Jorge E. Vidal, United States of America

University of Mississippi Medical Center Microbiology and Immunology

Presenter Of 2 Presentations

PNEUMOTAC: A HIGH-THROUGHPUT PLATFORM FOR SEROTYPING STREPTOCOCCUS PNEUMONIAE (ID 242)

Abstract

Background

Sensitive, high-throughput platforms, for pneumococcal serotyping are essential for vaccine effectiveness studies. We developed PneumoTAC, a TaqMan array card (TAC) platform for the rapid identification of most S. pneumoniae serotypes.

Methods

PneumoTAC reactions underwent a thorough analytical validation including studies of limit of detection (LOD) and sensitivity. The performance of PneumoTAC was investigated on blinded pneumococcal isolates (N=131), and in N=84 clinical specimens from pneumococcal disease episodes [cerebrospinal fluid samples (CSF, N=71), and blood cultures (N=13)].

Results

The LOD of assays in PneumoTAC spanned between 2 and 21.4 genome equivalents. A sensitivity of 100%, and 98.8%, was achieved for S. pneumoniae species confirmation using DNA from isolates, and clinical specimens, respectively. The sensitivity for the detection of the serotypes/serogroups was 94% using DNA from isolates. In clinical specimens PneumoTAC detected a serotype in N=80 (96.3%) of samples, whereas a multiplex (m)PCR approach detected a serotype in N=55 (66%) of specimens. The correlation between the serotype obtained by mPCR and that obtained by PneumoTAC was 96.3% (N=53).

Conclusions

We developed PneumoTAC a multi-target platform with increased serotype coverage, compared to other TAC assays and multiplex PCR approaches, to identify 94 S. pneumoniae serotypes/serogroups in a single run.

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TARGETING ERADICATION OF PNEUMOCOCCAL CARRIAGE WITH FDA-APPROVED DEOXYCHOLIC ACID (ID 828)

Abstract

Background

Nearly 1 billion children worldwide carry Streptococcus pneumoniae (Spn) in the upper airways. Human bile salts are known to dissolve the Spn cell wall of which deoxycholic acid (DoC) is approved by the FDA for aesthetic treatment (10 mg/ml). We investigated the antimicrobial effect of DoC against pneumococcal strains.

Methods

Dose-response and time-course studies were conducted with Spn reference strains (N=2) and MDR strains (N=3). We then challenged Spn strains (N=55), including vaccine types, and other oral bacterial species (N=21) with a MIC90 and viability was investigated. An in-vitro model of pneumococcal colonization assessed the antimicrobial activity in a life-like scenario.

Results

MIC90 was achieved with 0.5 mg/ml of DoC incubated for 2 h with vaccine and non-vaccine type strains and reference strains TIGR4 and D39. The pneumococcal colonization model demonstrated that DoC eradicated 107 cfu/ml of MDR strains bearing resistance to Penicillin, Meropenem, and/or Erythromycin within 20 min post-exposure. Viability of most oral bacterial species was not affected by a treatment with a Spn-MIC90.

Conclusions

DoC eradicated Spn strains with a MIC90 20-fold lower than the recommended human dose. It has the potential for prophylactic eradication of Spn carriage from the upper airways with no disturbance of other oral bacteria.

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Author Of 8 Presentations

IMPACT OF THE COM QUORUM SENSING SYSTEM ON THE TIGHT JUNCTION BELT DURING PNEUMOCOCCAL INVASION OF DIFFERENTIATED HUMAN BRONCHIAL EPITHELIAL CELLS (ID 681)

Abstract

Background

The tight junction (TJ) belt contributes to epithelial integrity during pneumococcal invasive disease. We investigated the effect of S. pneumoniae (Spn) invasion and translocation on TJ proteins and the role of Com, a quorum sensing system essential for the evolution of Spn.

Methods

A pseudostratified human airway epithelial tissue (HAE) model was developed with bronchial cells from donors. HAE cells were infected with TIGR4, or TIGR4ΔcomC, at different densities and incubated for up to 30 h. Pneumococci colonizing and those translocated to the basolateral side were quantified. Transepithelial electrical resistance (TEER), confocal/electron microscopy, ELISA for TJ proteins, and transcriptional analysis of genes encoding for ZO-1, claudin-4, and claudin-7, were performed.

