Extracellular vesicles expressing CEACAM proteins in the urine of bladder cancer patients.

Early detection and long-term monitoring are important for urothelial carcinoma of the bladder (UCB). Urine cytology and existing markers have insufficient diagnostic performance. Here, we examined medium-sized extracellular vesicles (EVs) in urine to identify specific markers for UCB and evaluated their usefulness as diagnostic material. To identify specific markers in urinary EVs derived from UCB, we undertook shotgun proteomics using urine from four UCB patients and four healthy subjects. Next, 29 healthy specimens, 18 noncancer specimens, and 33 UCB specimens, all from men, were analyzed for urinary EVs by flow cytometry to evaluate the diagnostic performance of UCB-specific EVs. Nanoparticle-tracking analysis indicated that the size of EVs extracted from urine was mostly <400 nm. By shotgun proteomics, we detected several proteins characteristic of UCB and found that carcinoembryonic antigen-related adhesion molecule (CEACAM) proteins were increased in patients. Flow cytometric analysis revealed that the degree of expression of CEACAM1, CEACAM5, and CEACAM6 proteins on the surface of EVs varied among patients. Extracellular vesicles expressing CEACAM proteins also expressed mucin 1, suggesting that they were derived from tumorigenic uroepithelial cells. The number of EVs expressing CEACAM1, 5, and 6 proteins was significantly increased in UCB (mean ± SD, 8.6 ± 13%) compared to non-UCB (0.69 ± 0.46) and healthy (0.46 ± 0.34) by flow cytometry. The results of receiver operating characteristic (ROC) analysis showed a good score of area under the ROC curve of 0.907. We identified EVs that specifically express CEACAM proteins in urine and have potential for diagnostic applications. These EVs are potential targets in a new liquid biopsy test for UCB patients.

Stepwise Evolution of a Klebsiella pneumoniae Clone within a Host Leading to Increased Multidrug Resistance.

Five blaCTX-M-14-positive Klebsiella pneumoniae isolates (KpWEA1, KpWEA2, KpWEA3, KpWEA4-1, and KpWEA4-2) were consecutively obtained from a patient with relapsed acute myeloid leukemia who was continuously administered antimicrobials. Compared with KpWEA1 and KpWEA2, KpWEA3 showed decreased susceptibility to antimicrobials, and KpWEA4-1 and KpWEA4-2 (isolated from a single specimen) showed further-elevated multidrug-resistance (MDR) phenotypes. This study aims to clarify the clonality of the five isolates and their evolutionary processes leading to MDR by comparison of these complete genomes. The genome comparison revealed KpWEA1 was the antecedent of the other four isolates, and KpWEA4-1 and KpWEA4-2 independently emerged from KpWEA3. Increasing levels of MDR were acquired by gradual accumulation of genetic alterations related to outer membrane protein expression: the loss of OmpK35 and upregulation of AcrAB-TolC occurred in KpWEA3 due to ramA overexpression caused by a mutation in ramR; then OmpK36 was lost in KpWEA4-1 and KpWEA4-2 by different mechanisms. KpWEA4-2 further acquired colistin resistance by the deletion of mgrB. In addition, we found that exuR and kdgR, which encode repressors of hexuronate metabolism-related genes, were disrupted in different ways in KpWEA4-1 and KpWEA4-2. The two isolates also possessed different amino acid substitutions in AtpG, which occurred at very close positions. These genetic alterations related to metabolisms may compensate for the deleterious effects of major porin loss. Thus, our present study reveals the evolutionary process of a K. pneumoniae clone leading to MDR and also suggests specific survival strategies in the bacteria that acquired MDR by the genome evolution. IMPORTANCE Within-host evolution is a survival strategy that can occur in many pathogens and is often associated with the emergence of novel antimicrobial-resistant (AMR) bacteria. To analyze this process, suitable sets of clinical isolates are required. Here, we analyzed five Klebsiella pneumoniae isolates which were consecutively isolated from a patient and showed a gradual increase in the AMR level. By genome sequencing and other analyses, we show that the first isolate was the antecedent of the later isolates and that they gained increased levels of antimicrobial resistance leading to multidrug resistance (MDR) by stepwise changes in the expression of outer membrane proteins. The isolates showing higher levels of MDR lost major porins but still colonized the patient's gut, suggesting that the deleterious effects of porin loss were compensated for by the mutations in hexuronate metabolism-related genes and atpG, which were commonly detected in the MDR isolates.