Correspondence: Takemasa Takii, Department of Molecular Health Sciences, Graduate School of Pharmaceutical Sciences, Nagoya City University, 3-1 Tanabe-Dori, Mizuho-Ku, Aichi, Nagoya 467-8603, Japan. Tel.: +81 52 836 3421; fax: +81 52 834 9309; e-mail: firstname.lastname@example.org
In order to evaluate the biochemical characteristics of 14 substrains of Mycobacterium bovis bacillus Calmette Guérin (BCG) – Russia, Moreau, Japan, Sweden, Birkhaug, Danish, Glaxo, Mexico, Tice, Connaught, Montreal, Phipps, Australia and Pasteur – we performed eight different biochemical tests, including those for nitrate reduction, catalase, niacin accumulation, urease, Tween 80 hydrolysis, pyrazinamidase, p-amino salicylate degradation and resistance to thiophene 2-carboxylic acid hydrazide. Catalase activities of the substrains were all low. Data for nitrate reduction, niacin accumulation, Tween 80 hydrolysis, susceptibility to hydrogen peroxide and nitrate, and optimal pH for growth were all variable among these substrains. These findings suggest that the heterogeneities of biochemical characteristics are relevant to the differences in resistance of BCG substrains to environmental stress. The study also contributes to the re-evaluation of BCG substrains for use as vaccines.
Biochemical tests are currently used as a technique for the identification of bacterial species. Recently, several studies have investigated the physiological meaning of the biochemical characters in the genus Mycobacterium. Sohaskey and colleagues reported variable nitrate production among Mycobacterium bovis bacillus Calmette Guérin (BCG) substrains in relation to survival in host cells (Sohaskey, 2008; Sohaskey & Modesti, 2009). Recycling of NAD and NAD-quinoline reductase relevant to the latent infection of Mycobacterium tuberculosis and resistance to oxidative stress, respectively, have also been reported (Boshoff et al., 2008). Mycobacterial phospholipase A (MPLA) catalyses the hydrolysis of lipids including Tween 80 (Parker et al., 2007), and this activity appears to contribute to survival under starvation at the dormant stage of growth (Jackson et al., 1989; Deb et al., 2009). Here, we analysed the biochemical characteristics and their relationship to susceptibility to environmental stress, such as oxidative stress, nitrosative stresses and pH changes, among BCG substrains.
Materials and methods
Mycobacterium bovis BCG strains Australia (ATCC 35739), Birkhaug (ATCC 35731), Connaught (ATCC 35745), Danish (ATCC 35733), Glaxo (ATCC 35741), Mexico (ATCC 35738), Montreal (ATCC 35735), Pasteur (ATCC 35734), Phipps (ATCC 35744), Tice (ATCC 35743), Russia (ATCC 35740) and M. tuberculosis strain H37Rv (ATCC 25618) were purchased from American Type Culture Collection (ATCC, Manassas, VA). BCG-Moreau, M. bovis (JATA) and Mycobacterium smegmatis were provided by Dr M. Takahashi (The Research Institute of Tuberculosis Japan Anti-tuberculosis Association, Kiyose, Tokyo, Japan). BCG-Japan (Tokyo 172) was purchased from Japan BCG Laboratory (Kiyose, Tokyo, Japan). BCG-Sweden (vaccine seed) was provided by Dr S. Yamamoto (Japan BCG Laboratory). Mycobacterium avium strains 724S and 2151SmO were kindly provided by Drs J. Inamine and E. Torsten (Colorado State University, Fort Collins, CO).
Tests for nitrate reduction, catalase, Tween 80 hydrolysis, urease, pyrazinamidase and resistance to thiophene 2-carboxylic acid hydrazide (TCH) were performed by standard procedures except as described below (Gangadharam & Jenkins, 1998). Nitrate reduction was performed by the classical procedure with liquid reagent. Pyrazinamidase activity was tested on Middlebrook 7H11 broth (BD, Franklin Lakes, NJ) instead of Dubos broth. Resistance to TCH was determined on solid Ogawa medium containing 1 or 10 μg mL−1 TCH. Niacin accumulation was detected using the Kyokuto Niacin Test (Kyokuto Pharmaceutical Industries, Tokyo, Japan) in accordance with the manufacturer's instruction. Degradation of p-amino salicylate (PAS) was determined according to Tsukamura (1961). Mycobacterium tuberculosis, M. bovis, M. avium and M. smegmatis were used as controls. In the urease test, urease-deficient recombinant BCG (Mukai et al., 2008) was used as a negative control.
Culture and differentiation of THP-1 cells
The human monocytic cell line THP-1 (ATCC TIB202) was purchased from ATCC and maintained in RPMI 1640 medium containing 100 U mL−1 penicillin G and 5% heat-inactivated fetal bovine serum (FBS). THP-1 cells were stimulated with 10 nM phorbol 12-myristate 13-acetate (PMA; Wako Pure Chemical Industries, Osaka, Japan) for 24 h to be differentiated to macrophages. Cells were washed three times with culture medium and used for the assays.
