Loss of Multicellular Behavior in Epidemic African Nontyphoidal Salmonella enterica Serovar Typhimurium ST313 Strain D23580

Abstract

Nontyphoidal Salmonella enterica serovar Typhimurium is a frequent cause of bloodstream infections in children and HIV-infected adults in sub-Saharan Africa. Most isolates from African patients with bacteremia belong to a single sequence type, ST313, which is genetically distinct from gastroenteritis-associated ST19 strains, such as 14028s and SL1344. Some studies suggest that the rapid spread of ST313 across sub-Saharan Africa has been facilitated by anthroponotic (person-to-person) transmission, eliminating the need for Salmonella survival outside the host. While these studies have not ruled out zoonotic or other means of transmission, the anthroponotic hypothesis is supported by evidence of extensive genomic decay, a hallmark of host adaptation, in the sequenced ST313 strain D23580. We have identified and demonstrated 2 loss-of-function mutations in D23580, not present in the ST19 strain 14028s, that impair multicellular stress resistance associated with survival outside the host. These mutations result in inactivation of the KatE stationary-phase catalase that protects high-density bacterial communities from oxidative stress and the BcsG cellulose biosynthetic enzyme required for the RDAR (red, dry, and rough) colonial phenotype. However, we found that like 14028s, D23580 is able to elicit an acute inflammatory response and cause enteritis in mice and rhesus macaque monkeys. Collectively, these observations suggest that African S. Typhimurium ST313 strain D23580 is becoming adapted to an anthroponotic mode of transmission while retaining the ability to infect and cause enteritis in multiple host species.

Importance: The last 3 decades have witnessed an epidemic of invasive nontyphoidal Salmonella infections in sub-Saharan Africa. Genomic analysis and clinical observations suggest that the Salmonella strains responsible for these infections are evolving to become more typhoid-like with regard to patterns of transmission and virulence. This study shows that a prototypical African nontyphoidal Salmonella strain has lost traits required for environmental stress resistance, consistent with an adaptation to a human-to-human mode of transmission. However, in contrast to predictions, the strain remains capable of causing acute inflammation in the mammalian intestine. This suggests that the systemic clinical presentation of invasive nontyphoidal Salmonella infections in Africa reflects the immune status of infected hosts rather than intrinsic differences in the virulence of African Salmonella strains. Our study provides important new insights into the evolution of host adaptation in bacterial pathogens.

FIG 1

D23580 is more invasive and cytotoxic for cultured cells than 14028s (A) Confluent monolayers of HeLa human cervical epithelial cells were infected with late-logarithmic-phase S. Typhimurium ST313 strain D23580 or ST19 strain 14028s cells at an MOI of ~100:1 for 10 min. Intracellular CFU were enumerated 1 h after internalization by lysis and plating. Bacterial invasion is expressed as the proportion of the starting inoculum. invA mutants were included as controls for reduced invasion. (B) RAW 264.7 murine macrophage-like cells were infected with late-logarithmic-phase S. Typhimurium D23580 or 14028s cells at an MOI of ~10:1. Four hours postinfection, culture supernatants were assayed for released lactate dehydrogenase as a measure of macrophage death. invA mutants were included as SPI1-deficient controls. (C) RAW 264.7 murine macrophage-like cells were infected with opsonized stationary-phase S. Typhimurium D23580 or 14028s cells at an MOI of ~10:1. Eighteen hours postinfection, macrophages were lysed and intracellular CFU were enumerated. Bacterial survival is expressed as a proportion of internalized bacteria remaining at 18 h postinfection. phoP mutants were included as controls with reduced intramacrophage survival. Strain D23580 MDS was included as a control to exclude effects from deletion of plasmid-borne antibiotic resistance determinants. Bars represent the means ± standard deviations. ND, not detected. Statistical significance was determined using a paired t test (*, P < 0.05; ns, not significant).

FIG 2

D23580 efficiently colonizes the murine intestine. CBA/J mice were pretreated with streptomycin for 24 h and infected i.g. with an ~1:1 mixture of S. Typhimurium ST313 strain D23580 or ST19 strain IR715. (A) On days 1 and 4 postinfection, coinfected mice (n = 10) were euthanized and CFU in Peyer’s patches (PP), mesenteric lymph nodes (mLN), colonic contents, liver, and spleen were enumerated. Values greater than 1 indicate a competitive advantage of D23580 over IR715. Bars represent the median values. Each symbol represents the result for 1 mouse. Statistical significance was determined using an unpaired Wilcoxon signed rank test (**, P < 0.01; ns, not significant). (B) Fecal pellets of i.g.-infected mice (n = 10 per group) were collected and homogenized, and CFU were enumerated on days 1, 2, and 4 postinfection. Values represent the medians ± ranges. Statistical significance was determined using a Mann-Whitney test (*, P < 0.05; ***, P < 0.001; ns, not significant). (C) Streptomycin-treated CBA/J mice were infected i.g. with S. Typhimurium D23580 or IR715. At 24 h postinfection, mice were euthanized, followed by removal of the cecum for histopathological analysis. Blinded detailed cecal pathology scores for D23580-, IR715-, and mock-infected mice (n = 4) were recorded. PMN, polymorphonuclear leukocyte. Each bar represents the result for 1 mouse. Statistical significance was determined using a Mann-Whitney test of the total histopathology scores (ns, not significant).

