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

Ashwani Kesarwani; Puja Nagpal; Alaknanda Mishra; Rana Zaidi; Pramod Upadhyay

doi:10.12688/f1000research.14467.1

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Method Article

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

[version 1; peer review: 2 approved]

Ashwani Kesarwani^1,2, Puja Nagpal¹, Alaknanda Mishra¹, Rana Zaidi², Pramod Upadhyay ¹

Ashwani Kesarwani^1,2, Puja Nagpal¹, [...] Alaknanda Mishra¹, Rana Zaidi², Pramod Upadhyay ¹

PUBLISHED 27 Apr 2018

Author details Author details

¹ National Institute of Immunology, New Delhi, 110067, India
² Department of Biochemistry, Jamia Hamdard, New Delhi, 110062, India

Ashwani Kesarwani
Roles: Investigation, Methodology, Validation, Writing – Original Draft Preparation

Puja Nagpal
Roles: Investigation, Methodology, Validation

Alaknanda Mishra
Roles: Investigation, Methodology

Rana Zaidi
Roles: Supervision

Pramod Upadhyay
Roles: Conceptualization, Methodology, Resources, Supervision, Validation, Writing – Original Draft Preparation

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Abstract

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.

Keywords

mycobacterium culture, antibiotics, customized antibiotics mix

Corresponding authors: Rana Zaidi, Pramod Upadhyay

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.

Copyright: © 2018 Kesarwani A et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Data associated with the article are available under the terms of the Creative Commons Zero "No rights reserved" data waiver (CC0 1.0 Public domain dedication).

How to cite: Kesarwani A, Nagpal P, Mishra A et al. An inexpensive and easy-to-make customized antibiotics mix for mycobacterium culture [version 1; peer review: 2 approved]. F1000Research 2018, 7:507 (https://doi.org/10.12688/f1000research.14467.1) First published: 27 Apr 2018, 7:507 (https://doi.org/10.12688/f1000research.14467.1) Latest published: 27 Apr 2018, 7:507 (https://doi.org/10.12688/f1000research.14467.1)

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 micro-organisms.

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.

Methods

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.

Table 1. Antibiotics and their concentration used for the preparation of CAM.

Serial number	Antibiotics/Name of commercial formulation	Concentration of antibiotics per pack	Concentration of working stock solution	Required amount antibiotic for 5 l agar media	Cost (INR) per pack* (for 5 l agar media)	Volume of working stock used for 5 l media
1.	Polymyxin B/POLY-B™	~500,000 IU per 580 mg	3.5 mg/1000 µl	30,000 IU	1600/pack (10)	1000 µl
2.	Amphotericin B/AMPHOTRET™	50 mg per pack	10 mg/1000 µl	3,000 µg	300/pack (60)	300 µl
3.	Nalidixic acid/GramoNeg®	500 mg per tab	1 tab/10 ml	12,000 µg	25/10Tab (2.5)	240 µl
4.	Trimethoprim/Bactrim®	160 mg per tab	1 tab/10 ml	3,000 µg	20/10 Tab (2)	187.5 µl
5.	Azlocilin/Azenam	1 g per pack	10 mg/1000 µl	3,000 µg	650/pack (6.5)	300 µl

*65 INR ≈ 1 USD.

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) of Polymyxin B (POLY-B^TM; Samarth Life Sciences Pvt. Ltd.), Amphotericin B (AMPHOTRET^TM; Bharat Serum and Vaccine Limited) and Azlocilin (Azenam; ARISTO Pharmaceuticals Pvt. Ltd.) were weighed and dissolved in water. The CAM was prepared by mixing appropriate volumes of working stock solutions and was filtered through a 0.2-µm syringe filter.

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 house 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).

Figure 1.

Mycobacterium bovis (BCG) colonies in the lung (A) and spleen (B) after immunization. At each indicated time point, the lung and spleen were isolated from immunized mice followed by single-cell suspension preparation and plating on 7H11 agar plates with Customized Antibiotic Mix (CAM) or BD-PANTA. The number of colonies were counted and compared between the two groups.

