- Email: [email protected]
Evaluation of liquid culture for quantitation of Mycobacterium tuberculosis in murine models
Available online at www.sciencedirect.com
Vaccine 25 (2007) 8203–8205
Letter to the Editor
Evaluation of liquid culture for quantitation of Mycobacterium tuberculosis in murine models
Abstract Quantitation of bacterial load in tissues is essential for experimental investigation of Mycobacterium tuberculosis infection and immunity. We have used an automated liquid culture system to determine the number of colony forming units (CFU) in murine tissues and compared the results to those obtained by conventional plating on Middlebrook agar. There is an overall good correlation between results obtained by the two methods. Although less consistency and more contamination was observed in the automated liquid culture, the method is more sensitive, less labour intensive and allows the processing of large numbers of samples. © 2007 Elsevier Ltd. All rights reserved. Keywords: Mycobacterium tuberculosis; Mouse; CFU/ml
The mouse model is used to study the pathogenesis of human tuberculosis, for drug evaluation and initial screening of vaccine candidates [1] and depends on quantitative bacteriology. Conventionally mycobacterial load has been determined by enumerating the colony forming units (CFU) of organ homogenates on Middlebrook agar [2], but this method is labour intensive, time consuming and occasionally subject to contamination. CFU are an estimate only of the cells which are able to form colonies under the growth conditions provided. Plating efficiency is not complete due to cording, the ability of virulent Mycobacterium tuberculosis to form serpentine cords of acid-fast bacilli in liquid media [3] which is undetectable in solid media, so the number of bacteria counted is usually much lower than the actual number in the culture [4]. Automated mycobacterial culture is more rapid with reduced labour and costs. With the automated BacT/ALERT 3D (bioM´erieux, Durham, NC) system, growth is measured by detecting CO2 production by changes in the pH which is then translated into reflectometric units by the automated fluorescence detector [5]. There is a good correlation between time to positivity as determined by the BACTEC system and CFU/ml as determined by viable counts [6] and between both methods when used for determining antibiotic pharmacodynamic parameters [7]. As it would be useful to have a reliable, high throughput, rapid and economical method for experimental studies we compared the BacT/ALERT 3D system with conventional plating on Middlebrook 7H11 agar for 0264-410X/$ – see front matter © 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.vaccine.2007.09.065
quantitation of the load of M. tuberculosis in murine lung and spleen. We compared 192 lung and spleen murine samples from three different aerosolised M. tuberculosis challenge models. Bacterial counts were determined by 7H11 Middlebrook agar plates containing oleic acid albumin dextrose catalase (OADC) and antibiotics azlocillin, nalidixic acid, polymyxin, trimethoprim and amphotericin (E & O Laboratories Ltd., Bonnybridge, UK) and BacT/ALERT 3D liquid media (bioM´erieux, Durham, NC). For the liquid BacT/ALERT 3D culture a standard curve was used to convert the time to positivity (TTP), as determined by the BacT/ALERT 3D system, of tissue homogenates to CFU/ml as determined by plating M. tuberculosis on Middlebrook 7H9 media (Fig. 1A). The CFU counts determined by both methods showed a significant correlation with Pearson coefficient of 0.729, p = 0.01 (Fig. 1B). Liquid culture had a shorter time to detection of M. tuberculosis than solid media. The mean TTP was 9.4 days (range 3.7–29.5) compared to 21 days for solid culture. Higher CFU counts were detected in liquid (average of 0.7 log) which is in agreement with other reports for faster growth in liquid media [8,9]. Contamination occurred in liquid culture (8%) but there was none detected on the solid cultures. These contaminants were identified by sequencing as Pasteurella, Haemophilus, Staphylococcus and Enterococcus species; all likely murine skin contaminants. A mild de-contamination step could be introduced to remove contamination with a minimal reduction in bacterial numbers. When comparing 29
8204
Letter to the Editor / Vaccine 25 (2007) 8203–8205
