The International Journal of Mycobacteriology

: 2022  |  Volume : 11  |  Issue : 2  |  Page : 159--166

Knockdown of the Type-II Fatty acid synthase gene hadC in mycobacterium fortuitum does not affect its growth, biofilm formation, and survival under stress

Ayushi Sharma, Jitendraa Vashistt, Rahul Shrivastava 
 Department of Biotechnology and Bioinformatics, Jaypee University of Information Technology, Solan, Himachal Pradesh, India

Correspondence Address:
Rahul Shrivastava
Department of Biotechnology and Bioinformatics, Jaypee University of Information Technology, Waknaghat, Solan - 173 234, Himachal Pradesh


Background: Mycobacterial fatty acid synthase Type-II (FAS-II) components are major virulence factors exploited as potential targets for developing novel antimycobacterial drugs. The FAS-II enzyme 3-hydroxyacyl-ACP dehydratase (HadC) is important for biofilm development and pathogenesis of Mycobacterium tuberculosis and other mycobacterial species. Methods: Literature review and homology search led to the identification of Mycobacterium fortuitum MFhadC gene. Functional interaction study of MFHadC protein was done using STRING. M. fortuitum MFhadC over-expressing (HS) and knockdown (HA) strains were constructed and validated by expression analysis using quantitative polymerase chain reaction. The strains were analyzed for growth behavior and surface spreading ability. Biofilm formation was assayed through crystal violet assay, viability count, and basic fuchsin staining. In addition, survival of the strains was studied under in vitro nutrient starvation and detergent stress. Results: STRING analysis showed the interaction of HadC with proteins involved in biofilm formation. The strains HS and HA showed spreading ability on the agarose surface, exhibiting translocation patterns similar to the vector control strain. All three strains showed a similar amount of biofilm formation when analyzed using crystal violet assay, viability count, and basic fuchsin staining. The strains showed no deviation in survival when incubated under nutrient starvation and detergent stress. Conclusion: Our results suggest that MFhadC may not be important for the formation and maintenance of biofilm, a factor critically important in M. fortuitum pathogenicity. However, not essential for survival and growth, MFhadC maintains the viability of M. fortuitum under a nutrient-starved environment. Collectively, MFhadC may not be used as a biofilm-specific marker for M. fortuitum.

How to cite this article:
Sharma A, Vashistt J, Shrivastava R. Knockdown of the Type-II Fatty acid synthase gene hadC in mycobacterium fortuitum does not affect its growth, biofilm formation, and survival under stress.Int J Mycobacteriol 2022;11:159-166

How to cite this URL:
Sharma A, Vashistt J, Shrivastava R. Knockdown of the Type-II Fatty acid synthase gene hadC in mycobacterium fortuitum does not affect its growth, biofilm formation, and survival under stress. Int J Mycobacteriol [serial online] 2022 [cited 2022 Jul 6 ];11:159-166
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Mycobacterium fortuitum is a rapidly growing human pathogenic nontuberculous mycobacteria (NTM).[1] It is one of the most frequent infection-causing agents among NTM in Asia, particularly in India, Iran, Korea, and Vietnam.[2],[3] Its ubiquitous environmental presence makes it a common reason behind hospital-acquired infections.[4] Nosocomial prevalence of M. fortuitum gives rise to major clinical manifestations such as skin, joint, traumatic, pulmonary infections, and biofilm-related infections.[5],[6] M. fortuitum is known for forming robust biofilms that are phenotypically different from the planktonic form of the bacteria and recalcitrant to medical treatment, requiring long duration chemotherapy with multiple drugs.[7] Increasing drug resistance shown by M. fortuitum biofilm-forming cells[6] presents a need to understand and analyze the complex array of genes associated with the development of biofilms.[8]

