Research ArticleDRUG RESISTANCE

Fitness cost of antibiotic susceptibility during bacterial infection

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Science Translational Medicine  22 Jul 2015:
Vol. 7, Issue 297, pp. 297ra114
DOI: 10.1126/scitranslmed.aab1621
  • Fig. 1. In vivo fitness of P. aeruginosa mutants with Tn insertions in four classes of genes annotated as VFs after 1, 6, or 24 hours of infection in the murine lung.

    The inner green circle represents RPKM changes between LB-cultured P. aeruginosa and 1 hour of lung infection; pink circle depicts changes from 1 to 6 hours of infection; and yellow circle represents changes from 6 to 24 hours of infection. The outer circle is the entire P. aeruginosa chromosome with each gene represented by one of six different shades of blue organized into a repetitive pattern. The genes associated with the identified VFs are represented at ×20 magnification in the outer circle. The VFs are depicted in four color-coded categories (green, orange, gray, and purple) in the green, pink, and yellow inner circles, and across a gradient of lighter to darker bars to differentiate the different gene clusters. A decrease in RPKM (bars pointing to circle’s center indicate a decreased fitness) of Tn insertions in genes encoding for all the known major VFs of P. aeruginosa was observed after 24 hours of lung infection (yellow circle) but not after 1 hour (green circle) or 6 hours (pink circle) of infection, except for Tn insertions in genes encoding LPS O-antigens (darker green).

  • Fig. 2. In vivo fitness of P. aeruginosa PA14 Tn library.

    (A) RPKM changes that occurred after five days of GI tract colonization by P. aeruginosa, as previously reported (3). (B to E) Relative ranking and absolute number of RPKM that changed for 5977 genes of P. aeruginosa PA14 during lung infection, comparing the RPKM in the LB input with those obtained 1 hour after infection (B) or comparing RPKM obtained 1 hour after infection with those obtained at 6 hours after infection, and 24 hours after infection with 32 to 48 hours after infection (labeled “Lung 48 h”) (C to E). Dots above the input lines indicate Tn insertions in genes with a positive fitness (increase in in vivo RPKM), whereas dots below the input line indicate those with a negative fitness (decrease in in vivo RPKM).

  • Fig. 3. Increased fitness of carbapenem (PA14_Tn-oprD)– and fosfomycin (PA14_Tn-glpT)–resistant P. aeruginosa Tn insertion mutants in murine lung infection.

    (A) Increases in RPKM in Tn-glpT or Tn-oprD P. aeruginosa strains from the TnSeq analysis in the murine model of pneumonia. (B) Virulence of P. aeruginosa PA14 glpT or oprD Tn insertion mutants in lung infections compared to wild-type (WT) and complemented strains (n = 12 mice per group; WT versus Tn-oprD: P < 0.0001, WT versus Tn-glpT: P < 0.0001, Tn-oprD versus Tn-oprD::PoprD: P = 0.0002, and Tn-glpT versus Tn-glpT::PglpT: P < 0.0001, log-rank test). (C) Killing of J774 macrophages by the fosfomycin-resistant glpT mutant compared to the WT or glpT-complemented strains. (D) Multiplication of P. aeruginosa PA14 WT, ΔglpT, and glpT-complemented mutant inside J774 macrophages. (E) Multiplication of the internalized ΔglpT compared with WT and glpT-complemented strains in MH-S alveolar macrophages after 24 hours. For (C) to (F), bars represent means of triplicate determinations, and error bars indicate the SD. *P < 0.05, Tukey’s post hoc test versus control. P < 0.05, overall analysis of variance (ANOVA) for each data set. PA14 background lacking the exoU gene was used in the in vitro studies to avoid cytotoxic effects of this effector of the type 3 secretion system.

  • Fig. 4. Effect of the loss of intrinsic antibiotic resistance genes in P. aeruginosa on fitness during GI tract colonization.

    The ability to colonize the murine GI tract was measured by the competitive index (CI). The CI is calculated by dividing the proportion of mutant cells at the end of the competition by the proportion at the start. (A to D) A CI ratio <1 in the left-hand panels indicates that the Tn insertion mutant is less fit. Right-hand panels depict the qRT-PCR analysis of the expression of the transcript for each indicated gene when P. aeruginosa was grown in LB, drinking water used for colonization, or recovered from the murine GI tract. (A) Resistance to amoxicillin–clavulanic acid (Tn-ampC). (B and C) Resistance to nalidixic acid, chloramphenicol, and cotrimoxazole (Tn-mexA and Tn-oprM). (D) Resistance to kanamycin (Tn-aph). Bars for CI represent means from four mice, and error bars represent the SD. Bars for qRT-PCR represent means of three individual experiments, and error bars represent the SD. *P < 0.05, one-sample t test (default = 1).

