using-human-induced-pluripotent-stem-cell-derived-intestinal

Using Human Induced Pluripotent Stem Cell-derived Intestinal Organoids to Study and Modify Epithelial Cell Protection Against <em>Salmonella</em> and Other Pathogens

The intestinal &#8216;organoid&#8217; (iHO) system, wherein 3-D structures representative of the epithelial lining of the human gut can be produced from ...

Wellcome Trust Sanger Institute

Colistin remains one of the few antibiotics effective against multi-drug resistant (MDR) hospital pathogens, such as Klebsiella pneumoniae. Yet resistance to this last-line drug is rapidly increasing. Characterized mechanisms of col in K. pneumoniae are largely due to chromosomal mutations in two-component regulators, although a plasmid-mediated col mechanism has recently been uncovered. However, the effects of intrinsic colistin resistance are yet to be characterized on a whole-genome level. Here, we used a genomics-based approach to understand the mechanisms of adaptive col acquisition in K. pneumoniae. In controlled directed-evolution experiments we observed two distinct paths to colistin resistance acquisition. Whole genome sequencing identified mutations in two colistin resistance genes: in the known col regulator phoQ which became fixed in the population and resulted in a single amino acid change, and unstable minority variants in the recently described two-component sensor crrB. Through RNAseq and microscopy, we reveal the broad range of effects that colistin exposure has on the cell. This study is the first to use genomics to identify a population of minority variants with mutations in a col gene in K. pneumoniae.

Morphological, genomic and transcriptomic responses of Klebsiella pneumoniae to the last-line antibiotic colistin.

Nontyphoidal  (NTS), particularly  serovar Typhimurium, is among the leading etiologic agents of bacterial enterocolitis globally and a well-characterized cause of invasive disease (iNTS) in sub-Saharan Africa. In contrast,  Typhimurium is poorly defined in Southeast Asia, a known hot spot for zoonotic disease with a recently described burden of iNTS disease. Here, we aimed to add insight into the epidemiology and potential impact of zoonotic transfer and antimicrobial resistance (AMR) in  Typhimurium associated with iNTS and enterocolitis in Vietnam. We performed whole-genome sequencing and phylogenetic reconstruction on 85 human (enterocolitis, carriage, and iNTS) and 113 animal  Typhimurium isolates isolated in Vietnam. We found limited evidence for the zoonotic transmission of  Typhimurium. However, we describe a chain of events where a pandemic monophasic variant of  Typhimurium (serovar I:4,[5],12:i:- sequence type 34 [ST34]) has been introduced into Vietnam, reacquired a phase 2 flagellum, and acquired an IncHI2 multidrug-resistant plasmid. Notably, these novel biphasic ST34  Typhimurium variants were significantly associated with iNTS in Vietnamese HIV-infected patients. Our study represents the first characterization of novel iNTS organisms isolated outside sub-Saharan Africa and outlines a new pathway for the emergence of alternative  variants into susceptible human populations. Typhimurium is a major diarrheal pathogen and associated with invasive nontyphoid  (iNTS) disease in vulnerable populations. We present the first characterization of iNTS organisms in Southeast Asia and describe a different evolutionary trajectory from that of organisms causing iNTS in sub-Saharan Africa. In Vietnam, the globally distributed monophasic variant of  Typhimurium, the serovar I:4,[5],12:i:- ST34 clone, has reacquired a phase 2 flagellum and gained a multidrug-resistant plasmid to become associated with iNTS disease in HIV-infected patients. We document distinct communities of  Typhimurium and I:4,[5],12:i:- in animals and humans in Vietnam, despite the greater mixing of these host populations here. These data highlight the importance of whole-genome sequencing surveillance in a One Health context in understanding the evolution and spread of resistant bacterial infections.

New Variant of Multidrug-Resistant  Serovar Typhimurium Associated with Invasive Disease in Immunocompromised Patients in Vietnam.

Intestinal epithelial cells (IECs) play a key role in regulating immune responses and controlling infection. However, the direct role of IECs in restricting pathogens remains incompletely understood. Here, we provide evidence that IL-22 primed intestinal organoids derived from healthy human induced pluripotent stem cells (hIPSCs) to restrict  serovar Typhimurium SL1344 infection. A combination of transcriptomics, bacterial invasion assays, and imaging suggests that IL-22-induced antimicrobial activity is driven by increased phagolysosomal fusion in IL-22-pretreated cells. The antimicrobial phenotype was absent in hIPSCs derived from a patient harboring a homozygous mutation in the  gene that inactivates the IL-22 receptor but was restored by genetically complementing the IL10RB deficiency. This study highlights a mechanism through which the IL-22 pathway facilitates the human intestinal epithelium to control microbial infection.

Article	Year
Morphological, genomic and transcriptomic responses of Klebsiella pneumoniae to the last-line antibiotic colistin. Scientific reports\| PubMed ID: 29959380	2018
New Variant of Multidrug-Resistant Serovar Typhimurium Associated with Invasive Disease in Immunocompromised Patients in Vietnam. mBio\| PubMed ID: 30181247	2018
Interleukin-22 promotes phagolysosomal fusion to induce protection against Typhimurium in human epithelial cells. Proceedings of the National Academy of Sciences of the United States of America\| PubMed ID: 30217896	2018

David Goulding

Biography

JoVE Journal Publications

Other Publications