Systematic Analysis of Mobile Genetic Elements Mediating ß-Lactamase Gene Amplification in Noncarbapenemase-Producing Carbapenem-Resistant Enterobacterales Bloodstream Infections.

Noncarbapenemase-producing carbapenem-resistant Enterobacterales (non-CP-CRE) are increasingly recognized as important contributors to prevalent carbapenem-resistant Enterobacterales (CRE) infections. However, there is limited understanding of mechanisms underlying non-CP-CRE causing invasive disease. Long- and short-read whole-genome sequencing was used to elucidate carbapenem nonsusceptibility determinants in Enterobacterales bloodstream isolates at MD Anderson Cancer Center in Houston, Texas. We investigated carbapenem nonsusceptible Enterobacterales (CNSE) mechanisms (i.e., isolates with carbapenem intermediate resistance phenotypes or greater) through a combination of phylogenetic analysis, antimicrobial resistance gene detection/copy number quantification, porin assessment, and mobile genetic element (MGE) characterization. Most CNSE isolates sequenced were non-CP-CRE (41/79; 51.9%), whereas 25.3% (20/79) were Enterobacterales with intermediate susceptibility to carbapenems (CIE), and 22.8% (18/79) were carbapenemase-producing Enterobacterales (CPE). Statistically significant copy number variants (CNVs) of extended-spectrum ß-lactamase (ESBL) genes (Wilcoxon Test; P-value < 0.001) were present in both non-CP-CR E. coli (median CNV = 2.6×; n = 17) and K. pneumoniae (median CNV = 3.2×, n = 17). All non-CP-CR E. coli and K. pneumoniae had predicted reduced expression of at least one outer membrane porin gene (i.e., ompC/ompF or ompK36/ompK35). Completely resolved CNSE genomes revealed that IS26 and ISEcp1 structures harboring blaCTX-M variants along with other antimicrobial resistance elements were associated with gene amplification, occurring in mostly IncFIB/IncFII plasmid contexts. MGE-mediated ß-lactamase gene amplifications resulted in either tandem arrays, primarily mediated by IS26 translocatable units, or segmental duplication, typically due to ISEcp1 transposition units. Non-CP-CRE strains were the most common cause of CRE bacteremia with carbapenem nonsusceptibility driven by concurrent porin loss and MGE-mediated amplification of blaCTX-M genes. IMPORTANCE Carbapenem-resistant Enterobacterales (CRE) are considered urgent antimicrobial resistance (AMR) threats. The vast majority of CRE research has focused on carbapenemase-producing Enterobacterales (CPE) even though noncarbapenemase-producing CRE (non-CP-CRE) comprise 50% or more of isolates in some surveillance studies. Thus, carbapenem resistance mechanisms in non-CP-CRE remain poorly characterized. To address this problem, we applied a combination of short- and long-read sequencing technologies to a cohort of CRE bacteremia isolates and used these data to unravel complex mobile genetic element structures mediating ß-lactamase gene amplification. By generating complete genomes of 65 carbapenem nonsusceptible Enterobacterales (CNSE) covering a genetically diverse array of isolates, our findings both generate novel insights into how non-CP-CRE overcome carbapenem treatments and provide researchers scaffolds for characterization of their own non-CP-CRE isolates. Improved recognition of mechanisms driving development of non-CP-CRE could assist with design and implementation of future strategies to mitigate the impact of these increasingly recognized AMR pathogens.

Enterocolitis induced by autoimmune targeting of enteric glial cells: a possible mechanism in Crohn's disease?

Early pathological manifestations of Crohn's disease (CD) include vascular disruption, T cell infiltration of nerve plexi, neuronal degeneration, and induction of T helper 1 cytokine responses. This study demonstrates that disruption of the enteric glial cell network in CD patients represents another early pathological feature that may be modeled after CD8(+) T cell-mediated autoimmune targeting of enteric glia in double transgenic mice. Mice expressing a viral neoself antigen in astrocytes and enteric glia were crossed with specific T cell receptor transgenic mice, resulting in apoptotic depletion of enteric glia to levels comparable in CD patients. Intestinal and mesenteric T cell infiltration, vasculitis, T helper 1 cytokine production, and fulminant bowel inflammation were characteristic hallmarks of disease progression. Immune-mediated damage to enteric glia therefore may participate in the initiation and/or the progression of human inflammatory bowel disease.

