Chlorpromazine, impregnated in foley catheter fragments, as an antibiofilm agent to control urinary tract infections

ABSTRACT

Background

The study proposes to evaluate the impregnation of Foley catheters, in different concentrations of CPZ, to control the formation of biofilms in vitro by the uropathogens most commonly associated with indwelling catheters.

Materials & methods

Minimum inhibitory concentrations (MICs) for CPZ and the effect of CPZ (at different concentrations) on biofilm formation were evaluated. Biofilm formation and the effect of CIP and MER on mature biofilms in CPZ-impregnated catheters were evaluated.

Results

The CPZ MIC was 312.5–625 µg/ml. CPZ significantly (p < 0.05) disrupted biofilm formation at all tested concentrations. In addition, the tested CPZ potentiated the action of CIP.

Conclusions

Our results suggest the use of CPZ by impregnation of catheters can prevent the formation of bacterial biofilms, preventing urinary infections through this route.

PLAIN LANGUAGE SUMMARY

This study was done to test a medicine called chlorpromazine. It is usually used to treat people with mental health problems. The researchers put this medicine into hospital tubes called catheters, which are placed in the body to help people urinate. The idea was to see if the medicine could stop bacteria from sticking together and causing infections. And it worked: the medicine was able to stop the bacteria from attaching to the tube. It also helped another medicine, an antibiotic called ciprofloxacin, to work better. Because of this, the scientists believe that using this medicine in the tube may help prevent infections.

1. Introduction

Urinary tract infections (UTIs) are one of the most common Healthcare-Related Infections (HAIs), especially when the patient uses an indwelling catheter, representing up to 60% of these cases [1,2]. The use of this medical device increases the risk of infection by between 3% and 10%, cumulatively, each day with the device in use and is directly associated with increased morbidity and mortality in hospitalized patients [3–5]. Several microorganisms are associated with catheter-associated UTI’s, but the main Gram negative bacteria are Escherichia coli, Proteus mirabilis, P. aeruginosa, Klebsiella pneumoniae [1,6,7].

Escherichia coli represents more than 80% of UTI isolates, mostly uncomplicated; however, when it comes to indwelling catheters, other enteropathogens such as P. mirabilis and K. pneumoniae appear more frequently [8]. Enteropathogens have a high resistance to antimicrobials and virulence factors that impair therapeutic management, leading in many cases to sepsis, in addition to being the main cause of UTI’s acquired in the hospital environment, mainly as an infectious agent of patients using long-term urinary catheters [9,10]. Pseudomonas aeruginosa is also a non-fermenting Gram negative, ubiquitous, opportunistic bacterium and is one of the main bacteria associated with Healthcare Associated Infections (HAI), being a predominantly hospital pathogen and being associated with a high mortality in these cases [11,12]. In several countries, both developed and developing, studies show the financial impact on health caused by UTI’s, with indwelling catheters being listed as the second most prevalent cause of infection in hospitals and P. aeruginosa being the microorganism most causing healthcare-associated infections, as a study in India shows [13,14].

Microorganisms use several virulence factors in the pathogenesis of UTI and the microenvironment can also favor its installation, such as pH, chemical composition and urine osmolarity [6]. An important virulence factor is the ability to form biofilm, making microorganisms more resistant to external stress and also being responsible for chronic infections and therapeutic failure [15]. In addition to the ability to form biofilms, microorganisms can decrease the permeability of the plasma membrane and increase the expression of efflux pumps. Efflux pumps are physiologically essential for bacteria, as they are responsible for maintaining homeostasis, efflux sugars and metabolites, are responsible for the efflux of self-inducing molecules (Ais), responsible for quorum-sensing, and are capable of extruding multiple drugs. The inhibition of these efflux pumps, in addition to promoting a break in quorum-sensing signaling and deregulating cellular homeostasis, also allows for a better antimicrobial action [16].

Several studies seek to elucidate the physiology of biofilms and ways to combat it, since microorganisms in their sessile form can be up to 1000x more resistant to antimicrobials than in their planktonic, free-living form [17,18]. Among these combat strategies, there is the redirection of medicines used clinically for other purposes, in the search for potential antimicrobial action [18]. A previous study showed that chlorpromazine (CPZ), an efflux pump inhibitor of the phenothiazine family, impregnated in Foley catheter fragments was able to reduce by up to 100% the biomass of E. coli, K. pneumoniae and P. mirabilis biofilms [7]. However, that study evaluated the use of CPZ at only one concentration and did not include P. aeruginosa, another important pathogen associated with indwelling catheters, in your analyses.

