Rifampicin and Isoniazid resistance in Mycobacterium tuberculosis is associated with cell envelope thickening

Abstract

Mycobacterium tuberculosis (Mtb) is a well-known Gram-positive pathogenic bacterium, which causes tuberculosis (TB) and accounts for 1.2 million deaths annually worldwide. First line of drugs used for treatment of TB includes rifampicin, isoniazid, ethambutol and streptomycin; these are bactericidal, less toxic and highly efficient. Outbreak of multidrug resistant (MDR) strains complicates the effective control of TB [1-2]. MDR-TB is defined as resistance to at least two major first line drugs, rifampicin and isoniazid. Resistance to these two first line of drugs is most commonly found. Emergence of drug resistant Mtb strains threaten the progress in TB containment. Mtb exhibits intrinsic resistance to many drugs and therefore, limits the number of drugs available for treatment. Independent cumulative changes in the Mtb bacterial cells may accompany the intrinsic (inherent) or acquired drug resistance phenotype [1-2]. This intrinsic resistance is the outcome of a multitude of mechanisms which include presence of thick, hydrophobic cell envelope, drug modifying and drug degrading enzymes.

The bacterial cell envelope is a complex multi-layered structure that protects cells from unpredictable and often hostile environment [1-3]. Mycobacterial cell envelope is composed of three entities, namely plasma membrane, cell wall and outer membrane called mycomembrane (MM) whose thickness is more than other bacterial species (Fig. 1) [1]. This mycomembrane is very tightly connected to peptidoglycan and arabinomannan inner layer of the cell wall. Studies reported that Mtb cell envelope exhibits several unusual characteristics, such as multiple cell envelope layers, asymmetric cell division, cell pole elongation, and intracytoplasmic lipid inclusion accumulation, which contribute to their survival under stress conditions [1, 4, 5]. This unusual composition of cell envelope of Mtb is of key importance for several physiological processes such as pathogenicity, virulence, protection from external stress as well as drug resistance [6]. Remarkable impermeability of cell envelope to anti-TB drugs protects the mycobacteria from drug action. In the present study, in order to establish a correlation between drug resistance and cell envelope architecture, the cell envelope thickness was investigated using Transmission Electron Microscopy (TEM). TEM has served as the most powerful tool for studying bacterial cell ultrastructure [7]. The Mtb isolates used in this study were obtained from the repository in National JALMA Institute for Leprosy and Other Mycobacterial Diseases (NJILOMD), Agra, India. The strain description is given in Table 1. Our results showed that the cell envelope thickness in the sensitive isolates were 27.1 ± 0.5 nm (JAL-14463) and 27.2 ± 1.2 nm (JAL-12842), whereas the cell envelope thickness in the resistant isolates were 34.9 ± 2.3 nm (JAL-16332) and 36.34 ± 2.8 nm (JAL-14606) (Fig. 2 A-D). For statistical significance, p-value was determined by the unpaired t-test and p-values ≤ 0.0001 were considered significant (Fig. 3).

The morphological change at the ultrastructural level has a definite role in the complexity of the disease. Several studies reported that cell wall thickness was a common feature of resistant isolates of various gram positive and gram negative bacterial pathogens e.g. E. coli, S. aureus, Enterococci [3,8-9]. It was observed that resistance to drugs was associated with distinct structural alterations of the cell envelope and cell wall thickening [8-9]. A study on M. avium reported that resistant variant with thicker cell wall was associated with enhanced pathogenicity and drug resistance [10,11-12]. It is probable that Mtb is impermeable to many anti-TB drugs due to its thick, multi-layered, and hydrophobic cell envelope, which might play a significant role in intrinsic resistance of mycobacteria to antibacterial drugs. The thickened cell envelope of Mtb appears to help the bacilli to overcome the action of anti-TB drugs. Thus, this study demonstrated that cell envelope thickness in resistant isolates might have evolved as one of the primary defense mechanisms adopted by Mtb against antimicrobials and that the observed resistance towards the two first line drugs, rifampicin and isoniazid might be attributed to increase in cell envelope thickness in resistant isolates. The cell envelope thickness holds significance in terms of drug selection for effective treatment of resistant isolates of Mtb.