An Easy New Modified Method for Detection of Antibacterial Susceptibility in Biofilm-Growing Bacteria

Background : Biofilms are a major challenge in treating infections. Clinically, biofilms are often associated with chronic infections, so rapid and accurate methods of investigating the antibiotic susceptibility of biofilm bacteria are very important for faster diagnosis and treatment. In this study, a new modified microplate method was proposed to evaluate the antibiotic susceptibility of alive bacteria in the biofilm. Materials and Methods: The study was conducted on clinical isolates of Pseudomonas aeruginosa and standard strain over a period of one year. The susceptibility test was conducted according to a modified version of the Calgary biofilm device method. In the last step, to study the living bacteria in the biofilm, oxidative-fermentative (OF) medium was used to measure the acid production following the use of glucose by the bacteria. The biofilm-growing bacteria was determined by observing the color changing and also measuring optical density (OD) at 427 nm of OF medium. Results: The method in this study could evaluate the effect of antibiotics on biofilm bacteria based on glucose metabolism. The results indicated that this method can quickly and easily identify alive bacteria in biofilm at a low cost and without the need for any special devices. Conclusion: Although biofilms are involved in most incurable clinical cases, there is currently no guideline to assist physicians in treating biofilm-related infections. Therefore, it is necessary to establish a specific sensitivity test for biofilm or to approve a new method for routine use in diagnostic laboratories.

Dis Diagn. Vol 11, No 3, 2022 96 http://ddj.hums.ac.ir http and evaluated in planktonic (free-living) mode. Biofilm pathogenic bacteria can be up to a thousand times more resistant to antimicrobial therapies. The problem of biofilm resistance makes it difficult to treat and eliminate them effectively. Hence, new strategies are needed to prevent, disperse, and treat bacterial biofilms (6). As a consequence of the interaction between planktonic bacteria and biofilms, bacteria can better survive in the host (in harsh conditions). In recent years, the number of patients in the health care sector who are prone to biofilm colonization has increased due to the use of implanted biomaterials. Further, biofilms are involved in chronic bacterial infections not related to the device. Treatment of these infections with conventional antimicrobial agents is not always successful, so it often results in surgical removal of the implant, which carries risks and problems (7). The amount of the minimum biofilm eradication concentration (MBEC) has been suggested as a laboratory assay to evaluate antimicrobial activity against mature biofilm (8). The Clinical Laboratory Standards Institute offers bacterial testing in the form of plankton and has no method of testing antimicrobial susceptibility to biofilm-related organisms. Different protocols have been developed to test antimicrobial sensitivity in biofilm including the MBEC assay (9). Biofilm detection of microorganisms is very important to conquer long-lasting infections, determining the antibiotic and antibacterial activity of the agents against the biofilm and the bacteria inside the biofilm as well. Hence, the present study tried to suggest an easy new method for the detection of antibiofilm activity of agents.

Sample Collection and Bacterial Screening
In this study, 151 samples consisted of urine, tracheal aspirates, wound, blood, and secretions obtained from Shahid Mohammadi Hospital, Bandar Abbas, Iran. The isolates of P. aeruginosa were identified using standard microbiological procedures (e.g., gram staining, oxidase test, and other biochemical tests). In addition, P. aeruginosa PTCC 1430 was used as reference strain of positive biofilm producer. Then, ciprofloxacin-resistant isolates were screened by 5µg ciprofloxacin disk (Floka, USA) using the disk diffusion method according to the Clinical Laboratory Standards Institute (2017) guideline.

Biofilm Formation Assay
The ability to produce biofilms in bacteria was assessed by the microtiter colorimetric method. Bacterial colonies were grown for 24 hours in the tryptic soy broth (TSB, Merck Darmstadt, Germany at 37 °C ). Bacterial suspensions (1:100) were diluted in a new TSB medium and 150 μL of this solution was added to 96-well polystyrene microtiter plates with a sterile flat bottom and incubated for 24 hours at 37°C, then the wells were gently washed three times with sterile distilled water. The wells were dried in an inverted position at room temperature and finally stained with 125 μL of 0.1% violet crystal solution in water for about 10-15 minutes. Crystal violet powder (Merck, Germany) was discarded. The wells were washed three times to remove excess crystal solution. Finally, violet crystals were released by adding 125 μL of 30% acetic acid (Merck, Germany). The optical density (OD) absorption of each well at 550 nm was measured using an ELISA reader (BioTek ELx800). All assays were repeated three times. As a control, an uninoculated medium was used to determine the background OD. The cut-off OD (ODc) was defined as three standard deviations above the mean of negatively controlled OD (10). 44 Biofilm forming ability of the strains was classified as follows: Non biofilm (OD test < ODc) Weak biofilm (ODc < OD < 2 × ODc) Moderate biofilm (2 × ODc < OD < 4 × ODc) Strong biofilm (4 × ODc < OD)

