In vitro activity of methylene blue on Mycobacterium tuberculosis complex isolates
doi • 10.5578/tt.20219819 Tuberk Toraks 2021;69(2):279-284
Geliş Tarihi/Received: 14.12.2020 • Kabul Ediliş Tarihi/Accepted: 30.05.2021
Deniz GAZEL1(ID) Özlem ZANAPALIOĞLU GAZEL2(ID)
1 Department of Medical Microbiology, Gaziantep University Faculty of Medicine, Gaziantep, Turkey
1 Gaziantep Üniversitesi Tıp Fakültesi, Tıbbi Mikrobiyoloji Anabilim Dalı, Gaziantep, Türkiye
2 Clinic of Infectious Diseases and Clinical Microbiology, Cengiz Gökçek Gynaecology, Obstetrics and Paediatrics State Hospital, Gaziantep, Turkey
2 Cengiz Gökçek Kadın Hastalıkları ve Doğum Hastanesi, Enfeksiyon Hastalıkları ve Klinik Mikrobiyoloji Kliniği, Gaziantep
SHORT REPORT KISA RAPOR
ABSTRACT
In vitro activity of methylene blue on Mycobacterium tuberculosis complex isolates
Methylene blue is used for bacterial staining in microbiology and as an anti- dote drug in medicine. In this study, we aimed to investigate the antimicrobi- al effects of methylene blue against Mycobacterium tuberculosis complex clinical isolates. Seventeen stored M. tuberculosis complex clinical isolates were included in the study. The isolates were inoculated into Mycobacteria Growth Indicator Tubes and incubated in Automated Mycobacterial Detection System. Mycobacteria Growth Indicator tubes containing methy- lene blue at critical concentrations of 0.2, 2, 20, 1000 µg ml-1 and control tube were prepared. Antimicrobial susceptibility testing was performed using Automated Mycobacterial Detection System which is gold standard for sec- ond line anti-tuberculosis drug testing. At the end of the study, six clinical isolates were susceptible to methylene blue at all critical concentrations. Five isolates were susceptible to only 1000 µg ml-1 methylene blue. Three isolates were susceptible to 1000 and 20 µg ml-1 methylene blue. Susceptibility rate was found as 94% when the critical proportion was accepted 400 GU (1/100 of control). Significant relationship was observed between the admin- istered methylene blue concentrations and bacterial survival rate in statistical analysis. We conclude that methylene blue may become a potential anti-tu- berculosis agent due to its well-known side effects and dosing regimens.
Key words: Mycobacterium tuberculosis complex; methylene blue; antimicro- bial; susceptibility; tuberculosis
Dr. Deniz GAZEL
Gaziantep Üniversitesi Tıp Fakültesi, Tıbbi Mikrobiyoloji Anabilim Dalı, GAZİANTEP - TÜRKİYE
e-mail: denizgazel@yahoo.com
Yazışma Adresi (Address for Correspondence)
Cite this article as: Gazel D, Zanapalıoğlu Gazel Ö. In vitro activity of methylene blue on Mycobacterium tuberculosis complex isolates. Tuberk Toraks 2021;69(2):279-284.
©Copyright 2021 by Tuberculosis and Thorax.
Available on-line at www.tuberktoraks.org.com
INTRODUCTION
The ongoing global burden of TB and the emergence of multi-drug-resistant (MDR) Mycobacterium tuber- culosis complex have led to increased use of sec- ond-line anti-TB drugs (1). Treatment of patients infected with primary anti-TB drug-resistant M. tuber- culosis complex microorganisms, including MDR isolates constitutes a real clinical challenge. Moreover, anti-tuberculosis agents against the MDR M. tubercu- losis complex remain very limited (2).
Methylene blue (MB), also known as methylthionini- um chloride, is a cationic thiazine dye which can be used as a drug for various indications in medicine (3,4). It is a medication in clinical science and a microbiological dye in laboratory medicine. It may be helpful in anaphylactic shock, and it has helped to treat hypotension related to lithium toxicity, ACE inhi- bition, and haemodialysis (3,4). In this study we aimed to investigate the inhibitory effect of MB com- pound against M. tuberculosis complex clinical iso- lates in our university hospital.
