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      Influence of probiotic culture supernatants on in vitro biofilm formation of staphylococci

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          Background: The effects of cell-free culture supernatants of probiotic Lactobacillus rhamnosus GG and Streptococcus salivarius K12 on replication and biofilm forming of Staphylococcus aureus and S. epidermidis were assessed in vitro.

          Methods: S. aureus and S. epidermidis strains were exposed to cell-free culture supernatants of L. rhamnosus GG and S. salivarius K12 at different concentrations starting at 0, 4, and 24 h after the onset of incubation. Bacterial amplification was measured on microplate readers, as well as biofilm growth after safranine staining. Scanning electron microscopy was performed for visualization of biofilm status.

          Results: The S. salivarius K12 culture supernatant not only reduced or prevented the formation and maturation of fresh biofilms but even caused a reduction of preformed S. epidermidis biofilms. The L. rhamnosus GG culture supernatant did not show clear inhibitory effects regardless of concentration or time of addition of supernatant, and even concentration-depending promotional effects on the planktonic and biofilm growth of S. aureus and S. epidermidis were observed.

          Conclusion: In particular, the inhibitory effects of the S. salivarius K12 culture supernatant on the formation of staphylococcal biofilms are of potential relevance for biofilm-associated diseases and should be further assessed by in vivo infection models.

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          Most cited references 54

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          Adherence of coagulase-negative staphylococci to plastic tissue culture plates: a quantitative model for the adherence of staphylococci to medical devices.

          The adherence of coagulase-negative staphylococci to smooth surfaces was assayed by measuring the optical densities of stained bacterial films adherent to the floors of plastic tissue culture plates. The optical densities correlated with the weight of the adherent bacterial film (r = 0.906; P less than 0.01). The measurements also agreed with visual assessments of bacterial adherence to culture tubes, microtiter plates, and tissue culture plates. Selected clinical strains were passed through a mouse model for foreign body infections and a rat model for catheter-induced endocarditis. The adherence measurements of animal passed strains remained the same as those of the laboratory-maintained parent strain. Spectrophotometric classification of coagulase-negative staphylococci into nonadherent and adherent categories according to these measurements had a sensitivity, specificity, and accuracy of 90.6, 80.8, and 88.4%, respectively. We examined a previously described collection of 127 strains of coagulase-negative staphylococci isolated from an outbreak of intravascular catheter-associated sepsis; strains associated with sepsis were more adherent than blood culture contaminants and cutaneous strains (P less than 0.001). We also examined a collection of 84 strains isolated from pediatric patients with cerebrospinal fluid (CSF) shunts; once again, pathogenic strains were more adherent than were CSF contaminants (P less than 0.01). Finally, we measured the adherence of seven endocarditis strains. As opposed to strains associated with intravascular catheters and CSF shunts, endocarditis strains were less adherent than were saprophytic strains of coagulase-negative staphylococci. The optical densities of bacterial films adherent to plastic tissue culture plates serve as a quantitative model for the study of the adherence of coagulase-negative staphylococci to medical devices, a process which may be important in the pathogenesis of foreign body infections.
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            Quantification of biofilm in microtiter plates: overview of testing conditions and practical recommendations for assessment of biofilm production by staphylococci.

            The details of all steps involved in the quantification of biofilm formation in microtiter plates are described. The presented protocol incorporates information on assessment of biofilm production by staphylococci, gained both by direct experience as well as by analysis of methods for assaying biofilm production. The obtained results should simplify quantification of biofilm formation in microtiter plates, and make it more reliable and comparable among different laboratories.
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              Growing and analyzing static biofilms.

              Many bacteria can exist as surface-attached aggregations known as biofilms. Presented in this unit are several approaches for the study of these communities. The focus here is on static biofilm systems, which are particularly useful for examination of the early stages of biofilm formation, including initial adherence to the surface and microcolony formation. Furthermore, most of the techniques presented are easily adapted to the study of biofilms under a variety of conditions and are suitable for either small- or relatively large-scale studies. Unlike assays involving continuous-flow systems, the static biofilm assays described here require very little specialized equipment and are relatively simple to execute. In addition, these static biofilm systems allow analysis of biofilm formation with a variety of readouts, including microscopy of live cells, macroscopic visualization of stained bacteria, and viability counts. Used individually or in combination, these assays provide useful means for the study of biofilms.

                Author and article information

                European Journal of Microbiology and Immunology
                Akadémiai Kiadó
                December 2018
                : 8
                : 4
                : 119-127
                [ 1 ]Institute for Medical Microbiology, Virology and Hygiene, University Medicine Rostock , Rostock, Germany
                [ 2 ]Department of Microbiology and Hospital Hygiene, Bundeswehr Hospital Hamburg , Hamburg, Germany
                [ 3 ]Kreth Lab, Department of Restorative Dentistry, Oregon Health & Science University , Portland, OR, USA
                Author notes

                Corresponding author: Department of Microbiology and Hospital Hygiene, Bundeswehr Hospital Hamburg, Bernhard Nocht Str. 74, 20359 Hamburg, Germany; E-mail: Frickmann@


                These authors contributed equally to the manuscript.

                © 2018 The Author(s)

                This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial 4.0 International License (, which permits unrestricted use, distribution, and reproduction in any medium for non-commercial purposes, provided the original author and source are credited, a link to the CC License is provided, and changes - if any - are indicated.

                Page count
                Pages: 9
                Original Research Paper


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