Abstract
Background:
Drug susceptibility testing (DST) protocol of omadacycline against non-tuberculous mycobacteria has not yet been established. We developed a method to accurately determine MIC omadacycline MIC against Mycobacterium abscessus (Mab), Mycobacterium avium-complex (MAC), and Mycobacterium kansasii (Mkn).
Methods:
First, we identified the oxyrase concentration not affecting Mab, MAC, and Mkn growth followed by omadacycline MIC experiments with and without oxyrase using reference and clinical strains.
Results:
Oxyrase 0.5 % (v/v) stabilized omadacycline in the culture medium. The median omadacycline MIC was 1 mg/L for Mab and 8 mg/L for Mkn. For MAC, the median omadacycline MIC was 2 mg/L for M. avium, 256 mg/L for M. intracellulare, and 4 mg/L for M. chimaera (p < 0.0001). Wilcoxon matched-pairs signed rank test revealed statistically lower MICs with oxyrase for all MAC subspecies (p < 0.0001), all Mab subspecies (p < 0.0001), and Mkn (p = 0.0002). The decrease in MICs with oxyrase was 17/18 of Mab, 14/19 of Mkn, 8/8 of M. avium, 4/5 M. chimera, but only 11/18 of M. intracellulare (p < 0.013).
Conclusion:
Use of 0.5 % oxyrase could be a potential solution to reliable and reproducible omadacycline MIC of Mab. However, oxyrase demonstrated a variable effect in reducing MICs against MAC and Mkn.
Keywords: Mycobacterium avium-complex, Mycobacterium kansasii, Mycobacterium abscessus, Oxyrase, Minimum inhibitory concentration
1. Introduction
Non-tuberculous mycobacteria (NTM) cause diseases that are recalcitrant to treatment with standard-of-care combination regimens, even with extremely long therapy duration [1-3]. Mycobacterium avium-complex (MAC), M. abscessus species (Mab) and M. kansasii (Mkn) account for >95 % of all NTMs. Tetracyclines are among the recommended drugs to treat Mab pulmonary disease [4,5]. Omadacycline, a third-generation tetracycline, has demonstrated inconsistent (both high and low) MICs for the rapidly growing mycobacterium (RGM), Mab, and high MICs for the slow-growing mycobacteria (SGM) MAC and Mkn, as well as a “trailing effect” [6-11]. Yet, pharmacokinetics/pharmacodynamics (PK/PD) studies where the drug was prepared fresh and dosed once daily demonstrated some of the best microbial kill seen with any agent in the hollow fiber system model of intracellular NTMs, in mouse models, in zebra-fish models, and in the clinic [8,9,12-18]. We traced this problem of high MICs versus good activity in preclinical models and patients and the “trailing effect” phenomenon to the rapid degradation of omadacycline (50 % decrease over 24 h) in broth versus the doubling time of both SGM and RGM. In other words, the drug degrades faster in the medium used for the drug susceptibility testing (DST) at either 30° C or 37° C compared to the doubling time of bacteria [7,8].
The problem of tetracyclines degradation and its impact on DST is not new [19-21]. There are three possible solutions to this: (i) either increase the doubling times of the mycobacteria to the 20 min seen for the commonly encountered bacteria in log-phase growth, (ii) supplement the drug’s concentration daily during the DST assay, or (iii) stabilize the drug so that it does not degrade. The first solution is problematic because the slower rates of growth of the mycobacteria are an accurate reflection of those in the pulmonary lesions of the patients. The second solution of daily supplementation is labor intensive and introduces a lot of opportunities to contaminate the cultures. Here, we opted for the third solution – stabilize omadacycline concentrations during the DST assay.
A biocatalytic oxygen-reducing agent, oxyrase, has been used in the past to optimize the drug susceptibility of macrolides and tetracyclines against Gram-positive and Gram-negative organisms that have a doubling time of minutes [20,21]. More recently, based on our re-analysis of previously published results, Noel et al. showed that the 50% omadacycline degradation in solution over 24 h could be completely prevented by using 2% and 5% oxyrase, which resulted in omadacycline MICs for Escherichia coli and Acinetobacter baumannii multiple-fold lower [22]. Given that because of slow doubling times, NTMs require co-incubation periods of 3 days for RGM and 7 days for SGM to read the MICs, we hypothesized that presence of oxyrase in solution could lead to more accurate MIC determination.