Fig. 1. (A) Standard curve relating time to positivity (TTP) to log10 colony forming units/ml in Mycobacterium tuberculosis. Serial 10-fold dilutions of M. tuberculosis (Erdman strain, provided by Dr. M. Brennan, Center for Biologics, Evaluation and Research, Food and Drug Administration, Maryland, USA), cultured to a 0.5 McFarland growth standard in Middlebrook 7H9 broth containing 3% Tween 80 (BDH, VWR International Ltd., Poole, England) and 10% (v/v) albumin dextrose catalase (ADC) (BD, Le Pont de Claix, France). Twenty microlitres of each dilution was spotted in duplicate onto Middlebrook 7H10 (Difco) agar plates, containing 10% OADC, and incubated at 37 ◦ C for 3 weeks. In parallel each dilution (0.5 ml) was inoculated into duplicate BacT/ALERT MP bottles, containing modified Middlebrook 7H9 broth with casein, catalase, and bovine serum albumin with an antibiotic supplement (amphotericin B, nalidixic acid, polymyxin B, trimethoprim, and vancomycin), and cultured in the BacT/ALERT 3D incubator at 37 ◦ C until the instrument indicated they were positive or until 42 days. The lower limit of detection was 10 microorganisms. The TTP was recorded and related to the standard curve to determine CFU/ml. When cultures signalled positive, an aliquot was plated onto Columbia agar with horse blood (Oxoid Ltd., Hampshire, UK) to check for non-mycobacterial contamination. The plates were incubated for 7 days and examined daily. (B) Comparison of CFU counts determined by liquid and solid cultures. Eight to 12 weeks female BALB/c or C57BL/6 mice were challenged by aerosol with M. tuberculosis (Erdman strain) using a modified Henderson apparatus [13]. Mice were sacrificed 4, 12 or 26 weeks after M. tuberculosis challenge. Spleens and lungs were homogenised in 1 ml PBS with 1 mm glass beads in a Mini-Beadbeater (Glen Mills Inc., Clifton, USA) at 4800 rpm for 60 s. The bacterial load for each tissue homogenate was determined by both plating on Middlebrook 7H11 agar and in the BacT/ALERT 3D culture system. The comparison of CFU counts determined by liquid and solid culture is shown. Pearson correlation of the log10 CFU determined by liquid and solid cultures for lung and spleen gives a coefficient of 0.729 and p = 0.01.
groups of results, we noted that the mean CFU/ml was always higher for liquid culture, the standard deviation ranged from 0.31 to 2.37 (liquid) and 0.22 to 1.65 (solid) and the coefficient of variation was 5.16–58.15% (liquid) and 4.28–47.33% (solid). Some samples (72/192) were frozen and analyzed 2 months later. For both liquid and solid culture methods there was a reduction in the CFU after freezing (0.7–1.6 log difference), but less contamination was detected when frozen samples were handled, most likely due to inactivation of the species previously detected. Again the liquid culture detected on average 0.7 logs more CFU per organ than the solid culture. There was a good correlation between these techniques on an organ by organ basis (Pearson correlation coefficient 0.716, p = 0.01 data not shown). Overall the results show that there is good correlation in bacterial counts enumerated by plating on Middlebrook agar and automated liquid BacT/ALERT 3D culture. Results are obtained generally faster with liquid culture, 9.4 days, than solid culture, 21 days. Although not observed in this study, the percentage recovery in liquid culture is better than in solid culture [10]. It has been shown previously that contamination rates are similar for solid and liquid culture [10–12]. However contamination was observed only in liquid culture in this study and could be addressed by introducing a mild de-contamination step. In conclusion the BacT/ALERT 3D system can be used for the quantitative recovery of mycobacteria from murine tissues. The liquid culture system was more sensitive than solid culture and this difference could be important when the number of surviving organisms is low. The liquid culture is the method of choice for high throughput handling of samples, but for experimental studies where accurate quantitation is important, the gold standard remains conventional agar plating and colony counting. References [1] Orme IM, Belisle JT. TB vaccine development: after the flood. Trends Microbiol 1999;7(10):394–5. [2] Kamath AT, Feng CG, Macdonald M, Briscoe H, Britton WJ. Differential protective efficacy of DNA vaccines expressing secreted proteins of Mycobacterium tuberculosis. Infect Immun 1999;67(4):1702–7. [3] Yagupsky PV, Kaminski DA, Palmer KM, Nolte FS. Cord formation in BACTEC 7H12 medium for rapid, presumptive identification of Mycobacterium tuberculosis complex. J Clin Microbiol 1990;28(6): 1451–3. [4] Mikell Jr AT, Smith CL, Richardson JC. Evaluation of media and techniques to enumerate heterotrophic microbes from karst and sand aquifer springs. Microb Ecol 1996;31:115–24. [5] Piersimoni C, Scarparo C, Callegaro A, Tosi CP, Nista D, Bornigia S, et al. Comparison of MB/Bact alert 3D system with radiometric BACTEC system and Lowenstein–Jensen medium for recovery and identification of mycobacteria from clinical specimens: a multicenter study. J Clin Microbiol 2001;39(2):651–7. [6] Joloba ML, Johnson JL, Namale A, Morissey A, Assegghai AE, Rusch-Gerdes S, et al. Quantitative bacillary response to treatment in Mycobacterium tuberculosis infected and M. africanum infected adults with pulmonary tuberculosis. Int J Tuberc Lung Dis 2001;5(6): 579–82.
