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
Global response to antibiotic access and resistance:
Central &
South America
Costa Rica
Cuba
Dominican
Republic
El Salvador
Guatemala
Honduras
Mexico
Nicaragua
Panama
Argentina
Bolivia
Brazil
Chile
Colombia
Ecuador
Paraguay
Peru
Uruguay
Venezuela
Europe
Azerbaijan
Austria
Belarus
Bulgaria
Croatia
Georgia
Greece
Italy
Kazakhstan
Kyrgyzstan
Moldova
Poland
Romania
Russia
Serbia
Montenegro
Spain
Sweden
Turkey
Ukraine
United Kingdom
Middle
East
Abu Dhabi
Lebanon
Africa
Ethiopia
Gambia
Ghana
Kenya
Mozambique
Namibia
Nigeria
Senegal
South Africa
Tanzania
Uganda
Zambia
Asia
Bangladesh
China
Fiji Islands
Indonesia
India
Nepal
Pakistan
Philippines
South Korea
Taiwan
Vietnam
Australia
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.
3
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
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.
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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)

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.
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Table 1. Species and country sites of ISRAR isolate collections
ISRAR ISOLATES
Aeromonas
Streptococcus/
Enterococcus
*Other
20
15
24
0
0
0
0
0
2
11
42
46
16
0
49
11
24
20
24
23
17
0
81
199
0
20
0
0
20
0
144
132
2
26
10
3
0
30
25
188
59
61
14
0
0
0
0
45
9
Vietnam
193
33
21
35
32
24
21
15
8
4
Total
1932
516
479
266
116
139
114
69
144
89
Total # of
Isolates
Received
Bangladesh
E. coli
Staphylococcus
Salmonella
Acinetobacter
Pseudomonas
Stenotrophomonas
131
24
0
2
23
23
Georgia
96
69
25
0
0
India
430
135
128
3
South Africa
172
22
31
South Korea
350
30
Turkey
372
Uganda
Origin
* Hafnia, Enterobacter, Providencia, Proteus, Citrobacter, Klebsiella, Micrococcus, Leuconostoc, Kluyvera, Aerococcus
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Table 2: ISRAR isolate sources
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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:
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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
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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 50 th 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)
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.
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POSTER (PRESENTED AT THE 2010 ICAAC, BOSTON, MA)
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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.
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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.
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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
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4. FIG. DEVELOPMENT AND IMPLEMENTATION OF A ROBUST
GEO-TEMPORAL USER INTERFACE
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