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This research was undertaken in accordance with the ethical and legal requirements of the Ministry of Environment and Natural Resources of Mexico (SEMARNAT), and was authorized by permit number SGPA/DGVS/07120/09. The University of Washington Institutional Animal Care and Use Committee approved the use of domestic dogs as part of our research team (protocol 2850-08).
We collected fecal samples from March to June 2010 during surveys of free-ranging populations of howler monkeys (Alouatta palliata) in Los Tuxtlas, and of howler monkeys (Alouatta palliata), spider monkeys (Ateles geoffroyi), felids [jaguars (Pantera onca), pumas (Puma concolor), jaguarundis (Puma yagouarundi), and ocelots (Leopardus pardalis)], and tapirs (Tapirus bardii) in Uxpanapa, which are both regions in the state of Veracruz, Mexico (Figure 1).
Figure data removed from full text. Figure identifier and caption: 10.1371/journal.pone.0107719.g001 Map showing the locations of fecal samples collected from primates, felids and tapirs in two study sites (Los Tuxtlas and Uxpanapa) in south-east Mexico.Villages are indicated with black dots. Dark green represents mature forest, light green secondary forest, yellow pasture, light brown citric plantations, and red rubber plantations. Los Tuxtlas supervised classification map based on freely available Landsat 2011 images (source: usgs.gov). Uxpanapa supervised classification map based on SPOT5 scenes obtained from ERMEX/SEMAR (2010; source: ERMEXS, Estación de Recepción México de la Constelación SPOT/Secretaría de Marina Armada de México. 2010. SPOT5 images).
Los Tuxtlas is located near the south of the state of Veracruz. The region has a long history of human occupation, dating back over 1,000 years to the earliest Mesoamerican civilization, the Olmecs [24]. The original forest of this region has been extensively transformed into pasture and agricultural landscapes. In the northern part of the region, where we conducted our sampling (18°28′−18°39′N, 93°02′−95°18′W), only 13% of the 75,000 ha of original rainforest remains and the landscape is composed of an archipelago of different sized forest patches that vary in degree of isolation and habitat quality [25]. The human population density in the region is 108.8 inhabitants/ha [26]. Uxpanapa is located approximately 150 km south east of Los Tuxtlas (17°04′−17°31′N, 93°46′−94°49′E); it is the northern limit of the Zoque Forest, which, at over 1,000,000 ha, is the largest remaining tract of tropical rainforest in Mexico. Compared to Los Tuxtlas, the region has a recent history of human occupation, starting in the late 1960’s [27]. Population density in this region is much lower (11.6 inhabitants/ha) [26] and, although deforestation in the region has been extensive in the past 40 years, large tracks of pristine tropical rainforest are still found there, inhabited by a diverse range of animal species.
Fecal sample collection and transport: Howler and spider monkeys are almost exclusively arboreal and occupy the mid and high strata of the forest. We observed these primates while walking along forest paths, often being alerted to their presence by their vocalizations and movement. Once a group was located, the sampling team waited for them to defecate, thus confirming the origin of the sample. Felids and tapirs are far more elusive and we did not directly observe them during our surveys. Therefore, we located their fecal samples with the assistance of a trained scat detection dog [28]. Scat detection dogs are able to find samples from multiple species simultaneously across large, remote areas and have a lower sampling bias than traditional wildlife detection methods [28]–[30]. When the dog detects the scat, it signals its whereabouts to the handler by sitting a short distance away from the sample. Therefore, the samples are not contaminated by the presence of the dog. We collected fresh fecal samples in transport swabs containing Stuart transport agar (Copan Diagnostics) only if free from environmental contamination (e.g., dust, mud). We kept samples on ice packs until refrigeration, which was no later than 4 days after sample collection. Additionally, for the terrestrial mammals (i.e., felids and tapirs), we collected duplicate samples for genetic analysis by swabbing the surface of the scat with sterile foam swabs soaked in phosphate buffered saline (PBS) solution. Genetic samples were then stored either in 90% ethanol (and later dried with desiccant) or in lysis buffer, and kept in a refrigerator, freezer, or on dry-ice in the field, as available.
Genetic typing of tapir and felid samples: We used mitochondrial DNA (mtDNA) markers to confirm the species origin of the felid and tapir samples, since felid scats cannot be distinguished from each other visually, and tapir feces can be confused with horse/donkey feces. Swabbing the surface of the scat samples minimizes downstream PCR inhibitors and maximizes epithelial cell DNA [31]. DNA was extracted from the swab samples using the tissue extraction protocol of the Qiagen Tissue Kit (Qiagen Inc.). We used a 175-bp sequence of the ATP6 ribosomal subunit gene [32], [33], which lays outside of the felid numt region that renders many mtDNA markers unreliable for felids. We then established the preliminary species assignment using NCBI’s BLAST search and we further confirmed this by aligning the sequences with in-house control sequences using the MEGA5 software [34]. For further corroboration of felid species identification, we used a second molecular marker, a species-specific fragment polymorphism of a different mtDNA region amplified using the HSF21 and LTPROB13 primers [35].
Detection and isolation of antibiotic resistant strains: We re-suspended swabs in 1 mL PBS by vigorous mixing, then plated 50 µL of the suspension on Mueller-Hinton plates containing antibiotics (ampicillin, sulfamethoxazol, nalidixic acid, gentamicin, tetracycline and chloramphenicol). We also plated 50 µL of the suspension on eosin-methylene-blue (EMB) agar to check for viability of enteric bacteria. We incubated plates aerobically at 35°C for 24 h. We isolated the colonies grown on antibiotic plates on antibiotic-free medium for further analyses, including identification based on gram staining and standard biochemical techniques.