2. Materials and methods
As a representative of the RGMs, ATCC19977 and 18 species-identified Mab clinical isolates were used in the experiments. For the SGM, ATCC700898 strain and 31 species identified clinical isolates of MAC and ATCC12478 and 19 clinical isolates of Mkn were used in the experiments. Omadacycline was purchased from BOC Sciences, NY, USA. The drug was first dissolved in 100% dimethyl sulfoxide (DMSO), followed by further dilution to desired drug concentrations in sterile water (final DMSO concentration <0.01% (v/v)). Oxyrase was purchased from Oxyrase, Inc. (Mansfield, OH). MIC experiments were performed using Cation-adjusted Mueller Hinton broth (CAMHB), whereas Middlebrook 7H10 agar supplemented with 10% OADC (herein termed 7H10 agar) was used for colony forming unit (CFU) determination.
In the first set of experiments, to determine the optimal concentration of oxyrase that does not affect the growth of Mab, MAC, and Mkn inoculum was prepared using the log-phase growth cultures by adjusting the turbidity to McFarland standard 0.5 followed by 1000-fold dilution with an intended bacterial burden of ~4.5 log10 CFU/mL. The oxyrase concentration (v/v) in the culture medium ranged between 0%, 0.5%, 1%, 2%, and 5%. The culture media for the SGM was supplemented with 5% oleic acid-albumin-dextrose-catalase (OADC). Mab cultures were incubated for 72 h at 30°C, whereas MAC and Mkn cultures were incubated at 37°C for 7 days. After the required incubation for RGM and SGM, cultures were washed twice with normal saline, 10-fold serially diluted, and inoculated on 7H10 agar for enumeration of bacterial burden. The CFUs for RGM were recorded 72 h of incubation at 30° C, whereas the data was recorded after 5–7 days of incubation at 37° C for the SGMs. Linear regression was used to calculate the bacterial growth rates, the exponential decline model was used for doubling time calculations, and a one-way analysis of variance was used to compare the growth rate under different experimental conditions. GraphPad Prism (v9) was used for data analysis and graphing.
In the second set of experiments, broth micro-dilution MIC experiment with standard ATCC19977 strain was performed under three different conditions – (i) omadacycline alone, (ii) omadacycline with 50% daily drug supplementation to account for the drug degradation, as reported previously [7], and (iii) omadacycline in the presence of 0.5% oxyrase but no omadacycline daily drug supplementation. The inoculum was prepared as described above. The omadacycline concentrations ranged between 0.25 mg/L to 32 mg/L in a two-fold dilution. At the end of 72 h of incubation at 30° C, 96-well plates were visually inspected, and the drug concentration completely inhibiting the bacterial growth was recorded as MIC. When a trailing effect was present, the concentration showing ~80% reduction in bacterial pellet compared to the nontreated controls was recorded as MIC [10].
In the third set of experiments, we performed omadacycline MIC, with and without 0.5% oxyrase, against the clinical isolates of Mab, MAC, and Mkn. The methods for inoculum preparation, and culture conditions were the same as described above. The drug concentration ranges from 0.25 mg/L to 32 mg/L for Mab and Mkn, whereas the concentration range was 0.25 mg/L to 512 mg/L for MAC. MICs were determined by visual inspection after 72 h of incubation at 30° C for Mab and after 7 days of incubation at 37°C for MAC and Mkn. Data was recorded using Microsoft Excel and used to calculate the omadacycline MIC50 and MIC90 among the NTM clinical isolates.
3. Results
Fig. 1 and Supplementary Online Table S1 shows the effect of different % (v/v) of oxyrase on the growth of each of the mycobacteria. For the Mab in Fig. 1A-as % of oxyrase increased, so did the doubling time, starting with 1% oxyrase. Fig. 1B shows the doubling time of MAC versus oxyrase concentration. The highest dose (5%) killed 92.23 ± 1.81% of 0 h MAC, so that the doubling time would be at infinity. For Mkn, even 0.5% oxyrase increased the doubling time, however, the increase was minimal. At 1% oxyrase, the increase in Mkn doubling time was more notable, and 5% oxyrase killed 23.40 ± 15.58% Mkn. Based on these results and the need for a single oxyrase concentration that could be used in both RGM and SGM experiments, the concentration of 0.5% oxyrase was chosen for MIC studies.
