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ABSTRACT Streptococcus dysgalactiae subspecies equisimilis (SDSE) is a Gram-positive bacterial pathogen that infects humans and is closely related to group A streptococcus (GAS). Compared with GAS, far less is known about SDSE pathobiology. Increased rates of invasive SDSE infections have recently been reported in many countries. One SDSE emm type (stG62647) is known to cause severe diseases, including necrotizing soft-tissue infections, endocarditis, and osteoarticular infections. To increase our understanding of the molecular pathogenesis of stG62647 SDSE isolates causing human infections, we sequenced to closure the genomes of 120 stG62647 SDSE isolates. The genomes varied in size from 2.1 to 2.24 Mb pairs. The great majority of stG62647 isolates had IS1548 integrated into the silB gene, thereby inactivating it. Regions of difference, such as mobile genetic elements, were the largest source of genomic diversity. All 120 stG62647 isolates were assayed for virulence using a well-established mouse model of necrotizing myositis. An unexpectedly wide range of virulence was identified (20% to 95%), as assessed by near-mortality data. To explore the molecular mechanisms underlying virulence differences, we analyzed RNAseq transcriptome profiles for 38 stG62647 isolates (comprising the 19 least and most virulent) grown in vitro. Genetic polymorphisms were identified from whole-genome sequence data. Collectively, the results suggest that these SDSE isolates use multiple genetic pathways to alter virulence phenotype. The data also suggest that human genetics and underlying medical conditions contribute to disease severity. Our study integrates genomic, mouse virulence, and RNAseq data to advance our understanding of SDSE pathobiology and its molecular pathogenesis.IMPORTANCEThis study integrated genomic sequencing, mouse virulence assays, and bacterial transcriptomic analysis to advance our understanding of the molecular mechanisms contributing to Streptococcus dysgalactiae subsp. equisimilis emm type stG62647 pathogenesis. We tested a large cohort of genetically closely related stG62647 isolates for virulence using an established mouse model of necrotizing myositis and discovered a broad spectrum of virulence phenotypes, with near-mortality rates ranging from 20% to 95%. This variation was unexpected, given their close genetic proximity. Transcriptome analysis of stG62647 isolates responsible for the lowest and highest near-mortality rates suggested that these isolates used multiple molecular pathways to alter their virulence. In addition, some genes encoding transcriptional regulators and putative virulence factors likely contribute to SDSE emm type stG62647 pathogenesis. These data underscore the complexity of pathogen–host interactions in an emerging SDSE clonal group.

Abstract Klebsiella pneumoniae has been classified into two types, classical K. pneumoniae (cKP) and hypervirulent K. pneumoniae (hvKP). cKP isolates are highly diverse and important causes of nosocomial infections; they include globally disseminated antibiotic-resistant clones. hvKP isolates are sensitive to most antibiotics but are highly virulent, causing community-acquired infections in healthy individuals. The virulence phenotype of hvKP is associated with pathogenicity loci responsible for siderophore and hypermucoid capsule production. Recently, convergent strains of K. pneumoniae, which possess features of both cKP and hvKP, have emerged and are cause of much concern. Here, we screen the genomes of 2,608 multidrug-resistant K. pneumoniae isolates from the United States and identify 47 convergent isolates. We perform phenotypic and genomic characterization of 12 representative isolates. These 12 convergent isolates contain a variety of antimicrobial resistance plasmids and virulence plasmids. Most convergent isolates contain aerobactin biosynthesis genes and produce more siderophores than cKP isolates but not more capsule. Unexpectedly, only 1 of the 12 tested convergent isolates has a level of virulence consistent with hvKP isolates in a murine pneumonia model. These findings suggest that additional studies should be performed to clarify whether convergent strains are indeed more virulent than cKP in mouse and human infections.

Abstract The ongoing evolution of SARS-CoV-2 into more easily transmissible and infectious variants has provided unprecedented insight into mutations enabling immune escape. Understanding how these mutations affect the dynamics of antibody-antigen interactions is crucial to the development of broadly protective antibodies and vaccines. Here we report the characterization of a potent neutralizing antibody (N3-1) identified from a COVID-19 patient during the first disease wave. Cryogenic electron microscopy revealed a quaternary binding mode that enables direct interactions with all three receptor-binding domains of the spike protein trimer, resulting in extraordinary avidity and potent neutralization of all major variants of concern until the emergence of Omicron. Structure-based rational design of N3-1 mutants improved binding to all Omicron variants but only partially restored neutralization of the conformationally distinct Omicron BA.1. This study provides new insights into immune evasion through changes in spike protein dynamics and highlights considerations for future conformationally biased multivalent vaccine designs.