Developmental regulation of intestinal epithelial hydrolase activity in human fetal jejunal xenografts maintained in severe-combined immunodeficient mice.

Intestinal epithelial brush border hydrolases are important and sensitive enzyme markers of gastrointestinal development and function. Little is know about the mechanisms that regulate the induction of these enzymes during human fetal development, as these events occur primarily in utero. The present work used ectopically grafted human fetal jejunal xenografts (median age,13.3 wk of gestation), maintained in severe-combined immunodeficient mice, to study the differential expression of five different hydrolases after 10 wk of xenotransplantation. The spatio-temporal distribution of brush border alkaline phosphatase, aminopeptidase-N, alpha-glucosidase, lactase-phlorizin hydrolase, and dipeptidyl peptidase IV enzyme activities were measured quantitatively using scanning microdensitometry along the crypt-villus axes of fetal, xenograft, and pediatric (median age, 34 mo) biopsies. Ectopic grafting of fetal jejunum closely recapitulated the development of these enzymes in utero, with alkaline phosphatase, aminopeptidase-N, alpha-glucosidase, and dipeptidyl peptidase IV enzyme activities closely matching the spatio-temporal distribution and levels recorded in pediatric duodenal biopsies. Lactase-phlorizin hydrolase was the only enzyme not to reach values recorded in pediatric brush border membranes, although activities were significantly (5.6-fold) higher than in pretransplanted fetal bowel. Human jejunal xenografts therefore demonstrate an appropriate developmental induction of brush border hydrolase activity and may represent a useful model to study trans-acting factors that promote human epithelial differentiation and function in vivo. Characterization of such agents may be of potential therapeutic use in the treatment of diseases associated with gastrointestinal immaturity, notably necrotizing enterocolitis.

Autoimmune enteropathy with distinct mucosal features in T-cell activation deficiency: the contribution of T cells to the mucosal lesion.

BACKGROUND: Autoimmune enteropathy is normally characterised by crypt hyperplastic villous atrophy with enterocyte autoantibodies, activation of mucosal lymphocytes and increased epithelial HLA-DR. This case involved a severely affected Portuguese infant who was found to have lymphocyte activation deficiency and demonstrated correspondingly distinct mucosal features. METHODS: A female infant of nonconsanguineous parents was treated for vomiting and diarrhoea, first with milk exclusion and then with parenteral nutrition. Lymphocyte subsets and immunoglobulin concentrations were normal, but in vitro testing showed no activation in response to phytohaemagglutinin, Candida, or purified protein derivative, although the response to interleukin (IL)-2 was intact. Interleukin-2 deficiency was excluded. Analysis of jejunal biopsy specimens revealed only mild villous blunting with absent goblet cells, normal epithelial proliferation, and no crypt hyperplasia. The dense infiltrate of CD8+ and CD4+ T lymphocytes showed normal CD2 and CD3 expression but no activation or proliferation markers. HLA-DR was not increased on epithelium or lymphocytes. Thus, in addition to in vitro evidence for lymphocyte activation deficiency, the mucosal specimens showed no evidence of in situ T-cell activation. RESULTS: After development of overwhelming septicaemia, the patient died at 18 months, just before a planned bone marrow transplant. CONCLUSIONS: These findings confirm significant heterogeneity within autoimmune enteropathy. Formal immune function testing should be performed in all affected infants to identify T-cell activation deficiencies. The distinct mucosal findings suggest that activated T cells usually induce the crypt hyperplastic villous atrophy characteristic of classic autoimmune enteropathy.

Characterization of candidate live oral Salmonella typhi vaccine strains harboring defined mutations in aroA, aroC, and htrA.

