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Necrotizing fasciitis and myositis are devastating infections characterized by high mortality. Group A streptococcus (GAS) is a common cause of these infections, but the molecular pathogenesis is poorly understood. We report a genome-wide analysis using serotype M1 and M28 strains that identified GAS genes contributing to necrotizing myositis in nonhuman primates (NHP), a clinically relevant model. Using transposon-directed insertion-site sequencing (TraDIS), we identified 126 and 116 GAS genes required for infection by serotype M1 and M28 organisms, respectively. For both M1 and M28 strains, more than 25% of the GAS genes required for necrotizing myositis encode known or putative transporters. Thirteen GAS transporters contributed to both M1 and M28 strain fitness in NHP myositis, including putative importers for amino acids, carbohydrates, and vitamins and exporters for toxins, quorum-sensing peptides, and uncharacterized molecules. Targeted deletion of genes encoding 5 transporters confirmed that each isogenic mutant strain was significantly (P < 0.05) impaired in causing necrotizing myositis in NHPs. Quantitative reverse-transcriptase PCR (qRT-PCR) analysis showed that these 5 genes are expressed in infected NHP and human skeletal muscle. Certain substrate-binding lipoproteins of these transporters, such as Spy0271 and Spy1728, were previously documented to be surface exposed, suggesting that our findings have translational research implications., Introduction Necrotizing fasciitis, commonly known as the flesh-eating disease, is an invasive infection with very high rates of human morbidity and mortality (1). In severe cases, contiguous muscle may be [...]

The identification of the molecular events responsible for strain emergence, enhanced virulence, and epidemicity has been a long-pursued goal in infectious diseases research. A recent analysis of 3,615 genomes of serotype M1 group A Streptococcus strains (the so-called 'flesh-eating' bacterium) identified a recombination event that coincides with the global M1 pandemic beginning in the early 1980s. Here, we have shown that the allelic variation that results from this recombination event, which replaces the chromosomal region encoding secreted NADase and streptolysin O, is the key driver of increased toxin production and enhanced infection severity of the M1 pandemic strains. Using isoallelic mutant strains, we found that 3 polymorphisms in this toxin gene region increase resistance to killing by human polymorphonuclear leukocytes, increase bacterial proliferation, and increase virulence in animal models of pharyngitis and necrotizing fasciitis. Genome sequencing of an additional 1,125 streptococcal strains and virulence studies revealed that a highly similar recombinational replacement event underlies an ongoing intercontinental epidemic of serotype M89 group A Streptococcus infections. By identifying the molecular changes that enhance upper respiratory tract fitness, increased resistance to innate immunity, and increased tissue destruction, we describe a mechanism that underpins epidemic streptococcal infections, which have affected many millions of people., Introduction One elusive goal in infectious diseases research is identification of the genetic changes and molecular mechanisms responsible for strain emergence, enhanced virulence, and epidemicity. This goal has practical importance [...]

Group A Streptococcus (GAS) is a gram-positive bacterial pathogen responsible for ≅600 million cases of pharyngitis each year worldwide (1). The widespread prevalence of this disease results in considerable costs, [...]

Infection with different strains of the same species of bacteria often results in vastly different clinical out-comes. Despite extensive investigation, the genetic basis of microbial strain-specific virulence remains poorly understood. [...]

Group A Streptococcus (GAS) causes human diseases ranging in severity from uncomplicated pharyngitis to life-threatening necrotizing fasciitis (1). GAS strains have traditionally been classified based on a serologic reaction against [...]

Streptococcus pyogenes is a strict human pathogen responsible for more than 700 million infections annually worldwide. Strains of serotype M28 S. pyogenes are typically among the five more abundant types causing invasive infections and pharyngitis in adults and children. Type M28 strains also have an unusual propensity to cause puerperal sepsis and neonatal disease. We recently discovered that a one-nucleotide indel in an intergenic homopolymeric tract located between genes Spy1336/R28 and Spy1337 altered virulence in a mouse model of infection. In the present study, we analyzed size variation in this homopolymeric tract and determined the extent of heterogeneity in the number of tandemly-repeated 79-amino acid domains in the coding region of Spy1336/R28 in large samples of strains recovered from humans with invasive infections. Both repeat sequence elements are highly polymorphic in natural populations of M28 strains. Variation in the homopolymeric tract results in (i) changes in transcript levels of Spy1336/R28 and Spy1337 in vitro, (ii) differences in virulence in a mouse model of necrotizing myositis, and (iii) global transcriptome changes as shown by RNAseq analysis of isogenic mutant strains. Variation in the number of tandem repeats in the coding sequence of Spy1336/R28 is responsible for size variation of R28 protein in natural populations. Isogenic mutant strains in which genes encoding R28 or transcriptional regulator Spy1337 are inactivated are significantly less virulent in a nonhuman primate model of necrotizing myositis. Our findings provide impetus for additional studies addressing the role of R28 and Spy1337 variation in pathogen-host interactions. [ABSTRACT FROM AUTHOR]

