article: Although several studies have associated Mycobacterium ulcerans (MU) infection, Buruli ulcer (BU), with slow moving water bodies, there is still no definite mode of transmission. Ecological and transmission studies suggest Variable Number Tandem Repeat (VNTR) typing as a useful tool to differentiate MU strains from other Mycolactone Producing Mycobacteria (MPM). Deciphering the genetic relatedness of clinical and environmental isolates is seminal to determining reservoirs, vectors and transmission routes. In this study, we attempted to source-track MU infections to specific water bodies by matching VNTR profiles of MU in human samples to those in the environment. Environmental samples were collected from 10 water bodies in four BU endemic communities in the Ashanti region, Ghana. Four VNTR loci in MU Agy99 genome, were used to genotype environmental MU ecovars, and those from 14 confirmed BU patients within the same study area. Length polymorphism was confirmed with sequencing. MU was present in the 3 different types of water bodies, but significantly higher in biofilm samples. Four MU genotypes, designated W, X, Y and Z, were typed in both human and environmental samples. Other reported genotypes were only found in water bodies. Animal trapping identified 1 mouse with lesion characteristic of BU, which was confirmed as MU infection. Our findings suggest that patients may have been infected from community associated water bodies. Further, we present evidence that small mammals within endemic communities could be susceptible to MU infections. M. ulcerans transmission could involve several routes where humans have contact with risk environments, which may be further compounded by water bodies acting as vehicles for disseminating strains.
  Buruli ulcer (BU) is a necrotizing skin disease which has been reported in over thirty countries. The most endemic countries include Ghana, Togo, Cote d’Ivoire and Benin with affected populations significantly being rural [1]. Mycobacterium ulcerans (MU), the causative agent of BU is an environmental mycobacteria. The mode of transmission to humans is still not clear although a few hypotheses have been advanced and tested [2–4]. Relying on advances in environmental microbiology and genotyping tools to identify habitats and reservoirs of M. ulcerans persistence and proliferation could aid in such transmission studies [5,6]. Human-to-human transmission is rare [7] and infection seems to be high among people with frequent contact to slow moving water bodies or wet lands, in endemic communities [5,8]. Within an aquatic environment, M. ulcerans can be found at the air-water interface, form biofilm on surfaces and probably occupy microhabitats not directly exposed to light but aerated [9]. From these biotopes, it is possible for the bacteria to infect susceptible hosts [10]. The viability of MU from environmental sources was proven with successful cultivation of the bacterium from an aquatic insect and subsequent establishment of infection in a mouse model [11]. Additionally, molecular data have correlated abundance of MU DNA from these environments with increasing BU cases [5]. We posit that patients are infected from MU-contaminated water bodies, hence MU genotypes from both sources should be identical. Earlier efforts have focused on comparing and differentiating human isolates within and from different geographical origins leaving out the environmental component [12–14]. An emerging development on the transmission of MU is the role small mammals could be playing in the ecology of the pathogen. M. ulcerans infection with clinical presentations similar to those in humans have been observed in koalas [15], possums [16,17] and in armadillos [18], in studies conducted in Australia. A study conducted by Durnez et al. in Benin, where small mammals were trapped and analyzed for mycobacterial infections, detected several species of mycobacteria but not M. ulcerans [19]. Experimental studies in Ghana have also shown indigenous grasscutters, Thryonomys swinderianus, to be susceptible to M. ulcerans infection [20]. Small mammals, living in close proximity to humans and commonly hunted animals, like grasscutters, rabbits and rats could therefore be potential reservoirs of M. ulcerans. Application of VNTR typing has revealed genetic differences among MU isolates collected from different patients and geographical regions [6,14,21–25]. Hilty et al. [14], identified three pathogenic MU genotypes in Ghana using this tool. Their findings were corroborated by ecological studies, investigating the distribution of M. ulcerans in endemic and non-endemic communities in Ghana [6]. The addition of other polymorphic loci will therefore increase the discrimination power in differentiating intra-species variation. Transmission of environmental mycobacteria is dependent on the overlapping habitats of the pathogen and humans [26]. Major overlap occurs in water where humans are exposed to mycobacteria through drinking, swimming, and bathing [26]. Thus, we source-tracked human MU infections to 10 water bodies, in four BU endemic communities, in Ghana. Using VNTR as an identifying tool, we uncovered additional genotypes and showed that patients were most likely infected from the water bodies they were frequently exposed to. We also assessed the role of small mammals as reservoirs of MU and suggest that they may be susceptible to MU infections and/or act as reservoirs. Using the ‘OneHealth’ concept, which seeks to define, manage and prevent diseases using a holistic approach of human, animal and environmental importance, we discussed a plausible transmission model, suggesting possible routes of MU infections from the environment.