Fig. 1. Oxyrase concentration versus doubling times of nontuberculous mycobacteria.
(A) Mycobacterium abscessus, (B) Mycobacterium avium-complex, (C) Mycobacterium kansasii. The vertical arrows shows that the doubling time with highest oxyrase concentration would be infinity due to the kill of bacteria.
Fig. 2 shows that 0.5% oxyrase eliminated the trailing effect observed for omadacycline MICs for Mab ATCC19977 and achieved a MIC similar to omadacycline with 50% daily drug supplementation.
Fig. 2. Omadacycline drug susceptibility testing using broth microdilution method.
The MIC of omadacycline was one tube dilution lower compared with 50% drug supplementation and in the presence of 0.5% oxyrase compared to omadacycline alone when drug degradation was not accounted for. NS, normal saline.
Omadacycline clinical isolate MIC results were as shown in Table 1 for Mab, Table 2 for MAC, and Table 3 for Mkn. First, MICs were lowest for the RGM compared to SGM, with or without 0.5% oxyrase. The median MIC was 1 mg/L for Mab and 8 mg/L for Mkn. For MAC, there was a species-dependent MIC distribution: the median MIC was 2 mg/L for M. avium, 256 mg/L for M. intracelhilare, and 4 mg/L for M. chimaera (Kruskal-Wallis p < 0.0001).
Table 1.
Omadacycline MIC (mg/L) of Mycobacterium abscessus alone and in the presence of 0.5% oxyrase.
| No | Organism | No Oxyrase | 0.5% Oxyrase |
|---|---|---|---|
| 1 | Mycobacterium abscessus | 1 | 0.25 |
| 2 | Mycobacterium abscessus | 1 | 0.5 |
| 3 | Mycobacterium abscessus subsp massilliense | 0.5 | ≤0.25 |
| 4 | Mycobacterium abscessus subsp massilliense | 1 | 0.5 |
| 5 | Mycobacterium abscessus subsp massilliense | 1 | 0.5 |
| 6 | Mycobacterium abscessus subsp bolletii | 1 | 0.5 |
| 7 | Mycobacterium abscessus subsp massilliense | 0.5 | ≤0.25 |
| 8 | Mycobacterium abscessus | 0.5 | ≤0.25 |
| 9 | Mycobacterium abscessus | 0.5 | ≤0.25 |
| 10 | Mycobacterium abscessus | 1 | 0.5 |
| 11 | Mycobacterium abscessus subsp bolletii | 1 T | 0.5 |
| 12 | Mycobacterium abscessus subsp massilliense | 2 | 1 |
| 13 | Mycobacterium abscessus subsp massilliense | 1 | 0.5 |
| 14 | Mycobacterium abscessus | 0.5 | 0.5 |
| 15 | Mycobacterium abscessus subsp massilliense | 0.5 | ≤0.25 |
| 16 | Mycobacterium abscessus | 1 | 0.5 |
| 17 | Mycobacterium abscessus | 2 | 1 |
| 18 | Mycobacterium abscessus subsp massilliense | 1 T | 0.5 |
T, trailing effect.
Table 2.
Omadacycline MIC (mg/L) Mycobacterium avium complex clinical isolates (n = 31) alone and in the presence of 0.5 % oxyrase.