Letter to the Editor / Vaccine 25 (2007) 8203–8205 [7] Zhanel GG, Saunders MH, Wolfe JN, Hoban DJ, Karlowsky JA, Kabani AM. Comparison of CO2 generation (BACTEC) and viable-count methods to determine the postantibiotic effect of antimycobacterial agents against Mycobacterium avium complex. Antimicrob Agents Chemother 1998;42(1):184–7. [8] Mirovic V, Lepsanovic Z. Evaluation of the MB/BacT system for recovery of mycobacteria from clinical specimens in comparison to Lowenstein–Jensen medium. Clin Microbiol Infect 2002;8(11): 709–14. [9] Dhillon J, Lowrie DB, Mitchison DA. Mycobacterium tuberculosis from chronic murine infections that grows in liquid but not on solid medium. BMC Infect Dis 2004;4:51. [10] Cruciani M, Scarparo C, Malena M, Bosco O, Serpelloni G, Mengoli C. Meta-analysis of BACTEC MGIT 960 and BACTEC 460 TB, with or without solid media, for detection of Mycobacteria. J Clin Microbiol 2004;42(5):2321–5. [11] Huang TS, Chen CS, Lee SS, Huang WK, Liu YC. Comparison of the BACTEC MGIT 960 and BACTEC 460TB systems for detection of mycobacteria in clinical specimens. Ann Clin Lab Sci 2001;31(3):279–83. [12] Somoskovi A, Kodmon C, Lantos A, Bartfai Z, Tamasi L, Fuzy J, et al. Comparison of recoveries of Mycobacterium tuberculosis using the automated BACTEC MGIT 960 system, the BACTEC 460 TB system, and Lowenstein–Jensen medium. J Clin Microbiol 2000;38(6):2395–7. [13] Phillpotts RJ, Brooks TJ, Cox CS. A simple device for the exposure of animals to infectious microorganisms by the airborne route. Epidemiol Infect 1997;118(1):71–5.