Bacilli count in 100�l of diluted tissue homogenate = plate count
Bacilli count in 100�l of tissue homogenate (before dilution) = plate count X 5
(Tissue homogenate was diluted 5 times.)
Bacilli count in whole (1000�l) of tissue homogenate = plate count X 5 X 10

			Lung
			Actual BCG colonies on plate1	Calculated BCG bacilli count	Actual BCG colonies on plate2	Calculated BCG bacilli count	Actual BCG colonies on plate3	Calculated BCG bacilli count

	Day 1
		BD-PANTA	32	1600	22	1100	18	900
		CAM	22	1100	29	1450	27	1350
	Day7
		BD-PANTA	37	1850	22	1100	22	1100
		CAM	43	2150	31	1550	24	1200
	Day 14
		BD-PANTA	26	1300	26	1300	51	2550
		CAM	36	1800	32	1600	28	1400
	Day 30
		BD-PANTA	32	1600	28	1400	10	500
		CAM	28	1400	28	1400	23	1150



			Spleen
			Actual BCG colonies on plate1	Calculated BCG bacilli count	Actual BCG colonies on plate2	Calculated BCG bacilli count	Actual BCG colonies on plate3	Calculated BCG bacilli count

	Day 1
		BD-PANTA	0	0	0	0	2	100
		CAM	1	50	1	50	0	0
	Day7
		BD-PANTA	1	50	2	100	0	0
		CAM	1	50	1	50	0	0
	Day 14
		BD-PANTA	8	400	0	0	1	50
		CAM	3	150	3	150	0	0
	Day 30
		BD-PANTA	4	200	0	0	4	200

Dataset 1.Number of BCG bacilli colony forming units (CFUs) on each plate from each experimental group.

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 antibiotic mix is highly economical, easy to prepare and can significantly reduce the total cost involved in mycobacterium culture.

Data availability

Dataset 1. Number of BCG bacilli colony forming units (CFUs) on each plate from each experimental group. The data show CFUs on each plate along with calculated bacilli load of the tissue. http://dx.doi.org/10.5256/f1000research.14467.d201182 (Kesarwani et al., 2018).

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.

Faculty Opinions recommended

References

Bhaskar S, Upadhyay P: Design and evaluation of an aerosol infection chamber for small animals. Int J Pharm. 2003; 255(1–2): 43–48. PubMed Abstract | Publisher Full Text
Brogden RN, Carmine AA, Heel RC, et al.: Trimethoprim: a review of its antibacterial activity, pharmacokinetics and therapeutic use in urinary tract infections. Drugs. 1982; 23(6): 405–430. PubMed Abstract | Publisher Full Text
Cardoso LS, Araujo MI, Góes AM, et al.: Polymyxin B as inhibitor of LPS contamination of Schistosoma mansoni recombinant proteins in human cytokine analysis. Microb Cell Fact. 2007; 6: 1. PubMed Abstract | Publisher Full Text | Free Full Text
Donovick R, Gold W, Pagano JF, et al.: Amphotericins A and B, antifungal antibiotics produced by a streptomycete. I. In vitro studies. Antibiot Annu. 1955; 3: 579–586. PubMed Abstract
Kesarwani A, Nagpal P, Mishra A, et al.: Dataset 1 in: An inexpensive and easy-to-make customized antibiotics mix for mycobacterium culture. F1000Research. 2018. Data Source
Lambrecht RS, Carriere JF, Collins MT: A model for analyzing growth kinetics of a slowly growing Mycobacterium sp. Appl Environ Microbiol. 1988; 54(4): 910–916. PubMed Abstract | Free Full Text
Mesa-Arango AC, Scorzoni L, Zaragoza O: It only takes one to do many jobs: Amphotericin B as antifungal and immunomodulatory drug. Front Microbiol. 2012; 3: 286. PubMed Abstract | Publisher Full Text | Free Full Text
O’Keeffe J, Doyle S, Kavanagh K: Exposure of the yeast Candida albicans to the anti-neoplastic agent adriamycin increases the tolerance to amphotericin B. J Pharm Pharmacol. 2003; 55(12): 1629–1633. PubMed Abstract | Publisher Full Text
Pommier Y, Leo E, Zhang H, et al.: DNA topoisomerases and their poisoning by anticancer and antibacterial drugs. Chem Biol. 2010; 17(5): 421–433. PubMed Abstract | Publisher Full Text
Sanders CC: Azlocillin: a new broad spectrum penicillin. J Antimicrob Chemother. 1983; 11(Suppl B): 21–31. PubMed Abstract | Publisher Full Text

