The Pneumococcus Blog: January 2015

Wednesday, January 28, 2015

Detection of co-colonization with Streptococcus pneumoniae by algorithmic use of conventional and molecular methods.

Vaccine. 2015 Jan 29;33(5):713-8. doi: 10.1016/j.vaccine.2014.11.040. Epub 2014 Dec 15.

Detection of co-colonization with Streptococcus pneumoniae by algorithmic use of conventional and molecular methods.

Saha S1, Modak JK1, Naziat H1, Al-Emran HM1, Chowdury M1, Islam M1, Hossain B1, Darmstadt GL2, Whitney CG3, Saha SK4.

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Abstract

Detection of pneumococcal carriage by multiple co-colonizing serotypes is important in assessing the benefits of pneumococcal conjugate vaccine (PCV). Various methods differing in sensitivity, cost and technical complexity have been employed to detect multiple serotypes of pneumococcus in respiratory specimens. We have developed an algorithmic method to detect all known serotypes that preserves the relative abundance of specific serotypes by using Quellung-guided molecular techniques. The method involves culturing respiratory swabs followed by serotyping of 100 colonies by either capsular (10 colonies) or PCR (90 colonies) reactions on 96-well plates. The method was evaluated using 102 nasal swabs from children carrying pneumococcus. Multiple serotypes were detected in 22% of carriers, compared to 3% by World Health Organization (WHO)-recommended morphology-based selection of 1 to 3 colonies. Our method, with a processing cost of $87, could detect subdominant strains making up as low as 1% of the population. The method is affordable, practical, and capable of detecting all known serotypes without false positive reactions or change in the native distribution of multiple serotypes.

KEYWORDS:

Carriage; Co-colonization; Pneumococcus; Quantitative detection

PMID: 25523524 [PubMed - in process]

Silver polyvinyl pyrrolidone nanoparticles exhibit a capsular polysaccharide influenced bactericidal effect against Streptococcus pneumoniae.

Front Microbiol. 2014 Dec 3;5:665. doi: 10.3389/fmicb.2014.00665. eCollection 2014.

Silver polyvinyl pyrrolidone nanoparticles exhibit a capsular polysaccharide influenced bactericidal effect against Streptococcus pneumoniae.

Bibbs RK1, Harris RD1, Peoples VA1, Barnett C2, Singh SR1, Dennis VA1, Coats MT3.

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Abstract

Streptococcus pneumoniae remains a leading cause of morbidity and mortality worldwide. The highly adaptive nature of S. pneumoniae exemplifies the need for next generation antimicrobials designed to avoid high level resistance. Metal based nanomaterials fit this criterion. Our study examined the antimicrobial activity of gold nanospheres, silver coated polyvinyl pyrrolidone (AgPVP), and titanium dioxide (TiO2) against various serotypes of S. pneumoniae. Twenty nanometer spherical AgPVP demonstrated the highest level of killing among the tested materials. AgPVP (0.6 mg/mL) was able to kill pneumococcal serotypes 2, 3, 4, and 19F within 4 h of exposure. Detailed analysis of cultures during exposure to AgPVP showed that both the metal ions and the solid nanoparticles participate in the killing of the pneumococcus. The bactericidal effect of AgPVP was lessened in the absence of the pneumococcal capsular polysaccharide. Capsule negative strains, JD908 and RX1, were only susceptible to AgPVP at concentrations at least 33% higher than their respective capsule expressing counterparts. These findings suggest that mechanisms of killing used by nanomaterials are not serotype dependent and that the capsular polysaccharide participates in the inhibition. In the near future these mechanisms will be examined as targets for novel antimicrobials.

KEYWORDS:

AgPVP; antimicrobial; nanoparticle; pneumococcus; serotype; titanium dioxide

PMID: 25520713 [PubMed] PMCID: PMC4253953

Streptococcus pneumoniae-Induced Oxidative Stress in Lung Epithelial Cells Depends on Pneumococcal Autolysis and Is Reversible by Resveratrol.

J Infect Dis. 2014 Dec 15. pii: jiu806. [Epub ahead of print]

Streptococcus pneumoniae-Induced Oxidative Stress in Lung Epithelial Cells Depends on Pneumococcal Autolysis and Is Reversible by Resveratrol.

Zahlten J1, Kim YJ1, Doehn JM1, Pribyl T2, Hocke AC1, García P3, Hammerschmidt S2, Suttorp N1, Hippenstiel S1, Hübner RH1.

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Abstract

BACKGROUND:

Streptococcus pneumoniae is the most common cause of community-acquired pneumonia worldwide. During pneumococcal pneumonia, the human airway epithelium is exposed to large amounts of H2O2 as a product of host and pathogen oxidative metabolism. Airway cells are known to be highly vulnerable to oxidant damage, but the pathophysiology of oxidative stress induced by S. pneumoniae and the role of nuclear factor erythroid 2-related factor 2 (Nrf2)-mediated antioxidant systems of the host are not well characterized.

METHODS:

For gluthation/gluthathion disulfide analysis BEAS-2B cells, primary broncho-epithelial cells (pBEC), explanted human lung tissue and mouse lungs were infected with different S. pneumoniae strains (D39, A66, R6x, H2O2/pneumolysin/LytA- deficient mutants of R6x). Cell death was proven by LDH assay and cell viability by IL-8 ELISA. The translocation of Nrf2 and the expression of catalase were shown via Western blot. The binding of Nrf2 at the catalase promoter was analyzed by ChIP.

