1. Introduction
Today, antimicrobial chemotherapy agents are used to control various infections; however, due to the emergence of antibiotic-resistant strains, researchers are looking for new solutions to solve this problem. In this regard, probiotics, including bacillus, have been more common [1, 2]. Nosocomial infection is one of the leading causes of death caused by antibiotic-resistant strains, including Acinetobacter baumannii [6, 7]. This bacterium is resistant to a wide range of antibiotics due to having properties that affect antibiotic resistance, including the ability to form strong biofilms [9, 10]. Outer membrane protein (ompA) plays an essential role in binding Acinetobacter baumannii to surfaces to form biofilms and causing antibiotic resistance [15, 16]. This study aims to identify the best probiotic with the ability to inhibit the growth and form a biofilm in Acinetobacter baumannii isolate with multiple antibiotic resistance by inhibiting the expression of ompA gene associated with biofilm formation in this isolate.  
2. Materials and Methods
The resistance of Lactobacillus rhamnosus, Bifidobacterium animalis, Pediococcus acidilactici [20, 21, 22], Bacillus subtilis, Bacillus coagulans, and Bacillus licheniformis [23, 24] isolates to acidic conditions, and bile salts were investigated. Then, the antimicrobial properties of supernatant of all isolates against Acinetobacter baumannii (A52), Acinetobacter baumannii (ATCC19606), Klebsiella pneumoniae (ATCC70063), and Pseudomonas aeruginosa (PAO1) hospital isolates were evaluated using the agar well diffusion method [32]. After determining the minimum inhibitory concentration (MIC) of Bacillus licheniformis supernatant against A. baumannii ATCC19606 and A52 [33], phenotypic inhibition of biofilm in these two strains was investigated and confirmed using the field emission scanning electron microscopy [34, 35]. In the next step, using the polymerase chain reaction (PCR) technique, the ompA gene effective in biofilm formation in A. baumannii A52 isolate was identified [31]. The expression of ompA gene was examined before and after treatment with B. licheniformis supernatant using the Real-Time PCR technique [37]. Finally, the PCR product of ompA gene in A. baumannii A52 isolate was sent to Pishgam Company for sequencing.
4. Results
In examining the resistance of isolates, the number of colonies counted in each isolate was more than 106 CFU/mL, and the inhibition coefficient of each isolate was <0.4. These results showed that all isolates were resistant to acids and bile salts. In studying the antimicrobial properties of supernatants extracted from all probiotic isolates against pathogens A. baumannii A52, A. baumannii ATCC19606, K. pneumonia ATCC70063, and P. aeruginosa PAO1, results showed that B. licheniformis isolate could inhibit the significant growth of these agents. The MIC of B. licheniformis isolate supernatant was 32 µg/mL. The B. licheniformis supernatant could reduce biofilm formation by 70% in A. baumannii A52 isolate. After performing PCR to investigate the presence of ompA gene in A. baumannii A52 isolate, a 578bp band was observed (Figure 1). Blast results showed that the sequence of PCR product of the ompA gene was 99.63% similar to the sequence of ompA in A. baumannii found in the National Center for Biotechnology and Information website. Evaluation of ompA gene expression using real-time PCR technique in A. baumannii before and after treatment with B. licheniformis supernatant showed that the expression of ompA gene was significantly decreased in A. baumannii ATCC19606 by 74.71% (P<0.0001) and in A. baumannii A52 by 69.72% (P<0.0001) (Figure 2). 5. Discussion and Conclusion
Nowadays, opportunistic pathogens, especially A. baumannii, in the hospital environment cause many health problems including nosocomial infections in people with weak immune systems due to the high resistance of antibiotics [38]. Given the importance of biofilm formation in developing this antibiotic resistance, the present study showed that the inhibition of ompA gene expression was effective in biofilm formation in A. baumannii after treatment with B. licheniformis supernatant compared to pre-treatment phase. In previous studies, researchers have used B. licheniformis supernatant to inhibit the growth and formation of biofilm pathogens, including candida albicans, pseudomonas aeruginosa, and staphylococcus aureus [39, 40]. In a similar study, considering the important role of ompA protein in creating antibiotic resistance in A. baumannii, it was attempted to inhibit ompA gene expression by chemical compounds. Finally, three compounds could inhibit the ompA gene expression, leading to a reduction in biofilm formation [44]. In another study, researchers used a plant-derived substance called Myrtenol to inhibit genes involved in biofilm formation, including pgaA, ompA, bap, csu A/B. They observed that, by suppression of these genes, the biofilm thickness was reduced in A. baumannii [45]. 
Based on the results of this novel study, it can be concluded that B. licheniformis has a good ability to inhibit the expression of ompA gene effective in biofilm formation in A. baumannii as a causative agent of nosocomial infections. The production of compounds with antimicrobial and anti-biofilm properties should be further identified in future studies. B. licheniformis can be used as a low-risk biomaterial to reduce the biofilm of pathogens with antibiotic resistance in therapeutic environments.

Ethical Considerations
Compliance with ethical guidelines
This research was approved by the Ethics Committee of the Faculty of Medical Sciences of Islamic Azad University of Qom (code: IR.IAU.QOM.REC.1398.004).

Funding
This article was extracted from the PhD dissertation of the first author in the Department of Microbiology, Faculty of Basic Sciences, Islamic Azad University, Qom Branch.

Authors' contributions
All authors have contributed to the research and preparation of this article.

Conflicts of interest
According to the authors, this article has no conflict of interest.

Acknowledgments
The authors would like to thank the personnel of Microbiology Research Laboratory, Islamic Azad University, Qom branch.


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