Genetic Features of Antarctic <em>Acinetobacter radioresistens</em> Strain A154 Harboring Multiple Antibiotic-Resistance Genes

Andrés Opazo-Capurro

Paul Higgins

Julia Wille

Harald Seifert

Camila Cigarroa

Paulina González-Muñoz

Mario Quezada-Aguiluz

Mariana Domínguez-Yévenes

Helia Bello-Toledo+2 authors

Bacterial Source

Acinetobacter spp. isolate A154 was recovered from ornitogenic soil (superficial layer) in Ardley Island (−62°12′60.00″S −58°55′59.99″W) in Fildes Peninsula, Antarctica, during a scientific expedition in 2013. Specifically, the isolate was recovered in a soil sample from a lagoon shore. Fildes Peninsula is located on King George Island and holds the largest number of scientific research bases in the Antarctic Treaty Area (Rabbia et al., 2016). The isolate was grown on trypticase soy agar incubated overnight at 30°C.

PCR Experiments

Total DNA was extracted by the boiling water method and utilized as template for preliminary species identification by the detection of the intrinsic chromosomally-encoded bla_{OXA−23−like} gene.

Susceptibility Testing

Susceptibility testing were performed by disc diffusion following the CLSI recommendations, for ampicillin (AMP, 10 μg), cefotaxime (CTX, 30 μg), ceftriaxone (CRO, 30 μg), ceftazidime (CAZ, 30 μg), cefepime (FEP, 30 μg), imipenem (IPM, 10 μg), meropenem (MEM, 10 μg), gentamicin (GEN, 10 μg), amikacin (AMK, 30 μg), kanamycin (KAN, 30 μg), streptomycin (STR, 10 μg), tetracycline (TET, 30 μg), and ciprofloxacin (CIP, 5 μg) (Clinical Laboratory Standards Institute, 2017). Minimum inhibitory concentrations (MICs) were determined by E-test and broth microdilution for AMP, CTX, IPM, MEM, GEN, AMK, and KAN (Clinical Laboratory Standards Institute, 2017).

Whole-Genome Sequencing

Total DNA for whole-genome sequencing (WGS) was extracted using the Wizard® Genomic DNA Purification kit (Promega, USA) following the manufacturer's protocol. DNA concentration and integrity were verified using a Take3 plate (BioTek Instruments). WGS was performed by the Illumina MiSeq platform (2 × 250 bp paired end reads) with libraries prepared by the NexteraXT kit (Illumina).

In silico Genome Analyses

De novo assembly was performed by using the SPades software version 3.9 available at the CGE server (https://cge.cbs.dtu.dk/services/SPAdes/), utilizing default values (Nurk et al., 2013). Species identification was carried out by the SpeciesFinder service, version 1.3 (https://cge.cbs.dtu.dk/services/SpeciesFinder/), which is based on the 16S rRNA sequence. Genome annotation was accomplished using the NCBI Prokaryotic Genome Annotation Pipeline (PGAP) web-service (http://www.ncbi.nlm.nih.gov/genome/annotation_prok). Moreover, the draft genome was analyzed by the Comprehensive Genome Analysis service (https://www.patricbrc.org/app/ComprehensiveGenomeAnalysis), which incorporates the RAST tool kit (RASTtk). The genetic contexts of the contigs in which plasmid-encoded ARGs were detected, were plotted using the Artemis software, version 18.0.2 (Carver et al., 2012).

In order to establish the taxonomic relatedness of A154, we used the Tetra Correlation Search (TCS), which allows to compare the draft genome of this isolate against the genomes reference database GenomesDB (http://jspecies.ribohost.com/jspeciesws/#genomesdb), which contains >30.000 whole and draft genomes with pre-calculated overall genome relatedness indices (Richter et al., 2015). In addition, we determined the acquired resistome, insertion sequences (ISs) content and toxin-antitoxin (TA) systems through ResFinder (https://cge.cbs.dtu.dk/services/ResFinder/), RASTtk and Pathosystems Resource Integration Center (PATRIC) platforms. Plasmid replicons were searched using the PlasmidFinder tool (Carattoli et al., 2014) and the PATRIC platform. The genome of the isolate A154 was deposited at DDBJ/EMBL/GenBank under the accession number {"type":"entrez-nucleotide","attrs":{"text":"PXJD00000000.1","term_id":"1361519317","term_text":"PXJD00000000.1"}}PXJD00000000.1.