* Context.--Ten years ago a bioterrorism event involving Bacillus anthracis spores captured the nation's interest, stimulated extensive new research on this pathogen, and heightened concern about illegitimate release of infectious agents. Sporadic reports have described rare, fulminant, and sometimes fatal cases of pneumonia in humans and nonhuman primates caused by strains of Bacillus cereus,a species closely related to Bacillus anthracis. Objectives.--To describe and investigate a case of rapidly progressive, fatal, anthrax-like pneumonia and the overwhelming infection caused by a Bacillus species of uncertain provenance in a patient residing in rural Texas. Design.--We characterized the genome of the causative strain within days of its recovery from antemortem cultures using next-generation sequencing and performed immunohistochemistry on tissues obtained at autopsy with antibodies directed against virulence proteins of B anthracis and B cereus. Results.--We discovered that the infection was caused by a previously unknown strain of B cereus that was closely related to, but genetically distinct from, B anthracis. The strain contains a plasmid similar to pXO1, a genetic element encoding anthrax toxin and other known virulence factors. Immunohistochemistry demonstrated that several homologs of B anthracis virulence proteins were made in infected tissues, likely contributing to the patient's death. Conclusions.--Rapid genome sequence analysis permitted us to genetically define this strain, rule out the likelihood of bioterrorism, and contribute effectively to the institutional response to this event. Our experience strongly reinforced the critical value of deploying a well-integrated, anatomic, clinical, and genomic strategy to respond rapidly to a potential emerging, infectious threat to public health. (Arch Pathol Lab Med. 2011; 135:1447-1459; doi: 10.5858/arpa.2011-0362-SA), On September 18, 2001, letters containing spores of the Ames strain of Bacillus anthracis were mailed to the offices of news media. Additional letters containing these spores, postmarked October 9, [...]

Background. Invasive group A Streptococcus (iGAS) disease caused by type emm89 strains has been increasing worldwide, driven by the emergence of an epidemic clonal variant (clade 3 emm89). The clinical characteristics of patients with emm89 iGAS disease, and in particular with clade 3 emm89 iGAS disease, are poorly described. Methods. We used population-based iGAS surveillance data collected in metropolitan Toronto, Ontario, Canada during the period 2000-2014. We sequenced the genomes of 105 emm89 isolates representing all emm89 iGAS disease cases in the area during the period and 138 temporally matched emm89 iGAS isolates collected elsewhere in Ontario. Results. Clades 1 and 2 and clade O, a newly discovered emm89 genetic variant, caused most cases of emm89 iGAS disease in metropolitan Toronto before 2008. After rapid emergence of new clade 3, previously circulating clades were purged from the population and the incidence of emm89 iGAS disease significantly increased from 0.14 per 100 000 in 2000-2007 to 0.22 per 100 000 in 2008-2014. Overall, emm89 organisms caused significantly more arthritis but less necrotizing fasciitis than strains of the more common type emm1. Other clinical presentations were soft tissue and severe respiratory tract infections. Clinical outcomes did not differ significantly between emm89 clades overall. However, clade 3 emm89 iGAS disease was more common in youth and middle-aged individuals. Conclusions. The rapid shift in emm89 iGAS strain genetics in metropolitan Toronto has resulted in a significant increase in the incidence of emm89 iGAS disease, with noticeably higher rates of clade 3 disease in younger patients. [ABSTRACT FROM AUTHOR]

