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Ó 2013 International Association for Ecology and Health Original Contribution Tracking Pathogen Transmission at the Human–WildlifeInterface: Banded Mongoose and Escherichia coli R. Pesapane,1 M. Ponder,2 and K. A. Alexander1,3 1Department of Fish and Wildlife Conservation, Virginia Tech, Virginia Polytechnic Institute and State University,Fish and Wildlife Conservation, 152 Cheatham Hall (0321), Blacksburg, VA 240612Department of Food Science and Technology, Virginia Tech, Blacksburg, VA3CARACAL: Centre for Conservation of African Resources Animals, Communities and Land Use, Kasane, Botswana Abstract: A primary challenge to managing emerging infectious disease is identifying pathways that allow pathogen transmission at the human–wildlife interface. Using Escherichia coli as a model organism, we eval- uated fecal bacterial transmission between banded mongoose (Mungos mungo) and humans in northern Botswana. Fecal samples were collected from banded mongoose living in protected areas (n = 87, 3 troops) and surrounding villages (n = 92, 3 troops). Human fecal waste was collected from the same environment (n = 46).
Isolates were evaluated for susceptibility to 10 antibiotics. Resistant E. coli isolates from mongoose were compared to human isolates using rep-PCR fingerprinting and MLST-PCR. Antimicrobial resistant isolates were identified in 57 % of the mongoose fecal samples tested (range 31–78% among troops). At least one individual mongoose fecal sample demonstrated resistance to each tested antibiotic, and multidrug resistance was highest in the protected areas (40.9%). E. coli isolated from mongoose and human sources in this study demonstrated an extremely high degree of genetic similarity on rep-PCR (AMOVA, FST = 0.0027, p = 0.18)with a similar pattern identified on MLST-PCR. Human waste may be an important source of microbial exposure to wildlife. Evidence of high levels of antimicrobial resistance even within protected areas identifies an emerging health threat and highlights the need for improved waste management in these systems.
Keywords: zoonotic, Escherichia coli, banded mongoose, human waste, emerging infectious disease, antibiotic reservoirs (Jones et al. ). Zoonotic disease is also anincreasing threat to humanity globally, crossing cultural As human populations grow and transform landscapes, and socioeconomic lines with developed and developing contact with wildlife concomitantly increases. Disease nations equally at risk (Mayer With increased travel emergence has been an important consequence of this and global interconnectedness, emergence of zoonotic escalation in interaction, with the majority of emerging disease in any one place has the potential to spark a global infectious diseases in humans now arising from wildlife pandemic, as seen with sudden acute respiratory syndrome(Dye and Gay ) and swine flu (H1N1 influenza)(Neumann et al. ). Indeed, some of the most devas- Correspondence to: K. A. Alexander, e-mail: [email protected] R. Pesapane et al.
tating and persistent human pathogens can be traced to Recent studies demonstrate the utility of Escherichia zoonotic origins, (i.e., human immunodeficiency virus/ coli as a model for examining transmission of fecal acquired immunodeficiency syndrome (Hahn et al. ) microorganisms between humans and wildlife (Singh and influenza (Schoub et al. Chapman et al. ; Skurnik et al. Pathogen transmission from humans to wildlife is also Goldberg et al. , ; Rwego et al. an important emerging threat, increasing wildlife conser- Farnleitner et al. Szekely et al. ). E. coli is a vation and management challenges (Deem et al. For bacterium found in the gastrointestinal tract and feces of example, emergence of human metapneumovirus morbidity all mammals. While the majority of E. coli are important and mortality in mountain gorillas (Gorilla beringei berin- commensals, some strains can be pathogenic (e.g., gei) in Rwanda is associated with human–gorilla contact Enterotoxigenic E. coli) in humans, domestic animals, and related to ecotourism activities (Palacios ). Similar wildlife. For example, E. coli is one of the leading causes studies demonstrate the increasing threat of human infec- of diarrheal-related deaths worldwide (Gaedirelwe tious diseases to non-human primate species (Nizeyi et al.
