Role of the microbiome in the normal and aberrant glycemic response

Multiple studies in the recent years suggest that the microbiome is critically important for normal host functions, while impaired host microbiome interactions contribute to the pathogenesis of numerous common disorders. Of these, much attention is recently given to the involvement of the microbiome in the pathogenesis of impaired glucose tolerance, type II diabetes mellitus (T2DM), and other metabolic disorders comprising the 'metabolic syndrome', including obesity, non-alcoholic fatty liver disease and their complications. In addition, alterations in the microbiome have been linked to the pathogenesis of type 1 diabetes mellitus (T1DM), an autoimmune disorder affecting the glycemic response, of distinct pathogenesis than T2DM. In this chapter we will discuss the roles of the microbiome in regulating the normal and impaired glycemic response in both mice and humans, and outline examples of underlying mechanisms by which the microbiome is contributing to diabetes mellitus. We will further discuss means by which the microbiome can be manipulated to develop future therapeutic interventions for hyperglycemia and its adverse effects

Microbiomes in physiology: insights into 21st century global medical challenges

New Findings: What is the topic of this review? The role of the gut microbiome in physiology and how it can be targeted as an effective strategy against two of the most important global medical challenges of our time, namely, metabolic diseases and antibacterial resistance. What advances does it highlight? The critical roles of the microbiome in regulating host physiology and how microbiome analysis is useful for disease stratification to enable informed clinical decisions and develop interventions such as faecal microbiota transplantation, prebiotics and probiotics. Also, the limitations of microbiome modulation, including the potential for probiotics to enhance antimicrobial resistance gene reservoirs, and that currently a ‘healthy microbiome’ that can be used as a biobank for transplantation is yet to be defined. Abstract: The human gut microbiome is a key factor in the development of metabolic diseases and antimicrobial resistance, which are among the greatest global medical challenges of the 21st century. A recent symposium aimed to highlight state-of-the-art evidence for the role of the gut microbiome in physiology, from childhood to adulthood, and the impact this has on global disease outcomes, ageing and antimicrobial resistance. Although the gut microbiome is established early in life, over time the microbiome and its components including metabolites can become perturbed due to changes such as dietary habits, use of antibiotics and age. As gut microbial metabolites, including short-chain fatty acids, secondary bile acids and trimethylamine-N-oxide, can interact with host receptors including G protein-coupled receptors and can alter host metabolic fluxes, they can significantly affect physiological homoeostasis leading to metabolic diseases. These metabolites can be used to stratify disease phenotypes such as irritable bowel syndrome and adverse events after heart failure and allow informed decisions on clinical management and treatment. While strategies such as use of probiotics, prebiotics and faecal microbiota transplantation have been proposed as interventions to treat and prevent metabolic diseases and antimicrobial resistance, caution must be exercised, first due to the potential of probiotics to enhance antimicrobial resistance gene reservoirs, and second, a ‘healthy gut microbiome’ that can be used as a biobank for transplantation is yet to be defined. We highlight that sampling other parts of the gastrointestinal tract may produce more representative data than the faecal microbiome alone.</p

Virulence Gene Profiling and Pathogenicity Characterization of Non-Typhoidal <em>Salmonella</em> Accounted for Invasive Disease in Humans

<div><p>Human infection with non-typhoidal <i>Salmonella</i> serovars (NTS) infrequently causes invasive systemic disease and bacteremia. To understand better the nature of invasive NTS (iNTS), we studied the gene content and the pathogenicity of bacteremic strains from twelve serovars (Typhimurium, Enteritidis, Choleraesuis, Dublin, Virchow, Newport, Bredeney, Heidelberg, Montevideo, Schwarzengrund, 9,12:l,v:- and Hadar). Comparative genomic hybridization using a <i>Salmonella enterica</i> microarray revealed a core of 3233 genes present in all of the iNTS strains, which include the <i>Salmonella</i> pathogenicity islands 1–5, 9, 13, 14; five fimbrial operons (<i>bcf, csg, stb, sth</i>, <i>sti</i>); three colonization factors <i>(misL, bapA, sinH</i>); and the invasion gene, <i>pagN</i>. In the iNTS variable genome, we identified 16 novel genomic islets; various NTS virulence factors; and six typhoid-associated virulence genes (<i>tcfA</i>, <i>cdtB</i>, <i>hlyE</i>, <i>taiA</i>, STY1413, STY1360), displaying a wider distribution among NTS than was previously known. Characterization of the bacteremic strains in C3H/HeN mice showed clear differences in disease manifestation. Previously unreported characterization of serovars Schwarzengrund, 9,12:l,v:-, Bredeney and Virchow in the mouse model showed low ability to elicit systemic disease, but a profound and elongated shedding of serovars Schwarzengrund and 9,12:l,v:- (as well as Enteritidis and Heidelberg) due to chronic infection of the mouse. Phenotypic comparison in macrophages and epithelial cell lines demonstrated a remarkable intra-serovar variation, but also showed that <i>S</i>. Typhimurium bacteremic strains tend to present lower intracellular growth than gastroenteritis isolates. Collectively, our data demonstrated a common core of virulence genes, which might be required for invasive salmonellosis, but also an impressive degree of genetic and phenotypic heterogeneity, highlighting that bacteremia is a complex phenotype, which cannot be attributed merely to an enhanced invasion or intracellular growth of a particular strain.</p> </div

Integrative Analysis of Salmonellosis in Israel Reveals Association of Salmonella enterica Serovar 9,12:l,v:− with Extraintestinal Infections, Dissemination of Endemic S. enterica Serovar Typhimurium DT104 Biotypes, and Severe Underreporting of Outbreaks

