Complete circular assembly of Histophilus somni PDS1537697-81-1 genome and phenotypic characterization of its antibiotic susceptibility

TL;DR

This paper reports the full genome assembly and antibiotic susceptibility testing of a Histophilus somni isolate from a healthy calf.

Contribution

The study provides a complete circular genome assembly and phenotypic characterization of an H. somni isolate.

Findings

A Histophilus somni isolate was obtained from a healthy calf.

The isolate's genome was fully sequenced and assembled in a circular form.

Antimicrobial susceptibility testing was performed on the isolate.

Abstract

A Histophilus somni isolate from a clinically healthy,
fall-placed calf was obtained upon arrival to a commercial feedlot.
Fall-placed calves are commonly viewed to be at high risk for the
development of bovine respiratory disease. The isolate was phenotyped for
antimicrobial susceptibility and sequenced to obtain a complete, circular,
genome assembly.

Tables

TABLE 1. Antibiotic susceptibility test results for Histophilus somni PDS1537697-81-1a

Antibiotic	Minimum inhibitory concentration (µg/mL)	Interpretation
Ampicillin	0.3	R (≥0.25)
Ceftioufur	0.3	S (≤2)
Chlortetracycline	0.5	NI
Clindamycin	1.0	NI
Danofloxacin	0.1	NI
Enrofloxacin	0.1	S (≤0.25)
Florfenicol	0.3	S (≤2)
Gentamicin	16.0	NI
Neomycin	32.0	NI
Oxytetracycline	4.0	NI
Penicillin	0.1	S (≤0.1)
Spectinomycin	64.0	I (=64)
Sulfadimethoxime	256.0	NI
Tiamulin	2.0	NI
Tilmicosin	8.0	NI
Trimethoprim/sulfa	2.0	NI
Tulathromycin	16.0	S (≤16)
Tylosin	8.0	NI

Full text

ANNOUNCEMENT

Histophilus somni is a significant bacterial pathogen in the bovine respiratory disease (BRD) complex (1). Genomics-based tools can enhance diagnostic support for antimicrobial stewardship in BRD management but require the establishment of comprehensive reference data on isolates with known antibiotic susceptibility phenotypes (2).

In December 2015, a deep nasopharyngeal swab (Tiegland Modified Culture Swab, Jorgensen Laboratories, LLC, Loveland, CO, USA) was collected from a clinically healthy auction-sourced steer on arrival to a commercial feedlot (University of Saskatchewan Animal Research Ethics Board Certificate of Approval AUP#20150075), placed in Amies transport medium (eSwab Transport System Copan Diagnostics Inc., Murrieta, CA, USA), and delivered on ice to Prairie Diagnostic Services (PDS; Saskatoon, SK, Canada). H. somni PDS1537697-81-1 was isolated by plating 10 µL of the transport medium on a Chocolate agar plate, incubating for 48 h at 35°C in 5% CO_2_, and identified by MALDI-TOF MS (Microflex LT system, Bruker Daltonics, Billerica, MA, USA) as previously described (3). The isolate was cryopreserved, in Tryptic Soy Broth (TSB, Difco Laboratories, Detroit, MI, USA) containing 15% glycerol at −80°C, after one passage. Recovery from cryopreservation was done by quadrant inoculation of a Chocolate agar plate and 18-h incubation at 35°C in 5% CO_2_. The culture purity and the organism identity were re-confirmed by analyzing single colonies of a characteristic morphology on a MALDI-TOF MS.

Antimicrobial susceptibility testing, as a single diagnostic submission, was performed by broth microdilution method (ThermoFisher Scientific, Nepean, ON, Canada) using 96-well Sensititre microtiter plates (BOPO6F, ThermoFisher Scientific; Table 1) as described previously (4).

Bacterial DNA was extracted from a fresh Chocolate agar plate after 18-h incubation at 35°C in 5% CO_2_ via MagAttract HMW DNA kit (Qiagen, Toronto, ON, Canada). Nucleic acid was confirmed to be of high molecular weight (peak concentration value > 60 kb) using a Genomic DNA Screen Tape on a 4200 TapeStation System (Agilent, Santa Clara, CA, USA). About 400 ng of the bacterial high molecular weight DNA was used for a sequencing library preparation with the Rapid Barcoding Sequencing gDNA kit as per the manufacturer’s protocol (SQK-RBK004, Oxford Nanopore Technologies, Oxford, United Kingdom). The prepared library was loaded on an FLO-PRO002 sequencing flow cell and sequenced on a PromethION P2Solo device connected to GridION X5 machine running on MinKNOW 23.11.3 software with real-time high accuracy basecalling. For de novo bacterial genome assembly, 12,000 reads/58.9 M bases (N50: 13,530, N75: 6,037, and N90: 2,415) and representing an estimated 27× coverage of the expected genome size of 2.18 Mb (5) were processed with the Dragonflye pipeline (v1.1.2)(6) with one round of medaka polishing (v1.11.3; using model r941_prom_hac_g507) (7), and one round of racon polisher (v.1.5.0) (8). The assembly resulted in a single circular 2,182,197 bp contig representing 20× coverage. Taxonomic classification of the assembled genome was performed using Kraken2 (minikraken reference database) (9) to confirm the isolate was H.somni. The assembly was annotated using PGAP (10) identifying 2,118 genes and a GC content of 37.3%. ABRicate (11) using the default database (12) identified the presence of tet(H), aadA3, sul2, aph(3″)-Ib, aph (6)-Id, and aph(3′)-Ia antimicrobial genes within the genome.

TABLE 1: Antibiotic susceptibility test results for Histophilus somni PDS1537697-81-1a