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1.  PBP2a Mutations Causing High-Level Ceftaroline Resistance in Clinical Methicillin-Resistant Staphylococcus aureus Isolates 
Antimicrobial Agents and Chemotherapy  2014;58(11):6668-6674.
Ceftaroline is the first member of a novel class of cephalosporins approved for use in the United States. Although prior studies have identified eight ceftaroline-resistant methicillin-resistant Staphylococcus aureus (MRSA) isolates in Europe and Asia with MICs ranging from 4 to 8 mg/liter, high-level resistance to ceftaroline (>32 mg/liter) has not been described in MRSA strains isolated in the United States. We isolated a ceftaroline-resistant (MIC > 32 mg/liter) MRSA strain from the blood of a cystic fibrosis patient and five MRSA strains from the respiratory tract of this patient. Whole-genome sequencing identified two amino acid-altering mutations uniquely present in the ceftaroline-binding pocket of the transpeptidase region of penicillin-binding protein 2a (PBP2a) in ceftaroline-resistant isolates. Biochemical analyses and the study of isogenic mutant strains confirmed that these changes caused ceftaroline resistance. Thus, we identified the molecular mechanism of ceftaroline resistance in the first MRSA strain with high-level ceftaroline resistance isolated in the United States.
PMCID: PMC4249384  PMID: 25155594
2.  Natural Variant of Collagen-Like Protein A in Serotype M3 Group A Streptococcus Increases Adherence and Decreases Invasive Potential 
Infection and Immunity  2015;83(3):1122-1129.
Group A Streptococcus (GAS) predominantly exists as a colonizer of the human oropharynx that occasionally breaches epithelial barriers to cause invasive diseases. Despite the frequency of GAS carriage, few investigations into the contributory molecular mechanisms exist. To this end, we identified a naturally occurring polymorphism in the gene encoding the streptococcal collagen-like protein A (SclA) in GAS carrier strains. All previously sequenced invasive serotype M3 GAS possess a premature stop codon in the sclA gene truncating the protein. The carrier polymorphism is predicted to restore SclA function and was infrequently identified by targeted DNA sequencing in invasive strains of the same serotype. We demonstrate that a strain with the carrier sclA allele expressed a full-length SclA protein, while the strain with the invasive sclA allele expressed a truncated variant. An isoallelic mutant invasive strain with the carrier sclA allele exhibited decreased virulence in a mouse model of invasive disease and decreased multiplication in human blood. Further, the isoallelic invasive strain with the carrier sclA allele persisted in the mouse nasopharynx and had increased adherence to cultured epithelial cells. Repair of the premature stop codon in the invasive sclA allele restored the ability to bind the extracellular matrix proteins laminin and cellular fibronectin. These data demonstrate that a mutation in GAS carrier strains increases adherence and decreases virulence and suggest selection against increased adherence in GAS invasive isolates.
PMCID: PMC4333440  PMID: 25561712
3.  Absence of Patient-to-Patient Intrahospital Transmission of Staphylococcus aureus as Determined by Whole-Genome Sequencing 
mBio  2014;5(5):e01692-14.
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.
PMCID: PMC4196229  PMID: 25293757
4.  Identification of Point Mutations in Clinical Staphylococcus aureus Strains That Produce Small-Colony Variants Auxotrophic for Menadione 
Infection and Immunity  2014;82(4):1600-1605.
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.
PMCID: PMC3993378  PMID: 24452687
5.  Clinical Laboratory Response to a Mock Outbreak of Invasive Bacterial Infections: a Preparedness Study 
Journal of Clinical Microbiology  2014;52(12):4210-4216.
Large hospital-based clinical laboratories must be prepared to rapidly investigate potential infectious disease outbreaks. To challenge the ability of our molecular diagnostics laboratory to use whole-genome sequencing in a potential outbreak scenario and identify impediments to these efforts, we studied 84 invasive serotype emm59 group A streptococcus (GAS) strains collected in the United States. We performed a rapid-response exercise to the mock outbreak scenario using whole-genome sequencing, genome-wide transcript analysis, and mouse virulence studies. The protocol changes installed in response to the lessons learned were tested in a second iteration. The initial investigation was completed in 9 days. Whole-genome sequencing showed that the invasive infections were caused by multiple subclones of epidemic emm59 GAS strains likely spread to the United States from Canada. The phylogenetic tree showed a strong temporal-spatial structure with diversity in mobile genetic element content, features that are useful for identifying closely related strains and possible transmission events. The genome data informed the epidemiology, identifying multiple patients who likely acquired the organisms through direct person-to-person transmission. Transcriptome analysis unexpectedly revealed significantly altered expression of genes encoding a two-component regulator and the hyaluronic acid capsule virulence factor. Mouse infection studies confirmed a high-virulence capacity of these emm59 organisms. Whole-genome sequencing, coupled with transcriptome analysis and animal virulence studies, can be rapidly performed in a clinical environment to effectively contribute to patient care decisions and public health maneuvers.