Drummond Stecher et al. ). While there are a ; Graczyk et al. ), elephants (Michalak et al. number of limitations to the use of E. coli (Desmarais sea birds (Steele et al. and reptiles (Wheeler et al.
et al. as a model organism, it is still regarded as ). These examples illustrate the bidirectional potential the global standard for detection of fecal contamination, for pathogen transmission at the human–wildlife interface.
indicating an increased possibility for the presence of Human modification of the environment is seen as a enteric pathogens.
primary driver of the emergence of zoonotic disease, pro- In northern Botswana, humans live in close association viding the opportunity for direct and indirect contact be- with a variety of wildlife species. One species in particular is tween humans and wildlife and increasing pathogen the banded mongoose (Mungos mungo). Banded mongoose exposure and transmission potential (Mayer ; Deem occur across protected and unprotected landscapes with et al. These changes can induce immediate as well as significant contact with humans in this system, coupled long-term effects on pathogen transmission dynamics, with a preference for denning and foraging in anthropo- modifying genetic and biological characteristics, biophysical genic features (Alexander et al. ). Banded mongoose elements, ecological dynamics, and socioeconomics, as well also utilize garbage resources and will search for insects in as host(s)–pathogen interactions (Smolinski et al. fecal waste (including human) found in the environment.
Human fecal waste is an anthropogenic disturbance that This species is also infected with two important emerging persists across human-occupied landscapes, both in devel- pathogens: the globally important zoonotic pathogen, lep- oped and developing nations, serving as an important tospirosis (Jobbins et al. ) and the newly discovered source of environmental pathogens (Cilimburg et al. ; Mycobacterium mungi, a member of the Mycobacterium Griffin et al. ). Sewage sludge and refuse hosts an array tuberculosis complex (Alexander et al. It is unclear at of pathogenic bacteria, viruses, protozoa, and helminthes present if M. mungi has the potential to infect humans or that can lead to outbreaks of disease (Hanks ; Burge other wildlife in the system. Banded mongoose's contact and Marsh ; Arthurson ). Contamination of with other wildlife and humans makes this species an ideal drinking water by sewage effluent is a recurring cause of candidate for evaluating microbial exchange at the human– gastroenteritis leading to morbidity and mortality world- wildlife interface.
wide (D'Antonio et al. ; Ashbolt This is espe- We determined the level of antimicrobial resistance of E. coli isolates collected from feces of banded mongoose populations often lack regulated waste management and occurring across protected and unprotected areas in sanitation infrastructure. While environmental fecal waste northern Botswana. We then evaluated the genetic rela- may be an important source of pathogen exposure for both tionships between antibiotic-resistant E. coli isolates from wildlife and humans, we still have a limited understanding mongoose and isolates from human fecal waste. We used of the complex process of pathogen spillover between these results to examine the potential for human fecal waste wildlife and humans. The relative infrequency of pathogen to act as a source gastrointestinal pathogen exposure for spillover events limits our ability to evaluate the complexity wildlife species living in both protected and unprotected of interacting and cascading factors driving this process.

Tracking Pathogen Transmission located within the Chobe National Park. In this region,there are no commercial chicken or livestock production systems and agriculture is, therefore, expected to contributeminimally to the accumulation of antibiotic resistance in This study was conducted in Chobe District in northern the region. In this same area, we have established a long- Botswana along the Chobe River, which traverses the Chobe term ecological study of banded mongoose (Mungos mun- National Park and the associated townships of Kasane and go) in both protected and unprotected land areas (Alex- Kazungula (Fig. ). Chobe District is 20, 800 km2 in size ander et al. ).
with a relatively low population of *23,387 people (Cen-tral Statistics Office BG largely restricted to villages surrounding the Chobe National Park. The rest of the landarea in the District is designated as protected (e.g., national The banded mongoose host is a small diurnal viverrid, parks, wildlife management areas, and forest reserves).
group-living, communal breeder, and a natural inhabitant Chobe District has rich and diverse wildlife resources.
of wildlife areas in proximity to permanent water. They Residential and tourism development occur near the pro- occur in groups or troops that can number between 8 and tected area network, as does a limited amount of subsis- 55 individuals and frequently cohabit with humans in tence agriculture. A few permanent tourism facilities are towns where they scavenge in human waste and feces.