Homology search is a useful method for matching evolutionarily-related gene sequences and identifying members of gene families. The literature search identified HadC (3-hydroxyacyl-ACP dehydratase) as a fatty acid synthase Type-II (FAS-II) contributing to biofilm development in Mycobacterium smegmatis mc2155.[9] Homology search showed that the hadC gene of M. smegmatis exhibited 84.71% similarity with the probable hadC gene of M. fortuitum. The gene has earlier been shown as accounting for the cording capacity, biofilm growth, and virulence of Mycobacterium tuberculosis.[10] The dehydratase has also been shown to maintain biofilm formation in M. smegmatis.[9]

Knockdown (antisense) strategy has been well established for identifying the pathogenic and virulent determinants in mycobacteria.[11],[12] Therefore, the present study was conducted to validate the role of hadC in M. fortuitum biofilm formation using knockdown strategy. For this, we constructed and characterized M. fortuitum hadC knockdown and over-expressing strains. The strategy combining homology and knockdown studies would help establish and validate M. fortuitum HadC as a target for antibiofilm drug development.

 Materials and Methods

Bacterial strains, plasmids, and culture conditions

M. fortuitum ATCC 6841 was grown at 37°C, at 160 rpm in Middlebrook 7H9 broth (HiMedia, India). Solid medium used for the culture of M. fortuitum was nutrient agar (NAT).[8] Escherichia coli DH5α was grown using Luria-Bertani broth and NAT. Plasmids pGEM-T Easy vector (Promega) and pMV261 were used in the present study. X-Gal and IPTG, both purchased from Sigma-Aldrich, India, were used for screening of the recombinant strains. Antibiotics were procured from HiMedia, India, and added at the following final concentrations: ampicillin (100 μg/mL) and kanamycin (30 or 100 μg/mL).

Selection of gene for the study

Literature related to mycobacterial genes involved in biofilm formation was searched on Google Scholar and PubMed, and the required mycobacterial gene sequences were extracted from NCBI. The sequences were input for BLAST search. High level of nucleotide similarity of hadC gene of M. smegmatis mc2155 with Mycolicibacterium fortuitum subsp. DSM 46621 = ATCC 6841 (84.71% (97% query coverage)) along with the significance of FAS-II components in mycobacterial biofilm formation[13] led to the selection of probable FAS-II gene hadC of M. fortuitum for further study.

Amplification and cloning of Mycobacterium fortuitum hadC gene

Primers were designed for polymerase chain reaction (PCR) amplification using gene sequence of probable hadC gene of M. fortuitum ATCC 6841 [Supplementary Table 1]. The probable hadC gene (510 bp) was amplified from genomic DNA of M. fortuitum ATCC 6841 using primers MFhadC_fwd and MFhadC_rev, having EcoRI site attached at the 5' ends. The PCR product was ligated into pGEM-T Easy vector (Promega), transformed into E. coli DH5α, and selected on agar plates containing 100 μg/mL ampicillin. The construct was confirmed for the presence of the hadC insert using restriction analysis and sequencing (BioKart, India).[INLINE:1]

Bioinformatics analysis

The sequence obtained was analyzed using BLASTN and translated using Open Reading Frame (ORF) finder ( It was submitted to GenBank and labeled as MFhadC. MFhadC gene and the corresponding protein sequence were subjected to sequence alignment using Clustal Omega ( and visualized using Jalview software.

Protein-protein interaction study

The MFHadC translated protein was subjected to in silico protein–protein interaction to identify the proteins jointly contributing to a shared function. HadC protein of M. tuberculosis H37Rv strain was chosen for the in silico study as M. fortuitum ATCC 6841 dataset was not available in STRING v. 10.5. The interaction confidence was set at 0.7 for the STRING network generation.

