Abstract
The main objective of this study was to compare in vitro activities of a novel fluoroquinolone (FQ), UB-8902, with ofloxacin (OFX), levofloxacin (LFX), and moxifloxacin (MOX) against Mycobacterium tuberculosis isolates. Eleven OFX-resistant and 11 drug-susceptible clinical isolates were studied. Individual minimum inhibitory concentrations of OFX, LFX, MOX, and UB-8902 were determined using Middlebrook 7H11 agar. The concentrations studied ranged from 0.125 to 128 μg/mL in twofold dilutions. UB-8902 was more active than LFX and similar to MOX for OFX-resistant M. tuberculosis isolates. In addition, UB-8902 and MOX showed equal activity against drug-susceptible isolates, both being more active than OFX and LFX. In conclusion, the new FQ, UB-8902, showed good activity against OFX-resistant isolates. Moreover, it showed better activity than OFX and LFX and was equivalent to MOX against FQ-susceptible clinical isolates. UB-8902 can be considered as a drug with potential antituberculous activity, similar to MOX.
Introduction
Multidrug resistant (MDR) tuberculosis (TB) and extensively drug resistant (XDR)-TB account for ∼450,000 cases/year, being an important problem for disease control. 1 Treatment of MDR-TB and XDR-TB requires an individualized regimen depending on the type of drug resistance, interactions, and toxicity. Fluoroquinolones (FQs) are synthetic molecules, which are considered to be bactericidal antibiotics, and are currently used as second-line drugs in TB treatment. Some FQs are recommended by the World Health Organization (WHO) for MDR-TB therapy, and they have even been investigated for first-line treatment of pulmonary TB.2–4 Among the FQs available, ciprofloxacin and ofloxacin (OFX) are the least active, whereas levofloxacin (LFX), gatifloxacin, and moxifloxacin (MOX) are the most active against Mycobacterium tuberculosis. 5
Quinolone antibiotics inhibit DNA synthesis by targeting two essential type II topoisomerases, DNA gyrase, and topoisomerase IV (Topo IV). Both targets allow one double-stranded DNA molecule to pass through another, followed by religation of the original strand, thereby changing the linking number of DNA by two in each enzymatic step. 6 UB-8902 is a novel FQ, a 7-(4-methyl)-piperazine ciprofloxacin derivate. 7 This compound has previously been shown to have potent activity against other MDR microorganisms such as Acinetobacter baumannii and Stenotrophomona maltophilia.7,8 In addition, UB-8902 has been tested in vitro in three antituberculous drug combinations against MDR and drug-susceptible clinical isolates of M. tuberculosis. 9 However, the effect of this novel FQ against OFX-resistant M. tuberculosis isolates remains unknown. Thus, the main objective of this study was to compare the in vitro antimycobacterial activity of UB-8902 with LFX and MOX against FQ-resistant M. tuberculosis clinical isolates and with OFX, LFX, and MOX against susceptible isolates.
Materials and Methods
M. tuberculosis clinical isolates and inoculum preparation
A total of 22 clinical isolates of M. tuberculosis (11 OFX-resistant and 11 susceptible to OFX and to first line antituberculous drugs) grown from samples obtained from tuberculous patients at the Hospital Clinic of Barcelona were studied. The FQ-resistant isolates analyzed in this study were selected according to their resistance to OFX determined using the proportions method with critical concentration (CC) of ≥1 μg/mL. 10 In addition, the reference strain H37Rv was included in the study as the 11th susceptible strain. The isolates were grown in Löwenstein–Jensen medium slants. Colonies were suspended with sterile distilled water with 5 mm glass beads (Afora, Spain) and vortexed for 45 sec. The supernatant was harvested and adjusted to 0.5 McFarland using a nephelometer and then diluted 1/1,000 to reach an inoculum of ∼5 × 105 colony forming units (CFU)/mL.
Antimicrobial agents
OFX, LFX, and MOX were obtained from Sigma-Aldrich (St. Louis, MO). UB-8902 was provided by Cenavisa S.A. Laboratories (Reus, Tarragona, Spain). All drugs were initially dissolved in 0.1 M NaOH and the solution was serially diluted twofold with distilled water to the appropriate concentrations. The stock solutions were sterilized by filtration and were stored at −20°C until use.
