Diversity of antibiotic-resistance genes in Canadian isolates of Aeromonas salmonicida
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1 Diversity of antibiotic-resistance genes in Canadian isolates of Aeromonas icida subsp. icida: dominance of psn54b and discovery of pasa8 Mélanie V. Trudel 1,,3*, Antony T. Vincent 1,,3*, Sabrina A. Attéré 1,,3, Myriam Labbé 1,,4, Nicolas Derome 1,5, Alexander I. Culley 1,,4 and Steve J. Charette 1,,3,# 1. Institut de Biologie Intégrative et des Systèmes (IBIS), Université Laval, Quebec City, QC, Canada, G1V 0A6. Département de biochimie, de microbiologie et de bio-informatique, Faculté des sciences et de génie, Université Laval, Quebec City, QC, Canada, G1V 0A6 3. Centre de recherche de l Institut universitaire de cardiologie et de pneumologie de Québec (IUCPQ), Quebec City, QC, Canada, G1V 4G5 4. Groupe de Recherche en Écologie Buccale (GREB), Faculté de médecine dentaire, Université Laval, Quebec City, QC, Canada, G1V 0A6 5. Département de biologie, Faculté des sciences et de génie, Université Laval, Quebec City, QC, Canada, G1V 0A6 # To whom correspondence should be addressed: Institut de Biologie Intégrative et des Systèmes (IBIS), Pavillon Charles-Eugène-Marchand, 1030 avenue de la Médecine, Université Laval, Quebec City, QC, Canada, G1V 0A6 steve.charette@bcm.ulaval.ca; Telephone: , ext. 6914; Fax: * These authors contributed equally to this work. Running title: A. icida: psn54b dominance and pasa8 discovery
2 Supplementary Table S1. Primers used in this study Primer Sequence (5-3 ) Reference PCR positive control (tapa gene on the chromosome) tapa F tapa R ORF101-strB_F1 a ORF101-strB_R1 a para-resa_f1 para-resa_r1 ORF397-tnp_F1 ORF397-tnp_R1 tnp-urf_f1 b tnp-urf_r1 b trac_f1 trac_r1 ORF1417_F1 ORF1417_R1 DD1-pAsa4-traG-F1 DD-pAsa4-traG-R1 Reg_1_F ACATGAAGAAGCAATCAGGC AGAGGTCATGCGTTAGCAG PCR for the detection of pab5s9 CTCGTGAATGTCGGTATCCTGTCT CTCGGTCGTGAGAACAATCTGATG CGTCAATGATGATGTGGTCGTACC CTGATCGACCAGGAAGGCTATAGC TGTTAGCACTCGGTTGCCTTCGAT GACATACACACCGTCGAGAAACAG PCR for the detection of psn54 CATTTCGGTCGACCATACGACGATC CTGCGTGTGCTGCAAGGAAATCC CTACGACAATGGCTTGATCCAAGAC GTACAGAATGGCAGGCAGTTGAGG CGAGACCTTCCCTTTTACTGATGC CAGACAGAGGACGAGTTGTAGAAC PCR for the detection of pasa4 AGGTTGCTCTGGAAAGCCTCTGAT TGTGGATGCCTGTGCTCTCCATTA PCR for the characterization of pras3 GGATAGTCGATCTGCTGGATACG 1 1
3 Reg_1_R Reg F1 Reg R1 CTACCCTGTGGAACACCTACATCT CGTTGTGGATGTGCTTCAGCAATC GAGTGGTTGAGCAATACAGGATGC PCR for the characterization of pasa8 Geno_pAsa8_F1b CATGATGGTCACACCTCGATACTC This study Geno_pAsa8_R1 GCAATCCACTACTCAGTAGGTGAG This study Geno_pAsa8_Fb GGAATCTGGTGGATGCGCATCTTC This study Geno_pAsa8_R CGTCTGATCCAAGAACAACCATCC This study Geno_pAsa8_F4 GTGATGATGTGAAATCTGCGTGGG This study Geno_pAsa8_R4 GCACTATCGCTTGCTTTCACCAGG This study Geno_pAsa8_F5 GCTTGCGGTCTACAGTGATATCTC This study Geno_pAsa8_R5 CGGTACTCAAATGCAACGCTACTAC This study a. The ORF101-strB primers were designed to give different amplicon sizes in pab5s9 and pab5s9b. b. The tnp-urf primers were specific to psn54b. References 1 Ebanks, R. O. et al. Expression of and secretion through the Aeromonas icida type III secretion system. Microbiology 15, , doi:15/5/175 [pii] /mic (006). Vincent, A. T. et al. Detection of variants of the pras3, pab5s9, and psn54 plasmids in Aeromonas icida subsp. icida: multidrug resistance, interspecies exchanges, and plasmid reshaping. Antimicrob Agents Chemother 58, , doi:10.118/aac (014).
