Draft genome sequences of two β-glucuronidase positive strains of Salmonella enterica subspecies salamae isolated from reptile feces in KwaZulu-Natal, South Africa

TL;DR

This study reports the genome sequences of two Salmonella strains from reptile feces in South Africa that show β-glucuronidase activity.

Contribution

The novelty lies in the genome sequencing and identification of two β-glucuronidase positive Salmonella salamae strains from reptiles.

Findings

Two Salmonella isolates from reptile feces showed β-glucuronidase activity.

Genome sequencing identified them as Salmonella enterica subspecies salamae with sequence type 1208 and serotype 42:r:-.

Abstract

Two Salmonella enterica isolates obtained from reptile feces displayed β-glucuronidase activity. Nearly complete genome sequences were obtained after shotgun sequencing and de novo genome assembly. By comparison to reference genomes, both isolates were identified as Salmonella enterica subspecies salamae with the sequence type identified as 1208 and the serotype as 42:r:-.

Figures

Midpoint-rooted phylogenetic tree inferred with FastME (2.1.6.1) from GBDP distances calculated from type strain genome sequences using the TYGS server. Branch lengths are scaled based on the GBDP distance formula d5; numbers shown are GBDP pseudo-bootstrap support values > 60 % (100 replications, average branch support = 63%).

Tables

TABLE 1. Genomic characteristics of two β-glucuronidase positive Salmonella enterica subsp. salamae strains isolated from reptile feces in South Africa

Strain	A199	G77
Origin, year	Agama feces, 2019	Gecko feces, 2021
Location	Pietermaritzburg, South Africa (29°36'S 30°23'E)	Pietermaritzburg, South Africa (29°36'S 30°23'E)
Identity	Salmonella enterica subsp. salamae	Salmonella enterica subsp. salamae
Digital DNA:DNA hybridization (dDDH, d4) best type strain genome match	Salmonella enterica subsp. salamae NCTC 5773T (83.2%)	Salmonella enterica subsp. salamae NCTC 5773T (83.2%)
Average nucleotide identity (ANIb) best type strain genome match	Salmonella enterica subsp. salamae CCUG 30039T (97.73%)	Salmonella enterica subsp. salamae CCUG 30039T (97.66%)
Predicted serotype	42:r:-	42:r:-
MLST sequence type	1208	1208
Total genome size	4,733,636 bp	4,733,809 bp
Completeness (BUSCO)	97.97%	97.97%
Number of reads (input)	10,679,960	7,179,091
Average read length	140	137
Number of contigs	59	59
N50	226,585	226,585
Largest contig	417,545	417,720
GC content	51.96%	51.96%
Protein coding sequences (CDS)	4,715	4,716
tRNA	73	73
rRNA	3	3
Predicted resistance genes	aac(6′)-Iaa	aac(6′)-Iaa
Salmonella pathogenicity islands	SESS-LEE, SPI-1, SPI-2, SPI-3, SPI-9	SESS-LEE, SPI-1, SPI-2, SPI-3, SPI-9
Human pathogen probability	0.926	0.926
Number of predicted intact prophages	1	1
Plasmids	Not detected	Not detected

Full text

ANNOUNCEMENT

β-Glucuronidase activity is typically present in Salmonella enterica subspecies diarizonae but can also occur in the subspecies enterica, salamae, and indica (1). Although human salmonellosis’ is mostly caused by Salmonella enterica subspecies enterica, infections by other non-enterica subspecies do occur but are usually rare (2–6). Salmonella enterica subspecies salamae, like all non-enterica subspecies, are typically associated with cold-blooded animals such as reptiles but can also be found in warm-blooded animals (3, 6–9).

The two Salmonella isolates, designated A199 and G77, were originally obtained from agama (2019) and gecko feces (2021). Fecal reptile pellets collected in a suburban garden in Pietermaritzburg, South Africa, were decimally diluted in sterile saline (0.85%). Subsamples of agama fecal dilutions (100 µL) were directly spread-plated onto XLD-Agar (24 h, 37°C). A portion of the pooled gecko feces dilution was pre-enriched in buffered peptone water (24 h, 37°C), followed by selective enrichment in Rappaport-Vassiliadis broth (41.5°C, 24 h) and spread-plating on XLD-Agar (37°C, 24 h). A purified (Nutrient agar) single colony was sub-cultivated in Nutrient broth to prepare glycerol stock cultures for storage at −20°C. Both isolates were confirmed using TSI- and Urea-Agar and by PCR-based detection of the invA gene (10). Sanger sequencing (CAF, Stellenbosch) of the 16S rRNA gene amplicon (primers fD1 + rP2) (11) further confirmed that both isolates belonged to the species Salmonella enterica (accession no. ON630378.1, ON630388.1). Notably, both isolates showed untypical colony morphology on improved Salmonella-Chromo-Select-Agar due to their β-glucuronidase activity, which was confirmed using TBX agar.

Washed cells of pure overnight cultures of A199 and G77 were transferred into DNA/RNA Shield Lysis Tubes (ZYMO). Extracted DNA (ZymoBIOMICS-96 MagBead kit) was used for shotgun genome sequencing (paired-end 2 × 150 bp, Illumina Nova Seq) by Zymo Research (Irvine, CA, USA) after creating a library (Nextera DNA Flex Library Prep Kit). Using the BV-BRC genome analysis pipeline (12), raw reads were quality filtered, trimmed (Trim Galore v0.6.5, Cutadapt v2.2), assembled (Unicycler v0.4.8, minimum contig size 500 bp), and annotated (RASTtk) (13–16). Default parameters were used for all bioinformatic tools unless otherwise stated. Resistance genes were detected using Resfinder (v.4.1) (17), prophages using Phaster (18), plasmids using Plasmidfinder (v2.1, https://cge.cbs.dtu.dk), and human pathogenicity was predicted using Pathogenfinder (v1.1) (19).

The results for the main genomic features of both isolates are summarized in Table 1. The genome assembly was estimated to be 97.97% complete using the universal single-copy ortholog benchmarking tool available at http://cab.cc.spbu.ru (20). Using ANIb (21) and the phylogenetic analysis tools available in TYGS (22) demonstrated that the two isolates (G77, A199) belonged to Salmonella enterica subspecies salamae (Fig. 1). Multilocus sequence typing (MLST v2.0) (23) identified both isolates as sequence type 1208, and the serotype was predicted as 42:r:- (SeqSero2) (24).

Midpoint-rooted phylogenetic tree inferred with FastME (2.1.6.1) from GBDP distances calculated from type strain genome sequences using the TYGS server. Branch lengths are scaled based on the GBDP distance formula d5; numbers shown are GBDP pseudo-bootstrap support values > 60 % (100 replications, average branch support = 63%).

Our results show that Salmonella enterica subspecies salamae strains of the same serotype (42:r:-) are prevalent in the feces of geckos and agama lizards in South Africa. As both Salmonella isolates showed a high probability of human pathogenicity, feces shed by these reptiles might be a source of Salmonella contamination and a potential health risk, especially for the most vulnerable YOPI (young-old-pregnant-immunocompromised) citizens.