Anthony R Flores

Publications (10)59.61 Total impact

• Article: Differential Virulence Gene Expression of Group A Streptococcus Serotype M3 in Response to Co-Culture with Moraxella catarrhalis.

  Suzanne J C Verhaegh, Anthony R Flores, Alex van Belkum, James M Musser, John P Hays
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  ABSTRACT: 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.
  PLoS ONE 01/2013; 8(4):e62549. · 4.09 Impact Factor
• Article: Full-genome dissection of an epidemic of severe invasive disease caused by a hypervirulent, recently emerged clone of group A Streptococcus.

  Nahuel Fittipaldi, Stephen B Beres, Randall J Olsen, Vivek Kapur, Patrick R Shea, M Ebru Watkins, Concepcion C Cantu, Daniel R Laucirica, Leslie Jenkins, Anthony R Flores, Marguerite Lovgren, Carmen Ardanuy, Josefina Liñares, Donald E Low, Gregory J Tyrrell, James M Musser
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  ABSTRACT: Group A Streptococcus (GAS) causes an exceptionally broad range of infections in humans, from relatively mild pharyngitis and skin infections to life-threatening necrotizing fasciitis and toxic shock syndrome. An epidemic of severe invasive human infections caused by type emm59 GAS, heretofore an exceedingly rare cause of disease, spread west to east across Canada over a 3-year period (2006 to 2008). By sequencing the genomes of 601 epidemic, historic, and other emm59 organisms, we discovered that a recently emerged, genetically distinct emm59 clone is responsible for the Canadian epidemic. Using near-real-time genome sequencing, we were able to show spread of the Canadian epidemic clone into the United States. The extensive genome data permitted us to identify patterns of geographic dissemination as well as links between emm59 subclonal lineages that cause infections. Mouse and nonhuman primate models of infection demonstrated that the emerged clone is unusually virulent. Transmission of epidemic emm59 strains may have occurred primarily by skin contact, as suggested by an experimental model of skin transmission. In addition, the emm59 strains had a significantly impaired ability to persist in human saliva and to colonize the oropharynx of mice, and seldom caused human pharyngitis. Our study contributes new information to the rapidly emerging field of molecular pathogenomics of bacterial epidemics and illustrates how full-genome data can be used to precisely illuminate the landscape of strain dissemination during a bacterial epidemic.
  American Journal Of Pathology 02/2012; 180(4):1522-34. · 4.89 Impact Factor
• Article: Polymorphisms in regulator of protease B (RopB) alter disease phenotype and strain virulence of serotype M3 group A Streptococcus.

  Randall J Olsen, Daniel R Laucirica, M Ebru Watkins, Marsha L Feske, Jesus R Garcia-Bustillos, Chau Vu, Concepcion Cantu, Samuel A Shelburne, Nahuel Fittipaldi, Muthiah Kumaraswami, Patrick R Shea, Anthony R Flores, Stephen B Beres, Maguerite Lovgren, Gregory J Tyrrell, Androulla Efstratiou, Donald E Low, Chris A Van Beneden, James M Musser
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  ABSTRACT: 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.
  The Journal of Infectious Diseases 01/2012; 205(11):1719-29. · 6.41 Impact Factor
• Article: Human disease isolates of serotype m4 and m22 group a streptococcus lack genes required for hyaluronic Acid capsule biosynthesis.

  Anthony R Flores, Brittany E Jewell, Nahuel Fittipaldi, Stephen B Beres, James M Musser
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  ABSTRACT: 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.
  mBio 01/2012; 3(6). · 5.31 Impact Factor
• Article: Group A Streptococcus emm gene types in pharyngeal isolates, Ontario, Canada, 2002-2010.

  Patrick R Shea, Amy L Ewbank, Javier H Gonzalez-Lugo, Alexandro J Martagon-Rosado, Juan C Martinez-Gutierrez, Hina A Rehman, Monica Serrano-Gonzalez, Nahuel Fittipaldi, Stephen B Beres, Anthony R Flores, Donald E Low, Barbara M Willey, James M Musser
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  ABSTRACT: 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.
  Emerging Infectious Diseases 11/2011; 17(11):2010-7. · 6.79 Impact Factor
• Article: Evolution of diversity in epidemics revealed by analysis of the human bacterial pathogen group A Streptococcus.

