Several lineages have been identified in the population of serogroup O1 Vibrio cholerae (V. cholerae) (1-3). The strains, which were responsible for the ongoing seventh cholera pandemic, were in Lineage 2. Nearly all the V. cholerae strains in this lineage carried genes coding cholera toxin (ctxAB) (1-2). Lineage 3b consists of strains isolated from different continents and the vast majority of strains in this lineage lack the ctxAB genes.
In China, toxigenic serogroup O1 V. cholerae strains were rarely isolated after 2010 (4). However, 2 serogroup of O1 V. cholerae that possessed the ctxAB genes were isolated from patients in June 2021. The first and second case were found after visiting doctors in the same hospital in Beijing on June 19 and 21, 2021, respectively. Both patients complained of abdominal pain and watery diarrhea about 5 times a day, which started on June 17 and 18, respectively. No erythrocytes and leukocytes were observed by stool microscopy. There was no epidemiological connection between these two cases. Neither patient had a common history of exposure or had a common travel history. The first patient stayed in Beijing and the second one traveled to Tianjin 3 days before onset of symptoms. No contacts of the two patients complained of having diarrhea symptoms. Both patients recovered after receiving antibiotic therapy. Real-time polymerase chain reaction (PCR) assay showed that the fecal specimens were positive for the ctxAB and rfb gene of O1 V. cholerae (5). V. cholerae strains (named BJVC202101 and BJVC202102) were obtained on June 20 and 22 from the specimens of the first and second cases, respectively, and both were serogroup O1, serotype Ogawa.
Genome sequencing analysis indicated that both of these 2 genomes harbored 2 tandem copies of CTX prophage (6), which both carried genes for rstRclass and ctxB10 (Figure 1C). Vibrio pathogenicity island 1 (VPI-1) and VPI-2 were detected but Vibrio seventh pandemic island 1 and 2 were absent in them. These 2 genome sequences were combined with a representative global serogroup O1 V. cholerae genome collection (1-3,7) for phylogenetic analysis using a pipeline in the China Pathogen Identification Net (China PIN). These 2 strains formed a cluster, and it was most closely related to the V. cholerae isolated in Ukraine and Russia (8) in the phylogenetic tree based on the non-repetitive, non-recombinant core genome single nucleotide polymorphisms (SNPs) (Figure 1A). We reconstructed a phylogenetic tree based on the non-repetitive, non-recombinant core genome SNPs of 18 (including these 2 genomes) most closely related genomes (Figure 1B). The SNP number between these 2 isolates was 8, and varied from 894 to 2,231 between this cluster and the Ukraine and Russia isolates. Therefore, these 2 strains were genetically closely related but remotely related to the Ukraine and Russia ones.
Figure 1. Phylogenetic analysis and CTX prophage structure of these two V. cholerae. (A) Maximum-likelihood tree constructed for SNPs identified in the non-repetitive, non-recombinant core-genome of the genome collection. (B) The maximum-likelihood tree was constructed on the SNPs identified in the non-repetitive, non-recombinant core-genome of the 18 most closely-related genomes including the 2 Beijing V. cholerae strains isolated in 2021. (C) Schematic diagram of the CTX prophages identified in V. cholerae BJVC202102.
Notes: Branches were colored according to the lineages they belonged to in Figure 1A. The isolation time was labelled and the tips were colored according to the isolation countries in Figure 1B. Schematic diagram of the CTX prophages were identified in V. cholerae BJVC202102. Abbreviations: SNPs=single nucleotide polymorphisms.
In conclusion, these 2 strains were most closely related to the genomes in Lineage 3b. We prefer to take these isolates as a new clone. The existence of ctxAB indicated the potential of this clone to cause cholera outbreaks. Moreover, no epidemiological connection between the 2 patients was observed, which may indicate the wide distribution of this clone of V. cholerae. Therefore, surveillance on this clone should be carried out.