Differential Response of the Cynomolgus Macaque Gut Microbiota to Shigella Infection Anna M. Seekatz 1 , Aruna Panda 2 , David A. Rasko 1,4 , Franklin R. Toapanta 3 , Emiley A. Eloe-Fadrosh 1 , Abdul Q. Khan 8 , Zhenqiu Liu 1,7 , Steven T. Shipley 2 , Louis J. DeTolla 2,6,7 , Marcelo B. Sztein 3,5 , Claire M. Fraser 1,4,6 * 1 Institute for Genome Sciences, University of Maryland School of Medicine, Baltimore, Maryland, United States of America, 2 Department of Pathology, University of Maryland School of Medicine, Baltimore, Maryland, United States of America, 3 Center for Vaccine Development, University of Maryland School of Medicine, Baltimore, Maryland, United States of America, 4 Department of Microbiology and Immunology, University of Maryland School of Medicine, Baltimore, Maryland, United States of America, 5 Department of Pediatrics, University of Maryland School of Medicine, Baltimore, Maryland, United States of America, 6 Department of Medicine, University of Maryland School of Medicine, Baltimore, Maryland, United States of America, 7 Department of Epidemiology and Public Health, University of Maryland School of Medicine, Baltimore, Maryland, United States of America, 8 Department of Microbiology and Immunology, Uniformed Services University of the Health Sciences, Bethesda, Maryland, United States of America Abstract Little is known about the role of gut microbiota in response to live oral vaccines against enteric pathogens. We examined the effect of immunization with an oral live-attenuated Shigella dysenteriae 1 vaccine and challenge with wild-type S. dysenteriae 1 on the fecal microbiota of cynomolgus macaques using 16 S rRNA analysis of fecal samples. Multi-dimensional cluster analysis identified different bacterial community types within macaques from geographically distinct locations. The fecal microbiota of Mauritian macaques, observed to be genetically distinct, harbored a high-diversity community and responded differently to Shigella immunization, as well as challenge compared to the microbiota in non-Mauritian macaques. While both macaque populations exhibited anti-Shigella antibody responses, clinical shigellosis was observed only among non-Mauritian macaques. These studies highlight the importance of further investigation into the possible protective role of the microbiota against enteric pathogens and consideration of host genetic backgrounds in conducting vaccine studies. Citation: Seekatz AM, Panda A, Rasko DA, Toapanta FR, Eloe-Fadrosh EA, et al. (2013) Differential Response of the Cynomolgus Macaque Gut Microbiota to Shigella Infection. PLoS ONE 8(6): e64212. doi:10.1371/journal.pone.0064212 Editor: Roy Martin Roop II, East Carolina University School of Medicine, United States of America Received January 11, 2013; Accepted April 8, 2013; Published June 5, 2013 This is an open-access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication. Funding: This study was supported by the National Institutes of Health grant U19 AI082655 (CCHI). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: cmfraser@som.umaryland.edu Introduction Shigella species represent a group of mucosally invasive bacteria that cause bacillary dysentery, or shigellosis, in humans and nonhuman primates (NHPs) [1]. Only a small inoculum of Shigella species is required for disease in humans (,10–100 bacteria), and the recent appearance of antibiotic-resistant strains and new serotypes is concerning. Worldwide, it is estimated that 90 million cases of food-borne illnesses and over 100,000 deaths are caused by Shigella each year [2]. Four different Shigella species cause human disease: Shigella sonnei, Shigella flexneri, Shigella boydii, and Shigella dysenteriae. While S. sonnei accounts for the majority of the cases in the developed world, S. dysenteriae 1 is capable of large endemic outbreaks in developing countries, especially within infants and children [1]. Ideally, a successful vaccine against Shigella would elicit high immunogenicity against all epidemiolog- ically relevant species without adverse side effects. While both live- attenuated Shigella strains and parenteral conjugate vaccine candidates have shown varying degrees of success in human volunteers and NHPs studies, licensed vaccines are not yet available [1]. Vaccine development against Shigella has been slowed by discrepancies observed in the efficacy of vaccines evaluated worldwide [1]. For example, the attenuated S. flexneri 2a vaccine strain SC602 demonstrated strong immunogenicity and evoked protection in North American volunteers, but it was found to be associated with minimal vaccine shedding and low immune stimulation in volunteers in Bangladesh [3–5]. Similar conflicting results between developed and underprivileged global populations have been observed in vaccine studies against polio, cholera, and rotavirus [1,6,7]. Development of a successful vaccine against Shigella has also been hampered by the lack of a small animal model that mimics human disease [1]. Mice, guinea pigs, and rabbits have been used to assess virulence and screen for vaccine candidates, but these model systems lack the ability to directly predict protection in humans [8]. A more clinically relevant model has been developed with S. flexneri in rhesus macaques, Macaca mulatta, [9] and S. dysenteriae 1 in cynomolgus macaques, Macaca fascicularis [10]. However, a large inoculum (,10 10 CFU) is necessary to cause bacillary dysentery in non-human primates as compared to humans. Multiple factors such as diet, nutrition, and host genetics may impact vaccine efficacy [11]. An additional factor that may PLOS ONE | www.plosone.org 1 June 2013 | Volume 8 | Issue 6 | e64212