Figure 1. Map of the study site. In northern Botswana, mongoose troops were sampled (triangles) in Chobe District in protected areas (Chobe National Park and Kasane Forest Reserve, hatched outline, troops living in the protected area are denoted with ‘‘+'') and towns of Kasane and Kazungula. The Chobe River flows through the Park to industrial, commercial, residential developments (gray circles) and commercial agricultural areas (light gray polygons). The sewage treatment facility is located in the Kazungula area (stripes). Tourism facilities are found within the developed and protected areas (stars). Sources of human waste such as feces and sewage (squares) found in the environment near study troops were sampled as well as the sewage treatment facility.
R. Pesapane et al.
Banded mongoose have a low-skew reproductive strategy; multiple females breed and there is little or no observed suppression of breeding in subordinates (Gilchrist ).
Dispersal can be infrequent and individuals may be re- tained within their natal group and breed with relatives upon sexual maturity (Cant et al. In our long-term study site, at least one or two animals in each study troop are radio-collared and monitored regularly with spatially referenced habitat, behavioral, clinical, and other relevant ecological data collected on the study population (Laver et al. Home range data were used to identify mongoose troops as occurring in protected areas or in surrounding villages. This study was conducted under a permit from the Botswana Ministry of Environ- ment, Wildlife, and Tourism and with approval of the Virginia Tech's Institutional Animal Care and Use Com- mittee (Protocol number 07-146-FIW and 10-154-FIW).
Fecal and Environmental Sample Collection Fecal samples were collected from banded mongoose living in protected areas (n = 87, 3 troops named CCL+, CGL+, HP+ with ‘‘+'' denoting protected area) and surrounding villages (n = 92, 3 troops, KUB, CSL, SEF, Fig. ). With the exception of eight individuals in one troop in the protected area (HP+), all troops had some level of range overlap with human populations. The CCL+ and CGL+ troop homerange included tourism facilities within the protected areas where some contact with humans and development would be experienced but at a much lower level than in the village troops. The HP+ troop occurred in a part of the protected area where there was no human infrastructural development or residences other than a dirt road. Feces from these 6 banded mongoose troops were collected following morning latrine behavior. Banded mongoose defecate individually at the same time period and in the same general location each morning upon leaving their den; it is, therefore, possible to collect fecal samples from individual mongoose in eachtroop without replication during that latrine event. In order to determine the required interval between troop resam- pling, such that each fecal sample could be considered an independent sampling point for the troop, we measured gut passage time. Colored dye was added to the diet of four captive mongoose (owned by CARACAL Biodiversity Center) and minimum gut fecal passage time was deter- mined to be approximately three days. Based on these re- sults, three sampling efforts were attempted at intervals of not less than 2 weeks to ensure independence of sampling Tracking Pathogen Transmission events. In this study, we pooled across sampling events,considering each fecal sample an independent sample given established clearance time and latrine behavior. Fecal sam- ples were collected in sterile tubes shortly after defecation and frozen at -20°C within 8 h of collection until pro- cessing (Roesch et al. Mongoose fecal samples aresmall and friable with large amounts of insect material,making sectioning of mongoose feces difficult. Mongoose also consume soil while foraging and thus sampling con-straints were relaxed and the whole fecal sample was taken and used without regard to exterior or interior components.
Human feces were collected within mongoose home ranges from bush latrines (e.g., on ground defecation) andsewage sludge or wastewater leakage identified in the study area (Fig. ). Only sections of human feces that did notcome in contact with soil were sampled to avoid environ- mental contamination. Additional samples of sewage were collected from the local wastewater treatment facility. In thecase of liquid samples such as wastewater, sterile gauze was swirled in the source to catch particulates along with a water sample in the same sterile tube. Soil samples werecollected from areas undisturbed by apparent human or animal waste and were used to provide a limited assessment of natural levels of antimicrobial resistance in the envi- Fecal and wastewater samples were homogenized in buf- fered peptone (0.1% BPW Sigma-Aldrich, St. Louis, MO USA), serially diluted 10-fold, and spread plated ontoMacConkey Agar (Becton Dickinson Company, FranklinLakes, NJ, USA) plates. After 24 h at 37°C, six putative E. coli colonies were picked from each mongoose fecalsample. Sewage waste represented an array of individual sources; therefore, 15 colonies were selected from human fecal samples to be used in subsequent analyses. All putative E. coli colonies were grown in tryptic soy broth (BectonDickinson Company) for further DNA extraction and antibiotic susceptibility testing.
