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Photo d Although most diarrheal diseases are self-resolving and easily treated with oral-rehydration therapy, it is common for antimicrobials to be used in isolated cases. Said agents will shorten the time of infection and thus limit the transmission of pathogens within in the environment. Throughout the past decade, however, there have been numerous studies published that suggest antimicrobial resistance (AMR) within enteric bacteria is not only high but increasing, especially in developing regions of the world. The objective of this study was to assess the prevalence of AMR within Ranomafana, Madagascar. We also evaluated behaviors potentially associated with the development of AMR using a comprehensive survey. We chose to place an emphasis on Salmonella Spp., Shigella Spp., & Vibrio cholera , as past studies have shown elevated prevalences. The results from this field season will help to support the existing health care framework, which Pivot (pivotworks.org) has established in the region by offering updated information on the effectiveness of antibiotic treatment regimens. Study Site Our study took place in and around Ranomafana National Park (RNP), a 43,500 hector World Heritage Site, well known for its high levels of species diversity and endemism. Sixteen communities located around the parks edge were selected for inclusion in the study, which represent a gradient of developed environments from Urban (Ranomafana proper) to rural (Ampitavanana). The study populations included domestic livestock: bovine ( Bos indicus ), porcine ( Sus domesticus ), poultry ( Gallus gallus, Anas platyrhyncha ) and humans from 80 randomly selected households. Diarrheal Disease Madagascar is one of the poorest developing regions of the world. While efforts have been made to increase the living conditions of the poor, only 50% of the population have access to clean water sources and less the 20% has access to proper sanitation facilities. As a result, diarrheal disease has become the leading cause of death in children under 5, with more than 1.5 billion cases a year, resulting in 3.3 million deaths per year. It is the second leading factor influencing morbidity across all age groups. Salmonella Spp., Shigella Spp., & Vibrio cholerae were selected as our pathogens of interest, as a pilot study conducted in 2011/12 suggests that these enteric pathogens are the most prevalent amongst the RPN populations. Objective 1 Assess the prevalence of Salmonella Spp., Shigella Spp., & Vibrio cholerae within the study populations in and around Ranomafana National Park Humans: All individuals within 80 households across 16 villages in the RNP region (n=243). Domestic Livestock: All bovine, porcine, and poultry from a given household included in the study (n=64). We thank the Madagascar Ministry of the Environment and MNP for allowing us to conduct research in Madagascar. We thank ICTE, MICET, and the Centre ValBio for essential logistic and research support. INSTAT for organizing and distributing the survey component of the project. K. Smith, A. Stumvoll, A. Benn, provided invaluable assistance in the field. This research was sponsored by the Gates Grand Challenges Program, Emory University’s: Global Health Institute, the International Research Experience for Science Students (IRES) and Scholarly Inquiry and Research at Emory (SIRE) programs, and PIVOT. For additional information please contact: Abstract Objectives and Hypotheses Study Design / Field Collection Laboratory Methods Background Preliminary Results Next Steps Acknowledgments and Contact Information Epidemiology and Antimicrobial Resistance of Pathogenic Enterobacteria in Ifanadiana District, Madagascar R.C. Giordano 1 , C.L. Rist 1,4 , M.B. Parsons 2 , J.B. Bliska 3 , M. Bonds 4,5 , T.R. Gillespie 1,4 1 Departments of Environmental Sciences and Environmental Health, Emory University and Rollins School of Public Health, Atlanta, GA USA; 2 Center for Emerging and Zoonotic Infectious Diseases, Centers for Disease Control and Prevention, Atlanta, GA; 3 School of Medicine, Stony Brook University, Stony Brook, NY; 4 PIVOT, Ranomafana, Madagascar; 5 Department of Global Health and Social Medicine, Harvard Medical School, Boston, MA Objective 2 Characterize the antimicrobial resistance profile within study populations through the use both antimicrobial susceptibility and genetic testing. Objective 3 Examine and assess the behavioral aspects associated with antimicrobial use that could be related to the observed levels resistance through comprehensive surveys/ interviews. H 0 : Salmonella Spp., Shigella Spp., & Vibrio cholerae are not present in the RNP region. H 0 : AMR does not exist within the study populations. H 0 : There are no human behavioral associations with AMR withing the study populations. Poultry is one of the major sources of protein in rural