Streptococcus pneumoniae is a facultative anaerobic pathogen. Although it maintains fermentative metabolism, during aerobic growth pneumococci produce high levels of H2O2, which can have adverse ...effects on cell viability and DNA, and influence pneumococcal interaction with its host. The pneumococcus is unusual in its dealing with toxic reactive oxygen species (ROS) in that it neither has catalase nor the global regulators of peroxide stress resistance. Previously, we identified pneumococcal thiol peroxidase (TpxD) as the key enzyme for enzymatic removal of H2O2, and showed that TpxD synthesis is up-regulated upon exposure to H2O2. This study aimed to reveal the mechanism controlling TpxD expression under H2O2 stress. We hypothesize that H2O2 activates a transcription factor which in turn up-regulates tpxD expression. Microarray analysis revealed a pneumococcal global transcriptional response to H2O2. Mutation of tpxD abolished H2O2-mediated response to high H2O2 levels, signifying the need for an active TpxD under oxidative stress conditions. Bioinformatic tools, applied to search for a transcription factor modulating tpxD expression, pointed toward CodY as a potential candidate. Indeed, a putative 15-bp consensus CodY binding site was found in the proximal region of tpxD-coding sequence. Binding of CodY to this site was confirmed by EMSA, and genetic engineering techniques demonstrated that this site is essential for TpxD up-regulation under H2O2 stress. Furthermore, tpxD expression was reduced in a ΔcodY mutant. These data indicate that CodY is an activator of tpxD expression, triggering its up-regulation under H2O2 stress. In addition we show that H2O2 specifically oxidizes the 2 CodY cysteines. This oxidation may trigger a conformational change in CodY, resulting in enhanced binding to DNA. A schematic model illustrating the contribution of TpxD and CodY to pneumococcal global transcriptional response to H2O2 is proposed.
Knowledge of the in vivo physiology and metabolism of Streptococcus pneumoniae is limited, even though pneumococci rely on efficient acquisition and metabolism of the host nutrients for growth and ...survival. Because the nutrient-limited, hypoxic host tissues favor mixed-acid fermentation, we studied the role of the pneumococcal pyruvate formate lyase (PFL), a key enzyme in mixed-acid fermentation, which is activated posttranslationally by PFL-activating enzyme (PFL-AE). Mutations were introduced to two putative pfl genes, SPD0235 and SPD0420, and two putative pflA genes, SPD0229 and SPD1774. End-product analysis showed that there was no formate, the main end product of the reaction catalyzed by PFL, produced by mutants defective in SPD0420 and SPD1774, indicating that SPD0420 codes for PFL and SPD1774 for putative PFL-AE. Expression of SPD0420 was elevated in galactose-containing medium in anaerobiosis compared to growth in glucose, and the mutation of SPD0420 resulted in the upregulation of fba and pyk, encoding, respectively, fructose 1,6-bisphosphate aldolase and pyruvate kinase, under the same conditions. In addition, an altered fatty acid composition was detected in SPD0420 and SPD1774 mutants. Mice infected intranasally with the SPD0420 and SPD1774 mutants survived significantly longer than the wild type-infected cohort, and bacteremia developed later in the mutant cohort than in the wild type-infected group. Furthermore, the numbers of CFU of the SPD0420 mutant were lower in the nasopharynx and the lungs after intranasal infection, and fewer numbers of mutant CFU than of wild-type CFU were recovered from blood specimens after intravenous infection. The results demonstrate that there is a direct link between pneumococcal fermentative metabolism and virulence.
Invasive pneumococcal disease (IPD) caused by Streptococcus pneumoniae serotype 2 (Sp2) is infrequent. Large-scale outbreaks were not been reported following pneumococcal conjugate vaccine (PCV) ...implementation. We describe a Sp2 IPD outbreak in Israel, in the PCV13 era, with focus on Sp2 population structure and evolutionary dynamics.
The data were derived from a population-based, nationwide active surveillance of IPD since 2009. PCV7/PCV13 vaccines were introduced in July 2009 and November 2010, respectively. Sp2 isolates were tested for antimicrobial susceptibility, multilocus sequence typing, and whole-genome sequencing (WGS) analysis.
Overall, 170 Sp2 IPD cases were identified during 2009-2019; Sp2 increased in 2015 and caused 6% of IPD during 2015-2019, a 7-fold increase compared with 2009-2014. The outbreak was caused by a previously unreported molecular type (ST-13578), initially observed in Israel in 2014. This clone caused 88% of Sp2 during 2015-2019. ST-13578 is a single-locus variant of ST-1504, previously reported globally including in Israel. WGS analysis confirmed clonality among the ST-13578 population. Single-nucleotide polymorphism-dense regions support a hypothesis that the ST-13578 outbreak clone evolved from ST-1504 by recombination. All tested strains were penicillin-susceptible (minimum inhibitory concentration <0.06 μg/mL). The ST-13578 clone was identified almost exclusively (99%) in the Jewish population and was mainly distributed in 3 of 7 Israeli districts. The outbreak is still ongoing, although it began declining in 2017.
To the best of our knowledge, this is the first widespread Sp2 outbreak since PCV13 introduction worldwide, caused by the emerging ST-13578 clone.
Highlights • A prospective surveillance was conducted in children <5 years in southern Israel. • We assessed the impact of PCV7/PCV13 sequential introduction on the dynamics of serotypes 6C/6D. • PCV ...introduction resulted in decreased carriage rates and conjunctivitis caused by 6C/6D. • Incidences of 6C/6D decreased in otitis media and dropped to zero in IPD in the PCV13 era. • We conclude that 6C/6D do not act as replacement serotypes for 6A/6B following PCV13 vaccination.
