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Cecum lymph node dendritic cells harbor slow-growing bacteria phenotypically tolerant to antibiotic treatment.

Kaiser P, Regoes RR, Dolowschiak T, Wotzka SY, Lengefeld J, Slack E, Grant AJ, Ackermann M, Hardt WD - PLoS Biol. (2014)

Bottom Line: High-dose ciprofloxacin treatment efficiently reduced pathogen loads in feces and most organs.The slow growth was sufficient to explain recalcitrance to antibiotics treatment.Thus, manipulating innate immunity may augment the in vivo activity of antibiotics.

View Article: PubMed Central - PubMed

Affiliation: Institute of Microbiology, Eidgenössische Technische Hochschule ETH, Zurich, Switzerland.

ABSTRACT
In vivo, antibiotics are often much less efficient than ex vivo and relapses can occur. The reasons for poor in vivo activity are still not completely understood. We have studied the fluoroquinolone antibiotic ciprofloxacin in an animal model for complicated Salmonellosis. High-dose ciprofloxacin treatment efficiently reduced pathogen loads in feces and most organs. However, the cecum draining lymph node (cLN), the gut tissue, and the spleen retained surviving bacteria. In cLN, approximately 10%-20% of the bacteria remained viable. These phenotypically tolerant bacteria lodged mostly within CD103⁺CX₃CR1⁻CD11c⁺ dendritic cells, remained genetically susceptible to ciprofloxacin, were sufficient to reinitiate infection after the end of the therapy, and displayed an extremely slow growth rate, as shown by mathematical analysis of infections with mixed inocula and segregative plasmid experiments. The slow growth was sufficient to explain recalcitrance to antibiotics treatment. Therefore, slow-growing antibiotic-tolerant bacteria lodged within dendritic cells can explain poor in vivo antibiotic activity and relapse. Administration of LPS or CpG, known elicitors of innate immune defense, reduced the loads of tolerant bacteria. Thus, manipulating innate immunity may augment the in vivo activity of antibiotics.

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LPS or CpG treatment reduces the number of ciprofloxacin-tolerant bacteria in the cLN.C57BL/6 mice were infected for 1 d or 3 d with wt S. Tm (5×107 cfu by gavage). Ciprofloxacin treatment (2×62 mg/kg/d) was started at day 1 p.i. and continued until the end of the experiment, as indicated. The indicated groups were treated with a single dose of LPS (5 µg intraperitoneally) or CpG (100 µg intraperitoneally) at day 2 p.i. Cfus in the cLN were analyzed by plating. For gut content and spleen loads, see Figure S14. Each symbol represents one mouse. Dashed line, detection limit. p, nonparametric statistical analysis (Mann–Whitney U test). n.s., not significant.
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pbio-1001793-g006: LPS or CpG treatment reduces the number of ciprofloxacin-tolerant bacteria in the cLN.C57BL/6 mice were infected for 1 d or 3 d with wt S. Tm (5×107 cfu by gavage). Ciprofloxacin treatment (2×62 mg/kg/d) was started at day 1 p.i. and continued until the end of the experiment, as indicated. The indicated groups were treated with a single dose of LPS (5 µg intraperitoneally) or CpG (100 µg intraperitoneally) at day 2 p.i. Cfus in the cLN were analyzed by plating. For gut content and spleen loads, see Figure S14. Each symbol represents one mouse. Dashed line, detection limit. p, nonparametric statistical analysis (Mann–Whitney U test). n.s., not significant.

Mentions: Finally, we were interested to identify a strategy for reducing loads of ciprofloxacin-tolerant pathogens in wild-type hosts. Earlier work had indicated that antibiotics and the host's immune system may cooperate in eliminating pathogens during antibiotic therapy [43],[44]. Furthermore, the antibiotic-treated lymph node displays a signature of reduced innate immune defense (Figure S13). Based on such observations, it has been speculated that triggering innate responses during the antibiotic treatment may allow reducing the number of persistent bacteria [1]. However, to the best of our knowledge, this has not been substantiated by experimental data in vivo. To test this hypothesis, we applied a PRR ligand. Mice were infected for 1 d with S. Tm and treated with ciprofloxacin alone (2×62 mg/kg per day), LPS alone (one dose of 5 µg at 48 h p.i.), or ciprofloxacin and LPS. Indeed, a single dose of LPS applied during the ciprofloxacin treatment elicited within 2 h an innate immune response in the cLN and significantly reduced the number of tolerant bacteria (Figure 6, Figure S13, Figure S14). In six animals the pathogen loads were reduced below the limit of detection (red arrowhead; Figure 6). Loads of tolerant bacteria were also reduced in the cecum wall and the cecal patch (Figure S15). However, it had remained unclear whether elimination of tolerant bacteria was attributable to direct or to indirect (paracrine) signaling to the infected host cell. In a first approach, we have generated mixed bone marrow chimeric mice. These mice displayed 50%–70% tlr4−/− cDC and 30%–50% wt cDC. Only the latter should directly respond to LPS, while both should respond to paracrine signaling. When these mice (or wt control animals) were infected with S. Tm(pAM34) and treated with ciprofloxacin and LPS, we observed the same strong reduction of the cLN loads (and pAM34 retention), as in wild-type mice (Figure S16). This suggested that LPS can affect tolerant S. Tm cells via an indirect, paracrine mechanism.


