Thursday, May 28, 2015

Streptococcus pneumoniae Enhances Human Respiratory Syncytial Virus Infection In Vitro and In Vivo.

PLoS One. 2015 May 13;10(5):e0127098. doi: 10.1371/journal.pone.0127098. eCollection 2015.
Streptococcus pneumoniae Enhances Human Respiratory Syncytial Virus Infection In Vitro and In Vivo.
Author information


Abstract
Human respiratory syncytial virus (HRSV) and Streptococcus pneumoniae are important causative agents of respiratory tract infections. Both pathogens are associated with seasonal disease outbreaks in the pediatric population, and can often be detected simultaneously in infants hospitalized with bronchiolitis or pneumonia. It has been described that respiratory virus infections may predispose for bacterial superinfections, resulting in severe disease. However, studies on the influence of bacterial colonization of the upper respiratory tract on the pathogenesis of subsequent respiratory virus infections are scarce. Here, we have investigated whether pneumococcal colonization enhances subsequent HRSV infection. We used a newly generated recombinant subgroup B HRSV strain that expresses enhanced green fluorescent protein and pneumococcal isolates obtained from healthy children in disease-relevant in vitro and in vivo model systems. Three pneumococcal strains specifically enhanced in vitro HRSV infection of primary well-differentiated normal human bronchial epithelial cells grown at air-liquid interface, whereas two other strains did not. Since previous studies reported that bacterial neuraminidase enhanced HRSV infection in vitro, we measured pneumococcal neuraminidase activity in these cultures but found no correlation with the observed infection enhancement in our model. Subsequently, a selection of pneumococcal strains was used to induce nasal colonization of cotton rats, the best available small animal model for HRSV. Intranasal HRSV infection three days later resulted in strain-specific enhancement of HRSV replication in vivo. One S. pneumoniae strain enhanced HRSV both in vitro and in vivo, and was also associated with enhanced syncytium formation in vivo. However, neither pneumococci nor HRSV were found to spread from the upper to the lower respiratory tract, and neither pathogen was transmitted to naive cage mates by direct contact. These results demonstrate that pneumococcal colonization can enhance subsequent HRSV infection, and provide tools for additional mechanistic and intervention studies.

PMID: 25970287 [PubMed - in process] PMCID: PMC4430531 

Vinpocetine Inhibits Streptococcus pneumoniae-Induced Upregulation of Mucin MUC5AC Expression via Induction of MKP-1 Phosphatase in the Pathogenesis of Otitis Media.

J Immunol. 2015 May 13. pii: 1401489. [Epub ahead of print]
Vinpocetine Inhibits Streptococcus pneumoniae-Induced Upregulation of Mucin MUC5AC Expression via Induction of MKP-1 Phosphatase in the Pathogenesis of Otitis Media.
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Abstract
Mucin overproduction is a hallmark of otitis media (OM). Streptococcus pneumoniae is one of the most common bacterial pathogens causing OM. Mucin MUC5AC plays an important role in mucociliary clearance of bacterial pathogens. However, if uncontrolled, excessive mucus contributes significantly to conductive hearing loss. Currently, there is a lack of effective therapeutic agents that suppress mucus overproduction. In this study, we show that a currently existing antistroke drug, vinpocetine, a derivative of the alkaloid vincamine, inhibited S. pneumoniae-induced mucin MUC5AC upregulation in cultured middle ear epithelial cells and in the middle ear of mice. Moreover, vinpocetine inhibited MUC5AC upregulation by inhibiting the MAPK ERK pathway in an MKP-1-dependent manner. Importantly, ototopical administration of vinpocetine postinfection inhibited MUC5AC expression and middle ear inflammation induced by S. pneumoniae and reduced hearing loss and pneumococcal loads in a well-established mouse model of OM. Thus, these studies identified vinpocetine as a potential therapeutic agent for inhibiting mucus production in the pathogenesis of OM.
Copyright © 2015 by The American Association of Immunologists, Inc.
PMID: 25972475 [PubMed - as supplied by publisher]


Feasibility and Safety of Local Treatment with Recombinant Human Tissue Factor Pathway Inhibitor in a Rat Model of Streptococcus pneumoniae Pneumonia.

PLoS One. 2015 May 18;10(5):e0127261. doi: 10.1371/journal.pone.0127261. eCollection 2015.
Feasibility and Safety of Local Treatment with Recombinant Human Tissue Factor Pathway Inhibitor in a Rat Model of Streptococcus pneumoniae Pneumonia.
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Abstract
Pulmonary coagulopathy is intrinsic to pulmonary injury including pneumonia. Anticoagulant strategies could benefit patients with pneumonia, but systemic administration of anticoagulant agents may lead to suboptimal local levels and may cause systemic hemorrhage. We hypothesized nebulization to provide a safer and more effective route for local administration of anticoagulants. Therefore, we aimed to examine feasibility and safety of nebulization of recombinant human tissue factor pathway inhibitor (rh-TFPI) in a well-established rat model of Streptococcus (S.) pneumoniae pneumonia. Thirty minutes before and every 6 hours after intratracheal instillation of S. pneumonia causing pneumonia, rats were subjected to local treatment with rh-TFPI or placebo, and sacrificed after 42 hours. Pneumonia was associated with local as well as systemic activation of coagulation. Nebulization of rh-TFPI resulted in high levels of rh-TFPI in bronchoalveolar lavage fluid, which was accompanied by an attenuation of pulmonary coagulation. Systemic rh-TFPI levels remained undetectable, and systemic TFPI activity and systemic coagulation were not affected. Histopathology revealed no bleeding in the lungs. We conclude that nebulization of rh-TFPI seems feasible and safe; local anticoagulant treatment with rh-TFPI attenuates pulmonary coagulation, while not affecting systemic coagulation in a rat model of S. pneumoniae pneumonia.