| Lab ID | Isolate | No Oxyrase |
0.5% Oxyrase |
|---|---|---|---|
| MAC_UTHCT_1 | Mycobacterium avium | 2 | 0.5 |
| MAC_UTHCT_2 | Mycobacterium avium | 2 | 0.5 |
| MAC_UTHCT_3 | Mycobacterium avium | 2 | 0.5 |
| MAC_UTHCT_4 | Mycobacterium avium | 4 | 2 |
| MAC_UTHCT_5 | Mycobacterium avium | 4 | 2 |
| MAC_UTHCT_6 | Mycobacterium avium | 2 | 2 |
| MAC_UTHCT_7 | Mycobacterium avium | 16 | 0.5 |
| MAC_UTHCT_8 | Mycobacterium avium | 2 | 1 |
| MAC_IC_UTHCT_9 | Mycobacterium avium intracellulare | 512 | 256 |
| MAC_IC_UTHCT_10 | Mycobacterium avium intracellulare | 512 | 256 |
| MAC_IC_UTHCT_11 | Mycobacterium avium intracellulare | 8 T | 4 |
| MAC_IC_UTHCT_12 | Mycobacterium avium intracellulare | 128 | 64 |
| MAC_IC_UTHCT_13 | Mycobacterium avium intracellulare | 256 | 256 |
| MAC_IC_UTHCT_14 | Mycobacterium avium intracellulare | 256 | 256 |
| MAC_IC_UTHCT_15 | Mycobacterium avium intracellulare | 512 | 256 |
| MAC_C_UTHCT_16 | Mycobacterium avium chimaera | 4 T | 2 |
| MAC_C_UTHCT_17 | Mycobacterium avium chimaera | 8 T | 2 |
| MAC_C_UTHCT_18 | Mycobacterium avium chimaera | 4 T | 2 |
| MAC_C_UTHCT_19 | Mycobacterium avium chimaera | 4 T | 2 |
| MAC_C_UTHCT_20 | Mycobacterium avium chimaera | 256 | 256 |
| MAC_CI_UVA | Mycobacterium avium intracellulare | 512 | 256 |
| MAC_C_UTHCT_21 | Mycobacterium avium intracellulare | 512 | 256 |
| MAC_C_UTHCT_22 | Mycobacterium avium intracellulare | 256 | 256 |
| MAC_C_UTHCT_23 | Mycobacterium avium intracellulare | 512 | 256 |
| MAC_C_UTHCT_24 | Mycobacterium avium intracellulare | 512 | 256 |
| MAC_C_UTHCT_25 | Mycobacterium avium intracellulare | 512 | 256 |
| MAC_C_UTHCT_26 | Mycobacterium avium intracellulare | 256 | 256 |
| MAC_C_UTHCT_27 | Mycobacterium avium intracellulare | 256 | 16 |
| MAC_C_UTHCT_28 | Mycobacterium avium intracellulare | 256 | 256 |
| MAC_C_UTHCT_29 | Mycobacterium avium intracellulare | 256 | 256 |
| MAC_C_UTHCT_30 | Mycobacterium avium intracellulare | 256 | 256 |
T, trailing effect.
Table 3.
Omadacycline MIC (mg/L) of Mycobacterium kansasii (n = 19) alone and in the presence of 0.5 % oxyrasea.
| Lab ID | Isolate | No Oxyrase | 0.5 % Oxyrase |
|---|---|---|---|
| UVA_2 | Mycobacterium kansasii | 8 | 4 |
| TY_2 | Mycobacterium kansasii | 8 T | 8 |
| TY_3 | Mycobacterium kansasii | 8 T | a |
| TY_4 | Mycobacterium kansasii | 8 T | 8 |
| TY_5 | Mycobacterium kansasii | 8 T | 4 |
| TY_6 | Mycobacterium kansasii | 16 T | 8 |
| TY_7 | Mycobacterium kansasii | 16 T | 8 |
| TY_8 | Mycobacterium kansasii | 8 T | 8 |
| TY_9 | Mycobacterium kansasii | 8 T | 8 |
| TY_10 | Mycobacterium kansasii | 16 T | 8 |
| TY_11 | Mycobacterium kansasii | 8 T | 4 |
| TY_12 | Mycobacterium kansasii | 8 T | 2 |
| TY_13 | Mycobacterium kansasii | 8 T | 2 |
| TY_14 | Mycobacterium kansasii | 8 T | 4 |
| TY_15 | Mycobacterium kansasii | 8 | 4 |
| TY_16 | Mycobacterium kansasii | 8 | 4 |
| TY_17 | Mycobacterium kansasii | 8 | 4 |
| TY_18 | Mycobacterium kansasii | 4 | 4 |
| TY_19 | Mycobacterium kansasii | 16 T | 8 |
Inconclusive MIC; T, trailing effect.