The properties of two candidate Salmonella typhi-based live oral typhoid vaccine strains, BRD691 (S. typhi Ty2 harboring mutations in aroA and aroC) and BRD1116 (S. typhi Ty2 harboring mutations in aroA, aroC, and htrA), were compared in a number of in vitro and in vivo assays. BRD1116 exhibited an increased susceptibility to oxidative stress compared with BRD691, but both strains were equally resistant to heat shock. Both strains showed a similar ability to invade Caco-2 and HT-29 epithelial cells and U937 macrophage-like cells, but BRD1116 was less efficient at surviving in epithelial cells than BRD691. BRD1116 and BRD691 were equally susceptible to intracellular killing within U937 cells. Similar findings were demonstrated in vivo, with BRD1116 being less able to survive and translocate to secondary sites of infection when inoculated into the lumen of human intestinal xenografts in SCID mice. However, translocation of BRD1116 to spleens and livers in SCID mice occurred as efficiently as that of BRD691 when inoculated intraperitonally. The ability of BRD1116 to increase the secretion of interleukin-8 following infection of HT-29 epithelial cells was comparable to that of BRD691. Therefore, loss of the HtrA protease in S. typhi does not seem to alter its ability to invade epithelial cells or macrophages or to induce proinflammatory cytokines such as IL-8 but significantly reduces intracellular survival in human intestinal epithelial cells in vitro and in vivo.

Apoptosis of human intestinal epithelial cells after bacterial invasion.

Epithelial cells that line the human intestinal mucosa are the initial site of host invasion by bacterial pathogens. The studies herein define apoptosis as a new category of intestinal epithelial cell response to bacterial infection. Human colon epithelial cells are shown to undergo apoptosis following infection with invasive enteric pathogens, such as Salmonella or enteroinvasive Escherichia coli. In contrast to the rapid onset of apoptosis seen after bacterial infection of mouse monocyte-macrophage cell lines, the commitment of human intestinal epithelial cell lines to undergo apoptosis is delayed for at least 6 h after bacterial infection, requires bacterial entry and replication, and the ensuing phenotypic expression of apoptosis is delayed for 12-18 h after bacterial entry. TNF-alpha and nitric oxide, which are produced as components of the intestinal epithelial cell proinflammatory program in the early period after bacterial invasion, play an important role in the later induction and regulation of the epithelial cell apoptotic program. Apoptosis in response to bacterial infection may function to delete infected and damaged epithelial cells and restore epithelial cell growth regulation and epithelial integrity that are altered during the course of enteric infection. The delay in onset of epithelial cell apoptosis after bacterial infection may be important both to the host and the invading pathogen since it provides sufficient time for epithelial cells to generate signals important for the activation of mucosal inflammation and concurrently allows invading bacteria time to adapt to the intracellular environment before invading deeper mucosal layers.

Mucin gene expression in human embryonic and fetal intestine.

BACKGROUND: The intestinal epithelium is covered by a continuous layer of mucus which is secreted by well differentiated epithelial cells. Disregulation of the expression of mucins has been reported to have possible implications in the neoplastic process which affects intestinal mucosae. It is well known that preneoplastic and neoplastic tissues can express fetal phenotypic characteristics. AIMS: To assess whether the expression of mucin genes in the intestinal tract is linked to the stage of cellular differentiation and tissue development, by studying the expression of six mucin genes in human fetal small intestine and colon, and also adult tissues. METHODS: In situ hybridisation was used to study mRNA expression of MUC2, MUC3, MUC4, MUC5B, MUC5AC, and MUC6 in 32 human embryos and fetuses (6.5-27 weeks gestation). Normal adult mucosae were used as controls. RESULTS: Three mucin genes, MUC2, MUC4, and MUC5AC, were differently expressed in fetal intestine compared with expression in normal adults. CONCLUSION: These differences in mucin gene expression suggest a possible regulatory role for these products in intestinal epithelial cell differentiation.

Fulminant jejuno-ileitis following ablation of enteric glia in adult transgenic mice.

