ABSTRACT
Nine Peruvian isolates of Vibrio parahaemolyticus were characterized through sequencing, revealing the presence of simple sequence repeat, Pir toxin-like genes, and genes associated with antibiotic resistance, toxic components, and transposable elements. These findings expand our understanding of the genetic diversity, disease resistance, and virulence in cultivated shrimp populations in Peru.
KEYWORDS: Vibrio parahaemolyticus, genetic diversity, shrimp cultures, PirAB genes, aquaculture health, pathogenic bacteria, antibiotic resistance, transposable elements, Peruvian farmed shrimp, disease control
ANNOUNCEMENT
Acute hepatopancreatic necrosis disease (AHPND) in Penaeus vannamei shrimp is specifically caused by strains of Vibrio parahaemolyticus (family Vibrionaceae) carrying a virulent plasmid pVA1, encoding the PirAvp and PirBvp toxins (1–3). In Latin America, pathogenic strains have been identified, but high mortalities as observed in Asia have not been reported. In this region, a chronic disease with unique tolerance to AHPND has been described in the Latin American shrimp population (4).
In this study, nine samples were collected, each containing five juvenile shrimp specimens. These samples were obtained from four epidemiological zones in Tumbes, Peru, between April, May, and August 2022. The epidemiological zones included North Zone 1 (samples Tumbes-M02, Tumbes-M04, and Tumbes-M05), South Zone 1 (Tumbes-M03), South Zone 2 (Tumbes-M06), and Central Zone (Tumbes-M07, Tumbes-M10, Tumbes-M11, and Tumbes-M12). Hepatopancreas tissue was collected and enriched in Trypticase soy broth medium with 2.5% NaCl, followed by a 16-hour incubation at 30°C. Subsequently, the bacterial culture was plated on CHROMagar plates. Mauve-colored colonies were selected and then inoculated onto thiosulfate citrate bile sucrose agar (TCBS) plates, from which green-colored colonies were chosen for further analysis.
DNA from the selected colonies was extracted through thermal shock, involving heating at 100°C for 10 minutes, followed by cooling at and −20°C for 2 minutes. Molecular detection of PirAB genes was carried out using PCR PirAB Duplex (4). Subsequently, nine confirmed colonies were selected for sequencing. Total DNA was isolated from bacterial cultures using the NucleoSpin tissue DNA kit (Macherey-Nagel, Germany) and treated with RNase. The isolated DNA was quantified using the Qubit4 fluorometer. The library was prepared using the Rapid Barcoding kit (SQK-RBK004) following the manufacturer’s instructions and loaded into an R9.4.1 flow cell. Sequencing was conducted on a MinION Mk1C device (Oxford Nanopore Technologies, UK) for a duration of 25 hours (1.58 million reads; 9 gigabases of passed basses; N50: 11.25 kb; average QScore: 11). Basecalling was performed using Guppy v5.0.12 (Fast basecalling).
The obtained sequences were processed using default parameters unless otherwise specified. In the Galaxy platform (5), the sequences were concatenated using the “Concatenate data sets” program (Galaxy Version 0.1.1) (6). De novo assemblies were performed using the Flye program (Galaxy Version 2.9.1+galaxy0) (7), and the taxonomic identity of the isolates was determined using Kraken2 (Galaxy Version 2.1.1+galaxy1) with the k2_standard_20210517 database (8). The results were visualized with Krona (Galaxy Version 2.6.1.1) (9), and annotations were conducted using the NCBI Prokaryotic Genomic Annotation Pipeline (PGAP) v.4.8 (10) and RASTtk (11) for functional analysis with the following options: “RASTtk: Automatically fix errors,” “Fix frameshifts,” and “Backfill gaps on YES.” During the analysis, genes associated with antibiotic resistance, toxic components, and transposable elements such as phage fragments and integrons were identified. Furthermore, the presence of SSR (TTGTTTTTC) with five and six repeats was found manually in plasmids carrying PirAB genes (1) (Table 1).
TABLE 1.