PMCID: PMC4313289  PMID: 25253790
6.  Natural Variation in the Promoter of the Gene Encoding the Mga Regulator Alters Host-Pathogen Interactions in Group A Streptococcus Carrier Strains 
Infection and Immunity  2013;81(11):4128-4138.
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.
PMCID: PMC3811816  PMID: 23980109
7.  Spread of Virulent Group A Streptococcus Type emm59 from Montana to Wyoming, USA 
Emerging Infectious Diseases  2014;20(4):679-681.
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.
PMCID: PMC3966365  PMID: 24655919
group A Streptococcus; GAS; bacteria; emm59; streptococci; invasive disease; virulence; genome sequencing; epidemiology; necrotizing fasciitis; Montana; Wyoming; United States
8.  Molecular Characterization of an Invasive Phenotype of Group A Streptococcus Arising During Human Infection Using Whole Genome Sequencing of Multiple Isolates From the Same Patient 
The Journal of Infectious Diseases  2013;209(10):1520-1523.
Invasive group A streptococcal (GAS) strains often have genetic differences compared to GAS strains from nonsterile sites. Invasive, “hypervirulent” GAS strains can arise from a noninvasive progenitor following subcutaneous inoculation in mice, but such emergence has been rarely characterized in humans. We used whole genome analyses of multiple GAS isolates from the same patient to document the molecular basis for emergence of a GAS strain with an invasive phenotype during human infection. In contrast to previous theories, we found that elimination of production of the cysteine protease SpeB was not necessary for emergence of GAS with an invasive, “hypervirulent” phenotype.
PMCID: PMC3997578  PMID: 24307742
group A Streptococcus; bacterial pathogenesis; virulence; whole genome sequencing; gene expression
9.  Mycobacterium tuberculosis Bacteraemia: Experience from a Non-Endemic Urban Center 
The isolation of Mycobacterium tuberculosis (Mtb) from blood culture specimens has been associated with HIV co-infection with variable impact on tuberculosis (TB) mortality reported. The overwhelming majority of Mtb bacteraemia cases were described in developing countries. We present a nested case-control analysis of clinical, sociodemographic and behavioral risk factors in patients with positive Mtb blood cultures compared with patients with negative blood cultures from a nine-year population-based active TB surveillance study conducted in Houston, Texas. There were 42 Mtb bacteraemic, 47 blood culture negative patients and 3,573 patients for whom no mycobacterial blood culture was requested. HIV infection was more common in patients for whom a mycobacterial blood culture was requested (79.8% vs. 15.1% P<0.001). Of the Mtb bacteraemic patients, 6 were HIV negative or had no documentation of HIV status including 5 with immunosuppressive conditions other than HIV. Patients with Mtb bacteraemia were more likely than patients with negative blood cultures to be deceased at diagnosis or die while on TB therapy (50.0% vs. 17.0%, P<0.01), to report Men Who-Have-Sex-With-Men behavior (MSM) (31.7% vs. 13.0%, P=0.03), to have renal failure (28.6% vs. 6.4%, P=0.01), and to have HIV RNA levels higher than 500,000 copies/ml (61.9% vs. 17.2%, P=<0.01). Requests for mycobacterial culture of blood specimens were more common in HIV-infected individuals, and the presence of Mtb bacteraemia was associated with a significant increase in mortality.
PMCID: PMC4004024  PMID: 23980760
tuberculosis; Mycobacterium tuberculosis bacteraemia; mortality; HIV; outcomes
10.  Asymptomatic Carriage of Group A Streptococcus Is Associated with Elimination of Capsule Production 
Infection and Immunity  2014;82(9):3958-3967.
Humans commonly carry pathogenic bacteria asymptomatically, but despite decades of study, the underlying molecular contributors remain poorly understood. Here, we show that a group A streptococcus carriage strain contains a frameshift mutation in the hasA gene resulting in loss of hyaluronic acid capsule biosynthesis. This mutation was repaired by allelic replacement, resulting in restoration of capsule production in the isogenic derivative strain. The “repaired” isogenic strain was significantly more virulent than the carriage strain in a mouse model of necrotizing fasciitis and had enhanced growth ex vivo in human blood. Importantly, the repaired isogenic strain colonized the mouse oropharynx with significantly greater bacterial burden and had significantly reduced ability to internalize into cultured epithelial cells than the acapsular carriage strain. We conducted full-genome sequencing of 81 strains cultured serially from 19 epidemiologically unrelated human subjects and discovered the common theme that mutations negatively affecting capsule biosynthesis arise in vivo in the has operon. The significantly decreased capsule production is a key factor contributing to the molecular détente between pathogen and host. Our discoveries suggest a general model for bacterial pathogens in which mutations that downregulate or ablate virulence factor production contribute to carriage.
PMCID: PMC4187823  PMID: 25024363
11.  Adhesin competence repressor (AdcR) from Streptococcus pyogenes controls adaptive responses to zinc limitation and contributes to virulence 
Nucleic Acids Research  2014;43(1):418-432.