To investigate the roles of astroglial cells, we targeted their ablation genetically. Transgenic mice were generated expressing herpes simplex virus thymidine kinase from the mouse glial fibrillary acidic protein (GFAP) promoter. In adult transgenic mice, 2 weeks of subcutaneous treatment with the antiviral agent ganciclovir preferentially ablated transgene-expressing, GFAP-positive glia from the jejunum and ileum, causing a fulminating and fatal jejuno-ileitis. This pathology was independent of bacterial overgrowth and was characterized by increased myeloperoxidase activity, moderate degeneration of myenteric neurons, and intraluminal hemorrhage. These findings demonstrate that enteric glia play an essential role in maintaining the integrity of the bowel and suggest that their loss or dysfunction may contribute to the cellular mechanisms of inflammatory bowel disease.

Human intestinal epithelial cells respond to Cryptosporidium parvum infection with increased prostaglandin H synthase 2 expression and prostaglandin E2 and F2alpha production.

Cryptosporidium parvum is an important cause of diarrhea in humans and several animal species. Prostaglandins play a central role in regulating intestinal fluid secretion in animal models of cryptosporidiosis, but their cellular sources and mechanisms of induction are unclear. Here, we show that C. parvum infection directly activates prostaglandin H synthase 2 expression and prostaglandin E2 and F2alpha production in human intestinal epithelial cells.

Cryptosporidium parvum infection of human intestinal epithelial cells induces the polarized secretion of C-X-C chemokines.

Cryptosporidium parvum infects intestinal epithelial cells and does not invade deeper layers of the intestinal mucosa. Nonetheless, an inflammatory cell infiltrate that consists of neutrophils and mononuclear cells is often present in the lamina propria, which underlies the epithelium. This study investigated the host epithelial cell response to C. parvum by assessing in vitro and in vivo the expression and production of proinflammatory cytokines by intestinal epithelial cells after infection. The human colon epithelial cell lines HCT-8 and Caco-2 and human intestinal xenografts in SCID mice were infected with C. parvum. The expression and secretion of the C-X-C chemokines interleukin-8 (IL-8) and GROalpha were determined by reverse transcription-PCR analysis and enzyme-linked immunosorbent assay. Our results demonstrate that upregulated expression and secretion of IL-8 and GROalpha after C. parvum infection of intestinal epithelial cells first occurred 16 to 24 h after infection and increased over the ensuing 1 to 2 days. The kinetics of C-X-C chemokine production by C. parvum-infected epithelial cells contrast markedly with the rapid but transient expression of C-X-C chemokines by epithelial cells infected with invasive enteric bacteria. C-X-C chemokine secretion in C. parvum-infected epithelial cells occurred predominantly from the basolateral surface in polarized monolayers of Caco-2 cells grown in Transwell cultures, whereas cell lysis occurred at the apical surface. The basolateral secretion of IL-8 and GROalpha from C. parvum-infected epithelial cells suggests that C-X-C chemokines produced by those cells contribute to the mucosal inflammatory cell infiltrate in the underlying intestinal mucosa.

Role of intestinal epithelial cells in the host secretory response to infection by invasive bacteria. Bacterial entry induces epithelial prostaglandin h synthase-2 expression and prostaglandin E2 and F2alpha production.

Increased intestinal fluid secretion is a protective host response after enteric infection with invasive bacteria that is initiated within hours after infection, and is mediated by prostaglandin H synthase (PGHS) products in animal models of infection. Intestinal epithelial cells are the first host cells to become infected with invasive bacteria, which enter and pass through these cells to initiate mucosal, and ultimately systemic, infection. The present studies characterized the role of intestinal epithelial cells in the host secretory response after infection with invasive bacteria. Infection of cultured human intestinal epithelial cell lines with invasive bacteria, but not noninvasive bacteria, is shown to induce the expression of one of the rate-limiting enzymes for prostaglandin formation, PGHS-2, and the production of PGE2 and PGF2alpha. Furthermore, increased PGHS-2 expression was observed in intestinal epithelial cells in vivo after infection with invasive bacteria, using a human intestinal xenograft model in SCID mice. In support of the physiologic importance of epithelial PGHS-2 expression, supernatants from bacteria-infected intestinal epithelial cells were shown to increase chloride secretion in an in vitro model using polarized epithelial cells, and this activity was accounted for by PGE2. These studies define a novel autocrine/paracrine function of mediators produced by intestinal epithelial cells in the rapid induction of increased fluid secretion in response to intestinal infection with invasive bacteria.

