Development of a novel luminescence reporter mycobacteriophage for rapid drug susceptibility testing of Mycobacterium tuberculosis
10.3760/cma.j.cn112309-20230626-00189
- VernacularTitle:新型分枝杆菌荧光报告噬菌体的构建及其在结核分枝杆菌药敏检测中的应用
- Author:
Chengcheng QIAN
1
;
Ruiqing MA
;
Mingquan GUO
;
Xiaoyong FAN
Author Information
1. 温州医科大学检验医学院、生命科学学院，温州 325035
- Keywords:
Mycobacterium tuberculosis;
Luminescent reporter mycobacteriophage;
Drug susceptibility testing;
Drug-resistant tuberculosis
- From:
Chinese Journal of Microbiology and Immunology
2023;43(10):749-755
- CountryChina
- Language:Chinese
-
Abstract:
Objective:To construct a new reporter mycobacteriophage, ΦFN, based on a nanoluciferase (Nluc) reporter system, and to analyze its ability to detect drug resistance in Mycobacterium tuberculosis ( Mtb). Methods:A Nluc reporter system controlled by P furAma promoter was constructed and integrated into Mycobacterium smegmatis ( Msm) genome to assess its reporting ability in mycobacteria. Then the P furAma- nluc reporter system was integrated into TM4 mycobacteriophage genome to construct a new phage termed ΦFN. A rapid procedure for detecting drug resistance in mycobacteria using ΦFN was established by adjusting conditions such as drug exposure time and time of infection. The susceptibility of 52 clinical isolates of Mtb with known drug resistance to three first-line anti-tuberculosis drugs were tested in 96-well plates. Results:The recombinant Msm mc 2155 expressing P furAma- nluc repoerter system could generate luminescence signal at a low limit of 100 colony-forming unit (CFU), which was lower than the previously reported nluc system controlled by Pleft promoter. The detection limit of ΦFN for mycobacteria reached 100 CFU within 24 h by luminescent microplate reader. After 48 h of antibiotic exposure and 24 h of phage incubation, no luminescence signal could be detected when susceptible strains were below 10 5 CFU, which could effectively distinguish susceptible strains and rapidly detect drug resistance. The drug susceptibility of 52 clinical isolates of Mtb to rifampin, isoniazid and streptomycin was tested using ΦFN, and the accuracy was 51/52, 48/52 and 49/52, respectively, by using the conventional drug susceptibility test, Lwenstein-Jensen culture medium assay, as reference. Conclusions:The new recombinant luminescent reporter phage, ΦFN, showed high accuracy in detecting the drug susceptibility of Mtb to rifampicin, isoniazid and streptomycin and it only took three days. It is expected to be a new simple assay for the rapid detection of drug susceptibility of Mtb.
