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Draft Genome Sequences of Three Ochrobactrum spp. isolated from Different Avian Hosts in Pakistan

ABSTRACT Here, we present the draft genome sequences of three Ochrobactrum sp. strains with multidrug-resistant properties, isolated in 2015 from a pigeon and two chickens in Pakistan.

There are few reports on the isolation of Ochrobactrum spp. from avian hosts. Ochrobactrum gallinifaecis has been isolated from chicken feces in Germany (7), and Ochrobactrum anthropi and Ochrobactrum pecoris have been isolated from the cecal contents of commercial turkeys (8). More recently, Ochrobactrum intermedium and Ochrobactrum tritici were recovered from broiler chickens (9), and a novel species has been reported in Nigeria (10).
Here, we present the draft genome sequences of three multidrug-resistant Ochrobactrum isolates from a pigeon and chickens that were coinfected with Newcastle disease virus. The distance between these and other members of the genus Ochrobactrum cannot be resolved using the 16S rRNA phylogeny (11), and therefore we examined the rpoB and dnaK sequences to distinguish the new isolates. The maximum similarity levels with rpoB and dnaK were 94.7% and 95.3%, with O. anthropi ATCC 49687 (GenBank accession no. CP008820) (12) and O. anthropi (GenBank accession no. LT671861), respectively, which distinguish these strains from other Ochrobactrum species. The average nucleotide identity among these isolates was 99.99% and varied between 96.96% and 97.05% with the five novel Ochrobactrum spp. recently reported from Nigeria (10,13). This 3 to 4% of genomic variation supports the finding that the Pakistani isolates belong to a novel avian Ochrobactrum spp. (10).
Oral swabs were plated onto Farrell's agar medium for purification as previously reported (10). Genomic DNA isolates were extracted using the blood and tissue genomic DNA extraction kit (Qiagen, Germantown, MD). Extracted DNA was quantified using the Qubit double-stranded DNA (dsDNA) high-sensitivity (HS) assay kit (Life Technologies, Inc., Waltham, MA). The libraries were prepared using the Nextera XT DNA library preparation kit and Nextera XT index primers (Illumina, San Diego, CA). The concentrations of the libraries were checked using the Qubit DNA HS assay kit in a Qubit fluorometer (Thermo Fisher Scientific, USA), and the fragment size distribution was checked using the Bioanalyzer 2100 with an Agilent high-sensitivity DNA kit (Agilent Technologies, Santa Clara, CA). The generated libraries were sequenced using MiSeq reagent kit version 3 (600 cycles), with a paired-end read length of 2 ϫ 300 bp on an Illumina MiSeq platform. Sequence data were assembled using MIRA version 3.4.1 (14) on the Galaxy platform (15). The genome sequence was annotated with the NCBI Prokaryotic Genome Annotation Pipeline (16). The statistics of library assembly and annotation are shown in Table 1.
The antibiotic resistance genes were identified using ARG-ANNOT (17). The isolates harbored genes conferring resistance to aminoglycosides, ␤-lactamase, tetracycline, and chloramphenicol. We also found additional antibiotic resistance genes conferring resistance to sulfonamide and trimethoprim.
Accession number(s). This whole-genome project has been deposited at DDBJ/ ENA/GenBank under BioProject number PRJNA407326, and the accession numbers are listed in Table 1.

ACKNOWLEDGMENTS
We thank Dawn Williams-Coplin and Tim Olivier for their technical assistance. This work was supported by the USDA CRIS grant 6612-32000-072-00D. The mention of trade names or commercial products in this article is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the U.S. Department of Agriculture or any of the authors.