PMID: 25992779 [PubMed - in process] 

Transfer of penicillin resistance from Streptococcus oralis to Streptococcus pneumoniae identifies murE as resistance determinant.

Mol Microbiol. 2015 May 25. doi: 10.1111/mmi.13070. [Epub ahead of print]
Transfer of penicillin resistance from Streptococcus oralis to Streptococcus pneumoniae identifies murE as resistance determinant.
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Abstract
Beta-lactam resistant clinical isolates of Streptococcus pneumoniae contain altered penicillin-binding protein (PBP) genes and occasionally an altered murM, presumably products of interspecies gene transfer. MurM and MurN are responsible for the synthesis of branched lipid II, substrate for the PBP catalyzed transpeptidation reaction. Here we used the high-level beta-lactam resistant S. oralis Uo5 as donor in transformation experiments with the sensitive laboratory strain S. pneumoniae R6 as recipient. Surprisingly, piperacillin resistant transformants contained no alterations in PBP genes but carried murEUo5 encoding the UDP-N-acetylmuramyl tripeptide synthetase. Codons 83-183 of murEUo5 were sufficient to confer the resistance phenotype. Moreover, the promoter of murEUo5 which drives a twofold higher expression compared to that of S. pneumoniae R6 could also confer increased resistance. Multiple independent transformations produced S. pneumoniae R6 derivatives containing murEUo5 , pbp2xUo5 , pbp1aUo5 and pbp2bUo5 , but not murMUo5 sequences; however, the resistance level of the donor strain could not be reached. S. oralis Uo5 harbors an unusual murM, and murN is absent. Accordingly, the peptidoglycan of S. oralis Uo5 contained interpeptide bridges with one L-Ala residue only. The data suggest that resistance in S. oralis Uo5 is based on a complex interplay of distinct PBPs and other enzymes involved in peptidoglycan biosynthesis.
This article is protected by copyright. All rights reserved.
KEYWORDS:
MurE; Penicillin-binding protein; Streptococcus oralis; Streptococcus pneumoniae; penicillin resistance

PMID: 26010014 [PubMed - as supplied by publisher]

Nonencapsulated Streptococcus pneumoniae causes otitis media during single-species infection and during polymicrobial infection with nontypeable Haemophilus influenzae.

Pathog Dis. 2015 Jul;73(5). pii: ftu011. doi: 10.1093/femspd/ftu011. Epub 2014 Dec 4.
Nonencapsulated Streptococcus pneumoniae causes otitis media during single-species infection and during polymicrobial infection with nontypeable Haemophilus influenzae.
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Abstract
Streptococcus pneumoniae strains lacking capsular polysaccharide have been increasingly reported in carriage and disease contexts. Since most cases of otitis media involve more than one bacterial species, we aimed to determine the capacity of a nonencapsulated S. pneumoniae clinical isolate to induce disease in the context of a single-species infection and as a polymicrobial infection with nontypeable Haemophilus influenzae. Using the chinchilla model of otitis media, we found that nonencapsulated S. pneumoniae colonizes the nasopharynx following intranasal inoculation, but does not readily ascend into the middle ear. However, when we inoculated nonencapsulated S. pneumoniae directly into the middle ear, the bacteria persisted for two weeks post-inoculation and induced symptoms consistent with chronic otitis media. During coinfection with nontypeable H. influenzae, both species persisted for one week and induced polymicrobial otitis media. We also observed that nontypeable H. influenzae conferred passive protection from killing by amoxicillin upon S. pneumoniae from within polymicrobial biofilms in vitro. Therefore, based on these results, we conclude that nonencapsulated pneumococci are a potential causative agent of chronic/recurrent otitis media, and can also cause mutualistic infection with other opportunists, which could complicate treatment outcomes.
© FEMS 2014. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.
KEYWORDS:
Haemophilus; coinfection; nonencapsulated; pneumococcus

PMID: 26014114 [PubMed - in process]

Frequency of Spontaneous Resistance to Peptide Deformylase Inhibitor GSK1322322 in Haemophilus influenzae, Staphylococcus aureus, Streptococcus pyogenes, and Streptococcus pneumoniae.

