Effect of Ciprofloxacin and Trimethoprim/Sulfamethoxazole on Biofilm Formation of Multi-Drug Resistant Uropathogenic Escherichia coli

2.1 Bacterial isolation and morphological diagnosis

A total of 76 UPEC isolates were collected from patients with urinary tract infections (UTIs) in various healthcare facilities across Baghdad province between December 2023 and February 2024. Patients were males (24) and females (52) at different ages ranging from 15 days to 85 years, and different-hospitalized status (Outpatients and Hospitalized care). Ethical approval for the study was obtained from the Iraqi Ministry of Health's Ethical Review Board. Metadata collected includes the patient’s gender and age. The isolates are suspected to be sourced from hospital-acquired and community-acquired UTI cases, according to the opinion of the specialist physician. Primary identification performed, considering morphological parameters and biochemical characteristics, including oxidase, catalase, indole, methyl-red, Voges-Proskauer, and citrate utilization [21]. Further confirmation was carried out with the Vitek®-2 compact system using Gram-negative identification (GNID) cards (BioMérieux, France).

2.2 MIC determination by Vitek-2 compact system

Antibiotic suitability test measured by using the Vitek®-2 compact system technique. Gram-negative antimicrobial susceptibility cards (AST) are intended to be used in the Vitek-2 system for determining susceptibility of aerobic gram-negative bacilli and to determine MICs for each isolate. Vitek®-2 compact system MIC levels depend on CLSI (2023). All E. coli isolates were cultured on MacConkey agar and incubated for 24 hours at 37℃. Following incubation, a single colony is taken to form a bacterial suspension with a turbidity of 0.5 cells/ml inside special tubes called Kan tubes. The bacterial suspension was placed inside the filling door of the Vitek®-2 compact system so that the suspension could be transferred from the Kan tubes to the susceptibility test kit.

2.3 MIC determination by the well diffusion method

Four UPEC isolates, K16, G33 (resistance), K42, and G27 (sensitive) used to determine the MICs, SMICs, and SSMICs of Ciprofloxacin and Trimethoprim/Sulfamethoxazole. Ciprofloxacin was diluted in sterilized distal water to make a series of dilutions ranging from (0.625-1024) µg/ml, while Trimethoprim/Sulfamethoxazole concentration ranged from (0.5-2024) µg/ml. By using the well diffusion method as mentioned in study [22], fresh cultures of each isolate were inoculated within 5 ml plastic tubes containing 3 ml of 0.85% normal saline and shaken using vortex. The bacterial suspensions corresponded to 0.5 McFarland (1.5×10⁸) CFU/ml, and a cotton swab was flooded at the bottom of each suspension and spread on Muller-Hinton agar. After leaving the plates to dry, a 5 mm sterilized cork borer was used to make four wells in each plate, and 100µL of each antibiotic concentration was transferred to each well. Plates were incubated overnight at 37℃.

2.4 Biofilm formation assay

The Crystal Violate (CV) biofilm assay is considered the gold standard to quantify biofilm formation in UPEC. Kowalska et al. [23] procedure carried out with modification included manipulating in type of media and supplements. Brain Heart Infusion broth (BHI) and Tryptic Soy Broth (TSB) provided by Himedia were used. BHI supplemented with 2% sucrose. TSB supplemented with 1% glucose. Briefly, 200 µl of each fresh bacterial suspension adjusted to 0.5 McFarland (1.5×10⁸) CFU/ml was inoculated in each well of a sterilized 96-well flat-bottom microtiter plate. Three-folds from each isolate were made, and three-folds of BHI and TSB were taken as Negative controls. The plates were incubated overnight at 37℃. After incubation, the contents of the plates were emptied and washed with three-fold of 200 µl of 0.98% Phosphate Buffer saline (PBS) and left to dry for 40 minutes to dry at room temperature. The attached cells in each well were stained with 200 µl of 0.1% CV for 10 minutes. The plates were then again emptied of the remaining stain and 200 µl of 95% Ethanol added to each well to solubilize the contents. Uninoculated broth of both BHI and TSB was taken as a Negative control. The optical density (OD) was measured at 620 nm using an ELISA plate reader in the Biotechnology Research Center/Al-Nahrain University.

