- Email: [email protected]
Draft genome sequence of a clinical Acinetobacter haemolyticus isolate from South Africa
Journal Pre-proof Draft Genome Sequence of a Clinical Acinetobacter haemolyticus A109R1B4 Isolated from South Africa Raspail Carrel Founou, Luria Leslie Founou, Mushal Allam, Arshad Ismail, Sabiha Yusuf Essack
PII:
S2213-7165(19)30269-3
DOI:
https://doi.org/10.1016/j.jgar.2019.10.015
Reference:
JGAR 1072
To appear in:
Journal of Global Antimicrobial Resistance
Received Date:
26 November 2018
Revised Date:
7 October 2019
Accepted Date:
15 October 2019
Please cite this article as: Founou RC, Founou LL, Allam M, Ismail A, Essack SY, Draft Genome Sequence of a Clinical Acinetobacter haemolyticus A109R1B4 Isolated from South Africa, Journal of Global Antimicrobial Resistance (2019), doi: https://doi.org/10.1016/j.jgar.2019.10.015
This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier.
Draft Genome Sequence of a Clinical Acinetobacter haemolyticus A109R1B4 Isolated from South Africa
Raspail Carrel Founou1,2*, Luria Leslie Founou1,3, Mushal Allam4, Arshad Ismail4, Sabiha Yusuf Essack1
Antimicrobial Research Unit, College of Health Sciences, University of KwaZulu-Natal,
ro of
1
Durban, South Africa 2
Department of Clinical Microbiology, Centre of Expertise and Biological Diagnotic of
3
-p
Cameroon (CEDBCAM), Yaoundé, Cameroon
Department of Food Safety and Environmental Health, Centre of Expertise and Biological
National Institute for Communicable Diseases Sequencing Core Facility, National Institute
lP
4
re
Diagnostic of Cameroon (CEDBCAM), Yaoundé, Cameroon
ur
na
for Communicable Diseases, Johannesburg, South Africa
Jo
Corresponding author:
Dr Raspail Carrel Founou Antimicrobial Research Unit College of Health Sciences University of KwaZulu-Natal Private Bag X54001
Durban, 4000 South Africa Tel: +237 675943567 [email protected]
Running title: Draft Genome Sequence of Clinical Acinetobacter haemolyticus in South
ro of
Africa
Abstract
Objective: The draft genome sequence of a clinical Acinetobacter haemolyticus A109R1B4
-p
isolated from a rectal swab of a hospitalised patient is described here.
re
Methods: Genomic DNA was sequenced using an Illumina MiSeq platform. Generated reads were de novo assembled using the Qiagen CLC Genomics Workbench. The assembled
lP
contigs were annotated and antibiotic resistance genes and sequence types were identified. Results: The genome comprised 281 contigs with a total assembly length of 3, 371,389 bp,
na
39.9% G+C content and N50 value of 58 196 bp and reference coverage was 93.08 while the coverage was 88.08. A total of 3,387 genes were detected, 3 292 coding sequences (CDS), 3
detected.
ur
175 coding genes and 95 RNA genes. The resistance genes blaOXA-264 and aac(6’)-Ig were
Jo
Conclusion: The genome sequence reported will serve as a reference point for molecular epidemiology of antibiotic resistant A. haemolyticus in Africa.
Keywords: Whole genome, antibiotic resistance, Acinetobacter haemolyticus, carriage, South Africa
Introduction The genus of Acinetobacter comprises of more than 50 non-pigmented, oxidase-positive and -negative Gram-negative coccobacilli of which the majority are non-pathogenic environmental organisms (1). These environmental isolates often harbour antibiotic resistance mechanisms, including extended-spectrum β-lactamases (ESBLs) and carbapenemases that may serve as significant environmental reservoirs for resistance genes that may be transferred to clinically relevant species (1). Although Acinetobacter baumannii is the most clinically
ro of
relevant Acinetobacter species such as A. calcoaceticus, A. lwoffii, and A. haemolyticus are
being increasingly occasionally reported in community- and hospital-acquired infections (2). We describe here the genome sequence of A. haemolyticus A109R1B4, isolated from a rectal
-p
swab of a patient admitted to a district hospital in uMgungundlovu district, South Africa.
re
Antimicrobial susceptibility testing revealed that this isolate exhibited resistance to β-lactams and WGS-based genomic analysis was performed to correlate resistance phenotypes and
lP
genotypes.
