Genome analysis of Salmonella enterica subsp. diarizonae isolates from invasive human infections reveals enrichment of virulence-related functions in lineage ST1256

Abstract

Background: Salmonella enterica subsp. diarizonae (IIIb) is frequently isolated from the environment, cold-blooded reptiles, sheep and humans; however only a few studies describe the isolation of this subspecies from invasive human infections. The factors contributing to this unusual behavior are currently unknown.

Results: We report here the genome features of two diarizonae strains, SBO13 and SBO27, isolated from endocervical tissue collected post-abortion and from cerebrospinal fluid of a newborn child, respectively, in the city of Santa Cruz, Bolivia. Although isolated six years apart, SBO27 in 2008 and SBO13 in 2014, both strains belong to the same sequence type 1256 (ST1256) and show a high degree of genome conservation sharing more than 99% of their genes, including the conservation of a ~ 10 kb plasmid. A prominent feature of the two genomes is the presence of 24 genomic islands (GIs), in addition to 10 complete Salmonella pathogenicity islands (SPI) and fragments of SPI-7, a pathogenicity island first reported in the human-adapted serovar Typhi. Some of the GIs identified in SBO13 and SBO27 harbor genes putatively encoding auto-transporters involved in adhesion, lipopolysaccharide modifying enzymes, putative toxins, pili-related proteins, efflux pumps, and several putative membrane cation transport related-genes, among others. These two Bolivian isolates also share genes encoding the type-III secretion system effector proteins SseK2, SseK3 and SlrP with other diarizonae sequence types (ST) mainly-associated with infections in humans. The sseK2, sseK3 and slrP genes were either absent or showing frameshift mutations in a significant proportion of genomes from environmental diarizonae isolates.

Conclusions: The comparative genomic study of two diarizonae strains isolated in Bolivia from human patients uncovered the presence of many genes putatively related to virulence. The statistically-significant acquisition of a unique combination of these functions by diarizonae strains isolated from humans may have impacted the ability of these isolates to successfully infect the human host.

Keywords: Comparative genomics; Invasive human infections; Salmonella enterica; Subspecies diarizonae; Type-III effectors; Virulence genes.

Fig. 1

Maximum likelihood pan-genome phylogeny of the genus Salmonella. The tree was estimated from the presence-absence matrix of 18,446 consensus homologous gene clusters found among 109 Salmonella spp. genomes with GET_HOMOLOGUES. Seventy-six of them correspond to a selection of S. enterica subsp. diarizonae sequences downloaded from ENTEROBASE, thirty-one to reference genomes from other species and serovars, and the remaining two to the Bolivian strains, SBO13 and SBO27. The matrix contains 4177 distinct patterns, 9276 parsimony-informative, 6963 singleton and 2207 constant sites. Nodal support values are color-coded as shown on the legend, the first value corresponding to approximate Bayesian support values and the second one to ultra-fast bootstrap values, as implemented in IQTREE. The scale represents the number of expected substitutions per site under the best-fitting GTR2 + F0 + R2 (binary) model. The phylogeny corresponds to the top-scoring tree (lnL = − 197,100.501) found among 10 independent IQTREE searches. It was rooted at the S. bongori clade. Labels for the 76 S. enterica subsp. diarizonae genomes fetched from ENTEROBASE indicate relevant ecological metadata and the sequence type (ST). Genomes in bold were selected for further analysis

Fig. 2

Genome comparison of S. enterica subsp. diarizonae SBO13 to nine genomes from representative Salmonella species, subspecies and serovars. (A) The innermost rings show diarizonae SBO13 genome position (kbp = kilo base pairs); G + C content (black). The remaining rings (from ring 3 to ring 12) correspond to the genomes of diarizonae strains SBO27, HZS154, MZ0080, SA20044251, 11–01855, 11–01854 and 11–01853; S. bongori NCTC-12419; Typhi CT18, and Typhimurium SL1344. BLASTN matches with an identity between 50 and 100% are colored in gradient, while nonmatching regions appeared as blank spaces in each ring. The outer ring contains genomic islands (GIs) predicted for diarizonae SBO13 with Islandviewer4 (http://www.pathogenomics.sfu.ca/islandviewer/). (B) Detail of a GI-6 fragment of the diarizonae SBO13 genome showing unique genes and genes shared exclusively with diarizonae HZS154, a human isolate reported in China. Highlighted with their corresponding gene numbers are the genes of this GI-6 cited in the text

Fig. 3

Heatmap of the SPI1-SPI5 regions in the S. enterica subsp. diarizonae genomes. TBLASTN searches, using the protein sequences from serovar Typhimurium SL1344 strain as query, were done to identify orthologous genes in S. bongori NCTC-12419, serovar Typhi CT-18, and 15 representative diarizonae genomes. Orthologous genes with 85% sequence coverage were considered present, and marked in blue. The color intensity represents the sequence identity from 50 to 100% (complete protein). Orthologous genes that did not fulfil the criteria outlined above, were considered absent, and marked in white. Phage remnants regions, SBO13 genomic islands (GI) and Salmonella pathogenic islands (SPI) are indicated in cyan, purple and dark blue, respectively

Fig. 4

Clustering map of the SPI-7 region in representative S. enterica subsp. diarizonae genomes. TBLASTN searches, using the protein sequences from serovar Typhi strain CT-18 as query, were done to identify orthologous genes in S. bongori NCTC-12419, serovar Typhimurium SL1344 and 15 representative diarizonae genomes. Orthologous genes with 85% sequence coverage and 40% sequence identity were considered present and marked in blue. Orthologous genes that did not fulfil the criteria outlined above, were considered absent, and marked in white. The SPI-7 island of Typhi CT-18 strain and its different regions are depicted below of the clustering map

Fig. 5

Distribution of virulence-related functions in representative S. enterica subsp. diarizonae strains, including SBO13 and SBO27. a TBLASTN searches using as queries all the virulence factors available from VFDB (http://www.mgc.ac.cn/VFs/) excluding SPI1, SPI2 and those presents in plasmids. Proteins with 70% sequence identity were considered present and marked in blue. The color intensity represents the sequence coverage from 50 to 100% (complete protein). Proteins that did not fulfil the criteria outlined above, were considered absent, and marked in white. b Schematic representation of SlrP sequences found are represented as purple bars on the right panel. The protein domains of SlrP are represented as coloured boxes: type III secretion system leucine rich repeat region (TTSSLRR; D1 in red), Leucine rich repeat region (LRR; D2 in green) and C-terminal novel E3 ligase domain (NEL; D3 in yellow)