2.5 Effect of antibiotics on biofilm formation

Four of the UPEC isolates were used to estimate the efficiency of ciprofloxacin and Trimethoprim/Sulfamethoxazole on biofilm formation. The selected isolates were K16 and G33 (resistant), K42 and G27 (sensitive). To dilute ciprofloxacin for the level of MICs, SMICs, and SSMICs, Rafaque et al. [24] proposed that antibiotics be diluted in Mueller-Hinton broth (MHB). Simply, stock solutions of each antibiotic are prepared, and then the concentrations are diluted in MHB. MICs of Ciprofloxacin were 4 µg/ml for both K16 and G33, while it was 8 µg/ml and 1 µg/ml for K42 and G27, respectively. The same previous dilution was carried out for preparing Trimethoprim/Sulfamethoxazole dilutions. MICs of Trimethoprim/Sulfamethoxazole were 256 µg/ml for both K16 and G33, while it was 64 µg/ml for K42 and G27. Two microtiter plates were used, one for the K16 and G33 (Resistance-Plate), the other for K42 and G27 (Sensitive-Plate). 100µ of K16 and G33 grown MHB added to wells and left to settle. Each isolate was taken as two two-fold replicates. Then, 100µL of each ciprofloxacin concentration (4 µg/ml, 2 µg/ml, and 1 µg/ml) was added to each well containing fresh bacterial isolates. The same procedure was followed in the Resistance plate for Trimethoprim/Sulfamethoxazole at concentrations: 256 µg/ml, 128 µg/ml, and 64 µg/ml. Sensitive plate carried out in the same way using K42 and G27. Ciprofloxacin was (8 µg/ml, 4 µg/ml, and 2 µg/ml) for K42 and (1 µg/ml, 0.5 µg/ml, and 0.125 µg/ml) for G27, while Trimethoprim/Sulfamethoxazole was (64 µg/ml, 32 µg/ml, and 16 µg/ml) for each isolate. All plates incubated overnight at 37℃, emptied and washed with 200µl (two-fold) and stained with 200µl of CV for 10 minutes, followed by 200µl of Ethanol 95% and O.D. measured at 620 nm. Free antibiotics, bacterial broths taken as positive control, and bacteria-free antibiotics dilutions taken as negative control. Each reading was compared to the tested wells analyzed to detect the efficiency of Ciprofloxacin and Trimethoprim/Sulfamethoxazole on biofilm formation by each isolate.

2.6 Effect of antibiotics on UPEC already formed biofilm

The effect of antibiotics ciprofloxacin and Trimethoprim/Sulfamethoxazole was tested on already formed UPEC biofilms. Four isolates of UPEC (K16, G33, K42, and G27) were selected. The same method described in study [23], which aimed to detect the effect of ciprofloxacin on biofilm formation by optimizing some steps (adding antibiotics after biofilm formation, amount of bacteria and antibiotic inoculation, and timing of using PBS and 0.1% CV). Briefly, each isolate was grown in MHB overnight at 37℃. Once again, two 96-well microtiter plates were used, one for resistance isolates and the other for sensitive isolates. 200 µl of each isolate was inoculated into the wells. Six replicates were taken for each isolate. The plates were incubated overnight at 37℃ to allow the isolates to form biofilms. After incubation, the contents of the plates were emptied to remove unattached cells. 100 µl of ciprofloxacin and Trimethoprim/Sulfamethoxazole concentrations were added to each well as two-fold replicates for each concentration. The same concentrations of antibiotic used to affect biofilm formation were used. After adding antibiotic concentration, plates were incubated for 24 hours at 37℃, emptied of unattached content with three-fold of 200µl of PBS, and left to dry for 40 minutes. Then, the plates were stained with 200µl of 0.1% CV for 10 minutes, followed by 200µl of 95% Ethanol. O.D. measured at 620 nm. Free antibiotics, bacterial broths taken as positive control, and bacteria-free antibiotics dilutions taken as negative control. Each reading was compared to the tested wells analyzed to detect the efficiency of Ciprofloxacin and Trimethoprim/Sulfamethoxazole on biofilm formation by each isolate.

