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

Abstract

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.

Epidemiological investigation and drug resistance characteristics of Riemerella anatipestifer strains from large-scale duck farms in Shandong Province, China from March 2020 to March 2022

“…β-Lactam resistance genes bla CTX , bla SHV , aminoglycoside resistance genes armA, rmtB , and other resistance genes were not detected. The current extensive use of tetracycline antibiotics has led to the widespread presence of tetracycline resistance genes in gram-negative bacteria, which poses a serious threat to human and animal health ( Zheng et al, 2022 ). The tet X resistance gene family is thought to degrade tetracyclines, and notably this resistance gene family degrades tigecycline, an antibiotic used in recent years to treat MDR bacterial infections in humans and animals ( Chen et al, 2022 ).…”

Review of hospital effluents: special emphasis on characterization, impact, and treatment of pollutants and antibiotic resistance

“…Therefore, the se antibiotics have activity against a wide spectrum of bacteria, being able to inhibit both Gram-negative and Gram-positive bacteria (Pavelquesi et al, 2021) to which are belonged both natural and semi-synthetic antibiotics. Bacterial resistance to tetracycline antibiotics is mediated by some mechanisms such as protection of ribosomes by large proteins, reduction of intracellular concentration, and enzymatic inactivation of the antibiotic (Pazda et al, 2019;Zheng et al, 2022). They are active against most Gram-positive and Gram-negative, anaerobic and aerobic bacteria (Pavelquesi et al, 2021).…”

Sensitive and rapid detection of tet(X2) ~ tet(X5) by loop-mediated isothermal amplification based on visual OTG dye

“…In recent years, a multitude of molecular methods have been developed for the detection of tet(X) and its mutants, such as RNA-based antibiotic susceptibility testing (RBAST) [ 11 ], multiplex polymerase chain reaction (PCR) [ 12 ], MALDI-TOF MS [ 13 ], the tetracycline inactivation method assay (TIM) [ 9 ], a TaqMan-based multiplex real-time PCR assay [ 14 ], a multiplex real-time SYBR green-based PCR assay [ 15 ], and combined with PCR and Sanger sequencing [ 16 ], etc. Although these methods exhibit enhanced detection in terms of rapidity, sensitivity, and specificity, they may not be suitable for large-scale applications given their complexity to operate, dependence on specialized and expensive equipment, lengthy detection time, and high costs.…”