Research ArticleCystic Fibrosis

Filamentous bacteriophages are associated with chronic Pseudomonas lung infections and antibiotic resistance in cystic fibrosis

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Science Translational Medicine  17 Apr 2019:
Vol. 11, Issue 488, eaau9748
DOI: 10.1126/scitranslmed.aau9748
  • Fig. 1 Pf phage is prevalent in the Pseudomonas Genome Database, a Danish CF cohort and the Stanford CF cohort.

    (A) The prevalence of the detection of the Pf prophage in P. aeruginosa logged in the Pseudomonas Genome Database, an internet- and community-based genome project containing the genome sequences and annotations of both clinical and environmental isolates of P. aeruginosa. (B) The prevalence of the detection of Pf prophage in P. aeruginosa isolates from the Danish CF cohort previously described by Marvig et al. (40). Four hundred seventy-four P. aeruginosa whole-genome sequences were collected from a cohort of 34 Danish patients with CF who were followed longitudinally. There are multiple isolates from each patient represented in this figure. (C) The prevalence of Pf phage detected by quantitative polymerase chain reaction (qPCR) in the sputum of patients also with P. aeruginosa in their sputum from the Stanford CF cohort. Each patient contributed only one sputum sample.

  • Fig. 2 In a Danish CF cohort, the prevalence of Pf phage rises with increasing patient age.

    (A) Patients were categorized as having all isolates with the Pf prophage detected (Pf phage +), intermittently having the Pf prophage detected (Pf phage intermittent), or all isolates with no Pf prophage detected (Pf phage −). (B) Mean age of patients with CF in each category. Age was unavailable for three subjects (n = 12, n = 9, n = 9). Boxes represent interquartile range (IQR) with whiskers showing 1.5 × IQR and dots as outliers. Comparison was made by Mann-Whitney test. The outliers on graph were included in analysis. Data are available in table S1. (C) Correlation between subject attained age at the date of the last isolate versus the proportion of total isolates in which Pf prophage was detected(r = 0.5098, P = 0.048). Data for n = 31 patients are included with each dot representing a specific age category and assessed by Pearson correlation.

  • Fig. 3 P. aeruginosa concentration is higher in patients with Pf phage detected in the sputum within the Stanford CF cohort.

    (A) Patients enrolled at the Stanford CF Center with detection of P. aeruginosa by both qPCR and respiratory culture and detection of Pf phage by qPCR. Of the 10 patients with P. aeruginosa detected by PCR but not grown on respiratory culture, 9 had history of P. aeruginosa on previous respiratory culture and 1 patient had no previous records available. Pa, P. aeruginosa. (B) P. aeruginosa concentrations in patients with Pf phage–positive sputum and Pf phage–negative sputum (P = 0.01). Boxes represent IQR with whiskers showing 1.5 × IQR and dots as outliers. Comparison was done by Wilcoxon ranked sum. Outliers on graph were included in analysis.

  • Fig. 4 Pf phage concentration correlates with P. aeruginosa burden, chronic infection and patient age in the Stanford CF cohort.

    (A) Correlation between the concentrations of Pf phage and P. aeruginosa in patients positive for Pf phage (r = 0.84, P < 0.001). Dotted line, line for 1:1 correlation for reference. All samples were run in duplicate. The area between dashed lines represents the 95% CI. Correlation calculated by Kendall correlation. (B) Percentage of patients meeting the Leeds criteria for chronic P. aeruginosa infection in Pf phage–positive and Pf phage–negative samples (P = 0.002). Comparison by Pearson’s χ2 was used. (C) Age of patients with P. aeruginosa–negative sputum, P. aeruginosa–positive and Pf phage–negative sputum, and P. aeruginosa–positive and Pf phage–positive sputum. Boxes represent IQR with whiskers showing 1.5 × IQR and dots as outliers. Comparison was done by Wilcoxon ranked sum. (D) Prevalence of Pf phage in adult versus pediatric patients with CF infected with P. aeruginosa (P = 0.03). Comparison by Pearson’s χ2. Outliers on graph were included in analysis.

  • Fig. 5 Patients positive for Pf phage have reduced lung function during exacerbations in the Stanford CF cohort.

    The difference between FEV1% predicted at time of sampling and at presentation of subsequent exacerbation in patients with Pf phage–positive sputum and in those with Pf phage–negative sputum (P = 0.03). Only patients who were enrolled while at baseline health and who had exacerbation after enrollment were included in this analysis (n = 11 patients positive for Pf phage, n = 20 patients negative for Pf phage). These data include only patients with P. aeruginosa detected in their sputum. Boxes represent IQR with whiskers showing 1.5 × IQR and dots as outliers. Comparisons were made by Mann-Whitney test. Outlier on graph was included in analysis. Data are available in table S2.

