Minimum Bactericidal Concentration Techniques in Mycobacterium tuberculosis : A Systematic Review

Abstract

Minimum bactericidal concentration (MBC) assay is an accepted parameter for evaluating new antimicrobial agents, and it is frequently used as a research tool to provide a prediction of bacterial eradication. To the best of our knowledge, there is no standardization among researchers regarding the technique used to detect a drug's MBC in Mycobacterium tuberculosis. Thus, the aim of this systematic review is to discuss the available literature in determining a drug's MBC in M. tuberculosis, to find the most commonly used technique and standardize the process. A broad and rigorous literature search of three electronic databases (PubMed, Web of Knowledge, and LILACS) was performed according to the PRISMA statement. We considered studies that were published from January 1, 1990 to February 19, 2019. Google Scholar was also searched to increase the number of publications. We searched for articles using the MeSH terms “microbiological techniques,” “Mycobacterium,” “antibacterial agents.” In addition, free terms were used in the search. The search yielded 6,674 publications. After filter application, 5,348 publications remained. Of these, we evaluated the full text of 187 publications. By applying the inclusion criteria, 69 studies were included in the present systematic review. In the literature analyzed, a great variety in the techniques used to determine a drug's MBC in M. tuberculosis was observed. The most common variability is related to the culture media used, culture incubation time, and the percentage of bacterial death for the drug to be considered as bactericidal. The most commonly used technique for drug's MBC determination was carried out using the drug's minimum inhibitory concentration (MIC) assay. Aliquots from prior MIC values were subcultured in Middlebrook agar and incubated for 4 weeks at 35°C for determining the colony forming unit (CFU) with relevance to detect 99.9% bacilli killed or reduction in 3 log₁₀ viable bacilli.

Introduction

Tuberculosis (TB) is still a global health problem with 10 million cases estimated in 2018.¹ The global disease context, in the world, has worsened in recent years due to the increase of multidrug resistance (MDR) (resistance to at least rifampicin and isoniazid) and extensively drug resistance (MDR-TB cases added of resistance to a fluoroquinolone and one of the three second-line injectable drugs, such as amikacin, kanamycin, or capreomycin). In 2018, an estimated half a million (417,000–556,000) new cases of rifampicin-resistant TB (with 78% multidrug-resistant TB) were related.¹

The increase of MDR-TB cases, intense drug side effects, and high rates of treatment abandonment pose difficulties to be overcome in the strategies to fight TB, and increase the need to look for new compounds that have anti-Mycobacterium tuberculosis activity.² For this purpose, the use of assays that are focused to determine the capacity of a tested compound to kill M. tuberculosis, as determination of minimum bactericidal concentration (MBC), is of paramount importance.³

MBC determination is an accepted parameter for evaluating new antimicrobial agents, and it is frequently used as a research tool to provide prediction of bacterial eradication.⁴ It requires subculture, in agar, of a bacterial suspension previously exposed to specific drugs and enumeration of survivor bacterial colonies during early drug screening. From this point of view, bactericidal screening would have greater physiological relevance, once the lack of viability is the real indicator of bacterial death.⁵ Specifically, in the case of TB, the bactericidal activity of drugs is their ability to kill the largest number of M. tuberculosis as soon as possible, leading to a decrease in the infectivity of a TB index case.⁵

In the literature, a variety of techniques are used to determine a drug's MBC in M. tuberculosis. The most common variability in the techniques is related to the use of micro- or macrodilutions before carrying out the MBC assay, the culture media, incubation times, and the percentage of bacterial death detection used. To the best of our knowledge, there is no standardization, among researchers, regarding a technique to be used to determine a drug's MBC in M. tuberculosis. Thus, the aim of this systematic review is to discuss the available literature in determining a drug's MBC in M. tuberculosis, to find the most commonly used assay and standardize the process.

Materials and Methods

Search strategy and selection criteria

A systematic review following the PRISMA guidelines (preferred reporting items for systematic reviews and meta-analyses) was performed.⁶ We included experimental and observational predictive study designs, in Portuguese, Spanish, or English, in which bactericidal techniques in M. tuberculosis were analyzed, from January 1, 1990 to February 19, 2019. We excluded systematic reviews and meta-analysis studies, case reports, case series, guidelines, editorial, comments, as well as conference abstracts.

The study domain was restricted to bactericidal techniques and bacterial time–kill (TK) curve. We compared the techniques used by each author, and excluded studies restricted to specific minimum inhibitory concentration (MIC) determination, studies with other bacteria, and those that used clinical samples (Fig. 1).

FIG. 1.