na
Methods
The isolate presented herein formed part of a bigger study investigating the molecular
ur
epidemiology of antibiotic resistance in clinical and carriage samples from patients admitted
Jo
to a rural, district hospital and an urban, tertiary hospital in the uMgungundlovu District in South Africa. A. haemolyticus A109R1B4 was isolated from a rectal swab of a 78 years old female patient admitted for cellulitis in the surgery ward. The isolate was identified using biochemical tests and antimicrobial susceptibility testing was performed by broth microdilution. The European Committee on Antimicrobial Susceptibility testing guidelines
(http://www.eucast.org/clinical_breakpoints/) was used for interpretation of the results and E. coli ATCC 25922 and K. pneumoniae ATCC700603 were used as controls. Genomic DNA (gDNA) was extracted using the GenElute bacterial genomic DNA kit (Sigma-Aldrich, St. Louis, MO, USA) according to the manufacturer’s instructions. Agarose gel electrophoresis, NanoDrop spectrophotometry and fluorimetric analysis (Qubit®) were used to verify the integrity of the gDNA. Samples were prepared with the Nextera XT DNA sample preparation kit (Illumina, San Diego, CA, USA) and whole genome sequencing
ro of
(WGS) analysis was undertaken using multiplex paired-end 2 by 300 bp reads with a
coverage of X 100 on an Illumina MiSeq machine (Illumina, San Diego, CA, USA). Quality check and read trimming were performed as previously described (3) with de novo assembly
-p
performed using automated parameters in CLC Genomics Workbench version 10 (CLC, Bio-
re
QIAGEN, Aarhus, Denmark). Genomes were annotated using the National Center for Biotechnology Information's Prokaryotic Genome Automated Pipeline. Resistance genes
lP
were identified using ResFinder (4) and the Comprehensive Antibiotic Resistance Database
na
(CARD)(5).
Results
ur
The A. haemolyticus A109R1B4 displayed phenotypical resistance with MID (μg/mL) of
Jo
ampicillin (≥512), cefoxitin (128), cefuroxime (≥256), ceftriaxone (32), cefotaxime (≥32), ceftazidime (≥32), gentamicin (8), amikacin (128), nitrofurantoin (≥512 µg/mL), trimethoprim (256), but was susceptible to imipenem (2), meropenem (1), ciprofloxacin (0.5), ofloxacin (0.5). The sequencing yielded 300bp reads and the de novo assembled genome produced 281 contigs with a total assembly length of 3, 371, 389 bp, G + C content of 39.9%, N50 value of 58,196 bps and reference coverage was 93.08 while the coverage was 88.08. A
total of 3 387 genes were detected, including 3 292 coding sequences (CDS), 3 175 coding genes and 95 RNA genes. ResFinder identified blaOXA-264 (contig 7) and aac(5’)-Ig (contig 6) which is consonant with the observed phenotypic profile.
Discussion The resistance genes encoding for β-lactams and aminoglycosides identified in this isolate
ro of
underscore the importance of this opportunist pathogen in the emergence of community- and hospital acquired-infections. The draft genome of this A. haemolyticus A109R1B4 will serve as a reference point for molecular epidemiology of antibiotic resistant A. haemolyticus in
-p
Africa and allow its monitoring as a potential source or reservoir of antibiotic resistance
lP
Nucleotide sequence accession no.
re
among Acinetobacter spp.
This Whole Genome Shotgun project PRJNA485584 of Acinetobacter haemolyticus
na
A109R1B4 has been deposited at DDBJ/EMBL/GenBank under accession no.
ur
QUST00000000. The version described in this paper is the first version QUST00000000.
Jo
Declarations
Author contributions RCF co-conceptualized the study, undertook sample collection, microbiological laboratory and data analyses, prepared tables and figures, interpreted results, contributed to bioinformatics analysis, and drafted the manuscript. LLF undertook sample collection, microbiological laboratory analyses, contributed to bioinformatics analysis and vetted the
results. MA contributed to bioinformatics analyses. AI performed whole genome sequencing analysis. SE co-conceptualized the study and undertook critical revision of the manuscript. All authors read and approve the final manuscript. Funding: This work was supported by the Antimicrobial Research Unit (ARU) and College of Health Sciences (CHS) of the University of KwaZulu-Natal. The National Research Foundation funded this study through the NRF Incentive Funding for Rated Researchers (Grant No.: 85595), the NRF Competitive Grant for Rated Researchers (Grant no.: 106063)
ro of
and the DST/NRF South African Research Chair in Antibiotic Resistance and One Health (Grant No.: 98342). The South African Medical Research Council through the Self-Initiated Research Grant also funded the study. Any opinions, findings and conclusions or
-p
recommendations expressed in this material are those of the author(s) and do not necessarily reflect the views of the organizations or agencies that provided support for the project. The
re
funders had no role in the study design, nor the decision to submit the work for publication.