Antimicrob Agents Chemother. 2015 May 26. pii: AAC.00484-15. [Epub ahead of print]
Frequency of Spontaneous Resistance to Peptide Deformylase Inhibitor GSK1322322 in Haemophilus influenzae, Staphylococcus aureus, Streptococcus pyogenes, and Streptococcus pneumoniae.
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Abstract
The continuous emergence of multidrug resistant pathogenic bacteria is compromising the successful treatment of serious microbial infections. GSK1322322, a novel peptide deformylase (PDF) inhibitor, shows good in vitro antibacterial activity and has demonstrated safety and efficacy in human proof-of-concept clinical studies. In vitro studies were performed to determine the frequency of resistance (FoR) to this antimicrobial agent in major pathogens causing respiratory tract and skin infections. Resistance to GSK1322322 occurred at high frequency through loss-of-function mutations in the formyl-methionyl transferase (FMT) protein in Staphylococcus aureus (4/4 strains) and Streptococcus pyogenes (4/4 strains), and via missense mutations in Streptococcus pneumoniae (6/21 strains), but these mutations are associated with a severe in vitro and/or in vivo fitness cost. The overall FoR to GSK1322322 was very low in Haemophilus influenzae with only one PDF mutant identified in one of four strains. No target-based mutants were identified from S. pyogenes, and only one or no PDF mutants were isolated in three of the four S. aureus strains studied. In S. pneumoniae, PDF mutants were only isolated from six of 21 strains tested and an additional 10 strains did not yield colonies on GSK1322322-containing plates. Most of the PDF mutants characterized from these three organisms (35/37), carried mutations in residues at or in close proximity to one of three highly conserved motifs, which are part of the active site of the PDF protein, with 30 of the 35 mutations occurring at position V71 (S. pneumoniae numbering system).
Copyright © 2015, American Society for Microbiology. All Rights Reserved.

PMID: 26014938 [PubMed - as supplied by publisher]

Thursday, May 14, 2015

Inclusion bodies and pH lowering: as an effect of gold nanoparticles in Streptococcus pneumoniae.

Metallomics. 2015 May 12. [Epub ahead of print]
Inclusion bodies and pH lowering: as an effect of gold nanoparticles in Streptococcus pneumoniae.
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Abstract
Streptococcus pneumoniae is a human pathogen whose principal virulence factor is its capsule. This structure allows the bacterium to evade the human immune system. Treatment of infections caused by this bacterium is based on antibiotics; however, the emergence of antibiotic-resistant strains makes this task increasingly difficult. Therefore, it is necessary to investigate new therapies, such as those based on gold nanoparticles, for which unfortunately the mechanisms involved have not yet been investigated. As far as we know, this study is the first that attempts to explain how gold nanoparticles destroy the bacterium Streptococcus pneumoniae. We found that the mean particle size was an important issue, and that the effect on the bacterium was dose-dependent. Cellular growth was inhibited by the presence of the nanoparticles, as was cell viability. The pH of the bacterial growth media was acidified, but interestingly the reactive species were not affected. A transmission electron microscopy analysis revealed the presence of inclusion bodies of gold nanoparticles within the bacterium. We present the first findings that attempt to explain how gold nanoparticles lyse Gram-positive bacteria.
PMID: 25966022 [PubMed - as supplied by publisher]


CXCL14 Displays Antimicrobial Activity against Respiratory Tract Bacteria and Contributes to Clearance of Streptococcus pneumoniae Pulmonary Infection.

J Immunol. 2015 May 11. pii: 1402634. [Epub ahead of print]
CXCL14 Displays Antimicrobial Activity against Respiratory Tract Bacteria and Contributes to Clearance of Streptococcus pneumoniae Pulmonary Infection.
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Abstract
CXCL14 is a chemokine with an atypical, yet highly conserved, primary structure characterized by a short N terminus and high sequence identity between human and mouse. Although it induces chemotaxis of monocytic cells at high concentrations, its physiological role in leukocyte trafficking remains elusive. In contrast, several studies have demonstrated that CXCL14 is a broad-spectrum antimicrobial peptide that is expressed abundantly and constitutively in epithelial tissues. In this study, we further explored the antimicrobial properties of CXCL14 against respiratory pathogens in vitro and in vivo. We found that CXCL14 potently killed Pseudomonas aeruginosa, Streptococcus mitis, and Streptococcus pneumoniae in a dose-dependent manner in part through membrane depolarization and rupture. By performing structure-activity studies, we found that the activity against Gram-negative bacteria was largely associated with the N-terminal peptide CXCL141-13. Interestingly, the central part of the molecule representing the β-sheet also maintained 62% killing activity and was sufficient to induce chemotaxis of THP-1 cells. The C-terminal α-helix of CXCL14 had neither antimicrobial nor chemotactic effect. To investigate a physiological function for CXCL14 in innate immunity in vivo, we infected CXCL14-deficient mice with lung pathogens and we found that CXCL14 contributed to enhanced clearance of Streptococcus pneumoniae, but not Pseudomonas aeruginosa. Our comprehensive studies reflect the complex bactericidal mechanisms of CXCL14, and we propose that different structural features are relevant for the killing of Gram-negative and Gram-positive bacteria. Taken together, our studies show that evolutionary-conserved features of CXCL14 are important for constitutive antimicrobial defenses against pneumonia.
Copyright © 2015 by The American Association of Immunologists, Inc.

PMID: 25964486 [PubMed - as supplied by publisher]

Streptococcus pneumoniae triggers progression of pulmonary fibrosis through pneumolysin.