Flowchart of the different phases of the systematic review. Source: Moher et al.⁸³

We developed the search strategy and definition of MeSH terms, in the first stage, by five researchers (A.L.I., E.G.S., H.C.C., N.C.d.S.S., TSS—Group 1). The descriptors were defined, independently, by groups of researchers 1 and 2, and validated by two specialist reviewers (J.J.V.T. and R.F.C.) by consensus.

The second step was to search for abstracts of potentially eligible articles, by Group 1 researchers, from the following databases: PubMed, Latin American and Caribbean Health Literature in Health Sciences (LILACS), Web of Science, and Google Scholar system. For PubMed, we used MeSH terms “microbiological techniques” (Block 1); “Mycobacterium” (Block 2); and “antibacterial agents” (Block 3). In addition, free terms were used in the search. Two specialist reviewers (J.J.V.T. and R.F.C.) approved the blocks. For Web of Science, we used a title search strategy with the same descriptors. For LILACS, the search was carried out with the following descriptors in Portuguese, providing total reproducibility: Block 1—técnicas microbiológicas [Descritor de assunto]; Block 2—Mycobacterium [Descritor de assunto] OR Mycobacterium tuberculosis [Descritor de assunto] and Block 3—agentes antibacterianos [Descritor de assunto]; to ensure greater accuracy. We then combined the three blocks to provide the largest number of abstracts. Duplicates were removed.

Data extraction and assessment of quality

The third stage consisted of screening the abstracts, getting the full-text articles PDFs, and performing bias assessments by Group 1 (A.L.I., E.G.S., H.C.C., N.C.S.S., T.S.S.) in pairs. Disagreements between two reviewers were discussed and solved by consensus. The extracted information of each publication was structured in the format of tables by Group 1 researchers. The following information was included in the tables: reference, publication year, kind of study, study with clinical isolates or reference strains, incubation time, culture medium, previous methodology, percentage of bacilli killed (%), and time of assay reading. In the fourth stage, five judges (L.D.G.L., P.A.Z.C.-S., R.B.L.S., V.L.D.S., and Group 2) independently validated the publications and extracted the most relevant features of the selected publications. Two specialist reviewers (J.J.V.T. and R.F.C.) conducted the final evaluation. To increase search sensitivity, all references in the selected articles have been checked.

Results and Discussion

To our knowledge, this is the first systematic review that addresses the research of techniques used in assays for determination of compounds' bactericidal activities in M. tuberculosis, focusing on their characteristics such as previous dilutions of compounds and bacillus to carry out the MBC assay, specific culture media, incubation conditions and percentage of bacterial death detection.

The initial database search about drug's MBC in M. tuberculosis yielded 6,674 publications. The application of “abstract”; “language Portuguese, English and Spanish”; and “publication date from January 1, 1990 to February 19, 2018” filters reduced the number of publications to 5,348. The search was refined based on title, abstract, and publication types, yielding 187 articles. Duplicated studies were removed. The full texts, in PDF, were evaluated, and 69 articles remained after the established inclusion and exclusion criteria were applied.

From the 69 articles that studied the in vitro determination of drug's MBC in M. tuberculosis (Supplementary Data), 37 performed only drug's MBC assays (Table 1),^5,9–44 30 only TK assays (Table 2),^46–75 and two studies performed both MBC and TK assays (Tables 1 and 2).^7,8 Of the 37 articles selected, which performed only drug's MBC in M. tuberculosis, 32 were carried out in the reference strain H₃₇Rv,^7–37 20 in clinical isolates,^{7,11–16,18,20,21,23,25,26,29,32,37–42} and a total of 9 in mycobacteria, such as M. tuberculosis H₃₇Ra,^8,43 M. tuberculosis Erdman,^13,14,33,44 Mycobacterium africanum,^15,16 Mycobacterium bovis,^15,16,24 and other mycobacteria species such as Mycobacterium smegmatis,^24,27,39 Mycobacterium avium^11,15,38 (Table 1).

Table 1.

Data Analysis of Minimum Bactericidal Concentration Techniques in Mycobacterium tuberculosis