Ranomafana. Chickens and ducks frequently have the ability to roam free during the day, increasing the risk of contact and, thus, disease transmission. Study Populations Humans: Once oral informed consent was obtained from the head of household, comprehensive household and individual surveys were administered by trained enumerators from the Institut National de la Statistique de Madagascar (INSTAT), a Malagasy consulting organization, using Malagasy, the local language. Surveys were designed to assess behaviors that could potentially lead to favorable conditions for AMR development as well gather socio-demographic (age, sex, profession, etc.) and health / hygiene data (diarrheal illness, water usage, defecation practices, etc.) Domestic Livestock: Fecal samples were collected non-invasively from all livestock of participating households with daily health survey. Study Design Samples were taken from all members of a given household to analyze for inter-household disease transmission. Cloaca swabs were taken from individual chickens and ducks. Fecal Samples were collected and preserved on site. Antibiotic Susceptibility Testing Molecular Analysis • Total nucleic acid was extracted from all fecal specimens (n= 309) preserved in RNAlater using the FastDNA SPIN Kit for Soil (MP Biomedicals, LLC, Solon, OH), following the manufacturer- recommended procedures. • Using conventional PCR we will screen the samples for S. enterica, Shigella spp. (flexneri, boydii, and dysenteriae),V. cholerae, along with several genes associated with AMR across a spectrum of antibiotic classes. • The genes we chose to amplify for entrobacteria included: ipaH (Shigella Spp.), invA (Salmonella enterica serovar Typhimurium), ctaX (Vibrio cholerae). • Various genes were chosen to screen for AMR within the following antimicrobial classes: β-Lactams, Aminoglycosides, Tetracycline, Trimethoprim, Sulfomamides, and Chloramphenicol. Streaking isolated S. dysenteriae on Mueller-Hinton agar slants. Robert Giordano [email protected] 631.603.9526 Thomas Gillespie [email protected] 404.727.7926 Photo Credit: Cassidy Rist Photo Credit: Cassidy Rist Figure 1: Amoxicillin and trimethoprim resistant S. dysenteriae Sample Type Samples Collected Positive for Shigella Spp. Domestic Animal 64 0 Bovine Porcine Poultry 16 9 39 0 0 0 Human 243 8 Total 309 8 Isolated Shigella Spp. Number of Isolates Table 1: Prevalence of Shigella Spp. relative to taxa Table 2: Diversity of Shigella Spp. isolates Table 3: Antimicrobial resistance profile of Shigella Spp. isolates from Ranomafana, Madagascar S. dysenteriae S. dysenteriae S. dysenteriae S. dysenteriae S. boydii S. boydii S. flexneri S. flexneri Sample ID Source Village Amoxicillin Ciprofloxacin Trimethoprim Antimicrobial Susceptibility Zone Diameter (mm) Ambatolahy Ambatolahy Tsararano Ampitavanana Afovoany Ampitavanana Ampitavanana Tsararano 8 (Resistant) 8 (Resistant) 8 (Resistant) 20 (Susceptible) 8 (Resistant) 24 (Susceptible) 12 (Resistant) 22 (Susceptible) 30 (Susceptible) 44 (Susceptible) 38 (Susceptible) 36 (Susceptible) 40 (Susceptible) 44 (Susceptible) 26 (Susceptible) 42 (Susceptible) 8 (Resistant) 8 (Resistant) 8 (Resistant) 8 (Resistant) 8 (Resistant) 8 (Resistant) 8 (Resistant) 23 (Susceptible) S. dysenteriae 4 S. boydii S. flexneri 2 2 • In-depth genetic analysis via PCR of all collected fecal samples for genetic confirmation of pathogenic Shigella Spp., Salmonella Spp., and Vibrio cholerae. • PCR analysis of all collected fecal samples for genetic markers associated with resistance to β-Lactams, Aminoglycosides, Tetracycline, Trimethoprim, Sulfomamides, and Chloramphenicol antimicrobal classes. • Analysis of survey results. • Report findings to all communities involved in study. • Write final report for masters thesis submission. • Submit results for publication. • Swabs from fecal samples were taken and preserved at -80°C in Cary-Blair transport medium until culturing. • A single swab from the Cary-Blair medium was inoculated on selective/ differential media (MacConkey, XLD, TCBS) for the isolation of suspicious colonies of Vibrio cholerae, Salmonella spp. and Shigella spp. • Additionally, a single fecal swab from the Cary-Blair was enriched for the isolation of Salmonella spp. in 10 mL of Selenite Cystine Broth and subsequently plated on XLD media. • Suspicious colonies were then tested in screening biochemicals and organism specific antisera to finalize the identity of isolated bacteria. • All suspicious isolates were then tested for Antibiotic susceptibility against Amoxicillin, Ciprofloxacin, and Trimethoprim using the disk- diffusion method on Mueller-Hinton Agar, according to the Clinical and Laboratory Standards Institute (CLSI) protocols and recommended quality controls and resistance cutoffs for the above-mentioned antibiotics (CLSI, 2013). Inoculation of differential/selective plates with fecal swabs. Photo Credit: Cassidy Rist
