Spatiotemporal distribution of Mycobacterium ulcerans and other mycolactone producing mycobacteria in southeastern United States

Abstract

Buruli ulcer (BU) is a chronic and debilitating skin disease caused by the environmental pathogen, Mycobacterium ulcerans (MU). The primary virulence determinant is mycolactone, a cytotoxic lipid compound unique to MU and its other mycolactone producing mycobacteria (MPM) ecological variants. Although BU prevalence is highest in West Africa and Australia, little is known about MU and other MPM distribution in non-endemic regions such as the Southeastern United States (US). In this study, environmental samples (water filtrand, plant biofilm, soil, aquatic invertebrates) were collected from nine freshwater sites across Louisiana, Mississippi and Alabama over three sampling periods (August 2020, November 2020, March 2021). Samples were screened for MU and MPM presence and abundance by PCR and genotyped using variable number tandem repeat (VNTR) profiling. All nine sites were positive for MU or other MPM DNA in at least one substrate, except invertebrates. Overall, mean concentrations were 4.3 × 10⁴ genome units (GU)/sample in August 2020, 1.26 GU/sample in November 2020, and 55.5 GU/sample in March 2021. Profiling by VNTR identified four MU (designated A-D) and one M. liflandii genotype(s), among environmental samples, with genotype frequencies varying by site and sampling time. Detection of MU and M. liflandii genotypes in Southeastern US aquatic environments, matching those from BU endemic regions, provides rationale for ongoing surveillance. Our findings broaden the known geographic range of MU and MPMs and offer baseline data to help predict and prevent and predict the possibility of zoonotic transmission in Southeastern US.

Keywords: Alabama; Buruli Ulcer; Louisiana; Mississippi; Mycobacterium liflandii; Mycobacterium ulcerans; Southeastern United States; mycolactone-producing mycobacteria.

Figure 1.

Map of sites sampled in August 2020, November 2020 and March 2021.

Figure 2.

Mycolactone producing mycobacteria mean positivity (A) and concentration (C) across sampling time. Sample positivity according to site and time and mean concentration (mean log10 GU of positive samples are shown in panels B and D. MS1: Mississippi Site 1; MS2: Mississippi Site 2; MS3: Mississippi Site 3; LA1: Louisiana Site 1; LA2: Louisiana Site 2; AL1: Alabama Site 1; AL2: Alabama Site 2; AL3: Alabama Site 3. x = Sites not sampled due to limited accessibility. Error bars denote standard error of the mean. *** represent p values <0.001, and ** represent p values <0.01.

Figure 3.

Mean Positivity (A) and Concentration (B) of positive water filter samples per Site and sampling time. Concentration was expressed as mean genome units across positive samples per site and sampling time. x = Sites not sampled due to limited accessibility. Error bars are standard error of the mean.

Figure 4.

Distribution of Mycobacterium ulcerans and M. liflandii VNTR profiles in Southeastern United States (Mississippi, Louisiana and Alabama). Sites in Louisiana are shown as purple diamonds, Mississippi as orange diamonds and Alabama as black diamonds. Genotypes identified from August 2020 samples are highlighted in yellow, November 2020 highlighted in red, and March highlighted in green. MUA: M. ulcerans VNTR profile A; MUB: M. ulcerans profile B; MUC: M. ulcerans profile C, MUD: M. ulcerans profile D; Mlif: M. liflandii profile.

Figure 5.

Minimum Spanning Tree of VNTR and VNTR-mycobacterial interspersed repetitive units (MIRU) profiles at four loci (Miru 1, Locus 6, ST1 and Locus 19). Samples include environmental samples collected from Southeastern United States (Mississippi (MS), Louisiana (LA) and Alabama (AL)), freezer stock controls from Africa (Ghana, Cote D’Ivoire, Cameroon and Benin), Asia (China and Japan), Australia and the Americas (Mexico, Surinam and French Guiana). Profiles from two previous studies were also included. Nodes represent similar genotypes. Number of individual samples are represented by the size of the node. Sites and Geographical origins of isolates are represented in the section of the node by colour. Three clusters were derived from this tree.