International Surveillance Of

Established 1981

International Surveillance of Reservoirs of Antibiotic Resistance (ISRAR)

1 PROJECT SYNOPSIS Background Antibiotic resistance continues to plague effective treatment of infectious diseases worldwide. US CDC, IDSA, and others consider antibiotic resistance to be a top public health threat. The interests of the biological defense and public health communities intersect strongly around the alarming increase of antimicrobial resistant organisms. The profile of a microorganism has profound implications both within the doctor’s office and during a biological attack. Multidrug resistance as a public health threat and the inherent bioterrorism opportunity has the scientific and medical communities alarmed. A number of reports of new superbugs have emerged in the past few years, including methicillin-resistant Staphylococcus aureus and the so-called ESKAPE organisms. “For the most part, these organisms owe their superbug status not to enhanced pathogenicity or virulence…but to their resistance to multiple antimicrobial agents.1” The spread of NDM-1 genes, for example, has been a widespread concern because of their resistance to all antimicrobial agents except the polymyxins.1 The goal of the ISRAR project is to better understand and characterize antibiotic resistance traits through a novel research approach which will accomplish understanding and development of countermeasures. This project was to serve a number of interconnected purposes: 1) to generate internal NBACC capability regarding antibiotic resistance and genetic analysis, 2) to acquire a diverse collection of antibiotic resistant bacterial isolates, 3) to impact the National Strategy against Biological Threats, and 4) to directly impact public health synergistically with biological defense. Most disease surveillance and response programs are designed for action after an epidemic. In contrast, these purposes all link to a primary goal of becoming proactive, versus reactive, in the battle against the spread of antibiotic resistance so that members of the biodefense community and the public health community can work cooperatively to predict the agents that will most likely threaten the health security of the United States. Resistance genes are not limited to human and animal pathogens. They are carried by bacteria in the environment, in those not causing disease – the so-called commensal flora. These are being increasingly recognized as the reservoirs for antibiotic resistance genes. A multitude of new, not-yet-characterized resistance genes are present in environmental bacteria throughout the world. These genes are probably different in different parts of the world, yet there are no collections or analyses of these major health-related organisms. The origins of antibiotic resistance genes and their location in organisms which are not causing disease are areas needing study if we are to control resistance emergence and spread. Commensals act as predictive features of resistance that could confront treatment. Based on a “one world/one health” model, this research focuses on environmental reservoirs of antibiotic resistance to enable earlier identification of antibiotic resistance threats and more rapid and effective DHS response.

The ISRAR project is novel, being the first of its kind to analyze bacteria of the commensal variety, rather than the clinical strains. The resistance traits among clinical strains are widely

1 Moellering, R.C. (2010). “NDM-1 – A Cause for Worldwide Concern.” New Engl J Med 363: 2377-2379.

2 known and studied. It is the nonclinical flora which bear the potential for new and devastating resistance determinants that have not, until now, been identified. The NDM-1 gene is just one of presumably many out there that, if recognized earlier, could have been contained and prevented from moving into bacteria of consequence to human health.

This project is unique, not only in focus, but also in the infrastructure organized for its performance. The ability to obtain many different kinds of bacteria from different parts of the world and analyze them for resistance determinants was a major undertaking, whose success adds a critically important dimension to the counter-threat measures of the U.S. government. APUA is uniquely situated with its international network of APUA Chapters for coordinating this venture. Fig. 1 APUA Global Chapter Map: An APUA international surveillance laboratory network adaptable for geographic expansion and other biodefense research

APUA has established affiliated chapters in 66 countries, including 30 resource-poor countries, and has an expanding network in over 10 sub-Saharan African countries. This global network of infectious disease experts supports country-based activities to control and monitor antibiotic resistance tailored to local needs and customs. The APUA network facilitates the exchange of objective, up-to-date scientific and clinical information among scientists, health care providers, consumers and policy makers worldwide. APUA chapters serve these vital functions:  Raising awareness about the problem of resistance within a country and about the dangers of incorrect antibiotic usage and faulty prescriptions;  Communicating information on proper antibiotic usage;  Fostering related research and educational projects;  Providing a multidisciplinary approach to interventions; fostering scientifically sound solutions;  Affording a local platform for input and feedback into global planning efforts;  Providing international networking opportunities to enhance their knowledge and effectiveness at the country level;  Working in partnership with Ministers of Health and public health organizations to improve antimicrobial use around the world