lP
Ethical Approval: Yes
Competing Interests: Professor Essack is a member of the Global Respiratory Infection Partnership sponsored by an unrestricted educational grant from Reckitt and Benckiser. All
na
other authors declare that there is no competing financial interest. Acknowledgement
ur
We are grateful to the NCBI GENBANK submission staff for help with genome upload,
Jo
decontamination and deposition procedures.
References 1.
Wong D, Nielsen TB, Bonomo RA, Pantapalangkoor P, Luna B, Spellberg B. Clinical and Pathophysiological Overview of Acinetobacter Infections: a Century of Challenges. Clin Microbiol Rev. 2017;30(1):409-47.
2.
Figueiredo S, Bonnin RA, Poirel L, Duranteau J, Nordmann P. Identification of the naturally occurring genes encoding carbapenem-hydrolysing oxacillinases from Acinetobacter haemolyticus, Acinetobacter johnsonii, and Acinetobacter calcoaceticus. Clin Microbiol Infect. 2012;18(9):907-13.
3.
Tyson GH, McDermott PF, Li C, Chen Y, Tadesse DA, Mukherjee S, et al. WGS
ro of
accurately predicts antimicrobial resistance in Escherichia coli. J Antimicrob Chemother. 2015;70(10):2763-9. 4.
Zankari E, Hasman H, Cosentino S, Vestergaard M, Rasmussen S, Lund O, et al.
Identification of acquired antimicrobial resistance genes. J Antimicrob Chemother.
5.
-p
2012;67(11):2640-4.
Jia B, Raphenya AR, Alcock B, Waglechner N, Guo P, Tsang KK, et al. CARD 2017:
re
expansion and model-centric curation of the comprehensive antibiotic resistance
Jo
ur
na
lP
database. Nucleic Acids Res. 2017;45(D1):D566-d73.
PII:
S2213-7165(19)30269-3
DOI:
https://doi.org/10.1016/j.jgar.2019.10.015
Reference:
JGAR 1072
To appear in:
Journal of Global Antimicrobial Resistance
Received Date:
26 November 2018
Revised Date:
7 October 2019
Accepted Date:
15 October 2019
Please cite this article as: Founou RC, Founou LL, Allam M, Ismail A, Essack SY, Draft Genome Sequence of a Clinical Acinetobacter haemolyticus A109R1B4 Isolated from South Africa, Journal of Global Antimicrobial Resistance (2019), doi: https://doi.org/10.1016/j.jgar.2019.10.015
This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier.
Draft Genome Sequence of a Clinical Acinetobacter haemolyticus A109R1B4 Isolated from South Africa
Raspail Carrel Founou1,2*, Luria Leslie Founou1,3, Mushal Allam4, Arshad Ismail4, Sabiha Yusuf Essack1
Antimicrobial Research Unit, College of Health Sciences, University of KwaZulu-Natal,
ro of
1
Durban, South Africa 2
Department of Clinical Microbiology, Centre of Expertise and Biological Diagnotic of
3
-p
Cameroon (CEDBCAM), Yaoundé, Cameroon
Department of Food Safety and Environmental Health, Centre of Expertise and Biological
National Institute for Communicable Diseases Sequencing Core Facility, National Institute
lP
4
re
Diagnostic of Cameroon (CEDBCAM), Yaoundé, Cameroon
ur
na
for Communicable Diseases, Johannesburg, South Africa
Jo
Corresponding author:
Dr Raspail Carrel Founou Antimicrobial Research Unit College of Health Sciences University of KwaZulu-Natal Private Bag X54001
Durban, 4000 South Africa Tel: +237 675943567 [email protected]
Running title: Draft Genome Sequence of Clinical Acinetobacter haemolyticus in South
ro of
Africa
Abstract
Objective: The draft genome sequence of a clinical Acinetobacter haemolyticus A109R1B4
-p
isolated from a rectal swab of a hospitalised patient is described here.