Abstract The emergence of a novel pathogen in a susceptible population can cause rapid spread of infection. High prevalence of SARS-CoV-2 infection in white-tailed deer (Odocoileus virginianus) has been reported in multiple locations, likely resulting from several human-to-deer spillover events followed by deer-to-deer transmission. Knowledge of the risk and direction of SARS-CoV-2 transmission between humans and potential reservoir hosts is essential for effective disease control and prioritisation of interventions. Using genomic data, we reconstruct the transmission history of SARS-CoV-2 in humans and deer, estimate the case finding rate and attempt to infer relative rates of transmission between species. We found no evidence of direct or indirect transmission from deer to human. However, with an estimated case finding rate of only 4.2%, spillback to humans cannot be ruled out. The extensive transmission of SARS-CoV-2 within deer populations and the large number of unsampled cases highlights the need for active surveillance at the human–animal interface.

Khan et al. use human blood and a macaque model of tuberculosis (TB) to assess whether macrophage heterogeneity contributes to TB immunity. They demonstrate that human macrophages show unique patterns of gene expression that enable differential control of TB after infection.

Gontu et al. conducted a longitudinal study in COVID patients in which they assessed the response trajectories of antibodies directed to the SARS-CoV-2 surface spike glycoprotein and in vitro SARS-CoV-2 live virus neutralizing titers. Their measurements demonstrate the presence of an optimum window for convalescent plasma donation as well as predictions of the most suitable donor type.

ABSTRACT Identification of genetic polymorphisms causing increased antibiotic resistance in bacterial pathogens traditionally has proceeded from observed phenotype to defined mutant genotype. The availability of large collections of microbial genome sequences that lack antibiotic susceptibility metadata provides an important resource and opportunity to obtain new information about increased antimicrobial resistance by a reverse genotype-to-phenotype bioinformatic and experimental workflow. We analyzed 26,465 genome sequences of Streptococcus pyogenes, a human pathogen causing 700 million infections annually. The population genomic data identified amino acid changes in penicillin-binding proteins 1A, 1B, 2A, and 2X with signatures of evolution under positive selection as potential candidates for causing decreased susceptibility to β-lactam antibiotics. Construction and analysis of isogenic mutant strains containing individual amino acid replacements in penicillin-binding protein 2X (PBP2X) confirmed that the identified residues produced decreased susceptibility to penicillin. We also discovered the first chimeric PBP2X in S. pyogenes and show that strains containing it have significantly decreased β-lactam susceptibility. The novel integrative reverse genotype-to-phenotype strategy presented is broadly applicable to other pathogens and likely will lead to new knowledge about antimicrobial agent resistance, a massive public health problem worldwide. IMPORTANCE The recent demonstration that naturally occurring amino acid substitutions in Streptococcus pyogenes PBP2X are sufficient to cause severalfold reduced susceptibility to multiple β-lactam antibiotics in vitro raises the concern that these therapeutic agents may become compromised. Substitutions in PBP2X are common first-step mutations that, with the incremental accumulation of additional adaptive mutations within the PBPs, can result in high-level resistance. Because β-lactam susceptibility testing is not routinely performed, the nature and extent of such substitutions within the PBPs of S. pyogenes are poorly characterized. To address this knowledge deficit, polymorphisms in the PBPs were identified among the most comprehensive cohort of S. pyogenes genome sequences investigated to date. The mutational processes and selective forces acting on the PBPs were assessed to identify specific substitutions likely to influence β-lactam susceptibility and to evaluate factors posited to be impediments to resistance emergence.

ABSTRACT The ARTIC Network provides a common resource of PCR primer sequences and recommendations for amplifying SARS-CoV-2 genomes. The initial tiling strategy was developed with the reference genome Wuhan-01, and subsequent iterations have addressed areas of low amplification and sequence drop out. Recently, a new version (V4) was released, based on new variant genome sequences, in response to the realization that some V3 primers were located in regions with key mutations. Herein, we compare the performance of the ARTIC V3 and V4 primer sets with a matched set of 663 SARS-CoV-2 clinical samples sequenced with an Illumina NovaSeq 6000 instrument. We observe general improvements in sequencing depth and quality, and improved resolution of the SNP causing the D950N variation in the spike protein. Importantly, we also find nearly universal presence of spike protein substitution G142D in Delta-lineage samples. Due to the prior release and widespread use of the ARTIC V3 primers during the initial surge of the Delta variant, it is likely that the G142D amino acid substitution is substantially underrepresented among early Delta variant genomes deposited in public repositories. In addition to the improved performance of the ARTIC V4 primer set, this study also illustrates the importance of the primer scheme in downstream analyses. IMPORTANCE ARTIC Network primers are commonly used by laboratories worldwide to amplify and sequence SARS-CoV-2 present in clinical samples. As new variants have evolved and spread, it was found that the V3 primer set poorly amplified several key mutations. In this report, we compare the results of sequencing a matched set of samples with the V3 and V4 primer sets. We find that adoption of the ARTIC V4 primer set is critical for accurate sequencing of the SARS-CoV-2 spike region. The absence of metadata describing the primer scheme used will negatively impact the downstream use of publicly available SARS-Cov-2 sequencing reads and assembled genomes.

