Genomic evolution and rearrangement of CTX-Φ prophage elements in Vibrio cholerae during the 2018-2024 cholera outbreaks in eastern Democratic Republic of the Congo

Abstract

ABSTRACTBetween 2018 and 2024, we conducted systematic whole-genome sequencing and phylogenomic analysis on 263 V. cholerae O1 isolates from cholera patients across four provinces in the Democratic Republic of Congo (North-Kivu, South-Kivu, Tanganyika, and Kasai Oriental). These isolates were classified into the AFR10d and AFR10e sublineages of AFR10 lineage, originating from the third wave of the seventh El Tor cholera pandemic (7PET). Compared to the strains analysed between 2014 and 2017, both sublineages had few genetic changes in the core genome but recent isolates (2022-2024) had significant CTX prophage rearrangement. AFR10e spread across all four provinces, while AFR10d appeared to be extinct by the end of 2020. Since 2022, most V. cholerae O1 isolates exhibited significant CTX prophage rearrangements, including a tandem repeat of an environmental satellite phage RS1 downstream the ctxB toxin gene of the CTX-Φ-3 prophage on the large chromosome, as well as two or more arrayed copies of an environmental pre-CTX-Φ prophage precursor on the small chromosome. We used Illumina data for mapping and coverage estimation to identify isolates with unique CTX-Φ genomic features. Gene localization was then determined on MinION-derived assemblies, revealing an organization similar to that of non-O1 V. cholerae isolates found in Asia (O139 VC1374, and environmental O4 VCE232), but never described in V. cholerae O1 El Tor from the third wave. In conclusion, while the core genome of AFR10d and AFR10e showed minimal changes, significant alterations in the CTX-Φ and pre-CTX-Φ prophage content and organization were identified in AFR10e from 2022 onwards.

Keywords: CTX-Φ rearrangement; DRC; Vibrio cholerae; genomic evolution; prophages; whole genome.

Figure 1.

Map of the DR Congo showing the localization of the sample collection by time and province.

Figure 2.

Phylogenomic analysis of V. cholerae O1 isolates (2014–2024) from four investigated DRC provinces, emphasizing the years of isolate collection (2A) and the collection province (2B). Notes: The trees were derived from 750 core genome SNPs mapped against the 7PET V. cholerae O1 biotype EL Tor N19691, which was used as reference genome and as an outgroup to root the tree.

Figure 3.

Temporal prevalence of AFR10d and AFR10e V. cholerae O1 isolates in the four DRC provinces under investigation. For the Kasai Oriental and Tanganyika provinces, strains were collected only in 2020 and 2022, respectively.

Figure 4.

(A) Representative organization of the CTX-Φ prophage and its satellite in V. cholerae isolates collected between 2018 and 2020 in the DRC. Notes: Block arrows represent genes present in each element of the CTX-Φ-3-phage and indicate the direction of transcription. RS1^ca and RS2^ca represent RS1 and RS2 canonical satellite phages. (B) Representative organization of the CTX-Φ prophage and its satellites in both the large and small chromosomes in the majority (125/147) of V. cholerae isolates collected since 2022 in DRC. Notes: Canonical sequences for each gene are shown in grey, while environmental sequences are shown in blue. (C) CTX-Φ-3 prophage organization observed in three V. cholerae isolates collected in 2023.