2.7 Statistical analyses

The data was tabulated in a datasheet of IBM SPSS version 25.0, which was utilized to do the statistical analysis. The mean and standard errors of continuous variables were reported, and significant differences were tested using the analysis of variance (ANOVA) test, followed by the least significant difference (LSD) test. The Pearson's correlation coefficient was utilized to determine the correlation between different parameters under study. The data were represented as (Mean±S.E.), and statistical significance was defined as a probability value (p≤ 0.05) [25].

According to our result, all UPEC were lactose fermenters, these isolates grew a dry to mucoid colonies on MacConkey agar. As for biochemical tests, the oxidase test was negative (no purple discoloration) and the catalase test positive (no formation of bubbles), both of which are criteria for the E. coli profile. Most UPEC isolates tested positive in the indole test, which is a classical characteristic of E. coli, which produces indole from tryptophan. Methyl red tested positive, while Voges-Proskauer tested negative. Typically, E. coli produces a mixture of acids during fermentation, leading to a positive methyl red result, while E. coli lac the ability to produce acetone, which results in a negative result in the Voges-Proskauer test as mentioned in study [21]. Vitek®- 2 compact system provides a high accuracy confirmation ranging between 93% - 99%. This system uses biochemical tests a vast array for the purpose of bacterial identification, and its accuracy supports rigor in E. coli identification. From the 76 isolates, it seems that only 70 were confirmed to be E. coli. The gender distribution in our study was 49 (70%) females and 21 (30%) males. This deviation to females agrees well with the epidemiology of Uropathogenic infections, which are most commonly seen in females. Al-Awkally et al. [26] demonstrate that females are more susceptible than men to urinary tract infection, which leads to many differences, like anatomical, physiological, hormonal, and the short urethra of females. AST performed by Vitek®-2 compact system provides us with valuable insights about the antibiotic resistance profiles of UPEC tested isolates. Our findings show that, highly significant resistance to Amoxicillin and Ampicillin. The high resistance reflects the truth of wildly spread of UPEC capable of producing β-lactamase, which hydrolyzes the β-lactam ring and makes these antibiotics ineffective. The absence of intermediate resistance within UPEC isolates is attributed to the complete efficacy of resistance mechanisms or full susceptibility either by Extended Spectrum β-lactamases (ESBLs) or variants of β-lactamase [2]. Low resistance demonstrated for Amoxicillin/Clavulanic acid. This indicates that the addition of an inhibitor of β-lactamase, such as Clavulanic acid, restores the efficacy [27]. High resistance rates were observed for each Ticarcillin and Piperacillin compared to Piperacillin/Tazobactam. The activity of Tazobactam, a β-lactamase inhibitor, underscores its effectiveness among UPEC isolates. Tazobactam was able to neutralize a wide range of β-lactamases, especially within infections caused by organisms that produce ESBLs [28]. For Cephalosporin, Cefazolin and Cefuroxime show a high resistance among UPEC isolates, while low resistance is noted for Cefoxitin and Cefixime. The variability in resistance rates is attributed to different resistance mechanisms that may be used by the UPEC, and these mechanisms may be due to β-lactamases, mutation in the target site, or efflux pumps [2, 29, 30]. High sensitivity observed for Carbapenems. All of Ertapenem, Imipenem, and Meropenem revealed high efficacy against UPEC isolates. Carbapenems are considered as the best option to treat ESBL UPEC [31], still, Carbapenems are considered the last choice for treating MDR UPEC infections because resistance to them is relatively rare [10]. The absence of Meropenem resistance suggests that it is considered a potent choice for infections caused by MDR UPEC. Very reduced resistance was noticed for Amikacin and Gentamicin. The reduced resistance reflects the utility of using aminoglycosides as treatment for UPEC infection. Aminoglycosides are effective against infections caused by gram-negative bacteria, and as a result of Amikacin's low resistance, it's still a viable option for the severe cases of MDR UPEC [32]. Resistance for ciprofloxacin and levofloxacin as fluoroquinolones was high. These came in line with the global trends in resistance of UPEC. This resistance is due to mutations in quinolone resistance-determining regions present in DNA gyrase and Topoisomerase IV [33, 34]. The absence of moderate sensitivity among UPEC isolates may reflect complete resistance or susceptibility. Nitrofurantoin exhibited low resistance by UPEC isolates. Nitrofurantoin effectiveness should be considered as the first line of choice for uncomplicated UTIs because it targets many pathways within bacterial cells after breaking down to its metabolites. These metabolites start to act as bacteriostatic agents by binding to bacterial ribosomes, leading to inhibition of protein synthesis as well as the citric cycle, and finally prevent DNA and RNA synthesis, thus making the resistance development very low [2, 35, 36].

