An inexpensive and easy-to-make customized antibiotics mix for mycobacterium culture

The cultivation of mycobacteria often requires the use of several antibiotics to limit the growth of other rapidly growing micro-flora present in the growth medium. This antibiotic cocktail is one of the most expensive reagents required for mycobacterium culture. Here we present a customized antibiotics mix that is easy to prepare at a fraction of the cost of the commercially available antibiotic mixture that protects against transient flora, which are normally present in lungs, without affecting mycobacterial colony number.


Introduction
Mycobacteria are slow-growing organisms (Lambrecht et al., 1988); to obtain visible colonies their culture has to continue for several days. Often the fluid to be examined for mycobacterium contains many other microflora and growth of this microflora has to be limited to allow the mycobacterium to grow. For this purpose, a cocktail of antibiotics is used in the culturing of mycobacterium. This antibiotics cocktail is one of the most expensive reagents (INR 4500 per pack, sufficient for 3 l of media) required for mycobacteria culture. We have formulated a Customized Antibiotics Mix (CAM), which is a mixture of antibiotics, to inhibit or reduce the growth of other microorganisms.
The CAM has the following antibiotic components. Polymyxin B is a mix of polymyxin B1 and B2, basic polypeptides obtained from strains of Bacillus polymyxa. Polymyxin B acts as a bactericidal against all Gram-negative bacilli except Proteus and Neisseria genera by binding with the cell membrane and increasing its permeability, changing its structure and causing a higher uptake of water, ultimately leading to cell death (Cardoso et al., 2007).
Amphotericin B is an antifungal drug first prepared from Streptomyces nodosus in 1955 (Donovick et al., 1955). This drug is also used to treat aspergillosis, blastomycosis, coccidioidomycosis, cryptococcosis and candidiasis. Amphotericin B causes the fungal cell to leak monovalent ions by binding with ergosterol, an integral part of fungal cell membrane, eventually causing fungal cell death (Mesa-Arango et al., 2012;O'Keeffe et al., 2003).
Nalidixic acid is a very weak organic acid used for the treatment of bacterial urinary tract infection such as Escherichia coli, Enterobacter, Klebsiella, Proteus and Shigella. It is a synthetic quinolone antibiotic, which is a group of antibiotics that inhibit bacterial growth by selectively blocking the DNA replication of these bacteria. Hence, it is also used for the study of regulation of bacterial division (Pommier et al., 2010).
Trimethoprim is a synthetic antibacterial drug mainly used for the treatment of bladder infections. It typically targets species such as Escherichia coli, Proteus mirabilis, Klebsiella pneumoniae and Enterobacter species. This drug inhibits the DNA synthesis of bacteria, hindering the reduction of dihydrofolic acid to tetrahydrofolic acid, which is a key precursor in the thymidine kinase pathway (Brogden et al., 1982).
Azlocilin is a semisynthetic broad-spectrum antibiotic used against a number of Gram-positive and -negative bacteria. Azlocilin weakens the cell wall of the bacteria by binding to the penicillin-binding protein located inside the bacterial cell wall, which in turn inhibit the crosslinking of peptidoglycan (Sanders, 1983).
All of these aforementioned reagents are among those commonly used in antibiotics formulations for treating infections.

Preparation of CAM
For the preparation of CAM, the aforementioned commercially available antibiotic formulations were procured and their working stocks were prepared in water. From the working stock, the calculated amount volume of antibiotics were mixed. The details for the preparation of working stock and the final volume used for the preparation of 5 l Middlebrook 7H11 agar medium, sufficient for around 200 culture plates, are given in Table 1.
Typically, one tablet each of nalidixic acid (GramoNeg®; Best laboratories Pvt. Ltd.) and Trimethoprim (Bactrim®; Piramal Enterprises Limited) tablets were dispersed in 10 ml water and kept on rocker shaker for 15-20 mins, the mixture was then centrifuged at 600g for 10 min. Supernatant was aspirated and used for the preparation of the CAM.
Required amounts (shown in Table 1)   Preparation of agar plates A total of 105 g Difco TM Mycobacteria 7H11 Agar (BD Biosciences, USA) was suspended in 4,500 ml water containing 25 ml glycerol. The medium was swirled on a hot magnetic plate to obtain a smooth suspension and autoclaved at 121°C for 15 min.
The medium was allowed to cool to 50-55°C in aseptic conditions. In the meantime, 25 g bovine serum albumin, 10 g dextrose, 15 mg catalase and 4.25 g sodium chloride (all Himedia, India) were dissolved in 500 ml water and filtered through a 0.2-µm filter. Next, 250 µl oleic acid (Himedia, India) was then added aseptically. This mix is commonly known as OADC (oleic acid, albumin, dextrose and catalase).
Ten vials of commercial BBL TM MGIT TM PANTA TM (BD-PANTA) antibiotic mixture (Becton, Dickson and company, USA) was then added to 5 l media. In another preparation of media, the BBL TM MGIT TM PANTA TM was replaced with the CAM, formulated as aforementioned.

Mice immunization with BCG
To compare the efficacy of the two antibiotic mixes, eight female B57BL/6J mice of 4-6 weeks age, weighing 20-25 g were immunized with Mycobacterium bovis (BCG) by the aerogenic route to establish around 1,000-2,000 bacilli of BCG in each mouse (Bhaskar & Upadhyay, 2003). Mice were housed in ventilated cages and fed with autoclaved acidified water and irradiated food ad libitum and were kept in 12 h light and 12 h dark conditions.
Use of animals in this investigation was approved by the Institutional animal ethical committee of National Institute of Immunology, New Delhi (IAEC#354/14).

Estimation of bacilli load
At every time point of day 1, 7, 14 and 30 post-immunization, two immunized mice were euthanized by an overdose of Ketamine and Xylazine given intraperitoneally. Typically, 35 mg ketamine and 3.5mg xylazine in 350 µl saline per mouse was used to euthanize a mouse.
Their lung and spleen were isolated aseptically and homogenized in 1 ml PBS using a tissue homogenizer (Polytron PT 1600E, Germany) at 30,000 rpm for 40 s. The homogenized mix was diluted 5 times in PBS and 100 µl diluted mix was spread on the aforementioned agar plates in triplicate.
At every time point the tissue homogenates were plated on media plates prepared using CAM and commercial BD-PANTA antibiotic mixture.
Plates were incubated at 37°C for 4 weeks.

Statistical analysis
GraphPad Prism 7 software was used to calculate p-values by two-way ANOVA.

Results
After incubation, BCG colonies present on plates were counted, the results of which are shown in Figure 1. The data (Dataset 1) confirm that on prepared agar plates BCG was able to selectively grow from a complex micro-flora of the lung and spleen. Similar number of BCG colonies were observed on CAM plates and BD-PANTA plates; the differences between the two were statically insignificant (Figure 1). The data show CFUs on each plate along with calculated bacilli load of the tissue.

Conclusions
The cost of above discussed customized antibiotics mix for preparing 5 l of agar media was around 80 INR (around 1.25 USD) which is almost 1/100 th of the cost of commercially available antibiotic formulation for the purpose. This antibiotics mix is highly economical, easy to prepare and can significantly reduce the total cost involved in mycobacterium culture.

Competing interests
No competing interests were disclosed.

Grant information
This work was supported by a grant received from Department of Science and Technology, Government of India (SB/SO/ HS/204/2013) and the core grant received from the Department of Biotechnology, Government of India to National Institute of Immunology, New Delhi.
The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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