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Version 1

VERSION 1 PUBLISHED 27 Apr 2018

Author details Author details

¹ National Institute of Immunology, New Delhi, 110067, India
² Department of Biochemistry, Jamia Hamdard, New Delhi, 110062, India

Ashwani Kesarwani
Roles: Investigation, Methodology, Validation, Writing – Original Draft Preparation

Puja Nagpal
Roles: Investigation, Methodology, Validation

Alaknanda Mishra
Roles: Investigation, Methodology

Rana Zaidi
Roles: Supervision

Pramod Upadhyay
Roles: Conceptualization, Methodology, Resources, Supervision, Validation, Writing – Original Draft Preparation

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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Published: 27 Apr 2018, 7:507

https://doi.org/10.12688/f1000research.14467.1

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© 2018 Kesarwani A et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Data associated with the article are available under the terms of the Creative Commons Zero "No rights reserved" data waiver (CC0 1.0 Public domain dedication).

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Kesarwani A, Nagpal P, Mishra A et al. An inexpensive and easy-to-make customized antibiotics mix for mycobacterium culture [version 1; peer review: 2 approved]. F1000Research 2018, 7:507 (https://doi.org/10.12688/f1000research.14467.1)

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Reviewer Report 04 Jun 2018

Bhupendra Singh, Division of Microbiology, CSIR-Central Drug Research Institute, Lucknow, India

Approved

https://doi.org/10.5256/f1000research.15748.r34517

The authors have addressed a very pertinent issue of growing mycobacterial culture using a customized cocktail of commonly available antibiotics instead of commercially available expensive antibiotics (PANTA), presently in use. They have performed whole experiments in a very simple way ... Continue reading

The authors have addressed a very pertinent issue of growing mycobacterial culture using a customized cocktail of commonly available antibiotics instead of commercially available expensive antibiotics (PANTA), presently in use. They have performed whole experiments in a very simple way and succinctly described the methods with appropriate conclusions. Since the findings obtained using CAM were comparable to the commercially available PANTA, this can be really useful to researchers in TB field.

Bypassing the routine culture and BACTEC methods authors tested CAM in animal based study, which gave ample scope to new mixture of antibiotics to eradicate any prevailing contamination carried forwarded through organ homogenate. However, a control carrying only organ homogenates on the culture media plates without any antibiotic mix would have given fair idea of prevailing microbial contamination and allowed authors to assess how effectively CAM exterminated the microbial flora under the experimental conditions used by authors.

Is the rationale for developing the new method (or application) clearly explained?

Yes
Is the description of the method technically sound?

Yes
Are sufficient details provided to allow replication of the method development and its use by others?

Yes
If any results are presented, are all the source data underlying the results available to ensure full reproducibility?

Yes
Are the conclusions about the method and its performance adequately supported by the findings presented in the article?

Yes

Competing Interests: No competing interests were disclosed.

I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.

