Killing of Serratia marcescens biofilms with chloramphenicol

Abstract Serratia marcescens is a Gram-negative bacterium with proven resistance to multiple antibiotics and causative of catheter-associated infections. Bacterial colonization of catheters mainly involves the formation of biofilm. The objectives of this study were to explore the susceptibility of S. marcescens biofilms to high doses of common antibiotics and non-antimicrobial agents. Biofilms formed by a clinical isolate of S. marcescens were treated with ceftriaxone, kanamycin, gentamicin, and chloramphenicol at doses corresponding to 10, 100 and 1000 times their planktonic minimum inhibitory concentration. In addition, biofilms were also treated with chemical compounds such as polysorbate-80 and ursolic acid. S. marcescens demonstrated susceptibility to ceftriaxone, kanamycin, gentamicin, and chloramphenicol in its planktonic form, however, only chloramphenicol reduced both biofilm biomass and biofilm viability. Polysorbate-80 and ursolic acid had minimal to no effect on either planktonic and biofilm grown S. marcescens. Our results suggest that supratherapeutic doses of chloramphenicol can be used effectively against established S. marcescens biofilms

YopE specific CD8+ T cells provide protection against systemic and mucosal Yersinia pseudotuberculosis infection.

Prior studies indicated that CD8+ T cells responding to a surrogate single antigen expressed by Y. pseudotuberculosis, ovalbumin, were insufficient to protect against yersiniosis. Herein we tested the hypothesis that CD8+ T cells reactive to the natural Yersinia antigen YopE would be more effective at providing mucosal protection. We first confirmed that immunization with the attenuated ksgA- strain of Y. pseudotuberculosis generated YopE-specific CD8+ T cells. These T cells were protective against challenge with virulent Listeria monocytogenes expressing secreted YopE. Mice immunized with an attenuated L. monocytogenes YopE+ strain generated large numbers of functional YopE-specific CD8+ T cells, and initially controlled a systemic challenge with virulent Y. pseudotuberculosis, yet eventually succumbed to yersiniosis. Mice vaccinated with a YopE peptide and cholera toxin vaccine generated robust T cell responses, providing protection to 60% of the mice challenged mucosally but failed to show complete protection against systemic infection with virulent Y. pseudotuberculosis. These studies demonstrate that vaccination with recombinant YopE vaccines can generate YopE-specific CD8+ T cells, that can provide significant mucosal protection but these cells are insufficient to provide sterilizing immunity against systemic Y. pseudotuberculosis infection. Our studies have implications for Yersinia vaccine development studies

YopE specific CD8⁺ T cells provide protection against systemic and mucosal <i>Yersinia pseudotuberculosis</i> infection - Table 1

YopE specific CD8⁺ T cells provide protection against systemic and mucosal <i>Yersinia pseudotuberculosis</i> infection - Table

<i>ksgA</i>^<i>-</i> immunization protects against recombinant <i>L</i>. <i>monocytogenes</i> expressing YopE.

<p>60 days after intravenous immunization with 200 CFU <i>ksgA</i>^<i>-</i> <i>Y</i>. <i>pseudotuberculosis</i> (<i>Yptb</i>), 200 CFU <i>ksgA</i>^<i>-</i> <i>yopE</i>^<i>-</i> <i>Yptb</i>, or 5.0 x 10⁷ CFU Δ<i>actA</i> Δ<i>plcB L</i>. <i>monocytogenes</i> (<i>Lm</i>) or nothing (naïve), C57BL/6 mice were intravenously challenged with either 5.0 x 10⁵ CFU Lm 1043s <b>(A)</b> 5.0 x 10⁵ CFU (<b>C, E</b>) or 2.0 x 10⁶ CFU (<b>B, D, F</b>) of virulent <i>L</i>. <i>monocytogenes</i> expressing ActAYopE_1-219OVA and followed for mortality (% survival <b>(A, B, E, F</b>) or morbidity (% weight change, <b>C, D</b>) over a four to six week time course. The data are representative of two independent experiments (n = 4–6 mice/experiment <b>C, D, E</b>, and <b>F</b>) or a single experiment (n = 8 mice/group <b>A, B</b>). For multiple group comparisons Dunn’s multiple-comparison post-test was used for weight change differences at selected time points: *, P ≤ 0.05, **, P ≤ 0.01, ***, P ≤ 0.001, ****, P ≤ 0.0001. Log rank Mantel-Cox test was used for survival curves.</p

Generation of YopE_69-77-specific CD8⁺ T cells in C57BL/6 mice exposed to attenuated <i>Y</i>. <i>pseudotuberculosis</i> strains.

<p>C57BL/6 mice were intravenously inoculated with <i>ksgA</i>^<i>-</i> (10² CFU) or Δ<i>yopK</i> (10³ CFU) bacteria, then sacrificed on day 8 and splenic cells analyzed by flow cytometry. (<b>A-C</b>) YopE_69-77-specific CD8⁺ T cells were detected by Kb/YopE_69-77 tetramer staining, as shown in representative FACS plots after gating for CD3⁺, CD4^- and CD8⁺ cells. The percent of Kb/YopE_69-77 tetramer⁺ cells in the CD8⁺ T cell population (<b>E</b>) is shown, with bars indicating mean values + S.E.M. The data are representative of two independent experiments (n = 4–5 mice). For multiple group comparisons Dunn’s multiple-comparison post-test was used: *, P ≤ 0.05, **, P ≤ 0.01, ***, P ≤ 0.001, ****, P ≤ 0.0001.</p

Mice immunized with attenuated YopE⁺ <i>L</i>. <i>monocytogenes</i> strains generate YopE_69-77-specific CD8⁺ T cells.

