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.
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.
Staphylococcus aureus small-colony variants (SCVs) are implicated in chronic and relapsing infections that are difficult to diagnose and treat. Despite many years of study, the underlying molecular mechanisms and virulence effect of the small-colony phenotype remain incompletely understood. We sequenced the genomes of five S. aureus SCV strains recovered from human patients and discovered previously unidentified nonsynonymous point mutations in three genes encoding proteins in the menadione biosynthesis pathway. Analysis of genetic revertants and complementation with wild-type alleles confirmed that these mutations caused the SCV phenotype and decreased virulence for mice.
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-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.
group A Streptococcus; GAS; bacteria; emm59; streptococci; invasive disease; virulence; genome sequencing; epidemiology; necrotizing fasciitis; Montana; Wyoming; United States
Altering zinc bioavailability to bacterial pathogens is a key component of host innate immunity. Thus, the ability to sense and adapt to the alterations in zinc concentrations is critical for bacterial survival and pathogenesis. To understand the adaptive responses of group A Streptococcus (GAS) to zinc limitation and its regulation by AdcR, we characterized gene regulation by AdcR. AdcR regulates the expression of 70 genes involved in zinc acquisition and virulence. Zinc-bound AdcR interacts with operator sequences in the negatively regulated promoters and mediates differential regulation of target genes in response to zinc deficiency. Genes involved in zinc mobilization and conservation are derepressed during mild zinc deficiency, whereas the energy-dependent zinc importers are upregulated during severe zinc deficiency. Further, we demonstrated that transcription activation by AdcR occurs by direct binding to the promoter. However, the repression and activation by AdcR is mediated by its interactions with two distinct operator sequences. Finally, mutational analysis of the metal ligands of AdcR caused impaired DNA binding and attenuated virulence, indicating that zinc sensing by AdcR is critical for GAS pathogenesis. Together, we demonstrate that AdcR regulates GAS adaptive responses to zinc limitation and identify molecular components required for GAS survival during zinc deficiency.
Staphylococcus aureus community-acquired pneumonia is often associated with influenza or an influenza-like syndrome. Morbidity and mortality due to methicillin-resistant S. aureus (MRSA) or influenza and pneumonia, which includes bacterial co-infection, are among the top causes of death by infectious diseases in the United States. We developed a non-lethal influenza A virus (IAV) (H3N2)/S. aureus co-infection model in cynomolgus macaques (Macaca fascicularis) to test the hypothesis that seasonal IAV infection predisposes non-human primates to severe S. aureus pneumonia. Infection and disease progression were monitored by clinical assessment of animal health; analysis of blood chemistry, nasal swabs, and X-rays; and gross pathology and histopathology of lungs from infected animals. Seasonal IAV infection in healthy cynomolgus macaques caused mild pneumonia, but unexpectedly, did not predispose these animals to subsequent severe infection with the community-associated MRSA clone USA300. We conclude that in our co-infection model, seasonal IAV infection alone is not sufficient to promote severe S. aureus pneumonia in otherwise healthy non-human primates. The implication of these findings is that comorbidity factors in addition to IAV infection are required to predispose individuals to secondary S. aureus pneumonia.
Staphylococcus aureus; influenza a virus; coinfection; USA300; MRSA; pneumonia
Despite 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 α2-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 fasC gene of the fasBCAX regulatory system and an inactivating polymorphism in the rivR regulator-encoding gene. The fasC and rivR mutations 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 fasC mutation in M3 GAS significantly enhanced levels of the small regulatory RNA FasX, which in turn enhanced streptokinase expression. Complementation of the rivR mutation in M3 GAS restored the regulation of grab mRNA abundance but did not alter capsule mRNA levels. While important, the fasC and rivR mutations 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 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.
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.
group A Streptococcus; GAS; bacteria; invasive disease; genome sequencing; epidemic; United States; Canada; streptococci
Determination of emm variations may help improve vaccine design.
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.