Recent advances in the scale and diversity of population genomic datasets for bacteria now provide the potential for genome-wide patterns of co-evolution to be studied at the resolution of individual bases. Here we describe a new statistical method, genomeDCA, which uses recent advances in computational structural biology to identify the polymorphic loci under the strongest co-evolutionary pressures. We apply genomeDCA to two large population data sets representing the major human pathogens Streptococcus pneumoniae (pneumococcus) and Streptococcus pyogenes (group A Streptococcus). For pneumococcus we identified 5,199 putative epistatic interactions between 1,936 sites. Over three-quarters of the links were between sites within the pbp2x, pbp1a and pbp2b genes, the sequences of which are critical in determining non-susceptibility to beta-lactam antibiotics. A network-based analysis found these genes were also coupled to that encoding dihydrofolate reductase, changes to which underlie trimethoprim resistance. Distinct from these antibiotic resistance genes, a large network component of 384 protein coding sequences encompassed many genes critical in basic cellular functions, while another distinct component included genes associated with virulence. The group A Streptococcus (GAS) data set population represents a clonal population with relatively little genetic variation and a high level of linkage disequilibrium across the genome. Despite this, we were able to pinpoint two RNA pseudouridine synthases, which were each strongly linked to a separate set of loci across the chromosome, representing biologically plausible targets of co-selection. The population genomic analysis method applied here identifies statistically significantly co-evolving locus pairs, potentially arising from fitness selection interdependence reflecting underlying protein-protein interactions, or genes whose product activities contribute to the same phenotype. This discovery approach greatly enhances the future potential of epistasis analysis for systems biology, and can complement genome-wide association studies as a means of formulating hypotheses for targeted experimental work. [ABSTRACT FROM AUTHOR]

Group A Streptococcus (GAS) causes a wide range of human infections, ranging from simple pharyngitis to life-threatening necrotizing fasciitis and toxic shock syndrome. A globally disseminated clone of M1T1 GAS has been associated with an increase in severe, invasive GAS infections in recent decades. The secreted GAS pore-forming toxin streptolysin O (SLO), which induces eukaryotic cell lysis in a cholesterol-dependent manner, is highly upregulated in the GAS M1T1 clone during bloodstream dissemination. SLO is known to promote GAS resistance to phagocytic clearance by neutrophils, a critical first element of host defense against invasive bacterial infection. Here, we examine the role of SLO in modulating specific neutrophil functions during their early interaction with GAS. We find that SLO at subcytotoxic concentrations and early time points is necessary and sufficient to suppress neutrophil oxidative burst, in a manner reversed by free cholesterol and anti-SLO blocking antibodies. In addition, SLO at subcytotoxic concentrations blocked neutrophil degranulation, interleukin-8 secretion and responsiveness, and elaboration of DNA-based neutrophil extracellular traps, cumulatively supporting a key role for SLO in GAS resistance to immediate neutrophil killing. A non-toxic SLO derivate elicits protective immunity against lethal GAS challenge in a murine infection model. We conclude that SLO exerts a novel cytotoxic-independent function at early stages of invasive infections (<30 min), contributing to GAS escape from neutrophil clearance. [ABSTRACT FROM AUTHOR]

Group A Streptococcus (GAS) causes a wide range of human infections, ranging from simple pharyngitis to life-threatening necrotizing fasciitis and toxic shock syndrome. A globally disseminated clone of M1T1 GAS has been associated with an increase in severe, invasive GAS infections in recent decades. The secreted GAS pore-forming toxin streptolysin O (SLO), which induces eukaryotic cell lysis in a cholesterol-dependent manner, is highly upregulated in the GAS M1T1 clone during bloodstream dissemination. SLO is known to promote GAS resistance to phagocytic clearance by neutrophils, a critical first element of host defense against invasive bacterial infection. Here, we examine the role of SLO in modulating specific neutrophil functions during their early interaction with GAS. We find that SLO at subcytotoxic concentrations and early time points is necessary and sufficient to suppress neutrophil oxidative burst, in a manner reversed by free cholesterol and anti-SLO blocking antibodies. In addition, SLO at subcytotoxic concentrations blocked neutrophil degranulation, interleukin-8 secretion and responsiveness, and elaboration of DNA-based neutrophil extracellular traps, cumulatively supporting a key role for SLO in GAS resistance to immediate neutrophil killing. A non-toxic SLO derivate elicits protective immunity against lethal GAS challenge in a murine infection model. We conclude that SLO exerts a novel cytotoxic-independent function at early stages of invasive infections (<30 min), contributing to GAS escape from neutrophil clearance. [ABSTRACT FROM AUTHOR]

Abstract: Advancements in high-throughput, high-volume data generating techniques increasingly present us with opportunities to probe new areas of biology. In this work we assessed the extent to which four closely related and genetically representative strains of group A Streptococcus causing epidemic disease have differentiated from one another. Comparative genome sequencing, expression microarray analysis, and proteomic studies were used in parallel to assess strain variation. The extent of phenotypic differentiation was unexpectedly large. We found significant associations between genetic polymorphisms and alterations in gene expression allowing us to estimate the frequency with which specific types of polymorphisms alter gene transcription. We identified polymorphisms in the gene (ropB) encoding the RopB regulator that associate with altered transcription of speB and production of the SpeB protein, a critical secreted protease virulence factor. Although these four epidemic strains are closely related, a key discovery is that accumulation of modest genetic changes has rapidly resulted in significant strain phenotypic differentiation, including the extracellular proteome that contains multiple virulence factors. These data provide enhanced understanding of genetic events resulting in strain variation in bacterial epidemics. [Copyright &y& Elsevier]