Construction of Mycobacterium fortuitum MFhadC knockdown and overexpressing strains

The pGEM-T-MFhadC construct was digested with EcoRI and purified using a DNA purification kit (Macherey-Nagel). The purified insert was ligated into pMV261 vector digested with EcoRI, followed by transformation into E. coli and selection on agar plates containing 30 μg/mL kanamycin. Gene orientation was determined by restriction analysis using BamHI and confirmed by sequencing (BioKart, India). [Supplementary Figure 1] details the strategy utilized for the construction of sense (over-expressing) and antisense (knockdown) constructs. pMV261 vector, pMV261

MFhadC-sense, and pMV261

MFhadC-antisense constructs' were individually electroporated into wild-type M. fortuitum[14] to generate three recombinant strains, M. fortuitum containing pMV261 without insert (vector control (VC)), M. fortuitum containing pMV261

MFhadC-sense (HS), and M. fortuitum containing pMV261

MFhadC-antisense (HA).[INLINE:3]

Quantitative polymerase chain reaction analysis of the recombinant strains

Expression of the MFhadC gene was determined by quantitative polymerase chain reaction (qPCR) analysis[15] using primers MFhadC_qPCR_fwd and MFhadC_qPCR_rev [Supplementary Table 1]. The expression was normalized using 16S rRNA internal control. Fold change was calculated according to the method detailed by Livak and Schmittgen.[16]

Growth curve analysis of Mycobacterium fortuitum MFhadC knockdown and over-expressing strains

Growth of the M. fortuitum MFhadC knockdown strain (HA) and the MFhadC overexpressing strain (HS) was compared with M. fortuitum VC by colony-forming units (CFU) and optical density (OD). The strains were inoculated into Middlebrook 7H9 medium containing 100 μg/mL kanamycin, and grown till OD600 reached to 0.6. M. fortuitum cells were pellet down and washed using 1×Phosphate Buffered Saline (PBS). The cell pellets were suspended in fresh medium and inoculated into 100 mL flasks to obtain a starting OD600 ~0.05. Culture OD600 and CFU were studied for 120 h at different time intervals.

Surface spreading assay

Middlebrook 7H9 medium was gelled using 0.3% agarose for performing the surface spreading assay as per the protocol of Martínez et al.[17] About 25 mL sterile medium was dispensed per Petri plate (Genaxy, India), and the plates were allowed to sit undisturbed for 24 h. Agarose plates were then individually inoculated with a single colony of the recombinant strain. Surface spreading was evaluated visually after incubating the plates for 10 days at 37°C in a humidified incubator.

Biofilm quantification assay

M. fortuitum planktonic cell culture grown to 2 × 105 cells/mL was used for biofilm formation. Biofilms were grown by inoculating 200 μL of the culture in 96-well polystyrene microtiter plates (Genaxy Scientific, India) for 96 h, at 37°C, without agitation.

The amount of biofilm formed by each M. fortuitum recombinant strain was quantified as described previously by Sharma et al.[18] For the Crystal Violet assay, biofilm-forming cells were stained with crystal violet (Loba Chemie) for 15 min. The cells were washed using sterile distilled water. Further, the stained cells were solubilized by 95% ethanol for 15 min. OD was determined at 570 nm using a Microtiter-plate Spectrophotometer (Thermo Fisher Scientific). For viability count assay, biofilm-forming cells were collected in microcentrifuge tubes and sonicated in an ultrasonic cleaner (Citizen) at 40 kHz for 5 min. The sonicated suspension was serially diluted and plated for CFU count. Further, biofilm-forming cells were stained using basic fuchsin (Merck) for 15 min and subsequently decolorized with 95% ethanol for visual quantification under a phase-contrast microscope (Nikon TS100, Japan) at ×100. The images acquired were analyzed by ImageJ software.

Survival study under in vitro nutrient starvation and detergent stress

M. fortuitum recombinant strains were exposed to nutrient starvation[19] and detergent stress[1] conditions. Briefly, the strains were initially grown to the mid-logarithmic phase (OD600-0.6). Cultures were centrifuged at 5500 × g for 10 min and washed using 1× PBS. The pellets were resuspended in Middlebrook 7H9 medium to achieve a cell count of 6 × 106 CFU/mL. 1 mL of the cell suspension was inoculated into 100 mL 1 × PBS (pH 7.4), and 100 mL Middlebrook 7H9 medium containing 0.05% SDS (HiMedia, India), for nutrient starvation and detergent stress studies, respectively. The flasks were incubated at 37°C, 160 rpm. Aliquots removed were spread on NAT plates containing 100 μg/mL kanamycin for CFU calculation.

