Lactobacillus reuteri and Enterococcus faecium from Poultry Gut Reduce Mucin Adhesion and Biofilm Formation of Cephalosporin and Fluoroquinolone-Resistant Salmonella enterica

Non-typhoidal Salmonella (NTS) can cause infection in poultry, livestock, and humans. Although the use of antimicrobials as feed additives is prohibited, the previous indiscriminate use and poor regulatory oversight in some parts of the world have resulted in increased bacterial resistance to antimicrobials, including cephalosporins and fluoroquinolones, which are among the limited treatment options available against NTS. This study aimed to isolate potential probiotic lactic acid bacteria (LAB) strains from the poultry gut to inhibit fluoroquinolone and cephalosporin resistant MDR Salmonella Typhimurium and S. Enteritidis. The safety profile of the LAB isolates was evaluated for the hemolytic activity, DNase activity, and antibiotic resistance. Based on the safety results, three possible probiotic LAB candidates for in vitro Salmonella control were chosen. Candidate LAB isolates were identified by 16S rDNA sequencing as Lactobacillus reuteri PFS4, Enterococcus faecium PFS13, and Enterococcus faecium PFS14. These strains demonstrated a good tolerance to gastrointestinal-related stresses, including gastric acid, bile, lysozyme, and phenol. In addition, the isolates that were able to auto aggregate had the ability to co-aggregate with MDR S. Typhimurium and S. Enteritidis. Furthermore, LAB strains competitively reduced the adhesion of pathogens to porcine mucin Type III in co-culture studies. The probiotic combination of the selected LAB isolates inhibited the biofilm formation of S. Typhimurium FML15 and S. Enteritidis FML18 by 90% and 92%, respectively. In addition, the cell-free supernatant (CFS) of the LAB culture significantly reduced the growth of Salmonella in vitro. Thus, L. reuteri PFS4, E. faecium PFS13, and E. faecium PFS 14 are potential probiotics that could be used to control MDR S. Typhimurium and S. Enteritidis in poultry. Future investigations are required to elucidate the in vivo potential of these probiotic candidates as Salmonella control agents in poultry and animal feed.

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Developments in techniques to test the efficacy of animal feed products

Developing animal feed products - Burleigh Dodds Series in Agricultural Science ◽

10.19103/as.2020.0083.11 ◽

2021 ◽

pp. 125-150

Author(s):

Gerhard Flachowsky ◽

◽

Ulrich Meyer ◽

Keyword(s):

Animal Feed ◽

Animal Health ◽

Feed Additives ◽

In Vivo Studies ◽

Animal Origin ◽

Food Of Animal Origin ◽

Key Issues ◽

Key Steps

Various animal feed products may influence animal health, conversion of animal feed into food of animal origin and the emissions caused by animals. All these matters are regulated in the directives of the European Food Safety Authority (EFSA). This chapter first discusses EFSA guidance on how to compile dossiers for feed additives. The chapter then discusses key issues and steps in demonstrating the efficacy of new animal feed products: reduction of nitrogen (N) excretion, reduction of feed contamination of by mycotoxins and, finally, reduction of methane emissions with feed additives. Key steps, such as the use of in vitro and in vivo studies, to test the effects of feed additives are discussed in detail.

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IN VITRO ASSESSMENT OF BINDING CAPACITY OF COMBINED ADSORBENT (BENTONITE WITH YEAST CELL WALL EXTRACTS) AND AFLATOXIN B1

Archives of Veterinary Medicine ◽

10.46784/e-avm.v13i1.65 ◽

2020 ◽

Vol 13 (1) ◽

pp. 41-52

Author(s):

Ksenija Nesic ◽

Sandra Jaksic ◽

Nenad Popov ◽

Milica Zivkov-Balos ◽

Marko Pajic ◽

...