Cecum lymph node dendritic cells harbor slow-growing bacteria phenotypically tolerant to antibiotic treatment.

Kaiser P, Regoes RR, Dolowschiak T, Wotzka SY, Lengefeld J, Slack E, Grant AJ, Ackermann M, Hardt WD - PLoS Biol. (2014)

LPS or CpG treatment reduces the number of ciprofloxacin-tolerant bacteria in the cLN.C57BL/6 mice were infected for 1 d or 3 d with wt S. Tm (5×107 cfu by gavage). Ciprofloxacin treatment (2×62 mg/kg/d) was started at day 1 p.i. and continued until the end of the experiment, as indicated. The indicated groups were treated with a single dose of LPS (5 µg intraperitoneally) or CpG (100 µg intraperitoneally) at day 2 p.i. Cfus in the cLN were analyzed by plating. For gut content and spleen loads, see Figure S14. Each symbol represents one mouse. Dashed line, detection limit. p, nonparametric statistical analysis (Mann–Whitney U test). n.s., not significant.
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Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC3928039&req=5

pbio-1001793-g006: LPS or CpG treatment reduces the number of ciprofloxacin-tolerant bacteria in the cLN.C57BL/6 mice were infected for 1 d or 3 d with wt S. Tm (5×107 cfu by gavage). Ciprofloxacin treatment (2×62 mg/kg/d) was started at day 1 p.i. and continued until the end of the experiment, as indicated. The indicated groups were treated with a single dose of LPS (5 µg intraperitoneally) or CpG (100 µg intraperitoneally) at day 2 p.i. Cfus in the cLN were analyzed by plating. For gut content and spleen loads, see Figure S14. Each symbol represents one mouse. Dashed line, detection limit. p, nonparametric statistical analysis (Mann–Whitney U test). n.s., not significant.
Mentions: Finally, we were interested to identify a strategy for reducing loads of ciprofloxacin-tolerant pathogens in wild-type hosts. Earlier work had indicated that antibiotics and the host's immune system may cooperate in eliminating pathogens during antibiotic therapy [43],[44]. Furthermore, the antibiotic-treated lymph node displays a signature of reduced innate immune defense (Figure S13). Based on such observations, it has been speculated that triggering innate responses during the antibiotic treatment may allow reducing the number of persistent bacteria [1]. However, to the best of our knowledge, this has not been substantiated by experimental data in vivo. To test this hypothesis, we applied a PRR ligand. Mice were infected for 1 d with S. Tm and treated with ciprofloxacin alone (2×62 mg/kg per day), LPS alone (one dose of 5 µg at 48 h p.i.), or ciprofloxacin and LPS. Indeed, a single dose of LPS applied during the ciprofloxacin treatment elicited within 2 h an innate immune response in the cLN and significantly reduced the number of tolerant bacteria (Figure 6, Figure S13, Figure S14). In six animals the pathogen loads were reduced below the limit of detection (red arrowhead; Figure 6). Loads of tolerant bacteria were also reduced in the cecum wall and the cecal patch (Figure S15). However, it had remained unclear whether elimination of tolerant bacteria was attributable to direct or to indirect (paracrine) signaling to the infected host cell. In a first approach, we have generated mixed bone marrow chimeric mice. These mice displayed 50%–70% tlr4−/− cDC and 30%–50% wt cDC. Only the latter should directly respond to LPS, while both should respond to paracrine signaling. When these mice (or wt control animals) were infected with S. Tm(pAM34) and treated with ciprofloxacin and LPS, we observed the same strong reduction of the cLN loads (and pAM34 retention), as in wild-type mice (Figure S16). This suggested that LPS can affect tolerant S. Tm cells via an indirect, paracrine mechanism.

Bottom Line: High-dose ciprofloxacin treatment efficiently reduced pathogen loads in feces and most organs.The slow growth was sufficient to explain recalcitrance to antibiotics treatment.Thus, manipulating innate immunity may augment the in vivo activity of antibiotics.

View Article: PubMed Central - PubMed

Affiliation: Institute of Microbiology, Eidgenössische Technische Hochschule ETH, Zurich, Switzerland.

ABSTRACT
In vivo, antibiotics are often much less efficient than ex vivo and relapses can occur. The reasons for poor in vivo activity are still not completely understood. We have studied the fluoroquinolone antibiotic ciprofloxacin in an animal model for complicated Salmonellosis. High-dose ciprofloxacin treatment efficiently reduced pathogen loads in feces and most organs. However, the cecum draining lymph node (cLN), the gut tissue, and the spleen retained surviving bacteria. In cLN, approximately 10%-20% of the bacteria remained viable. These phenotypically tolerant bacteria lodged mostly within CD103⁺CX₃CR1⁻CD11c⁺ dendritic cells, remained genetically susceptible to ciprofloxacin, were sufficient to reinitiate infection after the end of the therapy, and displayed an extremely slow growth rate, as shown by mathematical analysis of infections with mixed inocula and segregative plasmid experiments. The slow growth was sufficient to explain recalcitrance to antibiotics treatment. Therefore, slow-growing antibiotic-tolerant bacteria lodged within dendritic cells can explain poor in vivo antibiotic activity and relapse. Administration of LPS or CpG, known elicitors of innate immune defense, reduced the loads of tolerant bacteria. Thus, manipulating innate immunity may augment the in vivo activity of antibiotics.

Show MeSH
Related in: MedlinePlus