Scarless Removal of Large Resistance Island AbaR Results in Antibiotic Susceptibility and Increased Natural Transformability in Acinetobacter baumannii.

Anne-Sophie Godeux,Elin Svedholm,Maria-Halima Laaberki,Marisa Haenni,Xavier Charpentier,Agnese Lupo,Samuel Venner

doi:10.1128/aac.00951-20

Abstract

With a great diversity in gene composition, including multiple putative antibiotic resistance genes, AbaR islands are potential contributors to multidrug resistance in Acinetobacter baumannii However, the effective contribution of AbaR to antibiotic resistance and bacterial physiology remains elusive. To address this, we sought to accurately remove AbaR islands and restore the integrity of their insertion site. To this end, we devised a versatile scarless genome editing strategy. We performed this genetic modification in two recent A. baumannii clinical strains: the strain AB5075 and the nosocomial strain AYE, which carry AbaR11 and AbaR1 islands of 19.7 kbp and 86.2 kbp, respectively. Antibiotic susceptibilities were then compared between the parental strains and their AbaR-cured derivatives. As anticipated by the predicted function of the open reading frame (ORF) of this island, the antibiotic resistance profiles were identical between the wild type and the AbaR11-cured AB5075 strains. In contrast, AbaR1 carries 25 ORFs, with predicted resistance to several classes of antibiotics, and the AYE AbaR1-cured derivative showed restored susceptibility to multiple classes of antibiotics. Moreover, curing of AbaRs restored high levels of natural transformability. Indeed, most AbaR islands are inserted into the comM gene involved in natural transformation. Our data indicate that AbaR insertion effectively inactivates comM and that the restored comM is functional. Curing of AbaR consistently resulted in highly transformable and therefore easily genetically tractable strains. Emendation of AbaR provides insight into the functional consequences of AbaR acquisition.

Highlights

With a great diversity in gene composition, including multiple putative antibiotic resistance genes, AbaR islands are potential contributors to multidrug resistance in Acinetobacter baumannii
In contrast to other methods, it does not require cloning or prior genetic engineering of the target strain [17,18,19]. It relies on the use of chimeric PCR products and takes advantage of natural transformation, a phenotypic trait exhibited by most isolates of A. baumannii
A chimeric PCR product is assembled, consisting of the sacB_aacC4 cassette bearing a selection marker conferring resistance to apramycin, flanked by 2-kbp-long sequences identical to the target region (Fig. 1B). This chimeric PCR product is introduced by natural transformation, and A. baumannii transformants are selected using apramycin, an antibiotic to which most clinical isolates are sensitive

Summary

Introduction

With a great diversity in gene composition, including multiple putative antibiotic resistance genes, AbaR islands are potential contributors to multidrug resistance in Acinetobacter baumannii. We sought to accurately remove AbaR islands and restore the integrity of their insertion site To this end, we devised a versatile scarless genome editing strategy. Most AbaR islands are inserted into the comM gene involved in natural transformation. A potential contributor to multidrug resistance in A. baumannii is a genomic island, named AbaR, with a great diversity in gene content and carrying multiple putative antibiotic resistance genes [3, 4]. In A. baumannii, natural transformation appears as a conserved trait among clinical and nonclinical isolates of human and animal origin [13, 15, 16], providing the bacteria with a major route for acquisition of antibiotic resistance genes. We further confirm a role for the comM gene in natural transformation in A. baumannii

Methods

Results

Conclusion