Phenotypic and genetic alterations of Burkholderia pseudomallei in patients during relapse and persistent infections

Abstract

The bacterium Burkholderia pseudomallei is the causative agent of melioidosis, a severe tropical disease associated with high mortality and relapse and persistent infections. Treatment of melioidosis requires prolonged antibiotic therapy; however, little is known about relapse and persistent infections, particularly the phenotypic and genetic alterations of B. pseudomallei in patients. In this study, we performed pulsed-field gel electrophoresis (PFGE) to compare the bacterial genotype between the initial isolate and the subsequent isolate from each of 23 suspected recurrent and persistent melioidosis patients in Northeast Thailand. We used whole-genome sequencing (WGS) to investigate multilocus sequence types and genetic alterations of within-host strain pairs. We also investigated the bacterial phenotypes associated with relapse and persistent infections, including multinucleated giant cell (MNGC) formation efficiency and intracellular multiplication. We first identified 13 (1.2%) relapse, 7 (0.7%) persistent, and 3 (0.3%) reinfection patients from 1,046 survivors. Each of the 20 within-host strain pairs from patients with relapse and persistent infections shared the same genotype, suggesting that the subsequent isolates arise from the infecting isolate. Logistic regression analysis of clinical data revealed regimen and duration of oral antibiotic therapies as risk factors associated with relapse and persistent infections. WGS analysis demonstrated 17 within-host genetic alteration events in 6 of 20 paired isolates, including a relatively large deletion and 16 single-nucleotide polymorphism (stocktickerSNP) mutations distributed across 12 genes. In 1 of 20 paired isolates, we observed significantly increased cell-to-cell fusion and intracellular replication in the second isolate compared with the initial isolate from a patient with persistent infection. WGS analysis suggested that a non-synonymous mutation in the tssB-5 gene, which encoded an essential component of the type VI secretion system, may be associated with the increased intracellular replication and MNGC formation efficiency of the second isolate of the patient. This information provides insights into genetic and phenotypic alterations in B. pseudomallei in human melioidosis, which may represent a bacterial strategy for persistent and relapse infections.

Keywords: B. pseudomallei; melioidosis; persistent infection; recurrent infection; relapse infection; whole-genome sequencing; within-host alterations; within-host mutation.

FIGURE 1

Flow diagram of study procedures, number of patients, and bacterial isolates used in our cohort.

FIGURE 2

Multinucleated giant cell formation (MNGC) formation in A549 cells infected with paired Burkholderia pseudomallei isolates from 13 relapse and seven persistent patients. (A) Comparison of MNGC efficiency between the first and second isolates. (B) Comparison of MNGC size between the first and second isolates. (C) Giemsa stain of uninfected A549 cells, A549 cells infected with reference B. pseudomallei K96243, and A549 cells infected with primary DR60101A and persistent DREX512 strains from P17. Black arrows point to the MNGC formation area.

FIGURE 3

Intracellular survival and immunostaining of A549 cells infected with primary DR60101A and persistent DREX512 strains from P17 and reference Burkholderia pseudomallei K96243. (A) Intracellular survival at 2, 4, 6, 8, and 10 h post-infection by plate counting. (B) Number of bacterial cells per image at 8 h post-infection by confocal microscopy. (C) Visualization of infected staining A549 cells by confocal microscopy. Bacteria were stained in green, cytoplasm in red, and DNA in blue.

FIGURE 4

Characteristics of within-host mutations. (A) Read depth of alleles in the first and second isolates. (B) Frequency of alleles in the first and second isolates. (C) Mutational spectrum of within-host SNPs. (D) Within-host SNP counts vs. time between isolates collection.