PMC4843968_01
Phylogeny of S. Enteritidis gastroenteritis isolates and blood-isolates from the IL12 beta 1 receptor deficient patient. (A) Maximum likelihood tree of 17 S. Enteritidis isolates from uncomplicated gastroenteritis cases and eleven isolates from the blood of the patient, constructed using 5,087 SNPs with reference to S. Enteritidis P125109 whole genome sequence. Bootstrap values are indicated at each node and the scale bar indicates the estimated number of SNPs. (B) The number of SNPs from the root of the clade containing the patient isolates to each tip representing an isolate is plotted against the date of isolation. The broken line indicates the line of best fit for the data points extrapolated to intersect with the x-axis. (C) Bayesian time-dependent maximum clade credibility tree constructed using 2545 SNPs with reference to S. Enteritidis P125109 whole genome sequence. Grey bars indicate the 95% confidence intervals for the date of divergence for each ancestral node.
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Enteritidis phage type 4 (PT4) P125109, excluding SNPs within prophage elements, repetitive sequence and insertion elements (Figure 1A). Accumulation of SNPs along lineages within the patient isolate clade was linear and time-dependent, with a mean of 47 SNPs per genome per year (Figure 1B). A time-dependent phylogenetic reconstruction using Bayesian inference predicted a time of MRCA (tMRCA) around 1995 (+/-0.5 years) (Figure 1C). Within the immunocompromised patient clade, there were elevated G A and C T nucleotide substitutions (Supplementary Information Figure 1) relative to S.
Patient isolates have acquired a large number of pseudogenes. The total number of pseudogenes were determined for four of the patient isolates. (A) The number of pseudogenes acquired along specific branches of the phylogeny are given. (B) The overlap between the pseudogene repertoire in patient isolates and human-restricted serovars S. Typhi CT18 and S. Paratyphi A (ATCC 9150) is represented by a venn diagram where numbers of pseudogenes are indicated. The percentages of pseudogenes in (C) the whole genome, (D) in central anaerobic metabolism genes, and (E) genes encoding TTSS effectors are given for the patient isolates and other Salmonella serovars. Host-promiscuous serovars most commonly associated with gastro-intestinal disease are shown with grey bars, patient isolates with red bars and host-adapted serovars causing systemic infection with black bars. Calculations are based on the classification of pseudogenes according to Nuccio et. al.
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Information Figure 2), both of which were consistent with a defective mis-match repair mechanism. Indeed, the mutS gene in the patient isolates contained a 174 bp in-frame deletion encoding a truncated protein Each isolate had acquired, on average, 158 pseudogenes (Figure 2A) since the MRCA with PT4. This level of genome degradation is comparable to that reported for host-adapted Salmonella serovars (Figure 2C). Paratyphi A and at least one of the four patient isolates analyzed (Figure 2B). Importantly, the patient isolates have acquired pseudogenes in a large percentage of central anaerobic metabolism, colonization factor and secreted effector genes, three functional classes that promote growth and cellular invasion in the gut and are linked to pathogenesis (Figure 2D-F).
Colonisation of the murine host and interaction with epithelial-like cells, by patient isolates. (A) Invasion of HEp-2 epithelial-like cells inferred by protection from gentamicin killing. Invasion is expressed as the number of viable bacterial cells following lysis of tissue culture cells as a percentage of those added to each well. p-values calculated by Kruskal-Wallis test: * is p<=0.05, *** is p<=0.001. Colonisation of the liver, spleen and blood of C57BL/6 mice was determined five days post (B) oral or (C) intravenous inoculation with patient isolate MB4386 or S. Enterititidis P125109, n=5 per group. A, B, and C plotted mean +- SEM. Dashed lines indicate limit of detection. (D) Survival of mice following intravenous inoculation of C57BL/6 mice with either patient isolate MB4386 (squares) or S. Enterititidis P125109 (circles) n=4 per group.
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Enteritidis P125109 (0.001 SNPs per generation) (Supplementary Information Figure 3). Patient isolates B20062, MB760 and MB4386 were not able to invade the cultured epithelial-like cell line HEp-2 as efficiently as P125109 (Figure 3A). Colonization of the liver and spleen with MB4386 were below the limit of detection in most mice (Figure 3B). MB4386 was likewise attenuated with 1,000 to 10,000 fold lower liver and spleen colonization compared to P125109 (Figure 3C) and a higher survival rate. Mice intravenously infected with P125109 were moribund within five days, while all mice infected with MB4386 survived beyond day seven (Figure 3D).