DOI: 10.5061/dryad.4j0zpc8jt
title: Resistance to bacteriophage incurs a cost to virulence in drug resistant Acinetobacter baumannii

description: Introduction: Acinetobacter baumannii is a critical priority pathogen
 (World Health Organisation) because of the rise in nosocomial infections
 and its ability to evolve resistance to last resort antibiotics, which
 makes A. baumannii a priority target for phage therapy. Two strains of a
 novel, lytic bacteriophage (LemonAid and Tonic) able to infect
 carbapenem-resistant A. baumannii (strain NCTC 13420), were isolated from
 environmental water samples collected through a citizen science program.
 Methods: In vitro and in vivo assays, genomics and microscopy techniques
 were used to characterise the phages, determine mechanisms of phage
 resistance and the efficacy of the phages against A. baumannii. Results:
 A. baumannii developed resistance to both viruses, LemonAid and Tonic.
 Resistance came at a cost to virulence, with the resistant variants
 causing significantly reduced mortality in a Galleria mellonella larval in
 vivo model. A replicated 8bp insertion increased in frequency (~40% higher
 frequency than in the wildtype) within phage-resistant A. baumannii
 mutants, putatively resulting in early truncation of a protein of unknown
 function. Evidence from comparative genomics and an adsorption assay
 suggests this protein acts as a novel phage receptor site in A. baumannii.
 We find no evidence linking resistance to changes in capsule structure, a
 known virulence factor. LemonAid efficiently suppressed growth of A.
 baumanni in vitro across a wide range of titres. However, in vivo, while
 survival of A. baumannii infected larvae significantly increased with both
 remedial and prophylactic treatment with LemonAid (107 PFU/mL), the effect
 was weak and not sufficient to save larvae from morbidity and mortality.
 Conclusion: While LemonAid and Tonic did not prove effective as a
 treatment in a Galleria larvae model, there is potential to harness their
 ability to attenuate virulence in drug-resistant A. baumannii.
description: # Resistance to bacteriophage incurs a cost to virulence in drug resistant
 Acinetobacter baumannii
 [https://doi.org/10.5061/dryad.4j0zpc8jt](https://doi.org/10.5061/dryad.4j0zpc8jt) ## Description of the data and file structure There are 11 dataset provided here that were used in the analysis and production of figures for the manuscript entitled 'Resistance to bacteriophage incurs a cost to virulence in drug resistant Acinetobacter baumannii. The dataset can be split into three sections: The first section relates to the *in vitro* growth curve data (optical density recorded over 24 hours, across different treatments) and contains datasets 1-4. Two experiments were run in total: Experiment 1) *A. baumannii* wildtype was infected with different concentrations of phage (LemonAid and Tonic), the raw data for this is dataset 1. Area under the curve (AUC) and virulence was calculated from this dataset and these data are provided in dataset 2. Experiment 2) the growth curves of wildtype *A. baumannii* and phage resistant variants were compared (dataset 3), from this raw data further growth metrics were calculated using the R program Growthcurver (dataset 4, k = carrying capacity, r = growth and auc = area under the curve). For clarity, within the heading of each dataset I have included the manuscript figure number the dataset relates to. Column headings for datasets 1-4: Dataset 1 and 3 contain output data from 96 well plate optical density over time. well = location of sample on 96 well plate; time_hours = time in hours, mean_od = the mean (the software takes 4 measurements of each well at each time point, and takes a mean) optical density of each well per time point.  Dataset 2 contains the growth curve data converted into a measure of virulence: well = location of sample on 96 well plate; AUC_trap = the area under the curve per sample/time point, virulence = 1 – area under the curve (AUC) phage treatment/AUC (no phage) Dataset 4 contains the output from the R package growthcurver, that quantifies aspects of bateria growth: growth rate (r), carrying capacity (k) and AUC were calculated to compare the phage-resistant variant to the wildtype *A. baumannii*. The second section relates to the *Galleria mellonella* experiments and contains datasets 5-10, there were three experiments, two parameters were recorded for each experiment, survival and melanisation, and there are separate datasets for each.(thus 6 in total): Experiment 1) Comparing the virulence of the phage resistant variants to the wildtype *A.baumannii* and PBS negative controls (datasets 5 and 6), 2) Comparing the efficacy of LemonAid treatment (both remedial and prophylactic) of *A. baumannii* wildtype infected larvae (datasets 7 and 8), and 3) A dose-response experiment of wildtype *A.baumannii* (datasets 9 and 10), the figures of which are in the supplementary materials. Again, the figure number within the manuscript that each dataset relates to is within the title. Column headings for datasets 5-10: Datasets 5,7 and 9 contain survival data of Galleria infection experiments: well = the well within the specialised plate that each galleria occupied for imaging purposes (note, each treatment group was spread across the plate as described in the methods), batch = the experiment was repeated 3 times designated as batch 1,2 or 3; treatment = treatment, survivetime = length of time the galleria survived post treatment; status = dead or alive at the end of the experiment. Datasets 6,8 and 10 contain melanisation data of Galleria during infection experiments: time_hrs = time point measurement taken; batch = the experiment was repeated 3 times designated as batch 1,2 or 3; treatment = treatment; well = the well within the specialised plate that each galleria occupied for imaging purposes; well area and galleria area = predicted by software to determine melanisation; Melanisation = melanisation of galleria as measured by software; file path = file path to the galleria image being analysed; inv-melanisation = the inverse of the melanisation measure for intuitive plotting purposes. The third section contains a single dataset, dataset 11, containing the phage absorption assay data. Column headers: treatment = strain or control; time point = time point measurement taken; replicate = replicate of treatment; PFU/mL = the number of phage plaques per mL