the high potency of N-acetyl-l-cysteine and cysteamine against mixed biofilms of nonencapsulated S. pneumoniae and nontypeable H. influenzae Decades of clinical use mean that these compounds are safe to use, which may accelerate their evaluation in humans.

Pleiotropic effects of cell wall amidase LytA on Streptococcus pneumoniae sensitivity to the host immune response.

The complement system is a key component of the host immune response for the recognition and clearance of Streptococcus pneumoniae. In this study, we demonstrate that the amidase LytA, the main pneumococcal autolysin, inhibits complement-mediated immunity independently of effects on pneumolysin by a complex process of impaired complement activation, increased binding of complement regulators, and direct degradation of complement C3. The use of human sera depleted of either C1q or factor B confirmed that LytA prevented activation of both the classical and alternative pathways, whereas pneumolysin inhibited only the classical pathway. LytA prevented binding of C1q and the acute-phase protein C-reactive protein to S. pneumoniae, thereby reducing activation of the classical pathway on the bacterial surface. In addition, LytA increased recruitment of the complement downregulators C4BP and factor H to the pneumococcal cell wall and directly cleaved C3b and iC3b to generate degradation products. As a consequence, C3b deposition and phagocytosis increased in the absence of LytA and were markedly enhanced for the lytA ply double mutant, confirming that a combination of LytA and Ply is essential for the establishment of pneumococcal pneumonia and sepsis in a murine model of infection. These data demonstrate that LytA has pleiotropic effects on complement activation, a finding which, in combination with the effects of pneumolysin on complement to assist with pneumococcal complement evasion, confirms a major role of both proteins for the full virulence of the microorganism during septicemia.

In vitro biofilm formation by Streptococcus pneumoniae as a predictor of post-vaccination emerging serotypes colonizing the human nasopharynx.

The increasing use of the 7-valent pneumococcal conjugate vaccine has been accompanied by the rise of non-vaccine serotypes colonizing the human nasopharynx. The vast majority of infections are caused by microorganisms that grow in biofilms. It has recently been shown that the formation of Streptococcus pneumoniae biofilms in vivo and in vitro is hindered by the presence of capsular polysaccharide. The biofilm-forming capacity of pneumococcal clinical isolates with different types of capsular polysaccharide and various isogenic transformants was examined. Strains of serotypes 19A and 19F, but not 19B and 19C, formed ≥ 80% of the quantity of biofilm associated with a non-encapsulated control strain. Strains of serogroup 6 also showed significant biofilm-forming capacity. The capsules of serotypes 19A and 19F, and serogroup 6 contain the disaccharides α-D-Glcp-(1→2)-α-L-Rhap-(1→ and α-D-Glcp-(1→3)-α-L-Rhap-(1→. Serotype 18A and serotypes 18B/18C have very similar capsular disaccharides: α-D-GlcpNAc-(1→3)-ß-L-Rhap-(1→ and α-D-Glcp-(1→3)-ß-L-Rhap-(1→ respectively. However, the strains of serogroup 18 showed impaired biofilm formation. These results indicate that the chemical composition/structure of the capsular polysaccharide is crucial to the biofilm-forming capacity of pneumococcal serotypes. Testing of the in vitro biofilm-forming ability of isogenic transformants expressing different capsular polysaccharides may help predict the emergence of colonizing, non-vaccine serotypes.

Surveillance of invasive pneumococcal disease in Spain exploring the impact of the COVID-19 pandemic (2019-2023).

OBJECTIVES: Dynamic trends of invasive pneumococcal disease (IPD) including the evolution of prevalent serotypes are very useful to evaluate the impact of current and future pneumococcal conjugate vaccines (PCVs) and the rise of non-vaccine serotypes. In this study, we include epidemiological patterns of S. pneumoniae before and after COVID-19 pandemic. METHODS: We characterized all national IPD isolates from children and adults received at the Spanish Pneumococcal Reference Laboratory during 2019-2023. RESULTS: In the first pandemic year 2020, we found a general reduction in IPD cases across all age groups, followed by a partial resurgence in children in 2021 but not in adults. By 2022, IPD cases in children had returned to pre-pandemic levels, and partially in adults. In 2023, IPD rates surpassed those of the last pre-pandemic year. Notably, the emergence of serotype 3 is of significant concern, becoming the leading cause of IPD in both pediatric and adult populations over the last two years (2022-2023). Increase of serotype 4 in young adults occurred in the last epidemiological years. CONCLUSIONS: The COVID-19 pandemic led to a temporary decline in all IPD cases during 2020 attributable to non-pharmaceutical interventions followed by a subsequent rise. Employing PCVs with broader coverage and/or enhanced immunogenicity may be critical to mitigate the marked increase of IPD.

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.

Insight into the composition of the intercellular matrix of Streptococcus pneumoniae biofilms.

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.

Trends in invasive bacterial diseases during the first 2 years of the COVID-19 pandemic: analyses of prospective surveillance data from 30 countries and territories in the IRIS Consortium.

