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Improved in vitro assay for determining the mucin adherence of bacteria sensitive to Triton X-100 treatment

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Abstract

Mucin-associated microbiota are in relatively close contact with the intestinal epithelium and may thus have a more pronounced effect on host health. We have previously developed a simple mucin agar assay to simulate initial mucus colonization by intestinal microbial communities. Adherence of microbiota was estimated using flow cytometry after detachment with Triton X-100. In this study, the effect of this detergent on the cultivability of both virulent and commensal strains was investigated. Mucin attachment of selected strains was evaluated using the mucin adhesion assay. Bacteria were dislodged from the mucin surface by incubation with Triton or from the whole mucin agar layer using a stomacher. Mechanical extraction resulted in 1.24 ± 0.42, 2.69 ± 0.44, and 1.56 ± 0.85 log CFU/mL higher plate counts of Lactobacillus rhamnosus, Bacillus cereus, and Escherichia coli strains, respectively, than the chemical method. The sensitivity of bacteria to Triton varied among microbial species and strains. Among others, Triton inhibited the growth of Salmonella enterica LMG 10396 and Pseudomonas aeruginosa LMG 8029 on laboratory media, although these bacteria maintained their viability during this treatment. Only Gram-positive strains, Enterococcus hirae LMG 6399 and L. rhamnosus GG, were not affected by this detergent. Therefore, the mechanical method is recommended for the extraction of mucin-adhered bacteria that are sensitive to Triton, especially when followed by traditional cultivation techniques. However, this approach can also be recommended for strains that are not affected by this detergent, because it resulted in higher recovery of adhered L. rhamnosus GG compared to the chemical extraction.

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Abbreviations

AIEC:

Adhesive invasive Escherichia coli

BHI:

Brain heart infusion

CMC:

Critical micelle concentration

INRA:

National Institute for Agricultural Research

LGG:

Lactobacillus rhamnosus GG

LPS:

Lipopolysaccharide

A:

Absorbance

PG:

Peptidoglycan

PPBS:

Potassium phosphate buffered saline

PPS:

Physiological peptone solution

TX:

Triton X-100

VBNC:

Viable but nonculturable

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Acknowledgments

This work was financially supported by the Belgian Federal Public Service (FOD) of Health, Food Chain Safety and Environment (RT09/2 BACEREUS) and the Special Research Funds of Ghent University (B/09036/02 fund IV1 31/10/2008-31/10/2012). Pieter Van den Abbeele is a Postdoctoral Fellow belonging to the Fund for Scientific Research (Fonds Wetenschappelijk Onderzoek) of Flanders (Belgium). We thank Muhammad Lubowa and Jana De Bodt for the technical assistance and M.Sc. Frederiek-Maarten Kerckhof for the statistical analysis.

Conflict of interest

The authors have declared no conflicts of interest.

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Authors and Affiliations

  1. Laboratory of Microbial Ecology and Technology (LabMET), Department of Biochemical and Microbial Technology, Ghent University, Coupure Links 653, 9000, Ghent, Belgium

    Varvara Tsilia, Pieter Van den Abbeele & Tom Van de Wiele

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  1. Varvara Tsilia
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  2. Pieter Van den Abbeele
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  3. Tom Van de Wiele
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Correspondence to Tom Van de Wiele.

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Tsilia, V., Van den Abbeele, P. & Van de Wiele, T. Improved in vitro assay for determining the mucin adherence of bacteria sensitive to Triton X-100 treatment. Folia Microbiol 60, 435–442 (2015). https://doi.org/10.1007/s12223-015-0376-0

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