Keyword(s):

Aflatoxin B1 ◽

Animal Feed ◽

Binding Capacity ◽

Animal Husbandry ◽

Feed Additives ◽

Point Of View ◽

Feed Additive ◽

European Regulation

The contamination of animal feed with mycotoxins is a worldwide problem in the animal husbandry, but it also represents a serious threat for the whole food chain. The health of both animals and humans is potentially endangered. From this point of view aflatoxins are a class of mycotoxins especially well known. Therefore, new strategies to combat these natural contaminants are constantly being developed. The most applied method to protect animals against aflatoxicosis is the utilization of feed additives aimed to adsorb aflatoxins. In order to estimate adsorbing potential of feed additive “MycoStop DUPLO”, designed for the prevention and/or alleviation of adverse effects of aflatoxin B1 in animal nutrition, in vitro trial was conducted. As a result of the experiment, conducted at pH 5 during 120 minutes of incubation at 37°C, the optimal formulation of the adsorbent was revealed. This product, in low concentration and in the presence of high amounts of toxin, met the stringent European regulation requirements for minimum 90% aflatoxin binding efficiency (90.1% achieved with 0.02% adsorbent and 4 mg/L toxin concentration). In higher adsorbent (0.2%), and lower toxin (0.2 mg/L) conditions, adsorption was 99.6%. Such outcome indicated the validity of in vitro experimental approach which can serve as a reliable fast tool for triage of adsorbents and preselect them for in vivo tests.

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The use of an in vitro nitrogen digestibility screening method to assess the effect of protease pre-treatment of soyabean meals

Proceedings of the British Society of Animal Science ◽

10.1017/s1752756200001186 ◽

2000 ◽

Vol 2000 ◽

pp. 117-117

Author(s):

J.D. Beal ◽

P.H. Brooks

Keyword(s):

Animal Feed ◽

Screening Method ◽

Feed Additives ◽

Screening Methods ◽

Invasive Procedures ◽

Laboratory Screening ◽

Animal Trials ◽

Pre Treatment

The assessment of the potential usefulness of enzymes as animal feed additives or treatments requires an understanding not only of the effect an enzyme has on its target substrate but also on the digestibility of that substrate. In vivo digestibility studies are expensive, time consuming and often involve invasive procedures that necessitate premature slaughtering of experimental animals. Preliminary laboratory screening methods can be used to indicate potential beneficial treatments prior to undertaking animal trials. The objective of this study was to assess the effect of protease pre-treatment of four differently processed soyabean meals on the digestibility of protein using an in vitro digestibility of nitrogen (Boisen and Fernandez 1997) technique as a screening method. The assumption implicit in this objective was that, if protease pre-treatment did not improve in vitro digestibility, it was unlikely to improve in vivo digestibility.

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Glucose Oligosaccharide and Long-Chain Glucomannan Feed Additives Induce Enhanced Activation of Intraepithelial NK Cells and Relative Abundance of Commensal Lactic Acid Bacteria in Broiler Chickens

Veterinary Sciences ◽

10.3390/vetsci8060110 ◽

2021 ◽

Vol 8 (6) ◽

pp. 110

Author(s):

Nathalie Meijerink ◽

Jean E. de Oliveira ◽

Daphne A. van Haarlem ◽

Guilherme Hosotani ◽

David M. Lamot ◽

...

Keyword(s):

Immune System ◽

Nk Cells ◽

Relative Abundance ◽

Nk Cell ◽

Feed Additives ◽

Microbiota Composition ◽

In Ovo ◽

Long Chain

Restrictions on the use of antibiotics in the poultry industry stimulate the development of alternative nutritional solutions to maintain or improve poultry health. This requires more insight in the modulatory effects of feed additives on the immune system and microbiota composition. Compounds known to influence the innate immune system and microbiota composition were selected and screened in vitro, in ovo, and in vivo. Among all compounds, 57 enhanced NK cell activation, 56 increased phagocytosis, and 22 increased NO production of the macrophage cell line HD11 in vitro. Based on these results, availability and regulatory status, six compounds were selected for further analysis. None of these compounds showed negative effects on growth, hatchability, and feed conversion in in ovo and in vivo studies. Based on the most interesting numerical results and highest future potential feasibility, two compounds were analyzed further. Administration of glucose oligosaccharide and long-chain glucomannan in vivo both enhanced activation of intraepithelial NK cells and led to increased relative abundance of lactic acid bacteria (LAB) amongst ileum and ceca microbiota after seven days of supplementation. Positive correlations between NK cell subsets and activation, and relative abundance of LAB suggest the involvement of microbiota in the modulation of the function of intraepithelial NK cells. This study identifies glucose oligosaccharide and long-chain glucomannan supplementation as effective nutritional strategies to modulate the intestinal microbiota composition and strengthen the intraepithelial innate immune system.

