[Show abstract][Hide abstract] ABSTRACT: Group A streptococcus (GAS), the causative agent of pharyngitis and necrotizing fasciitis, secretes the potent cysteine protease SpeB. Several lines of evidence suggest that SpeB is an important virulence factor. SpeB is expressed in human infections, protects mice from lethal challenge when used as a vaccine, and significantly contributes to tissue destruction and dissemination in animal models. However, recent descriptions of mutations in genes implicated in SpeB production have led to the idea that GAS may be under selective pressure to decrease secreted SpeB protease activity during infection. Thus, two divergent hypotheses have been proposed. One postulates that SpeB is a key contributor to pathogenesis. Alternatively, GAS may be under selection to decrease SpeB during infection. To differentiate between these alternative hypotheses, we performed casein hydrolysis assays to measure SpeB protease activity secreted by 6,775 GAS strains recovered from infected humans. Results demonstrated that 84.3% have a wild-type SpeB protease phenotype. The availability of whole genome sequence data allowed us to determine the relative frequency of mutations in genes implicated in SpeB production. The most abundantly mutated genes were direct transcription regulators. We also sequenced the genome of 2,954 GAS isolates recovered from nonhuman primates with experimental necrotizing fasciitis. No mutations were identified that would result in a SpeB-deficient phenotype. Taken together, these data unambiguously demonstrate that the great majority of GAS recovered from infected humans secrete a wild-type level of SpeB protease activity. Our data confirm the important role of SpeB in GAS pathogenesis and help end a long-standing controversy.
Full-text · Article · Sep 2015 · Infection and immunity
[Show abstract][Hide abstract] ABSTRACT: The molecular mechanisms underlying pathogen emergence in humans is a critical but poorly understood area of microbiologic investigation. Serotype V group B Streptococcus (GBS) was first isolated from humans in 1975, and rates of invasive serotype V GBS disease significantly increased starting in the early 1990s. We found that 210 of 229 serotype V GBS strains (92%) isolated from the bloodstream of nonpregnant adults in the United States and Canada between 1992 and 2013 were multilocus sequence type (ST) 1. Elucidation of the complete genome of a 1992 ST-1 strain revealed that this strain had the highest homology with a GBS strain causing cow mastitis and that the 1992 ST-1 strain differed from serotype V strains isolated in the late 1970s by acquisition of cell surface proteins and antimicrobial resistance determinants. Whole-genome comparison of 202 invasive ST-1 strains detected significant recombination in only eight strains. The remaining 194 strains differed by an average of 97 SNPs. Phylogenetic analysis revealed a temporally dependent mode of genetic diversification consistent with the emergence in the 1990s of ST-1 GBS as major agents of human disease. Thirty-one loci were identified as being under positive selective pressure, and mutations at loci encoding polysaccharide capsule production proteins, regulators of pilus expression, and two-component gene regulatory systems were shown to affect the bacterial phenotype. These data reveal that phenotypic diversity among ST-1 GBS is mainly driven by small genetic changes rather than extensive recombination, thereby extending knowledge into how pathogens adapt to humans.
Full-text · Article · May 2015 · Proceedings of the National Academy of Sciences
[Show abstract][Hide abstract] ABSTRACT: -Clinical trials in heart failure have focused on the improvement in symptoms or decreases in the risk of death and other cardiovascular events. Little is known about the effect of drugs on the risk of clinical deterioration in surviving patients.
-We compared the angiotensin-neprilysin inhibitor LCZ696 (400 mg daily) with the angiotensinconverting enzyme inhibitor enalapril (20 mg daily) in 8399 patients with heart failure and reduced ejection fraction in a double-blind trial. The analyses focused on prespecified measures of nonfatal clinical deterioration. In comparison with the enalapril group, fewer LCZ696-treated patients required intensification of medical treatment for heart failure (520 versus 604; hazard ratio, 0.84; 95% confidence interval, 0.74-0.94; P=0.003) or an emergency department visit for worsening heart failure (hazard ratio, 0.66; 95% confidence interval, 0.52-0.85; P=0.001). The patients in the LCZ696 group had 23% fewer hospitalizations for worsening heart failure (851 versus 1079; P<0.001) and were less likely to require intensive care (768 versus 879; 18% rate reduction, P=0.005), to receive intravenous positive inotropic agents (31% risk reduction, P<0.001), and to have implantation of a heart failure device or cardiac transplantation (22% risk reduction, P=0.07). The reduction in heart failure hospitalization with LCZ696 was evident within the first 30 days after randomization. Worsening of symptom scores in surviving patients was consistently more common in the enalapril group. LCZ696 led to an early and sustained reduction in biomarkers of myocardial wall stress and injury (N-terminal pro-Btype natriuretic peptide and troponin) versus enalapril.