D.M. O’Sullivan Centre for Medical Microbiology, Department of Infection, Hampstead Campus, University College London, Rowland Hill Street, Hampstead, London NW3 2PF, UK
8205
C. Sander Centre for Clinical Vaccinology and Tropical Medicine, Oxford University, Churchill Hospital, Oxford OX3 7LJ, UK R.J. Shorten S.H. Gillespie Centre for Medical Microbiology, Department of Infection, Hampstead Campus, University College London, Rowland Hill Street, Hampstead, London NW3 2PF, UK A.V.S. Hill Centre for Clinical Vaccinology and Tropical Medicine, Oxford University, Churchill Hospital, Oxford OX3 7LJ, UK T.D. McHugh Centre for Medical Microbiology, Department of Infection, Hampstead Campus, University College London, Rowland Hill Street, Hampstead, London NW3 2PF, UK H. McShane E.Z. Tchilian ∗ Centre for Clinical Vaccinology and Tropical Medicine, Oxford University, Churchill Hospital, Oxford OX3 7LJ, UK ∗ Corresponding
author. Tel.: +44 1865 857 429; fax: +44 1865 857 471. E-mail address: [email protected] (E.Z. Tchilian) 12 September 2007 Available online 17 October 2007
Vaccine 25 (2007) 8203–8205
Letter to the Editor
Evaluation of liquid culture for quantitation of Mycobacterium tuberculosis in murine models
Abstract Quantitation of bacterial load in tissues is essential for experimental investigation of Mycobacterium tuberculosis infection and immunity. We have used an automated liquid culture system to determine the number of colony forming units (CFU) in murine tissues and compared the results to those obtained by conventional plating on Middlebrook agar. There is an overall good correlation between results obtained by the two methods. Although less consistency and more contamination was observed in the automated liquid culture, the method is more sensitive, less labour intensive and allows the processing of large numbers of samples. © 2007 Elsevier Ltd. All rights reserved. Keywords: Mycobacterium tuberculosis; Mouse; CFU/ml
The mouse model is used to study the pathogenesis of human tuberculosis, for drug evaluation and initial screening of vaccine candidates [1] and depends on quantitative bacteriology. Conventionally mycobacterial load has been determined by enumerating the colony forming units (CFU) of organ homogenates on Middlebrook agar [2], but this method is labour intensive, time consuming and occasionally subject to contamination. CFU are an estimate only of the cells which are able to form colonies under the growth conditions provided. Plating efficiency is not complete due to cording, the ability of virulent Mycobacterium tuberculosis to form serpentine cords of acid-fast bacilli in liquid media [3] which is undetectable in solid media, so the number of bacteria counted is usually much lower than the actual number in the culture [4]. Automated mycobacterial culture is more rapid with reduced labour and costs. With the automated BacT/ALERT 3D (bioM´erieux, Durham, NC) system, growth is measured by detecting CO2 production by changes in the pH which is then translated into reflectometric units by the automated fluorescence detector [5]. There is a good correlation between time to positivity as determined by the BACTEC system and CFU/ml as determined by viable counts [6] and between both methods when used for determining antibiotic pharmacodynamic parameters [7]. As it would be useful to have a reliable, high throughput, rapid and economical method for experimental studies we compared the BacT/ALERT 3D system with conventional plating on Middlebrook 7H11 agar for 0264-410X/$ – see front matter © 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.vaccine.2007.09.065
quantitation of the load of M. tuberculosis in murine lung and spleen. We compared 192 lung and spleen murine samples from three different aerosolised M. tuberculosis challenge models. Bacterial counts were determined by 7H11 Middlebrook agar plates containing oleic acid albumin dextrose catalase (OADC) and antibiotics azlocillin, nalidixic acid, polymyxin, trimethoprim and amphotericin (E & O Laboratories Ltd., Bonnybridge, UK) and BacT/ALERT 3D liquid media (bioM´erieux, Durham, NC). For the liquid BacT/ALERT 3D culture a standard curve was used to convert the time to positivity (TTP), as determined by the BacT/ALERT 3D system, of tissue homogenates to CFU/ml as determined by plating M. tuberculosis on Middlebrook 7H9 media (Fig. 1A). The CFU counts determined by both methods showed a significant correlation with Pearson coefficient of 0.729, p = 0.01 (Fig. 1B). Liquid culture had a shorter time to detection of M. tuberculosis than solid media. The mean TTP was 9.4 days (range 3.7–29.5) compared to 21 days for solid culture. Higher CFU counts were detected in liquid (average of 0.7 log) which is in agreement with other reports for faster growth in liquid media [8,9]. Contamination occurred in liquid culture (8%) but there was none detected on the solid cultures. These contaminants were identified by sequencing as Pasteurella, Haemophilus, Staphylococcus and Enterococcus species; all likely murine skin contaminants. A mild de-contamination step could be introduced to remove contamination with a minimal reduction in bacterial numbers. When comparing 29
8204
Letter to the Editor / Vaccine 25 (2007) 8203–8205