DNA was extracted from putative E. coli colonies using standard methods. Genera-specific primers for malB were used to confirm isolates as E. coli as previously described(Rekha et al. ). E. coli isolates were then tested for susceptibility to 10 antibiotics: ampicillin (10 lg), chlor- amphenicol (30 lg), ciprofloxacin (5 lg), doxycycline (30 lg), gentamycin (10 lg), neomycin (30 lg), strepto-mycin (10 lg), tetracycline (30 lg), sulfamethoxazole–

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Hierarchical Analyses of Molecular Variance (AMOVA) for E. coli Isolates Collected from Humans and Mongoose in the Chobe Region of Botswana.
Variance component Observed partition Mongoose and Humansa HP+ versus CSL, CGL+, SEF, KUB, CCL+ Isolates were analyzed from banded mongoose troops (n = 6) and human bush latrine sites as well as sewage effluent in the environment within the homeranges of study mongoose. Banding patterns of bacterial genotypes were converted to binary variables, identified and scored using the ‘‘bandmatch''procedure in the FPQuest, version 4.5 program (BioRad). The program Arlequin, version 3.1 (Excoffier et al. ) was used to perform AMOVA.
aHuman samples consisted of feces (n = 5, 41 isolates) and sewage (n = 14, 46 isolates).
Figure 2. Occurrence of resis- tant E. coli isolates in fecal samples collected from individ- ual banded mongoose by troop in Chobe District, Botswana.
Mongoose troops occurred in both the protected areas (+) and surrounding villages. All error bars represent 95% confidence trimethoprim (SXT) (25 lg), and ceftiofur (30 lg) (BBL, method with breakpoints indicated by the Clinical Labo- Becton Dickinson Company). Antimicrobials were chosen ratory Standards Institute (CLSI) In this modifi- based on their local availability and on previous studies on cation, a McFarland standard was not used to standardize microorganism transmission from ecotourism (Rolland turbidity. Instead, quality results were ensured in several et al. Skurnik et al. ; Wheeler ways. First, E. coli ATCC 25922 culture, a quality assurance et al. ). Ceftiofur was added to the panel as it is only indicator strain recommended by CLSI, was treated iden- used in livestock and poultry production and was not tically to sample isolates and results remained consistently available in the study area. Antibiotic susceptibility was within required limits for each test batch. Second, measured using a field-modified Kirby-Bauer disk diffusion any isolates demonstrating intermediate resistance were

Tracking Pathogen Transmission Figure 3. Prevalence of antibiotic resistance among total E. coli isolates collected from banded mongoose (n = 253), and human fecal waste (n = 75) in Chobe District Botswana. Resistance data were also extracted from routine laboratory microorganism susceptibility assessments conducted at the Kasane Primary Hosptial within the study site (2007–2011), on pre-established panels of antibiotics. Isolates would originate from patients from all villages in the District. Not every isolate was subjected to the same panel of antibiotics at the hospital and so the number of isolates screened for each drug varied. Antibiotics are listed as abbreviations followed by dosage (lg) in numbers, the number of human isolates tested at the hospital are noted in brackets (e.g., AM10 = ampicillin [n = 420], Te30 = tetracycline [n = 315], D30 = doxycycline, SXT = sulfamethoxazole-trimethaprim [n = 375], S10 = streptomycin, C30 = chloramphenicol [n = 256], XNL30 = cef- tiofur, GM10 = gentamycin [n = 521], N30 = neomycin, and CIP5 = ciprofloxacin). Black bars denote antibiotics that were not evaluated at health facilities in Chobe District during the assessment period.
accepted as susceptible for conservative reporting. Third, lates were evaluated. Genetic relationships were determined since stage of growth can affect resistance, all cultures tested by repetitive polymerase chain reaction (rep-PCR) and were >24 h of age (which corresponds to stationary phase) multi-locus sequence-type polymerase chain reaction in tryptic soy broth at the time of testing. We define (MLST-PCR). In summary, rep-PCR reactions were per- ‘‘multidrug resistant'' isolates as those resistant to 3 or formed in 25 lL reactions using 2 lL DNA template (of more of the antibiotics screened for susceptibility.