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Antibacterial Mechanisms and Efficacy of Sarecycline in Animal Models of Infection and Inflammation

Antibiotics ◽

10.3390/antibiotics10040439 ◽

2021 ◽

Vol 10 (4) ◽

pp. 439

Author(s):

Christopher G. Bunick ◽

Jonette Keri ◽

S. Ken Tanaka ◽

Nika Furey ◽

Giovanni Damiani ◽

...

Keyword(s):

Broad Spectrum ◽

Bacterial Resistance ◽

Antibiotic Use ◽

In Vivo Studies ◽

Infection Model ◽

Severe Acne ◽

Rat Paw Edema ◽

Infection And Inflammation

Prolonged broad-spectrum antibiotic use is more likely to induce bacterial resistance and dysbiosis of skin and gut microflora. First and second-generation tetracycline-class antibiotics have similar broad-spectrum antibacterial activity. Targeted tetracycline-class antibiotics are needed to limit antimicrobial resistance and improve patient outcomes. Sarecycline is a narrow-spectrum, third-generation tetracycline-class antibiotic Food and Drug Administration (FDA)-approved for treating moderate-to-severe acne. In vitro studies demonstrated activity against clinically relevant Gram-positive bacteria but reduced activity against Gram-negative bacteria. Recent studies have provided insight into how the structure of sarecycline, with a unique C7 moiety, interacts with bacterial ribosomes to block translation and prevent antibiotic resistance. Sarecycline reduces Staphylococcus aureus DNA and protein synthesis with limited effects on RNA, lipid, and bacterial wall synthesis. In agreement with in vitro data, sarecycline demonstrated narrower-spectrum in vivo activity in murine models of infection, exhibiting activity against S. aureus, but reduced efficacy against Escherichia coli compared to doxycycline and minocycline. In a murine neutropenic thigh wound infection model, sarecycline was as effective as doxycycline against S. aureus. The anti-inflammatory activity of sarecycline was comparable to doxycycline and minocycline in a rat paw edema model. Here, we review the antibacterial mechanisms of sarecycline and report results of in vivo studies of infection and inflammation.

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An In Vitro Coumarin-Antibiotic Combination Treatment of Pseudomonas aeruginosa Biofilms

Natural Product Communications ◽

10.1177/1934578x20987744 ◽

2021 ◽

Vol 16 (1) ◽

pp. 1934578X2098774

Author(s):

Jinpeng Zou ◽

Yang Liu ◽

Ruiwei Guo ◽

Yu Tang ◽

Zhengrong Shi ◽

...

Keyword(s):

Pseudomonas Aeruginosa ◽

Drug Resistance ◽

Combination Treatment ◽

Crude Extract ◽

Biofilm Formation ◽

Bacterial Resistance ◽

Antibacterial Properties ◽

Biofilm Growth ◽

Antibiotic Combination

The drug resistance of Pseudomonas aeruginosa is a worldwide problem due to its great threat to human health. A crude extract of Angelica dahurica has been proved to have antibacterial properties, which suggested that it may be able to inhibit the biofilm formation of P. aeruginosa; initial exploration had shown that the crude extract could inhibit the growth of P. aeruginosa effectively. After the adaptive dose of coumarin was confirmed to be a potential treatment for the bacteria’s drug resistance, “coumarin-antibiotic combination treatments” (3 coumarins—simple coumarin, imperatorin, and isoimperatorin—combined with 2 antibiotics—ampicillin and ceftazidime) were examined to determine their capability to inhibit P. aeruginosa. The final results showed that (1) coumarin with either ampicillin or ceftazidime significantly inhibited the biofilm formation of P. aeruginosa; (2) coumarin could directly destroy mature biofilms; and (3) the combination treatment can synergistically enhance the inhibition of biofilm formation, which could significantly reduce the usage of antibiotics and bacterial resistance. To sum up, a coumarin-antibiotic combination treatment may be a potential way to inhibit the biofilm growth of P. aeruginosa and provides a reference for antibiotic resistance treatment.

