FEMS Microbiology Letters, 362, 2015, fnv002 doi: 10.1093/femsle/fnv002 Advance Access Publication Date: 13 January 2015 Genome Announcement

G E N O M E A N N O U N C E M E N T – Pathogens & Pathogenicity

Draft genome sequences of two Aeromonas salmonicida subsp. salmonicida isolates harboring plasmids conferring antibiotic resistance Antony T. Vincent1,2,3 , Katherine H. Tanaka1,2,3,# , Melanie V. Trudel1,2,3,# , Michel Frenette3,4 , Nicolas Derome1,5 and Steve J. Charette1,2,3,∗ 1

´ ` ` Institut de biologie integrative et des systemes, Pavillon Charles-Eugene-Marchand, Universite´ Laval, 1030 ´ avenue de la Medecine, Quebec City, QC G1V 0A6, Canada, 2 Centre de recherche de l’Institut universitaire de ´ ˆ cardiologie et de pneumologie de Quebec (Hopital Laval), 2725 Chemin Sainte-Foy, Quebec City, QC G1V 4G5, ´ de biochimie, de microbiologie et de bio-informatique, Faculte´ des sciences et de Canada, 3 Departement ´ ´ genie, Universite´ Laval, 1045 avenue de la Medecine, Quebec City, QC G1V 0A6, Canada, 4 Groupe de Recherche ´ ´ en Ecologie Buccale (GREB), Faculte´ de medecine dentaire, Universite´ Laval, Quebec City, QC G1V 0A6, Canada ´ ´ ´ and 5 Departement de biologie, Faculte´ des sciences et de genie, Universite´ Laval, 1045 avenue de la Medecine, Quebec City, QC G1V 0A6, Canada ∗ Corresponding author: Steve J. Charette, Institut de Biologie Integrative ´ ` ` et des Systemes (IBIS), Pavillon Charles-Eugene-Marchand, 1030 avenue de la ´ Medecine, Universite´ Laval, Quebec City, QC G1V 0A6, Canada. Tel: 418-656-2131, ext. 6914; Fax: 418-656-7176; E-mail: [email protected] # These authors contributed equally to this work. One sentence summary: We present the draft genome sequences of two Aeromonas salmonicida isolates that harbor plasmids that confer resistance to several antibiotics. Editor: Juan Imperial

ABSTRACT The bacterium Aeromonas salmonicida is the etiological agent of furunculosis, a widespread fish disease causing important economic losses to the fish farming industry. Antibiotic treatments in fish farms may be challenging given the existence of multidrug-resistant isolates of this bacterium. Here, we report the draft genome sequences of the 2004-05MF26 and 2009-144K3 isolates, which harbor plasmids conferring antibiotic resistance. Both isolates also carry the large plasmid pAsa5, which is known to encode a type three secretion system (TTSS) and the pAsal1 plasmid which has the aopP gene producing a TTSS effector. These two isolates are good representatives of the plasmid diversity in A. salmonicida subsp. salmonicida. Key words: furunculosis; Aeromonas salmonicida; antibiotic resistance; genome; plasmid

AEROMONAS SALMONICIDA ISOLATES HAVING ANTIBIOTIC RESISTANCE Intensive fish farming causes stress in the fish populations (Barton and Iwama 1991), dramatically increasing their sus-

ceptibility to infections by opportunistic pathogens. The ubiquitous waterborne Gram-negative psychrophilic bacterium Aeromonas salmonicida subsp. salmonicida is the causative agent of furunculosis, a disease that affects fish farms worldwide

Received: 28 November 2014; Accepted: 24 December 2014  C FEMS 2015. All rights reserved. For permissions, please e-mail: [email protected]