As many agents of biological warfare have treatment windows of days to hours depending on the detection scenario, the timing and accuracy of the delivery of countermeasures is critical. This project was conceptualized to move the front lines in the battle against antimicrobial agents from the population attacked (or the patient in treatment), to the reservoir of commensal bacteria. It is at this level that new antimicrobial resistance genetic elements are being mobilized into pathogenic bacteria and clonally expanded prior to the shift to the clinic (or the hands of the biological terrorist). This proactive approach to identify newly emerging genes encoding antibiotic resistance allows a shift in our ability to predict public health impacts

3 and defend against the most current and advanced agents of biological terror. This project was designed to explore resistance patterns in organisms from 8 target countries with a focus on infectious disease hotspots to allow for more rapid understanding of antibiotic evolution and control. By shifting the focus from pathogen surveillance to commensals the project moves the epi curve to the left with the goal of identifying the early emergence of resistance.

HISTORY In 2008 the National Biodefense Analysis and Countermeasures Center (NBACC) antimicrobial resistance metagenomics project was initiated. APUA’s collaboration with NBACC was established with three interrelated objectives: 1) to establish an operational international laboratory network that collects, types, and identifies antibiotic resistant commensal and environmental bacteria in target countries, 2) to analyze these bacteria for novel genetic elements encoding antibiotic resistance, novel mobile genetic elements, or novel pairings of the two genetic structures, and 3) to elucidate the evolution of antibiotic resistance and its application for countermeasures by identifying patterns and trends of antibiotic resistance in commensal bacteria. The pilot project served to establish a “proof of principle” and proved successful, indicating the potential for a much larger operation.

Accomplishments of the ISRAR project from 2008-2010 are as follows:  Collection and transport protocols have been successfully developed and tested and an international field network of qualified laboratories has been engaged in 8 countries of interest. The network allows for potential expansion geographically through the APUA network of 66 country chapters and substantively by inclusion of other organisms including pathogens. Each laboratory has passed quality control tests at the Tufts University Center for Adaptation Genetics and Drug Resistance.  A total of 1932 isolates of Salmonella spp., Escherichia coli, Staphylococcus spp., Streptococcus spp., Aeromonas spp., Pseudomonas spp., Acinetobacter spp. and Stenotrophomonas spp., have been collected and processed from animal, plant, soil and water sources (See Tables 1 & 2 below). Confirmation of bacterial identities revealed ~85% recovery of target species by the site laboratories – an extraordinary accomplishment, as some countries are operating at a very basic level when compared to clinical diagnostic laboratories in the United States.  Meta-analysis of the cumulative data have begun, including an update of the Reservoirs of Antibiotic Resistance (ROAR) database to include GPS point data and a Google Maps interface (see Appendix)

 A poster was presented at the 2010 Interscience Conference on Antimicrobial Agents and Chemotherapy (ICAAC) held in Chicago, IL and was chosen by the meeting directors to be included in a Press release due to its impact on global health (see Appendix)

4  A project database has been designed and refined to allow input of massive amounts of data (www.roarproject.org). At this time, data from 422 isolates have been uploaded to the ROAR database (www.roarproject.org).  An ISRAR Advisory Committee comprised of top experts in antibiotic resistance has been formulated to assist in interpretation.

