Preliminary results of Raman spectroscopy of pure mycolic acid and Bacillus Calmette–Guérin vaccine: Implications toward medical diagnostics and immunotherapeutics

INTRODUCTION

Vibrational spectroscopy, like Raman spectroscopy, is an optical and non-contact mode of probing that enables molecular fingerprints of large biological molecules, including lipids, peptides, and proteins, to be identified in a simple, non-invasive way.^[1] Recently, the potential role of Raman spectroscopy in the diagnosis of human mycobacterial diseases has been evaluated.^[2,3] Various species of Mycobacteria are responsible for human infections, particularly of the lungs, skin, and soft tissue, with tuberculosis (caused by Mycobacterium tuberculosis) and leprosy (caused by Mycobacterium leprae) being of considerable public health importance.^[4,5] Mycobacterium bovis Bacillus Calmette–Guérin (BCG) is a bacterial species used as a model for mycobacterial infection, immunogenicity, and host-pathogen interaction studies. BCG immunotherapy is widely used in the treatment of non-muscle invasive bladder cancer (NMIBC)^[6] and in skin diseases such as common warts and melanoma.^[7,8] Diagnosis and therapeutic monitoring of mycobacterial infections currently depend on microscopy, culture, or nucleic acid amplification methods. Microscopic detection of acid-fast bacilli in biological specimens (sputum smears and slit-skin smears) still plays an important role in the diagnosis and monitoring of tuberculosis and leprosy.^[9,10] As the microscopy of smears is dependent on human expertise and therefore, partially subjective, there exists a need for methods for rapid detection of mycobacteria that are not operator-dependent, cost-effective, and with potential for rapid point of care diagnosis. Due to the high abundance of mycolic acid, a long-chain fatty acid, in the cell wall of mycobacterium (comprising up to 50% of the dry weight of the bacterium), it forms an attractive target for the detection of the bacteria.^[3] Although evolutionarily conserved, mycolic acid has chemical diversity, especially in terms of the chain length. The cell wall lipids of mycobacteria play important roles in pathogenesis, immunogenicity, and disease relapse.^[2] Keto-mycolic acid derived from BCG has been shown to trigger anti-tumour immune responses in rat bladder cancer cell lines.^[11] In addition, new drug candidates targeting the mycolic acid biosynthesis and other pathways are strategically important, especially with the current use of whole-cell screening.^[12] In this study, we present preliminary results of Raman spectroscopy of BCG and mycolic acid to explore the future feasibility of the detection of mycolic acid in biofluids. To the best of our knowledge, this is the first report of a spectroscopic study of mycolic acid and BCG in the complete Raman signal range, including that beyond the typical “fingerprint” region (usually up to 2000 cm⁻¹).

MATERIALS AND METHODS

RESULTS

The Raman peaks for BCG and purified mycolic acid are shown in Figure 1a and b, respectively. Both samples show a characteristic peak in the 2849–2882 cm^-1 region. These peaks correspond to carbon-hydrogen (CH) stretching vibrations in long-chain mycolic acids, distinct components of the mycobacterial cell envelope. The amplitude of the peak in this region was related to serial dilution of BCG (1 mg/mL, 0.001 mg/mL, and 0.0001 mg/mL concentration) as shown in Figure 1c, showing a decrease in amplitude and loss of the peaks at 2849–2881 cm^-1 on serial dilution of BCG. At 0.0001 mg/mL dilution, no detectable peak was observed.

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Figure 1:

(a) Raman analysis of onco-Bacillus Calmette-Guérin (BCG) as powder form (b) Raman analysis of lyophilized powder of mycolic acid (c) Raman analysis of different concentrations of BCG.

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DISCUSSION

BCG is strongly immunogenic, not only activating a wide variety of immune cells but also stimulating the immune system to better respond to unrelated antigens.^[13] Therefore, it is effective in both immune prophylaxis and immunotherapy. Although the BCG vaccine was developed to prevent serious forms of tuberculosis, it is also effective in the prevention of leprosy.^[14,15] In addition, BCG immunotherapy is also used in the treatment of bladder cancer. Weekly intravesical instillations of hundreds of millions of bacilli over 6 weeks, followed by maintenance treatment, have been used in NMIBC.^[16] Since 1976, BCG immunotherapy has proven to be an effective therapy for NMIBC.^[17] Adverse reactions such as cystitis, anaphylactoid purpura, hypersensitivity pneumonitis, granulomatous prostatitis, granulomatous lesions of the testicle and epididymis, and hepatitis are commonly associated with BCG therapy.^[18] Therefore, a simple, bedside method to determine the concentration of BCG in biofluids like urine is of crucial clinical significance.

