Genomic Islands in the Full-Genome Sequence of an NAD-Hemin-Independent Avibacterium paragallinarum Strain Isolated from Peru

Published: September 3, 2021

By: Luis Tataje-Lavanda 1; Ángela Montalván 1; Ricardo Montesinos 1; Vladimir Morales-Erasto 2; Mirko Zimic-Peralta 1,3; Manolo Fernández-Sánchez 1; Manolo Fernández-Díaz 1.

Summary

Author details:

1 Laboratorios de Investigación y Desarrollo, FARVET, Chincha Alta, Ica, Peru; 2 Centro de Investigación y Estudios Avanzados en Salud Animal, Facultad de Medicina Veterinaria y Zootecnia, Universidad Autónoma del Estado de México, Toluca, Mexico; 3 Laboratorio de Bioinformática y Biología Molecular, Facultad de Ciencias, Universidad Peruana Cayetano Heredia, Lima, Peru.

Here, we report the full-genome sequence of an NAD-hemin-independent Avibacterium paragallinarum serovar C-2 strain, FARPER-174, isolated from layer hens in Peru. This genome contained 12 potential genomic islands that include ribosomal protein-coding genes, a nadR gene, hemocin-coding genes, sequences of fagos, an rtx operon, and drug resistance genes.

Avibacterium paragallinarum is the etiologic agent of infectious coryza, an acute respiratory disease of chickens, which is globally distributed and causes serious economic losses in the poultry production industry. It is a Gram-negative, nonmotile, capsulated, facultative anaerobe belonging to the family Pasteurellaceae and is classified in 9 serovars distributed in 3 serogroups (A, B, and C) (1, 2). The study of its genome and virulence factors (hemagglutinin antigen, capsule, lipopolysaccharide, and RTX toxin) is important to better understand the pathogenesis of infectious coryza (3–6). Usually, virulence factor-coding genes are located in genomic islands (GIs) comprising clusters of genes suspected to have a horizontal origin (integrons, transposons, integrative and conjugative elements, and prophages) (7–9).

The strain FARPER-174 was isolated from nasal secretions of the paranasal sinuses of layer hens from an infectious coryza outbreak from the central rainforest region of Peru during 2015 and was grown in blood culture medium with X-(hemin) and V-(NAD) factors (10). FARPER-174 was subsequently cultured in brain heart infusion (BHI) agar without any factors at 37°C in a candle jar for 24 hours. The strain was identified as A. paragallinarum serovar C-2 using the morphology of the colony, biochemical tests (catalase test, oxidase test, urease test, peptone test, and carbohydrate fermentation, such as that of maltose, mannitol, lactose, and sorbitol) (11), specific PCR (12), and hemagglutination inhibition (HI) (13). The disk diffusion test (14) revealed resistance to colistin, ampicillin, sulfamethoxazoletrimethoprim, penicillin, neomycin, lincomycin, oxacillin, enrofloxacin, and gentamicin. The strain was susceptible to amoxicillin-clavulanic acid, doxycycline, streptomycin, florfenicol, tiamulin, levofloxacin, and gatifloxacin.

DNA was extracted from the fresh bacterial culture (500 ml of BHI without factors at 37°C with agitation at 180 rpm for 24 hours) centrifuged at 5,000 x g for 15 min. The pellet was resuspended in phosphate-buffered saline (PBS) for DNA isolation using the phenol-chloroform protocol (15). The genome was sequenced on the PacBio RS II platform (Pacific Biosciences, CA) using P6-C4 chemistry and assembled with Hierarchical Genome Assembly Process (HGAP) version 3.0 (16) with default parameters by Macrogen, Inc. (South Korea). A total of 92,198 reads (average length, 8,615 bp; N₅₀, 12,231 bp) generated a closed circular genome of 2,425,949 bp, with a G+C content of 40.87% and 147x average depth of coverage, and no plasmids.

Genomic Islands in the Full-Genome Sequence of an NAD-Hemin-Independent Avibacterium paragallinarum Strain Isolated from Peru - Image 1

The FARPER-174 strain genome was annotated with the NCBI Prokaryotic Genome Automatic Annotation Pipeline (PGAAP v4.7) (17). Some hypothetical proteins were similar to coding DNA sequences (CDSs) of A. paragallinarum previously published in GenBank; the annotations were, therefore, manually improved in this version.

A total of 2,327 genes, 2,248 CDSs, 106 pseudogenes, and 79 RNA genes, which included 56 tRNAs, 19 rRNAs (5S, 16S, and 23S), 1 transfer-messenger RNA (tmRNA), and 3 noncoding RNAs (ncRNAs), were identified. Six 16S rRNA genes compared by BLASTn with the 16S rRNA database of NCBI are from 97% to 98% similar to those of A. paragallinarum strain NCTC 11296 (GenBank accession number NR_042932). Genomic islands were predicted by submitting the PGAAP-generated GenBank file to the IslandViewer 4 tool (18). In total, 12 genetic islands, namely, I to XII, which include 220 genes (Fig. 1), were found.

Data availability. The complete Avibacterium paragallinarum strain FARPER-174 genome sequence is available in GenBank under the accession number CP034110. Raw data are available in SRA under BioSample number SAMN10471982 and SRA run number SRR8506728.

This article was originally published in Microbiology Resource Announcements 8:e00118-19. https://doi.org/10.1128/MRA.00118-19. This is an Open Access article distributed under the terms of the Creative Commons Attribution 4.0 International license.