standardized concentration 100–300 ng/lL) added to 2.5 U Human antimicrobial resistance data for the region were Phire Taq (Finnzymes) and 19 of 59 Phire Taq Buffer, extracted from the local primary hospital (all data were 1 lM of BOX AIR primer (50-CTACGGCAAGGCGACG anonymized, May 2007–July 2011). The research was con- CTGACG-30) and an additional 1.5 mM of MgCl2. Cycling ducted under permit from the Ministry of Health in Botswana was performed using a BioRad MyCycler (Bio-Rad) at 98°C and approval from the Virginia Tech Institutional Review for 3 min, followed by 35 cycles of 98°C for 1 min, 64°C Board (IRB# 11-573). Data were derived from routine testing for 8 min, and 71°C for 1 min. Final extension was 15 min of clinically isolated microorganisms from various fluids and at 71°C. PCR fragments (2 lL DNA template of stan- tested on pre-established antibiotic panels in order to deter- dardized concentration 100–300 ng/lL) were separated by mine susceptibility and treatment regimes. In this clinical electrophoresis in a 2% agarose gel using 19 TAE buffer at setting, intermediate susceptibility is reported as resistant to 45 V for 10 h at 4–8°C and visualized. Rep-PCR fingerprint ensure the most appropriate drug treatment regime.
profiles were generated using densitometric (curve-based)genotype determination (Goldberg et al. with theFPQuest ver 4.5 program (BioRad, Hercules, CA). Genetic Genetic Profiling of Mongoose and Human Bacte- diversity of fecal E. coli isolates were assessed using analysis rial E. coli Isolates of molecular variance (AMOVA) in the Arlequin program While all human E. coli isolates were analyzed for genetic version 3.0 as previously described (Excoffier et al. ).
relatedness, only antibiotic-resistant mongoose E. coli iso- Phylogenetic relationships were built through Pearson's R. Pesapane et al.
correlation coefficient and neighbor-joining algorithms Figure 4. Phylogram of rep-PCR fingerprint analysis of E. coli c with optimization parameters identified from previously isolates obtained from human and antibiotic-resistant banded published guidelines (Goldberg et al. ).
mongoose fecal samples (n = 74 mongoose, n = 87 human).
To confirm the genetic relationships generated from Comparisons were constructed using the Pearson's correlation and neighbor-joining algorithms. Values alongside branches correspond fingerprint analysis, a subset of isolates representing each to % confidence generated from 1,000 bootstraps. Isolates that did phylogenetic clade were amplified and sequenced for not visibly differ were not included. Tree is rooted with the MLST-PCR to construct a tree based on composite geno- fingerprint profile of E. coli K12 from the EcMLST database.
types at different loci in a single isolate (Archie et al. Seven housekeeping loci (aspC, clpX, fadD, icdA, lysP, mdh,and uidA) were PCR amplified and sequenced as previously production. Multidrug resistance (7.7–50%) was identified described (STEC ). Resulting DNA sequences were among a number of samples (Table Prevalence of mul- aligned and phylogenetic analysis was performed using the tidrug resistance among sampled mongoose feces was sig- program Geneious 5.4.2 (Biomatters) (Drummond nificantly different between sampled troops (p = 0.01, v2 Dendrograms were constructed using neighbor joining and test). Troop KUB had the lowest level of multidrug resistance pairwise similarity matrix methods. The tree was rooted among individual fecal samples (7.7% with 95% CI 0.2–36%) with composite sequence of the E. coli K12 from the while troops SEF (50% with 95% CI 1.3–98.7% n = 2) and EcMLST database ().
CGL+ (40.9% with 95% CI 20.7–63.6% n = 22) had thehighest levels. There were no significant differences in resis- tance to two or fewer antimicrobials (p = 0.11) among fecalsamples from study troops. Troop CGL+, located within the Fisher's exact and v2 tests were used to compare antibiotic protected area, did not have the highest prevalence of resis- resistance levels among groups using the statistical software tant isolates overall (Fig. but was the only troop where at JMP, version 8.0 (SAS Institute). Exact binomial 95% least one fecal isolate was resistant to each of the antibiotics confidence intervals were calculated using the Epitools screened (Fig. Table Troop HP+ had the highest package in R (Aragon and Enanoria prevalence of antibiotic-resistant isolates although thesewere resistant to ampicillin only (Fig. Table ).
