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As a newly emerging Klebsiella pathogen, more and more Klebsiella michiganensis drug resistant strains have been reported in recent years, which posed serious threats to public health. Here we first reported a multidrug-resistant K. michiganensis strain 12084 with two blaSIM-1 and one mcr-9.2 genes isolated from the sputum specimen of a patient in the Second Affiliated Hospital of Zhejiang University School of Medicine and analyzed its genetic basis and drug-resistance phenotypes. Genetic analysis showed that this strain harbored three different incompatibility groups (IncHI2, IncHI5, and IncFIIpKPHS2:IncFIB-4.1) of plasmids (p12084-HI2, p12084-HI5, and p12084-FII). A total of 26 drug-resistance genes belonging to 12 classes of antibiotics were identified, most of which (24) were located on two plasmids (p12084-HI2 and p12084-HI5). Interestingly, two blaSIM-1 genes were identified to locate on p12084-HI2 and p12084-HI5, respectively, both of which were embedded in In630, indicating their genetic homogeny. It was noting that one blaSIM-1 gene was situated in a novel unit transposon (referred to as Tn6733) on the p12084-HI5 plasmid. We also discovered an mcr-9.2 gene on the p12084-HI2 plasmid. To the best of our knowledge, this is the first report of a blaSIM-1 and mcr-9.2 harboring K. michiganensis strain. We then investigated the population structure/classification, and antibiotic resistance for all 275 availably global K. michiganensis genomes. Population structure revealed that K. michiganensis could be divided into two main clades (Clade 1 and Clade 2); the most popular ST29 was located in Clade 1, while other common STs (such as ST50, ST27, and ST43) were located in Clade 2. Drug-resistance analysis showed 25.5% of the K. michiganensis strains (70/275) harboring at least one carbapenemase gene, indicating severe drug resistance of K. michiganensis beyond our imagination; this is a dangerous trend and should be closely monitored, especially for ST27 K. michiganensis with the most drug-resistant genes among all the STs. Overall, we reported a blaSIM-1 and mcr-9.2 harboring K. michiganensis strain, and further revealed the population structure/classification, and drug-resistance of K. michiganensis, which provided an important framework, reference, and improved understanding of K. michiganensis.

Tuberculosis (TB) has been the leading lethal infectious disease worldwide since 2014, and about one third of the world’s population has a latent TB infection (LTBI). This is largely attributed to the difficulties in diagnosis and treatment of TB and LTBI patients. Exosomes offer a new perspective on investigation of the process of TB infection. In this study, we performed small RNA sequencing to explore small RNA profiles of serum exosomes derived from LTBI and TB patients and healthy controls (HC). Our results revealed distinct miRNA profile of the exosomes in the three groups. We screened 250 differentially expressed miRNAs including 130 specifically expressed miRNAs. Some miRNAs were further validated to be specifically expressed in LTBI (hsa-let-7e-5p, hsa-let-7d-5p, hsa-miR-450a-5p, and hsa-miR-140-5p) and TB samples (hsa-miR-1246, hsa-miR-2110, hsa-miR-370-3P, hsa-miR-28-3p, and hsa-miR-193b-5p). Additionally, we demonstrated four expression panels in LTBI and TB groups, and six expression patterns among the three groups. These specifically expressed miRNAs and differentially expressed miRNAs in different panels and patterns provide potential biomarkers for detection/diagnosis of latent and active TB using exosomal miRNAs. Additionally, we also discovered plenty of small RNAs derived from genomic repetitive sequences, which might play roles in host immune responses along with Mtb infection progresses. Overall, our findings provide important reference and an improved understanding about miRNAs and repetitive region-derived small RNAs in exosomes during the Mtb infectious process, and facilitate the development of potential molecular targets for detection/diagnosis of latent and active tuberculosis.

Tuberculosis (TB) has surpassed HIV as the leading infectious disease killer worldwide since 2014. The main pathogen, Mycobacterium tuberculosis (Mtb), contains ~4,000 genes that account for ~90% of the genome. However, it is still unclear which of these genes are primary/secondary, which are responsible for generality/individuality, and which interconvert during evolution. Here we utilized a pan-genomic analysis of 36 Mtb genomes to address these questions. We identified 3,679 Mtb core (i.e., primary) genes, determining their phenotypic generality (e.g., virulence, slow growth, dormancy). We also observed 1,122 dispensable and 964 strain-specific secondary genes, reflecting partially shared and lineage-/strain-specific individualities. Among which, five L2 lineage-specific genes might be related to the increased virulence of the L2 lineage. Notably, we discovered 28 Mtb “Super Core Genes” (SCGs: more than a copy in at least 90% strains), which might be of increased importance, and reflected the “super phenotype generality.” Most SCGs encode PE/PPE, virulence factors, antigens, and transposases, and have been verified as playing crucial roles in Mtb pathogenicity. Further investigation of the 28 SCGs demonstrated the interconversion among SCGs, single-copy core, dispensable, and strain-specific genes through copy number variations (CNVs) during evolution; different mutations on different copies highlight the delicate adaptive-evolution regulation amongst Mtb lineages. This reflects that the importance of genes varied through CNVs, which might be driven by selective pressure from environment/host-adaptation. In addition, compared with Mycobacterium bovis (Mbo), Mtb possesses 48 specific single core genes that partially reflect the differences between Mtb and Mbo individuality.

Tuberculosis (TB) has exceeded HIV as the most lethal infectious disease globally for two consecutive years. Moreover, one third of the world’s population is estimated to have latent tuberculosis infection (LTBI). This is mainly because of difficulties associated with diagnosis and treatment for both TB and LTBI patients. Exosomes provide a promising research tool for TB diagnosis and treatment because they are released from various cells containing valuable biochemical information related to disease. In this study, we performed RNA-sequencing analysis on exosomes derived from clinical specimens of healthy controls (HC), active tuberculosis (ATB) and LTBI patients. Our results revealed the distinct gene expression profiles of the exosomes from LTBI and ATB patients. 1) We identified many distinct up-regulated and down-regulated differentially expressed genes (DEGs) in LTBI and ATB samples, and further screened the top-20 DEGs which might provide a potential panel for differentiation of HC, LTBI, and ATB. 2) We classified all the DEGs into six expression patterns, screened the top-20 genes in each pattern, and mainly focused on those highly expressed in LTBI and ATB. 3) Some Mycobacterium tuberculosis (Mtb) RNAs were only enriched in the exosomes of LTBI samples. 4) Pathway and function analysis further indicated an increase in deteriorating healthy signals in LTBI and ATB samples, including down-regulated signaling pathways/immune response and up-regulated apoptosis/necrosis. Our findings indicate the selective packaging of RNA cargoes into exosomes under different stages of Mtb infection, while facilitating the development of potential targets for the diagnosis, prevention and treatment of tuberculosis.