Rapid visualized assessment of drug efficacy in live mice with a selectable marker-free autoluminescent Klebsiella pneumoniae.

Klebsiella pneumoniae is an opportunistic pathogen that is responsible for community acquired infections and nosocomial infections. Antibiotic-resistant K. pneumoniae and/or hypervirulent K. pneumoniae are emerging as a serious threat to public health. For the sake of alleviating and conquering current dilemma, discovery of effective new drugs against K. pneumoniae is a tough challenge. However, traditional anti-K. pneumoniae drug discovery methods cost considerable amount of time, animals, labor and so on. So an efficient technique for in vitro and in vivo drug screening with the least time duration, animals and labor cost is highly needed for the discovery of new effective compounds. Hence, in this study we constructed a selectable marker-free autoluminescent K. pneumoniae (SfAlKp) harboring luxCDABE by combining Tn7 transposon and Xer-dif system. SfAlKp can be used for discovery of new drugs via detecting luminescence intensity as a surrogate marker. The energy-consuming autoluminescent reaction catalyzed by the LuxAB enzymes which use the substrates produced by LuxCDE using the metabolites of the bacteria. Tn7 can insert exogenous genes into the bacterial genome and the DNA fragment in between dif sequences can be recognized and removed by endogenous XerCD recombinases of K. pneumoniae. The drug susceptibility and growth rate of SfAlKp are identical to its parent strain, meanwhile the luminescence intensity and stability are also significant characteristics of SfAlKp. Compared to conventional techniques, the autoluminescence-based measurement is more applicable to high throughput screening for compounds both in vitro as well as in vivo in animal model.

Advances in the development of molecular genetic tools for Mycobacterium tuberculosis.

Mycobacterium tuberculosis, a clinically relevant Gram-positive bacterium of great clinical relevance, is a lethal pathogen owing to its complex physiological characteristics and development of drug resistance. Several molecular genetic tools have been developed in the past few decades to study this microorganism. These tools have been instrumental in understanding how M. tuberculosis became a successful pathogen. Advanced molecular genetic tools have played a significant role in exploring the complex pathways involved in M. tuberculosis pathogenesis. Here, we review various molecular genetic tools used in the study of M. tuberculosis. Further, we discuss the applications of clustered regularly interspaced short palindromic repeat interference (CRISPRi), a novel technology recently applied in M. tuberculosis research to study target gene functions. Finally, prospective outcomes of the applications of molecular techniques in the field of M. tuberculosis genetic research are also discussed.