Refs.	Strains and clinical isolates	Incubation time (weeks)	Culture medium	Previous technique	Ability to kill (%)
Yamori et al.⁹	Mycobacterium tuberculosis H₃₇Rv (05001)	3	Ogawa egg medium	Proportion method	Reduction of inoculum
Mehta et al.¹⁰	M. tuberculosis H₃₇Rv (9 ATCC strains)	2	Middlebrook 7H10	MIC test plate	≥90
Dhople et al.¹¹	M. tuberculosis H₃₇Rv and Mycobacterium avium, strain 101 (serotype 1) and clinical isolates (10 M. avium, and 8 M. tuberculosis)	2	Middlebrook 7H11	MIC (BACTEC 460B)	≥99
Luna-Herrera et al.¹²	M. tuberculosis H₃₇Rv and 23 clinical isolates	3	Middlebrook 7H11	MIC (BACTEC-12B)	≥99
Mor et al.¹³	M. tuberculosis H₃₇Rv, Erdman, and a clinical isolate (Atencio)	NR	Middlebrook 7H12	MIC (BACTEC-12B)	≥90
Mor et al.¹⁴	M. tuberculosis H₃₇Rv, Erdman, and a clinical isolate (Atencio) and three clinical isolates of M. avium	NR	Middlebrook 7H11/7H12	MIC test plate	≥99
Rastogi et al.¹⁵	M. tuberculosis H₃₇Rv and nine clinical isolates (5 drug susceptible +4 drug resistant) as well as Mycobacterium africanum, Mycobacterium bovis, and M. bovis BCG	3	Middlebrook 7H11/7H12	MIC (BACTEC 12B)	≥90
Yamamoto et al.¹⁶	M. tuberculosis H₃₇Rv and nine clinical isolates, M. africanum, M. bovis, and M. bovis BCG	3	Silicone-coated slides Kirchner's medium	Suspension in petroleum benzene	≥99
Ho et al.¹⁷	M. tuberculosis H₃₇Rv	4	Löwestein-Jensen	Absolute concentration method and MIC test plate	≥99.90
Sato et al.¹⁸	M. tuberculosis H₃₇Rv and 54 clinical isolates	3–4	Middlebrook 7H11	MIC test plate	≥90
Lu and Drlica³⁸	M. tuberculosis Kurono, M. avium complex N-444, and clinical isolates	4–5	Middlebrook 7H9/7H10	Macrodilution	≥99
Ahmad et al.¹⁹	M. tuberculosis (Clinical isolate—TN6515) and Mycobacterium smegmatis mc2 155	4–5	Middlebrook 7H10	MIC test plate	≥99.99
Ahmad et al.³⁹	M. tuberculosis H₃₇Rv	4–5	Middlebrook 7H11	MIC test (Youman's medium)	≥99.99
Villar et al.²⁰	M. tuberculosis H₃₇Rv and clinical isolates	3	Middlebrook 7H9	MIC test plate	≥99
Hurdle et al.²¹	M. tuberculosis H₃₇Rv (ATCC 35801) and clinical isolates	4	Middlebrook 7H11	MIC test plate	≥99.90
Kinnings et al.²²	M. tuberculosis H₃₇Rv	2	Middlebrook 7H9	MIC test plate	≥99
Kirimuhuzya et al.⁴⁴	M. tuberculosis Erdman	8	Middlebrook 7H11	Fluorescence microscopy (rhodamine–auramine)	≥99
Freundlich et al.²³	M. tuberculosis rifampicin-sensitive 28-25271, M. tuberculosis rifampicin-resistant TMC-331 strain, and M. tuberculosis H₃₇Rv strain	3–6	Middlebrook 7H9/7H10	MIC test plate	No growth in tube
Bunalema et al.²⁴	M. smegmatis mc2 155, M. bovis BCG, and M. tuberculosis H₃₇Rv	6	Middlebrook 7H9	MIC test plate	≥99
Cremades et al.⁴⁰	16 Clinical isolates of M. tuberculosis	3–4	Middlebrook 7H11	Macroculture	≥99.9
Cremades et al.⁴¹	16 Clinical isolates of M. tuberculosis	3–4	Middlebrook 7H11	Macroculture	≥99.9
Tandon et al.²⁵	M. tuberculosis H₃₇Rv	3–4	Middlebrook 7H11	MIC test plate	≥99
Khare et al.²⁶	M. tuberculosis H₃₇Rv and two clinical isolates	4	Middlebrook 7H9/7H11	MIC test plate	≥99
Lechartier et al.²⁷	M. tuberculosis H₃₇Rv and M. smegmatis mc2155	3	Middlebrook 7H10	MIC and synergism	≥99
Vilchèze and Jacobs²⁸	M. tuberculosis H₃₇Rv and H₃₇Rv-BTZ-R mutation modified	3	Middlebrook 7H10	Macrodilution	≥99
Mbaveng et al.²⁹	M. tuberculosis H₃₇Rv and isolates of laboratory stocks	NR	Middlebrook 7H9	MIC test plate	No growth in tube
Zhang et al.³⁰	M. tuberculosis H₃₇Rv	4–5	Middlebrook 7H9/7H10	MIC test plate	Statistical study
Tekwu et al.³¹	M. tuberculosis H₃₇Rv	NR	Middlebrook 7H9/7H10	MIC test plate	≥99.90
Wilson et al.⁸	M. tuberculosis H₃₇Rv (ATCC 27294) and H₃₇Ra (ATCC 25177)	4	BACTEC 12B/Middlebrook 7H10	MIC test plate, synergism (BACTEC 12B)	≥99
Smolarz et al.³²	M. tuberculosis H₃₇Rv and laboratory stocks	2	Middlebrook 7H11	MIC test plate	≥99
Kumar et al.⁴³	M. tuberculosis H₃₇Ra	6	Middlebrook 7H9	MIC test plate	≥99
Khare et al.⁴²	M. tuberculosis H₃₇Rv TMC-102 and four clinical isolates	3–4	Middlebrook 7H11	MIC test plate	≥99
Sharma et al.³³	M. tuberculosis H₃₇Rv and Erdman	3–4	Middlebrook 7H10/7H9	MIC test plate	≥90
Muscia et al.³⁴	M. tuberculosis H₃₇Rv	2–3	Middlebrook 7H10	MIC test plate	≥99.90
Bellale et al.³⁵	M. tuberculosis H₃₇Rv (SRI 1345)	4–6	Middlebrook 7H11	MIC test plate	≥90
Nishanth Kumar and Mohandas⁷	M. tuberculosis H₃₇Rv and clinical isolates	6	Middlebrook 7H11	MIC test plate	No growth in tube
Singh et al.³⁶	M. tuberculosis H₃₇Rv ATCC 25618	2	Middlebrook 7H10	MIC test plate	Reduction in the CFU of the inoculum
Kaur et al.⁵	M. tuberculosis H₃₇Rv	3–4	Middlebrook 7H10	MIC test plate	≥90
Khara et al.³⁷	M. tuberculosis H₃₇Rv ATCC 27294 and 15 susceptible isolates and 5 drug-resistant isolates	2	Middlebrook 7H9/7H11	MIC test plate	≥99

