High-resolution single nucleotide polymorphism analysis distinguishes recrudescence and reinfection in recurrent invasive nontyphoidal Salmonella typhimurium disease

Abstract

Background: Bloodstream infection with invasive nontyphoidal Salmonella (iNTS) is common and severe among human immunodeficiency virus (HIV)-infected adults throughout sub-Saharan Africa. The epidemiology of iNTS is poorly understood. Survivors frequently experience multiply recurrent iNTS disease, despite appropriate antimicrobial therapy, but recrudescence and reinfection have previously been difficult to distinguish.

Methods: We used high-resolution single nucleotide polymorphism (SNP) typing and whole-genome phylogenetics to investigate 47 iNTS isolates from 14 patients with multiple recurrences following an index presentation with iNTS disease in Blantyre, Malawi. We isolated nontyphoidal salmonellae organisms from blood (n = 35), bone marrow (n = 8), stool (n = 2), urine (n = 1), and throat (n = 1) samples; these isolates comprised serotypes Typhimurium (n = 43) and Enteritidis (n = 4).

Results: Recrudescence with identical or highly phylogenetically related isolates accounted for 78% of recurrences, and reinfection with phylogenetically distinct isolates accounted for 22% of recurrences. Both recrudescence and reinfection could occur in the same individual, and reinfection could either precede or follow recrudescence. The number of days to recurrence (23-486 d) was not different for recrudescence or reinfection. The number of days to recrudescence was unrelated to the number of SNPs accumulated by recrudescent organisms, suggesting that there was little genetic change during persistence in the host, despite exposure to multiple courses of antibiotics. Of Salmonella Typhimurium isolates, 42 of 43 were pathovar ST313.

Conclusions: High-resolution whole-genome phylogenetics successfully discriminated recrudescent iNTS from reinfection, despite a high level of clonality within and among individuals, giving insights into pathogenesis and management. These methods also have adequate resolution to investigate the epidemiology and transmission of this important African pathogen.

Figure 1.

Maximum-likelihood phylogenetic trees based on single nucleotide polymorphisms (SNPs) showing the relationship between invasive nontyphoidal Salmonella (iNTS) isolates associated with recurrent invasive disease. A, Unrooted tree based on 45 497 chromosomal SNP loci showing relationship of 51 iNTS Typhimurium isolates and 4 iNTS Enteritidis isolates. B, Unrooted phylogenetic tree based on 1463 chromosomal SNP loci of 51 iNTS Typhimurium isolates. Branch lengths are indicative of estimated rate of substitution per variable site. Length of scale bar is 0.09 substitutions per variable site or ∼163 SNPs. Asterisks indicate branches of the trees that were recovered using 4 independent methods with bootstrap support and clade credibility values greater than 70%. Phylogenetic clusters 1, 2, and 3 are bound by colored ellipses. C, Zoomed-in view of cluster 1. D, Zoomed-in view of cluster 2. In C and D, black dashed lines represent links to the rest of the tree. Scale bar and asterisks are as described previously. Isolate groups for each participant are colored according to the key. Index events and follow-up visits were denoted respectively by the suffixes ’A’ and ’F1,’ ’F2,’ ’F3,’ etc. Isolates grown from blood culture samples have no further suffix. Isolates from bone-marrow, stool, urine, or throat swabs were denoted respectively by the suffixes _BM, _S, _U or _TH.

Figure 2.

Linear regression plots of single nucleotide polymorphism differences between recrudescent pairs (A) and reinfection pairs (B) against time to recurrence. R² values for A = 0.03; B = 0.0002. Abbreviation: SNP, single nucleotide polymorphism.