Streptococcus pyogenes; GAS; pharyngitis; group A streptococcus; Canada; emm; bacteria
Most biopsy and autopsy tissues are formalin-fixed and paraffin-embedded (FFPE), but this process leads to RNA degradation that limits gene expression analysis. The RNA genome of the 1918 pandemic influenza virus was previously determined in a 9-year effort by overlapping RT-PCR from post-mortem samples. Here, the full genome of the 1918 virus at 3000× coverage was determined in one high-throughput sequencing run of a library derived from total RNA of a 1918 FFPE sample after duplex-specific nuclease treatments. Bacterial sequences associated with secondary bacterial pneumonias were also detected. Host transcripts were well represented in the library. Compared to a 2009 pandemic influenza virus FFPE post-mortem library, the 1918 sample showed significant enrichment for host defence and cell death response genes, concordant with prior animal studies. This methodological approach should assist in the analysis of FFPE tissue samples isolated over the past century from a variety of diseases.
influenza A virus; pandemic; 1918 influenza virus; formalin-fixed; paraffin-embedded tissue; NGS
Infection with different strains of the same species of bacteria often results in vastly different clinical outcomes. Despite extensive investigation, the genetic basis of microbial strain-specific virulence remains poorly understood. Recent whole-genome sequencing has revealed that SNPs are the most prevalent form of genetic diversity among different strains of the same species of bacteria. For invasive serotype M3 group A streptococci (GAS) strains, the gene encoding regulator of proteinase B (RopB) has the highest frequency of SNPs. Here, we have determined that ropB polymorphisms alter RopB function and modulate GAS host-pathogen interactions. Sequencing of ropB in 171 invasive serotype M3 GAS strains identified 19 distinct ropB alleles. Inactivation of the ropB gene in strains producing distinct RopB variants had dramatically divergent effects on GAS global gene expression. Additionally, generation of isoallelic GAS strains differing only by a single amino acid in RopB confirmed that variant proteins affected transcript levels of the gene encoding streptococcal proteinase B, a major RopB-regulated virulence factor. Comparison of parental, RopB-inactivated, and RopB isoallelic strains in mouse infection models demonstrated that ropB polymorphisms influence GAS virulence and disease manifestations. These data detail a paradigm in which unbiased, whole-genome sequence analysis of populations of clinical bacterial isolates creates new avenues of productive investigation into the pathogenesis of common human infections.
The epidemiology of pediatric tuberculosis (TB) from 1995–2000 in Harris County, TX has been previously reported. This study was conducted to evaluate the continued trends of Mycobacterium tuberculosis (MTB) clustering and the role of genotyping in pediatric TB.
Data came from the Houston Tuberculosis Initiative, a prospective population-based active surveillance and molecular epidemiology project. The study population consisted of TB patients ≤ 18 years of age diagnosed in Harris County, TX from 2000 to 2004. Available MTB isolates were characterized by IS6110 restriction fragment length polymorphism and spoligotyping.
103 pediatric TB cases were enrolled in the Houston Tuberculosis Initiative study from 2000–2004. Sixty-one (59%) patients had potential source cases. MTB isolates were available and genotyped for 36 pediatric cases; 27 (75%) were clustered into 22 different genotypes. Of the 20 genotyped patients with a potential source case, 16 (80%) were clustered. Genotypes matched the potential source case in 12 cases. Eleven of the 16 (69%) genotyped patients without a potential source case were clustered.
Compared with pediatric cases in 1995–2000, there was a significant increase in the number of patients with unknown potential source cases that were clustered within the Houston Tuberculosis Initiative database. Since genotypic clustering is associated with recent transmission, there appears to be a failure in the identification of potential source cases through contact tracing. Reduced funding of public health departments forces more limited TB control activities and therefore could pose a threat to TB control.
tuberculosis; pediatric; genotype; cluster
Next-generation sequencing technology is available to many clinical laboratories; however, it is not yet widely used in routine microbiology practice. To demonstrate the feasibility of using whole-genome sequencing in a routine clinical microbiology workflow, we sequenced the genome of every organism isolated in our laboratory for 1 day.
Molecular pathogenomic analysis of the human bacterial pathogen group A Streptococcus has been conducted for a decade. Much has been learned as a consequence of the confluence of low-cost DNA sequencing, microarray technology, high-throughput proteomics, and enhanced bioinformatics. These technical advances, coupled with the availability of unique bacterial strain collections, have facilitated a systems biology investigative strategy designed to enhance and accelerate our understanding of disease processes. Here, we provide examples of the progress made by exploiting an integrated genome-wide research platform to gain new insight into molecular pathogenesis. The studies have provided many new avenues for basic and translational research.