There is mounting evidence of SARS-CoV-2 spillover from humans into many domestic, companion, and wild animal species. Research indicates that humans have infected white-tailed deer, and that deer-to-deer transmission has occurred, indicating that deer could be a wildlife reservoir and a source of novel SARS-CoV-2 variants. We examined the hypothesis that the Omicron variant is actively and asymptomatically infecting the free-ranging deer of New York City. Between December 2021 and February 2022, 155 deer on Staten Island, New York, were anesthetized and examined for gross abnormalities and illnesses. Paired nasopharyngeal swabs and blood samples were collected and analyzed for the presence of SARS-CoV-2 RNA and antibodies. Of 135 serum samples, 19 (14.1%) indicated SARS-CoV-2 exposure, and 11 reacted most strongly to the wild-type B.1 lineage. Of the 71 swabs, 8 were positive for SARS-CoV-2 RNA (4 Omicron and 4 Delta). Two of the animals had active infections and robust neutralizing antibodies, revealing evidence of reinfection or early seroconversion in deer. Variants of concern continue to circulate among and may reinfect US deer populations, and establish enzootic transmission cycles in the wild: this warrants a coordinated One Health response, to proactively surveil, identify, and curtail variants of concern before they can spill back into humans.

The mechanism by which environmental pH controls the virulence of the pathogen Streptococcus pyogenes is unclear. Here, Do et al. show that changes in pH affect the activity of the virulence regulator RopB via its interaction with a quorum-sensing peptide signal.

ABSTRACT We sequenced the genomes of 5,085 severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) strains causing two coronavirus disease 2019 (COVID-19) disease waves in metropolitan Houston, TX, an ethnically diverse region with 7 million residents. The genomes were from viruses recovered in the earliest recognized phase of the pandemic in Houston and from viruses recovered in an ongoing massive second wave of infections. The virus was originally introduced into Houston many times independently. Virtually all strains in the second wave have a Gly614 amino acid replacement in the spike protein, a polymorphism that has been linked to increased transmission and infectivity. Patients infected with the Gly614 variant strains had significantly higher virus loads in the nasopharynx on initial diagnosis. We found little evidence of a significant relationship between virus genotype and altered virulence, stressing the linkage between disease severity, underlying medical conditions, and host genetics. Some regions of the spike protein—the primary target of global vaccine efforts—are replete with amino acid replacements, perhaps indicating the action of selection. We exploited the genomic data to generate defined single amino acid replacements in the receptor binding domain of spike protein that, importantly, produced decreased recognition by the neutralizing monoclonal antibody CR3022. Our report represents the first analysis of the molecular architecture of SARS-CoV-2 in two infection waves in a major metropolitan region. The findings will help us to understand the origin, composition, and trajectory of future infection waves and the potential effect of the host immune response and therapeutic maneuvers on SARS-CoV-2 evolution. IMPORTANCE There is concern about second and subsequent waves of COVID-19 caused by the SARS-CoV-2 coronavirus occurring in communities globally that had an initial disease wave. Metropolitan Houston, TX, with a population of 7 million, is experiencing a massive second disease wave that began in late May 2020. To understand SARS-CoV-2 molecular population genomic architecture and evolution and the relationship between virus genotypes and patient features, we sequenced the genomes of 5,085 SARS-CoV-2 strains from these two waves. Our report provides the first molecular characterization of SARS-CoV-2 strains causing two distinct COVID-19 disease waves.

