Evaluation of Biofilm Formation and Anti-biofilm Properties of Peganum Harmala and Crocus Sativus in Shigella Flexneri Clinical Isolates

Evaluation of Biofilm Formation and Anti-biofilm Properties of Peganum Harmala and Crocus Sativus in Shigella Flexneri Clinical Isolates

The Open Microbiology Journal 31 Dec 2019 RESEARCH ARTICLE DOI: 10.2174/1874285801913010297



Biofilm formation causes many serious problems in the treatment of bacterial infections. In addition, chronic infections due to biofilm formation can pose a huge burden to the health care systems. Also, many bacteria are biofilm producers as an important strategy for pathogenicity. Furthermore, the traditional use of herbal medicines such as Peganum harmala and Crocus sativus in Iran is interesting.


The purpose of the current study was to investigate the biofilm formation in Shigella flexneri clinical isolates and to evaluate the anti-biofilm properties of P. harmala and C. sativus on Shigella flexneri clinical isolates.


For the study purpose, Thirty S.flexneri clinical isolates were collected from Ahvaz, Iran. Then, the collected bacteria were subjected to biofilm formation assay. Afterward, P. harmala and C. sativus were applied as an anti-biofilm formation in S. flexneri.

Results & Conclusion:

Our results demonstrated that a significant number of samples were identified as strong biofilm producers. Then, P. harmala and C . sativus in a concentration of 30μg/ml and 60μg/ml were able to eradicate a strong biofilm formation in S. flexneri, respectively. In addition, it seems that more extensive studies and in vivo research should be done to confirm their properties.

Keywords: Biofilm formation, Shigella flexneri, Peganum harmala, Crocus sativus, S. flexneri, P. harmala.


Despite, there are many virulence factors in bacteria; the role of biofilm is notable [1]. The biofilm structure has many complications on human health and environment [2].In addition , numerous chronic infectious diseases are caused by biofilm formation in bacteria [3]. Many bacteria are biofilm producers, among them some true pathogens are more considerable for their biofilm formation [4,5].

Though, biofilm formation was investigated in different species of bacteria, including Pseudomonas aeruginosa, Enterobacteriaceae etc [6], Shigella species is considered in some countries to have the ability to produce biofilm [7]. However, S. flexneri may also be one of the biofilm producers other than Shigella species. Many studies have investigated the intracellular survival and virulence factor in S. flexneri; but there is a big gap in the pathogenesis and intracellular survival of this bacterium [8].

Certainly, biofilm formation is a mechanism in bacteria, which can promote bacterial resistance [9]. Furthermore, the use of antibiotic treatments against the biofilm structure in bacteria is one of the main challenges in medical science [10]. New drug discovery can be a good choice for the eradication of biofilm formation. Also, many studies have shown that different types of medicinal plants can be considered as an effective weapon against infectious diseases [11]. In the meantime, P. harmala is originally a native Asian plant. This plant belongs to the Nitrariaceae family. P. harmala is traditionally used to treat many infectious diseases [12]. Besides that, Crocus sativus is a native plant of Iran and used in traditional medicine in this country. The essential oil of this plant has antibacterial effects [13]. Due to these reasons, in this study, biofilm formation by S. flexneri isolates was investigated and the anti-biofilm properties of P.harmala and C.sativus on S. flexenarii clinical isolates were evaluated.


2.1. Bacterial Collection and Identification

A total of thirty S. flexneri clinical isolates were prepared at the Microbiology Research Center, Ilam University of Medical Sciences. Ilam, Iran. Then, S. flexneri were obtained from Ahvaz, Iran, by a a standard method [14,15].

2.2. Cell Culture

The P. harmala and C. sativus ethanolic extracts were applied to determine their cytotoxic effect on a vero cell line. Then, the MTT assay was performed by the MTT assay kit (Sigma, United States).

2.3. Toxicity Assay

The cells were inoculated in 96-well microplates and cellular density was determined. Then, the cells encountered different concentrations of the P. harmala and C. sativus extracts. The MTT assay was performed and the absorbance of the transformed dye was measured at a 600nm wavelength.

2.4. Biofilm Formation Assay

Initially, 0.5 McFarland solution of S. flexneri was prepared. Then, we inoculated 200 uL of broth media (LB broth) with a 0.5 McFarland solution of S. flexneri in 96 microplates for the evaluation of biofilm formation. Henceforth, the culture incubated for 24 hours at 35°C, so, the experiment was performed in triplicate. LB broth without S. flexneri was a negative control.

2.5. Semi-quantification of Biofilm Biomass

In this study, we used the methodology defined by Mowat et al. [16].

