Molecular characterization of Vibrio cholerae responsible for cholera epidemics in Uganda by PCR, MLVA and WGS

Abstract

Background: For almost 50 years sub-Saharan Africa, including Uganda, has experienced several outbreaks due to Vibrio cholerae. Our aim was to determine the genetic relatedness and spread of strains responsible for cholera outbreaks in Uganda.

Methodology/principal findings: Sixty-three V. cholerae isolates collected from outbreaks in Uganda between 2014 and 2016 were tested using multiplex polymerase chain reaction (PCR), multi-locus variable number of tandem repeat analysis (MLVA) and whole genome sequencing (WGS). Three closely related MLVA clonal complexes (CC) were identified: CC1, 32% (20/63); CC2, 40% (25/63) and CC3, 28% (18/63). Each CC contained isolates from a different WGS clade. These clades were contained in the third wave of the 7th cholera pandemic strain, two clades were contained in the transmission event (T)10 lineage and other in T13. Analysing the dates and genetic relatedness revealed that V. cholerae genetic lineages spread between districts within Uganda and across national borders.

Conclusion: The V. cholerae strains showed local and regional transmission within Uganda and the East African region. To prevent, control and eliminate cholera, these countries should implement strong cross-border collaboration and regional coordination of preventive activities.

Fig 1. MLVA CC for V. cholerae associated with outbreaks in Uganda.

Each genotype is represented by five numbers indicating the number of repeats at the five loci, VC0147, VC0436-7 (intergenic), VC1650, VCA0171 and VCA0283. ‘N = ‘ reports the number of isolates with that genotype. The lines connecting the boxes indicate variation at a single locus. Part A is Clonal Complex 1, Part B is Clonal Complex 2, and Part C is Clonal Complex 3.

Fig 2

Shows the spatial distribution of MLVA clonal complexes and location of cholera outbreaks in Uganda: Part A during 2014, Part B during 2015, and Part C during 2016. Clonal Complex 1 are green circles, CC2 are purple stars, and CC3 are dark blue diamonds. The number of cases reported from each area varies by the year, yellow is the fewest number of cases, orange, then red-orange and red is the largest number of reported cases. Grey color indicates that there were no reported cases and blue indicates the Great Lakes of Africa.

Fig 3. Phylogram of V. cholerae WGS data.

Forty-one sequences from African isolates representing T10, T11 and T12 were included. Solid lines and black arrows demarcate the boundaries of the transmission events (T). Dotted lines and outlined arrows demarcate the boundaries of the clonal complexes (CC) in Uganda. Dashed arrows identify specific isolates from locations outside Uganda inferred to be examples of cross-border spread. The sequences within the Ugandan clades were less than five nucleotides apart. Those sequences in the Tanzanian clades were less than nine nucleotides from the Ugandan sequences of the closest clade. The radial lines are proportional to the number of nucleotide differences.