Isolation and culture of bone marrow-derived macrophages (BMMs)
Bone marrow was isolated from the tibias and femurs of C57BL/6J female mice at 4–8 weeks of age. Bone marrow cells haemolysed in 0.83% NH4Cl–Tris buffer were cultured in RPMI 1640 supplemented with 10% FBS, 100 U mL−1 penicillin G, 50 μM 2-mercaptoethanol and 10 ng mL−1 granulocyte-macrophage colony-stimulating factor (Wako) in 24-well culture plates; the culture medium was refreshed every 2 days. On day 7, adherent cells were collected and used for the assays.
Macrophages infected with bacilli at a multiplicity of infection (MOI) of 20 were incubated at 37 °C for 6 h. Extracellular bacilli were washed out three times and killed by 100 μg mL−1 amikacin treatment for 6 h. Interferon (IFN)-γ (final concentration of 100 U mL−1) was added to some of the wells as a stimulator. Following incubation, cells were washed three times and ruptured with 100 μL of sterile distilled water. To determine the number of intracellular live bacteria, the lysates were diluted and plated on 7H11 agar in triplicate. Colonies were counted after 3 weeks' incubation.
Tolerance test for hydrogen peroxide and nitric oxide
Bacilli (2 × 106 CFU) were incubated in 7H9 broth containing albumin, dextrose (without catalase) and 0–10 mM H2O2 for 6 h. In the same manner, bacilli were incubated in 7H9 broth supplemented with ADC (albumin, dextrose, catarase) and containing 0–10 mM NaNO2, as an NO donor, at pH 6.6, 6.0 or 5.5 for 3 days. Following incubation, bacilli were washed with 7H9 medium three times, diluted and plated on 7H11 agar. Plates were incubated for 3 weeks and the percentage of live bacilli relative to control (0 mM H2O2 or NaNO2) was calculated.
Determination of permissive pH range for growth of bacilli
Bacterial log-phase cultures in Middlebrook 7H9 (BD) supplemented with 10% ADC (BD) were adjusted to an OD of 0.1 at 530 nm and mixed with 100-fold volume of various pH-adjusted broths (pH 3, 4, 5, 5.4, 5.7, 6.2, 6.6, 7, 8, 9, 10, 11 and 12, adjusted with HCl or NaOH). Following incubation at 37 °C for 21 days, bacterial growth was evaluated by measuring OD at 530 nm.
Each experiment was repeated three times. Statistically significant differences between two series were assessed by Student's t-test or Aspin–Welch's t-test following an F-test assessment of variance.
Results and discussion
Eight different biochemical tests, nitrate reduction, niacin, catalase, Tween 80 hydrolysis, urease, pyrazinamidase, PAS degradation and resistance to TCH, were applied to 14 substrains of BCG, BCG-Russia, -Moreau, -Japan, -Sweden, -Birkhaug, -Danish, -Glaxo, -Mexico, -Tice, -Connaught, -Montreal, -Phipps, -Australia and -Pasteur (Table 1). BCG-Birkhaug was positive for nitrate reduction whereas BCG-Mexico, -Australia and -Pasteur were negative; the other BCG strains were weakly positive, although M. bovis, the parental strain of BCG, was negative. The nitrate respiration system may be responsible for the survival of M. tuberculosis under anaerobic conditions (Sohaskey, 2008), and the nitrate reductase gene narGHJI contributes to the virulence of BCG in immunodeficient mice (Weber et al., 2000). BCG-Russia and -Japan survived better both in THP-1 and in mouse BMMs than other substrains (Fig. 1 and Table 1). Although host M. bovis was negative for nitrate reduction, the viability in host cells was higher than BCG (Table 1 and Fig. 1). According to the standard method for the nitrate reductase test, the assay period was 2 h. Under different conditions, for example longer incubation times and anaerobic conditions, nitrite production has been found in some BCG strains (Weber et al., 2000; Sohaskey & Wayne, 2003; Stermann et al., 2003; Sohaskey & Modesti, 2009). Therefore, different incubation times could explain the discrepancy observed between nitrate reductase test results and intercellular survival. Nitrate reductase activity is not the sole explanation, but we believe it is partly responsible for the survival in host cells, as shown in previous reports (Weber et al., 2000; Sohaskey, 2008) and the present study.