ABSTRACT Carbapenemases confer resistance to nearly all β-lactam antibiotics. The extensive spread of carbapenemase-producing multidrug-resistant bacteria contributes significantly to hospital-acquired infections. We have developed a novel protein-based binding assay that identifies KPC β-lactamases from clinical isolates. We used the protein-protein interaction between KPCs and a soluble β-lactamase inhibitory protein (BLIP) variant, BLIPK74T/W112D, which specifically inhibits KPCs but not other β-lactamases. In this assay, BLIPK74T/W112D was allowed to form complexes with KPC-2 in bacterial cell lysates and then extracted using His tag binding resins. We demonstrated the presence of KPC-2 by monitoring the hydrolysis of a colorimetric β-lactam substrate. Also, to further increase the accuracy of the method, a BLIPK74T/W112D-mediated inhibition assay was developed. The binding and inhibition assays were validated by testing 127 Klebsiella pneumoniae clinical isolates with known genome sequences for the presence of KPC. Our assays identified a total of 32 strains as KPC-2 producers, a result in 100% concordance with genome sequencing predictions. To further simplify the assay and decrease the time to obtain results, the BLIPK74T/W112D protein was tested in combination with the widely used Carba-NP assay. For this purpose, the genome-sequenced K. pneumoniae strains were tested for the presence of carbapenemases with the Carba-NP test with and without the addition of BLIPK74T/W122D. The test accurately identified carbapenemase-producing strains and the addition of BLIPK74T/W112D allowed a further determination that the strains contain KPC carbapenemase. Thus, the BLIPK74T/W112D protein is an effective sensor to specifically detect KPC β-lactamases produced by clinical isolates. IMPORTANCE Infections caused by carbapenem-resistant Enterobacteriaceae are associated with high therapeutic failure and mortality rates. Thus, it is critical to rapidly identify clinical isolates expressing KPC β-lactamases to facilitate administration of the correct antibiotic treatment and initiate infection control strategies. To address this problem, we developed a protein-based, KPC-specific binding assay in combination with a cell lysate inhibition assay that provided a 100% identification rate of KPC from clinical isolates of known genomic sequence. In addition, this protein sensor was adapted to the Carba-NP assay to provide a rapid strategy to detect KPC-producing isolates that will facilitate informed treatment of critically ill patients.

ABSTRACT A fundamental goal of contemporary biomedical research is to understand the molecular basis of disease pathogenesis and exploit this information to develop targeted and more-effective therapies. Necrotizing myositis caused by the bacterial pathogen Streptococcus pyogenes is a devastating human infection with a high mortality rate and few successful therapeutic options. We used dual transcriptome sequencing (RNA-seq) to analyze the transcriptomes of S. pyogenes and host skeletal muscle recovered contemporaneously from infected nonhuman primates. The in vivo bacterial transcriptome was strikingly remodeled compared to organisms grown in vitro, with significant upregulation of genes contributing to virulence and altered regulation of metabolic genes. The transcriptome of muscle tissue from infected nonhuman primates (NHPs) differed significantly from that of mock-infected animals, due in part to substantial changes in genes contributing to inflammation and host defense processes. We discovered significant positive correlations between group A streptococcus (GAS) virulence factor transcripts and genes involved in the host immune response and inflammation. We also discovered significant correlations between the magnitude of bacterial virulence gene expression in vivo and pathogen fitness, as assessed by previously conducted genome-wide transposon-directed insertion site sequencing (TraDIS). By integrating the bacterial RNA-seq data with the fitness data generated by TraDIS, we discovered five new pathogen genes, namely, S. pyogenes 0281 (Spy0281 [dahA]), ihk-irr, slr, isp, and ciaH, that contribute to necrotizing myositis and confirmed these findings using isogenic deletion-mutant strains. Taken together, our study results provide rich new information about the molecular events occurring in severe invasive infection of primate skeletal muscle that has extensive translational research implications. IMPORTANCE Necrotizing myositis caused by Streptococcus pyogenes has high morbidity and mortality rates and relatively few successful therapeutic options. In addition, there is no licensed human S. pyogenes vaccine. To gain enhanced understanding of the molecular basis of this infection, we employed a multidimensional analysis strategy that included dual RNA-seq and other data derived from experimental infection of nonhuman primates. The data were used to target five streptococcal genes for pathogenesis research, resulting in the unambiguous demonstration that these genes contribute to pathogen-host molecular interactions in necrotizing infections. We exploited fitness data derived from a recently conducted genome-wide transposon mutagenesis study to discover significant correlation between the magnitude of bacterial virulence gene expression in vivo and pathogen fitness. Collectively, our findings have significant implications for translational research, potentially including vaccine efforts.