The life and times of an intestinal M cell.

Intestinal M cells are specialized epithelial antigen-sampling cells, strategically located over mucosal lymphoid follicles. They regulate the entry of antigen required for mucosal immune responses. There are several potential mechanisms involved in the induction of intestinal M cells. Both luminal antigen and B cells appear to have an influence on these altered epithelial differentiation pathways.

Epithelial cell proliferation in childhood enteropathies.

BACKGROUND/AIM: The aim of this study was to investigate epithelial cell turnover in childhood enteropathy to establish whether common disease related mechanisms operate. Levels of epithelial cell proliferation were measured in children with food intolerance (cows' milk protein intolerance and coeliac disease), and after infection with Giardia lamblia, Cryptosporidium, and enteropathogenic Escherichia coli. METHODS: Comparative measures of epithelial cell proliferation were performed by recording mitotic activity and MIB-1 immunoreactivity in proximal small intestinal biopsy specimens. RESULTS/CONCLUSIONS: A hyperplastic crypt response was evident in all of the disease states examined and was particularly pronounced in coeliac disease and in infection with enteropathogenic E coli, where mitotic and MIB-1 labelling indices were significantly raised above control values. In contrast with coeliac disease, increased crypt cell production rates in enteropathogenic E coli infection were also due to an expansion of the crypt proliferation compartment, without a comparable increase in crypt cell numbers. Crypt hyperplasia is therefore a common tissue response to mucosal damage in food allergy and infection, although disease specific mechanisms are evident.

Infection of human intestinal epithelial cells with invasive bacteria upregulates apical intercellular adhesion molecule-1 (ICAM)-1) expression and neutrophil adhesion.

The acute host response to gastrointestinal infection with invasive bacteria is characterized by an accumulation of neutrophils in the lamina propria, and neutrophil transmigration to the luminal side of the crypts. Intestinal epithelial cells play an important role in the recruitment of inflammatory cells to the site of infection through the secretion of chemokines. However, little is known regarding the expression, by epithelial cells, of molecules that are involved in interactions between the epithelium and neutrophils following bacterial invasion. We report herein that expression of ICAM-1 on human colon epithelial cell lines, and on human enterocytes in an in vivo model system, is upregulated following infection with invasive bacteria. Increased ICAM-1 expression in the early period (4-9 h) after infection appeared to result mainly from a direct interaction between invaded bacteria and host epithelial cells since it co-localized to cells invaded by bacteria, and the release of soluble factors by epithelial cells played only a minor role in mediating increased ICAM-1 expression. Furthermore, ICAM-1 was expressed on the apical side of polarized intestinal epithelial cells, and increased expression was accompanied by increased neutrophil adhesion to these cells. ICAM-1 expression by intestinal epithelial cells following infection with invasive bacteria may function to maintain neutrophils that have transmigrated through the epithelium in close contact with the intestinal epithelium, thereby reducing further invasion of the mucosa by invading pathogens.

Conventional patterns of human intestinal proliferation in a severe-combined immunodeficient xenograft model.

