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ABSTRACTTo obtain new information about Streptococcus pyogenesintrahost genetic variation during invasive infection, we sequenced the genomes of 2,954 serotype M1 strains recovered from a nonhuman primate experimental model of necrotizing fasciitis. A total of 644 strains (21.8%) acquired polymorphisms relative to the input parental strain. The fabTgene, encoding a transcriptional regulator of fatty acid biosynthesis genes, contained 54.5% of these changes. The great majority of polymorphisms were predicted to deleteriously alter FabT function. Transcriptome-sequencing (RNA-seq) analysis of a wild-type strain and an isogenic fabTdeletion mutant strain found that between 3.7 and 28.5% of the S. pyogenestranscripts were differentially expressed, depending on the growth temperature (35°C or 40°C) and growth phase (mid-exponential or stationary phase). Genes implicated in fatty acid synthesis and lipid metabolism were significantly upregulated in the fabTdeletion mutant strain. FabT also directly or indirectly regulated central carbon metabolism genes, including pyruvate hub enzymes and fermentation pathways and virulence genes. Deletion of fabTdecreased virulence in a nonhuman primate model of necrotizing fasciitis. In addition, the fabTdeletion strain had significantly decreased survival in human whole blood and during phagocytic interaction with polymorphonuclear leukocytes ex vivo. We conclude that FabT mutant progeny arise during infection, constitute a metabolically distinct subpopulation, and are less virulent in the experimental models used here.

ABSTRACTHumans 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 hasAgene 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 vivoin 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 vivoin the hasoperon. 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.

ABSTRACTDespite the public health challenges associated with the emergence of new pathogenic bacterial strains and/or serotypes, there is a dearth of information regarding the molecular mechanisms that drive this variation. Here, we began to address the mechanisms behind serotype-specific variation between serotype M1 and M3 strains of the human pathogen Streptococcus pyogenes(the group A Streptococcus[GAS]). Spatially diverse contemporary clinical serotype M3 isolates were discovered to contain identical inactivating mutations within genes encoding two regulatory systems that control the expression of important virulence factors, including the thrombolytic agent streptokinase, the protease inhibitor-binding protein-G-related a2-macroglobulin-binding (GRAB) protein, and the antiphagocytic hyaluronic acid capsule. Subsequent analysis of a larger collection of isolates determined that M3 GAS, since at least the 1920s, has harbored a 4-bp deletion in the fasCgene of the fasBCAXregulatory system and an inactivating polymorphism in the rivRregulator-encoding gene. The fasCand rivRmutations in M3 isolates directly affect the virulence factor profile of M3 GAS, as evident by a reduction in streptokinase expression and an enhancement of GRAB expression. Complementation of the fasCmutation in M3 GAS significantly enhanced levels of the small regulatory RNA FasX, which in turn enhanced streptokinase expression. Complementation of the rivRmutation in M3 GAS restored the regulation of grabmRNA abundance but did not alter capsule mRNA levels. While important, the fasCand rivRmutations do not provide a full explanation for why serotype M3 strains are associated with unusually severe invasive infections; thus, further investigation is warranted.

Group A Streptococcus(GAS) causes human infections that range in severity from pharyngitis ("strep-throat") to necrotizing fasciitis ("flesh-eating disease").  To facilitate investigation of the molecular basis of host-pathogen interactions, infection models capable of rapidly screening for differences in GAS strain virulence are needed.  To this end, we developed a Galleria mellonellalarvae (wax worm) model of invasive GAS infection and used it to compare the virulence of serotype M3 GAS strains.  We found that GAS causes severe tissue damage and kills wax worms in a dose-dependent manner.  The virulence of genetically distinct GAS strains was compared by Kaplan-Meier survival analysis and determining 50% lethal doses (LD50).  Host-pathogen interactions were further characterized using quantitative culture, histopathology and TaqMan assays.  GAS strains known to be highly pathogenic in mice and monkeys caused significantly lower survival and had significantly lower LD50s in wax worms than GAS strains associated with attenuated virulence or asymptomatic carriage.  Furthermore, isogenic inactivation of proven virulence factors resulted in a significantly increased LD50and decreased lesion size compared to the wild-type strain, a finding that also strongly correlates with animal studies.  Importantly, survival analysis and LD50determination in wax worms supported our hypothesis that a newly emerged GAS subclone that is epidemiologically associated with more human necrotizing fasciitis cases than its progenitor lineage has significantly increased virulence.  We conclude that GAS virulence in wax worms strongly correlates with the data obtained in vertebrate models, and thus, the Galleria mellonellalarva is a useful host organism to study GAS pathogenesis.