re
Methods: Genomic DNA was sequenced using an Illumina MiSeq platform. Generated reads were de novo assembled using the Qiagen CLC Genomics Workbench. The assembled
lP
contigs were annotated and antibiotic resistance genes and sequence types were identified. Results: The genome comprised 281 contigs with a total assembly length of 3, 371,389 bp,
na
39.9% G+C content and N50 value of 58 196 bp and reference coverage was 93.08 while the coverage was 88.08. A total of 3,387 genes were detected, 3 292 coding sequences (CDS), 3
detected.
ur
175 coding genes and 95 RNA genes. The resistance genes blaOXA-264 and aac(6’)-Ig were
Jo
Conclusion: The genome sequence reported will serve as a reference point for molecular epidemiology of antibiotic resistant A. haemolyticus in Africa.
Keywords: Whole genome, antibiotic resistance, Acinetobacter haemolyticus, carriage, South Africa
Introduction The genus of Acinetobacter comprises of more than 50 non-pigmented, oxidase-positive and -negative Gram-negative coccobacilli of which the majority are non-pathogenic environmental organisms (1). These environmental isolates often harbour antibiotic resistance mechanisms, including extended-spectrum β-lactamases (ESBLs) and carbapenemases that may serve as significant environmental reservoirs for resistance genes that may be transferred to clinically relevant species (1). Although Acinetobacter baumannii is the most clinically
ro of
relevant Acinetobacter species such as A. calcoaceticus, A. lwoffii, and A. haemolyticus are
being increasingly occasionally reported in community- and hospital-acquired infections (2). We describe here the genome sequence of A. haemolyticus A109R1B4, isolated from a rectal
-p
swab of a patient admitted to a district hospital in uMgungundlovu district, South Africa.
re
Antimicrobial susceptibility testing revealed that this isolate exhibited resistance to β-lactams and WGS-based genomic analysis was performed to correlate resistance phenotypes and
lP
genotypes.
na
Methods
The isolate presented herein formed part of a bigger study investigating the molecular
ur
epidemiology of antibiotic resistance in clinical and carriage samples from patients admitted
Jo
to a rural, district hospital and an urban, tertiary hospital in the uMgungundlovu District in South Africa. A. haemolyticus A109R1B4 was isolated from a rectal swab of a 78 years old female patient admitted for cellulitis in the surgery ward. The isolate was identified using biochemical tests and antimicrobial susceptibility testing was performed by broth microdilution. The European Committee on Antimicrobial Susceptibility testing guidelines
(http://www.eucast.org/clinical_breakpoints/) was used for interpretation of the results and E. coli ATCC 25922 and K. pneumoniae ATCC700603 were used as controls. Genomic DNA (gDNA) was extracted using the GenElute bacterial genomic DNA kit (Sigma-Aldrich, St. Louis, MO, USA) according to the manufacturer’s instructions. Agarose gel electrophoresis, NanoDrop spectrophotometry and fluorimetric analysis (Qubit®) were used to verify the integrity of the gDNA. Samples were prepared with the Nextera XT DNA sample preparation kit (Illumina, San Diego, CA, USA) and whole genome sequencing
ro of
(WGS) analysis was undertaken using multiplex paired-end 2 by 300 bp reads with a
coverage of X 100 on an Illumina MiSeq machine (Illumina, San Diego, CA, USA). Quality check and read trimming were performed as previously described (3) with de novo assembly
-p
performed using automated parameters in CLC Genomics Workbench version 10 (CLC, Bio-
re
QIAGEN, Aarhus, Denmark). Genomes were annotated using the National Center for Biotechnology Information's Prokaryotic Genome Automated Pipeline. Resistance genes
lP
were identified using ResFinder (4) and the Comprehensive Antibiotic Resistance Database
na
(CARD)(5).