The present work describes the pattern of human intestinal proliferation in an immunodeficient murine xenograft model, which we have shown to closely mimic cell division in normal paediatric gut. Cellular proliferation was measured using a double-label technique combining MIB-1 immunohistochemistry and [3H]thymidine autoradiography, to critically compare values for the tissue growth fraction (G1, G2, S- and M-phase cells) and DNA synthesizing (S-phase) cells in xenograft epithelium, lamina propria, muscularis externa and intraepithelial lymphocytes. The MIB-1 monoclonal antibody (which recognises the cell-cycle dependent nuclear antigen Ki-67) specifically labelled proliferating human cells within the xenografts and did not cross-react with dividing murine cells. This was confirmed using ultrastructural in situ hybridisation with human- and mouse-specific DNA probes to identify the genetic origin of proliferating cells. In general, we found a good tissue correlation between MIB-1 and [3H]thymidine labelling, the only exception being an apparent dysregulation of Ki-67 antigen expression in regenerating xenograft epithelium. In developed xenograft intestine, the highest levels of proliferation were consistently recorded within the crypt epithelium, where 15.7%-26.7% of cells were actively cycling and S-phase occupied approximately half of the cell cycle. The frequency distribution of proliferating epithelial cells within small and large intestinal xenograft crypts was clearly tissue-specific, showing typical patterns of cell division. Therefore, the presence of functional pluripotent epithelial stem cells and conventional spatio-temporal patterns in cellular proliferation, migration, de-cycling, lineage commitment and cytodifferentiation now makes this an attractive experimental model with which to study human intestinal crypt responses to various types of tissue manipulation, e.g. cytotoxic, radiotherapeutic, dietary, endocrine and gene-targeting therapy.

Human intestinal development in a severe-combined immunodeficient xenograft model.

The present work describes a severe-combined immunodeficient murine xenograft model used to investigate human gastrointestinal ontogenesis. Specifically, the study has tested whether carefully selected regions of human fetal gut are able to undergo region-specific morphogenesis and epithelial cytodifferentiation when transplanted subcutaneously into immunodeficient mice. In addition, double-label in situ hybridisation techniques, utilising specific human and mouse DNA probes, have been adopted to characterise host and donor cell types and to investigate the potential developmental roles for non-epithelial cells in the regulation of epithelial differentiation pathways in vivo. Human fetal small and large bowel developed to form a characteristic mucosa 10 weeks after transplantation, which displayed clear region-specific structural and functional gradients. The initial phase of xenograft epithelialisation closely resembled the stratified type of epithelium which is present during early fetal gastrointestinal development. Idiosyncratic epithelial differentiation pathways were recorded during xenograft regeneration, with an absence of Paneth cells and an abundance of enteroendocrine cells when compared with developed xenograft and paediatric intestine. Such differences may, therefore, be important in ensuring rapid and region-specific development in the absence of conventional luminal stimuli and hormonal changes that occur normally during pregnancy. In situ hybridisation demonstrated an exclusively human origin for the intestinal xenograft epithelium and muscularis mucosa and externa. Although the submucosa and lamina propria were comprised of a chimeric mixture, murine cells were rarely seen to contact with the epithelium, which interacted primarily with human myofibroblasts and human intraepithelial lymphocytes. It is proposed that a 'selection' process operates to maintain species-specific cellular interactions, and this mechanism may subsequently play an important role in regulating epithelial cell differentiation, orchestrated in part by juxtaposed non-epithelial cell types.

Novel insights into human intestinal epithelial cell proliferation in health and disease using confocal microscopy.

Measurement of intestinal epithelial cell proliferation has provided important information concerning tissue responses in neoplasia, enteropathy, and adaptation. This study reexamined current concepts regarding intestinal proliferation by using a novel confocal microscopical technique to map mitotic figures accurately within the intact three dimensional framework of the crypts of Lieberkühn. The ability of confocal microscopy to simultaneously measure crypt morphology and internal detail, without disrupting spatial cell arrangements, has provided important new data. These question the ability of existing methods to accurately measure and interpret proliferative changes in the gut. This work investigates intestinal proliferation in children with coeliac disease, a well defined hyperproliferative disorder, in comparison with the healthy intestine. These results show that crypt cell division occurs with an equal probability in health and disease. In addition, increased crypt cell production rates are largely caused by a change in crypt size rather than a change in cell cycle time or crypt growth fraction and, as such, alter our understanding of kinetic responses in gastrointestinal disease.