Thorax. 2015 May 11. pii: thoraxjnl-2014-206420. doi: 10.1136/thoraxjnl-2014-206420. [Epub ahead of print]
Streptococcus pneumoniae triggers progression of pulmonary fibrosis through pneumolysin.
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Abstract
RATIONALE:
Respiratory tract infections are common in patients suffering from pulmonary fibrosis. The interplay between bacterial infection and fibrosis is characterised poorly.
OBJECTIVES:
To assess the effect of Gram-positive bacterial infection on fibrosis exacerbation in mice.
METHODS:
Fibrosis progression in response to Streptococcus pneumoniae was examined in two different mouse models of pulmonary fibrosis.
MEASUREMENTS AND MAIN RESULTS:
We demonstrate that wild-type mice exposed to adenoviral vector delivery of active transforming growth factor-β1 (TGFß1) or diphteria toxin (DT) treatment of transgenic mice expressing the DT receptor (DTR) under control of the surfactant protein C (SPC) promoter (SPC-DTR) to induce pulmonary fibrosis developed progressive fibrosis following infection with Spn, without exhibiting impaired lung protective immunity against Spn. Antibiotic treatment abolished infection-induced fibrosis progression. The cytotoxin pneumolysin (Ply) of Spn caused this phenomenon in a TLR4-independent manner, as Spn lacking Ply (SpnΔply) failed to trigger progressive fibrogenesis, whereas purified recombinant Ply did. Progressive fibrogenesis was also observed in AdTGFβ1-exposed Ply-challenged TLR4 KO mice. Increased apoptotic cell death of alveolar epithelial cells along with an attenuated intrapulmonary release of antifibrogenic prostaglandin E2 was found to underlie progressive fibrogenesis in Ply-challenged AdTGFβ1-exposed mice. Importantly, vaccination of mice with the non-cytotoxic Ply derivative B (PdB) substantially attenuated Ply-induced progression of lung fibrosis in AdTGFβ1-exposed mice.
CONCLUSIONS:
Our data unravel a novel mechanism by which infection with Spn through Ply release induces progression of established lung fibrosis, which can be attenuated by protein-based vaccination of mice.
Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://group.bmj.com/group/rights-licensing/permissions.
KEYWORDS:
Bacterial Infection; Idiopathic pulmonary fibrosis; Respiratory Infection

PMID: 25964315 [PubMed - as supplied by publisher]

Development of a conjugate vaccine against invasive pneumococcal disease based on capsular polysaccharides coupled with PspA/family 1 protein of Streptococcus pneumoniae.

Microb Pathog. 2015 May 7. pii: S0882-4010(15)00073-X. doi: 10.1016/j.micpath.2015.04.006. [Epub ahead of print]
Development of a conjugate vaccine against invasive pneumococcal disease based on capsular polysaccharides coupled with PspA/family 1 protein of Streptococcus pneumoniae.
Lin H1, Peng Y2, Lin Z3, Zhang S4, Guo Y5.


Abstract
The efforts were focused on exploring alternative pneumococcal vaccine strategies, aimed at addressing the shortcomings of existing formulations, without compromising efficacy. Our strategy involved the use of the carrier protein, pneumococcal surface protein A (PspA), conjugated with capsular polysaccharides (CPS), to provide effective and non-serotype-dependent protection. In this study, we generated a stable Escherichia coli construct expressing functional PspA from a capsular serotype 6B strain and confirmed it belonging to family 1, which was conjugated with CPS. The distribution of anti-CPS antibody response was almost completely of IgG2a subclass followed by IgG3 and low level of IgG1 subclass, but that of anti-PspA IgG subclass antibodies was almost equal IgG1 and IgG2a subclasses. Though PspA was less conspicuous on the surface of pneumococci than the capsule, the antibodies induced with CPS-rPspA conjugate possessed more accessibility to the surface of Streptococcus pneumoniaeserotype 6B and 19F (the same family 1 PspA). By survival experiment, the result suggested that the level of cross-protection after immunized with the conjugate was more measurable within the same family 1. The CPS-rPspA conjugate not only induced CPS-specific protection but also provided PspA specific cross-protection.
Copyright © 2015. Published by Elsevier Ltd.
KEYWORDS:
Capsular polysaccharides; Conjugate; Family 1; PspA; Streptococcus pneumoniae
PMID: 25959527 [PubMed - as supplied by publisher]

Formation of Streptococcus pneumoniae choline-binding protein-DNA complexes in vitro. Implications for biofilm development.

Environ Microbiol Rep. 2015 May 7. doi: 10.1111/1758-2229.12295. [Epub ahead of print]
Formation of Streptococcus pneumoniae choline-binding protein-DNA complexes in vitro. Implications for biofilm development.
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Abstract
Extracellular DNA (eDNA) is an essential component of bacterial biofilm matrices, and is required in their formation and maintenance. eDNA binds to exopolysaccharides or extracellular proteins, affording biofilms greater structural integrity. Recently, we reported evidence of intercellular eDNA-LytC complexes in pneumococcal biofilms. The LytC lysozyme is a member of the choline-binding family of proteins (CBPs) located on the pneumococcal surface. The present work shows that other CBPs, i.e., LytA, LytB, Pce, PspC and CbpF, which also have a pI between 5 and 6, can bind DNA in vitro. This process requires the presence of divalent cations other than Mg2+ . This DNA binding capacity of CBPs appears to be independent of their enzymatic activity and, at least in the case of LytA, does not require the choline-binding domain characteristic of CBPs. Positively charged, surface-exposed, 25 amino acid-long peptides derived from the catalytic domain of LytB, were also found capable of DNA binding through electrostatic interactions. Confocal laser scanning microcopy revealed the existence of cell-associated LytB-eDNA complexes in Streptococcus pneumoniae biofilms. These and other findings suggest that these surface-located proteins of S. pneumoniae could play roles of varying importance in the colonization and/or invasion of human host where different environmental conditions exist.
This article is protected by copyright. All rights reserved.

PMID: 25950767 [PubMed - as supplied by publisher]

Temporal changes in nasopharyngeal carriage of Streptococcus pneumoniae serotype 1 genotypes in healthy Gambians before and after the 7-valent pneumococcal conjugate vaccine.