BACTEC is an automatic system for growth detection cultures; CFU, colony forming unit; MIC, minimum inhibitory concentration; NR, not reported.

Table 2.

Data Analysis of Time–Kill Curve Technique in Mycobacterium tuberculosis

Refs.	Strains and clinical isolates	Culture medium	Incubation time	Reading
Dickinson and Mitchison⁵²	Mycobacterium tuberculosis H₃₇Rv	Middlebrook 7H9 with Tween and Middlebrook 7H11	On day 3, an additional drug was added to increase the concentration. Bacterial cultivation was done at days 0, 3, and 7, and samples were removed at each time point. For 4 weeks.	Colonies count showed reduction to >2 log₁₀ compared with control
Herbert et al.⁶⁶	M. tuberculosis H₃₇Rv and drug susceptible clinical isolate (TS51476).	Middlebrook 7H9 Tween 80-albumin medium and Middlebrook 7H11 with polymixin B, amphotericin B, carbenicillin, and trimethoprim	On days 0, 2, 4, and 6, they were inoculated in the plates, and counting was conducted after 4 weeks incubation	Colonies were counted after 2 and 4 weeks of the incubation. Log₁₀ decrease in counting colonies was evaluated
Paramasivan et al.⁵³	M. tuberculosis H₃₇Rv	Middlebrook 7H9 and Middlebrook 7H11 containing polymixin B, amphotericin B, carbenicillin, and trimethoprim	On days 0, 2, 4, and 6, serial 10-fold dilutions of these cultures were inoculated on duplicate plates for 3–4 weeks incubated	Cultures were repeated with CFU counts determined at 2, 6, 12, 24, 36, 48, and 72 hr, since no CFU
Bhusal et al.⁵⁴	Ten clinical isolates of M. tuberculosis	Middlebrook 7H9 with OADC and Middlebrook 7H11	8 Days of incubation	Synergy was defined as two or more decrease in log₁₀ CFU/mL compared with those for the respective single agents
Malik and Drlica⁶⁷	M. tuberculosis H₃₇Rv	Middlebrook 7H9 medium containing albumin and Tween 80 and Middlebrook 7H10	0–5 days. Plates were incubated for 4–5 weeks	Determination of CFU
Rao et al.⁶⁸	M. tuberculosis H₃₇Rv ATCC 27294 e Erdman (ATCC 35801)	Middlebrook 7H9 with ADC, Middlebrook 7H11 with polymixin B, amphotericin B, and OADC	5 Days. The plates were incubated for 3 weeks	Viable CFU counts and evaluation of the decrease log₁₀
Shandil et al.⁵¹	M. tuberculosis H₃₇Rv (ATCC 27294)	BACTEC 7H12B media and Middlebrook 7H11	On days 1, 7, and 14 samples were collected and subcultured onto solid medium	3.2-log₁₀ CFU/mL reduction
Bueno-Sánchez et al.⁵⁵	M. tuberculosis H₃₇Rv (ATCC 27294)	Middlebrook 7H9 with OADC and Tween 80	6 Days	A bactericidal effect can be seen by a 3 log₁₀ (99.9% killing) decrease in CFU at the time specified
Kuete et al.⁵⁰	M. tuberculosis H₃₇Rv (ATCC 27294)	Middlebrook 7H9 and Middlebrook 7H12 medium with 14C-labeled substrate (palmitic acid)	The growth index was recorded until day 24	The sample was said to be bactericidal if delta G was ≤0 at the end of day 24. BACTEC 460 TB system was used
de Carvalho et al.⁵⁶	M. tuberculosis and M. bovis var. BCG	Middlebrook 7H9 at pH 6.6	4, 6, 8, and 10 days. Plates were incubated for 3 weeks	Reduction of CFU later incubation time ≥3 log₁₀
de Steenwinkel et al.⁵⁷	M. tuberculosis H₃₇Rv	Middlebrook 7H9 with OADC, glycerol, and Tween 20 and Middlebrook 7H10 with OADC	1, 2, 3, and 6 days. CFU values were determined after 21 days	Concentration-dependent bactericidal effect (≥99% killing) over time. Due to aggregation of the mycobacteria in the culture, at 6 days of exposure to some of the low concentrations, accurate CFU counts could not be performed