-Angiotensin-neprilysin inhibition prevents the clinical progression of surviving patients with heart failure more effectively than angiotensin-converting enzyme inhibition. Clinical Trial Registration-URL: http://www.clinicaltrials.gov. Unique identifier: NCT01035255.
[Show abstract][Hide abstract] ABSTRACT: Background: Over the past 20 years, the rates of invasive group B streptococcal (GBS) disease in non-pregnant adults have markedly increased with the majority of adult infections now due to serotype V strains. Methods: Multi-locus sequence typing (MLST) was performed on 233 serotype V GBS bloodstream isolates from non-pregnant adults in the United States and Canada collected from 1992 -2013. An ST-1 GBS genome was completed and used for whole genome comparative analyses of 201 ST-1 isolates. Analysis of gene specific polymorphism frequency was used to identify genes under positive selection. The impact of observed polymorphisms on strain phenotypic variation was assessed by capsule level measurements, pilus protein assays, or global transcriptome analysis using RNAseq. Results: 211/233 isolates (>90%) were ST-1 or a single nucleotide variant of ST-1. The completed ST-1 reference genome showed multiple, significant differences in cell surface components critical to GBS host-pathogen interaction compared to the previously published GBS serotype V ST-110 genome. Whole genome sequence analyses revealed significant recombination in 8 strains. ST-1 strains differed by an average of 97 single nucleotide polymorphisms (SNPs) over the 2.1MB genome. Phylogenetic analyses revealed a temporally dependent mode of genetic diversification consistent with the relatively recent introduction of ST-1 GBS into humans. Thirty-one GBS genes were found to be under positive selective pressure for variance including capsule, pilus proteins, and key transcriptional regulators. Antimicrobial resistance elements were widespread among ST-1 strains, with the majority of strains harboring both tetracycline (97%) and macrolide (70%) resistance determinants. Conclusion: These data provide the first dense genomic level insight into GBS population structure and reveal that phenotypic diversity among ST-1 GBS is mainly driven by small genetic changes rather than recombination. Our data provide novel information on GBS loci contributing to host-pathogen interaction in adult invasive infections which influences GBS preventive strategies, and more globally elucidate the molecular diversification of emerging bacterial pathogens recently introduced into humans.
[Show abstract][Hide abstract] ABSTRACT: Humans commonly carry pathogenic bacteria asymptomatically, but despite decades of study, the underlying molecular contributors
remain poorly understood. Here, we show that a group A streptococcus carriage strain contains a frameshift mutation in the
hasA gene resulting in loss of hyaluronic acid capsule biosynthesis. This mutation was repaired by allelic replacement, resulting
in restoration of capsule production in the isogenic derivative strain. The “repaired” isogenic strain was significantly more
virulent than the carriage strain in a mouse model of necrotizing fasciitis and had enhanced growth ex vivo in human blood. Importantly, the repaired isogenic strain colonized the mouse oropharynx with significantly greater bacterial
burden and had significantly reduced ability to internalize into cultured epithelial cells than the acapsular carriage strain.
We conducted full-genome sequencing of 81 strains cultured serially from 19 epidemiologically unrelated human subjects and
discovered the common theme that mutations negatively affecting capsule biosynthesis arise in vivo in the has operon. The significantly decreased capsule production is a key factor contributing to the molecular détente between pathogen
and host. Our discoveries suggest a general model for bacterial pathogens in which mutations that downregulate or ablate virulence
factor production contribute to carriage.
Full-text · Article · Jul 2014 · Infection and Immunity
[Show abstract][Hide abstract] ABSTRACT: The genetically diverse viridans group streptococci (VGS) are increasingly recognized as the cause of a variety of human diseases. We used a recently developed multilocus sequence analysis scheme to define the species of 118 unique VGS strains causing bacteremia in patients with cancer; Streptococcus mitis (68 patients) and S. oralis (22 patients) were the most frequently identified strains. Compared with patients infected with non-S. mitis strains, patients infected with S. mitis strains were more likely to have moderate or severe clinical disease (e.g., VGS shock syndrome). Combined with the sequence data, whole-genome analyses showed that S. mitis strains may more precisely be considered as >2 species. Furthermore, we found that multiple S. mitis strains induced disease in neutropenic mice in a dose-dependent fashion. Our data define the prominent clinical effect of the group of organisms currently classified as S. mitis and lay the groundwork for increased understanding of this understudied pathogen.