Determination of the minimum inhibitory concentration
The individual minimum inhibitory concentrations (MICs) of OFX, LFX, MOX, and UB-8902 were determined in Middlebrook 7H11 solid medium (Becton-Dickinson, Sparks, MD) supplemented with 10% oleic albumin dextrose catalase (Comercial Bellés, Tarragona, Spain) plates at concentrations ranging from 0.125 to 128 μg/mL in twofold dilutions and incubated at 37°C in 5% CO2 for 21 days. All the experiments were determined in duplicate. As a control, 100 μL of a 1/100 inoculum was seeded on an antibiotic-free Middlebrook 7H11 plate. An isolate was considered resistant if ≥1% of colonies were observed in the drug-containing medium compared with the drug-free medium. For the FQ studied, CCs of 2, 1, and 0.5 μg/mL were considered to be resistant to OFX, LFX, and MOX, respectively (WHO Manual). 10 No CC has been established for UB-8902.
Nucleotide sequencing
The gyrA and gyrB genes (GeneBank number L27512) were sequenced to detect mutations. In brief, 11 OFX-resistant isolates were suspended in water, inactivated at 95°C for 30 min, and amplified. PCR products were purified with Exo-SAP-IT (USB; Affymetrix, Inc., Cleveland, OH). Afterward, both DNA chains were sequenced using the ABI PRISM BigDye® terminator v3.1 cycle sequencing kit (Applied Biosystems, Inc., CA) on an ABI PRISM 3700 automatic sequencer (Applied Biosystems). Nucleotide sequences were analyzed using the Clustal W2 program.
Results
Among the drug-susceptible isolates, the MICs for each drug ranged from 0.5 to 1 μg/mL for OFX and LFX, from <0.125 to 0.5 μg/mL for MOX, and from 0.25 to 0.5 μg/mL for UB-8902 (Table 1). For OFX-resistant isolates, the MICs ranged from 4 to 128 μg/mL for OFX, from 1 to 16 μg/mL for LFX, from 0.25 to 8 μg/mL for MOX, and from 0.5 to 16 μg/mL for UB-8902 (Table 2).
In vitro Activity of the Four Compounds Against Drug-Susceptible Mycobacterium tuberculosis Isolates
S, drug-susceptible isolate.
LFX, levofloxacin; MIC, minimum inhibitory concentration; MOX, moxifloxacin; OFX, ofloxacin.
Minimum Inhibitory Concentration Values and Mutations in the gyrA and gyrB Genes Found in Fluoroquinolone-Resistant Isolates
R, FQ-resistant isolate; bgyrA95 polymorphism.
Five of the 11 (45.4%) OFX-resistant isolates were resistant (MIC > μg/mL) to LFX and UB-8902 and 4 (36.3%) were resistant to MOX. The MIC90 values in OFX-resistant isolates were 128 μg/mL for OFX, 16 μg/mL for LFX and UB-8902, and 4 μg/mL for MOX. The MIC90 values for susceptible isolates were 1 μg/mL for OFX and LFX and 0.25 μg/mL for UB-8902 and MOX.
Five FQ-resistant isolates (45.4%) showed a mutation in the gyrA gene. Three isolates presented a GAC-CAC change at position 94, one showed a GCG-GTG change at position 90, and the last a TCG-CCG change at position 91. The isolates with a mutation at position 94 were resistant to the four quinolones, showing a MIC at least eightfold the MIC90 value for susceptible isolates. The isolate with a mutation at position 90 was resistant to OFX, LFX, and UB-8902, whereas that with a mutation at position 91 was only resistant to OFX. In addition, four isolates showed a polymorphism at position 95 in the gyrA gene, two associated with a resistant mutation at positions 90 and 91, respectively. No mutations was found in the gyrB gene (Table 2).
