Detection of differentially culturable tubercle bacteria in sputum using mycobacterial culture filtrates

Abstract

Rapid detection of tuberculosis (TB) infection is paramount to curb further transmission. The gold standard for this remains mycobacterial culture, however emerging evidence confirms the presence of differentially culturable tubercle bacteria (DCTB) in clinical specimens. These bacteria do not grow under standard culture conditions and require the presence of culture filtrate (CF), from axenic cultures of Mycobacterium tuberculosis (Mtb), to emerge. It has been hypothesized that molecules such as resuscitation promoting factors (Rpfs), fatty acids and cyclic-AMP (cAMP) present in CF are responsible for the growth stimulatory activity. Herein, we tested the ability of CF from the non-pathogenic bacterium Mycobacterium smegmatis (Msm) to stimulate the growth of DCTB, as this organism provides a more tractable source of CF. We also interrogated the role of Mtb Rpfs in stimulation of DCTB by creating recombinant strains of Msm that express Mtb rpf genes in various combinations. CF derived from this panel of strains was tested on sputum from individuals with drug susceptible TB prior to treatment. CF from wild type Msm did not enable detection of DCTB in a manner akin to Mtb CF preparations and whilst the addition of RpfAB^Mtb and RpfABCDE^Mtb to an Msm mutant devoid of its native rpfs did improve detection of DCTB compared to the no CF control, it was not statistically different to the empty vector control. To further investigate the role of Rpfs, we compared the growth stimulatory activity of CF from Mtb, with and without Rpfs and found these to be equivalent. Next, we tested chemically diverse fatty acids and cAMP for growth stimulation and whilst some selective stimulatory effect was observed, this was not significantly higher than the media control and not comparable to CF. Together, these data indicate that the growth stimulatory effect observed with Mtb CF is most likely the result of a combination of factors. Future work aimed at identifying the nature of these growth stimulatory molecules may facilitate improvement of culture-based diagnostics for TB.

Figure 1

Strategy for construction of recombinant Msm strains expressing Mtb Rpfs in various combinations. The Mtb H37Rv wild type and Rpf defective mutant (Mtb ∆rpf) were used as a source of Mtb culture filtrates (CFs). Wild type Msm was used as a source of Msm Rpfs and this strain was transformed with a vector carrying RpfCD^Mtb to mimic the Rpf complement from Mtb. The Msm mutant defective for Rpfs (Msm ∆rpf) was used as a control and within this mutant background, vectors carrying various combinations of Mtb Rpfs were inserted to create strains that express Mtb Rpfs in Msm CF as a source of growth stimulatory molecules in MPN DCTB assays. The panel on the right shows the rpf gene complement present in the various Mtb and Msm strains. The vector diagram on the left depicts the strategy to construct vectors with different combinations of Mtb Rpfs that are stably carried in the resulting recombinant strains. The presence of the integrase on mycobacterial vectors that integrate phages at attachments sites, facilitates the first integration event but then can also lead to excision of the vector in the absence of antibiotic selection. For DCTB assays, CF that is free of antibiotic was required as antibiotics will inhibit the growth of sputum-derived bacteria. To facilitate this, the integrase was first removed from vectors expressing Mtb Rpfs and was provided in trans on another suicide vector during transformation. With this approach, the integrase is present for the first integration event and is then lost as it does not carry a mycobacterial origin of replication.

Figure 2

Assessment of Mtb and Msm rpf gene expression in recombinant strains. Transcriptional analysis was carried out on recombinant strains of Msm and the cognate parent strains. In all strains shown, transcriptional analysis was carried out for all five Mtb and four Msm rpf genes. Where these genes were deleted, no transcripts were detected. Transcript levels were assessed in each strain when axenic cultures reached mid-exponential growth (OD_600nm ~ 0.6). Gene expression was normalized against Msm sigA transcript levels. Data are representative of three independent biological repeats. Graph was generated using Graphpad Prism software.

Figure 3

Participant disposition flow chart for individuals recruited to this study. (A) Sputum specimens obtained from these individuals were decontaminated and the resulting bacteria grown in limiting dilution assays with various CF combinations. (B) Sputum specimens obtained from these individuals were decontaminated and the ability of the various fatty acids and cyclic-AMP to resuscitate the DCTB was assessed by MPN and CFU assays.

Figure 4

Bacterial yield in DCTB assays using CF from Msm strains expressing Mtb Rpfs in various combinations. (A) Assessment of DCTB recovery from sputum cultures using CF supplementation. DCTB were detected using the MPN limiting dilution assay. For comparison, CF containing and deficient in Mtb and Msm Rpf was used to assess the role of Rpfs in growth stimulation. To control for the effect of CF in growth stimulation, fresh Middlebrook media was used. CF from various recombinant Msm strains was used to assess if expression of Mtb Rpfs in Msm yields CF that can be used to stimulate DCTB in sputum specimens in a manner that is comparable to Mtb CF. To obtain the DCTB count, MPN values (with or without CF) were divided by CFU counts, which depict conventionally culturable bacteria. (B) Histogram depicting mean DCTB counts from assays using CF from Mtb and Msm. The mean DCTB counts were compared to that obtained from no CF assays using a Wilcoxon test to determine the growth stimulatory effect of the various CFs. Error bars depict the standard error of the mean. P-values depict the comparison of any given CF to the no CF or empty vector control. Graph was generated using Graphpad Prism software, Version 6.

Figure 5

Bacterial yield in DCTB assays using CF containing Fatty acids and cyclic-AMP. (A) Assessment of DCTB recovery from sputum cultures using CF, fatty acid and cyclic-AMP (CAMP) supplementation. DCTB were detected using the MPN limiting dilution assay. For comparison, CF containing and deficient in Mtb Rpfs was used to compare the growth stimulatory effects of fatty acids and cAMP. To control for the effect of CF in growth stimulation, fresh Middlebrook media was used, which standardly contains oleic acid. To obtain the DCTB count, MPN values (with or without CF) were divided by CFU counts, which depict conventionally culturable bacteria. (B) Histogram depicting mean DCTB counts from MPN assays supplemented with standard media containing cAMP or select fatty acids. Statistical comparisons were conducted between CFs and the no CF or cAMP/fatty acid assays. There was an increase in bacterial recovery from MPN assays containing CF, which was not significant, most likely due to the small samples size (n = 4), no significant increase in DCTB recovery was noted in the media control and cAMP or fatty acid assays. Error bars depict the standard error of the mean. (C) Histogram depicting mean DCTB counts from assays using CF from Mtb and standard media supplemented with fatty acids. The mean DCTB counts were compared to that obtained from the media controls assay using a Wilcoxon test to determine the growth stimulatory affect of fatty acids. P-values depict the comparison of the CFs to the no CF or fatty acid assays. Graphs were generated using Graphpad Prism software, Version 6.