References

1. Blackall PJ, Eaves LE, Rogers DG. 1990. Proposal of a new serovar and altered nomenclature for Haemophilus paragallinarum in the Kume hemagglutinin scheme. J Clin Microbiol 28:1185–1187.
2. Blackall PJ, Soriano-Vargas E. 2017. Infectious coryza and related bacterial infections, p. 859 – 873. In Swayne D (ed), Diseases of poultry. John
Wiley & Sons, Ltd, Chichester, United Kingdom.
3. Requena D, Chumbe A, Torres M, Alzamora O, Ramirez M, ValdiviaOlarte H, Gutierrez AH, Izquierdo-Lara R, Tataje-Lavanda L, Zavaleta
M, Tataje-Lavanda L, Best I, Fernández-Sánchez M, Icochea E, Zimic M,
Fernández-Díaz M, FARVET Research Group. 2013. Genome sequence and comparative analysis of Avibacterium paragallinarum. Bioinformation 9:528 –536. https://doi.org/10.6026/97320630009528.
4. Horta-Valerdi G, Sanchez-Alonso MP, Perez-Marquez VM, NegreteAbascal E, Vaca-Pacheco S, Hernandez-Gonzalez I, Gomez-Lunar Z,
Olmedo-Álvarez G, Vázquez-Cruz C. 2017. The genome sequence of
Avibacterium paragallinarum Strain CL has a large repertoire of insertion sequence elements. Genome Announc 5:e00152-17. https://doi.org/10
.1128/genomeA.00152-17.
5. Aguilar-Bultet L, Calderon-Copete SP, Frey J, Falquet L. 2013. Draft genome sequence of the virulent Avibacterium paragallinarum serotype
A strain JF4211 and identification of two toxins. Genome Announc
1:e00592-13. https://doi.org/10.1128/genomeA.00592-13.
6. Chen Y-C, Tan D-H, Shien J-H, Hsieh M-K, Yen T-Y, Chang P-C. 2014.
Identification and functional analysis of the cytolethal distending toxin gene from Avibacterium paragallinarum. Avian Pathol 43:43–50. https:// doi.org/10.1080/03079457.2013.861895.
7. Ho Sui SJ, Fedynak A, Hsiao WWL, Langille MGI, Brinkman FSL. 2009. The association of virulence factors with genomic islands. PLoS One 4:e8094. https://doi.org/10.1371/journal.pone.0008094.
8. Bertelli C, Tilley KE, Brinkman FSL. 2018. Microbial genomic island discovery, visualization and analysis. Brief Bioinform. https://doi.org/10
.1093/bib/bby042.
9. Dongsheng C, Hasan MS, Chen B. 2014. Identifying pathogenicity islands in bacterial pathogenomics using computational approaches. Pathogens
3:36 –56. https://doi.org/10.3390/pathogens3010036.
10. Falconi-Agapito F, Saravia LE, Flores-Pérez A, Fernández-Díaz M. 2015.
Naturally occurring -nicotinamide adenine dinucleotide–independent
Avibacterium paragallinarum isolate in Peru. Avian Dis 59:341–343. https://doi.org/10.1637/10969-110314-CaseR.
11. Blackall PJ, Christensen H, Beckenham T, Blackall LL, Bisgaard M. 2005.
Reclassification of Pasteurella gallinarum, [Haemophilus] paragallinarum,
Pasteurella avium and Pasteurella volantium as Avibacterium gallinarum gen. nov., comb. nov., Avibacterium paragallinarum comb. nov., Avibacterium avium comb. nov. and Avibacterium. Int J Syst Evol Microbiol
55:353–362. https://doi.org/10.1099/ijs.0.63357-0.
12. Chen X, Miflin JK, Zhang P, Blackall PJ. 1996. Development and application of DNA probes and PCR tests for Haemophilus paragallinarum. Avian
Dis 40:398 – 407. https://doi.org/10.2307/1592238.
13. Soriano VE, Blackall PJ, Dabo SM, Téllez G, García-Delgado GA, Fernández
RP. 2001. Serotyping of Haemophilus paragallinarum isolates from Mexico by the Kume hemagglutinin scheme. Avian Dis 45:680 – 683. https:// doi.org/10.2307/1592911.
14. Luna-Galaz GA, Morales-Erasto V, Peñuelas-Rivas CG, Blackall PJ, SorianoVargas E. 2016. Antimicrobial sensitivity of Avibacterium paragallinarum isolates from four Latin American countries. Avian Dis 60:673– 676. https://doi.org/10.1637/11398-022616-ResNote.1.
15. Sambrook J, Russell DW. 2001. Molecular cloning: a laboratory manual.
Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.
16. Chin C-S, Alexander DH, Marks P, Klammer AA, Drake J, Heiner C, Clum
A, Copeland A, Huddleston J, Eichler EE, Turner SW, Korlach J. 2013.
Nonhybrid, finished microbial genome assemblies from long-read SMRT sequencing data. Nat Methods 10:563–569. https://doi.org/10.1038/ nmeth.2474.
17. 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. https://doi
.org/10.1093/nar/gkw569.
18. Bertelli C, Laird MR, Williams KP, Simon Fraser University Research Computing Group, Lau BY, Hoad G, Winsor GL, Brinkman F. 2017. IslandViewer 4: expanded prediction of genomic islands for larger-scale datasets. Nucleic
Acids Res 45:W30 –W35. https://doi.org/10.1093/nar/gkx343.