Outbreaks of disease attributable to human error or natural causes can provide unique opportunities to gain new information about host-pathogen interactions and new leads for pathogenesis research. Poststreptococcal glomerulonephritis (PSGN), a sequela of infection with pathogenic streptococci, is a common cause of preventable kidney disease worldwide. Although PSGN usually occurs after infection with group A streptococci, organisms of Lancefield group C and G also can be responsible. Despite decades of study, the molecular pathogenesis of PSGN is poorly understood. As a first step toward gaining new information about PSGN pathogenesis, we sequenced the genome of Streptococcus equi subsp. zooepidemicus strain MGCS10565, a group C organism that caused a very large and unusually severe epidemic of nephritis in Brazil. The genome is a circular chromosome of 2,024,171 bp. The genome shares extensive gene content, including many virulence factors, with genetically related group A streptococci, but unexpectedly lacks prophages. The genome contains many apparently foreign genes interspersed around the chromosome, consistent with the presence of a full array of genes required for natural competence. An inordinately large family of genes encodes secreted extracellular collagen-like proteins with multiple integrin-binding motifs. The absence of a gene related to speB rules out the long-held belief that streptococcal pyrogenic exotoxin B or antibodies reacting with it singularly cause PSGN. Many proteins previously implicated in GAS PSGN, such as streptokinase, are either highly divergent in strain MGCS10565 or are not more closely related between these species than to orthologs present in other streptococci that do not commonly cause PSGN. Our analysis provides a comparative genomics framework for renewed appraisal of molecular events underlying APSGN pathogenesis. [ABSTRACT FROM AUTHOR]

Examines the phenotypic and genotypic changes of Staphylococcus aureus during in vitro serial passage. Presence of aconitase activity in S. coccus strains; Influence of in vitro passage on acotinase specific activity; Regulation of beta-hemolytic activity by cell density-sensing accessory gene regulatory.

Describes the serotype of Streptococcus agalactiae through the polymerase gene. Involvement of the genes in sialic acid synthesis, pentasaccharide repeating unit formation and oligosaccharide transport and polymerization; Transcription of the operon as a single polycistronic message; Serotype specificity of a single gene.

Streptococcus agalactiae (group B streptococcus [GBS]) is a major cause of infections in newborns, pregnant women, and immunocompromised patients. GBS strain CNCTC10/84 is a clinical isolate that has high virulence in animal models of infection and has been used extensively to study GBS pathogenesis. Two unusual features of this strain are hyperhemolytic activity and hypo-CAMP factor activity. These two phenotypes are typical of GBS strains that are functionally deficient in the CovR-CovS two-component regulatory system. A previous whole-genome sequencing study found that strain CNCTC10/84 has intact covR and covS regulatory genes. We investigated CovR-CovS regulation in CNCTC10/84 and discovered that a single-nucleotide insertion in a homopolymeric tract in the covR promoter region underlies the strong hemolytic activity and weak CAMP activity of this strain. Using isogenic mutant strains, we demonstrate that this single-nucleotide insertion confers significantly decreased expression of covR and covS and altered expression of CovR-CovS-regulated genes, including that of genes encoding ß-hemolysin and CAMP factor. This single-nucleotide insertion also confers significantly increased GBS survival in human whole blood ex vivo. [ABSTRACT FROM AUTHOR]

A series of postpartum Streptococcus pyogenes infections prompted an investigation to rule out potential transmission by a health care worker. None of the hospital staff screened were colonized. All isolates were determined to be unrelated by molecular methods, including whole-genome sequencing. Thus, nosocomial transmission was considered unlikely. [ABSTRACT FROM AUTHOR]

Necrotizing fasciitis and myositis are devastating infections characterized by high mortality. Group A streptococcus (GAS) is a common cause of these infections, but the molecular pathogenesis is poorly understood. We report a genome-wide analysis using serotype M1 and M28 strains that identified GAS genes contributing to necrotizing myositis in nonhuman primates (NHP), a clinically relevant model. Using transposon-directed insertion-site sequencing (TraDIS), we identified 126 and 116 GAS genes required for infection by serotype M1 and M28 organisms, respectively. For both M1 and M28 strains, more than 25% of the GAS genes required for necrotizing myositis encode known or putative transporters. Thirteen GAS transporters contributed to both M1 and M28 strain fitness in NHP myositis, including putative importers for amino acids, carbohydrates, and vitamins and exporters for toxins, quorum-sensing peptides, and uncharacterized molecules. Targeted deletion of genes encoding 5 transporters confirmed that each isogenic mutant strain was significantly (P < 0.05) impaired in causing necrotizing myositis in NHPs. Quantitative reverse-transcriptase PCR (qRT-PCR) analysis showed that these 5 genes are expressed in infected NHP and human skeletal muscle. Certain substrate-binding lipoproteins of these transporters, such as Spy0271 and Spy1728, were previously documented to be surface exposed, suggesting that our findings have translational research implications. [ABSTRACT FROM AUTHOR]