RESULTS:

We observed a significant induction of oxidative stress induced by S. pneumoniae in vivo, ex vivo, and in vitro. Upon stimulation, the oxidant-responsive transcription factor Nrf2 was activated, and catalase was upregulated via Nrf2. The pneumococci-induced oxidative stress was independent of S. pneumoniae-derived H2O2 and pneumolysin but depended on the pneumococcal autolysin LytA. The Nrf2 inducer resveratrol, as opposed to catalase, reversed oxidative stress in lung epithelial cells.

CONCLUSIONS:

These observations indicate a H2O2-independent induction of oxidative stress in lung epithelial cells via the release of bacterial factors of S. pneumoniae. Resveratrol might be an option for prevention of acute lung injury and inflammatory responses observed in pneumococcal pneumonia.

KEYWORDS:

Nuclear factor erythroid 2-related factor 2; Streptococcus pneumoniae; autolysin; glutathione; hydrogen peroxide; lung epithelial cells; pneumolysin

PMID: 25512625 [PubMed - as supplied by publisher]

The α-Tocopherol Form of Vitamin E Reverses Age-Associated Susceptibility to Streptococcus pneumoniae Lung Infection by Modulating Pulmonary Neutrophil Recruitment.

J Immunol. 2015 Feb 1;194(3):1090-9. doi: 10.4049/jimmunol.1402401. Epub 2014 Dec 15.

The α-Tocopherol Form of Vitamin E Reverses Age-Associated Susceptibility to Streptococcus pneumoniae Lung Infection by Modulating Pulmonary Neutrophil Recruitment.

Bou Ghanem EN1, Clark S1, Du X2, Wu D2, Camilli A3, Leong JM4, Meydani SN5.

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Abstract

Streptococcus pneumoniae infections are an important cause of morbidity and mortality in older patients. Uncontrolled neutrophil-driven pulmonary inflammation exacerbates this disease. To test whether the α-tocopherol (α-Toc) form of vitamin E, a regulator of immunity, can modulate neutrophil responses as a preventive strategy to mitigate the age-associated decline in resistance to S. pneumoniae, young (4 mo) and old (22-24 mo) C57BL/6 mice were fed a diet containing 30-PPM (control) or 500-PPM (supplemented) α-Toc for 4 wk and intratracheally infected with S. pneumoniae. Aged mice fed a control diet were exquisitely more susceptible to S. pneumoniae than young mice. At 2 d postinfection, aged mice suffered 1000-fold higher pulmonary bacterial burden, 2.2-fold higher levels of neutrophil recruitment to the lung, and a 2.25-fold higher rate of lethal septicemia. Strikingly, α-Toc supplementation of aged mice resulted in a 1000-fold lower bacterial lung burden and full control of infection. This α-Toc-induced resistance to pneumococcal challenge was associated with a 2-fold fewer pulmonary neutrophils, a level comparable to S. pneumoniae-challenged, conventionally fed young mice. α-Toc directly inhibited neutrophil egress across epithelial cell monolayers in vitro in response to pneumococci or hepoxilin-A3, an eicosanoid required for pneumococcus-elicited neutrophil trans-epithelial migration. α-Toc altered expression of multiple epithelial and neutrophil adhesion molecules involved in migration, including CD55, CD47, CD18/CD11b, and ICAM-1. These findings suggest that α-Toc enhances resistance of aged mice to bacterial pneumonia by modulating the innate immune response, a finding that has potential clinical significance in combating infection in aged individuals through nutritional intervention.

PMID: 25512603 [PubMed - in process]

Bright fluorescent Streptococcus pneumoniae for live cell imaging of host-pathogen interactions.

J Bacteriol. 2014 Dec 15. pii: JB.02221-14. [Epub ahead of print]

Bright fluorescent Streptococcus pneumoniae for live cell imaging of host-pathogen interactions.

Kjos M1, Aprianto R1, Fernandes VE2, Andrew PW2, van Strijp JA3, Nijland R3, Veening JW4.

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Abstract

Streptococcus pneumoniae is a common nasopharyngeal resident in healthy people, but at the same time one of the major causes of infectious diseases such as pneumonia, meningitis and sepsis. The shift from commensal to pathogen and its interaction with host cells is poorly understood. One of the major limitations for research on pneumococcal-host interactions is the lack of suitable tools for live cell imaging. To address this issue, we developed a generally applicable strategy to create genetically stable, highly fluorescent bacteria. Our strategy relies on fusing superfolder green fluorescent protein (GFP) or a far-red fluorescent protein (RFP) to the abundant histone-like protein HlpA. Due to efficient translation and limited cellular diffusion of these fusions, the cells are 25-fold brighter than the currently best available imaging S. pneumoniae strain. These novel bright pneumococcal strains are fully virulent and the GFP-reporter can be used for in situ imaging in mouse tissue. We used our reporter strains to study the effect of the polysaccharide capsule, a major pneumococcal virulence factor, on different stages of infection. By dual-color live cell imaging experiments, we show that unencapsulated pneumococci adhere significantly better to human lung epithelial cells compared to encapsulated strains, in line with previous data obtained by classical approaches. We also confirm with live cell imaging that the capsule protects pneumococci from neutrophil phagocytosis, demonstrating the versatility and usability of our reporters. The described imaging tools will pave the way for live cell imaging of pneumococcal infection and help understand the mechanisms of pneumococcal pathogenesis.

PMID: 25512311 [PubMed - as supplied by publisher]

Evaluation of a rapid immunochromatographic ODK0501 assay for detecting Streptococcus pneumoniae antigens in the sputum of pneumonia patients with positive S. pneumoniae urinary antigens.