BACKGROUND: The Invasive Respiratory Infection Surveillance (IRIS) Consortium was established to assess the impact of the COVID-19 pandemic on invasive diseases caused by Streptococcus pneumoniae, Haemophilus influenzae, Neisseria meningitidis, and Streptococcus agalactiae. We aimed to analyse the incidence and distribution of these diseases during the first 2 years of the COVID-19 pandemic compared to the 2 years preceding the pandemic. METHODS: For this prospective analysis, laboratories in 30 countries and territories representing five continents submitted surveillance data from Jan 1, 2018, to Jan 2, 2022, to private projects within databases in PubMLST. The impact of COVID-19 containment measures on the overall number of cases was analysed, and changes in disease distributions by patient age and serotype or group were examined. Interrupted time-series analyses were done to quantify the impact of pandemic response measures and their relaxation on disease rates, and autoregressive integrated moving average models were used to estimate effect sizes and forecast counterfactual trends by hemisphere. FINDINGS: Overall, 116 841 cases were analysed: 76 481 in 2018-19, before the pandemic, and 40 360 in 2020-21, during the pandemic. During the pandemic there was a significant reduction in the risk of disease caused by S pneumoniae (risk ratio 0·47; 95% CI 0·40-0·55), H influenzae (0·51; 0·40-0·66) and N meningitidis (0·26; 0·21-0·31), while no significant changes were observed for S agalactiae (1·02; 0·75-1·40), which is not transmitted via the respiratory route. No major changes in the distribution of cases were observed when stratified by patient age or serotype or group. An estimated 36 289 (95% prediction interval 17 145-55 434) cases of invasive bacterial disease were averted during the first 2 years of the pandemic among IRIS-participating countries and territories. INTERPRETATION: COVID-19 containment measures were associated with a sustained decrease in the incidence of invasive disease caused by S pneumoniae, H influenzae, and N meningitidis during the first 2 years of the pandemic, but cases began to increase in some countries towards the end of 2021 as pandemic restrictions were lifted. These IRIS data provide a better understanding of microbial transmission, will inform vaccine development and implementation, and can contribute to health-care service planning and provision of policies. FUNDING: Wellcome Trust, NIHR Oxford Biomedical Research Centre, Spanish Ministry of Science and Innovation, Korea Disease Control and Prevention Agency, Torsten Söderberg Foundation, Stockholm County Council, Swedish Research Council, German Federal Ministry of Health, Robert Koch Institute, Pfizer, Merck, and the Greek National Public Health Organization.

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.

Clearance of mixed biofilms of Streptococcus pneumoniae and methicillin-susceptible/resistant Staphylococcus aureus by antioxidants N-acetyl-L-cysteine and cysteamine.

Biofilm-associated infections are of great concern because they are associated with antibiotic resistance and immune evasion. Co-colonization by Staphylococcus aureus and Streptococcus pneumoniae is possible and a threat in clinical practice. We investigated the interaction between S. aureus and S. pneumoniae in mixed biofilms and tested new antibiofilm therapies with antioxidants N-acetyl-L-cysteine (NAC) and cysteamine (Cys). We developed two in vitro S. aureus-S. pneumoniae mixed biofilms in 96-well polystyrene microtiter plates and we treated in vitro biofilms with Cys and NAC analyzing their effect by CV staining and viable plate counting. S. pneumoniae needed a higher proportion of cells in the inoculum and planktonic culture to reach a similar population rate in the mixed biofilm. We demonstrated the effect of Cys in preventing S. aureus biofilms and S. aureus-S. pneumoniae mixed biofilms. Moreover, administration of 5 mg/ml of NAC nearly eradicated the S. pneumoniae population and killed nearly 94% of MSSA cells and 99% of MRSA cells in the mixed biofilms. The methicillin resistance background did not change the antioxidants effect in S. aureus. These results identify NAC and Cys as promising repurposed drug candidates for the prevention and treatment of mixed biofilms by S. pneumoniae and S. aureus.

Effect of pneumococcal conjugate vaccines and SARS-CoV-2 on antimicrobial resistance and the emergence of Streptococcus pneumoniae serotypes with reduced susceptibility in Spain, 2004-20: a national surveillance study.

