Persistence of Antibiotic Resistant Vibrio Spp. in Shellfish Hatchery Environment

Persistence of antibiotic resistant Vibrio spp. in shellfish hatchery environment.

Javier Dubert*, Carlos R. Osorio, Susana Prado and Juan L. Barja.

Department of Microbiology and Parasitology. CIBUS–Faculty of Biology and Aquaculture Institute. University of Santiago de Compostela. Santiago de Compostela, 15782. Spain.

*Corresponding author:

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SUPPLEMENTARY MATERIAL.

Table S1 PCR primer sets used for the detection of cat and tet genes.

Primer / Nucleotide sequence (5’–3’) / gene / Size of PCR
product (bp) / References
C–R / CCATCACATACTGCATGATG / Yoo et al.(16)
C–1 / GGTGATATGGGATAGTGTT / cat I / 349
C–2 / GATTGACCTGAATACCTGGAA / cat II / 567
C–3 / CCATACTCATCCGATATTGA / cat III / 275
C–4 / CCGGTAAAGCGAAATTGTAT / cat IV / 451
TETF / GCGCTNTATGCGTTGATGCA / Jun et al. (17)
TAR / ACAGCCCGTCAGGAAATT / tet(A) / 387
TBR / TGAAAGCAAACGGCCTAA / tet(B) / 171
TCR / CGTGCAAGATTCCGAATA / tet(C) / 631
TDR / CCAGAGGTTTAAGCAGTGT / tet(D) / 484
TER / ATGTGTCCTGGATTCCT / tet(E) / 246
TGR / ATGCCAACACCCCCGGCG / tet(G) / 803
tet(M)–FW / GTTAAATAGTGTTCTTGGAG / tet(M) / 656 / Kim et al. (18)
tet(M)–RV / CTAAGATATGGCTCTAACAA
tet(S)–FW / CATAGACAAGCCGTTGACC / tet(S) / 667
tet(S)–RV / ATGTTTTTGGAACGACAGAG

Table S2 Sequences accession numbers obtained in this study.

Strain / 16S rRNA / cat / tet
Vs–58 / HF937133 / HF937176 / HF937194
Vs–291 / HF937134 / HF937166 / HF937199
Vt–197 / HF937135 / HF937161 / HF937196
Vt–262 / HF937136 / HF937162 / HF937197
Vt–268 / HF937137 / HF937164 / HF937198
Vt–28 / HF937138 / HF937165 / HF937190
Vt–31 / HF937139 / HF937168 / HF937191
Vt–47 / HF937140 / HF937174 / HF937192
Vt–447 / HF937141 / HF937171 / HF937203
Vt–48 / HF937142 / HF937175 / HF937193
Vt–448 / HF937143 / HF937172 / HF937204
Vt–449 / HF937144 / HF937173 / HF937205
Vp–348 / HF937145 / HF937169 / HF937201
Vp–307 / HF937146 / HF937167 / HF937200
Vm–77 / HF937147 / HF937179 / HF937195
Vm–26 / HF937158 / HF937163 / HF937189
Vm–402 / HF937148 / HF937170 / HF937202
Vd–607 / HF937149 / HF937177 / HF937206
Vd–608 / HF937150 / HF937178 / HF937207
Vs–229 / HF937151 / HF937182 / –
Vs–63 / HF937152 / HF937181 / –
Vs–292 / HF937153 / HF937180 / –
Vd–3159 / HF937159 / HF937188 / –
Vd–3149 / HF937154 / HF937185 / –
Vd–3154 / HF937155 / HF937186 / –
Vd–3155 / HF937156 / HF937184 / –
Vd–3160 / HF937160 / HF937187 / –
Vd–3127 / HF937157 / HF937183 / –

Table S3 Antibiotic resistance phenotypes of the recipients and transconjugants, including the resistance genes detected after the mating assays 1, 2 and 3.

Mating assay: / 1 / 2 / 3
E. cola / A trb / B trb / V. tuba / Vd–3154 trc / Vs–58 trc / V. osta / Vs–63 trd
CHL / S / R / R / S / R / R / S / R
TET / S / R / S / S / S / R / S / S
AMX / S / R / S / S / S / R / R / R
STR / S / S / R / R / R / R / S / R
Resistance
gene / – / cat II
tet(D) / cat III / – / cat III / cat II
tet(D) / – / cat III

CHL, chloramphenicol (30 µg); TET, tetracycline (30 µg); AMX, amoxicillin (25 μg); STR, streptomycin (30 μg).

aRecipients: E. Coli KR CAG 18420, Vibrio tubiashii subsp. europaensis PP–638T and V. ostreicida PP–203T.

b Transconjugants of Group A and B.

c Transconjugants using as donor the strain Vd–3154 and Vs–58.

d Transconjugants using as donor the strain Vs–63.

Fig. S1 Phylogenetic tree based on partial 16S rRNA gene sequences of the isolates corresponding to the Groups A and B included in the Splendidus clade, constructed using the NJ algorithm. Vibrio cholerae CECT 514T was used as outgroup. GenBank sequence accession numbers are given in parentheses. The stability of the groupings was estimated by bootstrap percentages from 1000 replicates. Bar, 0.005 substitutions per nucleotide position.

Fig. S2 Phylogenetic tree based on partial 16S rRNA gene sequences of the isolates corresponding to Groups A and B included in the Harveyi clade, constructed using the NJ algorithm. Characteristics of the phylogenetic tree identical at FIG S1.

Fig. S3 Electrophoretic analyses of the cat(a) and tet (b) genes amplified by the specific PCR method.

M1: PCR Marker (Sigma, USA): 2000, 1500, 1000, 750, 500, 300, 150 and 50 bp.

M2: FastRuler Low Range DNA Ladder (Thermo Fisher Scientific, USA): 1500, 850, 400, 200 and 50 bp.

Fig. S4Phylogenetic tree based on partial cat III gene sequences of the isolates corresponding to the Group B constructed using the NJ algorithm. GenBank sequence accession numbers are given in parentheses. The stability of the groupings was estimated by bootstrap percentages from 1000 replicates. Bar. 0.1 substitutions per nucleotide position.

Fig. S5Purification of the R-plasmids isolatedfromE. coli transconjugants.

M: GeneRuler 1 kb DNA Ladder (Thermo Fisher Scientific, USA): 10000, 8000, 6000, 5000, 4000, 3500, 3000, 2500, 2000, 1500, 1000, 750, 500 and 250 bp.