Statistical analysis

Data shown represent the mean of readings from three independent experiments performed in technical duplicates. Error bars indicate standard deviation. Student's t-test was performed for checking the statistical variation with respect to the M. fortuitum VC, using SPSS 20 (Statistical Product and Service Solutions software, version 20, IBM Corp. Armonk, NY).


The study did not involve the use of animals or human samples; hence, no ethical clearance or patient consent was required.


Identification of 3-hydroxyacyl-ACP dehydratase (hadC) gene in Mycobacterium fortuitum as MFhadC

Literature review, homology search, and cloning of the selected gene, followed by sequencing identified a M. fortuitum homolog of M. smegmatis 3-hydroxyacyl-ACP dehydratase (hadC), labeled as “putative MFhadC.” The identified sequence was submitted to GenBank (ID: MW470668). Homology study of MFhadC showed 84.95% and 70.05% similarity with hadC gene of M. smegmatis mc2 155 and M. tuberculosis H37Rv, respectively; while the MFHadC translated protein showed 84.57% and 62.96% similarity with HadC protein of M. smegmatis mc2 155 and M. tuberculosis H37Rv, respectively. The sequence alignments are shown in [Figure 1].{Figure 1}

HadC showed functional interaction with proteins involved in biofilm formation

HadC protein of the pathogenic M. tuberculosis H37Rv strain (showing 62.96% homology with MFHadC) was chosen for the in silico study. The proteins predicted to be functionally associated with HadC protein include 3-oxoacyl-ACP synthase KasA and KasB, 3-hydroxyacyl-ACP dehydratase subunits HadA and HadB, 50S ribosomal protein L33 RpmG2, malonyl CoA-ACP transacylase FabD, conserved transmembrane proteins Rv3587c and Rv0039c, and enoyl-ACP reductase InhA [Figure 2]. Functions of the predicted interacting proteins were deduced from the literature and Mycobrowser database and are provided in [Supplementary Table 2]. Analysis of the results suggests that the majority of the proteins have association with biofilm formation.{Figure 2}[INLINE:2]

Construction of Mycobacterium fortuitum knockdown and overexpressing strains

The plasmids pMV261

MFhadC-sense and pMV261

MFhadC-antisense were constructed [Table 1] and confirmed by restriction analysis [Supplementary Figure 2]a and sequencing. Electroporation of the constructs into wild-type M. fortuitum generated three recombinant strains, M. fortuitum containing pMV261 without insert (VC), M. fortuitum containing pMV261:MFhadC-sense (HS), and M. fortuitum containing pMV261:MFhadC-antisense (HA), respectively.{Table 1}[INLINE:4]

Mycobacterium fortuitum MFhadC knockdown strain showed reduction in expression

Expression analysis of the constructed M. fortuitum recombinant strains by qPCR demonstrated a 49% reduction in expression of MFhadC gene in MFhadC knockdown strain (HA) in comparison to the VC strain, validating HA as the M. fortuitum knockdown strain. However, the analysis did not yield any remarkable difference in expression of MFhadC between VC and the MFhadC overexpressing strain (HS) [Supplementary Figure 2]b.