Group A streptococcus (GAS) is a Gram-positive human bacterial pathogen responsible for more than 700 million infections annually worldwide. Beta-lactam antibiotics are the primary agents used to treat GAS infections. Naturally occurring GAS clinical isolates with decreased susceptibility to beta-lactam antibiotics attributed to mutations in PBP2X have recently been documented. This prompted us to perform a genome-wide screen to identify GAS genes that alter beta-lactam susceptibility in vitro. Using saturated transposon mutagenesis, we screened for GAS gene mutations conferring altered in vitro susceptibility to penicillin G and/or ceftriaxone, two beta-lactam antibiotics commonly used to treat GAS infections. In the aggregate, we found that inactivating mutations in 150 GAS genes are associated with altered susceptibility to penicillin G and/or ceftriaxone. Many of the genes identified were previously not known to alter beta-lactam susceptibility or affect cell wall biosynthesis. Using isogenic mutant strains, we confirmed that inactivation of clpX (Clp protease ATP-binding subunit) or cppA (CppA proteinase) resulted in decreased in vitro susceptibility to penicillin G and ceftriaxone. Deletion of murA1 (UDP-N-acetylglucosamine 1-carboxyvinyltransferase) conferred increased susceptibility to ceftriaxone. Our results provide new information about the GAS genes affecting susceptibility to beta-lactam antibiotics. [ABSTRACT FROM AUTHOR]

The identification of the molecular events responsible for strain emergence, enhanced virulence, and epidemicity has been a long-pursued goal in infectious diseases research. A recent analysis of 3,615 genomes of serotype M1 group A Streptococcus strains (the so-called "flesh-eating" bacterium) identified a recombination event that coincides with the global M1 pandemic beginning in the early 1980s. Here, we have shown that the allelic variation that results from this recombination event, which replaces the chromosomal region encoding secreted NADase and streptolysin O, is the key driver of increased toxin production and enhanced infection severity of the M1 pandemic strains. Using isoallelic mutant strains, we found that 3 polymorphisms in this toxin gene region increase resistance to killing by human polymorphonuclear leukocytes, increase bacterial proliferation, and increase virulence in animal models of pharyngitis and necrotizing fasciitis. Genome sequencing of an additional 1,125 streptococcal strains and virulence studies revealed that a highly similar recombinational replacement event underlies an ongoing intercontinental epidemic of serotype M89 group A Streptococcus infections. By identifying the molecular changes that enhance upper respiratory tract fitness, increased resistance to innate immunity, and increased tissue destruction, we describe a mechanism that underpins epidemic streptococcal infections, which have affected many millions of people. [ABSTRACT FROM AUTHOR]

Group A Streptococcus (GAS) is an important human pathogen that causes many types of infections, including pharyngitis and severe invasive diseases. We recently sequenced the genome of a serotype M3 strain and identified a prophage-encoded secreted phospholipase A2 designated SlaA. To study SlaA structure-activity relationships, 20 site-specific mutants were constructed by alanine-replacement mutagenesis and purified to apparent homogeneity. Enzymatic activity was greatly reduced by alanine replacement of amino acid residues previously described as crucial in the catalytic mechanism of secreted phospholipase A2. Similarly, substitution of five residues in an inferred Ca2+binding loop and three residues in the inferred active site region resulted in loss of activity of 76.5% or greater relative to the wild-type enzyme. Analysis of enzyme substrate specificity confirmed SlaA as a phospholipase A2, with activity against multiple phospholipid head groups and acyl chains located at the sn-2 position. PCR analysis of 1,189 GAS strains representing 48 M protein serotypes commonly causing human infections identified the slaA gene in 129 strains of nine serotypes (M1, M2, M3, M4, M6, M22, M28, M75, and st3757). Expression of SlaA by strains of these serotypes was confirmed by Western immunoblot. SlaA production increased rapidly and substantially on co-culture with Detroit 562 human pharyngeal epithelial cells. Together, these data provide new information about a novel extracellular enzyme that participates in GAS-human interactions. [ABSTRACT FROM AUTHOR]