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Biofilm formation in the lung contributes to virulence and drug tolerance of Mycobacterium tuberculosis

Nature Communications ◽

10.1038/s41467-021-21748-6 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Poushali Chakraborty ◽

Sapna Bajeli ◽

Deepak Kaushal ◽

Bishan Dass Radotra ◽

Ashwani Kumar

Keyword(s):

Mycobacterium Tuberculosis ◽

Immune System ◽

Biofilm Formation ◽

Antimycobacterial Activity ◽

Drug Tolerance ◽

Host Immune System ◽

Tissue Sections ◽

Slow Growing

AbstractTuberculosis is a chronic disease that displays several features commonly associated with biofilm-associated infections: immune system evasion, antibiotic treatment failures, and recurrence of infection. However, although Mycobacterium tuberculosis (Mtb) can form cellulose-containing biofilms in vitro, it remains unclear whether biofilms are formed during infection in vivo. Here, we demonstrate the formation of Mtb biofilms in animal models of infection and in patients, and that biofilm formation can contribute to drug tolerance. First, we show that cellulose is also a structural component of the extracellular matrix of in vitro biofilms of fast and slow-growing nontuberculous mycobacteria. Then, we use cellulose as a biomarker to detect Mtb biofilms in the lungs of experimentally infected mice and non-human primates, as well as in lung tissue sections obtained from patients with tuberculosis. Mtb strains defective in biofilm formation are attenuated for survival in mice, suggesting that biofilms protect bacilli from the host immune system. Furthermore, the administration of nebulized cellulase enhances the antimycobacterial activity of isoniazid and rifampicin in infected mice, supporting a role for biofilms in phenotypic drug tolerance. Our findings thus indicate that Mtb biofilms are relevant to human tuberculosis.

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Eap1p, an Adhesin That Mediates Candida albicans Biofilm Formation In Vitro and In Vivo

Eukaryotic Cell ◽

10.1128/ec.00049-07 ◽

2007 ◽

Vol 6 (6) ◽

pp. 931-939 ◽

Cited By ~ 96

Author(s):

Fang Li ◽

Michael J. Svarovsky ◽

Amy J. Karlsson ◽

Joel P. Wagner ◽

Karen Marchillo ◽

...

Keyword(s):

Candida Albicans ◽

Cell Adhesion ◽

Biofilm Formation ◽

Fungal Infections ◽

Gene Dosage ◽

Venous Catheter ◽

Flow Chamber ◽

Dependent Manner

ABSTRACT Candida albicans is the leading cause of systemic fungal infections in immunocompromised humans. The ability to form biofilms on surfaces in the host or on implanted medical devices enhances C. albicans virulence, leading to antimicrobial resistance and providing a reservoir for infection. Biofilm formation is a complex multicellular process consisting of cell adhesion, cell growth, morphogenic switching between yeast form and filamentous states, and quorum sensing. Here we describe the role of the C. albicans EAP1 gene, which encodes a glycosylphosphatidylinositol-anchored, glucan-cross-linked cell wall protein, in adhesion and biofilm formation in vitro and in vivo. Deleting EAP1 reduced cell adhesion to polystyrene and epithelial cells in a gene dosage-dependent manner. Furthermore, EAP1 expression was required for C. albicans biofilm formation in an in vitro parallel plate flow chamber model and in an in vivo rat central venous catheter model. EAP1 expression was upregulated in biofilm-associated cells in vitro and in vivo. Our results illustrate an association between Eap1p-mediated adhesion and biofilm formation in vitro and in vivo.

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Xylitol enhances synthesis of propionate in the colon via cross-feeding of gut microbiota

Microbiome ◽

10.1186/s40168-021-01029-6 ◽

2021 ◽

Vol 9 (1) ◽

Author(s):

Shasha Xiang ◽

Kun Ye ◽

Mian Li ◽

Jian Ying ◽

Huanhuan Wang ◽

...

Keyword(s):