Molecular Typing

Additionally, from WGS data we extracted the seven alleles according to the Pasteur MLST scheme which were submitted to the PubMLST database (https://pubmlst.org/) in order to assign a specific sequence type (ST).

Plasmids Studies

Plasmids were isolated by the alkaline lysis method according to a standard protocol (Kado and Liu, 1981). Afterwards, DNA concentration of the plasmids extracts were measured by spectrophotometry (BioTek Instruments) and chromosomal contamination was evaluated by detecting the 16S rDNA gene, which is chromosomally encoded. After, ARGs previously detected in total DNA were screened using the pure DNA plasmids extracts. Moreover, we performed mating and plasmid curing experiments (Trevors, 1986; Leungtongkam et al., 2018).

Conflict of Interest Statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

^{Laboratorio de Investigación en Agentes Antibacterianos (LIAA), Departamento de Microbiología, Facultad de Ciencias Biológicas, Universidad de Concepción, Concepción, Chile}

^{Millennium Nucleus for Collaborative Research on Bacterial Resistance (MICROB-R), Santiago, Chile}

^{Institute for Medical Microbiology, Immunology and Hygiene, University of Cologne, Cologne, Germany}

^{German Center for Infection Research (DZIF), Partner Site Bonn-Cologne, Cologne, Germany}

^{Departamento de Ciencias Biológicas y Químicas, Facultad de Medicina y Ciencia, Universidad San Sebastián, Concepción, Chile}

Edited by: Xiangmin Lin, Fujian Agriculture and Forestry University, China

Reviewed by: Hossam Abdelhamed, Mississippi State University, United States; José Alejandro Di Conza, University of Buenos Aires, Argentina

*Correspondence: Gerardo González-Rocha lc.cedu@laznogg

This article was submitted to Molecular Bacterial Pathogenesis, a section of the journal Frontiers in Cellular and Infection Microbiology

Edited by: Xiangmin Lin, Fujian Agriculture and Forestry University, China

Reviewed by: Hossam Abdelhamed, Mississippi State University, United States; José Alejandro Di Conza, University of Buenos Aires, Argentina

Received 2019 Jun 14; Accepted 2019 Sep 2.

This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

Abstract

While antibiotic-resistant bacteria have been detected in extreme environments, including Antarctica, to date there are no reports of Acinetobacter species isolated from this region. Here, we characterized by whole-genome sequencing (WGS) the genetic content of a single antibiotic-resistant Acinetobacter spp. isolate (A154) collected in Antarctica. The isolate was recovered in 2013 from soil samples at Fildes Peninsula, Antarctica, and was identified by detection of the intrinsic OXA-23 gene, and confirmed by Tetra Correlation Search (TCS) and WGS. The antibiotic susceptibility profile was determined by disc diffusion, E-test, and broth microdilution methods. From WGS data, the acquired resistome and insertion sequence (IS) content were identified by in silico analyses. Plasmids were studied by the alkaline lysis method followed by pulsed-field gel electrophoresis and conventional PCR. The A154 isolate was identified as A. radioresistens by WGS analysis and displayed >99.9 of similarity by TCS in relation with the databases. Moreover, it was resistant to ampicillin, ceftriaxone, ceftazidime, cefepime, cefotaxime, streptomycin, and kanamycin. Likewise, in addition to the intrinsic bla_{OXA−23−like} gene, A154 harbored the plasmid-encoded antibiotic-resistance genes bla_PER−2, tet(B), aph(3′)-Vla, strA, and strB, as well as a large diversity of ISs. This is the first report of antibiotic-resistant A. radioresistens in Antarctica. Our findings show the presence of several resistance genes which could be either intrinsic or acquired in the region.

Keywords: antibiotic-resistance genes, Acinetobacter, Antarctica, whole-genome sequencing, resistance plasmid, ecotoxicology

Abstract

Acknowledgments

The authors highly appreciate the support from the Chilean National Commission for Scientific and Technological Research (CONICYT), the Chilean Antarctic Institute (INACH), and the Millennium Science Initiative of the Ministry of Economy, Development and Tourism, Chile.

Acknowledgments

Footnotes

Funding. This work was funded by the post-doctoral project N°3150286 from The National Fund for Scientific and Technological Development (FONDECYT) and the Chilean Antarctic Institute (INACH) project number RT_06_12.

Footnotes