ABSTRACTGenome sequencing of group A Streptococcus(GAS) has revealed that prophages account for the vast majority of gene content differences between strains. Serotype M28 strains are a leading cause of pharyngitis and invasive infections, but little is known about genetic diversity present in natural populations of these organisms. To study this issue, population-based samples of 568 strains from Ontario, Canada; Finland; and Houston, Texas, were analyzed. Special attention was given to analysis of variation in prophage-encoded virulence gene content by a PCR-based method. Thirty and 29 distinct prophage-encoded virulence gene profiles were identified among pharyngitis and invasive infection isolates. Thirteen profiles, representing the majority of the strains, were shared between these two classes of isolates. Significant differences were observed in the frequency of occurrence of certain prophage toxin gene profiles and infection type. M28 strains are highly diverse in prophage-encoded virulence gene content and integration site, supporting the key concept that prophages are critical contributors to GAS genetic diversity and population biology. Nucleotide sequence variation in the emmgene (encodes M protein) was also examined. Only three allelic variants were identified in the hypervariable portion of the emm28gene. All but one strain had the same inferred amino acid sequence in the first 100 amino acids of the mature M28 protein. In contrast, size differences in the emm28gene and inferred protein due to variable numbers of C-terminal repeats were common. The presence of macrolide resistance genes (mefA, ermB, and ermTR) was analyzed by PCR, and less than 2% of the strains were positive.

Streptococcus agalactiae(group B streptococcus, or GBS) is a common cause of bacteremia and sepsis in newborns, pregnant women, and immunocompromised patients. The molecular mechanisms used by GBS to survive and proliferate in blood are not well understood. Here, using a highly virulent GBS strain and transposon-directed insertion site sequencing (TraDIS), we performed genome-wide screens to discover novel GBS genes required for bacterial survival in human whole blood and plasma.

Resistance to macrolide antibiotics is a global concern in the treatment of Streptococcus pyogenes(group A Streptococcus[GAS]) infections. In Iceland, since the detection of the first macrolide-resistant isolate in 1998, three epidemic waves of macrolide-resistant GAS infections have occurred, with peaks in 1999, 2004, and 2008. We conducted whole-genome sequencing of all 1,575 available GAS macrolide-resistant clinical isolates of all infection types collected at the national reference laboratory in Reykjavik, Iceland, from 1998 to 2016.

Recently, two related Streptococcus pyogenesstrains with reduced susceptibility to ampicillin, amoxicillin, and cefotaxime, antibiotics commonly used to treat S. pyogenesinfections, were reported. The two strains had the same nonsynonymous (amino acid-substituting) mutation in the pbp2xgene, encoding penicillin-binding protein 2X (PBP2X). This concerning report led us to investigate our library of 7,025 genome sequences of type emm1, emm28, and emm89S. pyogenesclinical strains recovered from intercontinental sources for mutations in pbp2x.

ABSTRACTKlebsiella pneumoniaeis 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. pneumoniaestrains 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.IMPORTANCEKlebsiella pneumoniaecauses 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. pneumoniaestrains, we show that a relatively uncommon clonal type, CG307, caused the plurality of ESBL-producing K. pneumoniaeinfections 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.

ABSTRACTNosocomial 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 aureustransmission studies have focused on transmission of S. aureusbetween asymptomatic carriers or used low-resolution typing methods such as multilocus sequence typing (MLST) or spatyping. To identify patient-to-patient intrahospital transmission using high-resolution genetic analysis, we sequenced the genomes of a consecutive set of 398 S. aureusisolates from sterile-site infections. The S. aureusstrains 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. aureusbetween 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.IMPORTANCEPrevious studies have suggested that nosocomial transmission of S. aureusis common. Our data revealed an unexpected lack of evidence for intrahospital transmission of S. aureusbetween 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. aureusstrains exist in the community which may complicate outbreak investigations.