  • Fig. 5. Effect of the loss of intrinsic antibiotic resistance genes in P. aeruginosa on fitness in the murine lung infection model.

    (A) Survival curves of mice after lung infection with WT P. aeruginosa or strains carrying Tn insertions in oprM, mexA, ampC, or aph genes. A significant decrease in virulence was observed between WT and the various insertion mutants (oprM: P = 0.0036, ampC: P = 0.0162, mexA: P = 0.0288, and aph: P = 0.0050, log-rank test). (B to E) mRNA transcript expression levels of ampC, aph, mexA, and oprM as determined by qRT-PCR for the indicated P. aeruginosa mutant strain during infection of J774 macrophages for 1, 6, or 24 hours or in the murine lung for 1 hour. For each sample, transcript levels of ampC, aph, mexA, and oprM were assessed by relative quantification using the 2−ΔΔCt method. Expression of the rpsL gene was used as a housekeeping control gene.

  • Fig. 6. Fitness cost of antibiotic susceptibility in A. baumannii and V. cholerae.

    (A) Effect of Tn insertions in genes associated with constitutive antibiotic resistance on the virulence of A. baumannii in a murine lethal peritonitis infection setting. WT A. baumannii (AB) strain 5075 and isogenic strains with Tn insertions in genes homologous to A1S_1649 and A1S_1801 were used at a challenge dose of 5 × 109 CFU per mouse, administrated intraperitoneally. A significant decrease in virulence was observed when comparing the WT with either of the two Tn insertion mutants (AB5075-A1S_1649 and AB5075-A1S_A1S_1801; P = 0.01, log-rank test). (B) Effect of Tn insertions in genes tolC and lpp (tolC::Tn and lpp::Tn, respectively) on V. cholerae susceptibility to antibiotic polymyxin. V. cholerae C6706 WT, tolC::Tn, and lpp::Tn were grown in liquid culture (LB) in the presence of polymyxin (0.5 mg/liter) for 5 hours, and 10-fold serial dilutions were plated on agarose plates (LB). (C and D) Infant rabbit competition assays using WT, lpp::Tn, and tolC::Tn mutant V. cholerae strains. The in vivo CIs were determined phenotypically. V. cholerae C6706 WT strain had an insertion of TnFGL3 into lacZ (lacZ::Tn), whereas tolC::Tn and lpp::Tn produced β-galactosidase for differentiation when competing against the parental ΔlacZ WT strain. Inoculum used was 109 CFU per rabbit (C) or 107 CFU per rabbit (D). Significance was determined with the Student’s t test by comparing the colonization ratios of C6706 WT lacZ::Tn versus the tolC::Tn (P < 0.0001) or lpp::Tn (P < 0.01) mutant strains. Mean plus SEM is shown.

Supplementary Materials

  • www.sciencetranslationalmedicine.org/cgi/content/full/7/297/297ra114/DC1

    Materials and Methods

    Fig. S1. Evolution of the RPKM sequencing reads for Tn insertions in genes in each of 27 functional classes from LB to the lung after 1 hour (A), from 1 to 6 hours in the lung (B), and from 6 to 24 hours in the lung (C).

    Fig. S2. In vivo fitness of P. aeruginosa mutants with Tn insertions in genes from the functional class “motility and attachment” after 1, 6, or 24 hours of infection in the lung.

    Fig. S3. Comparative in vivo fitness (in the GI tract and lung) of bacterial strains with Tn insertions in genes needed to produce T4aP components.

    Fig. S4. Comparative in vitro fitness in LB and water of bacterial strains with Tn insertion mutants in genes needed to produce T4aP components.

    Fig. S5. Comparative in vivo fitness of bacterial strains with Tn insertions in genes that encode all of the annotated VFs of P. aeruginosa (after 1, 6, or 24 hours in the lung).

    Fig. S6. Evolution over time of the RPKM for Tn-interrupted genes in the LPS O-antigen locus.

    Fig. S7. Increased virulence of oprD mutant carbapenem-resistant clinical strains of P. aeruginosa (48.2 and 51.2) in lung infection compared to isogenic (48.1 and 51.1) and complemented strains (48.2::PoprD and 51.2::PoprD).

    Fig. S8. Evolution over time of the changes in the RPKM for Tn-interrupted genes associated with constitutive antibiotic resistance in P. aeruginosa in the GI tract, spleen, and lung.

    Fig. S9. Analysis of in vitro growth and survival of Tn mutants deficient in genes associated with constitutive antibiotic resistance in P. aeruginosa.

    Fig. S10. Effect of deletion of intrinsic antibiotic resistance genes on survival of P. aeruginosa in J774 macrophages.

    Table S1. Analysis of selective pressures detected by RPKM reads during P. aeruginosa lung infection.