Bacillus cereus strains harboring a pXO1-like virulence plasmid cause respiratory anthrax-like disease in humans, particularly in welders. We developed mouse models for intraperitoneal as well as aerosol challenge with spores of B. cereus G9241, harboring pBCXO1 and pBC218 virulence plasmids. Compared to wild-type B. cereus G9241, spores with a deletion of the pBCXO1-carried protective antigen gene (pagA1) were severely attenuated, whereas spores with a deletion of the pBC218-carried protective antigen homologue (pagA2) were not. Anthrax vaccine adsorbed (AVA) immunization raised antibodies that bound and neutralized the pagA1-encoded protective antigen (PA1) but not the PA2 orthologue encoded by pagA2. AVA immunization protected mice against a lethal challenge with spores from B. cereus G9241 or B. cereus Elc4, a strain that had been isolated from a fatal case of anthrax-like disease. As the pathogenesis of B. cereus anthrax-like disease in mice is dependent on pagA1 and PA-neutralizing antibodies provide protection, AVA immunization may also protect humans from respiratory anthrax-like death.
We previously demonstrated that the cell-surface lipoprotein MalE contributes to GAS maltose/maltodextrin utilization, but MalE inactivation does not completely abrogate GAS catabolism of maltose or maltotriose. Using a genome-wide approach, we identified the GAS phosphotransferase system (PTS) responsible for non-MalE maltose/maltotriose transport. This PTS is encoded by an open reading frame (M5005_spy1692) previously annotated as ptsG based on homology with the glucose PTS in Bacillus subtilis. Genetic inactivation of M5005_spy1692 significantly reduced transport rates of radiolabeled maltose and maltotriose, but not glucose, leading us to propose its reannotation as malT for maltose transporter. The ΔmalT, ΔmalE, and ΔmalE:malT strains were significantly attenuated in their growth in human saliva and in their ability to catabolize α-glucans digested by purified human salivary α-amylase. Compared to wild-type, the three isogenic mutant strains were significantly impaired in their ability to colonize the mouse oropharynx. Finally, we discovered that the transcript levels of maltodextrin utilization genes are regulated by competitive binding of the maltose repressor MalR and catabolite control protein A. These data provide novel insights into regulation of the GAS maltodextrin genes and their role in GAS host-pathogen interaction, thereby increasing the understanding of links between nutrient acquisition and virulence in common human pathogens.
Streptococcus; maltodextrin; transport; amylase; pharyngitis
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.
Inactivation of the Staphylococcus aureus tricarboxylic acid (TCA) cycle delays the resolution of cutaneous ulcers in a mouse soft tissue infection model. In this study, it was observed that cutaneous lesions in mice infected with wild-type or isogenic aconitase mutant S. aureus strains contained comparable inflammatory infiltrates, suggesting the delayed resolution was independent of the recruitment of immune cells. These observations led us to hypothesize that staphylococcal metabolism can modulate the host immune response. Using an in vitro model system involving RAW 264.7 cells, the authors observed that cells cultured with S. aureus aconitase mutant strains produced significantly lower amounts of nitric oxide (NO•) and an inducible nitric oxide synthase as compared to those cells exposed to wild-type bacteria. Despite the decrease in NO• synthesis, the expression of antigen-presentation and costimulatory molecules was similar in cells cultured with wild-type and those cultured with aconitase mutant bacteria. The data suggest that staphylococci can evade innate immune responses and potentially enhance their ability to survive in infected hosts by altering their metabolism. This may also explain the occurrence of TCA cycle mutants in clinical S. aureus isolates.
Staphylococcus aureus; Aconitase; Nitric oxide; RAW 264.7 cells; Immune evasion
Study of the maltose/maltodextrin binding protein MalE in Escherichia coli has resulted in fundamental insights into the molecular mechanisms of microbial transport. Whether gram-positive bacteria employ a similar pathway for maltodextrin transport is unclear. The maltodextrin binding protein MalE has previously been shown to be key to the ability of group A Streptococcus (GAS) to colonize the oropharynx, the major site of GAS infection in humans. Here we used a multifaceted approach to elucidate the function and binding characteristics of GAS MalE. We found that GAS MalE is a central part of a highly efficient maltodextrin transport system capable of transporting linear maltodextrins that are up to at least seven glucose molecules long. Of the carbohydrates tested, GAS MalE had the highest affinity for maltotriose, a major breakdown product of starch in the human oropharynx. The thermodynamics and fluorescence changes induced by GAS MalE-maltodextrin binding were essentially opposite those reported for E. coli MalE. Moreover, unlike E. coli MalE, GAS MalE exhibited no specific binding of maltose or cyclic maltodextrins. Our data show that GAS developed a transport system optimized for linear maltodextrins longer than two glucose molecules that has several key differences from its well-studied E. coli counterpart.