ABSTRACT Populations of a bacterial pathogen, whether recovered from a single patient or from a worldwide study, are often a heterogeneous mix of genetically and phenotypically divergent strains. Such heterogeneity is of value in changing environments and arises via mechanisms such as gene gain or gene mutation. Here, we identify an isolate of serotype M12 group A Streptococcus (GAS) (Streptococcus pyogenes) that has a natural mutation in rocA, which encodes an accessory protein to the virulence-regulating two-component system CovR/CovS (CovR/S). Disruption of RocA activity results in the differential expression of multiple GAS virulence factors, including the anti-phagocytic hyaluronic acid capsule and the chemokine protease SpyCEP. While some of our data regarding RocA-regulated genes overlaps with previous studies, which were performed with isolates of alternate GAS serotypes, some variability was also observed. Perhaps as a consequence of this alternate regulatory activity, we discovered that the contribution of RocA to the ability of the M12 isolate to survive and proliferate in human blood ex vivo is opposite that previously observed in M1, M3, and M18 GAS strains. Specifically, rocA mutation reduced, rather than enhanced, survival of the isolate. Finally, we also present data from an analysis of rocA transcription and show that rocA is transcribed in both mono- and polycistronic mRNAs. In aggregate, our data provide insight into the important regulatory role of RocA and into the mechanisms and consequences of GAS phenotypic heterogeneity. IMPORTANCE This study investigates the regulatory and phenotypic consequences of a naturally occurring mutation in a strain of the bacterial pathogen the group A Streptococcus (Streptococcus pyogenes). We show that this mutation, which occurs in a regulator-encoding gene, rocA, leads to altered virulence factor expression and reduces the ability of this isolate to survive in human blood. Critically, the blood survival phenotype and the assortment of genes regulated by RocA differ compared to previous studies into RocA activity. The data are consistent with there being strain- or serotype-specific variability in RocA function. Given that phenotypic variants can lead to treatment failures and escape from preventative regimes, our data provide information with regard to a mechanism of phenotypic variation in a prevalent Gram-positive pathogen.

ABSTRACT Streptococcus pyogenes (group A streptococcus [GAS]) causes 600 million cases of pharyngitis each year. Despite this considerable disease burden, the molecular mechanisms used by GAS to infect, cause clinical pharyngitis, and persist in the human oropharynx are poorly understood. Saliva is ubiquitous in the human oropharynx and is the first material GAS encounters in the upper respiratory tract. Thus, a fuller understanding of how GAS survives and proliferates in saliva may provide valuable insights into the molecular mechanisms at work in the human oropharynx. We generated a highly saturated transposon insertion mutant library in serotype M1 strain MGAS2221, a strain genetically representative of a pandemic clone that arose in the 1980s and spread globally. The transposon mutant library was exposed to human saliva to screen for GAS genes required for wild-type fitness in this clinically relevant fluid. Using transposon-directed insertion site sequencing (TraDIS), we identified 92 genes required for GAS fitness in saliva. The more prevalent categories represented were genes involved in carbohydrate transport/metabolism, amino acid transport/metabolism, and inorganic ion transport/metabolism. Using six isogenic mutant strains, we confirmed that each of the mutants was significantly impaired for growth or persistence in human saliva ex vivo. Mutants with an inactivated Spy0644 (sptA) or Spy0646 (sptC) gene had especially severe persistence defects. This study is the first to use of TraDIS to study bacterial fitness in human saliva. The new information we obtained will be valuable for future translational maneuvers designed to prevent or treat human GAS infections. IMPORTANCE The human bacterial pathogen Streptococcus pyogenes (group A streptococcus [GAS]) causes more than 600 million cases of pharyngitis annually worldwide, 15 million of which occur in the United States. The human oropharynx is the primary anatomic site for GAS colonization and infection, and saliva is the first material encountered. Using a genome-wide transposon mutant screen, we identified 92 GAS genes required for wild-type fitness in human saliva. Many of the identified genes are involved in carbohydrate transport/metabolism, amino acid transport/metabolism, and inorganic ion transport/metabolism. The new information is potentially valuable for developing novel GAS therapeutics and vaccine research. Podcast: A podcast concerning this article is available.

ABSTRACT Klebsiella pneumoniae is a major human pathogen responsible for high morbidity and mortality rates. The emergence and spread of strains resistant to multiple antimicrobial agents and documented large nosocomial outbreaks are especially concerning. To develop new therapeutic strategies for K. pneumoniae, it is imperative to understand the population genomic structure of strains causing human infections. To address this knowledge gap, we sequenced the genomes of 1,777 extended-spectrum beta-lactamase-producing K. pneumoniae strains cultured from patients in the 2,000-bed Houston Methodist Hospital system between September 2011 and May 2015, representing a comprehensive, population-based strain sample. Strains of largely uncharacterized clonal group 307 (CG307) caused more infections than those of well-studied epidemic CG258. Strains varied markedly in gene content and had an extensive array of small and very large plasmids, often containing antimicrobial resistance genes. Some patients with multiple strains cultured over time were infected with genetically distinct clones. We identified 15 strains expressing the New Delhi metallo-beta-lactamase 1 (NDM-1) enzyme that confers broad resistance to nearly all beta-lactam antibiotics. Transcriptome sequencing analysis of 10 phylogenetically diverse strains showed that the global transcriptome of each strain was unique and highly variable. Experimental mouse infection provided new information about immunological parameters of host-pathogen interaction. We exploited the large data set to develop whole-genome sequence-based classifiers that accurately predict clinical antimicrobial resistance for 12 of the 16 antibiotics tested. We conclude that analysis of large, comprehensive, population-based strain samples can assist understanding of the molecular diversity of these organisms and contribute to enhanced translational research. IMPORTANCE Klebsiella pneumoniae causes human infections that are increasingly difficult to treat because many strains are resistant to multiple antibiotics. Clonal group 258 (CG258) organisms have caused outbreaks in health care settings worldwide. Using a comprehensive population-based sample of extended-spectrum beta-lactamase (ESBL)-producing K. pneumoniae strains, we show that a relatively uncommon clonal type, CG307, caused the plurality of ESBL-producing K. pneumoniae infections in our patients. We discovered that CG307 strains have been abundant in Houston for many years. As assessed by experimental mouse infection, CG307 strains were as virulent as pandemic CG258 strains. Our results may portend the emergence of an especially successful clonal group of antibiotic-resistant K. pneumoniae.