Mycobacterium fortuitum recombinant strains exhibited similar growth behavior

Assessment of OD and CFU counts at different time intervals for 120 h showed no appreciable difference among the growth behavior of VC, HS, and HA strains [Figure 3].{Figure 3}

Mycobacterium fortuitum recombinant strains showed similar spreading patterns and amount of biofilm formation

The strains VC, HS, and HA when spread on the surface of semi-solid Middlebrook 7H9-enriched agarose surface generated halos originating at the inoculation point and showed similar translocation patterns [Figure 4].{Figure 4}

Analysis of the recombinant strains did not show any notable difference in the amount of biofilm formed. The crystal violet assay demonstrated comparable OD at 570 nm for the strains VC, HS, and HA [Figure 5]a. Nevertheless, a slight reduction was observed in the CFU count of biofilm-forming cells of HA, in comparison to VC [Figure 5]b. Microscopic analysis of biofilm using basic fuchsin also confirmed the results obtained by OD and CFU values. The strains VC, HS, and HA were able to maintain their biofilm structure over the 96 h period of study [Figure 5]c.{Figure 5}

MFhadC knockdown strain showed reduction in cell viability under nutrient starvation condition

All the M. fortuitum recombinant strains, VC, HS, and HA showed decreased CFU count when compared to their growth in the nutrient-sufficient medium control [Figure 3], as a consequence of the prevalent stress condition. However, it was observed that the ability of strain HA to sustain its growth marginally decreased post 4 h of incubation under nutrient-starved environment. A reduction of 0.7, 0.2, 0.4, and 0.6-log CFU was observed post 6, 12, 24, and 36 h of incubation, respectively, in HA, in comparison to VC [Figure 6]a.{Figure 6}

Mycobacterium fortuitum recombinant strains showed similar growth behavior under detergent stress

The strains VC, HS, and HA were not able to multiply at their regular division rates under the influence of SDS, in comparison to their growth in media control (without SDS) [Figure 3]. About 2-log decrease in CFU of the M. fortuitum recombinant strains was observed post 36 h of growth in Middlebrook 7H9 medium containing 0.05% SDS, relative to their growth in Middlebrook 7H9 medium devoid of SDS. However, growth behavior and CFU count showed no change with respect to under-expression of MFhadC in M. fortuitum HA strain [Figure 6]b.


NTM are well-known mycobacterial species capable of forming biofilms in natural and human-engineered environments. Mycobacterial biofilms exhibit increased resistance to antimicrobials and disinfectants, magnifying their virulence.[20] Biofilms are major form of M. fortuitum infections related to several invasive medical procedures such as endoscopy, bronchoscopy, hemodialysis, cardiac valve prosthesis, and catheter-associated infections.[21] Further, they assist in the survival of M. fortuitum despite the presence of drugs such as clarithromycin and amikacin, developed to kill the cells.[22]

The mycobacterial FAS-II system consists of core enzymes, β-hydroxyacyl-ACP dehydratases HadA, HadB, and HadC, a β-ketoacyl-ACP reductase MabA, β-ketoacyl-ACP synthases (KasA and KasB), and an enoyl-ACP reductase InhA. FAS-II plays an important role in the elongation of fatty acids and subsequent biosynthesis of mycolic acids in mycobacteria.[23] The 3-hydroxyacyl-ACP dehydratase (HadC) of the FAS-II system, encoded by hadC gene, is a crucial contributor of biofilm formation in M. tuberculosis and M. smegmatis.[9],[10] Isoxyl and thiacetazone, the prominent drugs used as clinical medicaments of tuberculosis, target the FAS-II enzyme HadC,[23] underscoring the prospect of HadC as a druggable target in related pathogenic mycobacteria. Role of the FAS-II gene MFhadC was hence explored as an antibiofilm drug target for M. fortuitum in the present study.

Homology search and bioinformatics analysis helped select FAS-II gene “MFhadC” for further study. Functional interaction of MFHadC with other proteins, predicted using a homologous protein HadC from M. tuberculosis H37Rv, demonstrated association of HadC with KasA, KasB, InhA, HadA, HadB, and RpmG2, the proteins involved in M. smegmatis mc2 155 biofilm formation.[9],[13],[24]. However, according to the present study, the FAS-II gene MFhadC exhibits no association with biofilm formation in M. fortuitum. Hence, M. tuberculosis H37Rv may not be a relevant organism for deciphering related functions of different proteins in M. fortuitum; warranting a specific dataset of M. fortuitum ATCC 6841 in the STRING database.

