The detection of mixed tuberculosis infections using culture filtrate and resuscitation promoting factor deficient filtrate

Abstract

Tuberculosis (TB) infected individuals harbor a heterogenous population of differentially culturable tubercle bacilli (DCTB). Herein, we describe how DCTB assays using culture filtrate either containing or deficient in resuscitation promoting factors can uncover mixed infections. We demonstrate that Mycobacterium tuberculosis (Mtb) strain genotypes can be separated in DCTB assays based on their selective requirement for growth stimulatory factors. Beijing mixed infections appear to be associated with a higher bacterial load and reduced reliance on growth stimulatory factors. These data have important implications for identifying mixed infections and hetero-resistance, which in turn can affect selection of treatment regimen and establishment of transmission links.

Keywords: Mycobacterium tuberculosis; differentially culturable tubercle bacteria; mixed infection; most probable number assay; resuscitation promoting factor.

Figure 1

Mycobacterial family strains identified in Most Probable Number (MPN) assays. (A) Shown is a schematic representation of the experimental workflow. Sputum specimens were decontaminated and the same specimen was subjected to three MPN assays in the presence of culture filtrate (CF) from wild type M. tuberculosis (CF^Mtb), CF from an Rpf-deficient mutant of M. tuberculosis (CF^Rpf-) and no CF. The populations detected in this experimental set up are shown. (B) Shown is the strain identified in the first well and last well of CF⁺MPN (red), Rpf ^– MPN (blue) and MPN no CF (green) assays. The absence of a MPN value reflects no growth under that condition. Wells in which Beijing strains were detected with another strain “Beijing Mixed” (top set) are delineated from those with only Non-Beijing strains “Non-Beijing mixed” (lower set) by the horizontal dashed line. Spoligotyping was used to genotype isolates present in the MPN wells. ND, not determined.

Figure 2

Detection of different strain types in MPN wells. Aliquots from the MPN wells from participant 54034 were plated to single colonies and 10 individual colonies were genotyped. Shown is the blot from the spoligotyping of these colonies.