MATERIAL and METHODS Isolate Selection
Seventeen non-repetitive M. tuberculosis complex clinical isolates which were obtained from tuberculo- sis patients in our university hospital were included in the study. During routine laboratory work, specimens were cultured using the BACTEC Mycobacteria Growth Indicator Tube (MGIT) 960 Automated Mycobacterial Detection System (AMDS) (Becton Dickinson, USA). Positive cultures were identified by microscopy (Ziehl-Neelsen stain) and the immune chromatographic method (detecting MPT64 antigen) using MGIT TBc Identification Test Kit (Becton
Dickinson, USA). Antibiogram tests for first-line drugs were also implemented by AMDS. All strains were isolated from the pulmonology clinic in our universi- ty hospital between 1st January 2018 and 1st January 2019 and stored at -20°C prior to the study.
Study Design
MGIT 960 AMDS system was modified for MB testing and 0.2, 2, 20 and 1000 µg ml-1 concentrations of MB were used instead of the critical concentrations of the four first-line anti-tuberculosis drugs (Figure 1).
In the cultivation stage, stored isolates were inoculat- ed into MGIT tubes and incubated until the AMDS gave a positive signal for growth. Then four MGIT tubes containing MB blue at concentrations of 0.2, 2, 20 and 1000 µg ml-1 were prepared (Figure 1). On the next day, four testing tubes were inoculated from the positive MGIT tube according to the manufactur- er’s recommendations in accordance with the MGIT protocol of susceptibility testing for first-line anti-tu- berculosis drugs (5). A control tube (1/100 dilution) was prepared in the same way. When the diluted control tube reached 400 growth units (GU) in the incubator, the AMDS gave a positive signal and the results were interpreted. If the growth rate was >100 GU in an MB tube, the isolate was determined to be resistant to this concentration of MB. If the growth rate was ≤100 GU, it was determined to be suscepti- ble (5). If the growth rate was between 100 and 400 GU, the result was determined as borderline. M.
tuberculosis H37Rv (ATCC 27294) standard strain was used as control in the study. Broth proportion method using AMDS (our method) is currently rec- ommended as the gold standard for second-line drug susceptibility testing by the World Health Organisation (WHO) (6).
ÖZ
Metilen mavisinin Mycobacterium tuberculosis kompleks izolatlarına in vitro etkisi
Metilen mavisi mikrobiyolojide bakteri boyası ve tıpta antidot ilaç olarak kullanmaktadır. Bu çalışmada, metilen mavisinin Mycobacterium tuberculosis kompleks klinik izolatlarına karşı antimikrobiyal etkilerinin araştırılması amaçlanmıştır. Önceden saklamaya alınmış 17 Mycobacterium tuberculosis kompleks klinik izolatı çalışmaya alındı. İzolatlar Mycobacteria Growth Indicator Tubes (MGIT) besiyerle- rine inoküle edildi ve akabinde Otomatize Mikobakteri Tanımlama Sistemi (OMTS)’nde enkübasyona kaldırıldı. Her izolat için 0.2, 2, 20 ve 1000 µg ml-1 kritik konsantrasyonlarında metilen mavisi içeren MGIT tüpleri ve kontrol tüpü hazırlandı. Antibiyogramlar, ikinci seçe- nek anti-tüberküloz ilaç duyarlılık testleri için altın standart kabul edilen OMTS kullanılarak yapıldı. Çalışma sonunda, altı klinik izolat tüm kritik konsantrasyonlarda metilen mavisine duyarlı bulundu. Beş izolat ise sadece 1000 µg ml-1 konsantrasyonda metilen mavisine duyarlı olarak tespit edildi. Üç izolat 1000 ve 20 µg ml-1 kritik konsantrasyonda metilen mavisine duyarlı bulundu. Kritik proporsiyon 400 GU (kontrolün 1/100’ü) kabul edildiğinde metilen mavisine duyarlılık oranı %94 olarak hesaplandı. İstatistiki analizde, uygulanan metilen mavisi konsantrasyonları ile bakteriyel sağkalım oranı arasında anlamlı bir ilişki gözlemlendi. Metilen mavisi, bilinen yan etkileri ve var olan doz rejim protokolleri nedeniyle, potansiyel bir anti-tüberküloz ajan olabilir.
Anahtar kelimeler: Mycobacterium tuberculosis kompleksi; metilen mavisi; antimikrobiyal; duyarlılık; tüberküloz
Statistical analysis
Univariate Binary Logistic Regression Analysis was used to investigate the relationship between MB con- centrations and bacterial resistance (survival). Relative risks were estimated with 95% confidence interval.
