key: cord-1050676-mcttgxde
authors: Gomez-Alvarez, Vicente; Boczek, Laura; Raffenberg, Ian; Revetta, Randy P.
title: Closed Genome and Plasmid Sequences of Legionella pneumophila AW-13-4, Isolated from a Hot Water Loop System of a Large Occupational Building
date: 2021-01-07
journal: Microbiol Resour Announc
DOI: 10.1128/mra.01276-20
sha: 2b405b28606e4b2e7d102e029f57af3ccebe48f2
doc_id: 1050676
cord_uid: mcttgxde

Unused water in unoccupied buildings can become stagnant, with reductions in temperature and levels of disinfectant resulting in increased microbial growth. We report the closed and complete genome and plasmid of Legionella pneumophila strain AW-13-4 (serogroup 1), which was isolated from a hot water loop system of a large building.

I n March 2020, state and local government officials in the United States issued shelter-in-place recommendations ("social distancing") and ordered the closing of highoccupancy venues (e.g., nonessential buildings) to stop the global pandemic caused by novel coronavirus (severe acute respiratory syndrome coronavirus 2 [SARS-CoV-2]) disease 2019 (COVID-19) (1-3). During this time, unoccupied and low-occupancy buildings might have experienced extended periods of low water demand without proper water management plans (i.e., mitigation) (4). As conditions allow, the workforce will steadily return to occupational buildings with their safety being a key priority for facility managers. It is important to understand the impact of the lockdown and mitigation efforts in the building water distribution system. Here, we report the assembled genome and plasmid of Legionella pneumophila strain AW-13-4, a Gram-negative bacterium and major causative agent of Legionnaires' disease (5, 6) .

Strain AW-13-4 was isolated in April 2019 from drinking water obtained from a hot water recirculating loop pipe in a large occupational building. At the time of publication, monthly sampling events confirmed the presence of L. pneumophila in the drinking water system. A 100-ml water sample was concentrated by membrane filtration (0.2 mm), and the subsequent selective recovery of L. pneumophila was performed as described by Gomez-Alvarez et al. (7) . Briefly, the concentrate was resuspended in 5 ml of Butterfield's phosphate buffer, and 100 ml was cultured on buffered CYE selective agar (catalogue number R110107; Remel, Lenexa, KS) for 5 days at 35°C. The agar is used for the selective recovery of L. pneumophila from potable water samples and contains vancomycin and anisomycin to suppress contaminating flora. A single colony was transferred to BCYE agar (catalogue number R01334; Remel) and incubated for 2 days at 35°C. The isolate was further characterized as serogroup 1 by latex agglutination (Oxoid Ltd., Basingstoke, UK). High-quality DNA was extracted using the UltraClean DNA microbial isolation kit (MO BIO Laboratories, Solana Beach, CA) following the manufacturer's instructions. A genomic library was prepared using the Nextera XT index kit (Illumina, Inc., San Diego, CA) and sequenced on the Illumina HiSeq 4000 platform (paired-end 150-nucleotide [nt] reads). Read processing was performed as described previously (7); short-read libraries were cleaned of adapters and phiX artifacts, error corrected, normalized (#100Â), and filtered to a minimum length of 100 nt (BBMap v38.67; ktrim=r k=23 mink=11 hdist=1 tbo tpe maxns=0 trimq=10 qtrim=r maq=12 minlength=100 ecco=t eccc=t ecct=t target=100). A long-read library was prepared Editor Julia A. Maresca, University of Delaware This is a work of the U.S. Government and is not subject to copyright protection in the United States. Foreign copyrights may apply.

using the ligation sequencing kit (1D SQK-LSK109) and the native barcoding kit (EXP-NBD104) on a MinION device (Oxford Nanopore Technologies [ONT], Inc., Oxford, UK). Fast5 reads were base called using Guppy v3.1.5 (ONT), trimmed with Porechop v0.2.3 (8) using a 90% identity threshold, and then filtered to a minimum length of 25,000 nt. Short-and long-read sequencing produced 45,606,672 reads (average length, 132 nt [range, 100 to 150 nt]; coverage, 1,599Â) and 10,313 reads (average length, 30,056 nt [range, 25,001 to 68,983 nt]; coverage, 142,495Â), respectively. A hybrid assembly approach was used to assemble the processed reads using Unicycler v0.4.8 (9) . Default parameters were used for all software unless otherwise specified.

