scholarly journals Novel ex vivo screening assay to preselect farm specific pre- and probiotics in pigs

2021 ◽  
pp. 1-16
Author(s):  
K. Zeilinger ◽  
J. Hellmich ◽  
J. Zentek ◽  
W. Vahjen
Keyword(s):  
Ex Vivo ◽  
Screening Method ◽  
Coli Strain ◽  
Screening Assay ◽  
Future Studies ◽  
E Coli ◽  
Feed Management ◽  
Specific Pathogen ◽  
On Farm ◽  
Ex Vivo Assay

A novel rapid ex vivo assay was developed as part of a concept to determine potential tailor-made combinations of pre- and probiotics for individual farms. Sow faecal slurries from 20 German pig farms were anaerobically incubated with pre- and probiotics or their combinations together with pathogenic strains that are of interest in pig production. Aliquots of these slurries were then incubated with media containing antibiotic mixtures allowing only growth of the specific pathogen. Growth was monitored and lag time was used to determine the residual fitness of the pathogenic strains. The background growth could be inhibited for an Escherichia coli- and a Clostridium difficile- but not for a Clostridium perfringens strain. The prebiotic fructo-oligosaccharides (FOS) and its combination with probiotics reduced the residual fitness of the E. coli strain in some farms. However, notable exceptions occurred in other farms where FOS increased the fitness of the E. coli strain. Generally, combinations of pre- and probiotics did not show additive effects on fitness for E. coli but displayed farm dependent differences. The effects of pre- and probiotics on the residual fitness of the C. difficile strain were less pronounced, but distinct differences between single application of prebiotics and their combination with probiotics were observed. It was concluded that the initial composition of the microbiota in the samples was more determinative for incubations with the C. difficile strain than for incubations with the E. coli strain, as the presumed fermentation of prebiotic products showed less influence on the fitness of the C. difficile strain. Farm dependent differences were pronounced for both pathogenic strains and therefore, this novel screening method offers a promising approach for pre-selecting pre- and probiotics for individual farms. However, evaluation of farm metadata (husbandry, feed, management) will be crucial in future studies to determine a tailor-made solution for combinations of pre- and probiotics for individual farms. Also, refinement of the ex vivo assay in terms of on-farm processing of samples and validation of unambiguous growth for pathogenic strains from individual farms should be addressed.

scholarly journals Human Neutrophil Chemotaxis Is Modulated by Capsule and O Antigen from an Extraintestinal Pathogenic Escherichia coli Strain

2003 ◽  
Vol 71 (11) ◽  
pp. 6435-6445 ◽  
Author(s):  
Thomas A. Russo ◽  
Bruce A. Davidson ◽  
Diana M. Topolnycky ◽  
Ruth Olson ◽  
Stacy A. Morrill ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Ex Vivo ◽  
Specific Antigen ◽  
Lavage Fluid ◽  
Coli Strain ◽  
Neutrophil Chemotaxis ◽  
Gram Negative ◽  
E Coli ◽  
O Antigen

ABSTRACT Gram-negative enteric bacilli, such as Escherichia coli, are common causes of nosocomial pneumonia. The interaction between pulmonary neutrophils and the infecting pathogen is a critical step in determining the outcome. Previous studies from our laboratory, for which a rat model of pneumonia was used, established that pulmonary neutrophil recruitment was modulated by the E. coli virulence factors capsule and O-specific antigen. To begin to understand the mechanism by which this recruitment occurs, we conducted in vitro and ex vivo chemotaxis assays, for which we used a clinically relevant E. coli isolate (CP9) and isogenic derivatives that were deficient in only the O antigen (CP921) or capsule (CP9.137) as chemoattractants with or without the high-affinity N-formylmethionyl-leucyl-phenylalanine receptor antagonist N-tert-butoxycarbonyl-methionine-leucine-phenylalanine (N-t-BOC). Given that only live E. coli was used for the initial in vitro chemotaxis assays, it was predicted that only N-t-BOC-sensitive chemotaxis would occur. However, both N-t-BOC-sensitive and -insensitive chemotaxis was observed. N-t-BOC-insensitive chemotaxis was mediated in part by interleukin 8, which was produced by neutrophils that had migrated toward E. coli. N-t-BOC-insensitive chemotaxis was only observed when live E. coli bacteria, not cell-free E. coli culture supernatants, were used as chemoattractants, suggesting that a direct E. coli-neutrophil interaction was necessary. The presence of both capsule and O antigen diminished total, N-t-BOC-sensitive, and N-t-BOC-insensitive neutrophil chemotaxis in vitro. The presence of capsule significantly decreased total, N-t-BOC-sensitive, and N-t-BOC-insensitive neutrophil chemotaxis ex vivo when cell-free bronchoalveolar lavage fluid from infected rats was used as the source of chemotactic factors. These effects of E. coli capsule and O antigen on neutrophil chemotaxis are novel, and they expand our understanding of the mechanisms by which these virulence traits contribute to the pathogenesis of gram-negative pneumonia and other extraintestinal infections.

