Soytone

Attenuated Salmonella Enteritidis strain CVD 1940 (pGEN206) (Tennant et al. 2011 (link)) and S. Typhi live oral vaccine strain CVD 909 (ΔaroC ΔaroD ΔhtrA Ptac-tviA) (Hone et al. 1991 (link); Levine et al. 1996 (link); Wang et al. 2000 (link)) were used to safely spike blood. Non-Salmonella strains used to determine specificity included Escherichia coli Bort, Pseudomonas aeruginosa PA01 and Klebsiella pneumoniae B5055 from collections at the Center for Vaccine Development (CVD). Salmonella spp., E. coli, P. aeruginosa and K. pneumoniae were grown in HS bacteriological medium (5 g sodium chloride, 10 g soytone (Teknova, Hollister, CA), 5 g Hy Yest 412 (Sigma Aldrich, St. Louis, MO) in 1 l distilled water) at 37°C. Media were supplemented with guanine (0·001% w/v) and 50 μg ml⁻¹ carbenicillin for growth of CVD 1940(pGEN206); culture medium for CVD 909 was supplemented with 2, 3-dihydroxybenzoate (DHB) (0·0001% w/v). Whole human blood with sodium heparin anticoagulant was purchased from Innovative Research (Novi, MI). For the buffy coat experiments, fresh blood was obtained from volunteers under the approval of the University of Maryland, Baltimore Institutional Review Board and used within 8 h. All blood donors provided written informed consent.

The strains used in this study are described (S1 Table). All strains were maintained in Hi-Soy (HS) bacteriological media (5 g/L sodium chloride, 10 g/L soytone [Teknova, CA], 5 g/L Hy-yest [Sigma Aldrich, MO]) at 37°C. Growth and preparation of bacteria for in-vitro analyses and in-vivo infection was conducted as described [18 (link)]. Growth media for all guaBA mutants were supplemented with guanine; kanamycin was additionally supplemented for CVD 1925 (pSEC10-wzzB) (S1 Table). STm CVD 1925 (with or without pSEC10-wzzB) (S1 Table) and CVD 1943 (S1 Table) were grown in fully chemically defined media (CDM) in a fermenter. STm D65 (S1 Table) was grown in shake flasks. Fermentation conditions were as follows: 50 mL of CDM supplemented with 0.004% guanine was inoculated with 3–5 colonies from an HS agar plate and grown for 12–18 h at 37°C in a shake flask with agitation at 80 rpm. This culture was then used to inoculate 500 mL of CDM supplemented with 0.004% guanine that was grown under equivalent conditions for 8–10 h. Four liters of CDM containing 0.025% guanine was then inoculated to an OD₆₀₀ nm of 0.15 with the 500 mL shake flask and maintained in a Biostat A-plus fermenter (Sartorius, Germany) culture, for 18–24 h at 400 rpm, 5 LPM ambient air, with an adjustment to pH 7 using 28% ammonium hydroxide.

Isolates used for whole-genome sequencing are listed in Table S1. Strains and plasmids used in this study are listed in Table S6 in the supplemental material. S. Paratyphi B, S. Paratyphi A, and S. Typhi strains were originally isolated from adults who presented with enteric fever symptoms in Santiago, Chile, in the 1980s. S. Paratyphi B strains were typed as Java or sensu stricto based on their ability to ferment d-tartrate. (Java strains are able to ferment d-tartrate, whereas sensu stricto strains do not.) We also confirmed the presence or absence of an intact d-tartrate fermentation gene by PCR as previously described (35 (link)). Bacteria were grown in animal-product-free Hy-Soy (HS) medium (1% [wt/vol] soytone [Teknova, Hollister, CA], 0.5% [wt/vol] Hy-Yest [Kerry Bioscience, Beloit, WI], 0.5% [wt/vol] NaCl [American Bio, Natick, MA]) at 37°C. Bacterial strains carrying ΔguaBA mutations were grown on media supplemented with 0.005% (wt/vol) guanine.

Invasive Salmonella Typhimurium ST313 (D65, Q55, S11) and Salmonella Typhimurium ST19 (I77, I41, S52) have previously been isolated from the blood of infants in Mali, West Africa [14 (link),15 (link),16 (link)]. These clinical strains were maintained on animal product-free Hy-Soy (HS) agar (0.5% Hy-yest [Kerry Biosciences, Beloit, WI], 1% Soytone [TEKNova, Hollister, CA], 0.5% NaCl [American Bio, Natick, MA]).

The strains used in this study are described in Table 1. All strains were maintained on HS bacteriological media (5 g l⁻¹ sodium chloride, 10 g l⁻¹ soytone [Teknova, Hollister, CA, USA], 5 g l⁻¹ Hy‐yest [Kerry, Beloit, WI, USA]) at 37°C. Growth medium for all guaBA mutants was supplemented with guanine (0·001% w/v). Kanamycin or carbenicillin was added at a final concentration of 50 μg ml⁻¹ for pSEC10 and pSE280 maintenance, respectively, in Salmonella spp. and Shigella spp. Carbenicillin was added at a final concentration of 250 μg ml⁻¹ for pUCP19 maintenance in P. aeruginosa strains PAO1, IATS O6 and IATS O11. Isopropyl β‐D‐1‐thiogalactopyranoside (IPTG; Teknova, Hollister, CA, USA) was added at 0·1 mol l⁻¹ final concentration for induction of wzz2 overexpression in P. aeruginosa strains.