We constructed two collections of targeted single gene deletion (SGD) mutants and two collections of targeted multi-gene deletion (MGD) mutants in Salmonella enterica sv Typhimurium 14028s. The SGD mutant collections contain (1), 3517 mutants in which a single gene is replaced by a cassette containing a kanamycin resistance (Kan^R) gene oriented in the sense direction (SGD-K), and (2), 3376 mutants with a chloramphenicol resistance gene (Cam^R) oriented in the antisense direction (SGD-C). A combined total of 3773 individual genes were deleted across these SGD collections. The MGD collections contain mutants bearing deletions of contiguous regions of three or more genes and include (3), 198 mutants spanning 2543 genes replaced by a Kan^R cassette (MGD-K), and (4), 251 mutants spanning 2799 genes replaced by a Cam^R cassette (MGD-C). Overall, 3476 genes were deleted in at least one MGD collection. The collections with different antibiotic markers permit construction of all viable combinations of mutants in the same background. Together, the libraries allow hierarchical screening of MGDs for different phenotypic followed by screening of SGDs within the target MGD regions. The mutants of these collections are stored at BEI Resources (www.beiresources.org) and publicly available.

Bloodstream infections caused by nontyphoidal Salmonella are a major public health concern in Africa, causing ~49,600 deaths every year. The most common Salmonella enterica pathovariant associated with invasive nontyphoidal Salmonella disease is Salmonella Typhimurium sequence type (ST)313. It has been proposed that antimicrobial resistance and genome degradation has contributed to the success of ST313 lineages in Africa, but the evolutionary trajectory of such changes was unclear. Here, to define the evolutionary dynamics of ST313, we sub-sampled from two comprehensive collections of Salmonella isolates from African patients with bloodstream infections, spanning 1966 to 2018. The resulting 680 genome sequences led to the discovery of a pan-susceptible ST313 lineage (ST313 L3), which emerged in Malawi in 2016 and is closely related to ST313 variants that cause gastrointestinal disease in the United Kingdom and Brazil. Genomic analysis revealed degradation events in important virulence genes in ST313 L3, which had not occurred in other ST313 lineages. Despite arising only recently in the clinic, ST313 L3 is a phylogenetic intermediate between ST313 L1 and L2, with a characteristic accessory genome. Our in-depth genotypic and phenotypic characterization identifies the crucial loss-of-function genetic events that occurred during the stepwise evolution of invasive S. Typhimurium across Africa.

Salmonella entericaserovar Typhi, the cause of typhoid fever, is host-adapted to humans and unable to cause disease in mice. Here, we show thatS.Typhi can replicate in vivo in nonobese diabetic (NOD)-scid IL2rγ^nullmice engrafted with human hematopoietic stem cells (hu-SRC-SCID mice) to cause a lethal infection with pathological and inflammatory cytokine responses resembling human typhoid. In contrast,S.Typhi does not exhibit net replication or cause illness in nonengrafted or immunocompetent control animals. Screening of transposon pools in hu-SRC-SCID mice revealed both known and previously unknownSalmonellavirulence determinants, includingSalmonellaPathogenicity Islands 1, 2, 3, 4, and 6. Our observations indicate that the presence of human immune cells allows the in vivo replication ofS.Typhi in mice. The hu-SRC-SCID mouse provides an unprecedented opportunity to gain insights intoS.Typhi pathogenesis and devise strategies for the prevention of typhoid fever.

Salmonella Typhimurium sequence type (ST) 313 causes invasive nontyphoidal Salmonella (iNTS) disease in sub-Saharan Africa, targeting susceptible HIV⁺, malarial, or malnourished individuals. An in-depth genomic comparison between the ST313 isolate D23580 and the well-characterized ST19 isolate 4/74 that causes gastroenteritis across the globe revealed extensive synteny. To understand how the 856 nucleotide variations generated phenotypic differences, we devised a large-scale experimental approach that involved the global gene expression analysis of strains D23580 and 4/74 grown in 16 infection-relevant growth conditions. Comparison of transcriptional patterns identified virulence and metabolic genes that were differentially expressed between D23580 versus 4/74, many of which were validated by proteomics. We also uncovered the S. Typhimurium D23580 and 4/74 genes that showed expression differences during infection of murine macrophages. Our comparative transcriptomic data are presented in a new enhanced version of the Salmonella expression compendium, SalComD23580: http://bioinf.gen.tcd.ie/cgi-bin/salcom_v2.pl. We discovered that the ablation of melibiose utilization was caused by three independent SNP mutations in D23580 that are shared across ST313 lineage 2, suggesting that the ability to catabolize this carbon source has been negatively selected during ST313 evolution. The data revealed a novel, to our knowledge, plasmid maintenance system involving a plasmid-encoded CysS cysteinyl-tRNA synthetase, highlighting the power of large-scale comparative multicondition analyses to pinpoint key phenotypic differences between bacterial pathovariants. Invasive nontyphoidal Salmonella (iNTS) is associated with a major and largely unreported tropical disease that is responsible for hundreds of thousands of deaths per year in Africa. The main causative agent is a pathovariant of Salmonella Typhimurium called ST313, which is closely related to the well-characterized ST19 sequence type of Salmonella that causes gastroenteritis globally. ST313 and ST19 vary by just 856 core genome single-nucleotide polymorphisms (SNPs). To understand how genetic changes generate phenotypic and mechanistic differences between African and global Salmonella, we used functional transcriptomic and proteomic approaches. By investigating the transcriptome of African and global S. Typhimurium in 17 growth conditions, we discovered that 677 genes and small RNAs were differentially expressed between strains D23580 (ST313) and 4/74 (ST19). A parallel proteomic approach linked gene expression differences to alterations at the protein level. We also identified differentially expressed genes during the actual infection of murine macrophages. Our data revealed the genetic basis of the loss of a carbon source utilization in African Salmonella and the discovery of a new mechanism for maintaining a plasmid in a bacterial population involving a plasmid-encoded essential bacterial gene.

Significance: Invasive nontyphoidal Salmonella disease is a major and previously neglected tropical disease responsible for an estimated ∼390,000 deaths per year in Africa, largely caused by a variant of Salmonella Typhimurium called ST313. Despite the availability of >100,000 Salmonella genomes, it has proven challenging to associate individual SNPs with pathogenic traits of this dangerous bacterium. Here, we used a transcriptomic strategy to identify a single-nucleotide change in a promoter region responsible for crucial phenotypic differences of African S. Typhimurium. Our findings show that a noncoding nucleotide of the bacterial genome can have a profound effect upon the pathogenesis of infectious disease.