PeerJ. 2015 Apr 30;3:e903. doi: 10.7717/peerj.903. eCollection 2015.
Temporal changes in nasopharyngeal carriage of Streptococcus pneumoniae serotype 1 genotypes in healthy Gambians before and after the 7-valent pneumococcal conjugate vaccine.
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Abstract
Streptococcus pneumoniae serotype 1 is one of the leading causes of invasive pneumococcal disease. However, this invasive serotype is hardly found in nasopharyngeal asymptomatic carriage and therefore large epidemiological studies are needed to assess the dynamics of serotype 1 infection. Within the context of a large cluster randomized trial conducted in rural Gambia to assess the impact of PCV-7 vaccination on nasopharyngeal carriage, we present an ancillary analysis describing the prevalence of nasopharyngeal carriage of pneumococcal serotype 1 and temporal changes of its more frequent genotypes. Nasopharyngeal swabs (NPS) were collected before PCV-7 vaccination (December 2003-May 2004) and up to 30 months after PCV-7 vaccination. The post-vaccination time was divided in three periods to ensure an equal distribution of the number of samples: (1) July 2006-March 2007, (2) April 2007-March 2008 and (3) April 2008-Feb 2009. S. pneumoniae serotype 1 were genotyped by MLST. Serotype 1 was recovered from 87 (0.71%) of 12,319 NPS samples collected. In the pre-vaccination period, prevalence of serotype 1 was 0.47% in both study arms. In the post-vaccination periods, prevalence in the fully vaccinated villages ranged between 0.08% in period 1 and 0.165% in period 2, while prevalence in partly vaccinated villages was between 0.17% in period 3 and 1.34% in period 2. Overall, four different genotypes were obtained, with ST3081 the most prevalent (60.71%), followed by ST618 (29.76%). ST3081 was found only in post-vaccination period 2 and 3, while ST618 had disappeared in post-vaccination period 3. Distribution of these major genotypes was similar in both study arms. Emergence of ST3081 and concomitant disappearance of ST618 may suggest a change in the molecular epidemiology of pneumococcal serotype 1 in this region. This change is not likely to be associated with the introduction of PCV-7 which lacks serotype 1, as it was observed simultaneously in both study arms. Future population-based epidemiological studies will provide further evidence of substantive changes in the pneumococcal serotype 1 epidemiology and the likely mechanisms.
KEYWORDS:
7-valent pneumococcal conjugate vaccine; Gambia; Invasive pneumococcal disease; Molecular epidemiology; Multilocus Sequence Typing; Nasopharyngeal Swab; Nasopharyngeal carriage; ST217 hyper virulent clonal complex; Sequence type; Streptococcus pneumoniae serotype 1

PMID: 25945306 [PubMed] PMCID: PMC4419557 

In vitro expression of Streptococcus pneumoniae ply gene in human monocytes and pneumocytes.

Eur J Med Res. 2015 May 7;20(1):52. doi: 10.1186/s40001-015-0142-4.
In vitro expression of Streptococcus pneumoniae ply gene in human monocytes and pneumocytes.
Author information


Abstract
BACKGROUND:
Streptococcus pneumoniae is one major cause of pneumonia in human and contains various virulence factors that contribute to pathogenesis of pneumococcal disease. This study investigated the role of pneumolysin, Ply, in facilitating S. pneumoniae invasion into the host blood stream.
METHODS:
S. pneumoniae strains were isolated from clinical blood and sputum samples and confirmed by PCR. Expression of ply gene was assessed by infecting human monocytes and pneumocytes.
RESULTS:
A total of 23 strains of S. pneumoniae isolated from blood (n = 11) and sputum (n = 12) along with S. pneumoniae ATCC49619 were used to infect human monocyte (THP-1) and Type II pneumocyte (A549) cell lines. All clinical strains of S. pneumoniae showed higher expression of ply mRNA than the American Type Culture Collection (ATCC) strain. Among the clinical strains, blood isolates showed higher expression of ply genes than sputum isolates, i.e., 2(1.5)- to 2(1.6)-folds in THP-1 and 2(0.4)- to 2(4.9)-folds in A549 cell lines.
CONCLUSIONS:
The data from the current study demonstrated that ply gene of blood- and sputum-derived S. pneumoniae is differentially expressed in two different cell lines. Under survival pressure, ply is highly expressed in these two cell lines for blood-derived S. pneumoniae, indicating that ply gene may facilitate S. pneumoniae invasion into the host blood system.

PMID: 25943628 [PubMed - in process] PMCID: PMC4426643 

Visualization of Streptococcus pneumoniae within Cardiac Microlesions and Subsequent Cardiac Remodeling.

J Vis Exp. 2015 Apr 7;(98). doi: 10.3791/52590.
Visualization of Streptococcus pneumoniae within Cardiac Microlesions and Subsequent Cardiac Remodeling.
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Abstract
During bacteremia Streptococcus pneumoniae can translocate across the vascular endothelium into the myocardium and form discrete bacteria-filled microscopic lesions (microlesions) that are remarkable due to the absence of infiltrating immune cells. Due to their release of cardiotoxic products, S. pneumoniae within microlesions are thought to contribute to the heart failure that is frequently observed during fulminate invasive pneumococcal disease in adults. Herein is demonstrated a protocol for experimental mouse infection that leads to reproducible cardiac microlesion formation within 30 hr. Instruction is provided on microlesion identification in hematoxylin & eosin stained heart sections and the morphological distinctions between early and late microlesions are highlighted. Instruction is provided on a protocol for verification of S. pneumoniae within microlesions using antibodies against pneumococcal capsular polysaccharide and immunofluorescent microscopy. Last, a protocol for antibiotic intervention that rescues infected mice and for the detection and assessment of scar formation in the hearts of convalescent mice is provided. Together, these protocols will facilitate the investigation of the molecular mechanisms underlying pneumococcal cardiac invasion, cardiomyocyte death, cardiac remodeling as a result of exposure to S. pneumoniae, and the immune response to the pneumococci in the heart.