Sharma et al.⁵⁸	M. tuberculosis H₃₇Rv (ATCC 27294)	Middlebrook 7H10 with OADC	Serial dilution technique at a single time point on alternate days for 10 days	Reduction of CFU later incubation time ≥3 log₁₀
Chopra et al.⁵⁹	M. tuberculosis H₃₇Rv	Middlebrook 7H9 with glycerol, Tween 80, and OADC, Middlebrook 7H10 with glycerol and OADC	5 Days	The plates were incubated for 28 days at 37°C, at which point the colonies were scored and a 3 log₁₀ decrease in CFU could be seen
Advani et al.⁶⁹	M. tuberculosis H₃₇Rv	Middlebrook 7H9 with ADC, glycerol, and Tween 80 and 7H11 with OADC	0, 3, 5, 7, and 10 days. Plates were incubated for 21 days	Viable CFU counts and comparison with control evaluation of the decrease log₁₀
Lim et al.⁴⁹	M. tuberculosis H₃₇Rv and clinical isolates	Middlebrook 7H9 and 7H10 broth supplemented with ADC and glycerol	21 Days of incubation. Plates were incubated for 4 weeks	Reduced initial bacterial viability up to six orders of magnitude (the limits of CFU detection)
Wilson et al.⁸	M. tuberculosis H₃₇Rv and laboratory stock	Middlebrook 7H9-OADC-tyloxapol-glycerol and 7H10-OADC-glycerol	4 Weeks	Growth index and CFU became undetectable
Arora et al.⁶⁰	M. tuberculosis H₃₇Rv and M. bovis BCG	Middlebrook 7H11	3 and 7 Days. Plates were incubated for 3 weeks	Considered 99% to kill compared with the drug-free control
de Knegt et al.⁶¹	M. tuberculosis Beijing-1585 (BE-1585)	Middlebrook 7H9 with OADC, glycerol, and Tween 20, and 7H10 with OADC and glycerol	The time-kill kinetics was incubated for a period of 6 days. On days 1, 3, and 6 samples (200 μL) were taken, provided that the mycobacterial suspension did not show visible aggregation, and plates were incubated for 2 weeks	Considered 99% ability to kill compared with the drug-free control
Hameed et al.⁷⁰	M. tuberculosis H₃₇Rv (ATCC 27294)	Middlebrook 7H9 broth supplemented with glycerol, Tween 80, and ADC and Middlebrook 7H11 agar plates	0, 3, 7, and 10 days after drug exposure. The plates were incubated for up to 28 days at 37°C, in 5% CO₂ in a humidified atmosphere	The bacterial colonies were enumerated and data expressed as the log₁₀ CFU for each drug concentration
Nishanth Kumar and Mohandas⁷	M. tuberculosis H₃₇Rv	Middlebrook 7H9 with OADC	The tubes were incubation at 37°C, and viable counts were performed in first, second, third, fourth, fifth, and sixth weeks of incubation	Bactericidal activity was defined as a reduction of 99.9% (>3 log₁₀) of the total number of CFU per milliliter in the original inoculum
Odingo et al.⁴⁸	M. tuberculosis H₃₇Rv	Middlebrook 7H9 with OADC and Tween 80	Time–kill: 2 weeks	Colony-forming units were enumerated by plating dilutions of cells onto compound-free plates at the indicated time points in reduction to >3 log₁₀ CFU
Bax et al.⁶²	M. tuberculosis H₃₇Rv (ATCC 27294)	Middlebrook 7H9 with OADC, glycerol, and Tween 20, and Middlebrook 7H10 with OADC and glycerol	On days 1, 2, 3, and 6, samples were collected, centrifuged, and subcultured onto solid medium The plates were incubated for 21 days to determine the number of CFU.	Determined the number of CFU and sterilization of M. tuberculosis after 6 days