E. coli were isolated from 41.3% (n = 46, feces and sewage) of human fecal waste samples assessed [human E. coli Isolation and Antibiotic Susceptibility feces found directly in the environment: (n = 5), sewagespill (n = 8), and sewage treatment pond samples (n = 6)].
E. coli were isolated from 49% (n = 179) of sampled mon- From these samples, 77 E. coli isolates were screened for goose feces. No E. coli isolates were obtained from soil antibiotic susceptibility and 80.3% (95% CI 69.5–88.5%) samples collected from environments expected to be free of were found to be resistant to at least one antibiotic. Of human or animal fecal contamination (n = 13). In mon- human clinical samples screened at the local hospital, goose fecal samples where E. coli were isolated, 57% dem- 89.9% (95% CI 87–92% for all years) of isolated micro- onstrated antimicrobial resistance. Resistance was identified organisms (various sources and bacteria species) were among individuals in all sampled troops (31–78%, Table resistant to at least one antibiotic. Human and banded No significant differences were observed in the prevalence of mongoose E. coli isolates collected in this study demon- antimicrobial resistant E. coli isolates across fecal sampling strated resistance to the same drugs as microorganisms events for each respective troop (p > 0.05) and data were isolated from patients at the Kasane Primary Hospital pooled by troop for analysis. At least one or more mongoose (Fig. Overall, resistance in banded mongoose was lower fecal samples demonstrated resistance to each antibiotic than that found in the human population.
tested in this study. Mongoose fecal isolates were mostcommonly resistant to ampicillin (AM10), followed by Repetitive BOX-PCR doxycyline (D30), tetracycline (Te30), streptomycin (S10),SXT, and chloramphenicol (C30) (Fig. Table ). A low Using AMOVA, significant variation was identified among number of isolates (n = 5) demonstrated resistance to cef- sampled mongoose feces (FST = 0.02, p = 0.01, Table ), tiofur (XNL30), an antibiotic used in livestock and poultry with the majority of the variation identified at the indi-

Tracking Pathogen Transmission vidual fecal sample level (98% of observed variation) rather living in an unmodified habitat (HP+) were indistin- than at the troop level (2% of the observed variation).
guishable genetically from those troops living in association However, E. coli strains isolated from the mongoose troop with humans in villages or at lodges in protected areas

R. Pesapane et al.
Figure 5. Phylogenetic tree based on E. coli composite sequences from seven housekeeping genes (uidA, mdh, lysP, icdA, clpX, aspC, and fadD) (n = 23). Values alongside branches correspond to % confi- dence generated from 1,000 boot- were built from seven consensus trees using Tamura-Nei distance model and neighbor-joining algo- rithms, and 70% support threshold.
The tree is rooted with composite sequence of E. coli K12 from the Scale is based on the number of base substitutions per site.
(CSL, CGL+, SEF, KUB, CCL+, FST = 0, p = 0.64, rep-PCR analysis revealed very slight genetic divergence Table When comparing humans and mongoose isolates, (maximum < 0.10 base substitutions per site) between a greater level of genetic diversity of E. coli was found isolates from banded mongoose and human fecal waste within each species (99.75% of the observed variation) than (Fig. E. coli isolated from banded mongoose and sources among species (0.25% of the observed variation). Mon- of human fecal waste consistently clustered together, goose and human associated E. coli demonstrated an forming one monophyletic tree. Genetic relationships extremely high degree of genetic similarity (FST = 0.0027, between isolates interpreted through branching patterns were not identical between fingerprint- and DNA sequence- Antibiotic-resistant isolates from all troops of mon- based analyses in this study, even when using optimization goose consistently clustered with those of humans and parameters published by previous studies (Goldberg et al.
human fecal waste in rep-PCR analysis (Fig. ). Clades containing exclusively mongoose or human isolates werenot observed. Mongoose isolates did not cluster signifi-cantly by troop, but instead were highly intermixed with isolates from human sources.