J Infect Chemother. 2014 Nov 20. pii: S1341-321X(14)00393-6. doi: 10.1016/j.jiac.2014.11.003. [Epub ahead of print]

Evaluation of a rapid immunochromatographic ODK0501 assay for detecting Streptococcus pneumoniae antigens in the sputum of pneumonia patients with positive S. pneumoniae urinary antigens.

Mukae H1, Yatera K2, Noguchi S3, Kawanami T2, Yamasaki K2, Tokuyama S4, Inoue N4, Nishida C2, Kawanami Y2, Ogoshi T2, Orihashi T3, Yoshii C5, Ishimoto H2.

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Abstract

BACKGROUND:

A novel, rapid and noninvasive test (ODK0501, RAPIRUN®Streptococcus pneumoniae) uses polyclonal antibodies to detect C polysaccharide of S. pneumoniae derived from sputum samples using an immunochromatographic assay. We evaluated its usefulness in Japanese patients with pneumonia who exhibited positive urinary antigen tests for S. pneumoniae (BinaxNOW®S. pneumoniae).

PATIENTS AND METHODS:

Forty adult patients with pneumonia treated between May 2011 and August 2013 were enrolled. Bacterial cultures, Gram staining and ODK0501 assays of sputum as well as urinary antigen tests for S. pneumoniae using urine samples obtained from the same patients were performed upon admission, the fourth day after starting antimicrobial treatment and at the end of the antimicrobial treatment.

RESULTS:

Twenty-seven of the 40 patients were positive for ODK0501, while a negative result for ODK0501 was associated with low-quality sputum samples according to the Geckler classification of sputum. The sensitivity and specificity of the ODK0501 assay in the 40 patients were 90.9% and 61.1%, respectively, based on the culture results. The results obtained with this kit were more favorable than those observed on Gram staining. The ODK0501 assay also showed a rapid reaction to the disappearance of S. pneumoniae in the sputum samples, while approximately 80% of the patients exhibited persistent positive results on the urinary antigen detection tests at the end of treatment.

CONCLUSIONS:

The ODK0501 test is a noninvasive, rapid and accurate tool for diagnosing respiratory infections caused by S. pneumoniae, although good quality sputum must be obtained prior to adequate treatment with antibiotics.

KEYWORDS:

ODK0501; Pneumococcal pneumonia; Sputum antigen detection; Streptococcus pneumoniae; Urinary antigen detection

PMID: 25511195 [PubMed - as supplied by publisher]

Proteomic analysis on the antibacterial activity of a Ru(II) complex against Streptococcus pneumoniae.

J Proteomics. 2014 Dec 10;115C:107-116. doi: 10.1016/j.jprot.2014.11.018. [Epub ahead of print]

Proteomic analysis on the antibacterial activity of a Ru(II) complex against Streptococcus pneumoniae.

Yang XY1, Zhang L2, Liu J3, Li N2, Yu G2, Cao K2, Han J2, Zeng G2, Pan Y4, Sun X5, He QY6.

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Abstract

Streptococcus pneumoniae is a Gram-positive pathogen that causes a variety of infection diseases in human. In this project, we determined the antibacterial activity of a Ru(II) complex X-03 against S. pneumoniae in vitro, by comparing its toxicity to host cells A549 and HBE. We performed two-dimensional gel electrophoresis (2-DE)-based proteomic analysis to characterize the protein alterations in S. pneumoniae after treatment with X-03. In total, 50 proteins exhibiting significant differential expressions were identified. RT-PCR was used to confirm the expression differences for selected proteins. Bioinformatics analysis on the proteomic alterations suggested that Ru(II) complex X-03 may obstruct bacterial fatty acid synthesis and oxidation-reduction process to suppress the growth of S. pneumoniae. Metal-uptake experiments revealed that iron-acquisition pathway in the bacterium may be interfered by X-03. These results provide useful clues for further investigations on the mechanism of the antibacterial action of metal compounds.

BIOLOGICAL SIGNIFICANCE:

The appearance of bacterial strains with broad antibiotic resistance is becoming an alarming global health concern. The development of novel efficient antibacterial compound is urgently needed. In the present study, we found that Ru(II) complex X-03 has a significant antibacterial activity and applied proteomic technology combined with bioinformatics analysis to investigate its antimicrobial mechanism in S. pneumoniae. Many proteins were found to be dysregulated, implicating that X-03 may affect various molecular pathways leading to the inhibition of bacterial growth. Metal-uptake experiments demonstrated that X-03 treatment reduced the iron content in the bacterium, suggesting the interference with iron acquisition systems by the complex. This disturbance in iron acquisition may directly or indirectly induce the proteomic response that involved many pathways. In addition, X-03 could selectively suppress Gram-positive bacteria but execute less cytotoxicity to Gram-negative bacteria, with almost no effect on human cells, implicating its potential to be developed as a specific antimicrobial agent. These results provide useful information for further investigations on the mechanism of the antibacterial action of metal drugs and development of efficient antibacterial drugs.

KEYWORDS:

Antibacterial activity; Proteomic profiling; Ru(II) complex; S. pneumoniae

PMID: 25497219 [PubMed - as supplied by publisher]

Increase in the nasopharyngeal carriage of non-vaccine serogroup 15 Streptococcus pneumoniae after introduction of children pneumococcal conjugate vaccination in Hong Kong.

Diagn Microbiol Infect Dis. 2015 Feb;81(2):145-8. doi: 10.1016/j.diagmicrobio.2014.11.006. Epub 2014 Nov 20.

Increase in the nasopharyngeal carriage of non-vaccine serogroup 15 Streptococcus pneumoniae after introduction of children pneumococcal conjugate vaccination in Hong Kong.

Ho PL1, Chiu SS2, Law PY3, Chan EL2, Lai EL3, Chow KH3.