  Ethical approval for patient recruitment into the study was sought and approved from the institutional review board of the Noguchi Memorial Institute for Medical Research (NMIMR), University of Ghana, (FWA 00001824; IRB00001276; IORG0000908). This also covered the administration of questionnaires. All participants signed a written informed consent form before recruitment into the study. All suspected cases were confirmed by PCR and results sent back to the District Health Directorate for treatment to commence. Approval for animal trapping and collection was under the permit of the Ashanti Regional Ministry of Health and the Wildlife Division of the Forestry Commission of Ghana (FCWD/GH-02), following its guidelines on animal husbandry. Dissection and harvesting of animal organs was adapted from Durnez et al. [19]. The study was carried out in Numereso, sub-district within the Amansie Central District of the Ashanti region, Ghana. The four selected communities for this study were Wromanso (N06. 03256 W001. 89761), Bepotenten (N06. 09213 W001. 96604), Monia-Gyaman (N06. 05113 W001. 92242) and Sukuumu (N06. 05190 W001. 94341). These communities were selected based on reported BU prevalence of 12. 1,7. 8,6. 6 and 6. 4 (per 1000 persons) for Bepotenten, Sukuumu, Monia-Gyaman and Wromanso respectively (Amansie District Health Directorate records). There was at least one working borehole with pump in each community, but inhabitants still used water from water bodies, for domestic and agricultural activities (S1 Table). We sought to identify among other things, livelihood strategies, hunting and animal rearing in households and perception on the modes of BU transmission, using a questionnaire we developed. Environmental sampling followed procedures described by Williamson et al. [6] with slight modifications. Soil, water filtrands and detritus were collected in triplicates and biofilm in quintuplicates. A sterile scalpel was used to collect about 5g of soil from the water floor and two from the riparian zone 5m apart into a 15ml falcon tube (BD Biosciences). This was preserved in 96% ethanol (Pharmacos). Detritus consisted of dead leaves, stems and grass blades within the water body. These were cut into a 15ml falcon tube and also preserved in 96% ethanol. Biofilm were taken from the surfaces of stems and leaves of dominant aquatic vegetation. Briefly, these parts were cut into Ziplock bags, 100ml of double distilled water was added, sealed and the biofilm material was dislodged by rubbing the bag vigorously several times. About 50ml of the resulting suspension was then poured out into a falcon tube for later analysis. For water filtrand, about 2L of water was scooped from the surface of the water body. Fifty milliliters of this was filtered through a 0. 45µm nitrocellulose filter (Whatman Inc). The water was then pumped through until the built resistance could not be overcome. The nitrocellulose filter was then removed and wrapped completely in aluminum foil. All samples were kept cool and transported to the laboratory where they were preserved at 4°C until processing. All materials were washed and decontaminated with 70% bleach and, sterilized with 90% ethanol and DNA away (Molecular BioProducts) between sampling sites. Active case searches were organized in the four communities and clinical samples taken from all suspected BU cases for confirmation and for the study. All participants signed a consent form before recruitment into the study. All suspected cases were confirmed by PCR and results sent back to the District Health Directorate for treatment to commence. Samples taken which were stored in a cooler included fine needle aspirates (FNA) for nodules and swabs for open lesions. Sherman livetraps (H. B. Sherman Traps, Inc.) were used in all small mammal collections. Traps, baited with a mixture of dried fish, groundnut paste and flour were set randomly in selected houses, farms and near water bodies. A total of 100 traps were set per community per night. Successful traps were labeled with the GPS coordinates of the site and all traps were washed with bleach between communities. Trapped animals were euthanized with chloroform and examined for external lesions and swellings before dissection. Organs including heart, lungs, liver, stomach, small intestines, caecum, kidneys, and spleen were harvested into separately labeled vials (2ml screw-cap tubes). Additionally, anal swabs, lesion swabs and tissue biopsies of lesions, if present, were taken. Animal carcasses were disinfected and buried 1. 