MALDI-TOF MS for rapid detection and differentiation between Tet(X)-producers and non-Tet(X)-producing tetracycline-resistant Gram-negative bacteria.

The extensive use of tetracycline antibiotics has led to the widespread presence of tetracycline-resistance genes in Gram-negative bacteria and this poses serious threats to human and animal health. In our previous study, we reported a method for rapid detection of Tet(X)-producers using MALDI-TOF MS. However, there have been multiple machineries involved in tetracycline resistance including efflux pump, and ribosomal protection protein. Our previous demonstrated the limitation in probing the non-Tet(X)-producing tetracycline-resistant strains. In this regard, we further developed a MALDI-TOF MS method to detect and differentiate Tet(X)-producers and non-Tet(X)-producing tetracycline-resistant strains. Test strains were incubated with tigecycline and oxytetracycline in separate tubes for 3 h and then analyzed spectral peaks of tigecycline, oxytetracycline, and their metabolite. Strains were distinguished using MS ratio for [metabolite/(metabolite+ tigecycline or oxytetracycline)]. Four control strains and 319 test strains were analyzed and the sensitivity was 98.90% and specificity was 98.34%. This was consistent with the results obtained from LC-MS/MS analysis. Interestingly, we also found that the reactive oxygen species (ROS) produced by tetracycline-susceptible strains were able to promote the degradation of oxytetracycline. Overall, the MALDI ^Tet(X)-plus test represents a rapid and reliable method to detect Tet(X)-producers, non-Tet(X)-producing tetracycline-resistant strains, and tetracycline-susceptible strains.