Cytotoxic Escherichia coli strains encoding colibactin isolated from immunocompromised mice with urosepsis and meningitis

Abstract

Immune-compromised mouse models allow for testing the preclinical efficacy of human cell transplantations and gene therapy strategies before moving forward to clinical trials. However, CRISPR/Cas9 gene editing of the Wsh/Wsh mouse strain to create an immune-compromised model lacking function of Rag2 and Il2rγ led to unexpected morbidity and mortality. This warranted an investigation to ascertain the cause and predisposing factors associated with the outbreak. Postmortem examination was performed on 15 moribund mice. The main lesions observed in these mice consisted of ascending urogenital tract infections, suppurative otitis media, pneumonia, myocarditis, and meningoencephalomyelitis. As Escherichia coli strains harboring polyketide synthase (pks) genomic island were recently isolated from laboratory mice, the tissue sections from the urogenital tract, heart, and middle ear were subjected to E. coli specific PNA-FISH assay that revealed discrete colonies of E. coli associated with the lesions. Microbiological examination and 16S rRNA sequencing confirmed E. coli-induced infection and septicemia in the affected mice. Further characterization by clb gene analysis and colibactin toxicity assays of the pks+ E. coli revealed colibactin-associated cytotoxicity. Rederivation of the transgenic mice using embryo transfer produced mice with an intestinal flora devoid of pks+ E. coli. Importantly, these barrier-maintained rederived mice have produced multiple litters without adverse health effects. This report is the first to describe acute morbidity and mortality associated with pks+ E. coli urosepsis and meningitis in immunocompromised mice, and highlights the importance of monitoring and exclusion of colibactin-producing pks+ E. coli.

Inulin and Galacto-oligosaccharides Increase the Genotoxic Effect of Colibactin Produced by pks+ Escherichia Coli Strains

“…We used the E. coli K-12 strain, which is psk-, and the pks + strain NC101 to infect the adenocarcinoma cell line Caco-2 and assessed cytotoxicity through the megalocytosis assay. As expected (25), Fig. 4A shows that cells infected with E. coli NC101 displayed enhanced megalocytosis compared to cells infected with the control E. coli K-12 strain.…”

Identification of a new strain of mouse kidney parvovirus associated with inclusion body nephropathy in immunocompromised laboratory mice

“…C57BL/6 c-Kit W–sh (B6.Cg- Kit W–sh /HNihrJaeBsmJ, Stock No:030764) mice were obtained from the Jackson Laboratory and maintained in the WI animal facility which is affiliated with Massachusetts Institute of Technology (Cambridge, MA, USA). C57BL/6 Il2rg −/– Rag2 −/– cKit W–sh mice, which is represented by B6.Il2rRag2W throughout the text, were generated via one-cell embryo injection of Cas9 mRNA and sgRNA into C57BL/6 c-Kit W–sh mice as previously described [ 8 ]. Additional laboratory mouse strains/stocks used in the present study included immunodeficient NSG (NOD.Cg- Prkdc scid Il2rg tm1Wjl /SzJ, Stock No: 001976) and immunocompetent UTX flox (B6;129S- Kdm6a tm1.1Kaig /J, stock No:024177) from Jackson Laboratory and Crl:CD1(ICR) mice from Charles River Laboratories.…”

“…We recently reported the occurrence of a sudden increase in morbidity and mortality in a colony of B6.Il2rRag2W mice associated with pks + E. coli-induced urosepsis and meningitis [8]. Retrospective evaluation of the kidneys from 14 of 15 B6.Il2rRag2W mice (5.5-22 months old) in this study [8] confirmed the presence of interstitial fibrosis and intranuclear inclusion bodies in convoluted tubules in 10 of 14 mice; these lesions are consistent with IBN described recently [5]. An additional 10 B6.Il2rRag2W mice (3.5-10 months old) submitted for necropsy after the pks+ E. coli outbreak also had IBN.…”

“…To improve human cell incorporation into the c-Kit W-sh mice and also to establish a system for human disease modelling, CRISPR (clustered regularly interspaced short palindromic repeat) and Cas (CRISPR-associated) proteins were applied to knockout interleukin 2 receptor subunit gamma (Il2rg) and recombination activating gene-2 (Rag2) in the c-Kit W-sh mice, to create Il2rg −/-Rag2 −/c-Kit W-sh/W-sh mice (aka Il2rg −/-Rag2 −/-W sh /W sh ). This newly created Il2rg −/-Rag2 −/c-Kit W-sh/W-sh mouse strain developed unexpected morbidity and mortality associated with infection of pks + Escherichia coli, urosepsis and meningitis [8]. In the present study, we identified and determined the complete genome sequence of a new strain of MKPV infecting Il2rg −/-Rag2 −/c-Kit W-sh/W-sh mice and selected additional mouse strains/stocks.…”

Cytotoxic Escherichia coli strains encoding colibactin, cytotoxic necrotizing factor, and cytolethal distending toxin colonize laboratory common marmosets (Callithrix jacchus)

“…As such, the prevalence of cyclomodulin-encoding E. coli in laboratory marmosets is currently unknown. We hypothesized that common marmosets, similar to laboratory rodents and other laboratory nonhuman primates 25 – 29 , are colonized by cyclomodulin-encoding E. coli , and the prevalence of pks +, cnf +, and cdt + E. coli in our population would vary significantly depending on the colony of origin, as has been demonstrated in laboratory rats 28 . In addition, we hypothesized that pks +, cnf +, and cdt + E. coli are present in animals afflicted with gastrointestinal disease with greater frequency than in clinically normal animals.…”