Journal of Current Biomedical Reports jcbior.com Volume 2, Number 2, 2021 eISSN: 2717-1906 1 Original research Detecting of biofilm formation in the clinical isolates of Pseudomonas aeruginosa and Escherichia coli: an evaluation of different screening methods Amin Mohsenzadeh1, Alireza Fazel1, Shirin Bavari2, Sanaz Borji3, Sahar Pourasghar3, Taher Azimi4, Hoda Sabati5,* 1Department of Microbiology, Ardabil Branch, Islamic Azad University, Ardabil, Iran 2Department of Microbiology, Faculty of Medicine, Iran University of Medical Sciences, Tehran, Iran 3Department of Biology, Faculty of Science, Rasht Branch, Islamic Azad University, Rasht, Iran 4Department of Pathobiology, Faculty of Public Health, Tehran University of Medical Sciences, Tehran, Iran 5Biotechnology and Biological Science Research Center, Shahid Chamran University of Ahvaz, Ahvaz, Iran Abstract Biofilm producer bacteria cause nosocomial, chronic, and recurrent human infections. It is hard to treat biofilm-embedded bacteria because they are more resistant to antimicrobials than planktonic bacteria. The present study aimed to investigate different methods for detecting biofilms in the clinical isolates of Escherichia coli and Pseudomonas aeruginosa. The study was carried out at the Department of Microbiology, Ardabil Branch, Islamic Azad University, Ardabil, Iran, from January 2019 to June 2019. A total of 320 clinical samples were collected from educational and medical centers in Tehran, Iran which from them 100 isolates of E. coli and P. aeruginosa were identified by standard microbiological procedures and subjected to biofilm detection methods. Biofilm detection was tested by Congo red agar (CRA), tube method (TM), microtiter plate assay (MTPA). The MTPA was considered to be superior to CRA and tube TM. From the total of 100 clinical isolates, MTPA detected 38 (38%) isolates as biofilm- positive phenotype, of which 30 as strong, and 8 as moderate biofilm-forming isolates. It can be concluded from the current study that the MTPA is a more quantitative and dependable assay for the detection of biofilm-forming microorganisms as compared to other methods, and it can be recommended as a general screening method for the detection of biofilm-producing bacteria in laboratories. Keywords: Biofilm detection, Microtiter plate assay, Congo red agar, Tube method 1. Introduction Microorganisms can live in one of two phenotypes: sessile or plankton. Planktonic are free- floating microorganisms. The sessile phenotype results from the binding of microorganisms to solid surfaces, their irreversible binding, and the development of exopolysaccharides with a gradual * Corresponding author: Hoda Sabati, MSc Biotechnology and Biological Science Research Center, Shahid Chamran University of Ahvaz, Ahvaz, Iran Tel/Fax: +98 922 6092051 Email: h.sabati@yahoo.com https://orcid.org/0000-0002-4473-7245 Received: March, 27, 2021 Accepted: April, 08, 2021 increase in cell secretions' thickness [1]. A biofilm is a collection of microbial cells that are irreversibly enclosed in a polysaccharide matrix and bonded together on biotic surfaces such as host cells or abiotic surfaces such as medical devices [2]. Microbial cells that grow in a biofilm are physiologically different from planktonic cells of the same organism, which, in © The Author(s) 2021 https://jcbior.com/ Mohsenzadeh et al. 2 contrast, are single-cell and may float or swim in a liquid environment [3]. Biofilms are considered a serious public health problem due to the increased resistance of the constituent organisms to antimicrobial drugs and the ability to cause infection in patients who have been implanted with medical devices [4, 5].The biofilm matrix blocks the penetration of antibiotics and prevents them from reaching the cells inside the biofilm [6]. Studies have shown that bacterial biofilms cause 80% of microbial infections that are very difficult to eradicate and treat with antibiotics [7]. One of the most important pathogens that produce strong biofilms is Escherichia coli. Previous studies on the survival of biofilm-related pathogens also indicate that biofilms may play an essential role in the persistence and release of stress-sensitive organisms into the environment. The results show that E. coli resists food deficiency conditions, and the interaction of this bacterium with Pseudomonas aeruginosa can increase E. coli growth in biofilm [8]. Biofilm formation contributes to the pathogenesis of P. aeruginosa in acute as well as chronic infections in clinical