Fluorescence-Based Flow Sorting in Parallel with Transposon Insertion Site Sequencing Identifies Multidrug Efflux Systems in Acinetobacter baumannii.
Multidrug efflux pumps provide clinically significant levels of drug resistance in a number of Gram-negative hospital-acquired pathogens. These pathogens frequently carry dozens of genes encoding putative multidrug efflux pumps. However, it can be difficult to determine how many of these pumps actually mediate antimicrobial efflux, and it can be even more challenging to identify the regulatory proteins that control expression of these pumps. In this study, we developed an innovative high-throughput screening method, combining transposon insertion sequencing and cell sorting methods (TraDISort), to identify the genes encoding major multidrug efflux pumps, regulators, and other factors that may affect the permeation of antimicrobials, using the nosocomial pathogen Acinetobacter baumannii A dense library of more than 100,000 unique transposon insertion mutants was treated with ethidium bromide, a common substrate of multidrug efflux pumps that is differentially fluorescent inside and outside the bacterial cytoplasm. Populations of cells displaying aberrant accumulations of ethidium were physically enriched using fluorescence-activated cell sorting, and the genomic locations of transposon insertions within these strains were determined using transposon-directed insertion sequencing. The relative abundance of mutants in the input pool compared to the selected mutant pools indicated that the AdeABC, AdeIJK, and AmvA efflux pumps are the major ethidium efflux systems in A. baumannii Furthermore, the method identified a new transcriptional regulator that controls expression of amvA In addition to the identification of efflux pumps and their regulators, TraDISort identified genes that are likely to control cell division, cell morphology, or aggregation in A. baumannii



Transposon-directed insertion sequencing (TraDIS) and related technologies have emerged as powerful methods to identify genes required for bacterial survival or competitive fitness under various selective conditions. We applied fluorescence-activated cell sorting (FACS) to physically enrich for phenotypes of interest within a mutant population prior to TraDIS. To our knowledge, this is the first time that a physical selection method has been applied in parallel with TraDIS rather than a fitness-induced selection. The results demonstrate the feasibility of this combined approach to generate significant results and highlight the major multidrug efflux pumps encoded in an important pathogen. This FACS-based approach, TraDISort, could have a range of future applications, including the characterization of efflux pump inhibitors, the identification of regulatory factors controlling gene or protein expression using fluorescent reporters, and the identification of genes involved in cell replication, morphology, and aggregation.
Department of Chemistry and Biomolecular Sciences, Macquarie University, Sydney, NSW, Australia karl.hassan@mq.edu.au.
Karl A
KA
Hassan
Wellcome Trust Sanger Institute, Hinxton, Cambridge, United Kingdom Liverpool School of Tropical Medicine, Malawi-Liverpool-Wellcome Trust Clinical Research Programme, Blantyre, Malawi.
Amy K
AK
Cain
Department of Chemistry and Biomolecular Sciences, Macquarie University, Sydney, NSW, Australia.
TaoTao
T
Huang
Department of Chemistry and Biomolecular Sciences, Macquarie University, Sydney, NSW, Australia.
Qi
Q
Liu
Department of Chemistry and Biomolecular Sciences, Macquarie University, Sydney, NSW, Australia.
Liam D H
LD
Elbourne
Wellcome Trust Sanger Institute, Hinxton, Cambridge, United Kingdom.
Christine J
CJ
Boinett
Department of Chemistry and Biomolecular Sciences, Macquarie University, Sydney, NSW, Australia.
Anthony J
AJ
Brzoska
Department of Chemistry and Biomolecular Sciences, Macquarie University, Sydney, NSW, Australia.
Liping
L
Li
Department of Chemistry and Biomolecular Sciences, Macquarie University, Sydney, NSW, Australia.
Martin
M
Ostrowski
The Hospital for Tropical Diseases, Wellcome Trust Major Overseas Programme, Oxford University Clinical Research Unit, Ho Chi Minh City, Vietnam School of Chemistry and Molecular Biosciences, The University of Queensland, Brisbane, Queensland, Australia.
Nguyen Thi Khanh
NT
Nhu
The Hospital for Tropical Diseases, Wellcome Trust Major Overseas Programme, Oxford University Clinical Research Unit, Ho Chi Minh City, Vietnam.
Tran Do Hoang
Tdo H
Nhu
The Hospital for Tropical Diseases, Wellcome Trust Major Overseas Programme, Oxford University Clinical Research Unit, Ho Chi Minh City, Vietnam Centre for Tropical Medicine, Nuffield Department of Clinical Medicine, Oxford University, Oxford, United Kingdom.
Stephen
S
Baker
Wellcome Trust Sanger Institute, Hinxton, Cambridge, United Kingdom.
Julian
0000-0002-7069-5958
J
Parkhill
Department of Chemistry and Biomolecular Sciences, Macquarie University, Sydney, NSW, Australia.
Ian T
IT
Paulsen
D040981:Acinetobacter baumannii / Q000737:chemistry* / Q000235:genetics*; D000890:Anti-Infective Agents / Q000378:metabolism*; D001693:Biological Transport, Active; D004251:DNA Transposable Elements; D024901:Drug Resistance, Multiple, Bacterial; D004996:Ethidium / Q000378:metabolism; D005434:Flow Cytometry; D005456:Fluorescent Dyes / Q000378:metabolism; D015723:Gene Library; D057166:High-Throughput Screening Assays; D026901:Membrane Transport Proteins / Q000235:genetics* / Q000378:metabolism*; D016254:Mutagenesis, Insertional; D017422:Sequence Analysis, DNA; D013194:Staining and Labeling
D000890:Anti-Infective Agents; D004251:DNA Transposable Elements; D005456:Fluorescent Dyes; D026901:Membrane Transport Proteins; D004996:Ethidium