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Abstract

This study assessed pneumococcal carriage in the early periods after routine use of 13-valent pneumococcal conjugate vaccine (PCV13) in Hong Kong. Nasopharyngeal swabs were obtained from 1110 children (<5 years) admitted with acute illness during September 2010-August 2013. Pneumococcal carriage rate was 13.5% in unvaccinated children, 14.1% in children who had ≥1 PCV dose and 15.3% in children who had ≥3 PCV doses. Nonv-PCV13 serotypes comprised 56.4% of all isolates. The most common serogroup/types were 15 (15A, 5.1%; 15B, 10.3%; 15C, 9.6%; 15F, 0.6%), 19F (17.9%), 6A (7.1%) and 6C (7.1%). Carriage of serogroup 15 was more common among vaccinated children (4.1% versus 0.6%, P = 0.033). Molecular typing revealed that expansion of several clones (clonal complex, CC63, CC199, CC1262, CC3397) was responsible for the increase in serogroup 15. Almost all CC63 and CC3397 isolates were nonsusceptible to both penicillin and erythromycin. The finding highlights the emergence of serogroup 15 following PCV13 use.

KEYWORDS:

Drug resistance; Prevalence; Serotype; Streptococcus pneumoniae

PMID: 25483278 [PubMed - in process]

Degradation Products of the Extracellular Pathogen Streptococcus pneumoniae Access the Cytosol via Its Pore-Forming Toxin.

MBio. 2015 Jan 20;6(1). pii: e02110-14. doi: 10.1128/mBio.02110-14.

Degradation Products of the Extracellular Pathogen Streptococcus pneumoniae Access the Cytosol via Its Pore-Forming Toxin.

Lemon JK1, Weiser JN2.

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Abstract

Streptococcus pneumoniae is a leading pathogen with an extracellular lifestyle; however, it is detected by cytosolic surveillance systems of macrophages. The innate immune response that follows cytosolic sensing of cell wall components results in recruitment of additional macrophages, which subsequently clear colonizing organisms from host airways. In this study, we monitored cytosolic access by following the transit of the abundant bacterial surface component capsular polysaccharide, which is linked to the cell wall. Confocal and electron microscopy visually characterized the location of cell wall components in murine macrophages outside membrane-bound organelles. Quantification of capsular polysaccharide through cellular fractionation demonstrated that cytosolic access of bacterial cell wall components is dependent on phagocytosis, bacterial sensitivity to the host's degradative enzyme lysozyme, and release of the pore-forming toxin pneumolysin. Activation of p38 mitogen-activated protein kinase (MAPK) signaling is important for limiting access to the cytosol; however, ultimately, these are catastrophic events for both the bacteria and the macrophage, which undergoes cell death. Our results show how expression of a pore-forming toxin ensures the death of phagocytes that take up the organism, although cytosolic sensing results in innate immune detection that eventually allows for successful host defense. These findings provide an example of how cytosolic access applies to an extracellular microbe and contributes to its pathogenesis. IMPORTANCE : Streptococcus pneumoniae (the pneumococcus) is a bacterial pathogen that is a leading cause of pneumonia. Pneumococcal disease is preceded by colonization of the nasopharynx, which lasts several weeks before being cleared by the host's immune system. Although S. pneumoniae is an extracellular microbe, intracellular detection of pneumococcal components is critical for bacterial clearance. In this study, we show that following bacterial uptake and degradation by phagocytes, pneumococcal products access the host cell cytosol via its pore-forming toxin. This phenomenon of cytosolic access results in phagocyte death and may serve to combat the host cells responsible for clearing the organism. Our results provide an example of how intracellular access and subsequent immune detection occurs during infection with an extracellular pathogen.

PMID: 25604786 [PubMed - in process] Free full text

Antimicrobial activity of solithromycin tested against serotyped macrolide-resistant Streptococcus pneumoniae collected from medical centers across the USA (2012).

Antimicrob Agents Chemother. 2015 Jan 20. pii: AAC.04568-14. [Epub ahead of print]

Antimicrobial activity of solithromycin tested against serotyped macrolide-resistant Streptococcus pneumoniae collected from medical centers across the USA (2012).

Farrell DJ1, Mendes RE2, Jones RN2.

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Abstract

Solithromycin, a fourth-generation macrolide and novel fluoroketolide, was tested against a 2012 collection of serotyped United States macrolide-resistant S. pneumoniae isolates associated with community-acquired bacterial pneumonia (CABP). Against all 272 isolates, solithromycin demonstrated high potency (MIC50/90, 0.06/0.25 μg/mL) and inhibited all strains at MIC values ≤0.5 μg/mL, including against the two most prevalent macrolide-resistant serotypes (19A and 35B). This data supports continued clinical development of solithromycin for the treatment of multidrug resistant CABP.

PMID: 25605359 [PubMed - as supplied by publisher]

Proteomic analysis of the copper resistance of Streptococcus pneumoniae.

Metallomics. 2015 Jan 22. [Epub ahead of print]

Proteomic analysis of the copper resistance of Streptococcus pneumoniae.

Guo Z1, Han J, Yang XY, Cao K, He K, Du G, Zeng G, Zhang L, Yu G, Sun Z, He QY, Sun X.