scholarly journals The Genome Sequence of Avian Pathogenic Escherichia coli Strain O1:K1:H7 Shares Strong Similarities with Human Extraintestinal Pathogenic E. coli Genomes

2007 ◽  
Vol 189 (8) ◽  
pp. 3228-3236 ◽  
Author(s):  
Timothy J. Johnson ◽  
Subhashinie Kariyawasam ◽  
Yvonne Wannemuehler ◽  
Paul Mangiamele ◽  
Sara J. Johnson ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Genomic Sequence ◽  
Epidemiological Studies ◽  
Coli Strain ◽  
Avian Pathogenic Escherichia Coli ◽  
Future Studies ◽  
E Coli ◽  
Food Borne ◽  
Pathogenic Escherichia Coli ◽  
Complete Genomic

ABSTRACT Escherichia coli strains that cause disease outside the intestine are known as extraintestinal pathogenic E. coli (ExPEC) and include human uropathogenic E. coli (UPEC) and avian pathogenic E. coli (APEC). Regardless of host of origin, ExPEC strains share many traits. It has been suggested that these commonalities may enable APEC to cause disease in humans. Here, we begin to test the hypothesis that certain APEC strains possess potential to cause human urinary tract infection through virulence genotyping of 1,000 APEC and UPEC strains, generation of the first complete genomic sequence of an APEC (APEC O1:K1:H7) strain, and comparison of this genome to all available human ExPEC genomic sequences. The genomes of APEC O1 and three human UPEC strains were found to be remarkably similar, with only 4.5% of APEC O1's genome not found in other sequenced ExPEC genomes. Also, use of multilocus sequence typing showed that some of the sequenced human ExPEC strains were more like APEC O1 than other human ExPEC strains. This work provides evidence that at least some human and avian ExPEC strains are highly similar to one another, and it supports the possibility that a food-borne link between some APEC and UPEC strains exists. Future studies are necessary to assess the ability of APEC to overcome the hurdles necessary for such a food-borne transmission, and epidemiological studies are required to confirm that such a phenomenon actually occurs.

scholarly journals Safety of Probiotic Escherichia coli Strain Nissle 1917 Depends on Intestinal Microbiota and Adaptive Immunity of the Host

2010 ◽  
Vol 78 (7) ◽  
pp. 3036-3046 ◽  
Author(s):  
Kerstin Gronbach ◽  
Ute Eberle ◽  
Martina Müller ◽  
Tobias A. Ölschläger ◽  
Ulrich Dobrindt ◽  
...  
Keyword(s):  
T Cells ◽  
Adaptive Immunity ◽  
Intestinal Microbiota ◽  
Adaptive Immune System ◽  
Coli Strain ◽  
Oral Challenge ◽  
Clinical Effects ◽  
E Coli ◽  
Specific Pathogen ◽  
Future Work