Table 1. Summary of characteristics of BCG substrains in vitro*
Heterogeneity of niacin accumulation was also observed among BCG substrains (Table 1). Recycling of NAD favours the latent infection of M. tuberculosis (Boshoff et al., 2008), and NAD-quinoline reductase is responsible for resistance to oxidative stress (Akhtar et al., 2006). These reports suggest that the activity of NAD metabolism is associated with the survival of BCG in macrophages or host cells. Whether the long or short survival of BCG in host cells favours the effectiveness of BCG has not been determined. However, the different characteristics of BCG substrains as reported here provide the basic information for further investigation of immunological characteristics and evaluation.
Parker et al. (2007) purified and characterized MPLA. MPLA is associated with cutinase, a serine esterase and catalyses the hydrolysis of lipids including Tween 80. MPLA activity was observed not only in pathogenic M. tuberculosis, but also in BCG-Pasteur. BCG-Pasteur was weakly positive for Tween 80 hydrolysis (Table 1). In fact, eight of the 14 substrains, namely BCG-Moreau, -Sweden, -Danish, -Connaught, -Montreal, -Phipps, -Australia and -Pasteur, were weakly positive. Mycobacteria are known to use this fatty acid as carbon source at the dormant stage. Therefore, this activity could contribute to survival under starvation conditions during dormancy (Jackson et al., 1989; Deb et al., 2009).
All BCG strains belong to the low-catalase group, although there were variations in the height of bubble column among them (Table 1). It was over 10 mm in BCG-Japan (14.8 mm) and -Birkhaug (11.8 mm) (Table 1). No mutation in the coding region of the ahpC gene among was observed among the substrains (data not shown). The differences between transcription of the genes and the activities have not yet been analysed. Catalase (katG) and peroxidase (ahpC) activities of M. tuberculosis are related to resistance to oxidative killing in human monocytes in vitro (Manca et al., 1999). The expression of katG is partially regulated by ferric uptake regulators (fur), and contributes to the virulence of M. tuberculosis (Lucarelli et al., 2008). Resistance to hydrogen peroxide of M. bovis, BCG-Russia and -Japan was higher than that of other BCG substrains (Fig. 1). This resistance relates well to survival in host cells, THP-1 and BMMs (Fig. 1). These findings suggest that resistance to H2O2 contributes to survival of BCG substrains in host cells and that enzyme activities other than of catalase could be relevant to the resistance to oxidative stress from host cells.
We next investigated the susceptibility of BCG substrains to nitrosative stress by exposing them to sodium nitrite for 3 days (Fig. 2b). BCG-Pasteur was tolerant to nitric oxide, and moderate susceptibility was observed in BCG-Japan, -Danish and -Glaxo. BCG-Russia, -Sweden, -Birkhaug, -Connaught and -Phipps were sensitive to NO. The parental strain of BCG, M. bovis, was able to tolerate NO. To assess NO production from the bacilli, reduction of pH of the media is required to generate NO from sodium nitrate (Darwin et al., 2003; MacMicking et al., 2003). Intriguingly, optimal pH levels were found to be different among the BCG substrains (Table 2). The optimal pH of BCG-Russia, -Moreau, -Japan, -Phipps, -Pasteur and M. bovis was 6.6. Optimal pH of BCG-Sweden and -Birkhaug was 8–9, and that of BCG-Danish, -Glaxo and -Connaught was 7–8. According to maturation state, pH in phagosomes decreases from about 6 to 4. All BCG strains were positive for urease (Table 1). The changes in pH of the culture broths for each BCG strain were not significantly different (data not shown). Therefore, these data indicate that the increasing pH of the culture broth, such as by generating ammonium, is not responsible for the tolerance of BCG strains to a reduction of pH. The precise mechanisms of adaptability to pH changes have not been elucidated.
Table 2. The range of pH permissive for growth of BCG and other mycobacteria
In summary, we have evaluated the usefulness of various biochemical tests currently used for identifying mycobacterial species. Surprisingly, there were differences in the results of these tests among BCG substrains. These differences could be generated during the long time of passage of BCG vaccine strains. Their characteristics are quality controlled by lyophilizing techniques. A good correlation between oxidative and nitrosative stress and survival in host cells were observed among BCG substrains. The relationship between antigen presentation and viability in host cells is not clear. The longer persistence of the bacilli in the host cells may favour antigen presentation by continuous supply of the antigens, while short persistent bacilli may stimulate antigen presentation through a different pathway (Grode L et al., 2005). Comparative analysis of BCG substrains on acquired immunity should be undertaken. This and our previous studies provide basic information on the biological characteristics and the effect on the innate immunological characteristics of BCG substrains, and these studies could contribute to the re-evaluation of BCG vaccine.
This study was supported in part by a Grant-in-Aid for Scientific Research from the Japan Society for the Promotion of Sciences, a grant for Research on Publicly Essential Drugs and Medical Devices, No. KHC1021, from the Japan Health Sciences Foundation, and a Grant-in-Aid for Scientific Research of the US–Japan Cooperative Medical Sciences Program, Ministry of Health, Labour and Welfare, Japan.