cases [9]. P. aeruginosa is currently considered the leading cause of ventilator-associated pneumonia (VAP) in the ICU. Infections caused by this bacterium occur mainly in patients with critical condition and immunodeficiency and increase the morbidity and mortality in these patients as well as increase the incidence of resistance of P. aeruginosa to antibiotics [10]. To control chronic and recurrent infections, it is essential to identify microorganisms' biofilms, maturity, and dispersion [11]. There are various laboratory methods to evaluate the production of biofilm in bacteria. Biofilm growth and observation with microscopes such as confocal scanning laser microscope (CLSM), quantitative method of closed systems under static conditions such as tissue culture plate (TCP) method and observation with non-specific color, floating biofilms (Air liquid interface (ALI)), bioluminescent assay, observation of bacteria colonies on the surface of a solid medium such as Congo red agar (CRA) and the simple tube method (TM) are among the biofilm measurement methods [12]. Microtiter plate assay (MTPA) is a screening method for comparing adhesion patterns. It is the most widely used quantitative method for detecting biofilm production. In the CRA method, a positive result is shown by the black colony and the dry crystal consistency. A pink colony appears in the absence of biofilm [13]. Laboratory study of biofilm production by bacteria can be affected by the choice of a specific culture medium. The conditions that the bacterium encounters during growth can encourage or suppress biofilms production or cause biofilms with unusual structures [14]. This study aimed to investigate the methods (microtiter plate methods, tube method, and Congo red agar) for the detection of biofilm formation in the clinical isolates of E. coli and P. aeruginosa. The sensitivity and specificity of the methods have been determined through statistical analysis. The MTPA method was considered the gold standard for this study and compared with data from TM and CRA assays. True positives were biofilm producers by MTPA, TM, and CRA assays. False-positive were biofilm producers by TM and CRA assay and not by MTPA method. False-negative were the isolates that were non-biofilm producers by TM and CRA but were producing biofilm by MTPA method. True negatives are those which were non-biofilm producers by all the methods. 2. Materials and Methods 2.1 Sample collection Out of 320 clinical samples collected from educational and medical centers in Tehran, Iran from January 2019 to June 2019, 50 isolates of E. coli and 50 isolates of P. aeruginosa were identified and subjected to biofilm formation detection. These isolates were identified by, colonial morphological analysis on Eosin Methylene Blue (EMB) Agar (for E. coli isolates) and Cetrimide Agar (for P. aeruginosa isolates) and standard microbiological procedures such as Gram staining, catalase test, cytochrome oxidase reaction, motility, and biochemical tests. These isolates were selected for biofilm formation evaluation. Reference strain of positive biofilm producer P. aeruginosa ATCC 9027, E. coli ATCC 25922 were used as controls. 2.2 Microtiter plate assay (MTPA) Each of the collected and reference strains was cultured on Tryptic Soy Agar (TSA) (Merck, Germany) medium and incubated overnight at 37°C. Fresh colonies were cultured in 10 ml Tryptic Soy Broth (TSB) (Merck, Germany) medium in a sterile tube and Mohsenzadeh et al. 3 incubated for 15-18 h at 37°C with a shake at 200 rpm. The optical absorption of each liquid culture was adjusted using a fresh medium with an OD of 0.1 at 620 nm. Bacterial cultures were diluted, and a suspension equivalent to half McFarland was prepared in TSB medium. 200 μl of the suspension prepared from each bacterial strain was transferred into 96 polystyrene plate wells. The plates were incubated at 37 °C for 96 h. The control organism was also incubated, diluted, and added to the culture plate. Negative control wells contained inoculated sterile broth. After this time, each well's contents were removed and washed twice with phosphate buffer solution (PBS) (pH 7). This removed free-floating bacteria. After drying, the wells were fixed with 95% Methanol for 10 minutes and then stained with Crystal violet (1%) for five minutes. The plates were emptied, and an excess stain was washed by sterile distilled water. The wells were filled with 100 μl of Glacial acetic acid solution (33%) and biofilm formation has been evaluated at 570 nm with an ELISA auto reader (Biotek ELx800, USA) [9]. 