Surviving Colonies of Pseudomonas aeruginosa Isolated In Vivo from Infected, Antibiotic-Treated Galleria mellonella Larvae Acquire an Antibiotic-Tolerant Phenotype

<b>Background:</b> The aim of this work was to induce the formation of antibiotic-tolerant and/or persister cells in vivo using antibiotic therapy on <i>Galleria mellonella</i> larvae infected with <i>P. aeruginosa</i>, isolate these surviving cells, and characterise their phenotype and genotype. <b>Methods</b>: Infected larvae were treated with effective doses of either ceftazidime or meropenem. Despite this, surviving <i>P. aeruginosa</i> colonies were isolated from living larvae, and antibiotic killing, fitness, virulence, antibiotic resistance and the whole genome sequence of a selection of these isolates were compared with their original parent strains. <b>Results</b>: The surviving isolates had an increased minimum duration to kill 99% of the population (MDK₉₉) upon exposure to ceftazidime or meropenem and decreased growth rates in culture, but they showed no change to the MIC or virulence-consistent with an antibiotic-tolerant phenotype. Long-read genome sequencing of selected isolates revealed only one single nucleotide polymorphism (SNP) within <i>bkdB</i>, encoding the lipoamide acyltransferase component of the branched-chain α-keto acid dehydrogenase, present in two independent isolates. However, time-kill assays with ceftazidime of <i>bkdB</i> knockout strains showed no significant change in the MDK₉₉. Concomitant with the antibiotic-tolerant phenotype, many of the isolates also had a reduced rate of killing when exposed to heat stress. <b>Conclusions</b>: <i>P. aeruginosa</i> cells that survived antibiotic therapy in vivo were found to be antibiotic-tolerant and thermotolerant but not antibiotic-resistant and had reduced growth rates under optimal conditions but unchanged virulence. In the absence of a convincing genetic explanation, the co-induction of enhanced thermotolerance with antibiotic tolerance indicated that both are conferred by a heritable phenotypic mechanism.