5ft below the ground. This paper presents data on the five animals with external lesions. Biofilm samples were concentrated with an optimized protocol developed in the lab. Briefly, the 50ml falcon tubes containing the biofilm in suspension were spun at 12,000 rpm for 5 minutes in a High Speed Refrigerated Centrifuge (Suprema 21, TOMY) at 4°C. Twenty milliliters of the supernatant was carefully decanted off. This procedure was repeated at 13,000 and 14,000 rpm and each time discarding 5mL of the resulting supernatant. The remaining 10ml was preserved at 4°C until further use. All other environmental samples were processed as previously described [7]. FNAs and swabs from patient samples were processed for culture, microscopy, DNA extraction and PCR using protocols from other studies [6,27,28]. The samples were cultured in two ways. In one set, serial dilutions were performed, plated on LJ media slants and incubated at 32ºC. In another set, samples were decontaminated using the Modified Petroffs method and plated on LJ media slants. Processing of animal samples was carried out in a biosafety cabinet (Clean Bench, Hitachi). About a half of each organ sample (entire biopsy was used) was homogenized on a glass slide using a scalpel. The homogenized tissue was then scraped into a vial containing 1ml of 1X PBS. The homogenate was then vortexed vigorously and 250µl was used for extraction. DNA extraction for human and animal samples were performed using the Qiagen Dneasy blood and tissue kit (QIAGEN) following the manufacturer’s protocol. For environmental samples, DNA extraction followed the protocol described by Williamson et al. [6]. Negative and positive controls were included for each PCR run. Additionally, for environmental samples spiked samples (2. 5µl each of, positive control and an environmental extract) were included to check for inhibitions. Bovine Serum Albumin (Promega) was also added to environmental samples to relieve PCR inhibition in the amplification of all target loci. All PCR reactions were performed in a 2720 Thermal Cycler (Applied Biosystems). All primer sequences used in this study are listed in S2 Table. To identify environmental mycobacteria, samples were first screened using mycobacterial 16S rRNA primer as previously described [29]. Samples were then screened for the insertion sequence IS2404 PCR in a nested PCR, adapted from Ablordey et al. [30]. New primer sets were designed to amplify a 476bp product on the MlsA domain, encodes enoyl reductase (ER), of pMUM001 plasmid. This was performed in a 25µl reaction containing 1X PCR buffer (Promega), 1. 5mM MgCl2,400µM each of deoxyribonucleotide (Promega), 160nM each of forward and reverse primers, 1U GoTaq polymerase (Promega) and 5µl of genomic DNA. Reaction was cycled at 95°C for 3mins followed by 40 cycles each of, denaturation at 94°C for 30s, annealing at 63°C for 35s and extension at 72°C for 45s. Final extension was at 72°C for 10mins and reaction held at 4°C. PCR for VNTRs was adapted from Williamson et al. and Hilty et al. [5,14] with slight modifications. Allelic profiles used were in the order: MIRU1, Locus 6, ST1, Locus 19. Briefly, thermal cycling for all four VNTR loci were increased by additional 5 cycles. Seven microliters (7uL) of PCR products were run on a 2% agarose gel (Sigma-Aldrich), stained with ethidium bromide (Sigma-Aldrich) and band sizes were estimated with 100bp ladder. Repeat numbers for all VNTR loci were calculated based on published data [6,14,21,23–25]. Representative amplicons of IS2404, ER, 16S and VNTRs were confirmed with sequencing. VNTR-profiling was performed for only IS2404 positive samples and repeats confirmed with sequencing randomized samples. PCR products (40uL), with varying repeat sizes for each locus, were sent for sequencing (Macrogen Inc, Netherlands). Multi sequence alignments (MSA) and phylogenetic analyses were performed within MEGA V5 [31]. Representative sequences for loci including IS2404, ER, MIRU1, ST1, Locus 6 and Locus 19 have been deposited in GenBank under the following accession numbers, KM459595, KM459596, KM459597, KM459598, KM459599, KM459600, KM459601, KM459602, KM459603 and KM459604.