FIG 3

D23580 elicits acute inflammation in the rhesus macaque intestine. Ligated ileal loops of anesthetized rhesus macaques (n = 4) were infected with S. Typhimurium ST313 strain D23580, ST19 strain IR715, or an equivalent volume of sterile LB broth. (A) Fluid accumulation in the infected ileal loops was measured 5 h after injection. Results are expressed as the volume of fluid that accumulated during infection compared to the volume of fluid that accumulated following injection of sterile LB broth into a loop from the same animal. wt, wild-type. (B) Expression of inflammatory cytokines in the ileal mucosa of S. Typhimurium D23580- or IR715-infected ileal loops 5 h after injection. Results shown are the levels of expression in infected ileal loops compared to the amount of expression in loops injected with sterile LB broth from the same animal. (C) Tissue-associated bacteria were measured 5 h after injection of the ileal loops. Bars represent the median values. Each symbol represents the results for 1 animal. Statistical significance (P > 0.05) was determined using a Wilcoxon matched-pairs signed rank test. ns, not significant.

FIG 4

D23580 exhibits deficient detoxification of hydrogen peroxide despite retaining σ^S-dependent gene expression. (A) Stationary-phase cultures of S. Typhimurium were grown for ~16 to 18 h before 20 µl of hydrogen peroxide was added to 1 ml of each culture. The height of the oxygen bubble column correlates with the ability of each strain to detoxify H₂O₂. S. Typhimurium strains 14028s rpoS*, 14028s ΔrpoS, and LT2 are known rpoS mutants and exhibit enhanced sensitivity to H₂O₂ (left). Multiple independent ST313 strains exhibit deficient H₂O₂ detoxification (right). D23580 MDS was included as a control to exclude effects from deletion of plasmid-borne antibiotic resistance determinants. wt, wild-type. *, the ST313 isolate A32793 was not sequenced and is of unknown lineage. (B) Stationary-phase cultures of S. Typhimurium were grown in the presence (open symbols) or absence (closed symbols) of 0.5 mM H₂O₂, and optical density was measured over time. A delay in the time to log phase indicates an enhanced susceptibility to H₂O₂. The data shown are average results for 3 biological replicates. (C) S. Typhimurium strains were grown to stationary phase prior to harvesting of RNA. Quantitative PCR (qPCR) was performed to determine relative levels of mRNA for the σ^S (RpoS)-regulated genes spvB (an ADP-ribosylating toxin), katE (stationary-phase catalase), and otsA (trehalose biosynthetic gene). Absolute qPCR values were normalized to values for the bacterial housekeeping gene rpoD and are expressed as the fold change over results for wild-type 14028s cells. Strain 14028s rpoS* was included as a control for the effects of reduced σ^S-dependent gene expression. Statistical significance was determined using a paired t test (ns, not significant).

FIG 5

D23580 fails to detoxify hydrogen peroxide due to a mutation in the stationary-phase catalase KatE. Stationary-phase cultures of S. Typhimurium were sonicated over ice to release intracellular KatE before the addition of hydrogen peroxide. Decay of H₂O₂ was monitored colorimetrically (based on the A₂₄₀) over time. Heat-treated lysates were incubated at 55°C for 15 min to inactivate KatG, the heat-sensitive Salmonella catalase, before assaying for KatE activity. The strain 14028s katE::tetRA contains a targeted mutation with an inactive katE. 14028s katE::tetRA is a KatE-null strain, a control for reduced stationary-phase catalase activity. D23580 MDS was included as a control to exclude effects of deleting plasmid-borne antibiotic resistance determinants. Statistical significance was determined using a paired t test (**, P < 0.01).

FIG 6

D23580 is unable to form RDAR colonies due to a mutation in the cellulose synthetic enzyme BcsG. S. Typhimurium strains were grown overnight in LB broth and plated onto LB agar containing the dyes Congo red and Coomassie blue without salt. Colonies were grown for 7 days at 25°C or 37°C. RDAR colonies characteristically form at 25°C and not 37°C. The images shown are representative examples.