    Table S2. Genes (116) whose loss shows an increased fitness for lung infection based on having Tn insertions with at least 100 reads after 24 hours and with reads further increasing more than twofold between 24 and 48 hours of infection.

    Table S3. Antibiotic sensitivity results for wild-type P. aeruginosa PA14 and strains deleted for genes encoding intrinsic antibiotic resistance.

    Table S4. Antibiotic sensitivity results for wild-type P. aeruginosa PA14 and strains deleted for genes encoding intrinsic antibiotic resistance (inhibition diameter).

    Table S5. Antibiotic sensitivity results for wild-type P. aeruginosa PA14 and strains deleted for genes encoding intrinsic antibiotic resistance (MIC as measured by E test).

    Table S6. Primers for genomic amplification used in this study.

    Table S7. Bacterial strains used in this study.

    Table S8. Plasmids used in this study.

    Data file S1. Analysis of the TnSeq data by functional classes of P. aeruginosa PA14.

    Data file S2. Fitness of flagellin mutants identified in the study.

    References (4959)

  • Supplementary Material for:

    Fitness cost of antibiotic susceptibility during bacterial infection

    Damien Roux, Olga Danilchanka, Thomas Guillard, Vincent Cattoir, Hugues Aschard, Yang Fu, Francois Angoulvant, Jonathan Messika, Jean-Damien Ricard, John J. Mekalanos, Stephen Lory, Gerald B. Pier,* David Skurnik*

    *Corresponding author. E-mail: gpier{at}bwh.harvard.edu (G.B.P.); dskurnik{at}rics.bwh.harvard.edu (D.S.)

    Published 22 July 2015, Sci. Transl. Med. 7, 297ra114 (2015)
    DOI: 10.1126/scitranslmed.aab1621

    This PDF file includes:

    • Materials and Methods
    • Fig. S1. Evolution of the RPKM sequencing reads for Tn insertions in genes in each of 27 functional classes from LB to the lung after 1 hour (A), from 1 to 6 hours in the lung (B), and from 6 to 24 hours in the lung (C).
    • Fig. S2. In vivo fitness of P. aeruginosa mutants with Tn insertions in genes from the functional class “motility and attachment” after 1, 6, or 24 hours of infection in the lung.
    • Fig. S3. Comparative in vivo fitness (in the GI tract and lung) of bacterial strains with Tn insertions in genes needed to produce T4aP components.
    • Fig. S4. Comparative in vitro fitness in LB and water of bacterial strains with Tn insertion mutants in genes needed to produce T4aP components.
    • Fig. S5. Comparative in vivo fitness of bacterial strains with Tn insertions in genes that encode all of the annotated VFs of P. aeruginosa (after 1, 6, or 24 hours in the lung).
    • Fig. S6. Evolution over time of the RPKM for Tn-interrupted genes in the LPS O-antigen locus.
    • Fig. S7. Increased virulence of oprD mutant carbapenem-resistant clinical strains of P. aeruginosa (48.2 and 51.2) in lung infection compared to isogenic (48.1 and 51.1) and complemented strains (48.2::PoprD and 51.2::PoprD).
    • Fig. S8. Evolution over time of the changes in the RPKM for Tn-interrupted genes associated with constitutive antibiotic resistance in P. aeruginosa in the GI tract, spleen, and lung.
    • Fig. S9. Analysis of in vitro growth and survival of Tn mutants deficient in genes associated with constitutive antibiotic resistance in P. aeruginosa.
    • Fig. S10. Effect of deletion of intrinsic antibiotic resistance genes on survival of P. aeruginosa in J774 macrophages.
    • Table S1. Analysis of selective pressures detected by RPKM reads during P. aeruginosa lung infection.
    • Table S2. Genes (116) whose loss shows an increased fitness for lung infection based on having Tn insertions with at least 100 reads after 24 hours and with reads further increasing more than twofold between 24 and 48 hours of infection.
    • Table S3. Antibiotic sensitivity results for wild-type P. aeruginosa PA14 and strains deleted for genes encoding intrinsic antibiotic resistance.
    • Table S4. Antibiotic sensitivity results for wild-type P. aeruginosa PA14 and strains deleted for genes encoding intrinsic antibiotic resistance (inhibition diameter).
    • Table S5. Antibiotic sensitivity results for wild-type P. aeruginosa PA14 and strains deleted for genes encoding intrinsic antibiotic resistance (MIC as measured by E test).
    • Table S6. Primers for genomic amplification used in this study.
    • Table S7. Bacterial strains used in this study.
    • Table S8. Plasmids used in this study.
    • Data file S1. Analysis of the TnSeq data by functional classes of P. aeruginosa PA14.
    • Data file S2. Fitness of flagellin mutants identified in the study.
    • References (4959)

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