Statistical analysis was performed using SPSS for Windows version 24.0 and a p value <0.05 was accepted as statistically significant.
RESULTS
According to previous AMDS antibiogram results which were obtained during routine laboratory work (before the study), two isolates (12%) were multi-drug-resistant, five isolates (29%) were mono-drug-resistant and ten isolates (59%) were sus- ceptible to all first-line anti-tuberculosis agents.
Antibiogram patterns for primary (first-line) anti-tu- berculosis drugs are shown in Table 1.
In the testing stage for MB, six isolates (two mono-drug-resistant and four susceptible) were found to be susceptible to MB at all concentrations (0.2 to 1000 µg ml-1). Five isolates (four susceptible and one mono-drug-resistant) were susceptible to only 1000 µg ml-1 of MB concentration. Three isolates (two mono-drug-resistant and one susceptible) were sus- ceptible to 1000 µg ml-1 and 20 µg ml-1 MB concen-
trations, and three isolates (two multi-drug-resistant and one susceptible) were resistant to all concentra- tions of MB if the critical proportion was accepted as 100 GU. In our study, 1000 µg ml-1 concentration of MB inhibited 82% of the Mycobacterium tuberculosis complex isolates. If the two borderline resistant clin- ical isolates (<400 GU and >100 GU) are accepted as susceptible, the rate of inhibition rises to 94% at the 1000 µg ml-1 concentration. M. tuberculosis H37Rv reference strain (control) was found susceptible to MB only at the 1000 µg ml-1 concentration. The results are summarised in Table 1.
A significant relationship was observed between the administered MB concentrations and bacterial resis- tance (survival) at the Univariate Binary Logistic Regression Analysis (p= 0.001). The risk of resistance development to MB (bacterial survival) was approxi- mately 23 times higher (RR= 22.86, 95% CI= 2.44- 214.55, p= 0.006) when 0.2 µg ml-1 MB concentra- tion is compared to 1000 µg ml-1 MB concentration.
The risk of resistance development was 18 times higher (RR= 18, 95% CI= 1.93-167.99, p= 0.011) when 2 µg ml-1 MB concentration was compared to 1000 µg ml-1 MB concentration. No significant differ- ence was observed between the 20 and 1000 µg ml-1 MB concentrations.
Figure 1. MGIT 960 antibiogram tubes containing increasing concentrations of methylene blue.
*MGIT 960 tubes containing 0.2, 2, 20, and 1000 µg ml-1 methylene blue and growth control (left to right).
DISCUSSION
Pal et al. have conducted a research on Candida albi- cans and Mycobacterium smegmatis and stated that MB alone inhibited the growth of Mycobacterium smegmatis at 15.62 µg ml-1 concentration in a bacte- riostatic manner similar to its fungistatic characteris- tic (Myco-bacteria means fungus-like bacteria in Greek) (7). They have also reported that MB was leading to impaired cell surface phenotypes, altered colony morphologies, and DNA damage in Mycobacteria. They have suggested performing fur- ther investigations on Mycobacterium tuberculosis since this pathogen contains unique cell envelope components with complex lipids providing pathoge- nicity (7). In a previous study, Walter-Sac et al. have reported that MB plasma concentration reached 2 µg ml-1 after oral intake of 500 mg MB and remained above this level for more than five hours in healthy individuals. They have also shown that MB plasma concentration reached 0.2 µg ml-1 after intravenous
injection of 50 mg MB and remained above this level for more than seven hours (8). In a study investigating the pharmacokinetics and organ distributions of intra- venous and oral methylene blue, it has been shown that much higher concentrations of MB were reached in some organs than in blood. In the animal models, 20-fold higher concentrations than in blood were found in the brain following administration of intrave- nous MB (9).
When we analysed the findings of our research, the critical concentration of MB at 2 µg ml-1 which inhib- ited 35% of our study isolates could have potential as an alternative anti-tuberculosis drug. When we accepted 20 µg ml-1 as a target critical MB concen- tration, 53% of the isolates was inhibited by adminis- tration of MB. If we took three isolates (isolate no: 7, 14, and 16) with borderline inhibition (103, 103, and 102 GU) into consideration, the rate of susceptibility rose to 70% at the 20 µg ml-1 critical concentration.