  Majority of the inhabitants in all four communities were farmers (S3 Table) but some youth were into small scale surface gold mining (galamsey) mainly along the Offin River (Fig. 1), which runs through all four communities. Preliminary analysis showed that although about 50% of inhabitants in a community used nearby surface water bodies for various purposes, swimming and bathing in these water bodies were the only activities (S1 Table) that were associated with an increased risk for BU infection (unpublished data). This informed our choice of specific water bodies to sample. In total, ten water bodies were selected after active case surveillance, where 2,4, 1 and 7 cases were detected in Wromanso, Monia-Gyaman, Bepotenten and Sukuumu respectively. Both microscopy and culture were attempted for FNA and swabs. However, acid-fast bacilli were not detected. There was over growth of other bacteria in the set that was serially diluted but not decontaminated. No growth was observed in the decontaminated set. Case confirmation was therefore based only on PCR (Table 1). All environmental samples (N = 140) were first screened for Mycobacterium spp using the mycobacterial 16S rRNA primers. Positive samples were then tested for IS2404. Total sample positivity was 38/140 (27%) and 25/38 (66%) for 16S and IS2404 respectively (Table 2). The Offin River at Sukuumu had the highest positivity for Mycobacterium spp and MPMs. Of the 14 samples from this river, eight contained mycobacteria out of which 5 were confirmed as containing MPMs. Mycobacteria was not detected in all 14 samples from Mon-Offin. At least one sample from all type of water body; River (Suk-Offin, Wro-Offin and Bep-Oda), Stream (Mon-Nkotia, Mon-Ampoma and Suk-Nkotia) or Pond (Wro-Bebonu, Suk-Twingun1 and Suk-Twingun2) tested positive for IS2404 (Table 2). The Offin River flows close to all four communities, but could not be sampled at Bepotenten as it was inaccessible due to galamsey (illegal surface gold mining) activities at the time of sampling (Fig. 1). Highest positivity for 16S was observed with biofilm 22/50 (44%) with the lowest being detritus 2/30 (7%). Differences in matrix positivity was statistically significant (P = 0. 0007) when tested with the Chi-square contingency table for independence (Table 2). AFB microscopy showed clumps of bacilli in biofilm samples as compared to single bacilli in detritus (S1 Fig.). Twenty-five of the thirty-eight (66%) 16S positive samples were positive for IS2404. All four 16S positive soil samples were IS2404 positive and 64% of 16S positive biofilm samples tested positive for IS2404. Fifteen suspected patients were included in this study following an active case surveillance. Fourteen were positive for 16S, IS2404 and ER (Table 1). ER sequence analysis showed >95% similarity to M. ulcerans except sample FY1 which had equal identities, 91%, to both M. ulcerans and M. liflandii. All 14 BU confirmed patients were recommended for treatment. VNTR-PCR was performed for IS2404 positive samples (14/15) and repeats confirmed with sequencing (Fig. 2). Allelic profiles were written as (MIRU1, Locus 6, ST1, Locus 19). Four genotypes, designated, W (1,1, 2,1), X (1,1, 2,2), Y (1,2, 2,1) and Z (1,2, 2,2), were observed for human samples. Locus 6 and 19 were the main determinants of a particular genotype because each gave two repeats, 1 or 2. MIRU1 and ST1, invariably gave repeats of 1 and 2, respectively. Four samples showed genotype X, 4 showed genotype Y, 3 typed genotype Z and 2 as genotype W. However, one patient (FS5) had MU infection with two genotypes, X and W (Table 1) with band sizes at 280bp and 340bp at locus 19 (S2 Fig.), differentiating these genotypes. Genotypes for FS3 and FS6 were indeterminate because there was no amplification at locus 19. Interestingly, sample