Fig. 1. (A) Standard curve relating time to positivity (TTP) to log10 colony forming units/ml in Mycobacterium tuberculosis. Serial 10-fold dilutions of M. tuberculosis (Erdman strain, provided by Dr. M. Brennan, Center for Biologics, Evaluation and Research, Food and Drug Administration, Maryland, USA), cultured to a 0.5 McFarland growth standard in Middlebrook 7H9 broth containing 3% Tween 80 (BDH, VWR International Ltd., Poole, England) and 10% (v/v) albumin dextrose catalase (ADC) (BD, Le Pont de Claix, France). Twenty microlitres of each dilution was spotted in duplicate onto Middlebrook 7H10 (Difco) agar plates, containing 10% OADC, and incubated at 37 ◦ C for 3 weeks. In parallel each dilution (0.5 ml) was inoculated into duplicate BacT/ALERT MP bottles, containing modified Middlebrook 7H9 broth with casein, catalase, and bovine serum albumin with an antibiotic supplement (amphotericin B, nalidixic acid, polymyxin B, trimethoprim, and vancomycin), and cultured in the BacT/ALERT 3D incubator at 37 ◦ C until the instrument indicated they were positive or until 42 days. The lower limit of detection was 10 microorganisms. The TTP was recorded and related to the standard curve to determine CFU/ml. When cultures signalled positive, an aliquot was plated onto Columbia agar with horse blood (Oxoid Ltd., Hampshire, UK) to check for non-mycobacterial contamination. The plates were incubated for 7 days and examined daily. (B) Comparison of CFU counts determined by liquid and solid cultures. Eight to 12 weeks female BALB/c or C57BL/6 mice were challenged by aerosol with M. tuberculosis (Erdman strain) using a modified Henderson apparatus [13]. Mice were sacrificed 4, 12 or 26 weeks after M. tuberculosis challenge. Spleens and lungs were homogenised in 1 ml PBS with 1 mm glass beads in a Mini-Beadbeater (Glen Mills Inc., Clifton, USA) at 4800 rpm for 60 s. The bacterial load for each tissue homogenate was determined by both plating on Middlebrook 7H11 agar and in the BacT/ALERT 3D culture system. The comparison of CFU counts determined by liquid and solid culture is shown. Pearson correlation of the log10 CFU determined by liquid and solid cultures for lung and spleen gives a coefficient of 0.729 and p = 0.01.
groups of results, we noted that the mean CFU/ml was always higher for liquid culture, the standard deviation ranged from 0.31 to 2.37 (liquid) and 0.22 to 1.65 (solid) and the coefficient of variation was 5.16–58.15% (liquid) and 4.28–47.33% (solid). Some samples (72/192) were frozen and analyzed 2 months later. For both liquid and solid culture methods there was a reduction in the CFU after freezing (0.7–1.6 log difference), but less contamination was detected when frozen samples were handled, most likely due to inactivation of the species previously detected. Again the liquid culture detected on average 0.7 logs more CFU per organ than the solid culture. There was a good correlation between these techniques on an organ by organ basis (Pearson correlation coefficient 0.716, p = 0.01 data not shown). Overall the results show that there is good correlation in bacterial counts enumerated by plating on Middlebrook agar and automated liquid BacT/ALERT 3D culture. Results are obtained generally faster with liquid culture, 9.4 days, than solid culture, 21 days. Although not observed in this study, the percentage recovery in liquid culture is better than in solid culture [10]. It has been shown previously that contamination rates are similar for solid and liquid culture [10–12]. However contamination was observed only in liquid culture in this study and could be addressed by introducing a mild de-contamination step. In conclusion the BacT/ALERT 3D system can be used for the quantitative recovery of mycobacteria from murine tissues. The liquid culture system was more sensitive than solid culture and this difference could be important when the number of surviving organisms is low. The liquid culture is the method of choice for high throughput handling of samples, but for experimental studies where accurate quantitation is important, the gold standard remains conventional agar plating and colony counting. References [1] Orme IM, Belisle JT. TB vaccine development: after the flood. Trends Microbiol 1999;7(10):394–5. [2] Kamath AT, Feng CG, Macdonald M, Briscoe H, Britton WJ. Differential protective efficacy of DNA vaccines expressing secreted proteins of Mycobacterium tuberculosis. Infect Immun 1999;67(4):1702–7. [3] Yagupsky PV, Kaminski DA, Palmer KM, Nolte FS. Cord formation in BACTEC 7H12 medium for rapid, presumptive identification of Mycobacterium tuberculosis complex. J Clin Microbiol 1990;28(6): 1451–3. [4] Mikell Jr AT, Smith CL, Richardson JC. Evaluation of media and techniques to enumerate heterotrophic microbes from karst and sand aquifer springs. Microb Ecol 1996;31:115–24. [5] Piersimoni C, Scarparo C, Callegaro A, Tosi CP, Nista D, Bornigia S, et al. Comparison of MB/Bact alert 3D system with radiometric BACTEC system and Lowenstein–Jensen medium for recovery and identification of mycobacteria from clinical specimens: a multicenter study. J Clin Microbiol 2001;39(2):651–7. [6] Joloba ML, Johnson JL, Namale A, Morissey A, Assegghai AE, Rusch-Gerdes S, et al. Quantitative bacillary response to treatment in Mycobacterium tuberculosis infected and M. africanum infected adults with pulmonary tuberculosis. Int J Tuberc Lung Dis 2001;5(6): 579–82.
