Sequential phage delivery can outperform cocktails by delaying cross-resistance evolution

Antimicrobial resistance has renewed interest in bacteriophage therapy,
yet bacterial evolution frequently undermines treatment efficacy.
Combination phage therapy is commonly implemented as simultaneous phage
cocktails, but whether this is optimal remains in question. Here, we
experimentally compare simultaneous versus sequential administration of
two phages, an evolved λ called ‘λtrn’ and T2, on Escherichia coli K-12
under controlled laboratory conditions. Across replicated experiments,
treatment outcome depended strongly on delivery strategy, dosing order,
and timing. Contrary to expectations, sequential delivery consistently
achieved greater and more sustained bacterial suppression than
simultaneous cocktails, although only when T2 initiated the sequence.
Phenotypic assays revealed that treatment differences are driven by the
accessibility and timing of cross-resistance evolution. λtrn-First
treatments rapidly selected for cross-resistant bacteria prior to exposure
to the second phage, rendering subsequent treatment ineffective. In
contrast, T2-First sequential treatments delayed or limited
cross-resistance and frequently produced single-phage resistance or
collateral sensitivity. Cocktail treatments showed intermediate dynamics,
with cross-resistance evolving more slowly but consistently. Whole-genome
sequencing identified distinct genetic routes to cross-resistance,
including regulatory mutations in envZ affecting expression of the phage
receptor OmpF, as well as envelope-modifying, mucoidy-associated
mutations. Engineering envZ mutations into unevolved backgrounds confirmed
the mutations sufficiency to confer low-cost cross-resistance. Together,
these results demonstrate that phage therapy efficacy depends not only on
phage composition but on how selection pressures are ordered in time,
highlighting evolutionary steering as a powerful principle for multi-phage
therapy design.