Hybrid assembled genomes of three Salmonella enterica isolates from municipal wastewater in Tennessee, USA

TL;DR

This study presents hybrid genome assemblies of three Salmonella strains isolated from wastewater in Tennessee, identifying their serotypes.

Contribution

The paper provides new hybrid genome assemblies and serotype identification for Salmonella isolates from municipal wastewater.

Findings

Three Salmonella enterica isolates were obtained from a wastewater treatment plant in Knoxville, Tennessee.

Hybrid assemblies revealed two isolates as serotype Bareilly and one as serotype Typhimurium.

Abstract

Three Salmonella enterica isolates were isolated from a municipal wastewater treatment plant in Knoxville, TN, USA. Genomic DNA was extracted and sequenced on the Illumina and Oxford Nanopore platforms. Annotated hybrid assemblies showed the strains were predicted to be serotypes Bareilly (n = 2) and Typhimurium (n = 1).

Tables

TABLE 1. Genomic sequence statistics and features of three S. enterica isolates from a local wastewater treatment plant

Strain	No. of Illumina reads	No. of ONT reads	N50 of ONT reads	Total length (bp)a	No. of chromosome contigsa	No. of plasmid contigsa	GC content (%)a	Predicted serotypeb	Illumina read coverage (×)c	Nanopore read coverage (×)c	No. of CDSsd	No. of tRNAsd	No. of CRISPR arrayse	Antibiotic resistance genesf
UTK W1-0001	2,419,110	5,000	19,045	4,964,787	1	1	52.17	Typhimurium	66	9	4,739	86	4	aac(6')-Iaa, aph (6)-Id, blaCMY-2, sul2, tet(A), floR
UTK W1-0003	3,066,142	8,000	17,340	4,843,513	1	1	52.05	Bareilly	90	14	4,648	85	3	aac(6')-Iaa
UTK W1-0011	2,943,720	7,000	17,533	5,032,162	1	2	52.14	Bareilly	83	12	4,871	86	2	aac(6')-Iaa

Full text

ANNOUNCEMENT

Salmonella enterica is a globally widespread bacterial pathogen, causing an estimated 100 million infections and over 200,000 deaths annually (1, 2). Reporting novel S. enterica strains from municipal wastewater contributes genomic resources for comparative analyses and genomic surveillance. This study presents the complete and draft genome sequences of three S. enterica strains (UTK W1-0001, UTK W1-0003, and UTK W1-0011) isolated from municipal wastewater samples collected at a local wastewater treatment plant in Knoxville, Tennessee, USA.

Samples were collected from the primary effluent of municipal wastewater on 9 October 2023. The collected wastewater samples were filtered and enriched for Salmonella spp. following the modified Food and Drug Administration’s Bacteriological Analytical Manual protocol (3, 4). Single colonies were isolated on selective media, and pure cultures were prepared. The presumptive Salmonella isolates were cultured overnight in brain heart infusion broth at 37°C with shaking at 200 rpm. DNA was extracted by the DNeasy Blood and Tissue Kit (Qiagen), assessed with Nanodrop (Thermo Fisher Scientific) and Qubit 4 (Thermo Fisher Scientific), and shipped to SeqCenter (Pittsburgh, PA, USA). Illumina short-read sequencing was performed using the Illumina DNA Prep kit and NovaSeq X Plus sequencer (Illumina), producing 151 bp paired-end reads (5). For Nanopore sequencing, DNA was extracted using the Zymo Quick-DNA HMW MagBead kit (Zymo Research Corp.). The library was prepared using the Oxford Nanopore rapid barcoding kit (SQK-RBK114.24) and sequenced with a MinION sequencer with Flongle (Oxford Nanopore Technologies). Basecalling was performed by MinKNOW (v24.06.5) using the fast basecalling model with adapter trimming, default quality score threshold, and no size-selection (6). Read quality was checked by FastQC v0.11.9 (7), and trimming was performed by Trimmomatic v0.39 (8). The hybrid assembly was performed using Unicycler v0.4.8 in bold mode, including overlap identification and trimming, with rotation to start at dnaA/repA (9). For all software, default parameters were used except where otherwise noted.

BLAST analysis (10) of the 7 kb contig in UTK W1-0011 assembly revealed 96.7% nucleotide identity across 81% of the query sequence length to plasmid NZ_CP128883.1. kSNP4 (v4.1) was used to identify core genome single-nucleotide polymorphism (SNP) among these three Salmonella isolates (11), revealing each was a distinct strain. Genome sequencing data and annotation features are summarized in Table 1. The genomes of strains UTK W1-0001, UTK W1-0003, and UTK W1-0011 were predicted to have 151-152 virulence factor genes. Key virulence determinants included fimbrial and nonfimbrial adherence determinants, macrophage inducible gene mig-14, and type III secretion system (T3SS) encoded by Salmonella pathogenicity island 1 (SPI-1) and 2 (SPI-2). Compared with previously isolated Salmonella enterica strains harboring 100-130 virulence genes identified by VFDB (12–15), the three newly isolated Salmonella enterica strains exhibit a notable virulence profile potentially indicative of aggressive pathogenic characteristics.