Precision targeting of food biofilm-forming genes by microbial scissors: CRISPR-Cas as an effective modulator

Precision targeting of food biofilm-forming genes by microbial scissors: CRISPR-Cas as an effective modulator

The abrupt emergence of antimicrobial resistant bacterial strains has been recognized as one of the biggest public health threats affecting the human race and food processing industries. One of the causes for the emergence of AMR is the ability of the microorganisms to form biofilm as a defense strategy that restricts the penetration of antimicrobial agents into bacterial cells. About eighty percent of human diseases are caused by biofilm-associated sessile microbes. Bacterial biofilm formation involves a cascade of genes that are regulated via the mechanism of quorum sensing and signaling pathways that control the production of the extracellular polymeric matrix, responsible for the three-dimensional architecture of the biofilm. Another defense strategy utilized commonly by various bacteria includes clustered regularly interspaced short palindromic repeats interference system that prevents the bacterial cell from viral invasion. Since multigenic signaling pathways and controlling systems are involved in each and every step of biofilm formation, the CRISPRi system can be adopted as an effective strategy to target the genomic system involved in biofilm formation. Overall, this technology enables site-specific integration of genes into the host enabling the development of paratransgenic control strategies to interfere with pathogenic bacterial strains. CRISPR-RNA-guided Cas nine endonuclease, being a promising genome editing tool, can be effectively programmed to re-sensitize the bacteria by targeting AMR-encoding plasmid genes involved in biofilm formation and virulence to revert bacterial resistance to antibiotics. CRISPRi-facilitated silencing of genes encoding regulatory proteins associated with biofilm production is considered by researchers as a dependable approach for editing gene networks in various biofilm-forming bacteria either by inactivating biofilm-forming genes or by integrating genes corresponding to antibiotic resistance or fluorescent markers into the host genome for better analysis of its functions both in vitro and in vivo or by editing genes to stop the secretion of toxins as harmful metabolites in food industries, thereby upgrading the human health status.

Introduction

The ever-increasing occurrence of bacterial biofilms in foods led to the emergence of a wide variety of outcomes, starting from negative outcomes like spoilage of food products and foodborne diseases to positive outcomes including preservation of food and improved health of the gut. Naturally, many matrices of food are composed of suitable conditions for the growth of bacteria due to the existence of important substrates, such as lipids, carbohydrates, minerals, proteins, and vitamins. A surplus of nutrients in addition to the presence of solid substrata will lead to the proliferation of microbial cells resulting in biofilm formation that is impenetrable to a wide variety of antimicrobials. This has resulted in the emergence of antimicrobial resistant strains that are difficult to remove.

The discovery of antibiotics is one of the biggest milestones achieved in medicines, which led to the prevention of a wide variety of diseases in humans. In contrast to this, misuse and overuse of antibiotics have led to the development of antibiotic resistance in bacteria. Multi-drug resistant bacteria are the major challenges that are being faced by the fields of medicine and food microbiology. In twenty seventeen, World Health Organization proposed a list of bacteria as some of the most dangerous and pathogenic, which includes Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumanii, Pseudomonas aeruginosa, and Enterobacter spp., and all these bacteria are responsible for hospital-acquired infections and some are also involved in causing food spoilage. This group of bacteria is known as superbugs because the available antibiotics are not sufficient to eradicate the infections caused by them. These superbugs contain resistant genes within their plasmids, thereby spreading the development of MDR at an alarming rate. Therefore, it is of the utmost need to develop some novel strategies to combat these bacteria by certain genome editing techniques, which will alter the resistant genes and make them more susceptible to antibiotic therapies or by inhibiting the genes involved in quorum sensing and thus hindering the formation of biofilm.

Over recent years, advancements in genomics and molecular biology have enhanced the ability of the food industries to manage and monitor the unfavorable food microbiota. Amongst these recent advancements, one of them is the technology of CRISPR-Cas nine. When external genetic elements like bacteriophages invade the bacterial cells, an immune response is mounted due to the presence of a defense mechanism, which is described as the CRISPR-Cas system. CRISPR and the different genes associated with it possess the capacity of providing a promising answer to emerging resistance against antibiotics. A typical system of CRISPR-Cas is composed of three constituents: an operon having a Cas gene group, a leader sequence, and an array of CRISPR DNA. The CRISPR-Cas technology has developed very rapidly as a tool for genome editing in different setups of experiments and types of cells. Results from experimental data have indicated its application in targeting antibacterial resistance genes possessing high rates of specificity and sensitivity.

The biology of CRISPR is embedded in the microbiology of food, which is confirmed by its function in adaptive immunity against Streptococcus thermophilus phages, the main starter culture for yogurt manufacturing. In the last decade, CRISPR research has exponentially grown mainly because of its capability to execute specific cleavage of DNA, so that it can be used for genome editing. With the emergence of terms like "CRISPR revolution" and "CRISPR craze," and the pace at which the scientific world has characterized and understood native systems of CRISPR-Cas, it is evident that this technology will be used in the form of genetic tools for gene insertion, deletion, and regulation in non-native systems. The utilization of CRISPR-associated technologies in prokaryotes that are used in the food industries is also vast.