Results

Infection with TIGR4 induced a dose-dependent decrease of TEER that correlated with invasion/translocation. Unexpectedly, a further decrease of TEER, with increased invasion, was induced by TIGR4ΔcomC compared to wt. Supernatant levels of ZO-1 and claudin-7 were increased when bacteria translocated, and were higher in HAE infected with TIGR4ΔcomC. Transcription of the ZO-1 gene was downregulated during infection with either strain, and further downregulated during the translocation stage.

Conclusions

S. pneumoniae invades, and translocates through, HAE cells by targeting TJ proteins. Knocking-down Com resulted in greater invasion and enhanced disturbance of the TJ.

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EFFICIENT DISSEMINATION OF INTEGRATIVE AND CONJUGATIVE ELEMENTS CONFERRING MULTIDRUG RESISTANCE IN STREPTOCOCCUS PNEUMONIAE IN AN EX VIVO HUMAN NASOPHARYNGEAL BIOFILM (ID 125)

Abstract

Background

Multidrug resistance in Streptococcus pneumoniae (Spn) has been increasingly attributed to dissemination of integrative and conjugative elements (ICEs), such as Tn2009 (23.5kb). The mechanism for Spn ICE dissemination has not been defined.

Methods

Recombination frequency (rF) for Tn2009 was investigated utilizing in vitro transformation or an ex vivo nasopharyngeal biofilm bioreactor. Recombinant lineage and extracellular DNA (eDNA) concentrations were determined by serotype-specific qPCR. Whole genome sequencing (WGS) identified putative junctions for Tn2009 recombination.

Results

In vitro transformation yielded no Tn2009-containing D39 recombinants (rF<10-9) while mutation-mediated streptomycin resistance was obtained (rF 10-6). However, in the bioreactor, Tn2009 transference from donor GA16833Tet/Ery (ST19F) to recipient D39Str (ST2) generated >90% D39Tet/Str recombinants with variably sized donor DNA fragments encompassing intact Tn2009 (rF 10-4), indicating varied recombination junctions. Tn2009 transference was prevented by DNaseI addition (rF<10-7). D39 competence mutants (ΔcomC/D/E) with GA16833 yielded reduced rFs (10-8-10-6) and nearly 100% ST19F recombinants acquiring Str resistance. Similar bacterial densities and eDNA concentrations from each strain were detected. D39ΔcomC with GA16833ΔcomC yielded no recombinants (rF<10-7).

Conclusions

Efficient Tn2009 dissemination among Spn strains occurs in an ex vivo nasopharyngeal biofilm and requires recipient competence development. Further, there is a com-mediated dominance for a specific Spn strain to acquire resistance.

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TARGETING ERADICATION OF PNEUMOCOCCAL CARRIAGE WITH FDA-APPROVED DEOXYCHOLIC ACID (ID 828)

Abstract

Background

Nearly 1 billion children worldwide carry Streptococcus pneumoniae (Spn) in the upper airways. Human bile salts are known to dissolve the Spn cell wall of which deoxycholic acid (DoC) is approved by the FDA for aesthetic treatment (10 mg/ml). We investigated the antimicrobial effect of DoC against pneumococcal strains.

Methods

Dose-response and time-course studies were conducted with Spn reference strains (N=2) and MDR strains (N=3). We then challenged Spn strains (N=55), including vaccine types, and other oral bacterial species (N=21) with a MIC90 and viability was investigated. An in-vitro model of pneumococcal colonization assessed the antimicrobial activity in a life-like scenario.

Results

MIC90 was achieved with 0.5 mg/ml of DoC incubated for 2 h with vaccine and non-vaccine type strains and reference strains TIGR4 and D39. The pneumococcal colonization model demonstrated that DoC eradicated 107 cfu/ml of MDR strains bearing resistance to Penicillin, Meropenem, and/or Erythromycin within 20 min post-exposure. Viability of most oral bacterial species was not affected by a treatment with a Spn-MIC90.

Conclusions

DoC eradicated Spn strains with a MIC90 20-fold lower than the recommended human dose. It has the potential for prophylactic eradication of Spn carriage from the upper airways with no disturbance of other oral bacteria.

Hide

PNEUMOTAC: A HIGH-THROUGHPUT PLATFORM FOR SEROTYPING STREPTOCOCCUS PNEUMONIAE (ID 242)

Abstract

Background

Sensitive, high-throughput platforms, for pneumococcal serotyping are essential for vaccine effectiveness studies. We developed PneumoTAC, a TaqMan array card (TAC) platform for the rapid identification of most S. pneumoniae serotypes.

