Genome sequence of carbapenem-resistant Citrobacter koseri carrying blaOXA-181 isolated from sewage sludge

Accepted Manuscript Title: Genome sequence of carbapenem-resistant Citrobacter koseri carrying blaOXA−181 isolated from sewage sludge Authors: Mutshiene Deogratias Ekwanzala, John Barr Dewar, Ilunga Kamika, Maggy Ndombo Benteke Momba PII: DOI: Reference:

S2213-7165(19)30177-8 https://doi.org/10.1016/j.jgar.2019.07.011 JGAR 989

To appear in: Received date: Accepted date:

24 June 2019 5 July 2019

Please cite this article as: Ekwanzala MD, Dewar JB, Kamika I, Momba MNB, Genome sequence of carbapenem-resistant Citrobacter koseri carrying blaOXA−181 isolated from sewage sludge, Journal of Global Antimicrobial Resistance (2019), https://doi.org/10.1016/j.jgar.2019.07.011 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. 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.

Genome sequence of carbapenem-resistant Citrobacter koseri carrying blaOXA-181 isolated from sewage sludge Running title: Carbapenem-resistant Citrobacter koseri carrying blaOXA-181

Mutshiene Deogratias Ekwanzala1* • John Barr Dewar2 • Ilunga Kamika3 • Maggy Ndombo

Department of Environmental, Water and Earth Sciences, Tshwane University of Technology,

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Benteke Momba1*

Arcadia Campus, Private BagX680, Pretoria 0001, South Africa

Department of Life and Consumer Sciences, University of South Africa, Florida Campus,

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2

Department of Environmental Sciences, University of South Africa, Science Campus,

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3

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Johannesburg, South Africa

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Johannesburg, South Africa

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✉ MNB Momba, Tel: +27123826335; E-mail: [email protected]

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Highlights

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MD Ekwanzala, Tel: +27123826631; E-mail: [email protected]

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No C. koseri genome has been reported to carry blaOXA-181; thus, C. koseri AS1 is the first of its kind. Genomes assessment revealed all C. koseri as putative human pathogens. This study also highlights the resistome contents of C. koseri genomes.

ABSTRACT Objectives: This study reports the resistome content of the sewage sludge-isolated carbapenem-resistant C. koseri carrying blaOXA-181 as well as provide a general phylogenomic

analysis highlighting antibiotic resistance genes (ARGs), plasmids and pathogenicity of C. koseri genomes. Methods: Carbapenem-resistant C. koseri AS1 strain was isolated from sewage sludge on CHROMagar™ mSuperCARBA™ media. The whole genome sequencing of C. koseri AS1 was performed using an HiSeq X™ Ten instrument. Additional C. koseri genomes were

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downloaded from NCBI. Phylogenomic analysis was established through the CSI Phylogeny.

PlasmidFinder 2.0 and PathogenFinder 1.1, respectively.

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Antibiotic resistance genes, plasmids and pathogenicity were identified using ResFinder 3.1,

Results: The phylogenomic tree indicates a polyclonal pattern of C. koseri genomes. Resistome

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analysis of C. koseri AS1 revealed β-lactam resistance genes (blaMAL-1 and blaOXA-181) as well as a fosfomycin resistance gene (fosA7). Three plasmids (ColKP3, ColRNAI and IncX30) were

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identified in the C. koseri AS1 genome. In addition, a total of 25 ARGs were found in

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downloaded genomes. Of these, clinically significant ARGs such as blaKPC-2 and blaOXA-48 were found in 2 and 4 genomes, respectively. Assessment of the genomes using PathogenFinder

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revealed all genomes as putative human pathogens. Conclusions: To the best of our knowledge, no C. koseri genome has been reported to carry

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blaOXA-181 and, thus, C. koseri AS1 is the first of its kind. This study also highlights the

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resistome contents of C. koseri genomes.

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Keywords: Citrobacter koseri; blaOXA-181; resistome; whole-genome sequencing

1. Introduction Citrobacter spp. are members of the Enterobacteriaceae. They are Gram-negative and are frequently found in water, soil, food and the intestines of animals and humans [1]. Among the 11 species of this genus, C. freundii, C. amalonaticus, C. youngae, C. braakii and C. koseri

(previously named C. diversus) are regarded as commensal in humans [2]. The two prominent opportunistic pathogens of Citrobacter spp. are C. freundii and C. koseri. The latter is known primarily to cause meningitis and central nervous system disease with abscess formation in new-borns [3]. It has also been associated with Harrington rod infection [1], infective endocarditis[4] and pneumonia and empyema [5]. Infections caused by C. koseri are treated

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with antibiotics such as aminoglycosides, carbapenems, cephalosporins, chloramphenicol and

quinolones [6]. However, the global spread of carbapenem-resistance encoding genes among

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Enterobacteriaceae, including C. koseri [2,7], is of significant public health concern as the only treatment option is polymyxin (colistin), antibiotics associated with neuro- and

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nephrotoxic side-effects [8].