Using molecular strain comparisons, E. coli isolated fromfeces of banded mongoose troops demonstrated an extremely high degree of genetic similarity to isolates MLST-PCR performed on a subset of E. coli isolates obtained from human fecal waste in these environments.
(n = 23) representing each of the clades observed in This was identified among troops with home ranges in both Tracking Pathogen Transmission protected and unprotected land areas. Other studies have greater diversity of drug resistance was identified and a applied similar molecular techniques demonstrating the higher prevalence of multidrug resistance occurred (Fig. potential for microorganism exchange to occur between Table ). Ampicillin resistance can be naturally acquired humans, wildlife, and livestock sharing the same land area.
from environmental sources (Martinez ). Patient iso- Comparisons between populations focused on the direct lates from the local hospital were also frequently resistant to collection of fecal material from human volunteers living in ampicillin (Fig. ). Other studies have observed a gradient these environments (Goldberg et al. ; effect of antimicrobial resistance among environments and ). While these examinations have provided substantial animals in relation to human proximity (Skurnik et al.
insight into the occurrence of microbial exchange at the Allen et al. ; Wheeler et al. ). Our study human–animal interface, the specific pathways where gas- design did not allow for this type of evaluation; however, trointestinal microbial transmission might occur remain our limited data suggest that this might be an important elusive. In this study, we focused our sampling strategy on area of future work.
environmental sources of human fecal waste and compared Multidrug resistance is increasingly identified as an isolated E. coli to that of isolates collected from mongoose important global health threat, involving all major micro- living in these same areas.
bial pathogens and antimicrobial drugs. Multidrug resis- Recent studies identify a high degree of genetic simi- tance was identified among isolates collected from troops larity among E. coli strains globally, regardless of human or living in proximity to humans, both in the National Park animal origin (Clermont et al. and suggest that and in village areas. Of these, CGL+ (Park troop) had the microbial exchange events implied by molecular genetic highest level, with half of all sampled mongoose demon- techniques should be viewed conservatively. However, in strating multidrug resistance among isolates. In some this study, we did find significant variation in E. coli by troops, multidrug-resistant isolates were aggregated to a species (human and mongoose, respectively). Our results few individual mongoose fecal samples (e.g., KUB, provide direct support for the possibility that direct human Kazungula Village), but in the CGL+ troop, multidrug fecal contamination of the environment is an important resistance was widespread across individuals and isolates potential source of microbial exposure and transmission to (40.9%, 95% CI 20.7–63.6%). This troop commonly den- wildlife living in these areas. This transmission appears to ned in the soak away of the septic tank servicing the occur even in protected wildlife areas.
employee accommodations and foraged around employee Antimicrobial resistance is an important marker for living quarters, including eating food remains from dishes human-source bacteria (Kabler et al. ) and previous left outside. Humans have also been observed feeding raw studies identified antimicrobial resistance in wild popula- meat waste from commercially produced chickens to tions living in association with human populations (Cole mongoose at this lodge, potentially contributing to the et al. Skurnik et al. ; Goldberg et al. ; levels of drug resistance in this troop. Antibiotic resistance Wheeler et al. ). In this study, we found to ampicillin and tetracycline has been identified in strains high levels of antimicrobial resistance including multidrug of Salmonella at commercial chicken farms in Botswana resistance among sampled mongoose across all sample (Gaedirelwe ) and may be an important source of troops irrespective of land use (protected and unprotected).
resistant microbiota to species fed raw animal byproducts.
Important in evaluating the significance of the anti- Alternatively, this resistance could be present in the human microbial resistance identified in wildlife is evaluating the population because of consumption of meat with antibiotic occurrence in the resident human population. In this study, residues. Wildlife could then be subsequently exposed resistance to the same antimicrobials were identified among through human fecal waste. Either or both of these mongoose isolates (although lower levels) as that identified mechanisms may have contributed to the low occurrence of in study-derived human E. coli isolates and patients pre- resistance to ceftiofur (XNL30, Table a broad-spectrum senting to the Kasane Primary Hospital (2007–2011, veterinary antibiotic not available in the study area. All of Fig. Most isolates obtained from the mongoose troop the study troops, with the exception of HP + , utilized living in a natural area of Chobe National Park (HP+) were garbage resources and would have access to such waste resistant to only ampicillin, with one isolate demonstrating albeit at varying levels depending on the facility. Uncon- additional drug resistance. This result was in contrast to the trolled and uncontained waste disposal provides a ubiqui- other sample troops living in contact with humans where a tous non-seasonal food resource in both peri-urban R. Pesapane et al.
