Personalized bacteriophage therapy outcomes for 100 consecutive cases: a multicentre, multinational, retrospective observational study

Abstract

In contrast to the many reports of successful real-world cases of personalized bacteriophage therapy (BT), randomized controlled trials of non-personalized bacteriophage products have not produced the expected results. Here we present the outcomes of a retrospective observational analysis of the first 100 consecutive cases of personalized BT of difficult-to-treat infections facilitated by a Belgian consortium in 35 hospitals, 29 cities and 12 countries during the period from 1 January 2008 to 30 April 2022. We assessed how often personalized BT produced a positive clinical outcome (general efficacy) and performed a regression analysis to identify functional relationships. The most common indications were lower respiratory tract, skin and soft tissue, and bone infections, and involved combinations of 26 bacteriophages and 6 defined bacteriophage cocktails, individually selected and sometimes pre-adapted to target the causative bacterial pathogens. Clinical improvement and eradication of the targeted bacteria were reported for 77.2% and 61.3% of infections, respectively. In our dataset of 100 cases, eradication was 70% less probable when no concomitant antibiotics were used (odds ratio = 0.3; 95% confidence interval = 0.127-0.749). In vivo selection of bacteriophage resistance and in vitro bacteriophage-antibiotic synergy were documented in 43.8% (7/16 patients) and 90% (9/10) of evaluated patients, respectively. We observed a combination of antibiotic re-sensitization and reduced virulence in bacteriophage-resistant bacterial isolates that emerged during BT. Bacteriophage immune neutralization was observed in 38.5% (5/13) of screened patients. Fifteen adverse events were reported, including seven non-serious adverse drug reactions suspected to be linked to BT. While our analysis is limited by the uncontrolled nature of these data, it indicates that BT can be effective in combination with antibiotics and can inform the design of future controlled clinical trials. BT100 study, ClinicalTrials.gov registration: NCT05498363 .

Fig. 1. Characteristics of the patient population involved in the 100 consecutive BT cases facilitated by the Belgian consortium.

a, Geographic location of the BT cases. b, Number of BT cases and their regulatory context, per year. SOC MP, standard-of-care with magistral bacteriophage preparations; DH, article 37 (unproven interventions in clinical practice) of the Declaration of Helsinki; SOC UM, standard-of-care with unlicensed medicines; ATU MP, ‘Autorisation Temporaire d’Utilisation’ of magistral preparations. c, Primary and secondary (concomitant) infection types. AbdI, abdominal infection; OPI, orthopaedic prostheses infection. d, Patient age and gender distribution. Boxplot shows the interquartile range of the age (years) of the patients (n = 90): first quartile (29.5), median (53) and third quartile (62). The whiskers extend from the quartiles to the last data point within 1.5 × the interquartile range. Data points plotted outside the boundary of the whiskers are outliers. Female patients are represented by purple filled circles and male patients by blue filled circles. e, Targeted bacterial species. In some cases, bacteriophages targeted two or three bacterial species (connected by lines) in one patient. Source data

Fig. 2. The in vivo emergence of bacteriophage resistance during BT.

Monitored by whole-genome analysis of sequential bacterial isolates in patients 30, 54, 64, 82 and 91 (in vivo emergence of bacteriophage resistance in patients 16 and 20 discussed in Table 2). a, Maximum likelihood phylogenetic tree of the genomes of the analysed sequential bacterial isolates. b–d, Circular chromosomic view (CCV) of the bacterial genomes of sequential isolates (Is) of Pseudomonas aeruginosa strains retrieved just before (Is 1, inner circle) and during BT (Is 2-n) from patients 54 (b), 30 (c) and 91 (d). Green rings display the genomes of bacteriophage-susceptible isolates, while the red rings display the genomes and relevant (for bacterial bacteriophage resistance) mutations in bacteriophage-resistant isolates. The two multicoloured outer rings display the protein annotations (categories) as present in the Clusters of Orthologous Groups of proteins (COGs) database. bp, basepairs; CDS, coding sequence; IS, insertion sequence; Mb, megabases; nt, nucleotide; PTM, post-translational modification.