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Reviewer Report 09 May 2018

Amit Singh, Department of Microbiology and Cell Biology, Centre for Infectious Disease Research, Indian Institute of Science, Bengaluru, Karnataka, India

Approved

https://doi.org/10.5256/f1000research.15748.r33548

This is a very simple paper based on common microbiological techniques. Authors have taken a few common antibiotics and made a cocktail which is cost-effective and gave results comparable to the existing commercially available antibiotics (PANTA). Experiments are planned well, appropriately described, ... Continue reading

This is a very simple paper based on common microbiological techniques. Authors have taken a few common antibiotics and made a cocktail which is cost-effective and gave results comparable to the existing commercially available antibiotics (PANTA). Experiments are planned well, appropriately described, and conclusions are justified. The comparable findings obtained using this highly cost-effective formulation relative to commercially available formulations can be of great help to researchers in TB field.

To check the reliability of the new formulation, authors have conducted animal experiments and enumerated bacillary load in the infected organs such as lungs and spleen. Authors detected no significant difference between commercial and the new mixture of antibiotics on the growth potential of the BCG strains isolated from the infected lungs. The statistical analysis was done using two-tailed ANOVA, which is most appropriate.

While experiments are reliable and results are presented well, I would have liked a control wherein authors plate organ homogenates on the culture media plates without any antibiotic mix. This would have confirmed that the normal microflora present in these organs are interfering with the outgrowth of BCG cells under the experimental conditions used by the authors. This way authors can bolster the efficacy of the antibiotic combination in reducing contamination in the mycobacterial cultures.

Is the rationale for developing the new method (or application) clearly explained?

Yes
Is the description of the method technically sound?

Yes
Are sufficient details provided to allow replication of the method development and its use by others?

Yes
If any results are presented, are all the source data underlying the results available to ensure full reproducibility?

Yes
Are the conclusions about the method and its performance adequately supported by the findings presented in the article?

Yes

Competing Interests: No competing interests were disclosed.

I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.

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Amit Singh, Indian Institute of Science, Bengaluru, India
Bhupendra Singh, CSIR-Central Drug Research Institute, Lucknow, India

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Reviewer Report

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04 Jun 2018 | for Version 1

Bhupendra Singh, Division of Microbiology, CSIR-Central Drug Research Institute, Lucknow, India

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Approved

The authors have addressed a very pertinent issue of growing mycobacterial culture using a customized cocktail of commonly available antibiotics instead of commercially available expensive antibiotics (PANTA), presently in use. They have performed whole experiments in a very simple way and succinctly described the methods with appropriate conclusions. Since the findings obtained using CAM were comparable to the commercially available PANTA, this can be really useful to researchers in TB field.

Bypassing the routine culture and BACTEC methods authors tested CAM in animal based study, which gave ample scope to new mixture of antibiotics to eradicate any prevailing contamination carried forwarded through organ homogenate. However, a control carrying only organ homogenates on the culture media plates without any antibiotic mix would have given fair idea of prevailing microbial contamination and allowed authors to assess how effectively CAM exterminated the microbial flora under the experimental conditions used by authors.

Is the rationale for developing the new method (or application) clearly explained?

Yes
Is the description of the method technically sound?

Yes
Are sufficient details provided to allow replication of the method development and its use by others?

Yes
If any results are presented, are all the source data underlying the results available to ensure full reproducibility?

Yes
Are the conclusions about the method and its performance adequately supported by the findings presented in the article?

Yes

Competing Interests

No competing interests were disclosed.

I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.

Respond to this report

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Reviewer Report

16 Views

09 May 2018 | for Version 1

Amit Singh, Department of Microbiology and Cell Biology, Centre for Infectious Disease Research, Indian Institute of Science, Bengaluru, Karnataka, India

16 Views Cite this report Responses(0)

Approved

This is a very simple paper based on common microbiological techniques. Authors have taken a few common antibiotics and made a cocktail which is cost-effective and gave results comparable to the existing commercially available antibiotics (PANTA). Experiments are planned well, appropriately described, and conclusions are justified. The comparable findings obtained using this highly cost-effective formulation relative to commercially available formulations can be of great help to researchers in TB field.

