Salmonella Typhimurium Diarrhea Reveals Basic Principles of Enteropathogen Infection and Disease-Promoted DNA Exchange

ISSN:1931-3128ISSN:1934-606

Peracetic Acid Treatment Generates Potent Inactivated Oral Vaccines from a Broad Range of Culturable Bacterial Species.

Our mucosal surfaces are the main sites of non-vector-borne pathogen entry, as well as the main interface with our commensal microbiota. We are still only beginning to understand how mucosal adaptive immunity interacts with commensal and pathogenic microbes to influence factors such as infectivity, phenotypic diversity, and within-host evolution. This is in part due to difficulties in generating specific mucosal adaptive immune responses without disrupting the mucosal microbial ecosystem itself. Here, we present a very simple tool to generate inactivated mucosal vaccines from a broad range of culturable bacteria. Oral gavage of 10(10) peracetic acid-inactivated bacteria induces high-titer-specific intestinal IgA in the absence of any measurable inflammation or species invasion. As a proof of principle, we demonstrate that this technique is sufficient to provide fully protective immunity in the murine model of invasive non-typhoidal Salmonellosis, even in the face of severe innate immune deficiency

Peracetic acid treatment generates potent inactivated oral vaccines from a broad range of culturable bacterial species

Our mucosal surfaces are the main sites of non-vector-borne pathogen entry, as well as the main interface with our commensal microbiota. We are still only beginning to understand how mucosal adaptive immunity interacts with commensal and pathogenic microbes to influence factors such as infectivity, phenotypic diversity, and within-host evolution. This is in part due to difficulties in generating specific mucosal adaptive immune responses without disrupting the mucosal microbial ecosystem itself. Here, we present a very simple tool to generate inactivated mucosal vaccines from a broad range of culturable bacteria. Oral gavage of 1010 peracetic acid-inactivated bacteria induces high-titer-specific intestinal IgA in the absence of any measurable inflammation or species invasion. As a proof of principle, we demonstrate that this technique is sufficient to provide fully protective immunity in the murine model of invasive non-typhoidal Salmonellosis, even in the face of severe innate immune deficiency

Microbiota stability in healthy individuals after single-dose lactulose challenge-A randomized controlled study.

BACKGROUND AND AIMS:Lactulose is a common food ingredient and widely used as a treatment for constipation or hepatic encephalopathy and a substrate for hydrogen breath tests. Lactulose is fermented by the colon microbiota resulting in the production of hydrogen (H2). H2 is a substrate for enteropathogens including Salmonella Typhimurium (S. Typhimurium) and increased H2 production upon lactulose ingestion might favor the growth of H2-consuming enteropathogens. We aimed to analyze effects of single-dose lactulose ingestion on the growth of intrinsic Escherichia coli (E. coli), which can be efficiently quantified by plating and which share most metabolic requirements with S. Typhimurium. METHODS:32 healthy volunteers (18 females, 14 males) were recruited. Participants were randomized for single-dose ingestion of 50 g lactulose or 50 g sucrose (controls). After ingestion, H2 in expiratory air and symptoms were recorded. Stool samples were acquired at days -1, 1 and 14. We analyzed 16S microbiota composition and abundance and characteristics of E. coli isolates. RESULTS:Lactulose ingestion resulted in diarrhea in 14/17 individuals. In 14/17 individuals, H2-levels in expiratory air increased by ≥20 ppm within 3 hours after lactulose challenge. H2-levels correlated with the number of defecations within 6 hours. E. coli was detectable in feces of all subjects (2 x 10(2)-10(9) CFU/g). However, the number of E. coli colony forming units (CFU) on selective media did not differ between any time point before or after challenge with sucrose or lactulose. The microbiota composition also remained stable upon lactulose exposure. CONCLUSION:Ingestion of a single dose of 50 g lactulose does not significantly alter E. coli density in stool samples of healthy volunteers. 50 g lactulose therefore seems unlikely to sufficiently alter growth conditions in the intestine for a significant predisposition to infection with H2-consuming enteropathogens such as S. Typhimurium (www.clinicaltrials.gov NCT02397512)

<i>S.</i> Tm^WITS infection and a modeling reveal slow growth of tolerant bacteria.