The combination of Trimethoprim/Sulfamethoxazole shows moderate resistance by UPEC isolates, and vast majority were sensitive. The resistance to this combination is linked to alterations in folic acid metabolism pathways [37]. All UPEC isolates were capable of biofilm formation. Biofilm formation was observed regardless of the media supplementation with sucrose or glucose, which both enhance the ability to form biofilm. Although the degree of biofilm formation was variable, it indicates the formation of it. According to studies [38, 39], E. coli isolated from urine samples are able to form biofilm at different rates. Also, study [40] indicated that 96.7% of the isolates were able to form biofilms, and 50% of them were moderate, while 46.7% of them were strong in forming biofilms. Tajbakhsh et al. [41] indicated that all E. coli isolates isolated from urinary tract infections were 100% biofilm formers, while the study [42] indicated that the biofilm production rate of multidrug resistance isolates was 80%, 29% of which were strong, 34% moderate, 17% weak, and 20% non-biofilm-forming. Also, study [43] indicated that 87.8% of E. coli isolated from patients of urinary tract infections from Ramadi city. The study [44], which was conducted to determine the effect of adding sugars on the process of biofilm formation showed that the process of adding both glucose at a concentration of 10% and sucrose at a concentration of 15% gave the same result, which is the formation of biofilm, as no differences were observed in the intensity of biofilm formation after adding sugars at different concentrations. The study [23], which was conducted to detect the ability of E. coli to form biofilm using brain heart infusion, supplemented with 2% sucrose, and Tryptone soy broth supplemented with 2% sucrose, showed that all bacterial isolates formed biofilm. Variability in biofilm formation was documented [45], that UPEC is able to form biofilm and is influenced by many genetic and environmental factors such as availability of nutrients and cell-to-cell communications, thus leading to differences in capacities of biofilm formation [46, 47]. In contrast, the use of sucrose as a supplement leads to the enhancement of biofilm formation. Enhancement of biofilm formation by sucrose is attributed to the use of sucrose as a carbon source. Using sucrose influences the production of extracellular polymeric substance, which is considered part of the biofilm matrix [48-50]. In another study, Katongole et al. [51] highlighted sucrose impact on UPEC biofilm formation and suggested that sucrose improves adherence and aggregation of bacterial cells, which makes the biofilm more robust. The use of different media leads to demonstrating that biofilm formation can be varied in its degree according to growth conditions and bacterial isolates, which reflects limitations. Biofilm formation can be varied, and it’s influenced by many factors such as bacterial strain and growth conditions, and this variation reflects limitations in biofilm formation under optimum and suboptimum conditions [52-54]. In addition, biofilm formation could be affected by many factors such as availability of nutrients, temperature, and pH [49, 55]. Differences in the values of the series of inhibitory concentrations for bacterial growth after determining them using the Vitek®-2 compact system and comparing them with the well diffusion method. As a difference was observed in the minimum inhibitory concentrations for the growth of E. coli using ciprofloxacin and Trimethoprim/Sulfamethoxazole, except for isolates K16 and G33, the minimum inhibitory concentration for bacterial growth was the same using Vitek®-2 system and well diffusion method, as the difference is due to the measurement method. This is what was indicated by study [56], as the minimum inhibitory concentration using the Broth Culture method differs from the minimum inhibitory concentration using the Disc diffusion method, and these differences are due to the difference in the state of bacteria in being planktonic cells and biofilm-forming cells. The cells in the biofilm state showed higher resistance to antibiotics compared to their state in planktonic cells. As for the antibiotic Trimethoprim/Sulfamethoxazole, no difference was observed in the minimum inhibitory concentrations for bacterial growth.