Pneumococcal conjugate vaccines have reduced the incidence of invasive pneumococcal disease, caused by vaccine serotypes, but non-vaccine-serotypes remain a concern. We used whole genome sequencing ...to study pneumococcal serotype, antibiotic resistance and invasiveness, in the context of genetic background.
Our dataset of 13,454 genomes, combined with four published genomic datasets, represented Africa (40%), Asia (25%), Europe (19%), North America (12%), and South America (5%). These 20,027 pneumococcal genomes were clustered into lineages using PopPUNK, and named Global Pneumococcal Sequence Clusters (GPSCs). From our dataset, we additionally derived serotype and sequence type, and predicted antibiotic sensitivity. We then measured invasiveness using odds ratios that relating prevalence in invasive pneumococcal disease to carriage.
The combined collections (n = 20,027) were clustered into 621 GPSCs. Thirty-five GPSCs observed in our dataset were represented by >100 isolates, and subsequently classed as dominant-GPSCs. In 22/35 (63%) of dominant-GPSCs both non-vaccine serotypes and vaccine serotypes were observed in the years up until, and including, the first year of pneumococcal conjugate vaccine introduction.
Penicillin and multidrug resistance were higher (p < .05) in a subset dominant-GPSCs (14/35, 9/35 respectively), and resistance to an increasing number of antibiotic classes was associated with increased recombination (R2 = 0.27 p < .0001). In 28/35 dominant-GPSCs, the country of isolation was a significant predictor (p < .05) of its antibiogram (mean misclassification error 0.28, SD ± 0.13).
We detected increased invasiveness of six genetic backgrounds, when compared to other genetic backgrounds expressing the same serotype. Up to 1.6-fold changes in invasiveness odds ratio were observed.
We define GPSCs that can be assigned to any pneumococcal genomic dataset, to aid international comparisons. Existing non-vaccine-serotypes in most GPSCs preclude the removal of these lineages by pneumococcal conjugate vaccines; leaving potential for serotype replacement. A subset of GPSCs have increased resistance, and/or serotype-independent invasiveness.
Trigger factor (TF) has a known cytoplasmic function as a chaperone. In a previous study we showed that pneumococcal TF is also cell-wall localized and this finding combined with the immunogenic ...characteristic of TF, has led us to determine the vaccine potential of TF and decipher its involvement in pneumococcal pathogenesis. Bioinformatic analysis revealed that TF is conserved among pneumococci and has no human homologue. Immunization of mice with recombinant (r)TF elicited a protective immune response against a pneumococcal challenge, suggesting that TF contributes to pneumococcal pathogenesis. Indeed, rTF and an anti-rTF antiserum inhibited bacterial adhesion to human lung derived epithelial cells, indicating that TF contributes to the bacterial adhesion to the host. Moreover, bacteria lacking TF demonstrated reduced adhesion, in vitro, to lung-derived epithelial cells, neural cells and glial cells. The reduced adhesion could be restored by chromosomal complementation. Furthermore, bacteria lacking TF demonstrated significantly reduced virulence in a mouse model. Taken together, the ability of rTF to elicit a protective immune response, involvement of TF in bacterial adhesion, conservation of the protein among pneumococcal strains and the lack of human homologue, all suggest that rTF can be considered as a future candidate vaccine with a much broader coverage as compared to the currently available pneumococcal vaccines.
Abstract
Background
IPD caused by Sp2 (non-PCV13 serotype) is relatively rare. However, Sp2 has a high potential for causing IPD including meningitis. Large-scale outbreaks of Sp2 IPD are rare and ...were not reported post-PCV implementation. We describe Sp2 IPD outbreak in Israel, in the PCV13 era, caused by a novel clone. Additionally, we analyzed the population structure and evolutionary dynamics of Sp2 during 2006–2018.
Methods
An ongoing, population-based, nationwide active surveillance, conducted since July 2009. PCV7/PCV13 were implemented in Israel in July 2009 and November 2010, respectively. All isolates were tested for antimicrobial susceptibility, PFGE, MLST and whole-genome sequencing (WGS). Results. Overall, 173 Sp2 IPD cases were identified; all isolates were analyzed by MLST (Figure 1). During 2016–2017, Sp2 caused 7.6% of all-IPD, a 7-fold increase compared with 2006–2015, and ranked second (after serotype 12F causing 12%) among IPD isolates. During 2006–2015, 98% (40/41) Sp2 IPD were caused by the previously reported global ST-1504 clone. The outbreak was caused by a novel clone ST-13578, not previously reported (Figure 2). WGS analysis confirmed that ST-13578 was related, but genetically distinct from ST-1504, observed exclusively before the outbreak. A single strain of clone ST-74 previously globally reported was identified in 2017–2018. An additional case was identified in an adult in the UK, following a family visit from Israel. The ST-13578 clone was identified only in the Jewish population and was mainly distributed in 3 of the 7 Israeli districts. All tested strains were penicillin-susceptible (MIC < 0.06 μg/mL).
Conclusion
To the best of our knowledge, this is the first widespread Sp2 outbreak since PCV13 introduction worldwide, caused by a novel clone ST-13578. The outbreak is still ongoing, although a declining trend was noted since 2017.
Disclosures
All Authors: No reported Disclosures.
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