Gut Microbiome ◽

Lactobacillus Reuteri ◽

Carbon Sources ◽

Short Chain Fatty Acids ◽

Microbial Composition ◽

Key Enzymes ◽

Rrna Sequencing ◽

Phosphate Acetyltransferase

Abstract Background Xylitol, a white or transparent polyol or sugar alcohol, is digestible by colonic microorganisms and promotes the proliferation of beneficial bacteria and the production of short-chain fatty acids (SCFAs), but the mechanism underlying these effects remains unknown. We studied mice fed with 0%, 2% (2.17 g/kg/day), or 5% (5.42 g/kg/day) (weight/weight) xylitol in their chow for 3 months. In addition to the in vivo digestion experiments in mice, 3% (weight/volume) (0.27 g/kg/day for a human being) xylitol was added to a colon simulation system (CDMN) for 7 days. We performed 16S rRNA sequencing, beneficial metabolism biomarker quantification, metabolome, and metatranscriptome analyses to investigate the prebiotic mechanism of xylitol. The representative bacteria related to xylitol digestion were selected for single cultivation and co-culture of two and three bacteria to explore the microbial digestion and utilization of xylitol in media with glucose, xylitol, mixed carbon sources, or no-carbon sources. Besides, the mechanisms underlying the shift in the microbial composition and SCFAs were explored in molecular contexts. Results In both in vivo and in vitro experiments, we found that xylitol did not significantly influence the structure of the gut microbiome. However, it increased all SCFAs, especially propionate in the lumen and butyrate in the mucosa, with a shift in its corresponding bacteria in vitro. Cross-feeding, a relationship in which one organism consumes metabolites excreted by the other, was observed among Lactobacillus reuteri, Bacteroides fragilis, and Escherichia coli in the utilization of xylitol. At the molecular level, we revealed that xylitol dehydrogenase (EC 1.1.1.14), xylulokinase (EC 2.7.1.17), and xylulose phosphate isomerase (EC 5.1.3.1) were key enzymes in xylitol metabolism and were present in Bacteroides and Lachnospiraceae. Therefore, they are considered keystone bacteria in xylitol digestion. Also, xylitol affected the metabolic pathway of propionate, significantly promoting the transcription of phosphate acetyltransferase (EC 2.3.1.8) in Bifidobacterium and increasing the production of propionate. Conclusions Our results revealed that those key enzymes for xylitol digestion from different bacteria can together support the growth of micro-ecology, but they also enhanced the concentration of propionate, which lowered pH to restrict relative amounts of Escherichia and Staphylococcus. Based on the cross-feeding and competition among those bacteria, xylitol can dynamically balance proportions of the gut microbiome to promote enzymes related to xylitol metabolism and SCFAs.

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Attenuation of Aeromonas hydrophila Infection in Carassius auratus by YtnP, a N-acyl Homoserine Lactonase from Bacillus licheniformis T-1

Antibiotics ◽

10.3390/antibiotics10060631 ◽

2021 ◽

Vol 10 (6) ◽

pp. 631

Author(s):

Mengfan Peng ◽

Wentao Tong ◽

Zhen Zhao ◽

Ling Xiao ◽

Zhaoyue Wang ◽

...

Keyword(s):

Virulence Factors ◽

Bacillus Licheniformis ◽

Aeromonas Hydrophila ◽

Biofilm Formation ◽

Carassius Auratus ◽

Quorum Quenching ◽

Inhibition Rate ◽

Sequence Identity

In this experiment, the quorum quenching gene ytnP of Bacillus licheniformis T-1 was cloned and expressed, and the effect against infection of Aeromonas hydrophila ATCC 7966 was evaluated in vitro and vivo. The BLAST results revealed a 99% sequence identity between the ytnP gene of T-1 and its homolog in B.subtilis sub sp. BSP1, and the dendroGram showed that the similarity in the YtnP protein in T-1 was 100% in comparison with B.subtilis 3610, which was categorized as the Aidc cluster of the MBL family. The AHL lactonase activity of the purified YtnP was detected as 1.097 ± 0.7 U/mL with C6-HSL as the substrate. Otherwise, purified YtnP protein could significantly inhibit the biofilm formation of A.hydrophila ATCC 7966 with an inhibition rate of 68%. The MIC of thiamphenicol and doxycycline hydrochloride against A. hydrophila reduced from 4 μg/mL and 0.5 μg/mL to 1 μg/mL and 0.125 μg/mL, respectively, in the presence of YtnP. In addition, YtnP significantly inhibited the expression of five virulence factors hem, ahyB, ast, ep, aerA of A. hydrophila ATCC 7966 as well (p < 0.05). The results of inhibition on virulence showed a time-dependence tendency, while the strongest anti-virulence effects were within 4–24 h. In vivo, when the YtnP protein was co-injected intraperitoneally with A. hydrophila ATCC 7966, it attenuated the pathogenicity of A. hydrophila and the accumulated mortality was 27 ± 4.14% at 96 h, which was significantly lower than the average mortality of 78 ± 2.57% of the Carassius auratus injected with 108 CFU/mL of A. hydrophila ATCC 7966 only (p < 0.001). In conclusion, the AHL lactonase in B. licheniformis T-1 was proven to be YtnP protein and could be developed into an agent against infection of A. hydrophila in aquaculture.

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