To check the reliability of the new formulation, authors have conducted animal experiments and enumerated bacillary load in the infected organs such as lungs and spleen. Authors detected no significant difference between commercial and the new mixture of antibiotics on the growth potential of the BCG strains isolated from the infected lungs. The statistical analysis was done using two-tailed ANOVA, which is most appropriate.

While experiments are reliable and results are presented well, I would have liked a control wherein authors plate organ homogenates on the culture media plates without any antibiotic mix. This would have confirmed that the normal microflora present in these organs are interfering with the outgrowth of BCG cells under the experimental conditions used by the authors. This way authors can bolster the efficacy of the antibiotic combination in reducing contamination in the mycobacterial cultures.

Is the rationale for developing the new method (or application) clearly explained?

Yes
Is the description of the method technically sound?

Yes
Are sufficient details provided to allow replication of the method development and its use by others?

Yes
If any results are presented, are all the source data underlying the results available to ensure full reproducibility?

Yes
Are the conclusions about the method and its performance adequately supported by the findings presented in the article?

Yes

Competing Interests

No competing interests were disclosed.

I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.

Respond to this report

Responses (0)

[1] Bhaskar S, Upadhyay P: Design and evaluation of an aerosol infection chamber for small animals. Int J Pharm. 2003; 255(1–2): 43–48. PubMed Abstract | Publisher Full Text

[2] Brogden RN, Carmine AA, Heel RC, et al.: Trimethoprim: a review of its antibacterial activity, pharmacokinetics and therapeutic use in urinary tract infections. Drugs. 1982; 23(6): 405–430. PubMed Abstract | Publisher Full Text

[3] Cardoso LS, Araujo MI, Góes AM, et al.: Polymyxin B as inhibitor of LPS contamination of Schistosoma mansoni recombinant proteins in human cytokine analysis. Microb Cell Fact. 2007; 6: 1. PubMed Abstract | Publisher Full Text | Free Full Text

[4] Donovick R, Gold W, Pagano JF, et al.: Amphotericins A and B, antifungal antibiotics produced by a streptomycete. I. In vitro studies. Antibiot Annu. 1955; 3: 579–586. PubMed Abstract

[5] Kesarwani A, Nagpal P, Mishra A, et al.: Dataset 1 in: An inexpensive and easy-to-make customized antibiotics mix for mycobacterium culture. F1000Research. 2018. Data Source

[6] Lambrecht RS, Carriere JF, Collins MT: A model for analyzing growth kinetics of a slowly growing Mycobacterium sp. Appl Environ Microbiol. 1988; 54(4): 910–916. PubMed Abstract | Free Full Text

[7] Mesa-Arango AC, Scorzoni L, Zaragoza O: It only takes one to do many jobs: Amphotericin B as antifungal and immunomodulatory drug. Front Microbiol. 2012; 3: 286. PubMed Abstract | Publisher Full Text | Free Full Text

[8] O’Keeffe J, Doyle S, Kavanagh K: Exposure of the yeast Candida albicans to the anti-neoplastic agent adriamycin increases the tolerance to amphotericin B. J Pharm Pharmacol. 2003; 55(12): 1629–1633. PubMed Abstract | Publisher Full Text

[9] Pommier Y, Leo E, Zhang H, et al.: DNA topoisomerases and their poisoning by anticancer and antibacterial drugs. Chem Biol. 2010; 17(5): 421–433. PubMed Abstract | Publisher Full Text

[10] Sanders CC: Azlocillin: a new broad spectrum penicillin. J Antimicrob Chemother. 1983; 11(Suppl B): 21–31. PubMed Abstract | Publisher Full Text

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

Abstract

Keywords

Introduction

Methods

Preparation of CAM

Table 1. Antibiotics and their concentration used for the preparation of CAM.

Preparation of agar plates

Mice immunization with BCG

Estimation of bacilli load

Statistical analysis

Results

Figure 1.

Conclusions

Data availability

Competing interests

Grant information

References

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