BACKGROUND: Epidemiological studies are necessary to explore the effect of current pneumococcal conjugate vaccines (PCVs) against antibiotic resistance, including the rise of non-vaccine serotypes that are resistant to antibiotics. Hence, epidemiological changes in the antimicrobial pattern of Streptococcus pneumoniae before and during the first year of the COVID-19 pandemic were studied. METHODS: In this national surveillance study, we characterised the antimicrobial susceptibility to a panel of antibiotics in 3017 pneumococcal clinical isolates with reduced susceptibility to penicillin during 2004-20 in Spain. This study covered the early and late PCV7 periods; the early, middle, and late PCV13 periods; and the first year of the COVID-19 pandemic, to evaluate the contribution of PCVs and the pandemic to the emergence of non-vaccine serotypes associated with antibiotic resistance. FINDINGS: Serotypes included in PCV7 and PCV13 showed a decline after the introduction of PCVs in Spain. However, an increase in non-PCV13 serotypes (mainly 11A, 24F, and 23B) that were not susceptible to penicillin promptly appeared. A rise in the proportion of pneumococcal strains with reduced susceptibility to ß-lactams and erythromycin was observed in 2020, coinciding with the emergence of SARS-CoV-2. Cefditoren was the ß-lactam with the lowest minimum inhibitory concentration (MIC)50 or MIC90 values, and had the highest proportion of susceptible strains throughout 2004-20. INTERPRETATION: The increase in non-PCV13 serotypes associated with antibiotic resistance is concerning, especially the increase of penicillin resistance linked to serotypes 11A and 24F. The future use of PCVs with an increasingly broad spectrum (such as PCV20, which includes serotype 11A) could reduce the impact of antibiotic resistance for non-PCV13 serotypes. The use of antibiotics to prevent co-infections in patients with COVID-19 might have affected the increased proportion of pneumococcal-resistant strains. Cefotaxime as a parenteral option, and cefditoren as an oral choice, were the antibiotics with the highest activity against non-PCV20 serotypes. FUNDING: The Spanish Ministry of Science and Innovation and Meiji-Pharma Spain. TRANSLATION: For the Spanish translation of the abstract see Supplementary Materials section.

Development of colorimetric sensors based on gold nanoparticles for SARS-CoV-2 RdRp, E and S genes detection.

We present a fast, reliable and easy to scale-up colorimetric sensor based on gold nanoparticles (AuNPs) to detect the sequences coding for the RdRp, E, and S proteins of SARS-CoV-2. The optimization of the system (so-called "the sensor") includes the evaluation of different sizes of nanoparticles, sequences of oligonucleotides and buffers. It is stable for months without any noticeable decrease in its activity, allowing the detection of SARS-CoV-2 sequences by the naked eye in 15 min. The efficiency and selectivity of detection, in terms of significative colorimetric changes in the solution upon target recognition, are qualitatively (visually) and quantitatively (absorbance measurements) assessed using synthetic samples and samples derived from infected cells and patients. Furthermore, an easy and affordable amplification approach is implemented to increase the system's sensitivity for detecting high and medium viral loads (≥103 - 104 viral RNA copies/µl) in patient samples. The whole process (amplification and detection) takes 2.5 h. Due to the ease of use, stability and minimum equipment requirements, the proposed approach can be a valuable tool for the detection of SARS-CoV-2 at facilities with limited resources.

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.

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.

In vitro destruction of Streptococcus pneumoniae biofilms with bacterial and phage peptidoglycan hydrolases.

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.

Vaccination with LytA, LytC, or Pce of Streptococcus pneumoniae Protects against Sepsis by Inducing IgGs That Activate the Complement System.

The emergence of non-vaccine serotypes of Streptococcus pneumoniae after the use of vaccines based in capsular polysaccharides demonstrates the need of a broader protection vaccine based in protein antigens and widely conserved. In this study, we characterized three important virulence factors of S. pneumoniae namely LytA, LytC, and Pce as vaccine candidates. These proteins are choline-binding proteins that belong to the cell wall hydrolases' family. Immunization of mice with LytA, LytC, or Pce induced high titers of immunoglobulins G (IgGs) of different subclasses, with IgG1, IgG2a, and IgG2b as the predominant immunoglobulins raised. These antibodies activated the classical pathway of the complement system by increasing the recognition of C1q on the surface of pneumococcal strains of different serotypes. Consequently, the key complement component C3 recognized more efficiently these strains in the presence of specific antibodies elicited by these proteins, activating, therefore, the phagocytosis. Finally, a mouse sepsis model of infection was established, confirming that vaccination with these proteins controlled bacterial replication in the bloodstream, increasing the survival rate. Overall, these results demonstrate that LytA, LytC, and Pce can be protein antigens to be contained in a future universal vaccine against S. pneumoniae.

Pneumococcal Choline-Binding Proteins Involved in Virulence as Vaccine Candidates.

Streptococcus pneumoniae is a pathogen responsible for millions of deaths worldwide. Currently, the available vaccines for the prevention of S. pneumoniae infections are the 23-valent pneumococcal polysaccharide-based vaccine (PPV-23) and the pneumococcal conjugate vaccines (PCV10 and PCV13). These vaccines only cover some pneumococcal serotypes (up to 100 different serotypes have been identified) and are unable to protect against non-vaccine serotypes and non-encapsulated pneumococci. The emergence of antibiotic-resistant non-vaccine serotypes after these vaccines is an increasing threat. Therefore, there is an urgent need to develop new pneumococcal vaccines which could cover a wide range of serotypes. One of the vaccines most characterized as a prophylactic alternative to current PPV-23 or PCVs is a vaccine based on pneumococcal protein antigens. The choline-binding proteins (CBP) are found in all pneumococcal strains, giving them the characteristic to be potential vaccine candidates as they may protect against different serotypes. In this review, we have focused the attention on different CBPs as vaccine candidates because they are involved in the pathogenesis process, confirming their immunogenicity and protection against pneumococcal infection. The review summarizes the major contribution of these proteins to virulence and reinforces the fact that antibodies elicited against many of them may block or interfere with their role in the infection process.