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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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Plasmid dilution experiment verifying the slow growth rate of tolerant S. Tm cells in vivo.(Left) Experimental strategy. (Right) Experimental data. Three groups of C57BL/6 mice were infected for 2 d or 3 d with S. Tm(pAM34), as indicated. The third group was treated with ciprofloxacin during the third day (2×62 mg/kg/d by gavage). Total S. Tm loads (closed symbols) and S. Tm(pAM34) (open symbols) in the cLN were determined by plating. Dashed line, detection limit. p, nonparametric statistical analysis. n.s., not significant.
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pbio-1001793-g005: Plasmid dilution experiment verifying the slow growth rate of tolerant S. Tm cells in vivo.(Left) Experimental strategy. (Right) Experimental data. Three groups of C57BL/6 mice were infected for 2 d or 3 d with S. Tm(pAM34), as indicated. The third group was treated with ciprofloxacin during the third day (2×62 mg/kg/d by gavage). Total S. Tm loads (closed symbols) and S. Tm(pAM34) (open symbols) in the cLN were determined by plating. Dashed line, detection limit. p, nonparametric statistical analysis. n.s., not significant.

Mentions: To confirm the slow growth rates of tolerant S. Tm cells in vivo, we employed a plasmid-dilution strategy (Figure 5, left side). pAM34 is an IPTG-addicted plasmid that stops replication as soon as IPTG is removed from the environment, including all mouse tissues analyzed (pAM34, AmpR, [42]). This type of plasmid can be used to verify the slow growth rate of the ciprofloxacin-tolerant S. Tm subpopulation. Such tolerant cells should survive the antibiotic and should feature a high fraction of cells retaining pAM34.


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)

Plasmid dilution experiment verifying the slow growth rate of tolerant S. Tm cells in vivo.(Left) Experimental strategy. (Right) Experimental data. Three groups of C57BL/6 mice were infected for 2 d or 3 d with S. Tm(pAM34), as indicated. The third group was treated with ciprofloxacin during the third day (2×62 mg/kg/d by gavage). Total S. Tm loads (closed symbols) and S. Tm(pAM34) (open symbols) in the cLN were determined by plating. Dashed line, detection limit. p, nonparametric statistical analysis. n.s., not significant.
© Copyright Policy
Related In: Results  -  Collection

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

pbio-1001793-g005: Plasmid dilution experiment verifying the slow growth rate of tolerant S. Tm cells in vivo.(Left) Experimental strategy. (Right) Experimental data. Three groups of C57BL/6 mice were infected for 2 d or 3 d with S. Tm(pAM34), as indicated. The third group was treated with ciprofloxacin during the third day (2×62 mg/kg/d by gavage). Total S. Tm loads (closed symbols) and S. Tm(pAM34) (open symbols) in the cLN were determined by plating. Dashed line, detection limit. p, nonparametric statistical analysis. n.s., not significant.
Mentions: To confirm the slow growth rates of tolerant S. Tm cells in vivo, we employed a plasmid-dilution strategy (Figure 5, left side). pAM34 is an IPTG-addicted plasmid that stops replication as soon as IPTG is removed from the environment, including all mouse tissues analyzed (pAM34, AmpR, [42]). This type of plasmid can be used to verify the slow growth rate of the ciprofloxacin-tolerant S. Tm subpopulation. Such tolerant cells should survive the antibiotic and should feature a high fraction of cells retaining pAM34.

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