Table 1. Species and country sites of ISRAR isolate collections

ISRAR ISOLATES

Strepto- Total # of Staphylo- Acineto- Pseudo- Stenotro- coccus/ Origin Isolates E. coli Salmonella Aeromonas *Other coccus bacter monas phomonas Entero- Received coccus

Bangladesh 131 24 0 2 23 23 20 15 24 0

Georgia 96 69 25 0 0 0 0 0 0 2

India 430 135 128 3 11 42 46 16 0 49

South Africa 172 22 31 11 24 20 24 23 17 0

South Korea 350 30 81 199 0 20 0 0 20 0

Turkey 372 144 132 2 26 10 3 0 30 25

Uganda 188 59 61 14 0 0 0 0 45 9

Vietnam 193 33 21 35 32 24 21 15 8 4

5 Total 1932 516 479 266 116 139 114 69 144 89

* Hafnia, Enterobacter, Providencia, Proteus, Citrobacter, Klebsiella, Micrococcus, Leuconostoc, Kluyvera, Aerococcus

6 Table 2: ISRAR isolate sources

7 SIGNIFICANCE AND FUTURE POTENTIAL This project has far ranging scope and impact for the greater biodefense community, and for public health. The project provides a unique opportunity to explore antibiotic resistance in the vast reservoirs of commensal organisms as a barometer of resistance emergence in clinical organisms. The focus on commensals as an early warning of emergence of antibiotic resistance is a measure of the vision and innovation demonstrated by the scientists at NBACC and DHS. The ISRAR project and infrastructure is adaptable for multiple biodefense purposes and offers the following value:

 Proven protocols and capacity to collect and analyze thousands of isolates within several months  Qualified international laboratory partners in over 30 developing countries  Tested protocols and contract models  A state of the art project bioinformatics database focused on antibiotic resistance  APUA specialized staff and a project advisory board committee including the world’s top experts in the field available for ongoing collaboration and advice

8 9 APPENDIX: 1. Abstract and Poster: An abstract on “Surveillance of antibiotic resistance determinants in commensal and environmental bacteria from international sources” was submitted and accepted for poster presentation at the 50th ICAAC 2010 in Boston, MA from September 12 through 15, 2010. 2. Press Release 3. Geo-temporal user interface 4. Extract of the APUA ROAR database (www.roarproject.org)

10 1. ABSTRACT AND POSTER Surveillance of antibiotic resistance determinants in commensal and environmental bacteria from international sources: 1,2B.M. Marshall, 1,2S.B.Levy, 2D.Ochieng, 3D.Gur, 3A. Nanuashvili, 3Y.S. Kim, 3D.K.Byarugaba, 3A.Okoh, 3A.T. Vo, 3S.K.Kashyap, 3H. Endtz, 2A.Sosa; 1Alliance for the Prudent Use of Antibiotics (APUA), Boston, MA, USA; Tufts University School of Medicine; 3APUA International Surveillance of Reservoirs of Antibiotic Resistance (ISRAR); Boston, MA, USA Background: Commensal and environmental flora form a large reservoir of mobile genetic elements, which under selective pressure from antibiotics, can potentially transfer resistance to pathogens. APUA, in conjunction with the National Biodefense Analysis and Countermeasures Center, has undertaken an international surveillance of the reservoirs of antibiotic resistance (ISRAR) in commensal bacteria to track geographically, and eventually temporally, the emergence and spread of resistance genes through its online Reservoirs of Antibiotic Resistance (ROAR) database. Methods: Protocols were developed to optimize isolation of nine targeted bacterial genera from healthy animals and environmental sites. Isolates obtained from 8 APUA country chapter (Bangladesh, Georgia, India, Turkey, Uganda, S. Africa, S. Korea, Vietnam) laboratories were speciated (API) and susceptibility tested (E-test) using appropriate classes of antibiotics. Results: There was 85% recovery of target species. Between 2008 and 2010, 1079 total isolates: E. coli, (389) Staphylococcus (329), Streptococcus/Enterococcus (79), Salmonella (121), Pseudomonas (49), Acinetobacter (49), Aeromonas (33) and Stenotrophomonas (50) were derived from healthy animals (807) water/sewage (193), plants (36) and soil (43). More than 70% of all isolates expressed resistance to one or more antibiotics; ~30% demonstrated multidrug resistance (>3 drugs). Resistance patterns were different among environmental sources and geographic sites. Conclusions: This study has revealed significant differences in the frequency of particular antibiotic resistance markers in bacteria isolated from geographically diverse environmental sites.