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Antibiotic-resistant C. koseri at a phenotypic level is well documented, including its intrinsic

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resistance to aminopenicillins and carboxypenicillins.[9] The phenotypic profile of resistant C.

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koseri has changed over time so that resistant C. koseri has acquired plasmid-mediated and chromosomal encoded genes that render them resistant to antibiotics such as ESBLs including

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carbapenems. At the genetic level, typed antibiotic resistance genes (ARGs) in C. koseri revealed the presence of genes encoding for extended-spectrum β-lactamase [6] and

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carbapenem-encoding genes blaKPC-2 and blaMAL-1 [2,7,10]. However, there is a paucity of information on the phylogenomics structure of C. koseri genomes and reports of C. koseri

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carrying blaOXA-181, a variant of blaOXA-48, are rare. Consequently, this study reports on the resistome content of a sewage sludge-isolated

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carbapenem-resistant C. koseri genome carrying blaOXA-181 and further provides a general phylogenomic analysis highlighting ARGs, plasmids and pathogenicity of C. koseri genomes.

2. Materials and methods

A carbapenem-resistant C. koseri AS1 strain was isolated from sewage sludge during a study targeting carbapenem-resistant Klebsiella pneumoniae [11] in wastewater treatment plants. The isolate grew on the chromogenic media CHROMagar™ mSuperCARBA™ (CHROMagar, MediaMage, South Africa). The strain was confirmed as C. koseri firstly using the MALDI Biotyper System (Bruker Daltonik, Bremen, Germany) at the MALDI-TOF Diagnostic Service

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of the University of Pretoria as well as an in-silico pipeline KmerFinder 3.1 [12]. To sequence the whole genome, the strain was grown overnight in tryptic soy agar at 37°C. Harvested cells

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were firstly washed using 1 × phosphate buffer solution before the whole genome DNA was extracted using the ZymoBIOMICS™ DNA Miniprep Kit (ZymoResearch, Inqaba Biotec,

South Africa). The quality and quantity of isolated whole genome DNA was assessed using a

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NanoDrop™ 2000 spectrophotometer (Thermo Scientific, South Africa). The whole genome

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DNA was then transported to Beijing Genomics Institute (BGI, Tai Po, Hong Kong) where a paired-end library was constructed using a Nextera® XT DNA Library Preparation Kit

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(Illumina Inc., San Diego, CA) and sequenced on a HiSeq X™ Ten platform (Illumina Inc.,

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San Diego, USA). Following whole genome sequencing, the quality generated reads were assessed using FastQC [13], and low-quality sequences were trimmed using Trimmomatic

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[14]. De novo assembly into contigs was performed using SPAdes 3.91. The assembled contigs were then annotated using Prokka v.1.3 (Seemann 2014) [15]. To compare the resistome

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content, phylogenetic relatedness and pathogenicity of the isolated strain against publicly available C. koseri genomes, FASTA files were downloaded from the NCBI FTP website2.

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Phylogenomic relatedness was established through the CSI Phylogeny pipeline3. Further visualisation and attributes annotation were added using the interactive Tree of life tool4. To

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https://cge.cbs.dtu.dk/services/SPAdes/ ftp.ncbi.nlm.nih.gov/genomes/refseq/bacteria/ 3 https://cge.cbs.dtu.dk/services/CSIPhylogeny/ 4 http://itol.embl.de/ 2

assess the ARGs and plasmids content, ResFinder 3.15 and PlasmidFinder 2.06 were used, respectively. Genomes potential as pathogen was putatively determined using PathogenFinder 1.17. Default settings were used in all software unless otherwise noted.

3. Results and discussion

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Whole-genome sequencing generated 1 587 134 reads accounting for 238 070 100 bp. The draft genome of C. koseri AS1 was assembled into 134 contigs with N50 of 97 580 and a total

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size of 5 011 791 bp (Table 1). The GC% content of isolate C. koseri AS1 was 53.7%. The genome contained 4 973 coding sequences, 95 RNAs and 409 number of subsystems. As

tabulated in Table 1, these genomic statistics are comparable to downloaded C. koseri genomes

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from NCBI.