The results of the current study provide a valuable vision on the efficacy of ciprofloxacin and trimethoprim/sulfamethoxazole against biofilm formation of variant isolates (antibiotic-resistant and antibiotic-sensitive) UPEC. Our findings reveal that there is complexities about inhibition of biofilm formation and how it varied according to antibiotic type, concentration type of bacterial isolate it was either resistant or sensitive and isolate source, which may be isolated from an adult or a child, as well as the variance may be attributed to the difference in the gender of the affected person, whether male or female, as it was noted that the effect was inhibitory through observing the decrease in light density rates. For the resistance isolates, ciprofloxacin demonstrated an effective activity to inhibit biofilm at low concentrations (SSMIC and SMIC) with significantly low optical density, despite their resistance to ciprofloxacin. These findings suggest that at higher concentrations of ciprofloxacin, it might lose its activity in inhibiting biofilm formation, and this may be due to stress responses of bacterial cells to survive. Ciprofloxacin on the sensitive isolates had robust activity. At the SSMIC and SIMC levels, the biofilm formation of K42 was significantly inhibited, while for G27, the SSMIC and SMIC were not effective in inhibiting biofilm formation, but rather stimulated it. These findings suggest that, although the G27 isolate is classified as sensitive according to MIC tests but it responds differently to ciprofloxacin, which this possibly due to strain-specific factors that enhance biofilm formation as a response to the antibiotic. Trimethoprim/Sulfamethoxazole was also effective in inhibiting biofilm formation of K42, with significantly reduced optical density. This activity mirrors ciprofloxacin activity. In contrast, its influence on biofilm formation of the G27 isolate, and this finding highlights the response to antibiotics by this isolate. The results of testing the effect of the antibiotic Ciprofloxacin and Trimethoprim/Sulfamethoxazole on the process of biofilm formation by antibiotic-sensitive and antibiotic-resistant isolates showed that the effect of the antibiotic varies depending on the source of the isolation, which may be isolated from an adult or a child. In addition, the variation may be attributed to the difference in the sex of the infected person, whether male or female, as it was noted that the effect was inhibitory, through observing the decrease in optical density rates. The study by Wojnicz and Tichaczek-Goska [57] aimed to determine the effect of sub-inhibitory concentrations (sub-MICs) of Ciprofloxacin, Amikacin, and Colistin on biofilm formation, motility, and villus formation of E. coli isolated from the urine of patients with various urinary tract infections. The results of the study indicated that all antibiotics used at sub-inhibitory concentrations reduced biofilm formation and decreased bacterial survival. This study suggests that Ciprofloxacin, Amikacin, and Colistin may be useful in the treatment of biofilm-associated infections caused by E. coli strains, as indicated by the study [58]. The possibility of inhibiting the biofilm of E. coli bacteria using Ampicilin, Cephalothin, Ceftriaxone, Ceftazidime, Amikacin, and Ciprofloxacin antibiotics at different concentrations after 48 hours of biofilm formation, as the study showed the ability of all antibiotics to inhibit the biofilm of E. coli bacteria except for the antibiotic Ampicilin, as these antibiotics work to reduce the mass of biofilms. According to study [59], the process of biofilm formation can be inhibited when using ¼ of the minimum inhibitory concentration (MIC) of the antibiotic Ciprofloxacin, as their study indicated that the antibiotic has an effect on the fim gene in addition to its effect on the gene locus pgaABC, as this site is an active part in the process of polysaccharide synthesis [60], while the fim gene encodes for the fimbriae type 1, which plays an essential role in the process of bacterial cell adhesion [51].