<p>C57BL/6 mice were intravenously inoculated with 5.0 x 10⁷ CFU of the attenuated <i>Lm</i> Δ<i>actA</i> Δ<i>plcB</i> strains, either expressing no OVA (Ø) or the indicated ActAYopEOVA fusions, or left naïve, and spleen cells analyzed day 8 post-inoculation by flow cytometry. YopE_69-77-specific CD8⁺ T cells were detected by K^b YopE_69-77 tetramer staining, and the % of K^b YopE_69-77 tetramer⁺ cells in the CD8⁺ T cell population (<b>A</b>). Quantitative representation of CD8⁺ T cell population percentages (<b>B</b>). Bars indicating mean values + S.E.M., and the data are representative of two independent experiments (n = 4–5 mice/experiment). Doted line represents naïve mice percentage. For multiple group comparisons Dunn’s multiple-comparison post-test was used: *, P ≤ 0.05.</p

Construction of recombinant <i>L</i>. <i>monocytogenes</i> expressing a secretable YopE-OVA fusion protein and impact of YopE sequence on acquisition/processing/presentation of OVA_257-264 peptide to OVA-specific CD8⁺ T cells.

<p>(<b>A</b>) Cartoon of engineered fusion proteins. Sequence encoding either full-length YopE (amino acids 1–219) or truncated YopE (amino acids 64–82) was inserted in-between sequence encoding the <i>actA</i> promoter and the first 100 amino acids of the ActA protein and sequence encoding OVA_254-265 in the <i>L</i>. <i>monocytogenes</i> integration vector pPL2 (Lauer et. al. 2002, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0172314#sec002" target="_blank">methods</a>). Resulting plasmids then were integrated into the <i>L</i>. <i>monocytogenes</i> genome. (<b>B, C</b>) Macrophages were incubated with <i>L</i>. <i>monocytogenes</i> expressing the ActA-YopE-OVA fusions, control bacteria expressing secreted OVA, the parental OVA-negative strain, or synthetic SIINFEKL peptide (i.e. OVA_257-264) at the indicated concentrations. After 12 hours, macrophages were incubated with B3Z T cells, a T cell hybridoma activated by the recognition of H-2K^b in association with OVA_257–264 peptide, and T cell activation measured using LacZ T cell hybridoma assay. Strain backgrounds were either the mouse-virulent 10403S (<b>B</b>) or mouse-attenuated 10403S Δ<i>actA</i> Δ<i>plcB</i> (<b>C</b>). Data is representative of two separate experiments. For multiple group comparisons Dunn’s multiple-comparison post-test was used: *, P ≤ 0.05, **, P ≤ 0.01.</p

Murine immunization with attenuated <i>L</i>. <i>monocytogenes</i> expressing the YopE epitope generates functional YopE-specific CD8⁺ T cells.

<p>C57BL/6 mice were immunized with 5.0 x 10⁷ CFU of Δ<i>actA</i> Δ<i>plcB L</i>. <i>monocytogenes</i> strains expressing the indicated fusion proteins. 8 days later, spleen cells were stimulated with OVA_257-264 peptide, YopE_69-77 peptide, or antibodies against CD3 and CD28 and stained for intracellular IFN-γ. Representative histograms are shown from one of two separate experiments. The data are representative of two independent experiments (n = 3–5 mice/experiment).</p

Immunization-mediated protection requires CD8+ T-cells.

<p>Mice (20) were immunized IV with 5.5 X 10⁷ CFU <i>L</i>. <i>monocytogenes ActAYopE</i>_<i>1-219</i> <i>OVA</i> and allowed to rest for 60 days. Mice were then treated IP with 200μg of anti-CD8 (2.43) or an isotype control (rat IgG2b LTF-2) two days prior to challenge and then every four days for the course of the experiment. Mice (9 from each treatment group) were challenged by oral gavage with 9 X10⁸ CFU of <i>Y</i>. <i>pseudotuberculosis</i> YPIII/pIB1 and survival was monitored for 20 days. <b>A)</b> Kaplan-Meier survival curve. Animals depleted of CD8 T-cells are represented by the solid line and those treated with the isotype control antibody by the dashed line. (p<0.0001, Mantel-Cox log-rank analysis). <b>B)</b> One mouse from each treatment group was analyzed by flow cytometry prior to challenge to ensure CD8+ cell depletion. Splenocytes were stained for CD45, CD4, and CD8. CD45+ cells were gated on and analyzed for the expression of CD4 and CD8. Essentially all CD45+ CD8+ cells were depleted with antibody treatment. These experiments were done once.</p

YopE-specific CD8⁺ T cells provide partial protection against virulent <i>Y</i>. <i>pseudotuberculosis</i> challenge via systemic or mucosal routes.

<p>60 days after immunization with either attenuated YopE-expressing bacteria (<b>A, A.1, B, C.1</b>) or cholera toxin plus YopE_69-77 peptide <b>(C, C.1</b>), mice were challenged with <i>Y</i>. <i>pseudotuberculosis</i> strain YPIII/pIB1 (fully mouse virulent), either 10⁹ CFU delivered orally (<b>A</b>) or 200 CFU delivered intravenously (<b>B, C</b>). Percent survival followed over a six week time course (A, B, C), mean time to death (A.1, B.1, C.1). Data are representative of two independent experiments (n = 3–6 mice/experiment). Log rank Mantel-Cox test was used for survival curves. For multiple group comparisons Dunn’s multiple-comparison post-test was used: *, P ≤ 0.05, **, P ≤ 0.01.</p