Methods

PneumoTAC reactions underwent a thorough analytical validation including studies of limit of detection (LOD) and sensitivity. The performance of PneumoTAC was investigated on blinded pneumococcal isolates (N=131), and in N=84 clinical specimens from pneumococcal disease episodes [cerebrospinal fluid samples (CSF, N=71), and blood cultures (N=13)].

Results

The LOD of assays in PneumoTAC spanned between 2 and 21.4 genome equivalents. A sensitivity of 100%, and 98.8%, was achieved for S. pneumoniae species confirmation using DNA from isolates, and clinical specimens, respectively. The sensitivity for the detection of the serotypes/serogroups was 94% using DNA from isolates. In clinical specimens PneumoTAC detected a serotype in N=80 (96.3%) of samples, whereas a multiplex (m)PCR approach detected a serotype in N=55 (66%) of specimens. The correlation between the serotype obtained by mPCR and that obtained by PneumoTAC was 96.3% (N=53).

Conclusions

We developed PneumoTAC a multi-target platform with increased serotype coverage, compared to other TAC assays and multiplex PCR approaches, to identify 94 S. pneumoniae serotypes/serogroups in a single run.

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PNEUMOCOCCAL COLONIZATION DENSITY PATTERNS OVER TIME IN YOUNG CHILDREN IN THE PERUVIAN ANDES (ID 246)

Abstract

Background

Factors associated with nasopharyngeal pneumococcal colonization density have not been comprehensively characterized. Age, immunization status, geographical location, population density, season, and/or acute respiratory illness (ARI) may play a role. We assessed longitudinal colonization density patterns in young rural Peruvian children.

Methods

Nasopharyngeal samples were collected monthly from children aged <3 years followed prospectively each week for ARI from May 2009-September 2011. PCV7 was introduced in the region in 2009. Longitudinally-collected samples from a convenience sample of children with >=1 pneumococcus-positive sample underwent density assessment by lytA qPCR. Density values were log10-transformed to reduce skewness. Assessments were stratified by enrollment age.

Results

Pneumococcus was detected in 471/625 (75%) samples from 30 children; 20/30 (67%) were enrolled before age 1. Variability was observed in colonization densities by calendar quarter and enrollment age, with substantial overlap in density levels and trajectories over time among age groups (Figure). Median densities during ARI episodes (n=62) were 5.60 (IQR 4.36-6.24) compared to non-ARI periods (5.00 [3.98-5.93], n=409, p=0.023). fig_1_01.13.20.jpg

Conclusions

Among these children, density varied by ARI status but did not clearly decrease with age. Future trajectory analyses will assess serotype-specific density colonization patterns and the association of serotype co-colonization, vaccination, and ARI status with pneumococcal density over time.

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PNEUMOCOCCAL CARRIAGE IN PCV-10 VACCINATED HEALTHY AMERINDIAN KICHWA CHILDREN FROM ECUADOR: PREVALENCE, SEROTYPES AND ANTIBIOTIC SUSCEPTIBILITY. (ID 1236)

STREPTOCOCCUS PNEUMONIAE GENERATES HYDROXYL RADICALS TO RAPIDLY INTOXICATE AND KILL STAPHYLOCOCCUS AUREUS STRAINS (ID 256)

Abstract

Background

Streptococcus pneumoniae (Spn) strains rapidly kills Staphylococcus aureus (Sau) by producing membrane-permeable hydrogen peroxide (H2O2). The exact mechanism by which the H2O2-mediated killing occurs is not well understood.

Methods

An in vitro model that mimicked Spn-Sau contact during colonization of the upper airways and whole genome sequencing was conducted. Different Spn H2O2 mutants were constructed to confirm the Sau killing mechanism.

Results

Sau killing required outcompeting densities of Spn. A collection of MRSA/MSSA strains showed a linear sensitivity (R2=0.95) for Spn killing but the same strains had different susceptibilities when challenged against pure H2O2. WGS of these MRSA/MSSA strains revealed no association between clonal complex and susceptibility, or resistance, to Spn, or H2O2,respectively. A sublethal dose (~1 mM) of pure H2O2 when incubated with TIGR4DspxB eradicated cultures of Sau strains suggesting that Spn converts H2O2 to the hydroxyl radical (·OH). Accordingly, Sau killing was completely blocked by incubating with scavengers of ·OH radicals, DMSO, or thiourea.

Conclusions

Spn produces H2O2 which is rapidly converted to a more potent oxidant, the ·OH radical. Hydroxyl radicals does not affect Spn viability but rapidly intoxicate Sau strains. The target(s) of the ·OH radicals represents an exciting new alternative for the development of therapeutics against Sau infections.

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