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Figure 1 shows the phylogenetic relatedness of the C. koseri genomes based on single

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nucleotide polymorphism (SNP) analysis. The generated SNP tree indicates a polyclonal

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pattern of C. koseri genomes. Thirteen genomes out of 30 including C. koseri AS1 were

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grouped in one phylogenetic cluster. A second clade included the reference genome C. koseri ATCC BAA 895 strain clustered 11 genomes with sub-clades.

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Resistome analysis of C. koseri AS1 revealed β-lactam resistance genes blaMAL-1 and blaOXAas well as a fosfomycin resistance gene fosA7. Interestingly, the presence of blaOXA-181 gene

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is an important finding of this study. To the best of our knowledge, no genome strain has been reported to carry blaOXA-181; thus, C. koseri AS1 is the first of its kind. Furthermore, the presence

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of C. koseri carrying blaOXA-181 isolated from a carbapenem-resistance medium contradict the finding of Shanti et al. [16] that blaOXA-48 or its variant blaOXA-181 cannot be considered as major mechanisms of carbapenem resistance. A total of 25 ARGs were found in downloaded

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https://cge.cbs.dtu.dk/services/ResFinder/ https://cge.cbs.dtu.dk/services/PlasmidFinder/ 7 https://cge.cbs.dtu.dk/services/PathogenFinder/ 6

genomes. Eighty-six per cent of genomes contained the blaMAL-1. The ARGs harboured in those genomes can confer resistance to aminoglycosides [aac(3)-IId, aac(6')-Ib-cr, aadA1, aadA2, ant(2'')-Ia, aph(3'')-Ib and aph(6)-Id)], β-lactamases (blaCKO-1, blaKPC-2, blaMAL-1, blaOXA-1, blaOXA-2, blaOXA-48, blaSHV-30 and blaTEM-1B), rifampicin (ARR-3), chloramphenicol (catA1), trimethoprim (dfrA12), fosfomycin (fosA7), macrolide [mph(A)], fluoroquinolones (qnrS1),

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sulphonamides (sul1 and sul2) and tetracycline [tet(A) and tet(B)]. Clinically important ARGs such as blaKPC-2 and blaOXA-48 were found in 2 and 4 of genomes, respectively. These findings

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emphasize the extensive drug-resistant C. koseri phenotypes reported elsewhere [6].

PlasmidFinder identified three plasmids (ColKP3, ColRNAI and IncX30) in the C. koseri AS1

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genome. Interestingly, the presence of ColKP3 in C. koseri has not been reported in C. koseri

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genomes. This plasmid has been reported to carry blaOXA-181 in carbapenem-resistant Klebsiella

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pneumoniae [17]. In addition, ColKP3 was also found to harbour blaOXA-232 which was not

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found in C. koseri AS1 genome [18]. A total of 13 resistance plasmids were found in downloaded C. koseri genomes with ColRNAI being the most abundant plasmid found in 14

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C. koseri genomes. Other plasmids included IncFIB(AP001918), IncFII, IncFII(29), IncFII(Yp), IncI1, IncI2, IncL/M(pMU407), IncL/M(pOXA-48), IncN, IncR and IncX3

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located in 13 C. koseri genomes.

PathogenFinder revealed all assessed genomes as putative human pathogens. C. koseri AS1

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had a 0.906 probability of being a human pathogen with all others showing a probability between 0.896 (C. koseri NCTC11074) to 0.931 (C. koseri YDC582) [19]. In addition to the

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known C. koseri genes encoding pathogenic proteins, homologous sequences of pathogenic proteins from Escherichia coli, Salmonella enterica, Yersinia pestis, Shigella dysenteriae and Klebsiella pneumoniae were found in studied C. koseri genomes. A further study analysing the virulence and pathogenicity of C. koseri genomes is recommended.

Data availability: The whole-genome shotgun sequencing project of this strain was deposited at DDBJ/ENA/GenBank under the BioProject number PRJNA543985 and BioSample accession number SAMN11793481. Raw sequences were deposited in the Sequence Read Archive (SRA) under the accession numbers SRR9099020.

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Declarations Funding: This project received funding from the National Research Foundation (Grant

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number: 112851) and the South African Research Chairs Initiative (SARChI) in Water Quality and Wastewater Management. Opinions expressed and conclusions arrived at are those of the authors and are not necessarily to be attributed to the funders.

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Competing Interests: The authors declare no competing interests.

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Ethical Approval: Not required.

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2018;6:1–2.

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Figure 1. C. koseri phylogenomic tree highlighting ARGs, plasmids and pathogenicity.