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.
PMCID: PMC4288194  PMID: 25510500
12.  Seasonal H3N2 influenza A virus fails to enhance Staphylococcus aureus co-infection in a non-human primate respiratory tract infection model 
Virulence  2013;4(8):707-715.
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.
PMCID: PMC3925702  PMID: 24104465
Staphylococcus aureus; influenza a virus; coinfection; USA300; MRSA; pneumonia
13.  Natural Disruption of Two Regulatory Networks in Serotype M3 Group A Streptococcus Isolates Contributes to the Virulence Factor Profile of This Hypervirulent Serotype 
Infection and Immunity  2014;82(5):1744-1754.
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.
PMCID: PMC3993421  PMID: 24516115
14.  Human Disease Isolates of Serotype M4 and M22 Group A Streptococcus Lack Genes Required for Hyaluronic Acid Capsule Biosynthesis 
mBio  2012;3(6):e00413-12.
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.
PMCID: PMC3487777  PMID: 23131832
15.  Genomic Analysis of emm59 Group A Streptococcus Invasive Strains, United States 
Emerging Infectious Diseases  2012;18(4):650-652.
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.
PMCID: PMC3309687  PMID: 22469010
group A Streptococcus; GAS; bacteria; invasive disease; genome sequencing; epidemic; United States; Canada; streptococci
16.  Group A Streptococcus emm Gene Types in Pharyngeal Isolates, Ontario, Canada, 2002–2010 
Emerging Infectious Diseases  2011;17(11):2010-2017.
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.
PMCID: PMC3310556  PMID: 22099088
Streptococcus pyogenes; GAS; pharyngitis; group A streptococcus; Canada; emm; bacteria
17.  Naturally occurring single amino acid replacements in a regulatory protein alter streptococcal gene expression and virulence in mice 
The Journal of Clinical Investigation  2011;121(5):1956-1968.
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.
PMCID: PMC3083769  PMID: 21490401
18.  High-throughput RNA sequencing of a formalin-fixed, paraffin-embedded autopsy lung tissue sample from the 1918 influenza pandemic 
The Journal of pathology  2013;229(4):535-545.
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.
PMCID: PMC3731037  PMID: 23180419
influenza A virus; pandemic; 1918 influenza virus; formalin-fixed; paraffin-embedded tissue; NGS
19.  A decade of molecular pathogenomic analysis of group A Streptococcus  
The Journal of Clinical Investigation  2009;119(9):2455-2463.
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.
PMCID: PMC2735924  PMID: 19729843
20.  Molecular characterization of group A Streptococcus maltodextrin catabolism and its role in pharyngitis 
Molecular microbiology  2008;69(2):436-452.
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.
PMCID: PMC2761070  PMID: 18485073
Streptococcus; maltodextrin; transport; amylase; pharyngitis
21.  Pediatric Tuberculosis: The Litmus Test for Tuberculosis Control 
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.
PMCID: PMC3473168  PMID: 22760534
tuberculosis; pediatric; genotype; cluster
22.  A Genomic Day in the Life of a Clinical Microbiology Laboratory 
Journal of Clinical Microbiology  2013;51(4):1272-1277.
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.
PMCID: PMC3666789  PMID: 23345298
23.  Vaccine Protection against Bacillus cereus-Mediated Respiratory Anthrax-Like Disease in Mice 
Infection and Immunity  2013;81(3):1008-1017.
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.
PMCID: PMC3584855  PMID: 23319564
24.  Polymorphisms in Regulator of Protease B (RopB) Alter Disease Phenotype and Strain Virulence of Serotype M3 Group A Streptococcus 
The Journal of Infectious Diseases  2012;205(11):1719-1729.
Whole-genome sequencing of serotype M3 group A streptococci (GAS) from oropharyngeal and invasive infections in Ontario recently showed that the gene encoding regulator of protease B (RopB) is highly polymorphic in this population. To test the hypothesis that ropB is under diversifying selective pressure among all serotype M3 GAS strains, we sequenced this gene in 1178 strains collected from different infection types, geographic regions, and time periods. The results confirmed our hypothesis and discovered a significant association between mutant ropB alleles, decreased activity of its major regulatory target SpeB, and pharyngitis. Additionally, isoallelic strains with ropB polymorphisms were significantly less virulent in a mouse model of necrotizing fasciitis. These studies provide a model strategy for applying whole-genome sequencing followed by deep single-gene sequencing to generate new insight to the rapid evolution and virulence regulation of human pathogens.
PMCID: PMC4447876  PMID: 22262791
25.  MalE of Group A Streptococcus Participates in the Rapid Transport of Maltotriose and Longer Maltodextrins▿ †  
Journal of Bacteriology  2007;189(7):2610-2617.
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.
PMCID: PMC1855798  PMID: 17259319

Results 1-25 (111)