Full-text · Article · May 2014 · Emerging Infectious Diseases
[Show abstract][Hide abstract] ABSTRACT: Invasive group A streptococcal (GAS) strains often have genetic differences compared to GAS strains from nonsterile sites.
Invasive, “hypervirulent” GAS strains can arise from a noninvasive progenitor following subcutaneous inoculation in mice,
but such emergence has been rarely characterized in humans. We used whole genome analyses of multiple GAS isolates from the
same patient to document the molecular basis for emergence of a GAS strain with an invasive phenotype during human infection.
In contrast to previous theories, we found that elimination of production of the cysteine protease SpeB was not necessary
for emergence of GAS with an invasive, “hypervirulent” phenotype.
Full-text · Article · Dec 2013 · The Journal of Infectious Diseases
[Show abstract][Hide abstract] ABSTRACT: Humans commonly carry pathogenic bacteria asymptomatically, but the molecular factors underlying microbial asymptomatic carriage
are poorly understood. We previously reported that two epidemiologically unassociated serotype M3 group A Streptococcus (GAS) carrier strains had an identical 12-bp deletion in the promoter of the gene encoding Mga, a global positive gene regulator.
Herein, we report on studies designed to test the hypothesis that the identified 12-bp deletion in the mga promoter alters GAS virulence, thereby potentially contributing to the asymptomatic carrier phenotype. Using allelic exchange,
we introduced the variant promoter into a serotype M3 invasive strain and the wild-type promoter into an asymptomatic carrier
strain. Compared to strains with the wild-type mga promoter, we discovered that strains containing the promoter with the 12-bp deletion produced significantly fewer mga and Mga-regulated gene transcripts. Consistent with decreased mga transcripts, strains containing the variant mga promoter were also significantly less virulent in in vivo and ex vivo models of GAS disease. Further, we provide evidence that the pleiotropic regulator protein CodY binds to the mga promoter and that the 12-bp deletion in the mga promoter reduces CodY-mediated mga transcription. We conclude that the naturally occurring 12-bp deletion in the mga promoter significantly alters the pathogen-host interaction of these asymptomatic carrier strains. Our findings provide new
insight into the molecular basis of the carrier state of an important human pathogen.
Full-text · Article · Aug 2013 · Infection and immunity
[Show abstract][Hide abstract] ABSTRACT: Streptococcus pyogenes (group A Streptococcus, GAS) and Moraxella catarrhalis are important colonizers and (opportunistic) pathogens of the human respiratory tract. However, current knowledge regarding colonization and pathogenic potential of these two pathogens is based on work involving single bacterial species, even though the interplay between respiratory bacterial species is increasingly important in niche occupation and the development of disease. Therefore, to further define and understand polymicrobial species interactions, we investigated whether gene expression (and hence virulence potential) of GAS would be affected upon co-culture with M. catarrhalis. For co-culture experiments, GAS and M. catarrhalis were cultured in Todd-Hewitt broth supplemented with 0.2% yeast extract (THY) at 37°C with 5% CO2 aeration. Each strain was grown in triplicate so that triplicate experiments could be performed. Bacterial RNA was isolated, cDNA synthesized, and microarray transcriptome expression analysis performed. We observed significantly increased (≥4-fold) expression for genes playing a role in GAS virulence such as hyaluronan synthase (hasA), streptococcal mitogenic exotoxin Z (smeZ) and IgG endopeptidase (ideS). In contrast, significantly decreased (≥4-fold) expression was observed in genes involved in energy metabolism and in 12 conserved GAS two-component regulatory systems. This study provides the first evidence that M. catarrhalis increases GAS virulence gene expression during co-culture, and again shows the importance of polymicrobial infections in directing bacterial virulence.
