Single-Cell Sequencing of a Bile Sample From an Acute Cholecystitis Patient.

Single-cell sequencing is a novel approach to genome sequencing of clinical samples. However, there are only few studies using single-cell sequencing of genomes for bacterial infections. A 71-year-old woman presented to the emergency department with epigastric pain, 38.5°C fever, and a history of hypertension and hyperuricemia. From blood test results, acute cholecystitis was suspected. The surgery went well and bilirubin calcium stones were found in the gallbladder. Single-cell sequencing was used to investigate a bile sample from a patient with acute cholecystitis. The sample, cultured on a MacConkey agar plate, produced four colonies, all identified as Escherichia coli by bacteriological and biochemical properties. Whole genome sequences of the four strains were determined using the single-cell amplified genome (SAG) sequencing technique. The average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) values of all four were 99.98-100% and 100%, respectively, indicating that they were the same bacterial species. Compared with type strains, these four strains were closest to Shigella sonnei (ANI 98.65-98.66%; dDDH 88.5%) than E. coli (ANI 96.79-96.80%; dDDH 74.2%), despite lacking stx1, stx2 and ipaH, which Shigella species harbor. 16S metagenome analysis identified E. coli as the predominant bacterial genome in the sample, comprising 93.15%. SAG raw data had a relatively high level of quality, with 98.4-98.7% of the read numbers used after quality trimming. However, the genome sequencing coverage was only 9.45-42.88% when compared to a complete genome of an isolate with a mapping quality set above 99%, resulting in gaps compared to conventional whole genome sequence data of these isolates. The procedures of the SAG sequencing technique should be revised to improve the sequencing coverage and reduce gaps in the sequence data. Nonetheless, single-cell genome sequencing can provide novel information for bacterial infections.