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(Dallaire-Dufresne et al., 2014). Antibiotherapies have, on several occasions, proven to be ineffective in curing or preventing furunculosis since many isolates were multidrug resistant (Sandaa and Enger 1994; Sørum et al., 2003; McIntosh et al., 2008). The 2004-05MF26 and 2009-144K3 A. salmonicida subsp. salmonicida isolates have recently been reported to bear plasmid variants conferring multidrug resistance (Vincent et al., 2014b). We provide the complete draft genome sequences of these two isolates. Total genomic DNA from both isolates was extracted using DNeasy Blood and Tissue Kit (Qiagen, Canada). The DNA from the 2009-144K3 isolate was pyrosequenced using a 454 GS-FLX+ apparatus (Roche, USA) with a 5-kb mate-pair library. The sequencing reads were de novo assembled using Newbler 2.5.3 (Margulies et al., 2005). A TruSeq shotgun library was prepared with DNA from the 2004-05MF26 isolate and was sequenced using Illumina technology on a MiSeq instrument (Illumina, USA). The sequencing reads were de novo assembled using the A5 pipeline (Tritt et al., 2012). The DNA from both isolates was se´ quenced at the Plateforme d’Analyse Genomique of the Institut ´ ` de Biologie Integrative et des Systemes (IBIS, Universite´ Laval). The draft sequences were annotated using the NCBI Prokaryotic Genome Annotation Pipeline. The de novo assembly of the 2009-144K3 DNA resulted in 214 contiguous sequences (contigs), the smallest and largest contigs were 200 and 218 040 bp, respectively and the total assembly had a N50 value of 64 305 bp and a mean G + C content of 58.35%. As previously reported, 2009-144K3 carries the pAB5S9b and pRAS3.3 plasmid variants (Vincent et al., 2014b). Chromosomes of several A. salmonicida subsp. salmonicida were recently shown to harbor variants of a genomic island (AsaGEI), which can be used as an indicator to track the geographic provenance of the isolates (Emond-Rheault et al., 2014). The 2009-144K3 isolate bears AsaGEI2a and an adjacent prophage, named prophage 3, which is related to its origin from New Brunswick, Canada. The de novo assembly of 2004-05MF26 resulted in 126 contigs, which is significantly less than for 2009-144K3. The smallest and largest contigs were 598 and 382 276 bp, respectively, and the assembly had a N50 value of 119 364 bp and a mean G + C con-

tent of 58.25%. As previously reported, this isolate has pSN254b, a large plasmid carrying several resistance genes against antibiotics, quaternary ammonium compounds and mercury (Table 1). The chromosome of this isolate also possessed AsaGEI2a and the prophage 3, which was expected since this isolate also comes from New Brunswick, Canada (Emond-Rheault et al., 2014). The AsaGEI2a and the prophage 3 sequences are identical (>99%) for both isolates (i.e. 2004-05MF26 and 2009-144K3). The putative chromosomal contig sequences were found by CONTIGuator (Galardini et al., 2011) analyses with default parameters using the chromosome sequence of the assembled strain A449 (Reith et al., 2008) as reference for both 2009144K3 and 2004-05MF26. The chromosomal contig sequences were concatenated for both isolates and compared against the A449 strain chromosome sequence by megablast using the NCBI server (http://blast.ncbi.nlm.nih.gov) with default parameters (word size of 28). These analyses resulted in 99% identity over 98% query cover and 99% identity over 99% query cover for 2004-05MF26 and 2009-144K3, respectively. The differences are due to the presence of the genomic island AsaGEI2a (53 088 bp) and the prophage 3 (37 358 bp). The alignment of 2009144K3 chromosomal contig sequences had a query cover of 1% more than for 2004-05MF26. The reason for this is that a contig from 2009-144K3 (contig00095) containing AsaGEI2a and a part (26.3%) of the prophage 3 (absent in the strain A449) was not initially mapped by CONTIGuator. Since it is well known that A. salmonicida subsp. salmonicida chromosome bears long repeated elements, mainly insertion sequences (ISs) and rRNA clusters, causing breaks in the assembly (Vincent et al., 2014a), it is impossible to verify if differences exist for these repeated elements between the new genomes and the one of A449. Both isolates harbor a pAsa5 plasmid (Table 1), which bears a type three secretion system (TTSS) (Reith et al., 2008). The TTSS is a major A. salmonicida subsp. salmonicida virulence factor. Growth in stressful conditions, such as high temperature (25◦ C), may result in the loss of the pAsa5 region encoding the TTSS (Daher et al., 2011). Preliminary bioinformatics analyses showed that pAsa5 in 2009-144K3 and 2004-05MF26 appear complete including the TTSS. However, it is premature to conclude on size