[Show abstract][Hide abstract] ABSTRACT: AIMS: Although the focus of therapeutic intervention has been on neurohormonal pathways thought to be harmful in heart failure (HF), such as the renin-angiotensin-aldosterone system (RAAS), potentially beneficial counter-regulatory systems are also active in HF. These promote vasodilatation and natriuresis, inhibit abnormal growth, suppress the RAAS and sympathetic nervous system, and augment parasympathetic activity. The best understood of these mediators are the natriuretic peptides which are metabolized by the enzyme neprilysin. LCZ696 belongs to a new class of drugs, the angiotensin receptor neprilysin inhibitors (ARNIs), which both block the RAAS and augment natriuretic peptides.Methods
Patients with chronic HF, NYHA class II-IV symptoms, an elevated plasma BNP or NT-proBNP level, and an LVEF of ≤40% were enrolled in the Prospective comparison of ARNI with ACEI to Determine Impact on Global Mortailty and morbidity in Heart Failure trial (PARADIGM-HF). Patients entered a single-blind enalapril run-in period (titrated to 10 mg b.i.d.), followed by an LCZ696 run-in period (100 mg titrated to 200 mg b.i.d.). A total of 8436 patients tolerating both periods were randomized 1:1 to either enalapril 10 mg b.i.d. or LCZ696 200 mg b.i.d. The primary outcome is the composite of cardiovascular death or HF hospitalization, although the trial is powered to detect a 15% relative risk reduction in cardiovascular death.PerspectivesPARADIGM-HF will determine the place of the ARNI LCZ696 as an alternative to enalapril in patients with systolic HF. PARADIGM-HF may change our approach to neurohormonal modulation in HF.Trial registrationNCT01035255.
Full-text · Article · Apr 2013 · European Journal of Heart Failure
[Show abstract][Hide abstract] ABSTRACT: Unlabelled:
Group A streptococcus (GAS) causes human pharyngitis and invasive infections and frequently colonizes individuals asymptomatically. Many lines of evidence generated over decades have shown that the hyaluronic acid capsule is a major virulence factor contributing to these infections. While conducting a whole-genome analysis of the in vivo molecular genetic changes that occur in GAS during longitudinal human pharyngeal interaction, we discovered that serotypes M4 and M22 GAS strains lack the hasABC genes necessary for hyaluronic acid capsule biosynthesis. Using targeted PCR, we found that all 491 temporally and geographically diverse disease isolates of these two serotypes studied lack the hasABC genes. Consistent with the lack of capsule synthesis genes, none of the strains produced detectable hyaluronic acid. Despite the lack of a hyaluronic acid capsule, all strains tested multiplied extensively ex vivo in human blood. Thus, counter to the prevailing concept in GAS pathogenesis research, strains of these two serotypes do not require hyaluronic acid to colonize the upper respiratory tract or cause abundant mucosal or invasive human infections. We speculate that serotype M4 and M22 GAS have alternative, compensatory mechanisms that promote virulence.
A century of study of the antiphagocytic hyaluronic acid capsule made by group A streptococcus has led to the concept that it is a major virulence factor contributing to human pharyngeal and invasive infections. However, the discovery that some strains that cause abundant human infections lack hyaluronic acid biosynthetic genes and fail to produce this capsule provides a new stimulus for research designed to understand the group A streptococcus factors contributing to pharyngeal infection and invasive disease episodes.
[Show abstract][Hide abstract] ABSTRACT: Group A Streptococcus (GAS) causes an exceptionally broad range of infections in humans, from relatively mild pharyngitis and skin infections to life-threatening necrotizing fasciitis and toxic shock syndrome. An epidemic of severe invasive human infections caused by type emm59 GAS, heretofore an exceedingly rare cause of disease, spread west to east across Canada over a 3-year period (2006 to 2008). By sequencing the genomes of 601 epidemic, historic, and other emm59 organisms, we discovered that a recently emerged, genetically distinct emm59 clone is responsible for the Canadian epidemic. Using near-real-time genome sequencing, we were able to show spread of the Canadian epidemic clone into the United States. The extensive genome data permitted us to identify patterns of geographic dissemination as well as links between emm59 subclonal lineages that cause infections. Mouse and nonhuman primate models of infection demonstrated that the emerged clone is unusually virulent. Transmission of epidemic emm59 strains may have occurred primarily by skin contact, as suggested by an experimental model of skin transmission. In addition, the emm59 strains had a significantly impaired ability to persist in human saliva and to colonize the oropharynx of mice, and seldom caused human pharyngitis. Our study contributes new information to the rapidly emerging field of molecular pathogenomics of bacterial epidemics and illustrates how full-genome data can be used to precisely illuminate the landscape of strain dissemination during a bacterial epidemic.
Full-text · Article · Feb 2012 · American Journal Of Pathology