4 Supplementary Table S. A. icida subsp. icida isolates used in this study Isolate Origin Date of Fish Region a Quebec subregion isolation Plasmids found by PCR genotyping Genes found by multiplex PCR Plasmids b Prophages c AsaGEI c Phenotypic assay d pasa1 pasa pasa3 pasal1 pasa5 1 3 A-layer Lipases Proteases A449 Brown trout France 1975 pasa4 cat, sul1, teta(e) None None B5 Brook trout Quebec (NW) NW-B 001 pasa4-like (trag+) sul1, teta(e) a B56 Brook trout Quebec (NW) NW-C a B516 Brook trout Quebec (NW) NW-H a Brook trout Quebec (NW) NW-B 007 psn54b flor, sui1, sul, teta a N/A e Quebec (NW) NW-B 007 psn54b flor, sui1, sul, teta a Atlantic Atlantic Quebec (NE) NE-D None a Quebec (NE) NE-D None a Brook trout Quebec (SW) SW-C 007 psn54b flor, sui1, sul, teta a Brook trout Quebec (NW) NW-B 008 psn54b flor, sui1, sul, teta a Brook trout Quebec (SW) SW-C 008 psn54b flor, sui1, sul, teta a Brook trout Quebec (NW) NW-B 008 psn54b flor, sui1, sul, teta a Atlantic Quebec (NE) NE-D None a m Brook trout Quebec (NW) NW-B 7-Sep a m Atlantic Quebec (NW) NW-B 04-aout-09 psn54b flor, sul1, sul, teta a + + +
5 m Brook trout Quebec (NW) NW-G 03-dec a m Brook trout Quebec (NE) NE-C 01-dec a m Brook trout Quebec (NW) NW-B 13-Oct a m Brook trout Quebec (NW) NW-B -Sep a m Atlantic Quebec (NE) NE-D 13-Jul a m Brook trout Quebec (SW) SW-C -Jul a m Brook trout Quebec (SW) SW-C 30-Jun a m Brook trout Quebec (SW) SW-C 17-Jun a m Brook trout Quebec (SW) SW-C 11-Jun-09 psn54b flor, sul1, sul, teta a m Brook trout Quebec (NW) NW-B 0-May a m Brook trout Quebec (NW) NW-B 8-Apr a m Brook trout Quebec (NW) NW-B 10-Jun a m Brook trout Quebec (NE) NE-C 7-Apr a m91-10 Brook trout Quebec (NE) NE-C 11-May a M Brook trout Quebec (NW) NW-B a M Brook trout Quebec (NW) NW-B 011 pasa8 flor, sul1, tetg, teta a M Brook trout Quebec (NW) NW-B 011 pasa8 flor, sul1, tetg, teta a M Brook trout Quebec (NW) NW-B a M Brook trout Quebec (NW) NW-H a + + +
6 M Brook trout Quebec (NW) NW-H a M Brook trout Quebec (SE) SE-A a M Brook trout Quebec (SE) SE-A a M Brook trout Quebec (SE) SE-B 011 psn54b flor, sul1, sul, teta a M Brook trout Quebec (SE) SE-B 011 psn54b flor, sul1, sul, teta a M Brook trout Quebec (SE) SE-B 011 psn54b flor, sul1, sul, teta a M Brook trout Quebec (SW) SW-C 011 psn54b flor, sul1, sul, teta a M Brook trout Quebec (SW) SW-C 011 psn54b flor, sul1, sul, teta a M Rainbow trout M Atlantic Quebec (SE) SE-B a Quebec (NE) NE-C a M Brook trout Quebec (NE) NE-C a M Brown trout Quebec (NW) NW-B None a M Brook trout Quebec (NW) NW-B 011 psn54b flor, sul1, sul, teta N/A N/A N/A N/A N/A a M Brook trout Quebec (SW) SW-C a M Brook trout Quebec (NW) NW-G a M Lumpfish Quebec (SE) SE-A None M Brook trout Quebec (NW) NW-B a M Brook trout Quebec (NW) NW-B 1-Jun-1 psn54b flor, sul1, sul, teta a M Brook trout Quebec (NE) NE-C 4-Oct a + + +