Table 1. Features of the 2009-144K3 and 2004-05MF26 plasmids. G + C (%)

Resistance genes

26 673 12 496 6165 5710 6478 6222 150 296

52,51 59,03 56,69 52,36 54,09 55,05 54,07

sul2, strA, strB, floR, tet(H), tetR tetA, tetR N/Ab N/A N/A N/A N/A

97 718

151 056

52,49

5614 5545 5790 4304 16 706

5614 5545 5790 6498 145 477

57,68 52,16 54,94 54,69 53,93

tetA, tetR, floR, strA, strB, sul1, sul2, blaCMY , aadA, qacEdelta1, sugE2, merE-D-B-A-P-T-R N/A N/A N/A N/A N/A

Strain

Plasmid

Contig number

2009-144K3

pAB5S9b pRAS3.3 pAsa1 pAsa2 pAsa3 pAsal1 pAsa5

2 1 1 1 1 1 12

21 878 12 496 6165 5710 6478 6222 16 390

2004-05MF26

pSN254b

2

pAsa1 pAsa2 pAsa3 pAsal1 pAsa5

1 1 1 3 12

a

N50 (bp)

Approximative length (bp)a

The plasmids length were approximated by bioinformatics analyses based on the total bases in the contigs composing the plasmids. Elements that can vary the approximate size compared to the actual size are the missing sequences at the ends of contigs caused by the repeated sequence elements found in the plasmids. In addition, the uncut ends of the three cryptic plasmids (pAsa1, pAsa2, and pAsa3) can potentially biased size. Actual size seems, in fact, similar to the one of cryptic plasmids found in A449. b N/A means none applicable.

Vincent et al.

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Figure 1. The contig sequences for pAsa5 of 2009-144K3 and 2004-05MF26 were mapped on the pAsa5 sequence of the A449 strain (GenBank: CP000646.1) (green arrows). The contigs for both strains have putatively broken because of the same repeated elements as ISs (red) and duplicated genes (blue).

differences between these plasmids, as they are unassembled and consequently with gaps between contigs that may differ in length. Further analyses are required to verify if both plasmids are really similar or with minor differences and if the TTSS is functional. The de novo assemblies of both pAsa5 resulted in the same contig number and approximately the same N50 value (Table 1). Contig mapping on the reference strain A449 with the webserver CONTIGuator (Galardini et al., 2011) and visualization with ACT (Carver et al., 2005) suggest that both de novo assemblies have broken at the same sites (Fig. 1). The A449 pAsa5 plasmid has 11 ISs (Tanaka et al., 2012), which means there is one repeated IS every 14.1 kbp as well as two traI and repA duplicated genes likely responsible for contig break in pAsa5 from 2004-05MF26 and 2009-144K3. The bacterium A. salmonicida subsp. salmonicida is well known to be challenging in de novo assembly, mainly due to the high number of repeated elements (Vincent et al., 2014a). Finally, it was determined experimentally (Trudel et al., 2013), and confirmed by the results of bioinformatics analyses presented here, that both isolates harbor three cryptic plasmids (pAsa1, pAsa2 and pAsa3) as well as pAsal1 (Table 1). The Whole Genome Shotgun projects were deposited in DDBJ/ENA/GenBank under accession numbers JRYV00000000 and JRYW00000000 for 2009-144K3 and 2004-05MF26, respectively.

ACKNOWLEDGEMENTS We thank the Aquatic Animal Health Department of Fisheries and Oceans Canada (NB, Canada) for the A. salmonicida subsp. salmonicida isolates.

FUNDING KHT received Alexander Graham Bell Canada graduate scholarships from the Natural Sciences and Engineering Research Council of Canada (NSERC). MVT received scholarships under ´ the CREATE program of Ressources Aquatiques Quebec (RAQ). This project was funded by an NSERC Discovery grant to SJC, ´ e´ de recherche et de developpement ´ a grant from the Societ en aquaculture continentale (SORDAC) to SJC and ND, and a grant ´ from Resources Aquatiques Quebec (RAQ) to MF. Conflict of interest statement. None declared.

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