The results of study [61] indicate that the use of high concentrations of the antibiotics Trimethoprim and Nitrofurantoin did not show any inhibitory effect on the biofilm formation process, while the antibiotic Ciprofloxacin showed an inhibitory effect on the formation of biofilms. The study attributed the inhibitory effect of the antibiotic Ciprofloxacin to the fact that it is responsible for inhibiting the replication of DNA. Rafaque et al. [24] indicated that the low concentration of Ciprofloxacin, lower than the minimum inhibitory concentration (SMIC), which is added or mediates the process of biofilm formation, enhance the formation of biofilms of E. coli isolated from cases of urinary tract infection, as five out of 6 isolates were stimulated to form biofilms at the level of the minimum inhibitory concentration (MIC) of the Ciprofloxacin. A recent study by Whelan et al. [2] refers to that biofilm formation can be inhibited by Ciprofloxacin at SMIC concentration, even if the bacterial strains are resistant, most likely due to the interference of biofilm-related pathways and the quorum-sensing system. The study also indicated that Trimethoprim/Sulfamethoxazole is known to inhibit folic acid synthesis and stop bacterial proliferation at low concentrations; however, it is similar to Ciprofloxacin in inhibiting biofilm formation but can also promote biofilm formation at higher concentrations. The effect of antibiotics on biofilm formation was variable, which may be due to the variation in the isolate strains that were resistant and susceptible and were isolated from males and females and at different age groups, as was the case in the study [2] which showed that the minimum inhibitory concentration (MIC) was effective in inhibiting E. coli biofilm. However, as shown in the results of the current study, it appears that the higher the concentration of the antibiotic Ciprofloxacin, the less effective it is in inhibiting biofilms. This decrease in effectiveness is attributed to the stress of bacterial cells in order to form biofilms as a survival mechanism. In addition, the effect of the minimum inhibitory concentration and sub-minimum inhibitory concentration on inhibiting biofilm formation for some isolates and activating biofilm for some isolates. There was no difference in the effect on biofilm formation between sensitive and resistant isolates, as indicated by study [24], while studies [62-64] indicated that bacterial cells communicate with each other using quorum sensing systems, which are responsible for regulating group behaviour such as biofilm formation. Sometimes, the concentration of the antibiotic at a concentration level below the SMIC may interfere with quorum sensing, leading to the promotion of biofilms rather than their inhibition. Several studies [46, 63, 65, 66] have indicated that the extracellular matrix of biofilms acts as a physical barrier, preventing the penetration of antibiotics. As a result, bacterial cells embedded within the matrix, especially in the deeper layers, are only exposed to non-lethal doses of antibiotics, which promote and maintain biofilm formation.