<p>(A) Schematic representation of the stochastic birth-death process modified by immigration and its parameterization (for details, see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001793#s4" target="_blank">Materials and Methods</a>). During the first 24 h p.i., we analyzed cLN colonization dynamics in absence of ciprofloxacin. Ciprofloxacin treatment was started by 24 h p.i. and continued until day 3, 5, or 10, as indicated. The model for analyzing the latter data is displayed on the right side. (B) Mice were infected with <i>S.</i> Tm^WITS and treated with ciprofloxacin (2×62 mg/kg/d by gavage) from day 1 p.i. until the indicated end of the experiment (day 1, <i>n</i> = 49; day 3, <i>n</i> = 28; day 5, <i>n</i> = 28; day 10, <i>n</i> = 28 data points). CFU determination and analysis of tag abundance using rtqPCR (experimental data, <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001793#pbio.1001793.s019" target="_blank">Table S2</a>) was used to fit the mathematical model. Graphic display of experimental data (x) and 100 simulations (grey lines). (C) Parameters (with confidence intervals) derived from fitting the mathematical model to our experimental data. <i>r</i>, replication rate; doubling time, ln2/<i>r</i>×h; <i>c</i>, clearance rate; half-life, ln0.5/<i>c</i>×h.</p

Plasmid dilution experiment verifying the slow growth rate of tolerant <i>S.</i> Tm cells in vivo.

<p>(Left) Experimental strategy. (Right) Experimental data. Three groups of C57BL/6 mice were infected for 2 d or 3 d with <i>S.</i> Tm(pAM34), as indicated. The third group was treated with ciprofloxacin during the third day (2×62 mg/kg/d by gavage). Total <i>S.</i> Tm loads (closed symbols) and <i>S.</i> Tm(pAM34) (open symbols) in the cLN were determined by plating. Dashed line, detection limit. <i>p</i>, nonparametric statistical analysis. n.s., not significant.</p

Ciprofloxacin treatment cannot clear <i>S.</i> Tm from the cLN.

<p>Cfus in the cLN of C57BL/6 mice 1 d and 3 d postinfection without therapy or 3 d, 5 d, and 10 d p.i. with ciprofloxacin therapy (2×62 mg/kg/d by gavage) starting 1 d p.i. Each symbol represents one mouse. (A), cfu in stool from cecum lumen; (B), cfu in spleen; (C), cfu in cLN; dashed line, detection limit. <i>p</i>, nonparametric statistical analysis compared to 1 d p.i.</p

<i>S.</i> Tm in cLN from ciprofloxacin-treated mice are sufficient for initiating an infection.

<p>C57BL/6 mice were infected for 1 d with <i>S.</i> Tm before ciprofloxacin therapy was started (2×62 mg/kg/d by gavage). Recipient mice remained uninfected and were also treated with ciprofloxacin. (Left) 3 d p.i. mice were sacrificed and single-cell suspensions of the cLN or spleen or cecal content were transferred into antibiotic-treated naïve recipient mice. Four days later, recipient mice were sacrificed and pathogen loads in the respective organs were determined. Transfer of cLN cells leads to significantly higher infection rates compared to transferred spleen cells, cecum content, or <i>S.</i> Tm cultured in LB (right part; Fisher's exact test, <i>p</i> = 0.00482). (Middle) Relapse control. Mice were infected for 1 d with <i>S.</i> Tm, treated with ciprofloxacin for 2 d, and left untreated for 4 additional days. Then, we analyzed pathogen loads in the cecum content, cLN, spleen, and liver.</p

Ciprofloxacin-tolerant bacteria reside within classical CD11c⁺ dendritic cells of the cLN.

<p>(A) CD11c-YFP mice were infected with <i>S.</i> Tm pDsRed and subjected to ciprofloxacin therapy (2×62 mg/kg/d by gavage; control group was treated with PBS). We prepared 20 µm cryo sections of the cLNs and analyzed them by fluorescence microscopy. Quantification shows accumulation of bacteria within CD11c^high cells. (B) Gating approach used for subsequent sorting by flow cytometry. Three nonlymphoid subsets were defined as (1) CX₃CR1⁺ iDCs: CD103⁻CX₃CR1⁺, (2) “classical” dendritic cells CD103⁺CX₃CR1⁻CD11c⁺Gr-1⁻ (cDC), and (3) CD103⁻CX₃CR1⁻CD11c⁻Gr-1⁺ “neutrophils.” (C) Frequency and respective bacterial load of <i>S.</i> Tm harboring CX₃CR1⁺ interstitial and classical DC subsets. Filled symbols, ciprofloxacin therapy; empty symbols, no therapy. (D) Depletion of CD11c⁺ cells in CD11c-DTR mice via DTX reduces the loads of tolerant bacteria (left side), whereas expansion of CD11c⁺ cells via FLT3-L injection (10 µg/mouse/d for 3 d) increases the loads of tolerant bacteria in the cLN of ciprofloxacin-treated mice (right side).</p