Raman spectroscopy analyses the vibrational modes of chemical bonds within molecules, generating a “fingerprint” of the molecule. Table 1 summarizes the common band assignment of mycolic acid, based on the nuclear magnetic resonance spectroscopy^[19] previous Raman spectroscopy studies of Mycobacteria^[1,20] and on the theoretical calculations of the Raman spectroscopy modes of mycolic acids.^[21,22]

Perumal et al. reported surface-enhanced Raman spectra of three major forms of mycolic acid.^[3] Prominent Raman bands were reported by the authors at 714, 857, 918, ~1,000, ~1,025, 1,130, 1,141, 1,231, 1,245, 1,314, 1,343, 1,450, 1,585, and 1,636 cm⁻¹ as multiple lipid components of mycolic acid derived from mycobacteria contribute to multiple peaks.^[3] Our results are similar to this study; the peak of mycolic acid and BCG beyond 2500 cm⁻¹ shows the peak around 2800 cm⁻¹ due to C-H stretching vibration is well preserved and can have diagnostic implications as per our preliminary studies. These peaks correspond to CH stretching vibrations in long-chain mycolic acids, which are characteristic components of the mycobacterial cell envelope. Our preliminary results suggest that Raman analysis is a feasible method of detection of BCG through peak identification of mycolic acid. This requires further detailed study. The conformational behaviour adopted by mycolates allows its detection by Raman spectroscopy and this has implications in biological sciences. It may also allow mycolates be studied in a similar manner by Fourier-transform infrared spectroscopy.^[23] Mycobacterial mycolic acid is involved in both innate and adaptive immune responses.^[24] Immune-mediated reaction is responsible for both therapeutic and associated adverse effects of BCG therapy in bladder cancer. Urinary side effects and dysuria are generally associated with inflammatory side effects and increase with successive BCG therapy. Real-time polymerase chain reaction targeting IS 6110 was used to detect BCG DNA in urine samples after intravesical immunotherapy with BCG for bladder cancer. BCG DNA was detected in 100% of urine samples collected 24 h after the administration of BCG, and in 24% of the specimens collected after 7 days.^[25] Similarly, the detection of mycolic acid in urine by Raman spectroscopy may be potentially used for detecting BCG in urine postintravesical therapy for optimization of the therapeutic regimen based on response. Apart from bladder cancer, further studies for the detection of mycolic acid in other biological specimens, such as slit-skin smears, sputum, synovial fluid, fine-needle aspiration specimens, ascitic fluid, and other biofluids, can be explored for rapid bedside diagnostics in the future. Raman spectroscopy has been investigated to study the nature of drug resistance in tubercular bacilli. A study by Ogunlade et al.^[26] reported a rapid, culture- and antibiotic-incubation-free method for determining tuberculosis drug resistance using Raman spectroscopy and machine learning. By analyzing Raman spectra of over 25,000 BCG cells, the method achieved >98% accuracy on dried samples and ~79% accuracy on sputum, identifying resistance to key antitubercular drugs.^[26] Another study demonstrates that spatially adjusted surface-enhanced Raman spectroscopy using a 785 nm diode laser can reveal molecular features distinguishing drug-sensitive and drug-resistant M. tuberculosis, with up to six distinct signal registration points per cell and key spectral biomarkers identified at 730 cm^-1, 747 cm^-1, and 1,170 ± 2 cm^-1 within the 400–1,800 cm^-1 fingerprint region.^[27] These findings collectively highlight the potential of Raman spectroscopy as a rapid, non-invasive tool for detecting mycolic acid in biofluids, offering both diagnostic utility and a means to monitor BCG-based immunotherapy responses. Furthermore, the integration of Raman spectral analysis with machine learning enables precise identification of drug resistance in M. tuberculosis, supporting its broader application in tuberculosis management and personalized therapeutic monitoring.

CONCLUSIONS

Detection of mycolic acid by Raman spectroscopy is a promising technology for monitoring BCG for optimization of the therapeutic regimen based on response. Apart from bladder cancer, further detailed studies for the detection of mycolic acid in other biological specimens, such as slit-skin smears, sputum, synovial fluid, fine-needle aspiration specimens, ascitic fluid, and other biofluids can be explored for rapid bedside diagnostics in the future.