Complete genome sequence of the pandrug-resistant Vibrio cholerae strain KBR06 isolated from a cholera patient in Bangladesh

ABSTRACT Vibrio cholerae poses a serious hazard to global health and causes cholera disease in humans. Here, we present the full-genome sequence of a pandrug-resistant V. cholerae strain KBR06 isolated from a cholera patient in Bangladesh that exhibited intermediate resistance to only two antibiotics out of 39 among 14 antibiotic categories.

T he fast rise of numerous antibiotic-resistant strains of Vibrio cholerae documented in Africa, Asia, and America (1-4) has presented challenges for antibiotic therapy in recent years.Globally, the rising trend of multidrug-resistant V. cholerae strains linked to severe diseases is posing a substantial public health risk (5)(6)(7).Whole-genome sequenc ing of a pandrug-resistant (PDR) V. cholerae KBR06 isolated from a cholera patient has been reported here.Based on the results (Table 1), the strain was defined as a PDR according to the experts' consensus (8).
The isolated strain grown on MacConkey agar was collected from the Dogma Hospital (Dhaka, Bangladesh).The hospital isolated it from the patient's stool according to the CDC guidelines (9).After collection, the strain was cultured on thiosulfate-citratebile salt-sucrose agar plates, and yellow-colored and shiny colonies were observed which indicated the strain to be Vibrio spp.The strain was then enriched by culturing in alkaline-peptone-water for 8 hours at 37°C, genomic DNA was extracted using a commercial kit (Norgen Biotek, Canada), and PCR amplified its 16S rDNA using bacte rial universal primers 27-forward (5′-AGAGTTTGATCCTGGCTCAG-3′) and 1,492-reverse (5′-CGGTTACCTTGTTACGACTT-3′) followed by sequencing.The obtained sequence was subjected to BLAST search at NCBI (https://blast.ncbi.nlm.nih.gov/Blast.cgi) for identifica tion of the strain's taxonomy.The sequence analysis results revealed that the isolate was V. cholera.For susceptibility testing, the discdiffusion method was performed using antibiotic discs (Oxoid Ltd., England) on Mueller-Hinton agar plates according to the CLSI guidelines (10).For amikacin and gentamicin, the broth dilution method was used.
The Nextera XT DNA library preparation kit (Illumina) was used to construct a sequencing library from 1 ng of genomic DNA.Whole-genome sequencing (WGS) was performed on the Illumina MiSeq platform using the Illumina MiSeq v3 reagent kit to generate paired-end sequence reads.Default settings were used for all software unless otherwise indicated.FastQC v0.11.9 (11) was used for checking the sequence quality, and Trimmomatic v0.40 (https://github.com/usadellab/Trimmomatic)was used for adapter trimming.Further trimming was performed using FastP v0.36 (12).The total number of reads was 502,534, and the length of reads was ≤151.The sequencing depth was found to be ~30×, and genome coverage was ~99.9%.Unicycler v0.5.0 (adding "rotate" option) was used to assemble the sequences (13).The NCBI Prokaryotic Genome Annotation Pipeline (PGAP) v5.3 (14) was used for annotation.The number of contigs and N 50 values were 67 and 168.2 kb, respectively.
Annotation of the V. cholerae KBR06 genome identified 3,552 coding sequences.Abricate v0.8.13 (https://github.com/tseemann/abricate)and CARD (https://card.mcmaster.ca/analyze/rgi) were used to identify the antibiotic resistance (ABR) genes.The sequence assembly of V. cholerae KBR06 yielded a single chromosome with a genome size of 3,964,744 bp, and the GC content was 47.5%.Yamamoto et al. reported a single chromosome of V. cholerae and explained its reason (15).Fifty-six genes were assigned to virulence, disease, and defense (Fig. 1) according to the subsystem category distribution of the genes assigned to various subsystems based on the RAST server-based annotation (16,17) of the V. cholerae KBR06 genome.Therefore, the genomic information of PDR V. cholerae KBR06 could be helpful to understand the genetic foundations of ABR and advance the study, management, and monitoring of ABR.

TABLE 1
Antibiogram of Vibrio cholerae KBR06 bThe diameters of the experimentally obtained and reference clear zones have been shown in millimeters (mm); R: resistant (mm), IR: intermediate resistant (mm), S: susceptible (mm), MD: mean diameter (mm) from three independent experiments, SD: standard deviation, and RA: responses of Vibrio cholerae KBR06 to the antibiotics.c -indicates no data available in CLSI guidelines and/or not applicable.
a Broth dilution method.b