2.6. Analysis of Biofilm Formation

The ability of biofilm formation in all S. flexneri isolates was measured by absorbance in the crystal violet stain. In addition, the capacity of all of the strains to form a biofilm was compared with biofilm-forming S. flexneri controls. Further- more, we measured biofilm formation for each sample by analyzing the absorbance of the crystal violet. In this process, each isolate can create a biofilm mass in 24 hours which is eventually compared with the control. Finally, the isolates were divided into three categories based on biofilm formation. These groups included biofilms with 75% of the biomass of the positive control, moderately adherent biofilms with 25-75% biomass or weak biofilms with 25% of the biomass of the positive control.

2.7. Determination of Anti-biofilm Properties of P. Harmala and C. Sativus

The bacterial suspension was inoculated in 96 microplates. Different concentrations of P. harmala (1-35μg/ml) and C. sativus (1-100μg/ml) were applied. Then, the biofilm formation assay was performed.


3.1. Biofilm Formation by S. flexneri

Initially, the bacteria were confirmed by phenotypic methods. Furthermore, we discovered biofilm formations as a significant factor in S. flexneri clinical isolates; while the largest number of clinical isolates with a strong biofilm structure (n=11). In some S. flexneri clinical isolates, a moderate biofilm formation was also significant (n=10). Nevertheless, S. flexneri isolate was also observed with a weak biofilm formation (n=8). In addition, strains with no biofilm were very low and negligible (n=1). These results are summarized in Fig (1).

Fig. (1). The biofilm formation in S. flexneri clinical isolates.

3.2. P. harmala as An Anti-biofilm Formation in S. flexneri

The IC50 of P. harmala was 35 μg/ml. In this study, eleven isolates (36.66 percentages of samples) were observed to be able to produce a strong biofilm. Different concentrations of P. harmala was tested for all of them. P. harmala in a concen- tration of 30μg/ml could eradicate the biofilm formation (Fig. 2).

Fig. (2). Anti-biofilm Properties of P. harmala for eleven isolates. The highest concentrations were reported as final concentrations.

3.3. C. sativus as An Anti-biofilm Formation in S. flexneri

The IC50 of C. sativus was 100 μg/ml. Different concen- trations of C. sativus were tested for eleven isolates (36.66 percentages of samples) with a strong biofilm formation. C. sativus in a concentration of 60μg/ml easily eradicated the biofilm of S. flexenary (Fig. 3).

Fig. (3). Anti-biofilm Properties of C.sativus for eleven isolates. The highest concentrations were reported as final concentrations.


The biofilm formation causes many serious problems in the development of effective therapies for the treatment of infectious diseases [17]. However, the inherent ability of biofilm production in some bacteria has created many challenges in medical science [18]. Also, biofilm formation can cause widespread complications in the treatment of diseases and in the maintainence of human health [19].

In addition, biofilm formation is a community of microorganisms, which results in many infections and diseases causing problems at biological and environmental level [20].


In fact, one of the main mechanisms of bacterial survival in different environments is the ability to produce biofilms [21]. Moreover, bacteria that have the capacity to create biofilms can escape the host immune system, and therefore, cause chronic infections [22]. Meanwhile, S. flexneri employs several strategies to escape the immune system; one of the most important strategies is the ability to produce a biofilm [23]. In some studies, the effective factor in biofilm formation by S. flexneri was investigated [24]. Also, in several studies, S. flexneri infection was investigated but there remained a huge and significant gap in our knowledge for how to make S. flexneri capable of surviving in stress conditions [25]. Our data demonstrated that biofilm formation is a significant factor in S. flexneri clinical isolates. Furthermore, our results declare that medicinal plants can be used as a suitable candidate for the treatment of biofilm formation caused by S. flexneri. However, it seems that in vivo studies and more extensive studies in the field are necessary.


The current research approved by Ethical committee of Ilam University of Medical Sciences, Iran.


Not applicable.


Not applicable.


Not applicable.




The authors declare no conflict of interest, financial or otherwise.


We would like to thank the clinical microbiology research center, Ilam University of medical sciences for the strains used in this study.