  • Fig. 6 Clinical isolates of P. aeruginosa from patients with Pf phage–positive sputum exhibit increased antibiotic resistance and are more likely to be mucoid.

    (A) Proportion of isolates that display mucoid phenotype in isolates from Pf phage–negative sputum and Pf phage–positive sputum (P = 0.03). Comparison by Pearson χ2 test. (B) The proportion of isolates that were resistant to antibiotics listed. n = 28 Pf phage–negative samples, n = 20 Pf phage–positive samples. Comparison was made by Pearson’s χ2 test. *P = 0.0499, **P = 0.002, ***P = 0.0009.

  • Fig. 7 Antibiotics aztreonam, amikacin, and meropenem can be sequestered by Pf phage.

    The magnitude of sequestered antibiotic (bacterial proliferation normalized to control conditions) is displayed for each antibiotic when run through dialysis cassette containing either Pf phage and DNA or DNA alone (control). Samples were run in triplicate (N = 2 experiments). Error bars indicate mean ± SEM. Comparisons were made by Student’s t test with an α level of 0.05. ****P < 0.0001, *P < 0.05.

  • Table 1 Patient characteristics and microbiologic data in the Stanford CF cohort.

    Wilcoxon ranked sum was used for analysis of continuous values, and Pearson’s χ2 was used for categorical values. P values for comparison of the two right columns, P. aeruginosa-positive Pf phage − patients versus P. aeruginosa-positive Pf phage-positive.

    P. aeruginosa-negative
    by qPCR
    P. aeruginosa-positive by qPCRP
    Pf phage-negativePf phage-positive
    Subjects, n183721
    Sex, % male72.2%51.4%52.4%0.94
    Age, years (±SD)23.2 ± 13.525.9 ± 11.033.4 ± 10.90.006
    FEV1, % predicted (±SD)50.3 ± 24.054.1 ± 20.158.7 ± 26.50.57
    BMI, Z score (±SD)−0.16 ± 1.00.8 ± 0.850.09 ± 0.90.37
    CFTR genotype
      Homozygous F508del, n (%)5 (27.8%)17 (46.0%)11 (54.5%)0.52
      Heterozygous F508del, n (%)8 (44.4%)14 (37.8%)5 (23.8%)
    P. aeruginosa, copies/ml
    (range)
    02.2 × 108
    (2.8 × 102–5.1 × 109)
    3.2 × 108
    (1.5 × 105–1.9 × 109)
    0.01
    Pf phage, copies/ml (range)004.4 × 109
    (1.3 × 106–3.1 × 1010)
  • Table 2 Phenotype and resistance patterns from respiratory culture from patients in the Stanford CF cohort.

    Antibiotic resistance was assessed in clinical P. aeruginosa isolates cultured from the same sputum samples in which Pf phage status was assessed in our cohort. N = 28 Pf phage–negative samples and 20 Pf phage–positive samples. Comparison by Pearson’s χ2 test.

    P. aeruginosa + by qPCRP
    Pf phage −Pf phage +
    Mucoid strain58.3%85.71%0.03
    Ciprofloxacin12 (33%)6 (29%)0.71
    Tobramycin7 (19%)7 (33%)0.24
    Aztreonam5 (14%)7 (33%)0.0499
    Amikacin8 (22%)14 (67%)0.0009
    Cefepime7 (19%)8 (38%)0.12
    Ceftazidime7 (19%)5 (23%)0.70
    Meropenem4 (11%)10 (48%)0.002
    Piperacillin-
    tazobactam
    7 (19%)5 (24%)0.70

Supplementary Materials

  • stm.sciencemag.org/cgi/content/full/11/488/eaau9748/DC1

    Fig. S1. Baseline characteristics of the Stanford CF cohort.

    Fig. S2. Patients with higher Pf phage concentrations have lower FEV1 percent predicted values.

    Fig. S3. Pf phage liquid crystal biofilm sequesters antibiotics leading to the development of antibiotic resistance and chronic infection.

    Table S1. Individual values for Fig. 2B.

    Table S2. Individual data points from Fig. 4 (C and D).

    Table S3. Resistance patterns in mucoid and nonmucoid isolates.

  • This PDF file includes:

    • Fig. S1. Baseline characteristics of the Stanford CF cohort.
    • Fig. S2. Patients with higher Pf phage concentrations have lower FEV1 percent predicted values.
    • Fig. S3. Pf phage liquid crystal biofilm sequesters antibiotics leading to the development of antibiotic resistance and chronic infection.
    • Table S1. Individual values for Fig. 2B.
    • Table S2. Individual data points from Fig. 4 (C and D).
    • Table S3. Resistance patterns in mucoid and nonmucoid isolates.

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