7 M Brook trout Quebec (NE) NE-C 1-Aug a M Brook trout Quebec (NW) NW-H 19-Jun a M895-1 Brook trout Quebec (NW) NW-G 5-Oct a M Brown trout Quebec (NW) NW-B 4-May-1 psn54b flor, sul1, sul, teta a M Brook trout Quebec (NW) NW-G 11-May a M Brook trout Quebec (SW) SW-C 3-Aug a M Brook trout Quebec (NW) NW-B 13-Jun a M Brook trout Quebec (NW) NW-C 4-Oct a M Brook trout Quebec (NW) NW-H 7-Jun-1 psn54b flor, sul1, sul, teta a M Brook trout Quebec (SE) SE-B 7-Jun-1 psn54b flor, sul1, sul, teta a M Atlantic Quebec (NE) NE-C 10-Jul a M Brook trout Quebec (NW) NW-H 7-Aug a M Brook trout Quebec (SE) SE-B 30-Jul-1 psn54b flor, sul1, sul, teta a SHY13-16 Brook trout Quebec (NE) NE-C 5-Jan a SHY Brook trout Quebec (NE) NE-C 5-Feb a + (+) + SHY Brook trout Quebec (NW) NW-H 9-May a SHY Brook trout Quebec (NW) NW-B 8-Jun-13 psn54b flor, sul1, sul, teta a SHY13- Brook trout Quebec (NE) NE-C 3-Jul a SHY13-57 Brook trout Quebec (NW) NW-G 8-Jul a + + +
8 SHY13-63 Brook trout Quebec (NW) NW-H 8-Jul a SHY Brook trout Quebec (SE) SE-B 1-Jul None SHY13-45 Brook trout Quebec (SW) SW-C -Jul None SHY Brook trout Quebec (NW) NW-B -Jul-13 psn54b flor, sul1, sul, teta a SHY Atlantic Quebec (NE) NE-D 31-Jul None a SHY13-67 Brook trout Quebec (NW) NW-H 9-Aug-13 pasa4-like (trag+) sul1, teta(e) a SHY Brook trout Quebec (NW) NW-B 9-Aug-13 psn54b flor, sul1, sul, teta a SHY13-85 Brook trout Quebec (SE) SE-B 0-Aug None SHY Brook trout Quebec (SW) SW-C -Aug None SHY Brook trout Quebec (NW) NW-H 7-Aug a SHY Brook trout Quebec (SW) SW-C 1-Sep a SHY Brook trout Quebec (NW) NW-B 1-Sep a SHY Brook trout Quebec (SW) SW-C 3-Oct-13 psn54b flor, sul1, sul, teta a SHY Brook trout Quebec (NW) NW-H 3-Oct a SHY Brook trout Quebec (NW) NW-H 3-Oct-13 pasa4-like (trag+) sul1, teta(e) a MF6 N/A New K Brook trout New 004 psn54b flor, sui1, sul, teta a a K9 Atlantic New a + + +
9 K5 Brook trout New 009 pras3 teta(c) a K9 Brook trout New 009 pras3 teta(c) a K3 Brook trout New 009 pab5s9b, flor, sul, teth, a pras3 teta(c) K18 Brook trout New 010 pab5s9b, flor, sul, teth, a pras3 teta(c) K5 Atlantic RS 1458 Rainbow trout Nova Scotia 004 psn54b flor, sui1, sul, teta a Ontario 1993 pasa4-like teta(e) a (trag+) RS 1706 Chinook Ontario a RS 1705 Brook trout Ontario a RS 1744 Coho Ontario a RS 175 Pumpkinseed Ontario b RS 1835 Coho Ontario a a. NW = northwest, SW = southwest, NE = northeast, SE = southeast (see Figure 4). b. See : Attere SA, et al. Diversity and Homogeneity among Small Plasmids of Aeromonas icida subsp. icida Linked with Geographical Origin. Front Microbiol 6: 174. (015) c. See: Emond-Rheault JG, et al. Variants of a genomic island in Aeromonas icida subsp. icida link isolates with their geographical origins. Vet Microbiol 175: (015) d. See: Daher, R. K. et al. Alteration of virulence factors and rearrangement of pasa5 plasmid caused by the growth of Aeromonas icida in stressful conditions. Vet Microbiol 15, (011). e. N/A: Not available
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