Results
ur
The A. haemolyticus A109R1B4 displayed phenotypical resistance with MID (μg/mL) of
Jo
ampicillin (≥512), cefoxitin (128), cefuroxime (≥256), ceftriaxone (32), cefotaxime (≥32), ceftazidime (≥32), gentamicin (8), amikacin (128), nitrofurantoin (≥512 µg/mL), trimethoprim (256), but was susceptible to imipenem (2), meropenem (1), ciprofloxacin (0.5), ofloxacin (0.5). The sequencing yielded 300bp reads and the de novo assembled genome produced 281 contigs with a total assembly length of 3, 371, 389 bp, G + C content of 39.9%, N50 value of 58,196 bps and reference coverage was 93.08 while the coverage was 88.08. A
total of 3 387 genes were detected, including 3 292 coding sequences (CDS), 3 175 coding genes and 95 RNA genes. ResFinder identified blaOXA-264 (contig 7) and aac(5’)-Ig (contig 6) which is consonant with the observed phenotypic profile.
Discussion The resistance genes encoding for β-lactams and aminoglycosides identified in this isolate
ro of
underscore the importance of this opportunist pathogen in the emergence of community- and hospital acquired-infections. The draft genome of this A. haemolyticus A109R1B4 will serve as a reference point for molecular epidemiology of antibiotic resistant A. haemolyticus in
-p
Africa and allow its monitoring as a potential source or reservoir of antibiotic resistance
lP
Nucleotide sequence accession no.
re
among Acinetobacter spp.
This Whole Genome Shotgun project PRJNA485584 of Acinetobacter haemolyticus
na
A109R1B4 has been deposited at DDBJ/EMBL/GenBank under accession no.
ur
QUST00000000. The version described in this paper is the first version QUST00000000.
Jo
Declarations
Author contributions RCF co-conceptualized the study, undertook sample collection, microbiological laboratory and data analyses, prepared tables and figures, interpreted results, contributed to bioinformatics analysis, and drafted the manuscript. LLF undertook sample collection, microbiological laboratory analyses, contributed to bioinformatics analysis and vetted the
results. MA contributed to bioinformatics analyses. AI performed whole genome sequencing analysis. SE co-conceptualized the study and undertook critical revision of the manuscript. All authors read and approve the final manuscript. Funding: This work was supported by the Antimicrobial Research Unit (ARU) and College of Health Sciences (CHS) of the University of KwaZulu-Natal. The National Research Foundation funded this study through the NRF Incentive Funding for Rated Researchers (Grant No.: 85595), the NRF Competitive Grant for Rated Researchers (Grant no.: 106063)
ro of
and the DST/NRF South African Research Chair in Antibiotic Resistance and One Health (Grant No.: 98342). The South African Medical Research Council through the Self-Initiated Research Grant also funded the study. Any opinions, findings and conclusions or
-p
recommendations expressed in this material are those of the author(s) and do not necessarily reflect the views of the organizations or agencies that provided support for the project. The
re
funders had no role in the study design, nor the decision to submit the work for publication.
lP
Ethical Approval: Yes
Competing Interests: Professor Essack is a member of the Global Respiratory Infection Partnership sponsored by an unrestricted educational grant from Reckitt and Benckiser. All
na
other authors declare that there is no competing financial interest. Acknowledgement
ur
We are grateful to the NCBI GENBANK submission staff for help with genome upload,
Jo
decontamination and deposition procedures.
References 1.
Wong D, Nielsen TB, Bonomo RA, Pantapalangkoor P, Luna B, Spellberg B. Clinical and Pathophysiological Overview of Acinetobacter Infections: a Century of Challenges. Clin Microbiol Rev. 2017;30(1):409-47.
2.
Figueiredo S, Bonnin RA, Poirel L, Duranteau J, Nordmann P. Identification of the naturally occurring genes encoding carbapenem-hydrolysing oxacillinases from Acinetobacter haemolyticus, Acinetobacter johnsonii, and Acinetobacter calcoaceticus. Clin Microbiol Infect. 2012;18(9):907-13.
3.
Tyson GH, McDermott PF, Li C, Chen Y, Tadesse DA, Mukherjee S, et al. WGS
ro of
accurately predicts antimicrobial resistance in Escherichia coli. J Antimicrob Chemother. 2015;70(10):2763-9. 4.
Zankari E, Hasman H, Cosentino S, Vestergaard M, Rasmussen S, Lund O, et al.
Identification of acquired antimicrobial resistance genes. J Antimicrob Chemother.
5.
-p
2012;67(11):2640-4.
Jia B, Raphenya AR, Alcock B, Waglechner N, Guo P, Tsang KK, et al. CARD 2017:
re
expansion and model-centric curation of the comprehensive antibiotic resistance
Jo
ur
na
lP
database. Nucleic Acids Res. 2017;45(D1):D566-d73.