ABSTRACT Serum opacity factor (SOF) is a cell surface virulence factor made by the human pathogen Streptococcus pyogenes. We found that S. pyogenes strains with naturally occurring truncation mutations in the sof gene have markedly enhanced beta-hemolysis. Moreover, deletion of the sof gene in a SOF-positive parental strain resulted in significantly increased beta-hemolysis. Together, these observations suggest that SOF is an inhibitor of beta-hemolysis. SOF has two major functional domains, including an opacification domain and a fibronectin-binding domain. Using a SOF-positive serotype M89 S. pyogenes parental strain and a panel of isogenic mutant derivative strains, we evaluated the relative contribution of each SOF functional domain to beta-hemolysis inhibition and bacterial virulence. We found that the opacification domain, rather than the fibronectin-binding domain, is essential for SOF-mediated beta-hemolysis inhibition. The opacification domain, but not the fibronectin-binding domain of SOF, also contributed significantly to virulence in mouse models of bacteremia and necrotizing myositis. Inasmuch as the opacification domain of SOF is known to interact avidly with host high-density lipoprotein (HDL), we speculate that SOF-HDL interaction is an important process underlying SOF-mediated beta-hemolysis inhibition and SOF-mediated virulence. IMPORTANCE Streptococcus pyogenes is a major human pathogen causing more than 700 million infections annually. As a successful pathogen, S. pyogenes produces many virulence factors that facilitate colonization, proliferation, dissemination, and tissue damage. Serum opacity factor (SOF), an extracellular protein, is one of the virulence factors made by S. pyogenes. The underlying mechanism of how SOF contributes to virulence is not fully understood. SOF has two major features: (i) it opacifies host serum by interacting with high-density lipoprotein, and (ii) it inhibits beta-hemolysis on blood agar. In this study, we demonstrate that the domain of SOF essential for opacifying serum is also essential for SOF-mediated beta-hemolysis inhibition and SOF-mediated virulence. Our results shed new light on the molecular mechanisms of SOF-host interaction.

Group A Streptococcus (GAS) is a Gram-positive human pathogen that causes a variety of diseases ranging from pharyngitis to life-threaten­ing streptococcal toxic shock syndrome. Recently, several global gene expression analyses have yielded extensive new information regarding the regulation of genes encoding known and putative virulence factors in GAS. A microarray analysis found that transcription of the GAS gene M5005_Spy_1343 was significantly increased in response to interaction with human polymorphonuclear leukocytes. M5005_Spy_1343 is predicted to encode a member of the LysR family of transcriptional regulators and is located upstream of a putative operon containing six genes. Five of these genes have sequence similarity to genes involved in short-chain fatty acid metabolism, whereas the sixth gene (luxS) is found in many bacterial species and is involved in quorum sensing. Unexpectedly, inactivation of the M5005_Spy_1343 gene resultedin hypervirulence in an intraperitoneal mouse model of infection. Increased virulence was not due to changes in luxS gene expression.We postulate that short-chain fatty acid metabolism is involved in GAS pathogenesis.

