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.

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.

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.

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.

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.

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.

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.

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.

Antibacterial activity of a DNA topoisomerase I inhibitor versus fluoroquinolones in Streptococcus pneumoniae.

The DNA topoisomerase complement of Streptococcus pneumoniae is constituted by two type II enzymes (topoisomerase IV and gyrase), and a single type I enzyme (topoisomerase I). These enzymes maintain the DNA topology, which is essential for replication and transcription. While fluoroquinolones target the type II enzymes, seconeolitsine, a new antimicrobial agent, targets topoisomerase I. We compared for the first time the in vitro effect of inhibition of topoisomerase I by seconeolitsine and of the type II topoisomerases by the fluoroquinolones levofloxacin and moxifloxacin. We used three isogenic non-encapsulated strains and five non-vaccine serotypes isolates belonging to two circulating pneumococcal clones, ST638 (2 strains) and ST1569V (3 strains). Each group contained strains with diverse susceptibility to fluoroquinolones. Minimal inhibitory concentrations, killing curves and postantibiotic effects were determined. Seconeolitsine demonstrated the fastest and highest bactericidal activity against planktonic bacteria and biofilms. When fluoroquinolone-susceptible planktonic bacteria were considered, seconeolitsine induced postantibiotic effects (1.00-1.87 h) similar than levofloxacin (1.00-2.22 h), but longer than moxifloxacin (0.39-1.71 h). The same effect was observed in sessile bacteria forming biofilms. Seconeolitsine induced postantibiotic effects (0.84-2.31 h) that were similar to those of levofloxacin (0.99-3.32 h) but longer than those of moxifloxacin (0.89-1.91 h). The greatest effect was observed in the viability and adherence of bacteria in the postantibiotic phase. Seconeolitsine greatly reduced the thickness of the biofilms formed in comparison with fluoroquinolones: 2.91 ± 0.43 µm (seconeolitsine), 7.18 ± 0.58 µm (levofloxacin), 17.08 ± 1.02 µm (moxifloxacin). When fluoroquinolone-resistant bacteria were considered, postantibiotic effects induced by levofloxacin and moxifloxacin, but not by seconeolitsine, were shorter, decreasing up to 5-fold (levofloxacin) or 2-fold (moxifloxacin) in planktonic cells, and up to 1.7 (levofloxacin) or 1.4-fold (moxifloxacin) during biofilm formation. Therefore, topoisomerase I inhibitors could be an alternative for the treatment of pneumococcal diseases, including those caused by fluoroquinolone-resistant isolates.

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.

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

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

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.

Widening the antimicrobial spectrum of esters of bicyclic amines: In vitro effect on gram-positive Streptococcus pneumoniae and gram-negative non-typeable Haemophilus influenzae biofilms.

Antibiotic resistance is a global current threat of increasing importance. Moreover, biofilms represent a medical challenge since the inherent antibiotic resistance of their producers demands the use of high doses of antibiotics over prolonged periods. Frequently, these therapeutic measures fail, contributing to bacterial persistence, therefore demanding the development of novel antimicrobials. Esters of bicyclic amines (EBAs), which are strong inhibitors of Streptococcus pneumoniae growth, were initially designed as inhibitors of pneumococcal choline-binding proteins on the basis of their structural analogy to the choline residues in the cell wall. However, instead of mimicking the characteristic cell chaining phenotype caused by exogenously added choline on planktonic cultures of pneumococcal cells, EBAs showed an unexpected lytic activity. In this work we demonstrate that EBAs display a second, and even more important, function as cell membrane destabilizers. We then assayed the inhibitory and disintegrating activity of these molecules on pneumococcal biofilms. The selected compound (EBA 31) produced the highest effect on S. pneumoniae (encapsulated and non-encapsulated) biofilms at very low concentrations. EBA 31 was also effective on mixed biofilms of non-encapsulated S. pneumoniae plus non-typeable Haemophilus influenzae, two pathogens frequently forming a self-produced biofilm in the human nasopharynx. These results support the role of EBAs as a promising alternative for the development of novel, broad-range antimicrobial drugs encompassing both Gram-positive and Gram-negative pathogens.

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

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

The Streptococcus pneumoniaeyefM-yoeB and relBE Toxin-Antitoxin Operons Participate in Oxidative Stress and Biofilm Formation.

Type II (proteic) toxin-antitoxin systems (TAs) are widely distributed among bacteria and archaea. They are generally organized as operons integrated by two genes, the first encoding the antitoxin that binds to its cognate toxin to generate a harmless protein⁻protein complex. Under stress conditions, the unstable antitoxin is degraded by host proteases, releasing the toxin to achieve its toxic effect. In the Gram-positive pathogen Streptococcus pneumoniae we have characterized four TAs: pezAT, relBE, yefM-yoeB, and phD-doc, although the latter is missing in strain R6. We have assessed the role of the two yefM-yoeB and relBE systems encoded by S. pneumoniae R6 by construction of isogenic strains lacking one or two of the operons, and by complementation assays. We have analyzed the phenotypes of the wild type and mutants in terms of cell growth, response to environmental stress, and ability to generate biofilms. Compared to the wild-type, the mutants exhibited lower resistance to oxidative stress. Further, strains deleted in yefM-yoeB and the double mutant lacking yefM-yoeB and relBE exhibited a significant reduction in their ability for biofilm formation. Complementation assays showed that defective phenotypes were restored to wild type levels. We conclude that these two loci may play a relevant role in these aspects of the S. pneumoniae lifestyle and contribute to the bacterial colonization of new niches.