ABSTRACT Probiotics are viable microorganisms that are increasingly used for treatment of a variety of diseases. Occasionally, however, probiotics may have adverse clinical effects, including septicemia. Here we examined the role of the intestinal microbiota and the adaptive immune system in preventing translocation of probiotics (e.g., Escherichia coli Nissle). We challenged C57BL/6J mice raised under germfree conditions (GF-raised C57BL/6J mice) and Rag1 −/− mice raised under germfree conditions (GF-raised Rag1 −/− mice) and under specific-pathogen-free conditions (SPF-raised Rag1 −/− mice) with probiotic E. coli strain Nissle 1917, strain Nissle 1917 mutants, the commensal strain E. coli mpk, or Bacteroides vulgatus mpk. Additionally, we reconstituted Rag1 −/− mice with CD4+ T cells. E. coli translocation and dissemination and the mortality of mice were assessed. In GF-raised Rag1 −/− mice, but not in SPF-raised Rag1 −/− mice or GF-raised C57BL/6J mice, oral challenge with E. coli strain Nissle 1917, but not oral challenge with E. coli mpk, resulted in translocation and dissemination. The mortality rate was significantly higher for E. coli strain Nissle 1917-challenged GF-raised Rag1 −/− mice (100%; P < 0.001) than for E. coli strain Nissle 1917-challenged SPF-raised Rag1 −/ − mice (0%) and GF-raised C57BL/6J mice (0%). Translocation of and mortality due to strain E. coli Nissle 1917 in GF-raised Rag1 −/− mice were prevented when mice were reconstituted with T cells prior to strain E. coli Nissle 1917 challenge, but not when mice were reconstituted with T cells after E. coli strain Nissle 1917 challenge. Cocolonization experiments revealed that E. coli mpk could not prevent translocation of strain E. coli Nissle 1917. Moreover, we demonstrated that neither lipopolysaccharide structure nor flagella play a role in E. coli strain Nissle 1917 translocation and dissemination. Our results suggest that if both the microbiota and adaptive immunity are defective, translocation across the intestinal epithelium and dissemination of the probiotic E. coli strain Nissle 1917 may occur and have potentially severe adverse effects. Future work should define the possibly related molecular factors that promote probiotic functions, fitness, and facultative pathogenicity.

scholarly journals Synthetic Peptides That Exert Antimicrobial Activities in Whole Blood and Blood-Derived Matrices

2002 ◽  
Vol 46 (12) ◽  
pp. 3883-3891 ◽  
Author(s):  
Michael R. Yeaman ◽  
Kimberly D. Gank ◽  
Arnold S. Bayer ◽  
Eric P. Brass
Keyword(s):  
Antimicrobial Activity ◽  
Antimicrobial Peptides ◽  
Whole Blood ◽  
Synthetic Peptides ◽  
Ex Vivo ◽  
Antimicrobial Activities ◽  
Biological Matrices ◽  
E Coli ◽  
Complex Fluid ◽  
Ex Vivo Assay

ABSTRACT Peptides that exert antimicrobial activity in artificial media may lack activity within blood or other complex biological matrices. To facilitate the evaluation of antimicrobial peptides for possible therapeutic utility, an ex vivo assay was developed to assess the extent and durability of peptide antimicrobial activities in complex fluid biomatrices of whole blood, plasma, and serum compared with those in conventional media. Novel antimicrobial peptides (RP-1 and RP-11) were designed based in part on platelet microbicidal proteins. RP-1, RP-11, or gentamicin was introduced into biomatrices either coincident with, or 2 h prior to, inoculation with an Escherichia coli target organism. Antimicrobial activities of peptides were assessed by quantitative culture 2 h after bacterial inoculation and compared to those of peptide-free and gentamicin controls. In whole blood and homologous plasma or serum, introduction of RP-1 or RP-11 coincident with E. coli was associated with a significant reduction in CFU per milliliter versus the respective peptide-free controls. Moreover, substantial antimicrobial activity remained when RP-1 or RP-11 was placed into whole blood or plasma 2 h prior to E. coli inoculation. These results suggest that the peptides were not rapidly inactivated within these biomatrices. Peptide antimicrobial activities were negatively affected by preincubation in serum or in heat-inactivated serum, compared with those of the respective controls. Peptides RP-1 and RP-11 were consistently effective at lower concentrations in biomatrices than in artificial media, indicating favorable antimicrobial interactions with components of blood or blood fractions. Collectively, these findings support the concept that synthetic peptides can be designed to exert potent antimicrobial activities in relevant and complex biological matrices.