ABSTRACT For over a century, a fundamental objective in infection biology research has been to understand the molecular processes contributing to the origin and perpetuation of epidemics. Divergent hypotheses have emerged concerning the extent to which environmental events or pathogen evolution dominates in these processes. Remarkably few studies bear on this important issue. Based on population pathogenomic analysis of 1,200 Streptococcus pyogenes type emm89 infection isolates, we report that a series of horizontal gene transfer events produced a new pathogenic genotype with increased ability to cause infection, leading to an epidemic wave of disease on at least two continents. In the aggregate, these and other genetic changes substantially remodeled the transcriptomes of the evolved progeny, causing extensive differential expression of virulence genes and altered pathogen-host interaction, including enhanced immune evasion. Our findings delineate the precise molecular genetic changes that occurred and enhance our understanding of the evolutionary processes that contribute to the emergence and persistence of epidemically successful pathogen clones. The data have significant implications for understanding bacterial epidemics and for translational research efforts to blunt their detrimental effects. IMPORTANCE The confluence of studies of molecular events underlying pathogen strain emergence, evolutionary genetic processes mediating altered virulence, and epidemics is in its infancy. Although understanding these events is necessary to develop new or improved strategies to protect health, surprisingly few studies have addressed this issue, in particular, at the comprehensive population genomic level. Herein we establish that substantial remodeling of the transcriptome of the human-specific pathogen Streptococcus pyogenes by horizontal gene flow and other evolutionary genetic changes is a central factor in precipitating and perpetuating epidemic disease. The data unambiguously show that the key outcome of these molecular events is evolution of a new, more virulent pathogenic genotype. Our findings provide new understanding of epidemic disease.

Group A Streptococcus (GAS) is a human-adapted pathogen that causes a variety of diseases, including pharyngitis and invasive infections. GAS strains are categorized by variation in the nucleotide sequence of the gene (emm) that encodes the M protein. To identify the emm types of GAS strains causing pharyngitis in Ontario, Canada, we sequenced the hypervariable region of the emm gene in 4,635 pharyngeal GAS isolates collected during 2002–2010. The most prevalent emm types varied little from year to year. In contrast, fine-scale geographic analysis identified inter-site variability in the most common emm types. Additionally, we observed fluctuations in yearly frequency of emm3 strains from pharyngitis patients that coincided with peaks of emm3 invasive infections. We also discovered a striking increase in frequency of emm89 strains among isolates from patients with pharyngitis and invasive disease. These findings about the epidemiology of GAS are potentially useful for vaccine research.

Full-genome sequencing showed that a recently emerged and hypervirulent clone of group A Streptococcus type emm59 active in Canada and parts of the United States has now caused severe invasive infections in rural northeastern Wyoming. Phylogenetic analysis of genome data indicated that the strain was likely introduced from Montana.

Tuberculosis (TB) rates in the United States are disproportionately high for certain ethnic minorities. Using univariate and multivariate analyses, we compared data for 1,318 US-born blacks with 565 US-born non-Hispanic whites who participated in the Houston TB Initiative (1995–2004). All available Mycobacterium tuberculosis isolates underwent susceptibility and genotype testing (insertion sequence 6110 restriction fragment length polymorphism, spoligotyping, and genetic grouping). TB in blacks was associated with younger age, inner city residence, HIV seropositivity, and drug resistance. TB cases clustered in 82% and 77% of blacks and whites, respectively (p = 0.46). Three clusters had >100 patients each, including 1 cluster with a predominance of blacks. Size of TB clusters was unexpectedly large, underscoring the ongoing transmission of TB in Houston, particularly among blacks.

ABSTRACT Strains of emm89 Streptococcus pyogenes have become one of the major causes of invasive infections worldwide in the last 10 years. We recently sequenced the genome of 1,125 emm89 strains and identified three major phylogenetic groups, designated clade 1, clade 2, and the epidemic clade 3. Epidemic clade 3 strains, which now cause the great majority of infections, have two distinct genetic features compared to clade 1 and clade 2 strains. First, all clade 3 organisms have a variant 3 nga promoter region pattern, which is associated with increased production of secreted cytolytic toxins SPN (S. pyogenes NADase) and SLO (streptolysin O). Second, all clade 3 strains lack the hasABC locus mediating hyaluronic acid capsule synthesis, whereas this locus is intact in clade 1 and clade 2 strains. We constructed isogenic mutant strains that produce different levels of SPN and SLO toxins and capsule (none, low, or high). Here we report that emm89 strains with elevated toxin production are significantly more virulent than low-toxin producers. Importantly, we also show that capsule production is dispensable for virulence in strains that already produce high levels of SPN and SLO. Our results provide new understanding about the molecular mechanisms contributing to the rapid emergence and molecular pathogenesis of epidemic clade 3 emm89 S. pyogenes. IMPORTANCE S. pyogenes (group A streptococcus [GAS]) causes pharyngitis (“strep throat”), necrotizing fasciitis, and other human infections. Serious infections caused by emm89 S. pyogenes strains have recently increased in frequency in many countries. Based on whole-genome sequence analysis of 1,125 strains recovered from patients on two continents, we discovered that a new emm89 clone, termed clade 3, has two distinct genetic features compared to its predecessors: (i) absence of the genes encoding antiphagocytic hyaluronic acid capsule virulence factor and (ii) increased production of the secreted cytolytic toxins SPN and SLO. emm89 S. pyogenes strains with the clade 3 phenotype (absence of capsule and high expression of SPN and SLO) are highly virulent in mice. These findings provide new understanding of how new virulent clones emerge and cause severe infections worldwide. This newfound knowledge of S. pyogenes virulence can be used to help understand future epidemics and conduct new translational research.