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Abstract

Streptococcus pneumoniae is a Gram-positive bacterial pathogen causing a variety of diseases, including otitis media, bacteraemia and meningitis. Although copper is an essential trace metal for bacterial growth, high intracellular levels of free-copper are toxic. Copper resistance has emerged as an important virulence determinant of microbial pathogens. In this study, we determined the minimum inhibition concentration of copper for the growth inhibition of S. pneumoniae. Two-dimensional-electrophoresis coupled with mass spectrometry was applied to identify proteins involved in copper resistance of S. pneumoniae. In total, forty-four proteins with more than 1.5-fold alteration in expression (p < 0.05) were identified. Quantitative reverse transcription PCR was used to confirm the proteomic results. Bioinformatics analysis showed that the differentially expressed proteins were mainly involved in the cell wall biosynthesis, protein biosynthesis, purine biosynthesis, pyrimidine biosynthesis, primary metabolic process, and the nitrogen compound metabolic process. Many up-regulated proteins in response to the copper treatment directly or indirectly participated in the cell wall biosynthesis, indicating that the cell wall is a critical determinant in copper resistance of S. pneumoniae.

PMID: 25608595 [PubMed - as supplied by publisher]

STREPTOCOCCUS PNEUMONIAE AND STAPHYLOCOCCUS AUREUS CARRIAGE IN HEALTHY SCHOOL-AGE CHILDREN AND ADOLESCENTS.

J Med Microbiol. 2015 Jan 22. pii: jmm.0.000029. doi: 10.1099/jmm.0.000029. [Epub ahead of print]

STREPTOCOCCUS PNEUMONIAE AND STAPHYLOCOCCUS AUREUS CARRIAGE IN HEALTHY SCHOOL-AGE CHILDREN AND ADOLESCENTS.

Esposito S1, Terranova L2, Ruggiero L2, Ascolese B2, Montinaro V2, Rios WP2, Galeone C3, Principi N2.

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Abstract

Streptococcus pneumoniae and Staphylococcus aureus are common commensals of the upper respiratory tract in children and adolescents. Understanding the relationship between these two pathogens, including their potential for mutual interference, is needed to evaluate their epidemiological status as well as the impact of related preventative measures. We obtained oropharyngeal and nasal swabs from 497 healthy subjects aged 6 to 17 years. S. pneumoniae detection and serotyping were performed using a real-time polymerase chain reaction, and S. aureus detection was performed using the RIDAGENE MRSA system. We found that 136 (27.3%) of the children were carriers of both pathogens, 121 (24.3%) of the children carried S. pneumoniae alone, and 128 (25.7%) of the children carried S. aureus alone. S. aureus carriage was similar between children who did vs. did not carry S. pneumoniae and was independent of age and vaccination with 7-valent pneumococcal conjugate vaccine (PCV7) also did not affect S. aureus carriage. Pneumococcal serotype also did not appear to affect S. aureus carriage. These findings suggest that the carriage of S. pneumoniae does not affect that of S. aureus in older children and adolescents, regardless of age, PCV7 vaccination, and pneumococcal serotype.

PMID: 25614277 [PubMed - as supplied by publisher]

An electrostatic interaction between BlpC and BlpH influences pheromone specificity in the control of bacteriocin production and immunity in Streptococcus pneumoniae.

J Bacteriol. 2015 Jan 26. pii: JB.02432-14. [Epub ahead of print]

An electrostatic interaction between BlpC and BlpH influences pheromone specificity in the control of bacteriocin production and immunity in Streptococcus pneumoniae.

Pinchas MD1, LaCross NC2, Dawid S3.

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Abstract

The blp locus of Streptococcus pneumoniae secretes and regulates bacteriocins, which mediate both intra- and interspecific competition in the human nasopharynx. There are four major alleles of the gene blpH, which encodes the receptor responsible for activating the blp locus when bound to one of four distinct peptide pheromones (BlpC). The allelic variation of blpH is presumably explained by a need to restrict cross talk between competing strains. The BlpH protein sequences have polymorphisms distributed throughout the entire sequence, making identification of the peptide binding site difficult to predict. To identify the pheromone binding sites that dictate pheromone specificity, we have characterized the four major variants and two naturally occurring chimeric versions of blpH in which recombination events appear to have joined two distinct blpH alleles together. Using these allelic variants, a series of lab generated chimeric blpH alleles, and site-directed mutants of both the receptor and peptide, we have demonstrated that BlpC binding to some BlpH types involves an electrostatic interaction between the oppositely charged residues of BlpC and the first transmembrane domain of BlpH. An additional recognition site was identified in the second extracellular loop. We identified naturally occurring BlpH types that have the capacity to respond to more than one BlpC type; however, this change in specificity results in a commensurate drop in overall sensitivity. These natural recombination events were presumably selected for to balance the need to sense bacteriocin secreting neighbors with the need to turn on bacteriocin production at a low density.

IMPORTANCE:

Bacteria use quorum sensing to optimize gene expression to accommodate for local bacterial density and diffusion rates. To prevent interception of quorum signals by neighboring strains, single species often encode strain-specific signal/receptor pairs. The blp locus in Streptococcus pneumoniae that drives bacteriocin secretion is controlled by quorum sensing that involves the interaction of the signal/receptor pair, BlpC/BlpH. We show that the pneumococcal population can be divided into several distinct BlpC/H pairs, however, there are examples of naturally occurring chimeric receptors that can bind to more than one BlpC type. The tradeoff for this broadened specificity is a loss of overall receptor sensitivity. This suggests that under certain conditions, the advantage of signal interception can trump the requirements for self-induction.

PMID: 25622617 [PubMed - as supplied by publisher]

PD-1 Suppresses Protective Immunity to Streptococcus pneumoniae through a B Cell-Intrinsic Mechanism.

J Immunol. 2015 Jan 26. pii: 1401673. [Epub ahead of print]

PD-1 Suppresses Protective Immunity to Streptococcus pneumoniae through a B Cell-Intrinsic Mechanism.