scholarly journals Synergistic Effects of Probiotics and Phytobiotics on the Intestinal Microbiota in Young Broiler Chicken

2019 ◽  
Vol 7 (12) ◽  
pp. 684 ◽  
Author(s):  
Hao Ren ◽  
Wilfried Vahjen ◽  
Temesgen Dadi ◽  
Eva-Maria Saliu ◽  
Farshad Goodarzi Boroojeni ◽  
...  
Keyword(s):  
Ex Vivo ◽  
Synergistic Effects ◽  
Coli Strain ◽  
Control Group ◽  
Quantitative Composition ◽  
Single Application ◽  
Bacterial Composition ◽  
Extended Spectrum Beta Lactamase ◽  
E Coli ◽  
Rdna Sequencing

Probiotics and phytobiotics have been studied as in-feed antibiotic alternatives for decades, yet there are no studies on their possible symbiotic effects. In the present study, newly hatched chickens were fed with feeds supplemented either with host-specific Lactobacillus strains (L. agilis and L. salivarius), commercial phytobiotics, or combinations of both. After 13 days of life, crops and caecums were analyzed for bacterial composition (16S rDNA sequencing, qPCR) and activity (bacterial metabolites). Crop and caecum samples were also used to study the ex vivo survival of a broiler-derived extended-spectrum beta-lactamase (ESBL) producing Escherichia coli strain. In the crop, combinations of probiotics and phytobiotics, but not their single application, increased the dominance of lactobacilli. The single application of phytobiotics reduced the metabolite concentrations in the crop, but certain combinations synergistically upregulated the metabolites. Changes in the qualitative and quantitative composition of the caecal microbiota were less pronounced than in the crop. Acetate concentrations were significantly lower for phytobiotics or the L. agilis probiotic strain compared to the control group, but the L. salivarius probiotic showed significantly higher acetate concentrations alone or in combination with one phytobiotic. The synergistic effects on the reduction of the ex vivo survival of an ESBL producing E. coli strain in crop or caecum contents were also observed for most combinations. This study shows the beneficial synergistic effects of probiotics and phytobiotics on the intestinal bacterial composition and their metabolic activity in young broilers. The reduced survival of potentially problematic bacteria, such as ESBL-producing E. coli further indicates that combinations of probiotics and phytobiotics may lead to a more enhanced functionality than their individual supplementation.

A Comparative Ex Vivo Study of Plasminogen Activators and Proteases for Fibrinolytic Activity and Side Effects in Rabbits

1977 ◽  
Vol 37 (01) ◽  
pp. 154-161 ◽  
Author(s):  
B. A Janik ◽  
S. E Papaioannou
Keyword(s):  
Side Effects ◽  
Effective Dose ◽  
Fibrinolytic Activity ◽  
Ex Vivo ◽  
Plasminogen Activators ◽  
Assay System ◽  
Coagulation Parameters ◽  
Ex Vivo Assay

SummaryUrokinase, streptokinase, Brinase, trypsin, and SN 687, a bacterial exoprotease, have been evaluated in an ex vivo assay system. These enzymes were injected into rabbits and the fibrinolytic activity as well as other coagulation parameters were measured by in vitro techniques. Dose-response correlations have been made using the euglobulin lysis time as a measure of fibrinolytic activity and the 50% effective dose has been determined for each enzyme. Loading doses, equal to four times the 50% effective dose, were administered to monitor potential toxicity revealing that Brinase, trypsin, and SN 687 were very toxic at this concentration.Having established the 50% effective dose for each enzyme, further testing was conducted where relevant fibrinolytic and coagulation parameters were measured for up to two days following a 50% effective dose bolus injection of each enzyme. Our results have demonstrated that urokinase and streptokinase are plasminogen activators specifically activating the rabbit fibrinolytic system while Brinase, trypsin and SN 687 increase the general proteolytic activity in vivo.The advantages of this ex vivo assay system for evaluating relative fibrinolytic potencies and side effects for plasminogen activators and fibrinolytic proteases have been discussed.