PMID: 25939051 [PubMed - in process]

A Fast and Reliable Pipeline for Bacterial Transcriptome Analysis Case study: Serine-dependent Gene Regulation in Streptococcus pneumoniae.

J Vis Exp. 2015 Apr 25;(98). doi: 10.3791/52649.
A Fast and Reliable Pipeline for Bacterial Transcriptome Analysis Case study: Serine-dependent Gene Regulation in Streptococcus pneumoniae.
Author information


Abstract
Gene expression and its regulation are very important to understand the behavior of cells under different conditions. Various techniques are used nowadays to study gene expression, but most are limited in terms of providing an overall picture of the expression of the whole transcriptome. DNA microarrays offer a fast and economic research technology, which gives a full overview of global gene expression and have a vast number of applications including identification of novel genes and transcription factor binding sites, characterization of transcriptional activity of the cells and also help in analyzing thousands of genes (in a single experiment). In the present study, the conditions for bacterial transcriptome analysis from cell harvest to DNA microarray analysis have been optimized. Taking into account the time, costs and accuracy of the experiments, this technology platform proves to be very useful and universally applicabale for studying bacterial transcriptomes. Here, we perform DNA microarray analysis with Streptococcus pneumoniae as a case-study by comparing the transcriptional responses of S. pneumoniae grown in the presence of varying L-serine concentrations in the medium. Total RNA was isolated by using a Macaloid method using an RNA isolation kit and the quality of RNA was checked by using an RNA quality check kit. cDNA was prepared using reverse transcriptase and the cDNA samples were labelled using one of two amine-reactive fluorescent dyes. Homemade DNA microarray slides were used for hybridization of the labelled cDNA samples and microarray data were analyzed by using a cDNA microarray data pre-processing framework (Microprep). Finally, Cyber-T was used to analyze the data generated using Microprep for the identification of statistically significant differentially expressed genes. Furthermore, in-house built software packages (PePPER, FIVA, DISCLOSE, PROSECUTOR, Genome2D) were used to analyze data.

PMID: 25938895 [PubMed - in process]

Long-term survival of Streptococcus pneumoniae, Haemophilus influenzae and Moraxella catarrhalis as isolates and in nasopharyngeal specimens in frozen STGG storage medium.

J Microbiol Methods. 2015 Apr 30;114:38-39. doi: 10.1016/j.mimet.2015.04.012. [Epub ahead of print]
Long-term survival of Streptococcus pneumoniae, Haemophilus influenzae and Moraxella catarrhalis as isolates and in nasopharyngeal specimens in frozen STGG storage medium.


Abstract
We evaluated survival in WHO-recommended STGG storage medium of bacteria causing respiratory-tract infection. Streptococcus pneumoniae and Moraxella catarrhalis survived as single and mixed isolates stored at -70°C for 12.5years, but Haemophilus influenzae less than 4years. All the bacteria survived in the nasopharyngeal specimens at -70°C for 11years.
Copyright © 2015 Elsevier B.V. All rights reserved.
KEYWORDS:
Deep-freeze; Long-term survival; Respiratory-tract bacteria; STGG storage medium
PMID: 25937246 [PubMed - as supplied by publisher]

MLVA-typing on Streptococcus pneumoniae serotype 1 isolated from meningitis cases in Niger before the introduction of PCV-13 revealed a low genetic diversity.

Trans R Soc Trop Med Hyg. 2015 May 1. pii: trv033. [Epub ahead of print]
MLVA-typing on Streptococcus pneumoniae serotype 1 isolated from meningitis cases in Niger before the introduction of PCV-13 revealed a low genetic diversity.
Author information


Abstract
BACKGROUND:
Serotype 1 was the most prevalent pneumococcal meningitis serotype encountered in Niger over the period 2003-2011 (pre-vaccination era), accounting for 45.3% of infections.
METHODS:
Multiple locus VNTR analysis (MLVA) was used to create a genotypic snapshot of a representative subset of the pneumococcal population of serotype 1.
RESULTS:
MLVA using 16 markers revealed a homogeneous genetic background of pneumococci serotype 1 from Niger, which clustered with few serotype 1 pneumococci from some African countries, while other African countries displayed different clonal complexes. DNA from Niger and from other African countries were different from pneumococci serotype 1 from European countries.
CONCLUSIONS:
MLVA-typing revealed a low genetic diversity among pneumococci serotype 1 from meningitis cases in Niger in the pre-vaccination era.
© The Author 2015. Published by Oxford University Press on behalf of Royal Society of Tropical Medicine and Hygiene. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.
KEYWORDS:
Genetic diversity; Multiple locus VNTR analysis; Niger; Serotype 1; Streptococcus pneumoniae

PMID: 25934981 [PubMed - as supplied by publisher]

Prevalence and impact of Streptococcus pneumoniae in adult cystic fibrosis patients: a retrospective chart review and capsular serotyping study.