Csl2, a novel chimeric bacteriophage lysin to fight infections caused by Streptococcus suis, an emerging zoonotic pathogen.

Streptococcus suis is a Gram-positive bacterium that infects humans and various animals, causing human mortality rates ranging from 5 to 20%, as well as important losses for the swine industry. In addition, there is no effective vaccine for S. suis and isolates with increasing antibiotic multiresistance are emerging worldwide. Facing this situation, wild type or engineered bacteriophage lysins constitute a promising alternative to conventional antibiotics. In this study, we have constructed a new chimeric lysin, Csl2, by fusing the catalytic domain of Cpl-7 lysozyme to the CW_7 repeats of LySMP lysin from an S. suis phage. Csl2 efficiently kills different S. suis strains and shows noticeable activity against a few streptococci of the mitis group. Specifically, 15 µg/ml Csl2 killed 4.3 logs of S. suis serotype 2 S735 strain in 60 min, in a buffer containing 150 mM NaCl and 10 mM CaCl2, at pH 6.0. We have set up a protocol to form a good biofilm with the non-encapsulated S. suis mutant strain BD101, and the use of 30 µg/ml Csl2 was enough for dispersing such biofilms and reducing 1-2 logs the number of planktonic bacteria. In vitro results have been validated in an adult zebrafish model of infection.

Fluorescence Imaging of Streptococcus pneumoniae with the Helix pomatia agglutinin (HPA) As a Potential, Rapid Diagnostic Tool.

Streptococcus pneumoniae is a common human pathogen and a major causal agent of life-threatening infections that can either be respiratory or non-respiratory. It is well known that the Helix pomatia (edible snail) agglutinin (HPA) lectin shows specificity for terminal αGalNAc residues present, among other locations, in the Forssman pentasaccharide (αGalNAc1→3ßGalNAc1→3αGal1→4ßGal1→4ßGlc). Based on experiments involving choline-independent mutants and different growth conditions, we propose here that HPA recognizes the αGalNAc terminal residues of the cell wall teichoic and lipoteichoic acids of S. pneumoniae. In addition, experimental evidence showing that pneumococci can be specifically labeled with HPA when growing as planktonic cultures as well as in mixed biofilms of S. pneumoniae and Haemophilus influenzae has been obtained. It should be underlined that pneumococci were HPA-labeled despite of the presence of a capsule. Although some non-pneumococcal species also bind the agglutinin, HPA-binding combined with fluorescence microscopy constitutes a suitable tool for identifying S. pneumoniae and, if used in conjunction with Gram staining and/or other suitable technique like antigen detection, it may potentially facilitate a fast and accurate diagnosis of pneumococcal infections.

p-Cymene Promotes Its Catabolism through the p-Cymene and the p-Cumate Pathways, Activates a Stress Response and Reduces the Biofilm Formation in Burkholderia xenovorans LB400.

p-Cymene is an aromatic terpene that is present in diverse plant species. The aims of this study were to study the p-cymene metabolism in the model aromatic-degrading bacterium Burkholderia xenovorans LB400, and its response to p-cymene. The catabolic p-cymene (cym) and p-cumate (cmt) genes are clustered on the LB400 major chromosome. B. xenovorans LB400 was able to grow on p-cymene as well as on p-cumate as a sole carbon and energy sources. LB400 growth attained higher cell concentration at stationary phase on p-cumate than on p-cymene. The transcription of the key cymAb and cmtAb genes, and p-cumate dioxygenase activity were observed in LB400 cells grown on p-cymene and on p-cumate, but not in glucose-grown cells. Diverse changes on LB400 proteome were observed in p-cymene-grown cells compared to glucose-grown cells. An increase of the molecular chaperones DnaK, GroEL and ClpB, the organic hydroperoxide resistance protein Ohr, the alkyl hydroperoxide reductase AhpC and the copper oxidase CopA during growth on p-cymene strongly suggests that the exposure to p-cymene constitutes a stress condition for strain LB400. Diverse proteins of the energy metabolism such as enolase, pyruvate kinase, aconitase AcnA, succinyl-CoA synthetase beta subunit and ATP synthase beta subunit were induced by p-cymene. Electron microscopy showed that p-cymene-grown cells exhibited fuzzy outer and inner membranes and an increased periplasm. p-Cymene induced diverse membrane and transport proteins including the p-cymene transporter CymD. Biofilm formation was reduced during growth in p-cymene in strain LB400 compared to glucose-grown cells that may be associated with a decrease of diguanylate cyclase protein levels. Overall, these results indicate active p-cymene and p-cumate catabolic pathways in B. xenovorans LB400. In addition, this study showed that p-cymene activated a stress response in strain LB400 and reduced its biofilm formation.

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.