Inhibition of biofilm formation by proteinase K and DNase I in <i>[P</i>.<i>] pneumotropica</i> biotypes Jawetz (A) and Heyl (B).

<p>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.</p

Dispersal of mature biofilms of <i>[P</i>.<i>] pneumotropica</i> biotypes Jawetz (A) and Heyl (B) by DNase I.

<p>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.</p

Characterization of Biofilm Formation in <i>[Pasteurella] pneumotropica</i> and <i>[Actinobacillus] muris</i> Isolates of Mouse Origin

<div><p><i>[Pasteurella] pneumotropica</i> biotypes Jawetz and Heyl and <i>[Actinobacillus] muris</i> are the most prevalent <i>Pasteurellaceae</i> 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 <i>in vitro</i> 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 <i>[P</i>.<i>] pneumotropica</i> biotypes but not <i>[A</i>.<i>] muris</i> are able to form robust biofilms <i>in vitro</i>, 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 <i>[P</i>.<i>] pneumotropica</i> 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 <i>[P</i>.<i>] pneumotropica</i> biotype Heyl and a majority of <i>[P</i>.<i>] pneumotropica</i> 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 <i>[P</i>.<i>] pneumotropica</i> 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 <i>[P</i>.<i>] pneumotropica</i>, which might contribute to elucidation of colonization and pathogenesis mechanisms for these obligate inhabitants of the mouse mucosa.</p></div

Physiological and anatomical aspects of the reproduction of mice with reduced Syndecan-1 expression

Abstract Background Syndecan-1 is a heparan sulfate proteoglycan acting as a co-receptor for cytokines and growth factors mediating developmental, immunological and angiogenic processes. In human, the uteroplacental localization of Syndecan-1 and its reduced expression in pregnancy-associated pathologies, such as the intrauterine growth restriction, suggests an influence of Syndecan-1 in embryo-maternal interactions. The aim of the present study was to identify the effect of a reduced expression of Syndecan-1 on the reproductive phenotype of mice and their progenies. Methods Reproductive characteristics have been investigated using animals with reduced Syndecan-1 and their wildtype controls after normal mating and after vice versa embryo transfers. Female mice were used to measure the estrus cycle length and the weight gain during pregnancy, as well as for histological examination of ovaries. Male mice were examined for the concentration, motility, viability and morphology of spermatozoa. Organs like heart, lung, liver, kidney, spleen, brain and ovaries or testes and epididymis of 6-month-old animals were isolated and weighed. Statistical analyses were performed using two-tailed students t-test with P < .05 and P < .02, chi square test (P < .05) and Fisher’s Exact Test (P < .05). A linear and a non-linear mixed-effects model were generated to analyze the weight gain of pregnant females and of the progenies. Results Focusing on the pregnancy outcome, the Syndecan-1 reduced females gave birth to larger litters. However, regarding the survival of the offspring, a higher percentage of pups with less Syndecan-1 died during the first postnatal days. Even though the ovaries and the testes of Syndecan-1 reduced mice showed no histological differences and the ovaries showed a similar number of primary and secondary follicles and corpora lutea, the spermatozoa of Syndecan-1 reduced males showed more tail and midpiece deficiencies. Concerning the postnatal and juvenile development the pups with reduced Syndecan-1 expression remained lighter and smaller regardless whether carried by mothers with reduced Syndecan-1 or wildtype foster mothers. With respect to anatomical differences kidneys of both genders as well as testes and epididymis of male mice with reduced syndecan-1 expression weighed less compared to controls. Conclusions These data reveal that the effects of Syndecan-1 reduction are rather genotype- than parental-dependent

Confocal laser scanning microscopy analysis of rodent <i>Pasteurellaceae</i> biofilms.

<p>The type reference strains of the three rodent <i>Pasteurellaceae</i> 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), <i>[P</i>.<i>] pneumotropica</i> biotype Heyl (B) and <i>[A</i>.<i>] muris</i> (C). The lower panels are orthogonal views of z-stacks of <i>[P</i>.<i>] pneumotropica</i> biotype Jawetz (D) and <i>[P</i>.<i>] pneumotropica</i> 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.</p

Reduction of cellular viability of planktonic and biofilm cells of <i>[P</i>.<i>] pneumotropica</i> after 3 hours exposure to antibiotics.

<p>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.</p

Minimal inhibitory concentrations (MICs) of amoxicillin and enrofloxacin for <i>[P</i>.<i>] pneumotropica</i> in planktonic and biofilm status.

<p>Minimal inhibitory concentrations (MICs) of amoxicillin and enrofloxacin for <i>[P</i>.<i>] pneumotropica</i> in planktonic and biofilm status.</p