BMC Pulm Med. 2015 May 2;15(1):49. [Epub ahead of print]
Prevalence and impact of Streptococcus pneumoniae in adult cystic fibrosis patients: a retrospective chart review and capsular serotyping study.


Abstract
BACKGROUND:
Cystic fibrosis (CF) is a genetic disease characterized by complex polymicrobial communities within the lower respiratory tract. S. pneumoniae, while a well-defined pathogen in the general population, has rarely been identified in CF. Furthermore, prevalence studies on Pneumococcus in CF have predominantly focused on the infant and pediatric populations, and outcome data is lacking.
METHODS:
Through a review of our comprehensive clinical and microbiologic database from a single adult CF center in Canada from 1978-2013 we sought to determine the incidence, prevalence, serotype and clinical impact of Pneumococcus in adults with CF.
RESULTS:
Only fifteen of 318 adult CF patients (5%) were ever found to have transient Pneumococcus colonization, and none developed persistent infection although length of carriage varied. As all isolates were stored, capsular serotyping could be performed using a multiplex PCR panel. Capsular serotyping revealed a varied distribution of several serotypes within these isolates. Lung function testing at time of incident Pneumococcus isolation was compared with values before and after isolation and showed no significant reduction in spirometry values, nor was there an increased need for rescue antibacterial therapy.
CONCLUSION:
Within our center, incident Pneumococcus infection is neither common, associated with a disproportionate clinical deterioration nor results in chronic infection.
PMID: 25930152 [PubMed - as supplied by publisher] 

Genome-wide association study of IgG1 responses to the choline-binding protein PspC of Streptococcus pneumoniae.

Genes Immun. 2015 Apr 30. doi: 10.1038/gene.2015.12. [Epub ahead of print]
Genome-wide association study of IgG1 responses to the choline-binding protein PspC of Streptococcus pneumoniae.
Author information


Abstract
Streptococcus pneumoniae causes invasive pneumococcal disease. Delayed development of antibodies to S. pneumoniae in infancy is associated with the development of atopy and asthma. Pneumococcal surface protein C (PspC) is a vaccine candidate and variation in its choline-binding region is associated with invasive disease. This study examined 523 060 single-nucleotide polymorphisms in The Western Australian Pregnancy Cohort (Raine) Study to find loci influencing immunoglobulin G1 (IgG1) responses to PspC measured at age 14 years (n=1152). Genome-wide significance (top SNP rs9275596; P=3.1 × 10-14) was only observed at human leucocyte antigen (HLA). Imputed HLA amino-acid polymorphisms showed the strongest associations at positions DRB1 47 (P=3.2 × 10-11), 13SRG (P=9.8 × 10-10) and 11SP (P=9.8 × 10-10), and at DQA1 34 (P=6.4 × 10-10), DQB1 167R (P=9.3 × 10-6) and HLA-B 95 W (P=1.2 × 10-9). Conditional analyses showed independent contributions from DRB1 47 and DQB1 167R to the signal at rs9275596, supported by an omnibus test showing a strong signal for the haplotype DRB1_47_DQB1_167 (P=9.02 × 10-15). In silico analysis showed that DRB1 four-digit allele groups defined by DRB1 47F bind to a greater complexity of core 9-mer epitopes compared with DRB1 47Y, especially across repeats in the C-term choline-binding region. Consequent differences in CD4 T-cell help for IgG1 to PspC could have implications for vaccine design. Further analysis in other cohorts is merited.Genes and Immunity advance online publication, 30 April 2015; doi:10.1038/gene.2015.12.

PMID: 25928883 [PubMed - as supplied by publisher]

Emerging, Non-PCV13 Serotypes 11A and 35B of Streptococcus pneumoniae Show High Potential for Biofilm Formation In Vitro.

PLoS One. 2015 Apr 30;10(4):e0125636. doi: 10.1371/journal.pone.0125636. eCollection 2015.
Emerging, Non-PCV13 Serotypes 11A and 35B of Streptococcus pneumoniae Show High Potential for Biofilm Formation In Vitro.
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Abstract
BACKGROUND:
Since the use of pneumococcal conjugate vaccines PCV7 and PCV13 in children became widespread, invasive pneumococcal disease (IPD) has dramatically decreased. Nevertheless, there has been a rise in incidence of Streptococcus pneumoniae non-vaccine serotypes (NVT) colonising the human nasopharynx. Nasopharyngeal colonisation, an essential step in the development of S. pneumoniae-induced IPD, is associated with biofilm formation. Although the capsule is the main pneumococcal virulence factor, the formation of pneumococcal biofilms might, in fact, be limited by the presence of capsular polysaccharide (CPS).
METHODOLOGY/PRINCIPAL FINDINGS:
We used clinical isolates of 16 emerging, non-PCV13 serotypes as well as isogenic transformants of the same serotypes. The biofilm formation capacity of isogenic transformants expressing CPSs from NVT was evaluated in vitro to ascertain whether this trait can be used to predict the emergence of NVT. Fourteen out of 16 NVT analysed were not good biofilm formers, presumably because of the presence of CPS. In contrast, serotypes 11A and 35B formed ≥45% of the biofilm produced by the non-encapsulated M11 strain.
CONCLUSIONS/SIGNIFICANCE:
This study suggest that emerging, NVT serotypes 11A and 35B deserve a close surveillance.