Chandrasekera et al.⁴⁷	M. tuberculosis H₃₇Rv	Middlebrook 7H9-Tw-OADC and Middlebrook 7H10 OADC	On days 0, 7, 14, and 21, samples were collected, and subcultured onto solid medium. The plates were incubated for 4 weeks to determine the number of CFU	Before incubation colonies were counted and determined bactericidal, if >3 log₁₀ reduction
van Breda et al.⁴⁵	M. tuberculosis H₃₇Ra (ATCC 25177)	Sauton media and Middlebrook 7H10 with glycerol and enriched with OADC	0, 1, 2, 3, and 6 days. Plates were incubated for 3–5 weeks	Colonies were counted every week until no more increase was determined to occur. >2 log₁₀ CFU/mL reduction in cell viability (>99% reduction) was observed and this was statistically different from all the other combinations
Caleffi-Ferracioli et al.⁷¹	M. tuberculosis H₃₇Rv (ATCC 27294)	Middlebrook 7H9 with OADC and Middlebrook 7H11 with OADC	On days 0, 1, 2, 3, 5, and 7, samples were collected, and subcultured onto solid medium. Plates were incubated for 21 days	The MBC was defined as reduction in 2 log₁₀ in 21 days
Machado et al.⁷²	M. tuberculosis H₃₇Rv	Middlebrook 7H9 and Middlebrook 7H11	1–3 days. Plates were incubated for 21 days	CFUs were predicted by the theoretical log₁₀ CFU based time to detection multiplied by the corresponding dilution factor. The killing effect of the compounds on in vitro growth of M. tuberculosis was defined as the lack of growth in MGIT tubes after 100 days of incubation
Kesicki et al.⁶³	M. tuberculosis H₃₇Rv (London Pride)	Middlebrook 7H9 with OADC and Tween 80	7 Days. Cultures were incubated at 37°C and serial dilutions plated on 7H10 agar plates to determine CFU/mL. Plates were incubated for 4 weeks before colonies were counted	The ability to kill, defined as 3 log reduction in 21 days
Ammerman et al.⁴⁶	M. tuberculosis H₃₇Rv and 4 MDR clinical isolates	Middlebrook 7H9 with 10% OADC, 0.5% glycerol, and 0.02% Tween 20	Removed samples from each vial after 1, 2, 7, and 14 days of incubation to determine CFU counts plated on 7H11 agar plate and incubated for 4 weeks	The EBA was defined as the decline in log₁₀ CFU/mL/day
Kokoczka et al.⁶⁴	M. tuberculosis H₃₇Rv (ATCC 25618)	Middlebrook 7H9 with OADC and Tween 80, and Middlebrook 7H10 with OADC	M. tuberculosis cultures were exposed for 10 days. Plates were incubated for 21 days	The ability to kill was defined as the minimum concentration required to effect in a 3 log kill in 21 days
de Knegt et al.⁷³	M. tuberculosis Beijing VN-2002-1585 (BE 1585)	Middlebrook 7H9 with 10% OADC, 0.5% glycerol, and 0.02% Tween 20; and Middlebrook 7H10 with 10% OADC, 0.5% glycerol	1, 2, 3, and 6 days. Plates were incubated for 28 days	Considered 99% ability to kill compared with the drug-free control
Maltempe et al.⁶⁵	M. tuberculosis H₃₇Rv (ATCC 27294) and clinical isolates	Middlebrook 7H9 with OADC and Middlebrook 7H11 with OADC	On days 3, 5, and 8, samples were collected, and subcultured onto solid medium. Plates were incubated for 21 days	The ability to kill was defined as reduction in 3 log in 21 days
Hegeto et al.⁷⁴	M. tuberculosis H₃₇Rv (ATCC 27294) and clinical isolates	Middlebrook 7H9 with OADC and Middlebrook 7H11 with OADC	On days 1, 2, 5, 7, and 10, samples were collected, and subcultured onto solid medium. Plates were incubated for 21 days	The ability to kill was defined as reduction in 2 log in 21 days