11 POSTER (PRESENTED AT THE 2010 ICAAC, BOSTON, MA)

12 2. PRESS RELEASE

ANTIBACTERIAL RESISTANCE FOUND IN NON-DISEASE-CAUSING BACTERIA, COULD SERVE AS AN EARLY WARNING OF DRUG RESISTANCE

BOSTON – September 13, 2010 – The Alliance for the Prudent Use of Antibiotics (APUA) today reported that an international surveillance system it established to track antibacterial resistance has found moderate to high frequencies of drug resistance in non-disease causing bacteria (commensals) of animals and environmental sources at diverse sites across the globe.

The findings, announced at the annual Interscience Conference on Antimicrobial Agents and Chemotherapy, mean that non-disease causing bacteria can serve as an early warning of antibiotic resistance in bacteria associated with disease in human populations, according to APUA.

“Knowledge about the antibacterial resistance arena is expanding greatly. Our findings mean that disease-causing bacteria can become resistant to antibiotics by interacting with commensals bearing resistance and not necessarily just with antibiotics,” said APUA President Stuart B. Levy.

The analysis was based on the first multi-year, systematic study to evaluate patterns of antibacterial resistance in non-disease carrying—or commensal—bacteria across diverse, global geographic sites. Drug resistance was found in commensal bacteria in Bangladesh, Georgia, India, South Africa, South Korea, Turkey, Uganda, and Vietnam. These data support pilot studies in the American and European continents which show resistance in non-disease-causing bacteria.

In 2009, the U.S. formulated a national strategy to counter biological threats, in part, by building international capacity to collect and detect infectious diseases threats worldwide. Since then, APUA, with the support of several private and governmental partners, has been working to establish an extensive international surveillance system to better understand the sources of the threat of antibiotic resistant organisms and potential interventions.

Rep. Stephen Lynch, of Massachusetts, vigorously endorsed APUA’s efforts, noting, “By tracking antibiotic resistance on a global scale, APUA is helping our nation monitor disease-resistant superbugs outside our borders to minimize the threat of dangerous, drug resistant bacteria to national and global security. The APUA is tackling one of those mounting global threats that for many governments remains below the radar screen at our collective peril.”

The new study was based on surveillance of commensal bacteria from a wide variety of animals, plants, water, and soils. Examination of more than 1000 isolates has identified substantial frequencies of resistance, with significant frequencies of multi-drug resistance and varying patterns of particular antibiotic resistance markers from geographically diverse environmental sites.

13 Commensal bacteria help keep humans healthy by helping to digest foods and acquire nutrients such as vitamins B and K, encouraging the immune system to develop, and preventing the colonization of bacterial pathogens that cause disease. Antibiotic resistance most often arises from the interaction of microorganisms with antibiotic substances, frequently through the misuse and overuse of antibiotics in humans, animals, and agriculture.

APUA, the Alliance for the Prudent Use of Antibiotics (www.apua.org), founded in 1981,is the leading, independent non-governmental organization with an extensive international field network dedicated to preserving the power of existing antibiotics and increasing access to needed agents. APUA conducts a multidisciplinary research program to provide evidence to inform and shape public policy. With chapters in over 60 countries, APUA works to improve antibiotic access and use with the goal of ensuring effective infectious disease treatments for generations to come.

14 15 3. DEVELOPMENT AND IMPLEMENTATION OF A ROBUST GEO-TEMPORAL USER INTERFACE A geo-temporal user interface has been designed that uses Google Maps. This feature displays all of the sites from where specimens were collected around the world. Additionally, each marker point contains information about the specimen site, strains collected, and links to information about those strains in the database. As an example, the map below depicts data on our current Bangladesh isolates, overlaid on a Google Map. Markers on the map identify each isolate that has GPS coordinates and clicking the marker reveals more information about that isolate, such as the collection date, organism, and testing data from the database.

Fig 1: GPS mapping of Bangladesh isolates

16 17 4. FIG. DEVELOPMENT AND IMPLEMENTATION OF A ROBUST GEO-TEMPORAL USER INTERFACE