PMID: 25927917 [PubMed - in process] PMCID: PMC4415931 

Streptococcus Pneumoniae: The Forgotten Microorganism in Neonatal Sepsis.

Fetal Pediatr Pathol. 2015 Apr 30. [Epub ahead of print]
Streptococcus Pneumoniae: The Forgotten Microorganism in Neonatal Sepsis.
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Abstract
Streptococcus pneumoniae is a rarely cause of neonatal sepsis. Its prevalence is low but with a mortality of 50%. Measures to prevent Streptococcus agalactiae transmission could help to increase Invasive Pneumococcal Disease (IPD) in newborns. Transmission could be from mother intrapartum; or in those cases of late onset sepsis, the community carriers. Systematic vaccination with PCV-7 and PCV-13 has reduced IPD rates. We present a case of a newborn with no perinatal risk factors for infection. In the first 24 hours after surgery of an ovarian cyst, the patient started with bad general condition with fever and regular perfusion. Empiric antibiotic treatment was started. Streptococcus pneumoniae was isolated in blood culture.
CONCLUSION:
In neonatal sepsis, we always think in Streptococcus agalactiae. Streptococcus pneumoniae is rare but with a high morbidity and mortality. Systematic vaccination is a measure that has demonstrated a reduction in the incidence of Invasive pneumococcal disease.
KEYWORDS:
Streptococcus pneumoniae; newborn; sepsis.

PMID: 25927681 [PubMed - as supplied by publisher]

Streptococcus pneumoniae GAPDH Is Released by Cell Lysis and Interacts with Peptidoglycan.

PLoS One. 2015 Apr 30;10(4):e0125377. doi: 10.1371/journal.pone.0125377. eCollection 2015.
Streptococcus pneumoniae GAPDH Is Released by Cell Lysis and Interacts with Peptidoglycan.
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Abstract
Release of conserved cytoplasmic proteins is widely spread among Gram-positive and Gram-negative bacteria. Because these proteins display additional functions when located at the bacterial surface, they have been qualified as moonlighting proteins. The GAPDH is a glycolytic enzyme which plays an important role in the virulence processes of pathogenic microorganisms like bacterial invasion and host immune system modulation. However, GAPDH, like other moonlighting proteins, cannot be secreted through active secretion systems since they do not contain an N-terminal predicted signal peptide. In this work, we investigated the mechanism of GAPDH export and surface retention in Streptococcus pneumoniae, a major human pathogen. We addressed the role of the major autolysin LytA in the delivery process of GAPDH to the cell surface. Pneumococcal lysis is abolished in the ΔlytA mutant strain or when 1% choline chloride is added in the culture media. We showed that these conditions induce a marked reduction in the amount of surface-associated GAPDH. These data suggest that the presence of GAPDH at the surface of pneumococcal cells depends on the LytA-mediated lysis of a fraction of the cell population. Moreover, we demonstrated that pneumococcal GAPDH binds to the bacterial cell wall independently of the presence of the teichoic acids component, supporting peptidoglycan as a ligand to surface GAPDH. Finally, we showed that peptidoglycan-associated GAPDH recruits C1q from human serum but does not activate the complement pathway.

PMID: 25927608 [PubMed - in process] PMCID: PMC4415926 

Modified MLVA for Genotyping Queensland Invasive Streptococcus pneumoniae.

PLoS One. 2015 Apr 29;10(4):e0121870. doi: 10.1371/journal.pone.0121870. eCollection 2015.
Modified MLVA for Genotyping Queensland Invasive Streptococcus pneumoniae.
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Abstract
BACKGROUND:
Globally, over 800 000 children under five die each year from infectious diseases caused by Streptococcus pneumoniae. To understand genetic relatedness between isolates, study transmission routes, assess the impact of human interventions e.g. vaccines, and determine infection sources, genotyping methods are required. The 'gold standard' genotyping method, Multi-Locus Sequence Typing (MLST), is useful for long-term and global studies. Another genotyping method, Multi-Locus Variable Number of Tandem Repeat Analysis (MLVA), has emerged as a more discriminatory, inexpensive and faster technique; however there is no universally accepted method and it is currently suitable for short-term and localised epidemiology studies. Currently Australia has no national MLST database, nor has it adopted any MLVA method for short-term or localised studies. This study aims to improve S. pneumoniae genotyping methods by modifying the existing MLVA techniques to be more discriminatory, faster, cheaper and technically less demanding than previously published MLVA methods and MLST.
METHODS:
Four different MLVA protocols, including a modified method, were applied to 317 isolates of serotyped invasive S. pneumoniae isolated from sterile body sites of Queensland children under 15 years from 2007-2012. MLST was applied to 202 isolates for comparison.
RESULTS:
The modified MLVA4 is significantly more discriminatory than the 'gold standard' MLST method. MLVA4 has similar discrimination compared to other MLVA techniques in this study). The failure to amplify particular loci in previous MLVA methods were minimised in MLVA4. Failure to amplify BOX-13 and Spneu19 were found to be serotype specific.
CONCLUSION:
We have modified a highly discriminatory MLVA technique for genotyping Queensland invasive S. pneumoniae. MLVA4 has the ability to enhance our understanding of the pneumococcal epidemiology and the changing genetics of the pneumococcus in localised and short-term studies.

PMID: 25923909 [PubMed - in process] PMCID: PMC4414480