Second, a trailing effect was seen in 2 of 18 (11.11%) isolates of the RGM (Mab) versus 19 of 50 (38.00%) of SGM (p = 0.04). When considered by NTM species, the proportion with trailing effect was similar between Mab (11.11%) and MAC (16.13%) (p = 0.628) but was highest in Mkn (73.68%) (p < 0.0001). Thus, the trailing effect could be eliminated in all three bacterial complexes by 0.5% oxidase, albeit subspecies differences exist.
Third, a pairwise comparison of omadacycline MICs without oxyrase versus with 0.5% oxyrase using the Wilcoxon matched-pairs signed rank test is shown in Table 4. Table 4 revealed a statistically lower MIC with the oxyrase for MAC across all species (p < 0.0001), all Mab subspecies (p < 0.0001), and Mkn (p = 0.0002). This means that 0.5% oxyrase consistently lowered MICs. The effect was seen across all subspecies. As regards to tube dilution changes, there was no instance in which 0.5% oxyrase increased MICs, but otherwise had either no effect in 13 of 63 MICs or decreased MICs in 50 of 63 cultures. The proportion of isolates in which there was a decrease in MICs with 0.5 % oxyrase by tube-dilution was 17/18 of Mab, 14/19 of Mkn, 9/9 of M. avium, 4/4 M. chimerea, but 12/21 of M. intracelhilare (p < 0.013). For MAC, there was a statistically significant tube dilution difference by species with the least changes in M. intracelhilare (p = 0.008).
Table 4.
Wilcoxon matched-pairs signed rank test for MICs with and without 0.5% oxyrase.
| Difference of medians | P | |
|---|---|---|
| All Mycobacterium avium complex (n = 31) | −2.0 | <0.0001 |
| M. avium (n = 8) | −1.5 | 0.0156 |
| M. intracellulare (n = 19) | −64.0 | 0.001 |
| M. chimaera (n = 5) | −2.0 | 0.06 |
| All Mycobacterium abscessus (n = 18) | −0.5 | <0.0001 |
| MAB subspecies abscessus (n = 8) | −0.5 | 0.0156 |
| MAB subspecies massilliense (n = 9) | −0.5 | 0.0039 |
| Mycobacterium kansasii (n = 18) | −4.0 | 0.0002 |
4. Discussion
For NTMs, MIC breakpoints have been established only for select drugs used to treat NTM [23,24]. Further, the correlation between the in vitro DST and clinical outcomes is often lacking. Complicating the situation are inconsistent treatment guidelines and recommendations from the American Thoracic Society-Infectious Diseases Society of America (ATS-IDSA) and the British Thoracic Society (BTS) on performing DST of the drugs. For example, as per the BTS guidelines, DST for Mab is recommended for clarithromycin, cefoxitin, and amikacin; and for tigecycline, imipenem, minocycline, doxycycline, moxifloxacin, linezolid, co-trimoxazole, and clofazimine if a validated method is available to guide the treatment regimens [4]. However, the ATS-IDSA guidelines do not recommend DST for minocycline, doxycycline, moxifloxacin, or co-trimoxazole [5,25].
Here, we show that 0.5% oxyrase did not change the doubling time of Mab, eliminated the trailing effects, and reduced omadacycline MICs for Mab clinical isolates. Thus, in the case of Mab and its subspecies, the approach used here can be incorporated into DST. Our findings reconfirm studies performed decades ago, showing a shift in MIC when oxyrase was used in the experiments with rapidly growing Gramnegative bacilli [21,26]. Therefore, “top-up” or daily supplementation of the antibiotic to compensate for the loss in the drug activity was suggested [26]. Elsewhere [7] and in this manuscript, we showed that 50% daily omadacycline supplementation resulted in lower MIC. However, daily drug supplementation for NTM DST may not be practical in clinical microbiology laboratories.