The final assembly contains a single chromosome spanning the complete 3.512-Mbp length of the genome (GC content of 38%) and a single 1.299-kbp plasmid (Fig. 1 ). Gene annotation with Prokka v1.14.6 (10) identified 3,100 coding sequences, 9 rRNAs, and 43 tRNAs. The average nucleotide identity (ANI) was calculated using FastANI v1.31 (11) , which indicated 99.99% ANI with L. pneumophila strain OLDA (12) . The in silico multilocus sequence typing (MLST) sequence type (13, 14) was determined using MentaLiST v0.2.3 (15) , and the presence of antimicrobial resistance (AMR) genes was determined with the RGI CARD Web-based tool (16) . AW-13-4 was identified as sequence type 1, and gene annotation confirmed the presence of lpeAB genes (Fig. 1) . Previous studies demonstrated the involvement of LpeAB in an efflux pump responsible for reduced sensitivity to azithromycin in L. pneumophila strains (17, 18) . The plasmid contained a class D b-lactamase (bla OXA-29 ) (19) . The VRprofile server (20) detected the presence of integrative and conjugative elements (ICEs) (21) (Fig. 1) , which are often associated with transfer/conjugative elements such as the type IV secretion system (T4SS) (22) .

Data availability. This whole-genome shotgun project has been deposited in DDBJ/ENA/GenBank under BioProject number PRJNA487286 with the accession numbers CP061840 (chromosome) and CP061841 (plasmid). The raw sequence reads have (23) . The rings, from outside to inside, denote AMR genes (ARG), secretion systems (T4SS), and rRNA coding genes on both the forward and reverse strands (rings 1 and 4), protein coding genes on both the forward and reverse strands (rings 2 and 3), and GC content (ring 5). Blue regions represent in silico identification of ICEs or mobile genetic elements (21) using the VRprofile server (20) . been submitted to the NCBI SRA under the accession numbers SRR13076822 and SRR13076823. The versions described in this paper are the first versions.

Thinking globally, acting locally: the U.S. response to COVID-19

The effect of large-scale anti-contagion policies on the COVID-19 pandemic

Public health response to the initiation and spread of pandemic COVID-19 in the United States

Considerations for large building water quality after extended stagnation

Epidemiology and clinical management of Legionnaires' disease

Legionnaires' disease

Draft genome sequences of seven Legionella pneumophila isolates from a hot water system of a large building

Completing bacterial genome assemblies with multiplex MinION sequencing

Unicycler: resolving bacterial genome assemblies from short and long sequencing read

Prokka: rapid prokaryotic genome annotation

High throughput ANI analysis of 90K prokaryotic genomes reveals clear species boundaries

Complete genome sequences of the historical Legionella pneumophila strains OLDA and Pontiac

Consensus sequence-based scheme for epidemiological typing of clinical and environmental isolates of Legionella pneumophila

Addition of neuA, the gene encoding N-acylneuraminate cytidylyl transferase, increases the discriminatory ability of the consensus sequence-based scheme for typing Legionella pneumophila serogroup 1 strains

MentaLiST: a fast MLST caller for large MLST schemes

CARD 2020: antibiotic resistome surveillance with the Comprehensive Antibiotic Resistance Database

Minimum inhibitory concentration (MIC) distribution among wild-type strains of Legionella pneumophila identifies a subpopulation with reduced susceptibility to macrolides owing to efflux pump genes

Macrolide resistance in Legionella pneumophila: the role of LpeAB efflux pump

Characterization of OXA-29 from Legionella (Fluoribacter) gormanii: molecular class D b-lactamase with unusual properties

VRprofile: gene-clusterdetection-based profiling of virulence and antibiotic resistance traits encoded within genome sequences of pathogenic bacteria

Integrative and conjugative elements (ICEs): what they do and how they work

More than 18,000 effectors in the Legionella genus genome provide multiple, independent combinations for replication in human cells

The CGView server: a comparative genomics tool for circular genomes

We thank Dawn King, Adin Pemberton, Stacy Pfaller, Mark Rodgers, and Jorge Santo Domingo for valuable assistance with this project. Special thanks go to Chris Bennett. This research was supported by EPA Office of Research and Development Safe and Sustainable Water Resources Program 7.2.1.The opinions expressed are those of the authors and do not necessarily reflect the official positions and policies of the U.S. EPA. Any mention of product or trade names does not constitute recommendation for use by the U.S. EPA.