Phage lysin to control the overgrowth of normal flora in processed sputum samples for the rapid and sensitive detection of Mycobacterium tuberculosis by luciferase reporter phage assay

Abstract

BackgroundPhage lysin, extracted from three bacteriophages was used in place of antibiotics to control the overgrowth of normal flora in processed sputum samples leading to the sensitive detection of Mycobacterium tuberculosis using diagnostic luciferase reporter phage assay (DLRPA).MethodsA total of 129 sputum samples were processed by modified Petroff’s method. Two Lowenstein Jensen slopes were inoculated from the processed sputum deposit thus obtained. The remaining deposits were transferred to 7 ml of Middlebrook 7H9 complete medium supplemented with phage lysin and incubated at 37°C. DLRPA was done using phAE129 at days 7, 9, 14 and 21. At the end of day 21, the samples were centrifuged and the pellets were inoculated on to 2 more LJ slopes to validate DLRPA results.ResultsThe sensitivity and specificity of DLRPA in detecting M. tuberculosis from sputum specimens was 90% and 81% respectively compared to conventional LJ culture. The agreement between the methods was 87%. The rate of contamination for DLRPA using phage lysin was 9.3%.ConclusionPhage lysin can be used to decontaminate sputum samples for the detection of M. tuberculosis by DLRPA directly from processed sputum specimens.

Phage lytic proteins: biotechnological applications beyond clinical antimicrobials

“…These authors used a pool of three endolysins to control the overgrowth of the normal microbiota in processed sputum samples in order to properly detect Mycobacterium tuberculosis. The rate of growth of normal microbiota for the detection method using the phage lysin pool was 9.3% compared with untreated samples (Subramanyam et al, 2013). Similar results were obtained when antibiotics were used to decontaminate sputum samples (Subramanyam et al, 2011); however, antibiotics also reduce the growth rate of mycobacteria.…”

“…In addition, LambdaSa2 endolysin and one of the Lactobacillus bacteriophage endolysins were able to reduce both L. fermentum and L. reuteri contaminants in mock fermentations of corn fiber hydrolysates (Roach et al, 2013). Another example of the control of bacterial growth using phage endolysins was proposed by Subramanyam et al (2013). These authors used a pool of three endolysins to control the overgrowth of the normal microbiota in processed sputum samples in order to properly detect Mycobacterium tuberculosis.…”

“…Similar results were obtained when antibiotics were used to decontaminate sputum samples (Subramanyam et al, 2011); however, antibiotics also reduce the growth rate of mycobacteria. In this context, endolysins demonstrated to be as effective as antibiotics without inhibiting M. tuberculosis growth (Subramanyam et al, 2013).…”

The Rebirth of Culture in Microbiology through the Example of Culturomics To Study Human Gut Microbiota

“…Phage therapy was previously used therapeutically in humans (201) and to eliminate the contamination of food by food-borne bacterial pathogens (202). Bacteriophages were previously used by Subramanyam et al to decontaminate sputum samples instead of using antiseptics and/or antibiotics before performance of Mycobacterium species culture (203,204). The use of T1, T4, and phiX174-like E. coli phages was initially undertaken to decrease the number of E. coli colonies adhering to the urothelium and causing persistent urinary tract infections (205).…”

Current and Past Strategies for Bacterial Culture in Clinical Microbiology

“…This noninvasive method obviates the requirement for gastric aspiration without a difference in pulmonary tuberculosis diagnosis (33). Finally, lytic phages have been used to decontaminate the normal flora from sputum before M. tuberculosis culture to replace the use of antibiotics (27,34).…”