Novel Conserved Genotypes Correspond to Antibiotic Resistance Phenotypes of E. coli Clinical Isolates

Abstract

Current efforts to understand antibiotic resistance on the whole genome scale tend to focus on known genes even as high throughput sequencing strategies uncover novel mechanisms. To identify genomic variations associated with antibiotic resistance, we employed a modified genome-wide association study; we sequenced genomic DNA from pools of E. coli clinical isolates with similar antibiotic resistance phenotypes using SOLiD technology to uncover single nucleotide polymorphisms (SNPs) unanimously conserved in each pool. The multidrug-resistant pools were genotypically similar to SMS-3-5, a previously sequenced multidrug-resistant isolate from a polluted environment. The similarity was evenly spread across the entire genome and not limited to plasmid or pathogenicity island loci. Among the pools of clinical isolates, genomic variation was concentrated adjacent to previously reported inversion and duplication differences between the SMS-3-5 isolate and the drug-susceptible laboratory strain, DH10B. SNPs that result in non-synonymous changes in gyrA (encoding the well-known S83L allele associated with fluoroquinolone resistance), mutM, ligB, and recG were unanimously conserved in every fluoroquinolone-resistant pool. Alleles of the latter three genes are tightly linked among most sequenced E. coli genomes, and had not been implicated in antibiotic resistance previously. The changes in these genes map to amino acid positions in alpha helices that are involved in DNA binding. Plasmid-encoded complementation of null strains with either allelic variant of mutM or ligB resulted in variable responses to ultraviolet light or hydrogen peroxide treatment as markers of induced DNA damage, indicating their importance in DNA metabolism and revealing a potential mechanism for fluoroquinolone resistance. Our approach uncovered evidence that additional DNA binding enzymes may contribute to fluoroquinolone resistance and further implicate environmental bacteria as a reservoir for antibiotic resistance.

A comparative analysis of methylome profiles of Campylobacter jejuni sheep abortion isolate and gastroenteric strains using PacBio data

“…Flagellar basal-body rod protein (Konkel et al, 2004 ), DNA gyrase subunit A and putative multidrug efflux transporter (Iovine, 2013 ), and putative ferredoxin (Van Vliet et al, 2001 ) are known virulence genes in Campylobacter . These and all other genes in the major hypomethylated regions of IA3902 (with the exception of selenocysteine synthase and hypothetical protein) are also associated with virulence in other bacterial species (Klose and Mekalanos, 1997 ; Ran Kim and Haeng Rhee, 2003 ; Sjöblom et al, 2008 ; Kawai et al, 2011 ; Schweizer, 2012 ; Swick et al, 2013 ). The hypomethylation of a large number of genes in a concentrated area of the genome (major hypomethylated region) may suggest a role in gene or virulence expression.…”

‘Supermutators’ found amongst highly levofloxacin-resistant E. coli isolates: a rapid protocol for the detection of mutation sites

“…There may be additional resistance mechanisms in such supermutator isolates, including the following mechanisms: mutations in mutM , ligB and recG encoding other putative DNA binding enzymes; 30 mutations in tolC encoding the outer part of the AcrAB-TolC efflux pump; 31 the acquisition of plasmid-borne genes qnr and aac(6′)-Ib-cr that encode drug-blocking and drug-modifying proteins, respectively; the overexpression of quorum-sensing regulator SdiA; mutations in robA encoding the global regulator RobA; and mutations in gyrB encoding the B subunit of the gyrase enzyme. 4 Such resistance mechanisms present themselves as interesting locations for investigation in future studies.…”

Oxidative DNA damage is instrumental in hyperreplication stress-induced inviability of Escherichia coli

“…This suggests an additive effect of 8-oxoG repair to the DSB-induced death by fluoroquinolone exposure. Fluoroquinolone resistant clinical isolates of E. coli frequently carry mutations in mutM ( 55 ).…”