Genomic characterization of Vibrio parahaemolyticus isolates from the Tumbes regiona
| Isolate | Assemblies | Subsystem feature counts | |||||
|---|---|---|---|---|---|---|---|
| N° contigs | Length (bp) | Cov. | Circ. | %GC | Resistance to antibiotics and toxic compound | Transposable elements | |
| Tumbes-M02 (JAUIQE000000000) | contig_2 | 3,329,736 | 262 | Y | 45 | Copper homeostasis (18) Cobalt-zinc-cadmium resistance (8) Resistance to fluoroquinolones (2) Copper homeostasis: copper tolerance (9) Resistance to chromium compounds (2) Multidrug resistance efflux pumps (19) |
Phage replication (3) Prophage-encoded Rst operon (2) Phage DNA synthesis (3) Integrons (2) |
| contig_1 | 1,906,514 | 201 | Y | 45 | |||
| contig_7c | 54,879 | 766 | N | 43 | |||
| contig_8 | 51,645 | 825 | N | 44 | |||
| contig_3 | 23,633 | 575 | N | 52 | |||
| contig_6 | 4,672 | 830 | Y | 41 | |||
| contig_5 | 4,083 | 134 | N | 50 | |||
| Tumbes-M03 (JAUIQF000000000) | contig_1 | 3,366,142 | 262 | Y | 45 | Copper homeostasis (17) Cobalt-zinc-cadmium resistance (7) Resistance to fluoroquinolones (2) Copper homeostasis: copper tolerance (10) Resistance to chromium compounds (1) Multidrug resistance efflux pumps (16) |
Phage replication (7) Phage DNA synthesis (3) Integrons (2) |
| contig_3 | 1,958,396 | 204 | Y | 45 | |||
| contig_5b | 73,251 | 681 | Y | 45 | |||
| contig_4 | 57,864 | 749 | Y | 47 | |||
| contig_2 | 21,938 | 638 | N | 52 | |||
| Tumbes-M04 (JAUIQG000000000) | contig_2 | 3,363,672 | 72 | Y | 45 | Copper homeostasis (15) Cobalt-zinc-cadmium resistance (7) Resistance to fluoroquinolones (2) Copper homeostasis: copper tolerance (9) Resistance to chromium compounds (1) Multidrug resistance efflux pumps (12) |
Phage DNA synthesis (3) Integrons (2) |
| contig_5 | 1,958,928 | 58 | Y | 45 | |||
| contig_4b | 73,294 | 95 | Y | 46 | |||
| contig_1 | 57,904 | 81 | Y | 46 | |||
| contig_3 | 21,947 | 432 | N | 52 | |||
| Tumbes-M05 (JAUIQH000000000) | contig_1 | 3,366,948 | 113 | Y | 45 | Copper homeostasis (15) Cobalt-zinc-cadmium resistance (8) Resistance to fluoroquinolones (2) Copper homeostasis: copper tolerance (9) Resistance to chromium compounds (1) Multidrug resistance efflux pumps (14) |
Phage replication (6) Phage DNA synthesis (3) Integrons (2) |
| contig_3 | 1,958,861 | 85 | Y | 45 | |||
| contig_4b | 73,299 | 210 | Y | 46 | |||
| contig_2 | 57,869 | 229 | Y | 46 | |||
| contig_5 | 21,930 | 506 | N | 52 | |||
| Tumbes-M06 (JAUIQI000000000) | contig_1 | 3,366,917 | 119 | Y | 45 | Copper homeostasis (15) Cobalt-zinc-cadmium resistance (8) Resistance to fluoroquinolones (2) Copper homeostasis: copper tolerance (9) Resistance to chromium compounds (1) Multidrug resistance efflux pumps (14) |
Phage replication (6) Phage DNA synthesis (3) Integrons (2) |
| contig_3 | 1,957,184 | 95 | Y | 45 | |||
| contig_4b | 73,304 | 243 | Y | 46 | |||
| contig_5 | 57863 | 275 | Y | 46 | |||
| contig_2 | 21937 | 537 | N | 52 | |||
| Tumbes-M07 (JAUIQJ000000000) | contig_1 | 3367212 | 74 | Y | 45 | Copper homeostasis (15) Cobalt-zinc-cadmium resistance (7) Resistance to fluoroquinolones (2) Copper homeostasis: copper tolerance (9) Resistance to chromium compounds (1) Multidrug resistance efflux pumps (15) |
Phage replication (5) Phage DNA synthesis (3) Integrons (2) |
| contig_3 | 1957361 | 59 | Y | 45 | |||
| contig_5b | 74470 | 161 | N | 46 | |||
| contig_4 | 35928 | 168 | N | 43 | |||
| Tumbes-M10 (JAUIQK000000000) | contig_1 | 3366564 | 181 | Y | 45 | Copper homeostasis (17) Cobalt-zinc-cadmium resistance (7) Resistance to fluoroquinolones (2) Copper homeostasis: copper tolerance (9) Resistance to chromium compounds (1) Multidrug resistance efflux pumps (17) |
Phage replication (5) Phage DNA synthesis (3) Integrons (2) |
| contig_3 | 1958634 | 141 | Y | 45 | |||
| contig_4b | 73267 | 375 | Y | 46 | |||