Stauch-White K, Srinivasan VN, Camilla Kuo-Dahab W, Park C, Butler CS. The role of inorganic nitrogen in successful formation of granular biofilms for wastewater treatment that support cyanobacteria and bacteria. AMB Express 2017; 7(1): 146.
Foong K, Allen F, Islam I, Srivastava N, Seneviratne CJ. Microbial Biofilms: An Introduction to Their Development, Properties and Clinical Implications Microbial Biofilms 2017; 1-32.
Wu H, Moser C, Wang H-Z, Høiby N, Song Z-J. Strategies for combating bacterial biofilm infections. Int J Oral Sci 2015; 7(1): 1-7.
Neupane S, Pant ND, Khatiwada S, Chaudhary R, Banjara MR. Correlation between biofilm formation and resistance toward different commonly used antibiotics along with extended spectrum beta lactamase production in uropathogenic Escherichia coli isolated from the patients suspected of urinary tract infections visiting Shree Birendra Hospital, Chhauni, Kathmandu, Nepal. Antimicrob Resist Infect Control 2016; 5(1): 5.
Aboualigalehdari E, Sadeghifard N, Taherikalani M, Zargoush Z, Tahmasebi Z, Badakhsh B, et al. Anti-biofilm properties of P. harmala against Candida albicans. Oso Pub Health Res Perpect 2016; 7(2): 116-8.
Ciofu O, Rojo Molinero E, Macià MD, Oliver A. Antibiotic treatment of biofilm infections. Apmis 2017; 125(4): 304-19.
Karimi S, Ghafourian S, Taheri Kalani M, Azizi Jalilian F, Hemati S, Sadeghifard N. Association between toxin-antitoxin systems and biofilm formation. Jundishapur J Microbiol 2014; 8(1)e14540
Hu L, Grim CJ, Franco AA, et al. Analysis of the cellulose synthase operon genes, bcsA, bcsB, and bcsC in Cronobacter species: Prevalence among species and their roles in biofilm formation and cell-cell aggregation. Food Microbiol 2015; 52: 97-105.
Feng J, Shen Q, Wu J, Dai Z, Wang Y. Naked-eyes detection of Shigella flexneri in food samples based on a novel gold nanoparticle-based colorimetric aptasensor. Food Control 2019; 98: 333-41.
Kang J, Liu L, Liu M, Wu X, Li J. Antibacterial activity of gallic acid against Shigella flexneri and its effect on biofilm formation by repressing mdoH gene expression. Food Control 2018; 94: 147-54.
Oreopoulou A, Tsimogiannis D, Oreopoulou V. Extraction of polyphenols from aromatic and medicinal plants: An overview of the methods and the effect of extraction parameters Polyphenols in Plants 2019; 243-59.
Hamid IS, Elsidig IME. In vitro susceptibility of Isolated Shigella flexneri and Shigella dysenteriae to the Ethanolic extracts of Trachyspermum ammi and P harmala 2019.
Ahmadi Shadmehri A, Miri H, Namvar F, Nakhaei Moghaddam M, Yaghmaei P. Cytotoxicity, antioxidant and antibacterial activities of crocus sativus petal extract. Int J Res App Basic Med Sci 2019; 5(1): 69-76.
Chattaway MA, Greig DR, Gentle A, Hartman HB, Dallman TJ, Jenkins C. Whole-genome sequencing for national surveillance of shigella flexneri. Front Microbiol 2017; 8: 1700.
McGuire E, Tiberi S, Ciesielczuk H, Melzer M. Shigellosis and toxic megacolon secondary to Shigella flexneri serotype 3a: The challenges of laboratory diagnosis. Int J Infect Dis 2018; 70: 104-6.
Mowat E, Rajendran R, Williams C, et al. Pseudomonas aeruginosa and their small diffusible extracellular molecules inhibit Aspergillus fumigatus biofilm formation. FEMS Microbiol Lett 2010; 313(2): 96-102.
Wenzel RP. Health care–associated infections: Major issues in the early years of the 21st century. Clinical infectious diseases 2007; 45(Supplement_1): S85-8.
Díaz De Rienzo MA, Banat IM, Dolman B, Winterburn J, Martin PJ. Sophorolipid biosurfactants: Possible uses as antibacterial and antibiofilm agent. N Biotechnol 2015; 32(6): 720-6.
Høiby N, Bjarnsholt T, Moser C, et al. ESCMID guideline for the diagnosis and treatment of biofilm infections 2014. Clin Microbiol Infect 2015; 21(Suppl. 1): S1-S25.
Davey ME, O’toole GA. Microbial biofilms: From ecology to molecular genetics. Microbiol Mol Biol Rev 2000; 64(4): 847-67.
Hall-Stoodley L, Costerton JW, Stoodley P. Bacterial biofilms: from the natural environment to infectious diseases. Nat Rev Microbiol 2004; 2(2): 95-108.
Pavithra D, Doble M. Biofilm formation, bacterial adhesion and host response on polymeric implants-issues and prevention. Biomed Mater 2008; 3(3)034003
Reddick LE, Alto NM. Bacteria fighting back: How pathogens target and subvert the host innate immune system. Mol Cell 2014; 54(2): 321-8.
Crawford RW, Gibson DL, Kay WW, Gunn JS. Identification of a bile-induced exopolysaccharide required for Salmonella biofilm formation on gallstone surfaces. Infect Immun 2008; 76(11): 5341-9.
Köseoğlu VK, Hall CP, Rodríguez-López EM, Agaisse H. The Autotransporter IcsA promotes shigella flexneri biofilm formation in the presence of bile salts. Infect immun 2019; 87(7): e00861-18.