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The increasing incidence and emergence of multi-drug resistant (MDR) Acinetobacter baumannii has become a major global health concern. Colistin is a historic antimicrobial that has become commonly used as a treatment for MDR A. baumannii infections. The increase in colistin usage has been mirrored by an increase in colistin resistance. We aimed to identify the mechanisms associated with colistin resistance in A. baumannii using multiple high-throughput-sequencing technologies, including transposon-directed insertion site sequencing (TraDIS), RNA sequencing (RNAseq) and whole-genome sequencing (WGS) to investigate the genotypic changes of colistin resistance in A. baumannii. Using TraDIS, we found that genes involved in drug efflux (adeIJK), and phospholipid (mlaC, mlaF and mlaD) and lipooligosaccharide synthesis (lpxC and lpsO) were required for survival in sub-inhibitory concentrations of colistin. Transcriptomic (RNAseq) analysis revealed that expression of genes encoding efflux proteins (adeI, adeC, emrB, mexB and macAB) was enhanced in in vitro generated colistin-resistant strains. WGS of these organisms identified disruptions in genes involved in lipid A (lpxC) and phospholipid synthesis (mlaA), and in the baeS/R two-component system (TCS). We additionally found that mutations in the pmrB TCS genes were the primary colistin-resistance-associated mechanisms in three Vietnamese clinical colistin-resistant A. baumannii strains. Our results outline the entire range of mechanisms employed in A. baumannii for resistance against colistin, including drug extrusion and the loss of lipid A moieties by gene disruption or modification.

Colistin remains one of the few antibiotics effective against multi-drug resistant (MDR) hospital pathogens, such as Klebsiella pneumoniae. Yet resistance to this last-line drug is rapidly increasing. Characterized mechanisms of col R in K. pneumoniae are largely due to chromosomal mutations in two-component regulators, although a plasmid-mediated col R mechanism has recently been uncovered. However, the effects of intrinsic colistin resistance are yet to be characterized on a whole-genome level. Here, we used a genomics-based approach to understand the mechanisms of adaptive col R acquisition in K. pneumoniae. In controlled directed-evolution experiments we observed two distinct paths to colistin resistance acquisition. Whole genome sequencing identified mutations in two colistin resistance genes: in the known col R regulator phoQ which became fixed in the population and resulted in a single amino acid change, and unstable minority variants in the recently described two-component sensor crrB. Through RNAseq and microscopy, we reveal the broad range of effects that colistin exposure has on the cell. This study is the first to use genomics to identify a population of minority variants with mutations in a col R gene in K. pneumoniae.

Background: The emergence of Neisseria gonorrhoeae with decreased susceptibility to extended spectrum cephalosporins raises the prospect of untreatable gonorrhoea. In the absence of new treatments, efforts to slow the increasing incidence of resistant gonococcus require insight into the factors that contribute to its emergence and spread. We assessed the relatedness between isolates in the USA and reconstructed likely spread of lineages through different sexual networks., Methods: We sequenced the genomes of 236 isolates of N gonorrhoeae collected by the Centers for Disease Control and Prevention's Gonococcal Isolate Surveillance Project (GISP) from sentinel public sexually transmitted disease clinics in the USA, including 118 (97%) of the isolates from 2009-10 in GISP with reduced susceptibility to cefixime (cef(RS)) and 118 cefixime-susceptible isolates from GISP matched as closely as possible by location, collection date, and sexual orientation. We assessed the association between antimicrobial resistance genotype and phenotype and correlated phylogenetic clustering with location and sexual orientation., Findings: Mosaic penA XXXIV had a high positive predictive value for cef(RS). We found that two of the 118 cef(RS) isolates lacked a mosaic penA allele, and rechecking showed that these two were susceptible to cefixime. Of the 116 remaining cef(RS) isolates, 114 (98%) fell into two distinct lineages that have independently acquired mosaic penA allele XXXIV. A major lineage of cef(RS) strains spread eastward, predominantly through a sexual network of men who have sex with men. Eight of nine inferred transitions between sexual networks were introductions from men who have sex with men into the heterosexual population., Interpretation: Genomic methods might aid efforts to slow the spread of antibiotic-resistant N gonorrhoeae through augmentation of gonococcal outbreak surveillance and identification of populations that could benefit from increased screening for asymptomatic infections., (Copyright © 2014 Elsevier Ltd. All rights reserved.)