Community-based geographical distribution of Mycobacterium ulcerans VNTR-genotypes from the environment and humans in the Nyong valley, Cameroon

Abstract

Background: Genotyping is a powerful tool for investigating outbreaks of infectious diseases and it can provide useful information such as identifying the source and route of transmission, and circulating strains involved in the outbreak. Genotyping techniques based on variable number of tandem repeats (VNTR) are instrumental in detecting heterogeneity in Mycobacterium ulcerans (MU) and also for discriminating MU from other mycobacteria species. Here, we describe and map the distribution of MU genotypes in Buruli ulcer (BU) endemic communities of the Nyong valley in Cameroon. We also tested the hypothesis of whether the suspected animal reservoirs of BU that share the human microhabitat are shedding contaminated fecal matters and saliva into their surrounding environments.

Methods: Environmental samples from suspected MU-risk factors and lesion swabs from human patients were sampled in BU-endemic communities and tested for the presence of MU by qPCR targeting three independent sequences (IS2404, IS2606, KR-B). Positive samples to MU were further genotyped by VNTR with confirmation by sequencing of four loci (MIRU1, Locus 6, ST1, Locus 19).

Results: MU was detected in environmental samples including water bodies (23%), biofilms (14%), detritus (10%), and in human patients (73%). MU genotypes D, W, and C were found both in environmental and human samples. The micro geo-distribution of MU genotypes from communities showed that genotype D is found both in environmental and human samples, while genotypes W and C are specific to environmental samples and human lesions, respectively. No obvious focal grouping of MU genotypes was observed at the community scale. An additional survey in the human microhabitat suggests that domestic and wild animals do not shed MU in their saliva and feces in sampled communities.

Conclusions: VNTR typing uncovered different MU genotypes circulating in the endemic communities of the Akonolinga district. A MU environmental genotype was found in patients, yet the mechanism of contamination remains to be investigated; and recovering MU in culture from the environment remains key priority to enable a better understanding of the mode of transmission of BU. We also conclude that excretions from suspected animals are unlikely to be major sources of MU in the Nyong Valley in Cameroon.

Keywords: Environmental samples; Locus repeat; Mycobacterium ulcerans infection; VNTR-profiling.

Fig. 1

Two water bodies and two suspected BU-like lesions that were sampled at Edjom. a Nyong river (Nlong). b Fishing activities on the Nyong river. c Suspected BU-lesion (a circular ulcer of the left foot at the granulation phase on an articulation. This is a venous ulcer with a dark background and edema of the foot, found in a 27-year-old fisher. d Suspected BU-lesion (a plate of the left elbow with the onset of ulceration and a fibrinous background, found in a 14-year-old boy). These patients declared to work in close contact with the Nyong river for fishing and agricultural activities

Fig. 2

Community-based geographical distribution of M. ulcerans genotypes from the environment and humans. Blue square callouts contain genotypes found only in the environment (water bodies and biofilms); Pink square callouts have genotypes found only in human beings; Genotype E corresponds to M. marinum; Genotype D overlaps between the environment and human populations; blue lines represent water bodies (streams and rivers); Sampled water bodies, biofilms, detritus, and suspected BU patients are represented by colored dots as defined in the legend

Fig. 3

Comparison of MU VNTR profiles between the environment and humans. The relatedness between the genotypes was inferred using the UPGMA method in MEGA 7. Bootstrapping values for 1000 replicates are shown in percentage next to the branches. MIRU1 reference orthologs for M. marinum (the ancestor of MU), M. liflandii (another MPM), and M. ulcerans Agy99 (MU reference strain) were retrieved from GenBank with accession numbers given in the tree. MU: M. ulcerans; Sample test IDs and MU genotypes C, D, W are shown for environmental samples (B1NY, B6YE, W2YE, W7YE, W6AC) and BU lesions (H2ED, H3YE). The figure reveals clustering of MU genotypes from the environment and humans