  Ronan K Carroll, Stephen B Beres, Izabela Sitkiewicz, Leif Peterson, Risë K Matsunami, David A Engler, Anthony R Flores, Paul Sumby, James M Musser
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  ABSTRACT: Advancements in high-throughput, high-volume data generating techniques increasingly present us with opportunities to probe new areas of biology. In this work we assessed the extent to which four closely related and genetically representative strains of group A Streptococcus causing epidemic disease have differentiated from one another. Comparative genome sequencing, expression microarray analysis, and proteomic studies were used in parallel to assess strain variation. The extent of phenotypic differentiation was unexpectedly large. We found significant associations between genetic polymorphisms and alterations in gene expression allowing us to estimate the frequency with which specific types of polymorphisms alter gene transcription. We identified polymorphisms in the gene (ropB) encoding the RopB regulator that associate with altered transcription of speB and production of the SpeB protein, a critical secreted protease virulence factor. Although these four epidemic strains are closely related, a key discovery is that accumulation of modest genetic changes has rapidly resulted in significant strain phenotypic differentiation, including the extracellular proteome that contains multiple virulence factors. These data provide enhanced understanding of genetic events resulting in strain variation in bacterial epidemics.
  Epidemics. 09/2011; 3(3-4):159-70.
• Article: Rapidly progressive, fatal, inhalation anthrax-like infection in a human: case report, pathogen genome sequencing, pathology, and coordinated response.

  Angela M Wright, Stephen B Beres, Erin N Consamus, S Wesley Long, Anthony R Flores, Roberto Barrios, G Stefan Richter, So-Young Oh, Gabriella Garufi, Hannah Maier, Ashley L Drews, Kathryn E Stockbauer, Patricia Cernoch, Olaf Schneewind, Randall J Olsen, James M Musser
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  ABSTRACT: Ten years ago a bioterrorism event involving Bacillus anthracis spores captured the nation's interest, stimulated extensive new research on this pathogen, and heightened concern about illegitimate release of infectious agents. Sporadic reports have described rare, fulminant, and sometimes fatal cases of pneumonia in humans and nonhuman primates caused by strains of Bacillus cereus , a species closely related to Bacillus anthracis. To describe and investigate a case of rapidly progressive, fatal, anthrax-like pneumonia and the overwhelming infection caused by a Bacillus species of uncertain provenance in a patient residing in rural Texas. We characterized the genome of the causative strain within days of its recovery from antemortem cultures using next-generation sequencing and performed immunohistochemistry on tissues obtained at autopsy with antibodies directed against virulence proteins of B anthracis and B cereus. We discovered that the infection was caused by a previously unknown strain of B cereus that was closely related to, but genetically distinct from, B anthracis . The strain contains a plasmid similar to pXO1, a genetic element encoding anthrax toxin and other known virulence factors. Immunohistochemistry demonstrated that several homologs of B anthracis virulence proteins were made in infected tissues, likely contributing to the patient's death. Rapid genome sequence analysis permitted us to genetically define this strain, rule out the likelihood of bioterrorism, and contribute effectively to the institutional response to this event. Our experience strongly reinforced the critical value of deploying a well-integrated, anatomic, clinical, and genomic strategy to respond rapidly to a potential emerging, infectious threat to public health.
  Archives of pathology & laboratory medicine 08/2011; 135(11):1447-59. · 2.58 Impact Factor
• Article: Naturally occurring single amino acid replacements in a regulatory protein alter streptococcal gene expression and virulence in mice.

  Ronan K Carroll, Samuel A Shelburne, Randall J Olsen, Bryce Suber, Pranoti Sahasrabhojane, Muthiah Kumaraswami, Stephen B Beres, Patrick R Shea, Anthony R Flores, James M Musser
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  ABSTRACT: 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 Journal of clinical investigation 05/2011; 121(5):1956-68. · 15.39 Impact Factor
• Article: Distinct signatures of diversifying selection revealed by genome analysis of respiratory tract and invasive bacterial populations.