Fig. 3. Results of the in vitro evaluation of the combined effects of bacteriophages and concomitantly applied antibiotics on the targeted bacterial strains.

Determined by an OmniLog system for patients 54, 71, 82, 91 and 92 (those for patients 9, 20, 21, 27 and 43 are discussed in Table 2). Bacterial proliferation is presented through relative units of cellular respiration. a,b, No additive effect of colistin and bacteriophages PNM (a) and 14-1 (b) for patient 54. c, Additive effect (delayed bacterial growth) of ceftazidime and bacteriophage PT07 for patient 71. d,e, Synergistic effect of levofloxacin and bacteriophages 4P (d) and DP1 (e) for patient 82. f,g, No additive effect of tobramycin and bacteriophages 4P (f) and DP1 (g) for patient 82. h,i, Synergistic effect of bacteriophage PT07 and the antibiotics meropenem (h) and colistin (i) for patient 91. j–l, Synergistic effect of bacteriophage ISP and the antibiotics clindamycin (j), vancomycin (k) and ceftarolin (l). Pa, Pseudomonas aeruginosa; Sa, Staphylococcus aureus. Source data

Fig. 4. Emergence of bacteriophage immune neutralization.

a–e, Chronological bacteriophage immune neutralization (BIN) activity against the applied bacteriophages in sera collected before, during and after BT in patients 9 (a), 13 (b), 20 (c), 66 (d) and 92 (e). The evolution over time of the serum BIN activity against the applied bacteriophages is shown as % bacteriophage titre loss (compared to pre-BT control sera) after incubation of the bacteriophages with sequential serum samples for 30 min. BIN activity appeared 1–5 weeks after BT initiation. Data are presented as mean ± s.d. of three biological replicates. ABCONCOM, concomitant antibiotherapy; admin, administration; CI, clinical improvement; ERADIC, eradication; IL, intralesional; INDICATI, indication; i.v., intravenous; Kp, Klebsiella pneumoniae; Ma, Mycobacterium abscessus; Nebul, nebulization; Pa, Pseudomonas aeruginosa; Sa, Staphylococcus aureus; Sm, Stenotrophomonas maltophilia; TARGET, targeted bacterial species. Bacteriophage cocktail BFC 1 contains bacteriophages ISP, 14-1 and PNM. Source data

Extended Data Fig. 1

The Phage Therapy Coordination Centre’s patient selection process for bacteriophage therapy.

Extended Data Fig. 2. Missense mutations in the pre-adapted variant of bacteriophage ISP, as compared to the original clone (before adaptation).

HHpred (https://www.sciencedirect.com/science/article/pii/S0022283617305879), HMMR (https://nar.oxfordjournals.org/content/46/W1/W200), and Phyre (https://www.nature.com/articles/nprot.2009.2) were used for functional prediction. ESI-MS, electrospray ionization mass spectrometry.

Extended Data Fig. 3. Kaplan-Meier plots and activity scores of Galleria mellonella larvae post-infection.

Ten larvae in each group were either inoculated with phosphate buffered saline (PBS, control), with the initial bacteriophage-susceptible isolates (wild type, wt), or with the in vivo selected bacteriophage-insensitive mutants of the Pseudomonas aeruginosa strains isolated from patient (P) 91, 54, and 30. a-b, P54 (Is1 and 4). c-d, P30 (Is1 and 3). e-f, P91 (Is1 to 6). Mean values of activity scores are represented by a dot symbol. P values were calculated using the log-rank test with Bonferroni correction for multiple comparisons. Is, isolate; mut, mutation. Source data

Extended Data Fig. 4. Results of the in vitro evaluation of the influence of serial multiplicities of infection (MOIs) on the virulence and on the resistance suppression of Pseudomonas aeruginosa bacteriophage 4 K on the bacterial host strain CN573, as determined in liquid culture, using an OmniLog^® system.

Bacterial proliferation is presented through relative units of cellular respiration over time (72 h). Source data