environments and protected areas, the latter in association bial exchange and antibiotic resistance accumulation in with ecotourism enterprises (Rolland et al. While mongoose may extend through food webs. Mongoose are ecotourism developments are important for conservation eaten by a large number of avian, reptile, and mammalian and economic growth, associated human waste (both gar- predators including domestic dogs in this system. Thus, the bage and feces) remains a primary negative consequence. It cascading effects of exposure of wildlife species to human is unclear how garbage utilization may contribute to waste-associated microbes can impact an array of suscep- microbial exchange and accumulation of antimicrobial tible species across an ecosystem and in turn increase resistance. Given the increasing nature of this anthropo- human exposure, coupling humans and natural systems in genic landscape modification, it is a crucial area for con- complicated ways.
tinued research.
Human fecal waste and garbage are widespread across There was a high level of resistance among human many environments and are an increasingly global threat to isolates collected in this study and those tested at the pri- animal and public health (Rushton Cantalupo et al.
mary hospital. This may appear surprising at first given This phenomenon extends to protected areas, where poverty and expectations of diminished access to health increasing pressure to develop economic enterprises based care and pharmaceuticals that is assumed for much of on tourism brings people and their waste even to remote Africa. However, antibiotics are widely available to patients areas of a protected area network. In order to control the visiting government hospitals in Botswana, where health threat of potential pathogen transmission, closed sewage care is largely free, as well as local pharmacies where few systems should be required. Feeding of poultry and live- controls on the dispensing of antibiotics are identified.
stock products from kitchen waste should be actively pro- Decreased regulation of antibiotic use and access is an hibited to decrease the potential for transmission of important contributor to the epidemic spread of resistance resistant bacteria. Waste receptacles need to be completely (Okeke et al. , and has likely contributed to enclosed and ‘‘wildlife-proofed'' to discourage wildlife the occurrence of increased microbial resistance in this access and exposure.
human population.
Transmission of pathogens through fecal waste is likely A number of study limitations could have influenced to be amplified in places such as Africa, where sanitation our results. For example, the potential for contamination of and water treatment facilities are often poor, and fecal sewage samples with non-human fecal material and soil is a contamination of the environment and limited surface possibility. It is likely, however, that such contamination, if water resources represents a growing problem. Common it occurred, would be present at exceptionally low levels water resources may represent an important exposure relative to the volume of human sewage. Sewage and direct mechanism, increasing the potential for bidirectional fecal contamination of the only surface water in the region microbial traffic and increased health risks to both humans creates another vehicle for microorganism transmission and wildlife living in these systems. The intimate connec- between humans and wildlife and could be an important tions between human and animal populations are mechanism of E. coli exchange between humans and increasingly evident, as is the need to better understand mongoose. Antibiotics from local human use may accu- these system coupling points where human and animal mulate in both the soil (Tolls ) and contaminated health are interlinked.
surface water (Kummerer potentially increasing thespread of antibiotic resistance.
We would like to thank the Botswana Ministry of Health andthe Ministry of Environment Wildlife, and Tourism for Our study suggests that environmentally associated human permission to conduct this study. We thank M. Vandewalle fecal waste may serve as an important source of microbial and P. Laver for their invaluable assistance in this study and transmission to mongoose. Furthermore, widespread E. Hallerman and J. Fox for comments and assistance on accumulation of antimicrobial resistance is identified this manuscript. This project was funded under the among mongoose even in protected areas considered to be National Science Foundation Coupled Human Environmental relatively free from human impacts. The impact of micro- Systems Award CNH#1114953, Morris Animal Foundation Tracking Pathogen Transmission Award # D10Z0-828A, and the WildiZe Foundation. The Desmarais TR, Solo-Gabriele HM, et al. (2002) Influence of soil National Science Foundation S-STEM Program (DUE-0850198) on fecal indicator organisms in a tidally influenced subtropicalenvironment.
provided partial financial support for R. Pesapane.
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