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

Sager M, Benten WP, Engelhardt E, Gougoula C, Benga L - PLoS ONE (2015)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Central Animal Research Facility, Heinrich-Heine-University, University Hospital, Düsseldorf, Germany.

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.

No MeSH data available.


Related in: MedlinePlus

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.
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pone.0138778.g005: 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.

Mentions: Concentrations of 100 μg/ml of proteinase K were necessary to achieve a statistically significant disruption of the biofilm produced by both [P.] pneumotropica type strains in comparison with the non-treated controls (S1 Fig). Incubation of the mature biofilms from the field isolates of [P.] pneumotropica with 100 μg/ml proteinase K caused significant detachment in nearly all strains tested (Fig 5). By four strains of [P.] pneumotropica biotype Jawetz and eight strains of [P.] pneumotropica biotype Heyl no statistically significant (p—values between 0.06 and 0.14 and one value of 0.22) proteinase K dispersal of the biofilms was achieved, although visual differences could be recognized.


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

Sager M, Benten WP, Engelhardt E, Gougoula C, Benga L - PLoS ONE (2015)

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.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4592018&req=5

pone.0138778.g005: 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.
Mentions: Concentrations of 100 μg/ml of proteinase K were necessary to achieve a statistically significant disruption of the biofilm produced by both [P.] pneumotropica type strains in comparison with the non-treated controls (S1 Fig). Incubation of the mature biofilms from the field isolates of [P.] pneumotropica with 100 μg/ml proteinase K caused significant detachment in nearly all strains tested (Fig 5). By four strains of [P.] pneumotropica biotype Jawetz and eight strains of [P.] pneumotropica biotype Heyl no statistically significant (p—values between 0.06 and 0.14 and one value of 0.22) proteinase K dispersal of the biofilms was achieved, although visual differences could be recognized.

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Central Animal Research Facility, Heinrich-Heine-University, University Hospital, Düsseldorf, Germany.

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.

No MeSH data available.


Related in: MedlinePlus