Salmonella enterica is the leading etiologic agent of bacterial food-borne outbreaks worldwide. This ubiquitous species contains more than 2,600 serovars that may differ in their host specificity, clinical manifestations, and epidemiology. To characterize salmonellosis epidemiology in Israel and to study the association of nontyphoidal Salmonella (NTS) serovars with invasive infections, 48,345 Salmonella cases reported and serotyped at the National Salmonella Reference Center between 1995 and 2012 were analyzed. A quasi-Poisson regression was used to identify irregular clusters of illness, and pulsed-field gel electrophoresis in conjunction with whole-genome sequencing was applied to molecularly characterize strains of interest. Three hundred twenty-nine human salmonellosis clusters were identified, representing an annual average of 23 (95% confidence interval [CI], 20 to 26) potential outbreaks. We show that the previously unsequenced S. enterica serovar 9,12:l,v:- belongs to the B clade of Salmonella enterica subspecies enterica, and we show its frequent association with extraintestinal infections, compared to other NTS serovars. Furthermore, we identified the dissemination of two prevalent Salmonella enterica serovar Typhimurium DT104 clones in Israel, which are genetically distinct from other global DT104 isolates. Accumulatively, these findings indicate a severe underreporting of Salmonella outbreaks in Israel and provide insights into the epidemiology and genomics of prevalent serovars, responsible for recurring illness

Presence and expression of typhoid-associated virulence factors among NTS serovars.

<p>(A) PCR analysis was used to confirm CGH results and to determine the distribution of three typhoid-associated genes in 35 clinical isolates from the 12 NTS serovars. PCR amplicons of 353-bp, 294-bp, and 335-bp indicate the presence of <i>cdtB</i>, <i>hlyE</i> and <i>tcfA</i>, respectively. Tested isolates are numbered from 1–35 according to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0058449#pone.0058449.s001" target="_blank">Table S1</a>, with the isolates that were characterized in mice and subjected to CGH analysis in bold. <i>S.</i> Typhi CT18 (CT18) and <i>S.</i> Typhimurium SL1344 (SL1344) were used as positive and negative controls, respectively. (B) Reverse transcription-PCR was applied to examine expression of <i>cdtB</i>, <i>hlyE, taiA</i> and <i>tcfA</i> genes in serovars Schwarzengrund, Montevideo, 9,12:l,v:- and Bredeney. Bacterial RNA was extracted from <i>Salmonella</i> cultures grown to late logarithmic phase in LB, followed by treatment with DNase I and reverse transcription. cDNA was used as template for PCR amplification using the primers listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0058449#pone.0058449.s002" target="_blank">Table S2</a>. <i>Salmonella</i> RNA without a reverse transcriptase treatment (-RT) and purified gDNA were used as negative and positive controls, respectively.</p

Persistent infection of iNTS strains in the mouse model.

<p>Following challenge with ∼5×10³ CFU of the bacteremic strains, fecal pellets were collected from the C3H\HeN mice at the indicated time points during 40 days (or until the animal was sacrificed). Pellets were weighed, homogenized in PBS and plated onto XLD-agar plates to determine the number of CFU/g stool. Shedding of <i>S.</i> Choleraesuis (D) and <i>S.</i> Dublin (E) is shown only until 3 days p.i., as the mice had to be euthanized. Dots represent independent CFU counts in pellets of individual mice.</p

Distribution of virulence genes across NTS bacteremic isolates.

<p>The presence-absence of genes associated with <i>Salmonella</i> virulence were determined in five <i>S</i>. Typhimurium stool isolates (115043, 88359, 93561, 98001, 130100) and five blood isolates (103259, 111682, 116449, 93130, 98666) in addition to 11 invasive strains from serovars Schwarzengrund (124983), 9,12:l,v:- (94293), Bredeney (96115), Choleraesuis (90958), Dublin (74007), Enteritidis (122205), Hadar (121851), Heidelberg (78646), Montevideo (111072), Newport (91532) and Virchow (103033). Where not stated otherwise, presence was determined by CGH using the <i>Salmonella</i> STv7E microarray. <i>a</i>, presence was determined or confirmed by southern blot; <i>b</i>, presence was determined or confirmed by PCR. A plus sign indicates the presence of the gene; white blocks indicate an absence; a variable presence among the blood or stool isolates of <i>S</i>. Typhimurium is shown by the number of positive isolates/ total number of isolates (N = 5); ND, no data (CGH was not conclusive).</p

Pathogenicity of iNTS strains in mice.

<p>Twelve groups of 4–5 female C3H\HeN mice were challenged i.p. with ∼5×10³ CFU of bacteremic strains from serovars Schwarzengrund (isolate number 124983), 9,12:l,v:- (94293), Bredeney (96115), Choleraesuis (90958), Dublin (74007), Enteritidis (122205), Hadar (121851), Heidelberg (78646), Montevideo (111072), Newport (91532), Typhimurium (103259), and Virchow (103033). Survival of the mice during 40 days post-infection is shown (A). At end-points (as shown in A), harvested organs were homogenized and serial dilutions were plated onto XLD-agar plates for CFU count. Bacterial load in each mouse is represented as CFU/organ by individual dots in the liver (B), spleen (C), ileum (D), cecum (E), and colon (F). Geometric mean for each serovar in the different sites is shown by a horizontal line.</p

Intracellular growth of invasive and enteritis strains in epithelial cells.

<p>Clinical isolates (N = 41) from blood and stool sources were grown to late logarithmic phase in LB medium and used to infect epithelial HeLa cells at a MOI of ∼100∶1. Intracellular replication (ratio between recovered CFU at 24 h/CFU at 2 h p.i.) is shown in relation to the stool isolate of each serovar. In <i>S</i>. Typhimurium, replication is presented relative to median value of the stool isolates (isolate 88359). Indicated values present the mean and the SEM of at least 4 independent infections.</p