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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

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.
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pone.0138778.g003: 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.

Mentions: To date, it is not known whether the three rodent Pasteurellaceae species included in this study are able to produce biofilms. To address this, biofilm formation was quantified by a standard microtiter plate crystal violet assay as well as by a glass tube assay in a various collection of reference and field isolates of [P.] pneumotropica biotypes Jawetz and Heyl and [A.] muris. Fig 1 shows the amount of biofilm formed after 24 h in BHI as recorded photometrical using the crystal violet technique. There is an obvious difference in the biofilm formation of [P.] pneumotropica species belonging to both biotypes in comparison to the [A.] muris isolates which can be categorized as no biofilm producers. In sharp contrast to [A.] muris, all [P.] pneumotropica strains tested were capable to produce robust biofilms, even though one can differentiate among them strong and weak biofilm producers. The results obtained by the glass tubes biofilm formation assays correlated with the results obtained by the crystal violet microtiter plate assays, showing as well the ability of [P.] pneumotropica biotypes Jawetz and Heyl but not of [A.] muris to form biofilms (Fig 2). Confocal microscopy analyses using the type reference strains of [P.] pneumotropica biotypes Jawetz and Heyl and of [A.] muris showed that indeed only [P.] pneumotropica strains, but not [A.] muris, were able to produce consistent biofilms (Fig 3).


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)

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.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0138778.g003: 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.
Mentions: To date, it is not known whether the three rodent Pasteurellaceae species included in this study are able to produce biofilms. To address this, biofilm formation was quantified by a standard microtiter plate crystal violet assay as well as by a glass tube assay in a various collection of reference and field isolates of [P.] pneumotropica biotypes Jawetz and Heyl and [A.] muris. Fig 1 shows the amount of biofilm formed after 24 h in BHI as recorded photometrical using the crystal violet technique. There is an obvious difference in the biofilm formation of [P.] pneumotropica species belonging to both biotypes in comparison to the [A.] muris isolates which can be categorized as no biofilm producers. In sharp contrast to [A.] muris, all [P.] pneumotropica strains tested were capable to produce robust biofilms, even though one can differentiate among them strong and weak biofilm producers. The results obtained by the glass tubes biofilm formation assays correlated with the results obtained by the crystal violet microtiter plate assays, showing as well the ability of [P.] pneumotropica biotypes Jawetz and Heyl but not of [A.] muris to form biofilms (Fig 2). Confocal microscopy analyses using the type reference strains of [P.] pneumotropica biotypes Jawetz and Heyl and of [A.] muris showed that indeed only [P.] pneumotropica strains, but not [A.] muris, were able to produce consistent biofilms (Fig 3).

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