Letter to the Editor / Vaccine 25 (2007) 8203–8205 [7] Zhanel GG, Saunders MH, Wolfe JN, Hoban DJ, Karlowsky JA, Kabani AM. Comparison of CO2 generation (BACTEC) and viable-count methods to determine the postantibiotic effect of antimycobacterial agents against Mycobacterium avium complex. Antimicrob Agents Chemother 1998;42(1):184–7. [8] Mirovic V, Lepsanovic Z. Evaluation of the MB/BacT system for recovery of mycobacteria from clinical specimens in comparison to Lowenstein–Jensen medium. Clin Microbiol Infect 2002;8(11): 709–14. [9] Dhillon J, Lowrie DB, Mitchison DA. Mycobacterium tuberculosis from chronic murine infections that grows in liquid but not on solid medium. BMC Infect Dis 2004;4:51. [10] Cruciani M, Scarparo C, Malena M, Bosco O, Serpelloni G, Mengoli C. Meta-analysis of BACTEC MGIT 960 and BACTEC 460 TB, with or without solid media, for detection of Mycobacteria. J Clin Microbiol 2004;42(5):2321–5. [11] Huang TS, Chen CS, Lee SS, Huang WK, Liu YC. Comparison of the BACTEC MGIT 960 and BACTEC 460TB systems for detection of mycobacteria in clinical specimens. Ann Clin Lab Sci 2001;31(3):279–83. [12] Somoskovi A, Kodmon C, Lantos A, Bartfai Z, Tamasi L, Fuzy J, et al. Comparison of recoveries of Mycobacterium tuberculosis using the automated BACTEC MGIT 960 system, the BACTEC 460 TB system, and Lowenstein–Jensen medium. J Clin Microbiol 2000;38(6):2395–7. [13] Phillpotts RJ, Brooks TJ, Cox CS. A simple device for the exposure of animals to infectious microorganisms by the airborne route. Epidemiol Infect 1997;118(1):71–5.
D.M. O’Sullivan Centre for Medical Microbiology, Department of Infection, Hampstead Campus, University College London, Rowland Hill Street, Hampstead, London NW3 2PF, UK
8205
C. Sander Centre for Clinical Vaccinology and Tropical Medicine, Oxford University, Churchill Hospital, Oxford OX3 7LJ, UK R.J. Shorten S.H. Gillespie Centre for Medical Microbiology, Department of Infection, Hampstead Campus, University College London, Rowland Hill Street, Hampstead, London NW3 2PF, UK A.V.S. Hill Centre for Clinical Vaccinology and Tropical Medicine, Oxford University, Churchill Hospital, Oxford OX3 7LJ, UK T.D. McHugh Centre for Medical Microbiology, Department of Infection, Hampstead Campus, University College London, Rowland Hill Street, Hampstead, London NW3 2PF, UK H. McShane E.Z. Tchilian ∗ Centre for Clinical Vaccinology and Tropical Medicine, Oxford University, Churchill Hospital, Oxford OX3 7LJ, UK ∗ Corresponding
author. Tel.: +44 1865 857 429; fax: +44 1865 857 471. E-mail address: [email protected] (E.Z. Tchilian) 12 September 2007 Available online 17 October 2007