The results of testing Ciprofloxacin and Trimethoprim/Sulfamethoxazole on already formed biofilms by E. coli show alarming results. The results showed that the antibiotics at the MBC, MIC, SMIC, and SSMIC concentrations, instead of inhibiting biofilms, work to enhance biofilms, as an increase in optical density rates was observed at all concentrations. Biofilms contain multiple and different alternatives or forms of bacterial cells, which cannot be reached by antibiotics and are known as persistent cells. Persistent cells are characterized by being metabolically inactive, and they can survive for a long time because they are protected and cannot be reached by antibiotics, and thus work to renew biofilms after the antibiotic activity has disappeared [46, 63, 66]. We note from the results of the experiment that ciprofloxacin and Trimethoprim/Sulfamethoxazole at concentrations lower than the minimum inhibitory concentrations led to the inhibition of biofilm formation. This is due to its effect on the suspended cells, while it led to the activation of the biofilm formed by not affecting the cells due to their surrounding materials that prevent the access of antibiotics. The studies [67, 68] confirm that E. coli resistant to the antibiotic Ciprofloxacin shows significant differences in their behavior from bacteria sensitive to the antibiotic in the case of planktonic cells and biofilm cells when exposed to the antibiotic Ciprofloxacin. Planktonic cells, which are metabolically active and free-floating, show higher sensitivity to the antibiotic Ciprofloxacin because the antibiotic targets the enzymes DNA gyrase and topoisomerase IV responsible for the process of DNA replication in bacterial cells, which leads to effective killing of bacteria at lower concentrations. In contrast, E. coli cells forming biofilms are significantly less susceptible to the antibiotic Ciprofloxacin, as the extracellular polymeric substance (EPS) matrix surrounding the biofilm cells acts as a barrier, which hinders the penetration of antibiotics. In addition, biofilm cells are in a state of inactivity or slow growth, which reduces the effectiveness of the antibiotic Ciprofloxacin, which targets active cellular processes. The study also indicates that concentrations below the minimum inhibitory concentration promote the survival of biofilms and allow for genetic exchange, such as horizontal gene transfer, which enables the spread of resistance. This is what indicated by study [69] that the antibiotic Ciprofloxacin appears to be effective in killing planktonic cells, which effectively reduces their survival over time, while its effect on biofilm cells shows that these cells appear the opposite of what planktonic cells show, as they have a higher ability to tolerate the antibiotic Ciprofloxacin. The study [70] also indicated that the acquisition of resistance in planktonic cells occurs at a slower rate, and thus, strains appear that are inhibited at lower concentrations compared to biofilm cells, because they need long periods and high concentrations of the antibiotic Ciprofloxacin to show the resistance trait. As for biofilm cells, they showed rapid development of resistance to the antibiotic Ciprofloxacin when exposed for long periods and to high concentrations of the antibiotic Ciprofloxacin, in addition to their need for higher minimum inhibitory concentrations compared to planktonic cells. This rapid development of resistance is attributed to the protective environment of biofilms, which facilitates the occurrence of genetic mutations and horizontal gene transfer, enhancing the ability of bacteria to withstand the pressure of antibiotics. As for the antibiotic Trimethoprim/Sulfamethoxazole, the study [71], which indicated that the antibiotic Trimethoprim/Sulfamethoxazole is effective against planktonic cells, effectively inhibiting their growth, while its effectiveness appears less in biofilm cells, which was also indicated by the study [72], that it is effective in inhibiting the growth of planktonic cells and biofilm cells at a concentration of 50 μg/ml. In the study of [73], the antibiotic Trimethoprim/Sulfamethoxazole was effective in inhibiting planktonic cells, but its effect decreased in the case of biofilm cells. The effectiveness of ciprofloxacin and trimethoprim/sulfamethoxazole depends on many factors such as timing, dosage, and adherence to the prescribed treatment strategy. Ciprofloxacin and trimethoprim/sulfamethoxazole should be administered during active infection to eliminate biofilm formation, due to these biofilms provide protection to these bacterial cells against antibiotics, forming a barrier similar to immune response to these therapeutic agents, which eventually makes the infection harder to heal. If these antibiotics (ciprofloxacin and trimethoprim/sulfamethoxazole) are used incorrectly, such as at their suboptimal doses or during insufficient durations, bacterial cells easily form biofilm, reducing drug penetration and leading to persistence of infection. Therefore, restricting adherence to the prescribed antibiotic protocol is currently essential to prevent biofilm-associated resistance.