Genomic analysis of type emm59 group A Streptococcus invasive strains isolated in the United States discovered higher than anticipated genetic heterogeneity among strains and identified a heretofore unrecognized monoclonal cluster of invasive infections in the San Francisco Bay area. Heightened monitoring for a potential shift in the epidemic behavior of emm59 group A Streptococcus is warranted.

ABSTRACT Nosocomial transmission of pathogens is a major health care challenge. The increasing spread of antibiotic-resistant strains represents an ongoing threat to public health. Previous Staphylococcus aureus transmission studies have focused on transmission of S. aureus between asymptomatic carriers or used low-resolution typing methods such as multilocus sequence typing (MLST) or spa typing. To identify patient-to-patient intrahospital transmission using high-resolution genetic analysis, we sequenced the genomes of a consecutive set of 398 S. aureus isolates from sterile-site infections. The S. aureus strains were collected from four hospitals in the Houston Methodist Hospital System over a 6-month period. Importantly, we discovered no evidence of transmission of S. aureus between patients with sterile-site infections. The lack of intrahospital transmission may reflect a fundamental difference between day-to-day transmission events in the hospital setting and the more frequently studied outbreak scenarios. IMPORTANCE Previous studies have suggested that nosocomial transmission of S. aureus is common. Our data revealed an unexpected lack of evidence for intrahospital transmission of S. aureus between patients with invasive infections. This finding has important implications for hospital infection control and public health efforts. In addition, our data demonstrate that highly related pools of S. aureus strains exist in the community which may complicate outbreak investigations.

Serotype M1 group A Streptococcus, the most common cause of invasive disease in many case series, generally have resisted extensive molecular subtyping by standard techniques (e.g., multilocus enzyme electrophoresis, pulsed-field gel electrophoresis). We used automated sequencing of the sic gene encoding streptococcal inhibitor of complement and of a region of the chromosome with direct repeat sequences to unambiguously differentiate 30 M1 isolates recovered from 28 patients in Texas with invasive disease episodes temporally clustered and thought to represent an outbreak. Sequencing of the emm gene was less useful for M1 strain differentiation, and restriction fragment length polymorphism analysis with IS1548 or IS1562 as Southern hybridization probes did not provide epidemiologically useful subtyping information. Sequence polymorphism in the direct repeat region of the chromosome and IS1548 profiling data support the hypothesis that M1 organisms have two main evolutionary lineages marked by the presence or absence of the speA2 allele encoding streptococcal pyrogenic exotoxin A2.

Research in bacterial population genetics has increased in the last 10 years. Population genetic theory and tools and related strategies have been used to investigate bacterial pathogens that have contributed to recent episodes of temporal variation in disease frequency and severity. A common theme demonstrated by these analyses is that distinct bacterial clones are responsible for disease outbreaks and increases in infection frequency. Many of these clones are characterized by unique combinations of virulence genes or alleles of virulence genes. Because substantial interclonal variance exists in relative virulence, molecular population genetic studies have led to the concept that the unit of bacterial pathogenicity is the clone or cell line. Continued new insights into host-parasite interactions at the molecular level will be achieved by combining clonal analysis of bacterial pathogens with large-scale comparative sequencing of virulence genes.

ABSTRACT 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. IMPORTANCE 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.

ABSTRACT Staphylococcus aureus is a human commensal bacterium and a prominent cause of infections globally. The high incidence of S. aureus infections is compounded by the ability of the microbe to readily acquire resistance to antibiotics. In the United States, methicillin-resistant S. aureus (MRSA) is a leading cause of morbidity and mortality by a single infectious agent. Therapeutic options for severe MRSA infections are limited to a few antibiotics to which the organism is typically susceptible, including vancomycin. Acquisition of high-level vancomycin resistance by MRSA is a major concern, but to date, there have been only 12 vancomycin-resistant S. aureus (VRSA) isolates reported in the United States and all belong to a phylogenetic lineage known as clonal complex 5. To gain enhanced understanding of the genetic characteristics conducive to the acquisition of vancomycin resistance by S. aureus, V. N. Kos et al. performed whole-genome sequencing of all 12 VRSA isolates and compared the DNA sequences to the genomes of other S. aureus strains. The findings provide new information about the evolutionary history of VRSA and identify genetic features that may bear on the relationship between S. aureus clonal complex 5 strains and the acquisition of vancomycin resistance genes from enterococci.