We tested susceptibility towards 8 antibiotics: ampicillin (AM), amoxicillin-clavulanate (AMC), cefotaxime (CTX), gentamicin (G), tetracycline (TE), chloramphenicol (CML), ciprofloxacin (CIP) and sulfadiazine (SUL) using the disk diffusion method (BBL disks on Mueller-Hinton agar). We interpreted the resulting inhibitory halos according to Clinical and Laboratory Standards Institute guidelines. Following Över et al. [36], we applied further antibiotic susceptibility testing to all G-resistant isolates, which included assaying a set of 12 aminoglycoside compounds designed to assess the underlying mechanism of resistance. Briefly, we used disks containing 12 different aminoglycosides (6 of them not used clinically) for typical disk-diffusion susceptibility testing. Each known aminoglycoside-modifying enzyme, and most common combinations, yield a distinct inhibitory halo profile, while a uniform reduction of the activity of all 12 is interpreted as a result of decreased permeability. Although varying levels of enzyme expression can produce atypical profiles, the method can reliably distinguish between enzyme-mediated and permeability-mediated aminoglycoside resistance, which was the main goal here.
Class I integrons are bacterial genetic elements that play a role in the acquisition and dissemination of antibiotic resistance genes. Little is known about the distribution or abundance of integrons outside of the clinical context [13]. However, evidence suggests that the prevalence of class 1 integrons is directly related to exposure to human environments [37]. Therefore, we attempted to amplify the intI1 integrase gene of class-1 integrons for all E. coli isolates using the PCR primers (intI1.F: 5′-GGGTCAAGGATCTGGATTTCG-3′; and intI1.R: 5′-ACATGGGTGTAAATCATCGTC-3′) and conditions reported in [38].
We recorded the location of each sample using a GPS unit. In Uxpanapa, we later calculated the shortest distance to the nearest anthropogenic habitat (pasture, plantation, orchard, etc. ), shortest distance to the nearest human settlement, and the number of human settlements within a 2.5 km, 5 km and 10 km radius, using a classified 2008 Landsat satellite image (1∶20,000 scale) of the study area, the ArcView GIS software (version 3.1), and the Patch Analyst 2.2 extension for ArcView [39]. We did not perform these analyses for the samples collected in Los Tuxtlas given that they all belonged to groups of howler monkeys living in forest fragments in close proximity to human settlements (within 1 km distance).
We calculated four ATBR parameters: 1) the proportion of samples resistant to at least one antibiotic (rS); 2) the number of isolates per sample that where resistant to at least one of the drugs tested (rO); 3) the total number of antibiotic resistance phenotypes detected per sample (rP); and 4) the average number of antibiotics each isolate per sample was resistant to (rA). For example, if a sample had two different isolates, an Escherichia coli and a Pseudomononas sp., and the first isolate was resistant to AM and SUL, and the second to CIP, CML and G, the resistance parameters for this sample would have been: rO = 2, rP = 5 and rA = 2.5. We excluded resistance phenotypes deemed “intrinsic” (i.e., not being selected by antibiotics: AM, AMC and CTX resistance in Pseudomonas; AM and AMC resistance in Acinetobacter; CTX resistance in enterococci), from all calculations. We included in our analysis those traits that are usually thought of as “intrinsic resistance”, such as chromosomally-encoded AmpC beta-lactamases in Enterobacter and Klebsiella, and aminoglycoside-resistance due to decreased accumulation in Pseudomonas aeruginosa, if they were not present in all our isolates of a given taxon, as this would suggest that they were selected for by antibiotics or related agents. Also, we calculated the prevalence of different ATBR for each host species, that is, the number of samples resistant to a given antibiotic. Finally, since we did not process swabs to reveal the total composition of the fecal microbiota, but only to isolate resistant organisms, we were unable to assess the resistance rate per bacterial species. However, since E. coli was present in all samples, as inferred from the characteristic metallic green hue on EMB plates, we were able to determine the prevalence of resistance in this species (Table 1).
Table data removed from full text. Table identifier and caption: 10.1371/journal.pone.0107719.t001 Antibiotic resistance prevalence (N [%]) in E. coli isolates. N represents the total number of samples analyzed.
We used a Z test for proportions (independent groups) to compare rS among howler monkey samples collected in Los Tuxtlas and Uxpanapa, among samples from different species, between terrestrial and arboreal species in Uxpanapa, and between samples collected ≤2.5 km away from humans settlements and >2.5 km away. Due to the non-parametric nature of rO, rP and rA, for samples in our study, we used a Mann-Whitney U test to compare these parameters between samples collected ≤2.5 km from a human settlement and those collected further away in Uxpanapa, and between howler monkey samples collected in Los Tuxtlas and Uxpanapa. For the samples collected in Uxpanapa, we used a Kruskal-Wallis H test to analyze the existence of overall differences in rO, rP and rA among the different study species, and a Mann-Whitney U test to conduct pairwise comparison of these parameters between the different species, as well as to compare terrestrial and arboreal species. Finally, we analyzed the relationship between rO, rP and rA parameters and the distance to the nearest human settlement and pasture in Uxpanapa using linear regression analyses. All analyses were carried out in SPSS Version 20.0, considering p<0.05 as significant.
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