Intrapopulation variability in mutator prevalence among urinary tract infection isolates of Escherichia coli  A. Couce, N. Alonso-Rodriguez, C. Costas, A. Oliver, J. Blázquez  Clinical Microbiology and Infection  Volume 22, Issue 6, Pages 566.e1-566.e7 (June 2016) DOI: 10.1016/j.cmi.2016.03.008 Copyright © 2016 European Society of Clinical Microbiology and Infectious Diseases Terms and Conditions

Fig. 1 Intrapopulation variability on mutant frequency among urinary tract infection (UTI) isolates. Frequency of rifampicin-resistant mutants among 24 independent strains per patient from a set of 80 patients. Data points are the median of four replicate experiments. Panels represent each patient (numbered from 1 to 80). The horizontal dashed line indicates the cut-off value used to consider strains as potential hypermutators (median mutant frequency ≥1 × 10−7). A total of 55 candidates coming from 12 patients exceeded this cut-off and were subjected to a second round of assays. Only isolates from populations 9, 42, 73 and 74 were confirmed as true hypermutators. This large false-positive rate (∼64%) is largely explained by a combination of the well-known fluctuating nature of mutagenesis data with the inevitability of systematic errors in large experiments, highlighting the necessity of double-checking when screening for hypermutable phenotypes. Clinical Microbiology and Infection 2016 22, 566.e1-566.e7DOI: (10.1016/j.cmi.2016.03.008) Copyright © 2016 European Society of Clinical Microbiology and Infectious Diseases Terms and Conditions

Fig. 2 Distribution of mutant frequency and antibiotic resistance among urinary tract infection (UTI) isolates. (a) Distribution of the data from Fig. 1. The median of the distribution is 1.1 × 10−8 (interquartile range 6.5 × 10−9–1.9 × 10−8, n = 1920). The vertical dashed line indicates the cut-off value used to select strains for further investigation (roughly a ten-fold increase relative to the median). (b) Distribution of the number of resistances against nine antibiotics commonly prescribed in UTIs of a representative sample of 240 isolates (see Materials and Methods). Resistance was defined according to EUCAST breakpoints. Mutators and non-mutators exhibited on average 2.75 and 2.02 resistances respectively, a difference statistically non-significant (p 0.185, permutation test). Clinical Microbiology and Infection 2016 22, 566.e1-566.e7DOI: (10.1016/j.cmi.2016.03.008) Copyright © 2016 European Society of Clinical Microbiology and Infectious Diseases Terms and Conditions

Fig. 3 Complementation assays for candidate hypermutable isolates. Bars represent the median mutant frequency to rifampicin resistance (error bars indicate interquartile range, n = 3) of confirmed candidates. Each panel shows the results for one hypermutable strain from patients 9, 42, 73 and 74 (a, b, c and d, respectively). Complementation was assayed with plasmids harbouring well-known DNA repair or damage avoidance genes. Appropriate open-reading framess from confirmed candidates were then PCR-amplified and submitted for sequencing. Clinical Microbiology and Infection 2016 22, 566.e1-566.e7DOI: (10.1016/j.cmi.2016.03.008) Copyright © 2016 European Society of Clinical Microbiology and Infectious Diseases Terms and Conditions