Analysis of microbial epidemics has been revolutionized by whole-genome sequencing. We recently sequenced the genomes of 601 type emm59 Group A Streptococcus (GAS) organisms responsible for an ongoing epidemic of invasive infections in Canada and some of the United States. The epidemic has been caused by the emergence of a genetically distinct, hypervirulent clone that has genetically diversified. The ease of obtaining genomic data contrasts with the relatively difficult task of translating them into insightful epidemiological information. Here, we sequenced the genomes of 90 additional invasive Canadian emm59 GAS organisms, including 80 isolated recently in 2010–2011. We used an improved bioinformatics pipeline designed to rapidly process and analyze whole-genome data and integrate strain metadata. We discovered that emm59 GAS organisms are undergoing continued multiclonal evolutionary expansion. Previously identified geographic patterns of strain dissemination are being diluted as mixing of subclones over time and space occurs. Our integrated data analysis strategy permits prompt and accurate mapping of the dissemination of bacterial organisms in an epidemic wave, permitting rapid generation of hypotheses that inform public health and virulence studies.
bacterial epidemics; Canada; group A Streptococcus; invasive disease; United States; whole-genome sequencing
Decreasing the time to species identification and antibiotic susceptibility determination of strains recovered from patients with bacteremia significantly decreases morbidity and mortality. Herein, we validated a method to identify Gram-negative bacteria directly from positive blood culture medium using the Bruker MALDI Biotyper and to rapidly perform susceptibility testing using the BD Phoenix.
Sepsis continues to pose a clear challenge as one of the most difficult and costly problems to treat and prevent. Sepsis is caused by systemic or localized infections that damage the integrity of microcirculation in multiple organs. The challenge of sepsis and its long-term sequelae was addressed by the National Institutes of Health National Heart Lung and Blood Institute Division of Blood Diseases and Resources. Defining sepsis as severe endothelial dysfunction syndrome that causes multiorgan failure in response to intravascular or extravascular microbial agents, the National Heart Lung and Blood Institute panel proposed the concept of genome wars as a platform for new diagnostic, therapeutic, and preventive approaches to sepsis.
Low G+C Gram-positive bacteria typically contain multiple LacI/GalR regulator family members which often have highly similar amino-terminal DNA binding domains suggesting significant overlap in target DNA sequences. The LacI/GalR family regulator catabolite control protein A (CcpA) is a global regulator of the Group A Streptococcus (GAS) transcriptome and contributes to GAS virulence in diverse infection sites. Herein, we studied the role of the maltose repressor (MalR), another LacI/GalR family member, in GAS global gene expression and virulence. MalR inactivation reduced GAS colonization of the mouse orophyarnx but did not detrimentally affect invasive infection. The MalR transcriptome was limited to only 25 genes, and a highly conserved MalR DNA-binding sequence was identified. Variation of the MalR binding sequence significantly reduced MalR binding in vitro. In contrast, CcpA bound to the same DNA sequences as MalR but tolerated variation in the promoter sequences with minimal change in binding affinity. Inactivation of pulA, a MalR regulated gene which encodes a cell-surface carbohydrate binding protein, significantly reduced GAS human epithelial cell adhesion and mouse oropharyngeal colonization but did not affect GAS invasive disease. These data delineate a molecular mechanism by which hierarchical regulation of carbon source utilization influences bacterial pathogenesis in a site-specific fashion.
Streptococcus; maltodextrin; pharyngitis; adhesion
is a human commensal bacterium and a prominent cause of infections globally. The high incidence of
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
strains. The findings provide new information about the evolutionary history of VRSA and identify genetic features that may bear on the relationship between
clonal complex 5 strains and the acquisition of vancomycin resistance genes from enterococci.