FY1 showed a partial repeat for locus 19 (Fig. 2) leading to the profile Z. Six MU genotypes, W, X, Y, Z, A and B, were observed for environmental samples (Table 3), in IS2404 positive samples. M. marinum DL genotype E (1,2, 1,2) was observed in a biofilm sample from Twingun pond 2. A biofilm sample, BAB-4, collected from the Oda River, Bepotenten, showed a VNTR profile Y (1,2, 2,1) similar to a patient sample, FB1 (Table 1 and S2 Fig.), from the same community. Additional bands were observed, suggesting presence of other MPMs. A similar observation was made for BAF-3, which had ST1 repeat of 3 and was confirmed with sequencing as M. marinum M strain (Fig. 2). Also, we observed a unique genotype, OTS (1,2, 7, ND) in soil and biofilm samples. Genotypes W, X, Y, A, B and E were observed in Sukuumu. Genotypes X and Y, OTS and Y, Z and Y, were observed in Wromanso, Monia-Gyaman and Bepotenten respectively. Twingun 2 pond had the most diverse genotypes (n = 5). Four MU genotypes, W (1,1, 2,1), X (1,1, 2,2), Y (1,2, 2,1) and Z (1,2, 2,2) were identified from both human and environmental samples. Comparing them to published MU genotypes, repeat variation (1 or 2), for Locus 6 and 19, were similar (S4 Table). MIRU1and ST1 had conserved repeats of 1 and 2 respectively, for all human MU genotypes in this study. This again was consistent with the published MU genotypes, except for Bb, C, and the two Amansie MU strains 2 and 3. Amansie MU strains 1,2 and 3, were profiles from Amansie West, Ghana [14]. This is adjacent to the current study area. Furthermore, Genotype X had similar VNTR profile as MU genotype D [32]. M. liflandii with reported genotype F (1,2, 2,1) [7] was identical to current study genotype Y (1,2, 2,1). Overlapping the VNTR profiles of two Ghanaian isolates, the Amansie West strain 1 and Ghana sequence strain, gives a complete VNTR profile X (1,1, 2,2). Other genotypes observed, A (1,1, 1,2), B (3,1, 1,2) and E (1,2, 1,2), were identical to published genotypes, however, they were typed to environmental samples. Phylogenetic analysis clustered human (FW1, FY1 and FW2) and environmental (BAB4 and SKB5) samples with reference M. ulcerans ScoA ortholog suggesting genetic relatedness as predicted with the VNTR profiles (Fig. 2). In separate analysis we performed multi sequence alignment of the MIRU repeat consensus sequences from all sequenced amplicons with the corresponding repeat sequence published [14]. We observed a 100% sequence match, thus, supporting the observed clustering of our profiles with reference MU. Similarly, a separate phylogenetic analysis using IS2404 sequences showed a similar clustering with reported M. ulcerans orthologs (S3 Fig.). However, we observed two clusters. Our samples formed one cluster with two M. ulcerans orthologs and the other cluster was formed by IS2404 orthologs of other MPMs. A total of 78 small mammals were collected over 4 collection nights. Lesions (Fig. 3) were observed on only five animals (6. 41%). Identified lesions were on the thigh, tail (2 animals), ear and abdomen. Table 4 gives the PCR and sequence data analysis for the organs and lesions of the five small mammals. All five had at least one sample positive for 16S-PCR. However, sequences of the amplified 16S rRNA confirmed Microthrix parvicella, Corynebacterium mastitidis and Corynebacterium macginleyi in 3 animals (Table 4). 16S rRNA sequence of the lesion sample from a mouse, Mastomys spp, with Test ID S11, showed sequence similarity at 81% to the 16S rRNA of M. ulcerans Agy99 (accession Nº NR 074861, GenBank). Additionally, sequencing of the IS2404 amplicon revealed 99% identity to M. ulcerans Agy99 partial, plasmid, pMUM001, GenBank accession Nº CP000325. 