2.3 Congo red agar (CRA) The Congo red indicator (8 g/L) was prepared separately from the other medium constituents as a concentrated aqueous solution and autoclaved (121°C for 10 minutes). Then it was added to the autoclaved Muller Hinton agar medium (Merck, Germany) (10 g/ml) with sucrose (50 g/L) at 55°C. Test organisms were cultured on the CRA medium and incubated aerobically for 24 - 48 h at 37 °C. Black colonies with a dry crystalline consistency indicated biofilm production [1]. 2.4 Tube method (TM) A loopful of the bacterial suspension was transferred to a tube containing 10 ml of LB broth (Merck, Germany) medium with glucose (2%). The tubes were incubated for 24 h at 37°C. The contents of the tubes were then removed and washed with PBS (pH 7.3). Then the dried tubes were stained with Crystal violet (1%). Excess dye was washed with sterile distilled water. Tubes were dried in the inverted position. Isolates forming slime or biofilm-based on Crystal violet color's thickness on the tube's bottom and the wall was observed and recorded. The observation of a visible thin layer on the wall and bottom of the tube was considered a moderate biofilm. The observation of a thick layer visible on the wall and bottom of the tube was considered a strong biofilm. The lack of a visible layer on the wall and bottom of the tube was considered non-biofilm formation and negative. The experiment was performed in three replications [3]. 2.5 Statustical analysis The results were entered into Microsoft Excel 2016 and were analyzed by using SPSS version 25 (SPSS Inc., Chicago, IL, USA). Spearman's correlation test was used to define the direction of association. P- value <0.05 was considered statistically significant. 3. Results Of the 100 E. coli and P. aeruginosa isolates, 38 (38%) isolates showed a biofilm-positive phenotype under the optimized conditions in MTPA (Figure 1) and isolates were further classified as strong, moderate, weak, and no biofilm (Table 1). CRA assay led to different results, only 26.3% of isolates showed black colonies with dry crystalline morphology after 24-48 hours (Table 2). Though the CRA assay can well detect biofilm-producing strains, weak producers were difficult to discriminate from biofilm negative isolates. No direct correlation between colony morphology on CRA and MTPA results was observed. In TM, strong biofilm producers could be easily detected, whereas it was difficult to differentiate between moderate and weak biofilm-forming isolates which affected its performance in terms of sensitivity and specificity. Statistical analysis of TM and CRA methods for detection of biofilm formation in isolates using MTPA as a gold standard (n=100) showed that TM assay has more sensitivity and specificity than the CRA method (Table 3). Biofilm formation in the isolates by either of the methods was 16% and 60% for E. coli and P. aeruginosa, respectively. Discussion Biofilm-producing bacteria cause many chronic and recurrent infections that are difficult to treat. Treatment and control of biofilm-associated infections is an essential issue in hospitals and medical care. These bacteria exhibit resistance by different assays such as restricted penetration of antibiotics into biofilms and expression of resistance genes by facilitating the exchange of plasmids responsible for drug resistance due to proximity of cells within Mohsenzadeh et al. 4 biofilms. There are a lot of different assays for detecting biofilm [15, 16]. In this study, we evaluated 100 isolates by three screening methods for their ability in biofilm formation. In the MTPA, the number of isolates showing biofilm formation was 38 (38%) (30 strong and 8 moderate) and no weak biofilm producers were detected. A previous study showed from the 50 clinical Figure 1. Biofilm formation detection in Pseudomonas aeruginosa by microtiter plate assay 0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8 O p ti c a l d e n si ty ( O D ) a t 5 7 0 n m No. of isolate control+ S54 S66 S57 S62 S40 S59 S43 S35 S53 S63 S45 S46 S52 S19 S61 S15 S14 S48 S49 S4 S21 S18 S8 S64 S22 S47 S34 S16 S26 control - Table 1. Interpretation of biofilm formation by average OD value in microtiter plate assay Biofilm formation OD isolate (at 570 nm) ODc Isolates Strong OD isolate>0.929 OD isolate>4×ODc E41, E42, E43, E45, E48, E58, E60, S66, S57, S62, S40, S59, S35, S53, S63, S46, S34, S16, S52, S19, S61, S15, S48, S4, S21, S18, S8, S26, positive controls Moderate 0.464