Response surface modeling integrated microtiter plate assay for Mycobacterium fortuitum biofilm quantification

Abstract

In this study, the effects of agitation, temperature, and pH on biofilm formation by Mycobacterium fortuitum were studied and quantified through response surface modeling. The microtiter plate assay was optimized to achieve conditions favoring maximum mycobacterial biofilm quantification. Optical density (OD) measurement using a crystal violet assay was performed to estimate the amount of biofilm formed. Response surface methodology (RSM) results revealed an R² value of 96.18%, exhibiting a maximum OD of 2.119 (λ_570 nm) at a temperature of 37 °C and pH 7.0, under a static environment. The conditions were experimentally validated. Statistically significant results showed that the maximum biofilm was produced 96 h after mycobacterial inoculation. Thus, the results provide a basis for using RSM as an efficient optimization method for M. fortuitum biofilm assays. This approach can also be incorporated into strategies for screening anti-biofilm compounds, synthetic chemicals, drugs, or inhibitors against pathogenic mycobacteria.

Keywords:

• Mycobacterium fortuitum
• response surface methodology
• biofilm assay
• nosocomial infection
• in vitro biofilm model

Acknowledgements

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 a fellowship to Ms Ayushi Sharma. The authors are also grateful to CDRI, Lucknow, for providing the bacterial strain.

Disclosure statement

No conflict of interest was declared by the authors.

Supplemental material

Supplemental Figure 1. Flowchart of experimental measures undertaken in this study.

Additional information

Notes on contributors

Ayushi Sharma

Ayushi Sharma is a Ph.D student and teaching assistant in the Biotechnology and Bioinformatics Department at Jaypee University of Information Technology. Her research work focuses on studies related to biofilm formation by nontuberculous mycobacterial species, and identification of novel drug targets and inhibitors thereof.

Jitendraa Vashistt

Jitendraa Vashistt is an associate professor at Jaypee University of Information Technology, where he teaches metabolic engineering, proteomic technologies, biochemistry, cell biology, molecular biology, and clinical trials to undergraduate and postgraduate students. He specializes in molecular diagnostics, studies on bacterial resistance mechanisms, and clinical proteomics. His work has appeared in Journal of Proteome Research, PLoS One, and Infection Genetics and Evolution. He is a life member of ‘The Indian Biophysical Society’ and ‘DNA Society of India’.

Rahul Shrivastava

Rahul Shrivastava is an associate professor at Jaypee University of Information Technology, where he teaches antibody engineering technologies, clinical diagnostics, diagnostics and vaccine manufacture technologies, immunotechnology, and microbiology to undergraduate and postgraduate students. His research work is related to identification of new treatment targets against Mycobacterium tuberculosis. His work has appeared in Journal of Antimicrobial Chemotherapy, Microbial Pathogenesis, Biochemical and Biophysical Research Communications, and BMC Infectious Diseases. His current research interests include host-pathogen interactions related to fast growing mycobacterial species together with the identification of drug targets, inhibitors, and interacting partners involved in their virulence. He is a life member of ‘Indian Science Congress’, ‘Indian Society of Cell Biology’ and ‘The Association of Microbiologists of India’.