Since the proportion method accepts the resistance Table 1. Antibiogram profiles of the isolates for first-line drugs and MB compound via Automated Mycobacterial Detection System
First line drugs
Growth units of isolates at critical concentrations of methylene blue
No: STR INH RIF ETA 1000 µg ml-1 20 µg ml-1 2 µg ml-1 0.2 µg ml-1
1 S S S S 0 0 0 0
2 S S S S 6 0 0 0
3 R R R R 160 400 400 400
4 S S S S 0 0 0 0
5 S R R S 400 400 400 400
6 R S S S 0 8 143 357
7 S R S S 7 103 400 400
8 S S R S 0 0 0 8
9 R S S S 0 9 400 400
10 S S S S 7 0 6 0
11 S R S S 0 1 0 66
12 S S S S 0 400 400 400
13 S S S S 0 400 400 400
14 S S S S 0 103 400 400
15 S S S S 305 400 400 400
16 S S S S 8 102 136 400
17 S S S S 62 85 400 400
ATCC 27294 S S S S 0 400 400 400
*STR: Streptomycin, INH: Isoniazid, RIF: Rifampicin, ETA: Ethambutol.
** ATCC 27294: M. tuberculosis H37Rv strain.
*** Green boxes: Susceptible (≤100 GU), Yellow boxes: Intermediate (>100 GU and <400 GU), Red boxes: Resistant (≥400 GU).
**** S: Susceptible, R: Resistant.
breakpoint as 1% (equal to 400 GU) of the control population, these borderline values may be accepted as susceptible (10). Second, the talent of MB to cross the blood-brain barrier and diffuse inside the brain at higher concentrations than blood could make MB attractive as a potential therapeutic agent for central nervous system infections such as TB meningitis or TB encephalitis (9). Comparing the doses of topical MB preparations (11) with our results, 1000 µg ml-1 con- centration of MB inhibited 82% of the M. tuberculo- sis complex isolates in our study. If the two borderline resistant clinical isolates (<400 GU and >100 GU) are accepted as susceptible, the rate of inhibition rises to 94% at 1000 µg ml-1 concentration. So, top- ical preparations containing higher MB concentra- tions could be investigated for treatment of cutane- ous infections due to M. tuberculosis complex in the future.
Our study has some limitations to be mentioned. As a limitation, when we continued incubating the iso- lates in AMDS for one more week, we observed re-growth of bacteria in the MGIT tubes which were evaluated as MB susceptible. So, the effect of MB could be a bacteriostatic effect which may limit the use of this agent against most of the tuberculosis cases. In addition, our study isolates were collected from patients with pulmonary tuberculosis disease and may be different than the isolates causing cuta- neous tuberculosis. Another problem is that most of the cutaneous infections are caused by Mycobacteria other than tuberculosis which were excluded in our study. The treatment of cutaneous infections is car- ried out systemically for a long period of time; how- ever, the long-term side effects of MB are not yet clear. Finally, the results that we found in our study are preliminary data which need further investigation in the aspects of safety use and in vivo effectiveness of MB. Thus, novel combination studies with more isolates should be performed to see the real anti-tu- berculosis activity of methylene blue compound.
CONCLUSION
In our study, we found significant relationship between the MB concentrations and bacterial surviv- al. In addition, when the critical proportion was accepted as 400 GU, MB was found effective on 94% of the isolates. Further in vitro and in vivo inves- tigations are needed to understand the effects and potential of methylene blue as a novel anti-tubercu- losis drug in the future.
Acknowledgements
Competing interests: The author declares there are no competing interests. Funding: No public or private funding/grant was received for this study. Assistance:
I would like to thank the laboratory technicians, Şükran Buğur and Betül Gürler for their technical assistance during the study and Prof. Dr. Gönül Aslan for providing us M. tuberculosis reference strain.
Proofreading: Proofreading was performed by Anchor English Editing Service.
Ethical Committee Approval: Ethical approval was obtained from the Clinical Research Ethics Committee of Gaziantep University (Decision No: 2018/381, Date: 18.12.2018).
CONFLICT of INTEREST
The authors reported no conflict of interest related to this article.
AUTHORSHIP CONTRIBUTIONS Concept/Design: DG
Analysis/Interpretation: DG, ÖZG Data Acqusition: DG
Writing: DG, ÖZG
Clinical Revision: DG, ÖZG Final Approval: DG, ÖZG
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