An 83-year-old patient originally from Turkey presented with progressive light-headedness, and dyspnoea with minimal exertion over 4 days, on a background of diabetes mellitus, congestive cardiac failure, chronic kidney disease, and hypertension. Blood tests on presentation revealed a microcytic anaemia (haemoglobin 49 g l-1, MCV 66 fL), which was treated with blood transfusion. The patient spiked a single fever of 38.1  C on day six of admission for which blood was collected into BACTEC Plus Aerobic medium (BD Diagnostics) and BACTEC Lytic Anaerobic medium (BD Diagnostics). Concurrently, a non-contrast CT of chest, abdomen and pelvis was performed to investigate deranged liver function tests (with a predominantly cholestatic pattern) and hypoechoic liver lesions seen on abdominal ultrasound. The CT scan demonstrated caecal wall thickening, and endoscopic biopsies were taken. Adenocarcinoma of the proximal ascending colon without lymphovascular or perineural invasion was seen on histopathological examination. The patient proceeded to have an urgent open right hemicolectomy given ongoing transfusion-dependent blood loss. Histopathologic examination of operative tissue confirmed adenocarcinoma. Chronic inflammatory infiltrate at the advancing edge was also seen.
Gram-positive cocci and Gram-negative rods were detected in the anaerobic (17 h) and aerobic (66 h) cultures respectively and were each isolated. Reliable identification on the MALDI-TOF (MALDI Biotyper smart with a Security Library Extension; Bruker) could not be obtained due to identification discordance, with acceptable scores for   Streptococcus mitis   (score: 2.32) and   S. oralis   (score: 2.29) for the Gram-positive cocci recovered anaerobically, and   Yersinia pestis   (score: 2.25) and   Y. pseudotuberculosis   (score: 2.13) for the Gram-negative rods recovered aerobically. Viridians group streptococci were deemed not clinically significant, as trans-thoracic echocardiogram did not demonstrate a valvular vegetation and two subsequent blood cultures had no growth. However, discordant   Yersinia   results triggered a genomic analysis for identification and the patient was treated with ciprofloxacin with subsequent testing by BD Phoenix AST indicating in vitro susceptibility (MIC <=0.125). Repeat blood cultures were negative.
Nanopore sequencing on a MinION (Oxford Nanopore Technologies) was performed for 20 h with a 'two-pronged' approach to gather multiple lines of evidence at the reads and assembly level (Fig. 1a; Supplementary File 1, available in the online version of this article). Of the 336 622 trimmed reads, 81.7 % were classified as   Y. pseudotuberculosis   as opposed to 1.74 % as   Y. pestis   (Fig. 1b). Assembly resulted in in a single, circular 4.79 Mb contig. In silico Multi Locus Sequence Typing (MLST) against the McNally MLST scheme returned a   Y. pseudotuberculosis   associated sequence type (ST) of ST96. In addition, core genome MLST (cgMLST) showed that this isolate clustered together with other   Y. pseudotuberculosis   genomes (Fig. 1c, Supplementary File 2). The entire process, which took approximately 29 h (20 h of sequencing and 9 h of downstream offline base-calling and analysis), indicated that the isolate was   Y. pseudotuberculosis  . Retrospective analysis showed that data generated within 2 h would return the same results on both the read and assembly level. (Fig. 2). A contiguous assembly, albeit 59 bases shorter, could be generated from reads 10 h into sequencing with genomic difference localised to 141 'SNPs' (Fig. 2), 446 insertions and 456 deletions. Confirmatory reference biochemical tests were subsequently performed which yielded results expected for   Y. pseudotuberculosis   (Table 1). Concordant genomic and phenotypic results allowed for the reclassification from   Y. pestis   to   Y. pseudotuberculosis  .