McKay JT1, Egan RP1, Yammani RD1, Chen L2, Shin T3, Yagita H4, Haas KM5.

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Abstract

Despite the emergence of the programmed cell death 1 (PD-1):PD-1 ligand (PD-L) regulatory axis as a promising target for treating multiple human diseases, remarkably little is known about how this pathway regulates responses to extracellular bacterial infections. We found that PD-1-/- mice, as well as wild-type mice treated with a PD-1 blocking Ab, exhibited significantly increased survival against lethal Streptococcus pneumoniae infection following either priming with low-dose pneumococcal respiratory infection or S. pneumoniae-capsular polysaccharide immunization. Enhanced survival in mice with disrupted PD-1:PD-L interactions was explained by significantly increased proliferation, isotype switching, and IgG production by pneumococcal capsule-specific B cells. Both PD-L, B7-H1 and B7-DC, contributed to PD-1-mediated suppression of protective capsule-specific IgG. Importantly, PD-1 was induced on capsule-specific B cells and suppressed IgG production and protection against pneumococcal infection in a B cell-intrinsic manner. To our knowledge, these results provide the first demonstration of a physiologic role for B cell-intrinsic PD-1 expression in vivo. In summary, our study reveals that B cell-expressed PD-1 plays a central role in regulating protection against S. pneumoniae, and thereby represents a promising target for bolstering immunity to encapsulated bacteria.

PMID: 25624454 [PubMed - as supplied by publisher]

High Throughput Screen Identifies Natural Product Inhibitor of Phenylalanyl-tRNA Synthetase from Pseudomonas Aeruginosa and Streptococcus Pneumoniae.

Curr Drug Discov Technol. 2015 Jan 20. [Epub ahead of print]

High Throughput Screen Identifies Natural Product Inhibitor of Phenylalanyl-tRNA Synthetase from Pseudomonas Aeruginosa and Streptococcus Pneumoniae.

Bullard JM1.

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Abstract

Pseudomonas aeruginosa and Streptococcus pneumoniae are causative agents in a wide range of infections. Genes encoding proteins corresponding to phenylalanyl-tRNA synthetase (PheRS) were cloned from both bacteria. The two forms of PheRS were kinetically evaluated and the Km's for P. aeruginosa PheRS with its three substrates, phenylalanine, ATP and tRNAPhe were determined to be 48, 200, and 1.2 μM, respectively, while the Km's for S. pneumoniae PheRS with respect to phenylalanine, ATP and tRNAPhe were 21, 225 and 0.94 μM, respectively. P. aeruginosa and S. pneumoniae PheRS were used to screen a natural compound library and a single compound was identified that inhibited the function of both enzymes. The compound inhibited P. aeruginosa and S. pneumoniae PheRS with IC50's of 2.3 and 4.9 μM, respectively. The compound had a Ki of 0.83 and 0.98 μM against P. aeruginosa and S. pneumoniae PheRS, respectively. The minimum inhibitory concentration (MIC) of the compound was determined against a panel of Gram positive and negative bacteria including efflux pump mutants and hyper-sensitive strains. MICs against wild-type P. aeruginosa and S. pneumoniae cells in culture were determined to be 16 and 32 μg/ml, respectively. The mechanism of action of the compound was determined to be competitive with the amino acid, phenylalanine, and uncompetitive with ATP. There was no inhibition of cytoplasmic protein synthesis, however partial inhibition of the human mitochondrial PheRS was observed.

PMID: 25601215 [PubMed - as supplied by publisher]

Trivalent pneumococcal protein recombinant vaccine protects against lethal Streptococcus pneumoniae pneumonia and correlates with phagocytosis by neutrophils during early pathogenesis.

Vaccine. 2015 Jan 15. pii: S0264-410X(15)00033-X. doi: 10.1016/j.vaccine.2015.01.014. [Epub ahead of print]

Trivalent pneumococcal protein recombinant vaccine protects against lethal Streptococcus pneumoniae pneumonia and correlates with phagocytosis by neutrophils during early pathogenesis.

Xu Q1, Surendran N1, Verhoeven D1, Klapa J1, Ochs M2, Pichichero ME3.

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Abstract

OBJECTIVE:

Due to the fact that current polysaccharide-based pneumococcal vaccines have limited serotype coverage, protein-based vaccine candidates have been sought for over a decade to replace or complement current vaccines. We previously reported that a trivalent Pneumococcal Protein recombinant Vaccine (PPrV), showed protection against pneumonia and sepsis in an infant murine model. Here we investigated immunological correlates of protection of PPrV in the same model.

METHODS:

C57BL/6J infant mice were intramuscularly vaccinated at age 1-3 weeks with 3 doses of PPrV, containing pneumococcal histidine triad protein D (PhtD), pneumococcal choline binding protein A (PcpA), and detoxified pneumolysin mutant PlyD1. 3-4 weeks after last vaccination, serum and lung antibody levels to PPrV components were measured, and mice were intranasally challenged with a lethal dose of Streptococcus pneumoniae (Spn) serotype 6A. Lung Spn bacterial burden, number of neutrophils and alveolar macrophages, phagocytosed Spn by granulocytes, and levels of cytokines and chemokines were determined at 6, 12, 24, and 48h after challenge.

RESULTS:

PPrV vaccination conferred 83% protection against Spn challenge. Vaccinated mice had significantly elevated serum and lung antibody levels to three PPrV components. In the first stage of pathogenesis of Spn induced pneumonia (6-24h after challenge), vaccinated mice had lower Spn bacterial lung burdens and more phagocytosed Spn in the granulocytes. PPrV vaccination led to lower levels of pro-inflammatory cytokines IL-6, IL-1β, and TFN-α, and other cytokines and chemokines (IL-12, IL-17, IFN-γ, MIP-1b, MIP-2 and KC, and G-CSF), presumably due to a lower lung bacterial burden.

