Draft Genome Sequences of Four Citrobacter Isolates Recovered from Wild Australian Shorebirds

Citrobacter is a ubiquitous bacterial genus whose members inhabit a variety of niches. Some species are clinically important for both antimicrobial resistance (AMR) carriage and as the cause of nosocomial infections. Surveillance of Citrobacter species in the environment can provide indicators of the spread of AMR genes outside clinical spaces. In this study, we present draft genome sequences of four Citrobacter isolates obtained from three species of wild Australian shorebirds.

T he genus Citrobacter comprises 11 species. They occupy a broad range of habitats, play a key role in the nitrogen cycle, and are frequently found in food and in the gut of animals, including humans (1). Citrobacter is an opportunistic pathogen of humans, most commonly associated with infant meningitis, urinary tract infections, sepsis, and pneumonia (2). The species most commonly isolated from clinical specimens are C. koseri, C. freundii, C. youngae, C. braakii, and C. amalonaticus (3). Citrobacter species can act as reservoirs for antimicrobial resistance (AMR) genes and can transfer these genes to other pathogenic bacteria (4,5). Citrobacter spp. have been isolated from both healthy (6) and diseased birds (7).
We present here draft genomes of four Citrobacter isolates collected from Australian shorebirds through 2017 to 2018.
Four Citrobacter isolates were recovered from cloacal swabs collected from healthy Australian shorebirds (Table 1). The cloacal swabs were preenriched by incubating overnight in brain heart infusion broth at 35°C, followed by a secondary enrichment by transferring 100 ml into Mannitol broth and again incubating overnight at 35°C. The broths were subsequently subcultured onto MacConkey II agar (Oxoid) and incubated overnight at 35°C.
Phenotypic testing of antimicrobial resistance was conducted using the disk diffusion method (8). For DNA extraction, organisms were grown overnight on nutrient agar. Genomic DNA (gDNA) was extracted using a Qiagen DNeasy kit and quantified using an Invitrogen Qubit 2 fluorometer. Sequencing was conducted at the Australian Genome Research Facility using the Illumina MiSeq platform, with Illumina gDNA shotgun library preparation with the bead size selection protocol generating 150-bp pairedend reads.
The raw reads were uploaded to the Galaxy Web platform, and the data were analyzed via the public server at usegalaxy.org, version 20.01 (9). The genomes were assembled de novo, read quality control was performed using Unicycler version 0.4.8.0 (10), and the genome assembly quality was analyzed using QUAST version 5.0.21galaxy1 (11). The genomes were uploaded to NCBI and annotated using PGAP version 4.12 (12). Further information on the genome parameters is given in Table 1.
ResFinder version 3.2 (13), hosted by the Centre for Genomic Epidemiology (http:// www.genomicepidemiology.org/), was used to identify AMR genes. The AMR gene identified can be seen in Table 1. This gene is thought to have originated in C. freundii (14).
Data availability. The whole-genome sequences, assemblies, and raw reads for this project have been deposited in GenBank under the BioProject accession number PRJNA602163. Raw reads are available in the SRA under the accession numbers SRR11613026 (C. braakii), SRR11613020 (C. freundii), SRR11613006 (C. freundii), and SRR11612996 (C. amalonaticus).

ACKNOWLEDGMENTS
Funding for this work was provided by the Holsworth Wildlife Research Endowment (grant HLS-18-005) and the Stuart Leslie Bird Research Award provided by Birdlife Australia (grant FOST-18-416). We thank GFN's major funders, the Netherlands Organisation Spinoza Premium Prize for Scientific Research to Theunis Piersma (2014 to 2017), WWF Netherlands (2010 to 2018), and MAVA, Fondation pour la Nature (2018).
We thank the Australasian Wader Study Group (AWSG), Rosalind Jessop, and the late Clive Minton for their help with this project. We acknowledge the Yawuru People, via the offices of Nyamba Buru Yawuru Limited, for permissions granted to AWSG to capture birds on the shores of Roebuck Bay, the traditional lands of the Yawuru people.