Table 1. Genome features of C. koseri used in this study. Organis

Strain

BioSample

BioProject

m name

Size

GC

Numb

(Mb)

%

er

of

genes

er koseri

895

Citrobact

AS1

BAA- SAMN02603912 PRJNA1271

4.7353

53.8

6

6

0

SAMN1179348

PRJNA5439

5,0117

1

85

91

4538

Citrobact

FDAARGOS_2 SAMN06173300 PRJNA2312

4.9346

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ATCC

er koseri

87

6

0

Citrobact

FDAARGOS_3 SAMN07312437 PRJNA2312

4.8844

53.8

er koseri

93

5

0

Citrobact

AR_0025

4.9232

53.8

N

Citrobact

8

5

4.9288

53.7

01

7

0

4.581

53.9

21

SAMN04014866 PRJNA2929

er koseri

01

AR_0024

SAMN04014865 PRJNA2929

A

Citrobact

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21

M

er koseri

FDAARGOS_5 SAMN10163225 PRJNA2312

er koseri

30

Citrobact

GED7778C

er koseri DNF00568

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Citrobact

ED

Citrobact

er koseri

21

SAMN03842474 PRJNA2721 12

SAMN03842473 PRJNA2573 74

53.8

4.8121

53.8

7

0

4.8823

53.7

8

0

4.7273

53.8

er koseri

6

6

0

Citrobact

FDAARGOS_1 SAMN03996311 PRJNA2312

4.9852

53.6

er koseri

64

3

0

4.6356

53.8

5

0

4.6529

53.8

4

0

A

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FDAARGOS_8 SAMN02934540 PRJNA2312

Citrobact

B1B

er koseri Citrobact er koseri

21 SAMN05371063 PRJNA3284 18

YDC582

SAMN07974444 PRJNA4169 08

4876

4779

4853

4862

4393

0

Citrobact

21

4973

0

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er koseri

53.7

4688

4779

4577

4879

4486

4550

Citrobact

CK778

SAMN09104667 PRJNA4707 69

53.7

6

0

4738

Citrobact

FDAARGOS_5 SAMN10163220 PRJNA2312

5.1808

53.5

er koseri

26

3

0

Citrobact

BL23

4.8262

53.9

79

8

0

PRJEB6403

4.9944

53.7

7

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er koseri

4.7563

21 SAMN11126861 PRJNA5270

er koseri NCTC10770

er koseri

68 NCTC10768

1 NCTC11076

er koseri NCTC11074

NCTC10769

er koseri Citrobact

NCTC10771

Citrobact

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NCTC10849

L168

HAMBI_1287

A

er koseri

Citrobact

ICR003207

er koseri Citrobact er koseri

8

0

4.7353

53.9

1

0

4.8832

53.7

4

0

4.8216

53.7

1

0

4.8374

53.8

2

0

4.9156

53.6

2

9

0

SAMN09270542 PRJNA4732

4.8451

53.8

SAMEA103924

PRJEB6403

SAMEA104338

SAMEA104338

PRJEB6403

PRJEB6403

SAMEA270901

PRJEB6403

5

er koseri

Citrobact

53.6

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NCTC11077

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er koseri

5.0485

PRJEB6403

398

er koseri Citrobact

0

401

er koseri Citrobact

7

SAMEA344377

SAMEA258031

PRJEB6403

97 SAMN09428653 PRJNA4762 09 SAMN10847465 PRJNA5179

SAMN10847462 PRJNA5179 17

4850

4883

4949

4635

4975

4789

4764

4785

4841

0 4.6854

53.8

3

0

4.934

53.8

17 ICR003204

4996

0

53.7

407 NCTC10811

53.6

4.9282

8

er koseri Citrobact

PRJEB6403

6

er koseri Citrobact

SAMEA322207

U

Citrobact

4.8983 3

N

Citrobact

PRJEB6403

4669

0

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er koseri

SAMEA270901

A

Citrobact

SAMEA804566

M

Citrobact

5088

4615

4820

0 4.7670

53.8

2

0

4612

0123A_53_520

SAMN05509313 PRJNA3271

er koseri Citrobact

NCTC11075

er koseri Citrobact

53.9

06

9

0

PRJEB6403

4.7857

53.7

6

0

4.6614

53.8

6

0

4.7327

53.8

3

5 NCTC10786

er koseri Citrobact

SAMEA322207

4.7524

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Citrobact

SAMEA266513

PRJEB6403

2 NCTC10810

PRJEB6403

3

0

A

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ED

M

A

N

U

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er koseri

SAMEA266513

-

5737

6514

5429