| contig_2 | 57878 | 332 | Y | 46 | |||
| contig_5 | 21927 | 539 | N | 52 | |||
| Tumbes-M11 (JAUIQL000000000) | contig_1 | 3391395 | 189 | Y | 45 | Copper homeostasis (17) Cobalt-zinc-cadmium resistance (6) Resistance to fluoroquinolones (2) Fosfomycin resistance (1) Copper homeostasis: copper tolerance (9) Resistance to chromium compounds (1) Multidrug resistance efflux pumps (17) |
Phage replication (4) Phage packaging machinery (2) Phage DNA synthesis (3) Phage tail proteins (11) Phage tail proteins 2 (11) Prophage-encoded Rst operon (2) Phage capsid proteins (1) Phage tail fiber proteins (2) Integrons (2) |
| contig_2 | 1907405 | 152 | Y | 45 | |||
| contig_7 | 124986 | 280 | Y | 44 | |||
| contig_6b | 73218 | 335 | Y | 46 | |||
| contig_20 | 42402 | 272 | Y | 48 | |||
| contig_18 | 34908 | 372 | Y | 46 | |||
| contig_17 | 21189 | 795 | N | 53 | |||
| contig_16 | 19712 | 504 | N | 42 | |||
| contig_11 | 13187 | 575 | N | 40 | |||
| contig_8 | 13063 | 700 | N | 46 | |||
| contig_22 | 11506 | 564 | Y | 41 | |||
| contig_14 | 9335 | 588 | Y | 41 | |||
| contig_10 | 7614 | 265 | N | 41 | |||
| contig_21 | 7127 | 586 | Y | 42 | |||
| contig_15 | 5836 | 317 | N | 39 | |||
| contig_3 | 2494 | 54 | N | 45 | |||
| contig_9 | 1747 | 67 | N | 42 | |||
| Tumbes-M12 (JAUIQM000000000) | contig_15 | 3391992 | 101 | Y | 45 | Copper homeostasis (17) Cobalt-zinc-cadmium resistance (7) Resistance to fluoroquinolones (2) Fosfomycin resistance (1) Copper homeostasis: copper tolerance (10) Resistance to chromium compounds (1) Multidrug resistance efflux pumps (12) |
Phage tail proteins (6) Phage replication (3) Phage packaging machinery (2) Prophage-encoded Rst operon (2) Phage tail proteins 2 (6) Phage tail fiber proteins (2) Phage DNA synthesis (3) Integrons (1) |
| contig_21 | 1907735 | 84 | Y | 45 | |||
| contig_5 | 125091 | 133 | Y | 44 | |||
| contig_6b | 46703 | 314 | N | 43 | |||
| contig_22 | 42405 | 432 | Y | 48 | |||
| contig_3 | 29192 | 273 | N | 42 | |||
| contig_18 | 21103 | 421 | N | 53 | |||
| contig_17 | 20017 | 34 | Y | 40 | |||
| contig_1 | 17447 | 882 | Y | 46 | |||
| contig_12 | 13510 | 314 | N | 40 | |||
| contig_10 | 13068 | 599 | N | 46 | |||
| contig_20 | 11502 | 510 | Y | 41 | |||
| contig_4 | 10880 | 347 | N | 41 | |||
| contig_16 | 9330 | 585 | Y | 41 | |||
| contig_2 | 4240 | 129 | N | 44 | |||
| contig_19 | 3716 | 90 | N | 55 | |||
| contig_14 | 3593 | 819 | Y | 42 | |||
| contig_7 | 2372 | 89 | N | 42 | |||
This table presents a comprehensive genomic analysis of Vibrio parahaemolyticus isolates obtained from the Tumbes region (Peru). Essential details are provided, including isolate names, contig names, contig lengths in base pairs (bp), coverage (cov.), circularity (circ.), GC content (%GC), resistance to antibiotics and toxic compounds, and transposable elements.
The presence of the PIrA gene and the simple sequence repeat (SSR) repetitive sequence (TTGTTTTTC) five times is indicated.
Additionally, the presence of the PIrAB gene and the SSR repetitive sequence six times is also indicated.
This study contributes to the understanding of the pathogenicity and evolution of these bacteria in the context of diseases in farmed shrimps. These findings may have significant implications for the development of strategies for disease control and prevention in aquaculture.
ACKNOWLEDGMENTS
This project received funding from the National Program for Innovation in Fisheries and Aquaculture (PNIPA) of the Ministry of Production of Peru, under contract number 004-2020-PNIPA-SUBPROJECT-SANIPES "Surveillance of exotic, emerging, and disease-causing pathogenic microbial communities in farmed and wild Penaeus vannamei shrimp to strengthen aquaculture in Tumbes and Piura regions."
Contributor Information
Rodolfo Velazco-Peña, Email: rodolfo.velazco@sanipes.gob.pe.
