One of the concerns with opportunistic pathogens in human is antibiotic resistance in biofilm-producing Pseudomonas aeruginosa [1, 2]. So, antibiotic-resistant biofilms are the major cause of P. aeruginosa-associated infections and lead to increased morbidity and mortality. Biofilms are a surface-associated bacterial community that plays an important role in chronic infections, such as cystic fibrosis, burn wounds, bacterial keratitis, urinary tract infections, and peritoneal dialysis catheter infections, as well as acute infections [3].

Bacteria such as P. aeruginosa are protected by biofilms from various environmental stresses such as antimicrobial agents and antibiotics [4] So, due to increasing resistance to antimicrobial agents, P. aeruginosa still remains an infectious disease when it forms biofilms or is absorbed into a host. Biofilms are also able to adhere to surfaces, making them harder to remove. This means that P. aeruginosa can persist in the environment and be a source of recurrent infections. Therefore, it is important to take preventive measures to contain the spread of this pathogen.

hence, it is important to develop strategies to prevent and control of antibiotic resistance in biofilm-producing Pseudomonas aeruginosa.

The use of nanotechnology to produce new nanomaterials for use in medicine has opened up a new world of possibilities [5].A metal nanoparticles has a variety of properties that make it suitable for medical applications, which is why it is considered an effective antibacterial agent. Therefore, ZnO NPs have been extensively studied due to their extensive biological activity. Zinc oxide is the most commonly used zinc nanoparticle [6] because it has less toxic properties and is more effective against resistant microbial pathogens. They also have selective toxicity against bacteria [7]. There are several noteworthy properties of this nanoparticle, including its chemical and physical stability, high catalytic activity, and effective antibacterial activity. Moreover, some metal nanoparticles of ZnO NPs were reported to possess anti-biofilm properties [8, 9].

The antibacterial and biofilm inhibitory properties of ZnO NP have been widely reported against a variety of microbes, including P. aeroginosa, Streptococcus pneumoniae, Listeria monocytogens, Salmonella enteritidis, and E. coli [10].

Due to their potent antimicrobial activity, these NPs can reduce microbial adhesion, proliferation, and biofilm growth. ZnO NPs damage bacterial cells through the formation of reactive oxygen species (ROS) [11].

Alginate is a polysaccharide that plays an important role in biofilm formation in P. aeruginosa. Alginate is also involved in antibiotic resistance of biofilms and helps to protect bacteria from antibiotics [12]. Structurally, alginate forms a polymer containing α-L-guluronic acid and β-D-mannuronic acid, which is encoded by algD. algD is located in a large operon and is necessary for alginate production, and its expression is fully controlled [12, 13]. It means that AlgD is the key enzyme that catalyzes the formation of alginate polymers. Thus, AlgD plays a central role in the formation of alginate polymers, making it a key enzyme for alginate production.

According to a number of studies, P. aeruginosa has TA systems that regulate biofilm-associated genes such as MqsR/MqsA [14]. In addition, hundreds of genes are differentially regulated during biofilm development, including quorum sensing (lasIR, rhlIR), psl, and pel.

The bacterial TA systems have many physiological functions such as apoptosis, growth arrest, gene regulation, and survival. These systems have retained the genetic element as an addiction module [15].TA systems consist of two genes in an operon encoding a stable toxin moiety and a labile antitoxin moiety encoded on either extrachromosomal units or chromosomal units. A chromosomally encoded TA system is critical for cell viability and plasmid stability, whereas an extrachromosomally encoded TA system is important for biofilm formation, persister cell formation, growth arrest, and tolerance to multiple drugs. The chromosomal and extrachromosomal TA systems consist of the same two components, but in different configurations. Both modules are necessary for the proper functioning of the cell [16]. Moreover, TA systems are classified into different types according to the nature and mode of action of the antitoxin; the type II TA systems is the most common compared with the other types [16]. The type II TA system is responsible for most cases of antibiotic resistance, making it an important target for research and development of new antibiotics.

It is also an important tool to gain insight into the functioning of other TA systems. The MqsR/MqsA pair was the first TA system associated with biofilm formation and regulated biofilm formation [17, 18]. The role of genes of the TA system in biofilm formation, such as MazEF, RelBE, higBA, has also been investigated [18],On the other hand, antibiotics targeting P. aeruginosa are not effective against its ability to form a biofilm. Therefore, the present study investigated the effect of ZnO NPs as antimicrobial agents on P. aeruginosa biofilm formation and also the correlation of gene expression of TA systems among clinical isolates.