For SGM, there was inhibition of MAC growth starting at oxyrase concentrations of 1%, while 5% oxyrase killed MAC. For Mkn, even 0.5% oxyrase increased the doubling time up to 2.4-fold, while 5% killed Mkn. The overall omadacycline MICs were higher for SGM than for the RGM’s MIC; as these were read at 7 days and beyond. When coincubated with 0.5% oxyrase, the median MIC decrease in tube dilutions was one for Mkn, with a moderate percent coefficient of variation (%CV). For MAC, there were differences in omadacycline MIC by species, with the least changes seen with M. intracellulare, which also had a high %CV. This means that M. intracellulare exhibited a heterogenous MIC response to 0.5% oxyrase. Taken together, our results suggest that the protective effects of 0.5% oxyrase on omadacycline degradation do not last for the duration of the assay (which is read on day 7 and beyond) for the SGM. This means that further work needs to be done to identify other chemical inhibitors for omadacycline degradation that can be applied to SGM.
Another important finding was the species differences for omadacycline MICs encountered within the MAC complex. The high omadacycline MICs in MAC were driven by M. intracellulare which had median MICs of 256 mg/L versus 2 mg/L for M. avium, and 4 mg/L for M. chimaera. Further, the addition of 0.5% oxyrase demonstrated no effect in 38.1% of M. intracellulare versus 0% of other species; the decrease in MIC was most heterogeneous in M. intracellulare as well. These findings suggest that either this species is more resistant to omadacycline compared to other MAC species or, more likely, the interplay of M. intracellulare growth rates versus omadacycline degradation rate. This also means that M. intracellulare will need special DST assays developed for it, not only for omadacycline but also for other tetracyclines.
One limitation of our study is that we did not test how long oxyrase could prevent omadacycline degradation in the culture medium. Likely, the effect is short-lived due to denaturation of the enzyme itself in CAMHB or decreased biocatalytic oxygen-reduction with time. Such information could improve omadacycline DST of M. tuberculosis, a pathogenic mycobacterium that requires even longer incubation compared to the SGM MIC we recorded after seven days of incubation. Second, even at 0.5% concentration, the enzyme had an “antibacterial” effect on SGM, so the omadacycline MIC is really more of a drug interaction index (additivity) similar to the checkerboard method.
In summary, omadacycline that has shown excellent antimicrobial activity against Mab, MAC, and Mkn in vitro, in vivo, and in clinics, still lacks a reliable DST method due to drug degradation. We propose the addition of 0.5% oxyrase in the culture medium as a solution to produce reliable and reproducible omadacycline MICs for Mab.
Supplementary Material
Acknowledgement
We thank Barbara Brown-Elliot and Kavya Somji, Mycobacteria Nocardia Lab UT Health Science Center at Tyler, Texas, USA, for providing the species-identified NTM clinical isolates. We also thank Drs. Alisa Serio and Holly Hoffman for an impromptu discussion on oxyrase with Shashikant Srivastava that prompted him to design this study.
FUNDING source
Shashikant Srivastava is supported by funding from the Eunice Kennedy Shriver National Institute of Child Health and Human Development (1R01HD099756), 1R21AI148096 and 1R01AI179827 from the National Institute of Allergy and Infectious Diseases, KANT23G0 from the Cystic Fibrosis Foundation, the University of Texas System (STARS award #250439/39411), and NTM Education and Research funding support from the Vice President of Research, University of Texas at Tyler.
Footnotes
CRediT authorship contribution statement
Gunavanthi D. Boorgula: Writing – review & editing, Visualization, Methodology, Data curation. Tawanda Gumbo: Writing – review & editing, Writing – original draft, Validation, Formal analysis. Sanjay Singh: Writing – review & editing, Validation, Methodology, Data curation. Pamela J. McShane: Writing – review & editing. Julie V. Philley: Writing – review & editing. Shashikant Srivastava: Writing – review & editing, Writing – original draft, Validation, Supervision, Resources, Project administration, Funding acquisition, Formal analysis, Conceptualization.
Declaration of competing interest
Tawanda Gumbo founded and is president and CEO of Praedicare Inc., a System of Systems Drug Development company, and founded Praedicare Africa, a clinical contract research organization. Julie V Philley is an advisor for Insmed, Paratek and AN2; a research investigator for Insmed, Paratek, AN2 and Zambon. All other authors have nothing to declare.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.org/10.1016/j.tube.2024.102519.
Data availability statement
Upon a reasonable request, the raw data for the results presented in the manuscript is available with the corresponding authors.
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Data Availability Statement
Upon a reasonable request, the raw data for the results presented in the manuscript is available with the corresponding authors.