CONCLUSION:

Trivalent PPrV vaccination results in increased serum and lung antibody levels to the vaccine components, a reduction in Spn induced lethality, enhanced early clearance of Spn in lungs due to more rapid and thorough phagocytosis of Spn by neutrophils, and correspondingly a reduction in lung inflammation and tissue damage.

KEYWORDS:

Alveolar macrophages; Infant murine model; Neutrophils; Phagocytosis; Pneumococcal protein vaccine; Streptococcus pneumoniae

PMID: 25597944 [PubMed - as supplied by publisher]

Compound 48/80 acts as a potent mucosal adjuvant for vaccination against Streptococcus pneumoniae infection in young mice.

Vaccine. 2015 Jan 14. pii: S0264-410X(15)00032-8. doi: 10.1016/j.vaccine.2015.01.013. [Epub ahead of print]

Compound 48/80 acts as a potent mucosal adjuvant for vaccination against Streptococcus pneumoniae infection in young mice.

Zeng L1, Liu Y2, Wang H2, Liao P3, Song Z2, Gao S2, Wu Y2, Zhang X2, Yin Y2, Xu W4.

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Abstract

Streptococcus pneumoniae, a major respiratory pathogen, is a leading cause of death among children worldwide. Mucosal vaccination is a recommended method to prevent respiratory infection. However, development of mucosal vaccination is usually hindered due to the lack of safe and effective mucosal adjuvants. Mast cell activator compound 48/80 (C48/80) has been used as a mucosal adjuvant in immunization of adult mice, but its adjuvanticity is not clear in the immunization of young mice. In this study, the adjuvanticity of C48/80 was evaluated when intranasally co-administrated with a pneumococcal vaccine candidate strain SPY1 in a young mice model in comparison with a classical mucosal adjuvant cholera toxin (CT) and a relatively safe mucosal adjuvant Pam2CSK4. All three adjuvants enhanced antibody responses, whereas serum IgG titers were maintained at a stable level during the 3 months after the last immunization only in the SPY1+C48/80 and SPY1+CT groups. Furthermore, both the SPY1+CT group and the SPY1+C48/80 group induced strong Th17 immune response. Notably, C48/80 showed the exceptional ability to promote the clearance of nasal pneumococcal colonization which CT and Pam2CSK4 did not show. We found that C48/80's ability to induce protection against nasal pneumococcal colonization depended on B cells and IL-17A. Additionally, C48/80, as a mucosal adjuvant, showed a greater ability to protect young mice against lethal pneumococcal infection than CT. In comparison with CT, C48/80 also showed a favorable safety. These results reveal a promising perspective for using C48/80 as a mucosal adjuvant to improve protection against pneumococcal diseases early in life.

KEYWORDS:

Mucosal adjuvant; Nasal colonization; Protective immunity; Streptococcus pneumoniae

PMID: 25595867 [PubMed - as supplied by publisher]

Enhanced inflammation in aged mice following infection with Streptococcus pneumoniae is associated with decreased IL-10 and augmented chemokine production.

Am J Physiol Lung Cell Mol Physiol. 2015 Jan 16:ajplung.00141.2014. doi: 10.1152/ajplung.00141.2014. [Epub ahead of print]

Enhanced inflammation in aged mice following infection with Streptococcus pneumoniae is associated with decreased IL-10 and augmented chemokine production.

Williams AE1, José RJ2, Brown JS2, Chambers RC3.

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Abstract

Streptococcus pneumoniae is the most common cause of severe pneumonia in the elderly. However, the impact of ageing on the innate inflammatory response to pneumococci is poorly defined. We compared the innate immune response in old versus young adult mice following infection with S. pneumoniae. The accumulation of neutrophils recovered from bronchoalveolar lavage fluid and lung homogenates was increased in aged compared to young adult mice, although bacterial outgrowth was similar in both age groups, as were markers of microvascular leak. Aged mice had similar levels of IL-1β, TNF, IFN-γ, IL-17 and G-CSF following S. pneumoniae infection, compared to young mice, but increased levels of CXCL9, CXCL12, CCL3, CCL4, CCL5, CCL11 and CCL17. Moreover, levels of IL-10 were significantly lower in aged animals. Neutralization of IL-10 in infected young mice was associated with increased neutrophil recruitment but no decrease in bacterial outgrowth. Furthermore, IL-10 neutralization resulted in increased levels of CCL3, CCL5 and CXCL10. We conclude that ageing is associated with enhanced inflammatory responses following S. pneumoniae infection as a result of a compromised immunomodulatory cytokine response.

KEYWORDS:

Pneumonia; ageing; chemokine; inflammation; neutrophil

PMID: 25595646 [PubMed - as supplied by publisher]

Measuring the effects of pneumococcal conjugate vaccine (PCV7) on Streptococcus pneumoniae carriage and antibiotic resistance: The Palestinian-Israeli Collaborative Research (PICR).

Vaccine. 2015 Jan 12. pii: S0264-410X(15)00004-3. doi: 10.1016/j.vaccine.2015.01.003. [Epub ahead of print]

Measuring the effects of pneumococcal conjugate vaccine (PCV7) on Streptococcus pneumoniae carriage and antibiotic resistance: The Palestinian-Israeli Collaborative Research (PICR).

Daana M1, Rahav G2, Hamdan A3, Thalji A4, Jaar F5, Abdeen Z6, Jaber H2, Goral A7, Huppert A8, Raz M9, Regev-Yochay G10; for the PICR study group.

