In Vitro Activity of Tetracycline Analogs against Multidrug-resistant and Extensive drug resistance Clinical Isolates of Mycobacterium tuberculosis

Background Multidrug-resistant tuberculosis (MDR-TB) has become a big threaten to global health . The current strategy for treatment of MDR-TB and extensive drug resistant tuberculosis (XDR-TB) is with low efficacy and high side effect. While new drug is fundamental for cure MDR-TB, repurposing the Food and Drug Administration (FDA)-approved drugs represents an alternative soluation with less cost. Methods The activity of 8 tetracycline-class antibiotics against mycobacterium tuberculosis ( M.tb ) were determined by Minimum Inhibitory Concentration (MIC) in vitro. A transposon M.smeg libraries was generated by using the Harm phage and then used to isolate the conditional growth mutants in doxycycline containing plate. 11 mutants were isolated and genomic DNAs were extracted using the cetyltrimethyl ammonium bromide (CTAB) method and analyzed by whole genome sequencing. Results We found that three of eight drugs efficiently inhibited mycobacteria growth under the peak plasma concentration in the human body. Further tests showed these three tetracycline analogs (demeclocycline, doxycycline and methacycline) had antimicrobial activity against seven clinical isolates, including MDR and XDR strains. Among them, Doxycycline had the lowest MICs in all mycobacteria strains tested in this study. By using a transposon library, we identify the insertion of transposon in two genes, porin and MshA, associate with the resistant to doxycycline. Conclusions Our findings show that tetracycline analogs such as doxycycline, has bactericidal activity against not only drug sensitive M.tb , but also clinical MDR and XDR strains, provided proof of concept to repurpose doxycycline to fight MDR-TB and XDR-TB. Further investigations are warranted to clarify the underlying mechanism and optimize the strategy in combination with other anti-TB drugs.

11 mutants were isolated and genomic DNAs were extracted using the cetyltrimethyl ammonium bromide (CTAB) method and analyzed by whole genome sequencing.
Results We found that three of eight drugs efficiently inhibited mycobacteria growth under the peak plasma concentration in the human body. Further tests showed these three tetracycline analogs (demeclocycline, doxycycline and methacycline) had antimicrobial activity against seven clinical isolates, including MDR and XDR strains. Among them, Doxycycline had the lowest MICs in all mycobacteria strains tested in this study. By using a transposon library, we identify the insertion of transposon in two genes, porin and MshA, associate with the resistant to doxycycline.
Conclusions Our findings show that tetracycline analogs such as doxycycline, has bactericidal activity against not only drug sensitive M.tb , but also clinical MDR and XDR strains, provided proof of concept to repurpose doxycycline to fight MDR-TB and XDR-TB. Further investigations are warranted to clarify the underlying mechanism and optimize the strategy in combination with other anti-TB drugs. Tetracycline antibiotic was first reported in 1948 and found widespread clinical use shortly thereafter [4][5][6][7][8]. Tetracyclines were known as broad-spectrum antibacterial agents which have a good inhibitory effect on gram-positive and gram-negative bacteria, rickettsia, filtered virus, spirochetes and even protozoa [9,10].
Tetracyclines inhibit bacterial protein synthesis by binding to the 16S rRNA of the 30S bacterial ribosome subunit, preventing accommodation of incoming aminoacyl-tRNAs at the acceptor site [11,12]. Both synthetic and semisynthetic tetracycline analogs have been shown effective against wide ranges of pathogens and different inflammatory diseases and conditions [10], which are the most prescribed oral antibiotics, with a long satisfactory track record of efficacy, low price and safety [13]. Previous studies have showed that doxycycline, a second-generation tetracycline, could efficacy reduce M.tb growth both in macrophage and guinea pig TB model via inhibiting host MMPs activity [14]. Doxycycline has been used in vitro against M.tuberculosis [15], and combination with amikacin significantly inhibited 18 of the 29 MDR and XDR TB strains replications [16]. Some of the older members of the tetracycline family also showed activity against M. tuberculosis strains in animal models [17].
However, very few studies have been carried out to assess all three generation of tetracyclines activity in mycobacteria. Furthermore, the underlying mechanism of doxycycline direct killing activity is still unclear. Here, we investigated the potential anti-mycobactirum activity of several tetracyclines including demeclocycline, tetracycline, chlortetracycline, tigecycline, oxytetracycline, methacycline, tetracycline and doxycycline in vitro. We further identified two mutants which were resistant to doxycycline by using a transposon M.smeg library.