Frank J. Stewart, Montana State University, Bozeman, Montana, USA
DATA AVAILABILITY
The Nanopore raw reads for this sequencing project (PRJNA980916) are available under the following accession numbers: SRR24899490, SRR24899239, SRR24899244, SRR24898281, SRR24899242, SRR24899243, SRR24898079, SRR24899231, SRR24902115. The assembled contigs can be accessed using the following accession numbers: JAUIQE000000000, JAUIQF000000000, JAUIQG000000000, JAUIQH000000000, JAUIQI000000000, JAUIQJ000000000, JAUIQK000000000, JAUIQL000000000, JAUIQM000000000.
REFERENCES
- 1. Han JE, Tang KFJ, Lightner DV. 2015. Genotyping of virulence plasmid from Vibrio parahaemolyticus isolates causing acute hepatopancreatic necrosis disease in shrimp. Dis Aquat Organ 115:245–251. doi: 10.3354/dao02906 [DOI] [PubMed] [Google Scholar]
- 2. Han JE, Tang KFJ, Tran LH, Lightner DV. 2015. Photorhabdus insect-related (Pir) toxin-like genes in a plasmid of Vibrio parahaemolyticus, the causative agent of acute hepatopancreatic necrosis disease (AHPND) of shrimp. Dis Aquat Organ 113:33–40. doi: 10.3354/dao02830 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 3. Lee C-T, Chen I-T, Yang Y-T, Ko T-P, Huang Y-T, Huang J-Y, Huang M-F, Lin S-J, Chen C-Y, Lin S-S, Lightner DV, Wang H-C, Wang A-J, Wang H-C, Hor L-I, Lo C-F. 2015. The opportunistic marine pathogen Vibrio parahaemolyticus becomes virulent by acquiring a plasmid that expresses a deadly toxin. Proc Natl Acad Sci USA 112:10798–10803. doi: 10.1073/pnas.1503129112 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 4. Aranguren Caro LF, Mai HN, Noble B, Dhar AK. 2020. Acute hepatopancreatic necrosis disease (VPAHPND), a chronic disease in shrimp (Penaeus vannamei) population raised in Latin America. J Invertebr Pathol 174:107424. doi: 10.1016/j.jip.2020.107424 [DOI] [PubMed] [Google Scholar]
- 5. de Koning W, Miladi M, Hiltemann S, Heikema A, Hays JP, Flemming S, van den Beek M, Mustafa DA, Backofen R, Grüning B, Stubbs AP. 2020. NanoGalaxy: nanopore long-read sequencing data analysis in galaxy. Gigasci 9:1–7. doi: 10.1093/gigascience/giaa105 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 6. Bjoern A. 2014. Galaxy tool wrappers. Available from: https://github.com/bgruening/galaxytools
- 7. Kolmogorov M. 2021. Flye assembler. Available from: https://github.com/fenderglass/Flye
- 8. Wood DE, Salzberg SLS, Venter C, Remington K, Heidelberg J, Halpern A, Rusch D, Eisen J, Wu D, Paulsen I, et al. 2014. Kraken: ultrafast metagenomic sequence classification using exact alignments. Genome Biol 15:R46. doi: 10.1186/gb-2014-15-3-r46 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 9. Ondov BD, Bergman NH, Phillippy AM. 2011. Interactive metagenomic visualization in a web browser. BMC Bioinform 12:385. doi: 10.1186/1471-2105-12-385 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 10. Tatusova T, DiCuccio M, Badretdin A, Chetvernin V, Nawrocki EP, Zaslavsky L, Lomsadze A, Pruitt KD, Borodovsky M, Ostell J. 2016. NCBI prokaryotic genome annotation pipeline. Nucleic Acids Res 44:6614–6624. doi: 10.1093/nar/gkw569 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 11. Brettin T, Davis JJ, Disz T, Edwards RA, Gerdes S, Olsen GJ, Olson R, Overbeek R, Parrello B, Pusch GD, Shukla M, Thomason JA, Stevens R, Vonstein V, Wattam AR, Xia F. 2015. RASTtk: a modular and extensible implementation of the RAST algorithm for building custom annotation pipelines and annotating batches of genomes. Sci Rep 5:8365. doi: 10.1038/srep08365 [DOI] [PMC free article] [PubMed] [Google Scholar]
Associated Data
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Data Availability Statement
The Nanopore raw reads for this sequencing project (PRJNA980916) are available under the following accession numbers: SRR24899490, SRR24899239, SRR24899244, SRR24898281, SRR24899242, SRR24899243, SRR24898079, SRR24899231, SRR24902115. The assembled contigs can be accessed using the following accession numbers: JAUIQE000000000, JAUIQF000000000, JAUIQG000000000, JAUIQH000000000, JAUIQI000000000, JAUIQJ000000000, JAUIQK000000000, JAUIQL000000000, JAUIQM000000000.