Transport of escherichia coli through soil to groundwater traced by randomly amplified polymorphic DNA (RAPD)

1997 ◽  
Vol 35 (11-12) ◽  
pp. 351-357 ◽  
Author(s):  
R. Rothmaier ◽  
A. Weidenmann ◽  
K. Botzenhart
Keyword(s):  
Negative Impact ◽  
Random Amplified Polymorphic Dna ◽  
Coli Strain ◽  
Soil Samples ◽  
Test Field ◽  
Liquid Manure ◽  
E Coli ◽  
Rapd Pattern ◽  
Geological Situation ◽  
Different Strains

Isolates (50) of E. coli obtained from liquid manure (20 bovine, 20 porcine) were genotyped using random amplified polymorphic DNA (RAPD). Typing revealed 9 and 14 different strains in bovine and porcine liquid manure respectively with no strains in common. One porcine strain, showing a simple RAPD pattern, was subcultured and spread on a test field (1.5l/m2 at 1010 cfu/l) in a drinking water protection zone with loamy to sandy sediments in the Donauried area, Baden-Wurttemberg. Soil samples and groundwaters were collected at monthly intervals October 1994 – June 1995 during which 114 E. coli isolates were recovered. The first occurrence and maximum concentration of E. coli in soil samples taken from more than 20cm depth was in January 1995, declining rapidly with depth and time. All isolates from soil and only one from groundwater showed the RAPD pattern of the spread E. coli strain. The results could not demonstrate a severe negative impact of the spreading of liquid manure on the bacteriological quality of the groundwater in the given geological situation. The distinct strain patterns found in different kinds of liquid manure suggest that genotyping of E. coli by RAPD may be an adequate tool for tracing sources of faecal contamination.

PAF increases vascular permeability without increasing pulmonary arterial pressure in the rat

2001 ◽  
Vol 90 (1) ◽  
pp. 261-268 ◽  
Author(s):  
Leonardo C. Clavijo ◽  
Mary B. Carter ◽  
Paul J. Matheson ◽  
Mark A. Wilson ◽  
William B. Wead ◽  
...  
Keyword(s):  
Arterial Pressure ◽  
Pulmonary Edema ◽  
Pulmonary Arterial Pressure ◽  
Ex Vivo ◽  
Platelet Activating Factor ◽  
Microvascular Permeability ◽  
Blue Dye ◽  
E Coli ◽  
Pulmonary Arterial

In vivo pulmonary arterial catheterization was used to determine the mechanism by which platelet-activating factor (PAF) produces pulmonary edema in rats. PAF induces pulmonary edema by increasing pulmonary microvascular permeability (PMP) without changing the pulmonary pressure gradient. Rats were cannulated for measurement of pulmonary arterial pressure (Ppa) and mean arterial pressure. PMP was determined by using either in vivo fluorescent videomicroscopy or the ex vivo Evans blue dye technique. WEB 2086 was administered intravenously (IV) to antagonize specific PAF effects. Three experiments were performed: 1) IV PAF, 2) topical PAF, and 3) Escherichia coli bacteremia. IV PAF induced systemic hypotension with a decrease in Ppa. PMP increased after IV PAF in a dose-related manner. Topical PAF increased PMP but decreased Ppa only at high doses. Both PMP (88 ± 5%) and Ppa (50 ± 3%) increased during E. coli bacteremia. PAF-receptor blockade prevents changes in Ppa and PMP after both topical PAF and E. coli bacteremia. PAF, which has been shown to mediate pulmonary edema in prior studies, appears to act in the lung by primarily increasing microvascular permeability. The presence of PAF might be prerequisite for pulmonary vascular constriction during gram-negative bacteremia.

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