MIC determination
The anti-mycobacterium activity was tested using M.smeg, H37Ra, H37Rv and 7 clinical isolates. The bacterial culture was diluted with 7H9-OADC to an OD = 0.01 and transferred to a 96-well microtiter plate for the drug sensitivity assay. Serial dilutions of tetracyclines were dispensed into 96-well microtiter plates containing bacteria strains. The 96-well microtiter plates were incubated at 37℃ with slowly horizontal shaking. The OD600 was measured in a microplate spectrophotometer (BioTek) in indicated times and the growth rate was calculated. The MIC is defined as the lowest concentration of the antimicrobial agent that prevents visible growth of a microorganism under the compound-containing plates [18].

Whole Genome Sequencing of Transposon Mutants
The transposon M.smeg library was constructed with donor phagemid φMycoMarT7 which includes a transposon that encodes a kanamycin resistance gene [19]. The entire sequence of the φMycoMarT7 transposon has been deposited in GenBank

Statistical analysis
Statistical analysis was performed using GraphPad Prism 7 software. One-way ANOVA was used to assess the effects between more than two groups. Student's ttest was used to assess the effects of only one parameter.

Results
Tetracycline analogs showed bactericidal activity against mycobacteria in vitro 8 tetracycline analogs were selected to investigate the antimicrobial effect against Mycobacterium. We first test the effect of these analogs on M.smeg and results showed that seven of the tested tetracycline analogs significantly inhibited M.smeg growth (Fig 1 A). Previously studies have showed that tetracycline [22] and tigecycline [23] could significantly inhibited mycobacterial strains growth, which is consistent with our results. We further determined the MIC of four tetracyclines, demeclocycline, chlortetracycline, methacycline and doxycycline. We found that the MICs of M.smeg in 7H9 broth were 0.067μg/mL, 0.34μg/mL, 0.051μg/mL and 0.064 μg/mL for demeclocycline, chlortetracycline, doxycycline and methacycline (Fig 1 B and Table 2), respectively. The MICs of the M.tb strain H37Ra were 1.25 μg/mL, 5.15 μg/mL, 1.03 μg/mL, 2.39μg/mL for demeclocycline, chlortetracycline, doxycycline and methacycline (Fig 1 C and Table 2), respectively. We noted that the MICs of those tetracyclines in M.tb strains were about 20-fold higher than that in M.smeg.
The results above demonstrated that some of tetracycline analogs could significantly inhibited mycobacteria growth in vitro.

Activity of Tetracyclines against drug-resistant clinical isolates
The MICs of demeclocycline, doxycycline and methacycline are lower than that in the peak plasma concentration [24,25]. We next assessed the activities of these tetracyclines in clinical isolates.
All the clinical isolates were collected from Shenzhen third people's hospital, China.
Three analogs shown anti-Mycobacterium activity for most of the isolates (Table3).
The MIC result of three drugs was from 1.28 to 5.13 μg/mL for doxycycline, from 0.25 to 25.06 μg/mL for demeclocycline and 0.59 to 23.9 μg/mL for methacycline (Fig 2 and Table 3 We further tested the MIC of doxycycline with these two mutants. Our results showed both mutants showed increasing resistant to doxycycline and the MICs were 0.51μg/mL and 0.25μg/mL for MSMEG_0965 and MSMEG_0933 mutant strains, respectively (Table 5). However, both MshA and porin are non-essential genes [28, 34,35], it was unlikely to encode the target. The mechanism underlying these two mutants resistant to doxycycline needs to be further investigated. discussion With rising levels of antibiotic resistance eliminating the drug classes available for treating MDR-and XDR-TB, identifying new drug classes serves a critical need.
Additionally, concerning the rising of heritable resistance to PZA [36], novel molecules that are bactericidal against M.tb will be important components of muchneeded new cocktails for shorter treatment without relapse. In this study, we examined the activity of tetracycline analogs, including three generations of tetracyclines, against mycobacteria. We identified 6 tetracycline analogs (demeclocycline, tetracycline, chlortetracycline, methacycline, tigecycline and doxycycline) that were efficacious against Mycobacteria while oxytetracycline showed poor activity. Our results is consistent with the previously reports on doxycycline and tigecycline [14,23,37,38]. We have found that demeclocycline, doxycycline and methacycline were effective against clinical MDR-or XDR-isolates.
When comparing the MICs of efficacious tetracyclines, doxycycline had lower MICs among all tetracyclines and is a pre-existing drug approved for the treatment of various infections by gram-positive and gram-negative bacteria, aerobes and anaerobes [39].
Doxycycline is a well-tolerated broad-spectrum oral antibiotic that is used in therapy for many infections.iIn this study, 6 of 7 clinical isolates were susceptibility to doxycycline. The result is similar as previously reported doxycycline resistant rate in Russia clinical isolates [15].