MFhadC over-expressing strain (HS) and MFhadC knockdown strain (HA) were constructed and validated by expression analysis using qPCR. The recombinant strains did not show any deviation on growth curve and viability analysis, in comparison to VC strain, indicating no prominent function of MFhadC in cell division and multiplication. Slama et al.[10] also proposed the nonessentiality of hadC for viability of M. tuberculosis.

Previous reports suggest that mycobacterial spreading on surfaces and biofilm formation requires similar genetic elements.[25],[26] Hence, surface spreading ability and biofilm formation of the recombinant strains were studied. The ability to translocate on surfaces reduces friction between cells and the surface, resulting in cell mobility.[26] VC, HS, and HA showed similar surface spreading. Surface translocation in mycobacteria is related to glycopeptidolipids.[17],[25] The results obtained from the knockdown strain HA suggest that MFhadC does not play a key role in glycopeptidolipid biosynthesis of M. fortuitum. Observations from the surface spreading assay comply with earlier report of Sousa et al.[26] who showed that M. fortuitum exhibits the ability to translocate on agar surface.

The strains, VC, HS, and HA showed a similar amount of biofilm formation and maintenance throughout the 96 h duration of the study. The observation was in accordance with the report of Recht et al.[25] and coincides with the results of our surface spreading study. No substantial variation between the recombinant strains' mobility and biofilm-forming pattern shows that knockdown of MFhadC does not contribute to the development of biofilm by M. fortuitum. In contrast, mutation in the hadC gene has been shown to impact biofilm development in M. tuberculosis[10] and M. smegmatis.[9] The intricate relation between the abundance of glycopeptidolipids, biofilm development, and mycobacterial pathogenesis[27] enables us to predict the inessentiality of MFhadC for M. fortuitum in vivo infection.

FAS-II gene hadC shows downregulation in M. tuberculosis H37Rv under in vitro nutrient starvation.[28] We, therefore, investigated whether knockdown of the FAS-II gene MFhadC has any effect on M. fortuitum survival under in vitro nutrient starvation. Similar growth behavior of VC and HS was observed under nutrient starvation condition, with a slight reduction in cell survival shown by HA. Taken together, the results indicate that MFhadC maintains viability of M. fortuitum but may not be important for survival under the nutrient-starved environment. Further, observation on the survival of M. fortuitum recombinant strains under detergent stress conditions showed that HA exhibits CFU counts similar to VC, indicating no impact of the knockdown of MFhadC. Contradictory to our results, Jamet et al.[9] showed that the M. smegmatis hadC mutants exhibit a comparatively increased resistance to detergent stress.


Target-based drug screening increasingly focuses on the FAS-II components, suggesting these as potential mycobacterial therapeutic targets.[23] We identified a novel gene MFhadC in M. fortuitum, submitted to GenBank as putative 3-hydroxyacyl-ACP dehydratase gene. Our results suggest noninvolvement of MFhadC in the growth and survival of M. fortuitum under stress. The study shows that MFhadC may not be important for the formation and maintenance of M. fortuitum biofilm.

Limitation of the study

In vitro studies show that MFhadC may not be a potential target for antibiofilm drug development for M. fortuitum, albeit, in vivo studies are needed to confirm its role in pathogenesis and virulence.


The authors would like to acknowledge the administration of Jaypee University of Information Technology, Waknaghat, Solan (Himachal Pradesh), India, for providing the research facility and fellowship to Ms. Ayushi Sharma. The authors are also grateful to CDRI, Lucknow, India, for providing bacterial strains and plasmids.

Ethical clearance

The study did not involve use of animals or human samples, hence, no ethical clearance or patient consent was required.

Financial support and sponsorship


Conflicts of interest

There are no conflicts of interest.


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