1 (S3 Fig.), and was also positive for ER. We have not been able to determine the VNTR profile of this sample yet. We overlapped human and environmental VNTR profiles within our study communities to observe MU genotype distribution (Fig. 4). Genotype Y intersects both environment and human populations in all communities. Genotype Z was detected only in environmental sources. A similar situation was observed in Monia where genotypes W and X were found in humans but not in environmental samples. Genotypes X and Y were detected in both the environment and in human infections in Wromanso. Sukuumu presented the most diverse genotypes, W, X and Y in both human and environmental samples and Z, A, B and E in the environment but not humans. The MU positive lesion sample, on the small mammal, was also detected in Sukuumu. In grouping genotypes from the three types of water bodies, we observed that ponds harboured 6/8 genotypes (X, Y, Z, A, B and E), rivers had 3/8 (Y, Z and OTS) and streams with 3/8 (W, Y and OTS). However, mycobacteria presence, particularly MU, was independent of the type of water body (P = 0. 8081) when tested using the Chi-square contingency test for independence.
  VNTR typing confirmed repeats previously reported by other studies and resolved the apparent homogeneity in MU isolates, in Ghana. In this study, we identified four M. ulcerans genotypes (W, X, Y, & Z) both in humans and sampled water bodies in the Amansie Central District of the Ashanti region, Ghana. Other previously reported M. ulcerans genotypes (A & B) and M. marinum DL, genotype E, were detected only in water bodies. Confirmation of repeat numbers by sequencing showed that certain MU strains harboured partial repeats and other MPMs had repeats not previously reported. Genetic comparisons and geo-distribution of genotypes were used to source track MU infections to specific water bodies. Our findings suggest that patients may have been infected from local water body sources, which also serve as reservoirs and vehicles for the dissemination of MU strains and other MPMs in endemic communities. Transmission of MU could involve several routes where humans have contact with risk environments. Further, we present evidence that small mammals within endemic communities could be susceptible to MU infections and may be acting as reservoirs. Thus, we support the hypothesis that transmission of MPMs, particularly, M. ulcerans, is dependent on the overlapping habitats of the pathogen and humans.
summary: Buruli ulcer is a skin disease, which is endemic in over thirty countries, mostly in West Africa, with affected populations being largely rural. The causative organism, Mycobacterium ulcerans (MU), is an environmental mycobacterium and although transmission is unclear, frequent exposure to these MU-contaminated environments have been suggested as risk factors. We conducted this study on the premise that if patients are infected from MU-contaminated water bodies, then the genotype of MU strains in these patients should be identical to those in their community associated water bodies and wetlands. Using Variable Number Tandem Repeat (VNTR) as a genetic tool, we determined the genotypes of MU from both water bodies and patient samples. Comparison and overlap of these genotypes, within each community, suggest that patients were possibly infected from at least one water body. Additionally, we present evidence that small mammals within endemic communities could be susceptible to MU infections and may be acting as reservoirs. Our findings suggest that future ecological and molecular studies in the hope of elucidating a definite transmission route, should focus on source-tracking MU infections to community associated risk environments while employing a OneHealth approach in the process.
title: Source Tracking Mycobacterium ulcerans Infections in the Ashanti Region, Ghana