Mycobacterium tuberculosis H₃₇Rv is a reference strain.

ADC, albumin, dextrose, and catalase; CFU, colony forming unit; EBA, early bactericidal activity; MBC, minimum bactericidal concentration; MGIT, mycobacteria growth indicator; OADC, oleic acid, albumin, dextrose, and catalase.

There were publications on 36 drug's MBC that described the culture incubation time, which ranged from 2 to 8 weeks, with an average of 4–5 weeks, depending on the culture medium used and some previous techniques applied, such as MIC determination. A minimum incubation time of 8 weeks was achieved by Kirimuhuzya et al.,⁴⁴ who detected Lantana camara's MBC in M. tuberculosis, by visual fluorescence microscopy using rhodamine–auramine stain.

The most commonly used culture media for drug's MBC determination were Middlebrook 7H10, 7H11, and 7H12, supplemented with oleic acid, albumin, dextrose, and catalase (OADC) or albumin, dextrose, and catalase enrichments. Former studies used other culture media.^9,17 Yamori et al.⁹ used Dubos liquid medium in shaking culture (amplitude 8 cm; 56 strokes/min) and in the sequence, the inoculation onto Ogawa egg medium. Ho et al.¹⁷ subcultured aliquots from broth medium, with no visible growth, onto Löwenstein–Jensen medium (LJ) and incubated at 35°C. The colony counting was carried out after 3–4 weeks of incubation. Then, the number of colonies allowed for a 99.9% MBC endpoint was determined (ratio of the colony-forming unit [CFU] in a drug-containing medium and the number of CFUs in the control medium without drug).

Ogawa and LJ have been the most used media for the conventional culture of M. tuberculosis,^75–77 mainly because of their low cost. However, these media are traditionally prepared in tubes, making the colony counting difficult for a drug's MBC determination, once bacterial growth is sometimes confluent.^77,78

A very simple assay for a drug's MBC determination was proposed by Yamamoto et al.¹⁶ A silicone-coated slide containing Kirchner's medium supplemented with 10% bovine serum was used. The prepared slide was immersed in a M. tuberculosis bacillary suspension, previously standardized by visual comparison of turbidity equivalent to McFarland scale 1, and incubated for 3 weeks at 35°C.¹⁶ The drug's MBC was defined as the lowest concentration of the drug, in which no visible bacilli growth was observed macroscopically on the slide medium containing the drug, when compared with a slide medium with no drug. Silicone-coated slides were used by researchers in the 60s for counting viable colonies, but nowadays this technique is no longer used.^79,80

In the selected publications, the drug's MBC determination was usually carried out using MIC assay. Three studies^28,40,41 did not report the use of previous methodology to perform MBC. Cremades et al.^40,41 used a macrotechnique (10 mL), in which the drug's MBC was without previous MIC determination. They exposed the bacillus in its exponential growth to drug dilution and incubated for 48 hr at 37°C. After that, aliquots (100 μL) were subcultured on Middlebrook 7H11 to establish the MBC point. Vilchèze and Jacobs²⁸ initially performed twofold serial dilutions of the studied compounds/drugs in Middlebrook 7H9 medium. The bacillus inoculum tested was diluted (10⁵–10⁶ CFU/mL), and 100-μL aliquot was added to the medium containing the compounds/drugs dilutions. The tubes were incubated by shaking at 37°C for 2 weeks, and 100 μL bacillus aliquots were plated onto Middlebrook 7H10 plates. MBC was determined as the compound concentration that reduced the initial bacterial load by 99%.²⁸

Six studies did not report the bacilus killed percentage as criteria for considering a drug's MBC.^{7,9,23,29,30,36} Of these, Zhang et al.³⁰ analyzed the bactericidal activity by analysis of variance, CFU counting was log₁₀ transformed before analysis, expressed as mean log₁₀ CFU ± standard deviation, and compared using unpaired Student's t test in the Prism version 5.0 (Graph Pad). However, Yamori et al.⁹ and Singh et al.³⁶ compared the CFU score obtained from a drug-containing medium against the CFU score obtained from a medium without the drug. In other words, the ratio between the CFU observed in the drug-containing medium and the CFU in the control medium (no drug) was calculated. Three other studies described the drug's MBC when no growth in medium containing the studied drug was observed.^7,23,29

TK is a very efficient technique used in drug screening studies, which can monitor the dynamic of drug activity during a determined time.³ In this sense, it is possible to identify if a studied drug has bacteriostatic or bactericidal effect. TK can be used to study both concentration-dependent and time-dependent bactericidal activities of a specific antimicrobial agent.³