Antimicrobial Susceptibility Test of Biofilm-Growing Bacteria
The susceptibility test was conducted according to a modified version of the Calgary biofilm device method (11). In brief, 20 µL 10 8 CFU/mL −1 bacterial pellets were resuspended in 180 µL TSB containing 1% glucose and aliquoted into the 96-well culture plate (TPP Techno Plastic Products) and incubated overnight at 37°C without shaking. After washing 3 times with sterile saline solution, plates were incubated at room temperature for 15 minutes. Then, microplates were incubated with each antimicrobial agent at a serial twofold dilution in Mueller-Hinton broth for 24 hours at 37°C (11,8). Again, plates were washed 3 times with sterile saline solution, and oxidativefermentative (OF) medium Merck (Germany) was added for 24 hours at 37°C. OF medium and medium antibioticfree containing bacteria are considered as negative and positive controls, respectively. The MBEC was determined by observing the color-changing (qualitative) and also measuring OD at a wavelength of 427 nm using an ELISA reader (BioTek ELx800) of OF medium (quantitative) in the microplate wells ( Figure 1). Finally, all the assays were repeated three times.

Statistical analysis
All data analyses were done with SPSS (version 16, SPSS Inc, USA), and GraphPad Prism 6 software (GraphPad Software, Inc., USA) was used to prepare graphs. A p-value less than 0.05 (two-tailed) were considered statistically significant.

Susceptibility Testing of Biofilm-Growing Bacteria
The OF test was applied to indicate the oxidation or fermentation of glucose by gram-negative rods. The concentration of acid produced during the process of glucose metabolism turned the bromothymol blue indicator from green to yellow in OF medium. Therefore, if the bacteria are alive and they use the glucose in the medium, they will change the color from green to yellow.   Therefore, yellow wells were considered positive. Table 1 presents the MBEC qualitative results, where the highest frequency for ciprofloxacin was 2560 μg/mL, and MBEC50 of ciprofloxacin was calculated at 1280 μg/mL. According to Figure 3, the cut-off for bacterial growth was obtained by calculating the mean ± standard deviation of the negative control at a wavelength of 427 nm, which is the maximum adsorption of bromothymol blue reagent in an acidic state (yellow). Therefore, absorptions higher than the cut-off value were considered positive wells. Table 2 represents the data related to two samples. For example, in isolate No. 71 with strong biofilm, the adsorption rate in the well with a concentration of 640 μg/mL was equal to 0.957 nm, which was higher than the cut-off value. Therefore, a slightly isolated MBEC of 71 was recorded for the antibiotic ciprofloxacin 1280 μg/mL. A small MBEC was also recorded for isolate No. 75 with an average biofilm of 640 μg/mL.

Discussion
To screen the efficacy of antibiotics against P.aeruginosa biofilms, we optimized a microtiter-based protocol. This methodology was selected because it requires few steps and is user-friendly. This method is based on cellular metabolism as well as the production of acid from sugar. The method does not require special materials and equipment, and all materials are available in any laboratory, so it is more economical. It also takes less time to perform the test compared to other methods. It can be examined qualitatively (observationally), and the results can be quantitatively confirmed. Another method used in this study is the modified calgary apparatus. In this method, to check for live bacteria, each well should be cultured on an agar medium at the end of the assay. The advantage of this method is that the number of colonies can be measured in each concentration. In our method, the number of steps and days of testing was less. In addition, as illustrated in Table 3, like the method mentioned, it did not require special devices such as ultrasonic water baths (1). Comparing the results of biofilm-growing bacteria by qualitative and quantitative methods using OF medium, no significant difference was found.
The microbial community (biofilm) can produce extracellular polysaccharides after binding to nonbiological surfaces. Microbial cells that grow in biofilms are physiologically different from planktonic cells of the same organism (1). The researchers have challenges with biofilm complexity and developmental mechanisms (12). Due to the lack of reliable and accurate methods for testing new antibiotics, their development is hampered. One of the methods of evaluation and determination of MBEC for different antibiotics is the resazurin method which has both advantages and disadvantages. Using this method as a screen test requires ptimizing resazurin for each bacterial strain. The test results must also be interpreted carefully because this method is less valuable. This method cannot detect the number of cells less than 10 6 CFU per biofilm, so a supplementary method is required (13). Based on current instructions or antibiotic treatments, Plankton   bacteria are often unable to successfully deliver a pathway for the treatment of biofilm infections. The biofilm bacteria are inherently more tolerant to antibiotics than planktonic cells. Therefore, the determination of specific biofilm antibiotics is an essential step for predicting treatment success (14). In the study on different methods of antibiofilm, it is necessary to examine the bacteria present in the biofilm. Both the matrix and the bacteria in it are essential for the formation and maintenance of a mature biofilm. When a potential treatment only destroys the matrix, the remaining bacteria can create a new biofilm by producing new extracellular matrix components (15).
For the results to be generalizable, other methods must be evaluated, but due to the time limitation of the study, it was decided to carry out them in future studies.

Conclusion
In this study, OF medium microtiter plate test has been developed and optimized to detect antimicrobial effect of drugs on palanktomic cell in biofilms. As such, OF medium, which was a new, simple, and reproducible method, was used to study biofilm growing bacteria. Moreover, this procedure can be used to investigate the antimicrobial activity of a wide range of biocides.