Discussion
This study demonstrates that the novel FQ UB-8902 is more active than OFX and LFX, and similar to MOX for OFX-resistant isolates, as for drug-susceptible M. tuberculosis isolates.
The results of this study confirm the findings reported for other resistant and susceptible microorganisms. UB-8902 showed intracellular activity against FQ-resistant Staphylococcus aureus isolates. 12 López-Rojas et al. 8 evaluated not only the in vitro activity but also the in vivo efficacy of UB-8902 against A.baumannii isolates in a murine model of pneumonia and peritoneal sepsis. The pharmacokinetic parameters of UB-8902 were similar to MOX and were lower than those for ciprofloxacin, whereas the pharmacodynamic parameters were better than ciprofloxacin. Interestingly, this novel FQ has been tested against MDR and drug-susceptible M. tuberculosis isolates in three antituberculous drug combinations, using an in vitro adaptation of the chequerboard assay. 9 No differences was found between MDR and drug-susceptible isolates and the three combinations were equally effective against M. tuberculosis. 9 According to the results obtained with susceptible isolates (Table 1), we suggest the CC of UB-8902 be established at 0.5 μg/mL, since the MICs obtained were similar to those of MOX.
The acquisition of FQ resistance is mainly due to chromosomal mutations in the gyrA and gyrB genes encoding the DNA gyrase. Amino acid substitutions were described at different positions in the quinolone resistance-determining region of the gyrA and gyrB genes.11–16 Mutations in codons 90 and 94 in the gyrA gene are the most commonly found and are associated with a high level of resistance as was observed in our isolates.11,13,15 Several other substitutions in gyrA (codons 88 and 95) were described as polymorphisms not related to phenotypic resistance.11,13 Four of the isolates in our study presented a gyrA95 polymorphism, and two of them also presented a mutation in codons 90 and 91, respectively. Cross-resistance has been described among FQs. 16 Based on the selection criteria of this study, all the 11 resistant strains were OFX resistant. However, LFX, UB-8902, and MOX were active against OFX-resistant isolates, 6/11 (54.5%) and 7/11 (63.6%), respectively. Mutations at position 94 of the gyrA gene conferred cross-resistance to all four FQs, whereas mutations at positions 90 and 91 of gyrA showed differences among these compounds. GyrB mutations were initially described in an in vitro selected FQ-resistant strain, 17 but they were not reported in M. tuberculosis clinical isolates; however, with the increased use of FQ, some gyrB substitutions have been reported. 18 We did not find any mutation in the gyrB gene.
In conclusion, the new FQ, UB-8902, showed good activity against FQ-resistant isolates and could be considered as a drug with potential antituberculous activity, similar to MOX.
Footnotes
Disclaimer
All of the authors belong to the Study Group of Mycobacterial Infections (GEIM) of the Sociedad Española de Enfermedades Infecciosas y Microbiología Clínica (SEIMC), the Spanish Network for the Research in Infectious Diseases (REIPI) and the research team awarded for quality control by Agència de Gestió d'Ajuts Universitaris i de Recerca (AGAUR, 2014SGR653). ISGlobal is a member of the CERCA Programme, Generalitat de Catalunya.
Disclosure Statement
No competing financial interests exist.
Funding Information
This study was supported by the Ministerio de Economía y Competitividad, Instituto de Salud Carlos III, cofinanced by European Regional Development Fund (ERDF, FEDER) “A Way to Achieve Europe,” the Spanish Ministry of Health (Grant Nos. FIS13/01752, PI16/01047), Planes Nacionales de I+D+i 2013-2016 and Instituto de Salud Carlos III, Subdirección General de Redes y Centros de Investigación Cooperativa, Ministerio de Economía y Competitividad, Spanish Network for Research in Infectious Diseases (REIPI, RD16/0016/0010), cofinanced by European Development Regional Fund “A way to achieve Europe” and operative program Intelligent Growth 2014-2020. This study was also supported by Grant 2017SGR0809 from the Departament d'Universitats, Recerca i Societat de la Informació de la Generalitat de Catalunya and Sociedad Española de Neumología y Cirugía Torácica (grant: SEPAR 210/2015).