Genome sequencing and annotation of a Campylobacter coli strain isolated from milk with multidrug resistance

As the most prevalent bacterial cause of human gastroenteritis, food-borne Campylobacter infections pose a serious threat to public health. Whole Genome Sequencing (WGS) is a tool providing quick and inexpensive approaches for analysis of food-borne pathogen epidemics. Here we report the WGS and annotation of a Campylobacter coli strain, FNW20G12, which was isolated from milk in the United States in 1997 and carries multidrug resistance. The draft genome of FNW20G12 (DDBJ/ENA/GenBank accession number LWIH00000000) contains 1, 855,435 bp (GC content 31.4%) with 1902 annotated coding regions, 48 RNAs and resistance to aminoglycoside, beta-lactams, tetracycline, as well as fluoroquinolones. There are very few genome reports of C. coli from dairy products with multidrug resistance. Here the draft genome of FNW20G12, a C. coli strain isolated from raw milk, is presented to aid in the epidemiology study of C. coli antimicrobial resistance and role in foodborne outbreak.


Introduction
Campylobacteriosis is the primary cause of bacterial infections resulting in food-borne gastroenteritis with symptoms ranging from abdominal cramping, fever, to bloody diarrhea. In the United States, there are over 2.4 million people infected by Campylobacter each year [1]. Besides raw meat, primary risk factors of campylobacteriosis include consumption of raw milk, contaminated produce and water. Recently the role of C. coli in human disease has been increasingly recognized as it accounts for up to 25% of the total Campylobacter-associated diarrhea and has been reported to cause bacteremia, meningitis, spontaneous abortion and community acquired inflammatory enteritis [2][3][4]. Despite the application of Whole Genome Sequencing (WGS) on Campylobacter isolates, especially C. jejuni from clinical patients, genome reports of C. coli from dairy products with multidrug resistance are scarce. Here we announce the WGS and annotation of C. coli FNW20G12 strain, which was isolated from milk in the United States in 1997.

Experimental methods and results
The FNW20G12 strain was isolated and previously characterized according to the FDA Bacteriological Analytical Manual [5]. Prepared Campylobacter selective agar (catalog# B21727X, ThermoFisher Scientific Inc., Waltham, MA) and GasPak EZ Container System with Gas Sachets (Becton Dickinson Co., Franklin Lakes, NJ) were used to achieve microaerobic growth of FNW20G12. Genomic DNA was extracted with a QIAcube instrument (QIAGEN Inc., Valencia, CA) and quantified with a Qubit 3.0 fluorimeter (ThermoFisher Scientific Inc.). A library was then prepared with the Nextera XT sample preparation kit (Illumina, San Diego, CA) following manufacturer's instructions.
The genome of FNW20G12 was sequenced using a MiSeq instrument (Illumina) with 251 reading cycles. A total of 460,796,326 nucleotides were sequenced and 2,061,344 paired-end raw reads were produced with overall coverage of 312 ×. CLC Genomics Workbench v8.5.1 (QIAGEN Inc.) was employed to trim the index primers and a total of 2,059,260 filtered high quality reads were yielded with the median PHRED score greater than 30, showing the overall accuracy of base

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Genomics Data  Fig. 1, the genome of FNW20G12 strain was annotated with a total of 312 subsystems, 1902 coding sequences (CDSs), and 48 RNAs with Rapid Annotation using Subsystem Technology (RAST) [6][7][8]. RAST also identified C. coli RM2228 as the closest neighbor of FNW20G12. Among the CDSs, there were conserved genes in metabolism and biosynthesis subsystems, as well as those involved in adhesion, virulence, and response to oxidation stress, such as cadF, flaC, and sodABC. With RAST and ResFinder-2.1 [9], genes responsible for multidrug resistance, aph(3′)-III, blaOXA-61, tetO and gyrAB were identified in FNW20G12 with predicted resistance to aminoglycoside, beta-lactams, tetracycline and fluoroquinolones, respectively. Additionally, we found genes encoding efflux pumps and transporters (RND, MFS, DMT and MATE) which are associated with increased multidrug resistance. There was no plasmid replicon region identified in FNW20G12 genome using PlasmidFinder [10].

Accession number of nucleotide sequence
The draft genome assembled form Whole Genome Shotgun Sequencing of C. coli FNW20G12 strain has been deposited at DDBJ/ENA/ GenBank under the accession LWIH00000000 with the version LWIH01000000.

Conflict of interest
The authors declare that no conflict of interest exists about the work published in this paper.

Disclaimer
The views presented in this work do not necessarily reflect those of the Food and Drug Administration, nor do the authors specifically endorse the listed instrumentation or products.