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Abstract

BACKGROUND:

The Palestinian-Israeli Collaborative Research (PICR) cross-conflict setting provided a unique opportunity to study overall and indirect effects of pneumococcal conjugate vaccine (PCV7), in two closely related Palestinian populations governed by two distinct health authorities with distinct vaccination policies. Here, PCV7 effects on pneumococcal carriage, serotype distribution and antibiotic resistance are reported.

METHODS:

Annual cross-sectional surveys of pneumococcal carriage were performed during 2009-2011 among Palestinian children (≤5 years) (a) under Palestinian-Authority (PA) health policy (Ramallah, Nablus and Bethlehem), where PCV7 was unlicensed (b) under Israeli health policy (East-Jerusalem (EJ)) where PCV7 was rapidly implemented from July 2009. Clinical data were collected, pneumococci identified and characterized for antibiotic susceptibilities and serotype. Analyses included multivariate logistic models with an interaction term for PCV7-effect.

RESULTS:

Altogether, 2755 children from PA (n=1772) and EJ (n=983) were enrolled, of which ∼30% were pneumococcal carriers. While overall carriage was not affected by vaccination policy, carriage of vaccine-type (VT7) strains decreased from 52% to 22% (p<0.001) in EJ, where PCV was implemented, but not in PA. This was accompanied by an increase in non-VT13 strains from 34% to 65% (p<0.001) in EJ, but not in PA. Furthermore, within two years post-PCV7 introduction, proportion of multi-drug resistant strains, which was initially 23% in both populations, decreased significantly in EJ, to 10%, while simultaneously it increased in PA to 33% (p<0.001). Similar trends were observed for resistance to most antibiotic groups. The proportion of resistant isolates among non-VT13 strains did not change during the study period.

CONCLUSIONS:

The unique study design distinguishes secular and seasonal effects from true vaccine effects. While PCV7 did not affect overall pneumococcal carriage rate, VT7 strains, many of which were antibiotic resistant decreased and were replaced by non-VT13 strains, which were mostly not antibiotic resistant, resulting in a net decrease in antibiotic resistance.

KEYWORDS:

Antibiotic resistance; PCV7 effects; Pneumococcal carriage; Population-based study

PMID: 25593104 [PubMed - as supplied by publisher]

Thursday, January 22, 2015

Importance of bacterial replication and alveolar macrophage-independent clearance mechanisms during early lung infection with Streptococcus pneumoniae.

Infect Immun. 2015 Jan 12. pii: IAI.02788-14. [Epub ahead of print]

Importance of bacterial replication and alveolar macrophage-independent clearance mechanisms during early lung infection with Streptococcus pneumoniae.

Camberlein E1, Cohen JM2, José R1, Hyams CJ1, Callard R3, Chimalapati S1, Yuste J4, Edwards LA1, Marshall H1, van Rooijen N5, Noursadeghi M6, Brown JS7.

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Abstract

Although the importance of alveolar macrophages for host immunity during early Streptococcus pneumoniae lung infection is well established, the contribution and relative importance of other innate immunity mechanisms and of bacterial factors is less clear. We have used a murine model of S. pneumoniae early lung infection with wild-type, unencapsulated and para-amino benzoic acid auxotroph mutant TIGR4 strains to assess the effects of inoculum size, bacterial replication, the capsule, and alveolar macrophage-dependent and -independent clearance mechanisms on bacterial persistence within the lungs. Alveolar macrophage-dependent and -independent (calculated indirectly) clearance half-lives and bacterial replication doubling times were estimated using a mathematical model. In this model after infection with a high dose inoculum of encapsulated S. pneumoniae alveolar macrophage-independent clearance mechanisms were dominant, with a clearance half-life of 24 minutes compared to 135 minutes for alveolar macrophage-dependent clearance. In addition, after a high dose inoculum successful lung infection required rapid bacterial replication, with an estimated S. pneumoniae doubling time of 16 minutes. The capsule had wide effects on early lung clearance mechanisms, with reduced half-lives of 14 minutes for alveolar-independent and 31 minutes for alveolar macrophage-dependent clearance of unencapsulated bacteria. In contrast, with a lower dose inoculum, the bacterial doubling time increased to 56 minutes and the S. pneumoniae alveolar macrophage-dependent clearance half-life improved to 42 minutes and was largely unaffected by the capsule. These data demonstrate the large effects of bacterial factors (inoculum size, the capsule, rapid replication) and alveolar macrophage-independent clearance mechanisms during early lung infection with S. pneumoniae.

PMID: 25583525 [PubMed - as supplied by publisher]

Measuring the effects of pneumococcal conjugate vaccine (PCV7) on Streptococcus pneumoniae carriage and antibiotic resistance: The Palestinian-Israeli Collaborative Research (PICR).

Vaccine. 2015 Jan 12. pii: S0264-410X(15)00004-3. doi: 10.1016/j.vaccine.2015.01.003. [Epub ahead of print]

Measuring the effects of pneumococcal conjugate vaccine (PCV7) on Streptococcus pneumoniae carriage and antibiotic resistance: The Palestinian-Israeli Collaborative Research (PICR).

Daana M1, Rahav G2, Hamdan A3, Thalji A4, Jaar F5, Abdeen Z6, Jaber H2, Goral A7, Huppert A8, Raz M9, Regev-Yochay G10; for the PICR study group.

Author information

Abstract

BACKGROUND:

METHODS:

RESULTS:

CONCLUSIONS:

KEYWORDS:

Antibiotic resistance; PCV7 effects; Pneumococcal carriage; Population-based study

PMID: 25593104 [PubMed - as supplied by publisher]