  Patrick R Shea, Stephen B Beres, Anthony R Flores, Amy L Ewbank, Javier H Gonzalez-Lugo, Alexandro J Martagon-Rosado, Juan C Martinez-Gutierrez, Hina A Rehman, Monica Serrano-Gonzalez, Nahuel Fittipaldi, Stephen D Ayers, Paul Webb, Barbara M Willey, Donald E Low, James M Musser
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  ABSTRACT: Many pathogens colonize different anatomical sites, but the selective pressures contributing to survival in the diverse niches are poorly understood. Group A Streptococcus (GAS) is a human-adapted bacterium that causes a range of infections. Much effort has been expended to dissect the molecular basis of invasive (sterile-site) infections, but little is known about the genomes of strains causing pharyngitis (streptococcal "sore throat"). Additionally, there is essentially nothing known about the genetic relationships between populations of invasive and pharyngitis strains. In particular, it is unclear if invasive strains represent a distinct genetic subpopulation of strains that cause pharyngitis. We compared the genomes of 86 serotype M3 GAS pharyngitis strains with those of 215 invasive M3 strains from the same geographical location. The pharyngitis and invasive groups were highly related to each other and had virtually identical phylogenetic structures, indicating they belong to the same genetic pool. Despite the overall high degree of genetic similarity, we discovered that strains from different host environments (i.e., throat, normally sterile sites) have distinct patterns of diversifying selection at the nucleotide level. In particular, the pattern of polymorphisms in the hyaluronic acid capsule synthesis operon was especially different between the two strain populations. This finding was mirrored by data obtained from full-genome analysis of strains sequentially cultured from nonhuman primates. Our results answer the long-standing question of the genetic relationship between GAS pharyngitis and invasive strains. The data provide previously undescribed information about the evolutionary history of pathogenic microbes that cause disease in different anatomical sites.
  Proceedings of the National Academy of Sciences 03/2011; 108(12):5039-44. · 9.68 Impact Factor
• Article: Estimated burden of rotavirus-associated diarrhea in ambulatory settings in the United States.

  Anthony R Flores, Peter G Szilagyi, Peggy Auinger, Susan G Fisher
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  ABSTRACT: Two vaccines to prevent disease from rotavirus among young children were recently approved in the United States. Although previous studies of the burden of rotaviral disease have focused on hospitalizations, the assessment of baseline disease burden in ambulatory settings is useful for evaluating the overall impact of the vaccine. Outpatient and emergency department (ED) data for 1994-2006 were analyzed from 2 nationally representative databases: the National Ambulatory Medical Care Survey and the National Hospital Medical Care Survey. Visits by children (younger than 5 years) with acute gastroenteritis (AGE) were identified by using a defined set of International Classification of Diseases, Ninth Revision, Clinical Modification codes. Two previously described methods (the winter-residual-excess [WRE] and Brandt methods) were used to estimate the proportion of AGE attributable to rotavirus and to determine the annual number of visits, annual average visits, and annual visit rates in each setting. The estimated average annual number of rotavirus-associated visits over the 13-year period was 782 453 outpatient visits and 164 261 ED visits from the WRE method and 665 773 outpatient visits and 205 206 ED visits from the Brandt method. This resulted in an average of 39.1 and 33.3 outpatient visits per 1000 children and 8.2 and 10.3 ED visits per 1000 children for both the WRE and Brandt methods, respectively. The average annual proportion of visits for AGE attributed to rotavirus was 34.2% (29.1% from the Brandt method) in the outpatient setting and 21.8% (27.1% from the Brandt method) in the ED, with wide variations in individual years. Before the rotavirus vaccine, rotavirus seemed to be associated with a large number of outpatient and ED visits among young children. Rotavirus vaccine has the potential to reduce many outpatient and ED visits.
  PEDIATRICS 02/2010; 125(2):e191-8. · 4.47 Impact Factor