In this review, we observed 32 studies that used TK to investigate a drug's MBC. Among these studies, two performed MBC and TK assays.^7,8 The MBC assays were used to complement the TK results and confirm the drug's bactericidal effect. In these studies, the drug's MBC was determined using different techniques. Wilson et al.⁸ used BACTEC based, while Nishanth Kumar and Mohandas⁷ used a microdilution assay. The other 30 studies, which performed TK, are summarized in Table 2. In all TK assays performed, the bacilli inoculums were kept shaking for all assay time to favor aerial growth before the bacilli inoculums were subcultured in an agar plate.^81,82

According to Garcia and Isenberg,³ the bacterial inoculum concentration must be standardized carefully to avoid variations in the TK assay result. An additional limitation of the TK assay is that relatively few drug concentrations are subject to CFU determination in the presence of drug, differently from other assays, such as drug's MBC determination carried out using the drug's MIC assay. TK assay is more labor intensive and time-consuming when compared with the MBC assay. In a study performed by White et al.,⁸² making a comparison of three techniques to detect the synergy of a drug, differences among bacterial inoculums, drug concentrations used, and different definitions of synergy in the studies were observed. These discrepancies clearly showed effect on the growth curve, which turned difficult to distinguish between synergy and additive effect.

In general, standardization of the culture media used to perform the TK assays was done. Middlebrook media variation (7H9, 7H10, and 7H11) was the most commonly used. In only one study⁴⁵ Sauton medium was used for initial bacillus drug exposure, and Middlebrook 7H10 supplemented with OADC was used for subculture and M. tuberculosis CFU counting. The authors explained their protocol assay difference to avoid antagonism with polymixin in Middlebrook 7H9 supplemented with OADC, which could induce false MIC and MBC results.⁴⁵

A variation in drug exposure time was also observed. In some studies, TK assays did not show a significant reduction in the number of live bacilli until the sixth day of incubation, and the time of drug exposure was prolonged to 14 days.^46–51 The observance of a longer time for drug exposure was sometimes necessary, as noted by Ammerman et al.,⁴⁶ who conducted a study with clofazimine that found to have delayed anti-M. tuberculosis activity. In the 7 days of clofazimine exposure, no significant decrease in CFU counting was observed. However, after 14 days of clofazimine exposure, a considerable reduction in viable bacilli was observed.⁴⁶

Most of the TK assays determined the drug's bactericidal effect considering the CFU reduction ≥3 log₁₀ (>99%) compared with the CFU in the initial bacillus inoculum in the medium without drug.^{7,8,45–48,50–65}

Few authors have discussed bias in the selected individual studies,^8,30 whereas in eight articles the limitations were highlighted.^{13,17,24,33,48,61,62,65}

In conclusion, the most common technique for a drug's MBC determination was the drug's MIC assay. Aliquots from prior MIC values were subcultured in Middlebrook 7H10 or 7H11 and incubated for 4 weeks for determining the CFU with relevance to determine 99.9% bacilli killed, or reduction in 3 log₁₀ viable bacilli. However, we could observe that there are many variables in the previous MIC assays performed before the MBC assay, since different culture media, incubation times, and the percentage of bacilli killed used were related to each laboratory protocol. In the case of TK assay, the main standardization consists in the drug exposure time for 7 or 14 days, in which aliquots are plated, generally in Middlebrook solid medium, at a predefined time according to the drug tested, and CFU counting is carried out after 28 days of incubation at 35°C.

Strengths and limitations

The present systematic review searched three databases and the Google Scholar System. This strategy allowed us to increase the sensitivity and accuracy of the publications that were obtained. The main findings were analyzed and organized as tables, based on the consensus of the researchers. In the literature analyzed, a great variety of the techniques used to determine drug's MBC in M. tuberculosis were observed. The relatively large number of publications on the topic hinders discussion and definitive conclusion. We selected studies discussing MBC and TK techniques to help find which was most commonly used. For this proposal, some exclusion criteria were considered as limitations to insert the publication in our study, such as in vivo studies, with other mycobacteria species, and the lack of specific information about the studied topic. We searched individually and in pairs to identify texts in the selected articles that highlighted bias or limitations. We observed that both limitations and biases of the selected studies were not sufficiently validated and discussed by the authors. Getting an efficient and easy MBC assay to apply in research laboratories, which studies new anti-TB compounds, is of paramount importance. The use of a standardized drug's MBC assay for investigating new drugs will help in the development of new therapeutics to treat TB with greater safety and efficacy.

Footnotes

Acknowledgments

We acknowledge the collaborators of the Laboratory of Medical Bacteriology of the State University of Maringa.

Disclosure Statement

No competing financial interests exist.

Funding Information

No funding was received for this article.

Supplementary Material

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