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. 2015 Oct 2;10(10):e0138778.
doi: 10.1371/journal.pone.0138778. eCollection 2015.

Characterization of Biofilm Formation in [Pasteurella] pneumotropica and [Actinobacillus] muris Isolates of Mouse Origin

Martin Sager  1 , W Peter M Benten  1 , Eva Engelhardt  1 , Christina Gougoula  1 , Laurentiu Benga  1
Affiliations

Characterization of Biofilm Formation in [Pasteurella] pneumotropica and [Actinobacillus] muris Isolates of Mouse Origin

Martin Sager et al. PLoS One. .

Abstract

[Pasteurella] pneumotropica biotypes Jawetz and Heyl and [Actinobacillus] muris are the most prevalent Pasteurellaceae species isolated from laboratory mouse. However, mechanisms contributing to their high prevalence such as the ability to form biofilms have not been studied yet. In the present investigation we analyze if these bacterial species can produce biofilms in vitro and investigate whether proteins, extracellular DNA and polysaccharides are involved in the biofilm formation and structure by inhibition and dispersal assays using proteinase K, DNase I and sodium periodate. Finally, the capacity of the biofilms to confer resistance to antibiotics is examined. We demonstrate that both [P.] pneumotropica biotypes but not [A.] muris are able to form robust biofilms in vitro, a phenotype which is widely spread among the field isolates. The biofilm inhibition and dispersal assays by proteinase and DNase lead to a strong inhibition in biofilm formation when added at the initiation of the biofilm formation and dispersed pre-formed [P.] pneumotropica biofilms, revealing thus that proteins and extracellular DNA are essential in biofilm formation and structure. Sodium periodate inhibited the bacterial growth when added at the beginning of the biofilm formation assay, making difficult the assessment of the role of β-1,6-linked polysaccharides in the biofilm formation, and had a biofilm stimulating effect when added on pre-established mature biofilms of [P.] pneumotropica biotype Heyl and a majority of [P.] pneumotropica biotype Jawetz strains, suggesting that the presence of β-1,6-linked polysaccharides on the bacterial surface might attenuate the biofilm production. Conversely, no effect or a decrease in the biofilm quantity was observed by biofilm dispersal using sodium periodate on further biotype Jawetz isolates, suggesting that polysaccharides might be incorporated in the biofilm structure. We additionally show that [P.] pneumotropica cells enclosed in biofilms were less sensitive to treatment with amoxicillin and enrofloxacin than planktonic bacteria. Taken together, these findings provide a first step in understanding of the biofilm mechanisms in [P.] pneumotropica, which might contribute to elucidation of colonization and pathogenesis mechanisms for these obligate inhabitants of the mouse mucosa.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. Biofilm formation in different rodent Pasteurellaceae strains.
The biofilm formation was recorded after 24 h of growth by a standard crystal violet microtiter plate assay and measuring the absorbance at 540 nm (y-axis). Strains tested are shown on the x-axis and grouped by species. Bars represent the average absorbance + standard deviation from three independent experiments.
Fig 2
Fig 2. Biofilm formation by rodent Pasteurellaceae in glass tubes.
Representative pictures of biofilm formation in glass tubes by [P.] pneumotropica biotype Jawetz (A.), [P.] pneumotropica biotype Heyl (B.) and [A.] muris (C.) after 24 h incubation at 37°C and staining with crystal violet. The strain number is pasted on the corresponding tube. The picture of a tube containing sterile BHI was included in panel A for comparison purposes.
Fig 3
Fig 3. Confocal laser scanning microscopy analysis of rodent Pasteurellaceae biofilms.
The type reference strains of the three rodent Pasteurellaceae species studied were allowed to produce biofilms for 24 h on glass coverslips and then examined by CLSM as described in Materials and Methods. The upper panel images are two-dimensional images of the biofilms formed by [P.] pneumotropica biotype Jawetz (A), [P.] pneumotropica biotype Heyl (B) and [A.] muris (C). The lower panels are orthogonal views of z-stacks of [P.] pneumotropica biotype Jawetz (D) and [P.] pneumotropica biotype Heyl biofilms (E) where the larger panel is a “bird´s eye” view of the biofilms whereas the right and the upper panels are side views of x- and y-axis sections respectively.
Fig 4
Fig 4. Inhibition of biofilm formation by proteinase K and DNase I in [P.] pneumotropica biotypes Jawetz (A) and Heyl (B).
The bacterial strains indicated along the x-axis were grown statically for 24 h in BHI broth alone (black bars) or in BHI broth supplemented with 100 μg/ml proteinase K (grey bars) or 50 μg/ml DNase I (white bars). The biofilm formation was then quantified photometrical at 540 nm after staining with crystal violet. Bars represent mean values + standard deviation of at least three independent experiments. Asterisks (*) designate a p-value less than 0.05 between the treated group and the corresponding control.
Fig 5
Fig 5. Dispersal of mature biofilms of [P.] pneumotropica biotypes Jawetz (A) and Heyl (B) by proteinase K.
The supernatants of 24 h old biofilms of the strains indicated along the x-axis were replaced for 2 h by buffer alone (black bars) or buffer containing 100 μg/ml proteinase K (grey bars). Biofilm quantity was then recorded using a standard crystal violet assay by measuring the absorbance at 540 nm. Bars represent mean values + standard deviation of at least three independent experiments. Asterisks (*) designate a p-value less than 0.05 between the treated group and the corresponding control.
Fig 6
Fig 6. Dispersal of mature biofilms of [P.] pneumotropica biotypes Jawetz (A) and Heyl (B) by DNase I.
The supernatants of 24 h old biofilms of the strains indicated along the x-axis were replaced for 2 h by buffer alone (black bars) or buffer containing 50 μg/ml DNase I (grey bars). Biofilm quantity was then recorded using a standard crystal violet assay by measuring the absorbance at 540 nm. Bars represent mean values + standard deviation of at least three independent experiments. Asterisks (*) designate a p-value less than 0.05 between the treated group and the corresponding control.
Fig 7
Fig 7. Dispersal of mature biofilms of [P.] pneumotropica biotypes Jawetz (A) and Heyl (B) by sodium periodate.
The supernatants of 24 h old biofilms of the strains indicated along the x-axis were replaced for 2 h by buffer alone (black bars) or buffer containing 10 mM sodium periodate (grey bars). Biofilm quantity was then measured by a standard crystal violet assay and measuring the absorbance at 540 nm. Bars represent mean values + standard deviation of at least three independent experiments. Asterisks (*) designate a p-value <0.05 between the treated group and the corresponding control.
Fig 8
Fig 8. Reduction of cellular viability of planktonic and biofilm cells of [P.] pneumotropica after 3 hours exposure to antibiotics.
The y-axis indicates the log10 cfu/ml recovered from the controls without antibiotics (black bars) in comparison to bacteria treated with amoxicillin (grey bars) or enrofloxacin (white bars) in planktonic or biofilm status. Bars represent mean values + standard deviation of three independent experiments.
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