Thus, continuing the previously carried out study [7], this study proposes to evaluate the impregnation of Foley catheters with different concentrations of CPZ to control in vitro biofilm formation by the P. aeruginosa and others uropathogens most commonly associated with indwelling catheter.

2. Materials and methods

2.1. Microorganisms

In this research, 6 strains of P. aeruginosa were used, 5 clinical isolates and 1 ATCC strain for in vitro assays of sensitivity and antibiofilm activity of chlorpromazine. Regarding the tests using impregnated catheters, the following strains were used: E. coli (n = 2, ATCC 25,922 and enteroaggregative E. coli), K. pneumoniae (n = 3, ATCC 600,703, KL1S e KL2M) e P. mirabilis (n = 2, ATCC 29,906 and PRMR1). All isolates belong to the culture collection of the Applied Group in Medical Microbiology (GrAMM) and were identified and evaluated for antimicrobial sensitivity using the VITEK 2 System (bioMérieux^TM). Thus, the criterion used to choose the isolates was different sensitivity profiles, both sensitive to all drugs and multiresistant strains (resistant to more than 3 classes of antimicrobials).

2.2. Planktonic susceptibility to chlorpromazine, ciprofloxacin and meropenem

P. aeruginosa isolates (n = 6) were tested to assess their susceptibility to chlorpromazine (CPZ, 5 mg/mL, HYPOFARMA, Brazil) and to the antimicrobials ciprofloxacin (CIP, Sigma, Brazil) and meropenem (MER, Sigma, Brazil). To validate the experiment, the test was performed following the recommendations of the Clinical Laboratory Standards Institute document M100-S29 for the determination of the minimum inhibitory concentration (MIC) for each drug [19]. Drugs were tested at the following concentrations 2.44–1250 µg/mL for CPZ, 0.008–32 µg/mL for CIP and 0.0625–16 mg/L for meropenem. The minimum inhibitory concentration (MIC) was defined as the lowest concentration capable of causing 100% inhibition of microbial growth [7].

2.3. Biofilm susceptibility to chlorpromazine

To determine the activity of chlorpromazine on the mature biofilms of the 6 P. aeruginosa isolates in the study, the biofilm of each isolate was grown in 96-well polystyrene plates with flat bottom, according to Sidrim et al. [7], with adaptations. The inoculums were prepared using the McFarland turbidity scale of 0.5 and the biofilms were mounted in a total volume of 200 µL, in a ratio of 1:7, with 25 µL of inoculum and 175 µL of BHI-1% glucose broth. At the end of the 48 hours of incubation, all wells were washed with sterile PBS. Then, increasing drug concentrations were added to each well along with BHI +1% glucose, totaling a solution of 200 µL per well. CPZ was tested at concentrations ranging from 2.44 to 1.250 µg/mL. Plates were incubated at 37°C for another 24 h. Subsequently, the metabolic activity was evaluated using the rezasurin metabolic assay [20]. The minimum concentration required to completely inhibit the metabolic activity of the biofilm was defined as the minimum biofilm eradication concentration (MBEC) [20]. The drug-free biofilm growth control was included for each isolate tested. Tests were performed at two different times.

2.4. Effect of chlorpromazine on biofilm formation

The action of CPZ on the formation of biofilms by P. aeruginosa isolates was evaluated, following the protocol previously used by Sidrim et al. [7]. The CPZ concentrations used were MIC, MIC/2 and MIC/4 for each isolate, which were incorporated into the broth at the time of induction of biofilm formation. The biofilms were mounted in a total volume of 200 µL, following the inoculum:culture medium ratio of 1:7, as mentioned above, and incubated at 37°C for 48 hours for biofilm formation. The quantification of the biomass was performed using the crystal violet staining technique [20]. Drug-free wells were included as growth control and bacterial inoculum-free wells as sterility control. All conditions were tested in triplicate, at two different times.

2.5. Impregnation of catheter fragments with chlorpromazine

The impregnation process of latex catheter fragments (number 12, silicone-coated latex catheters, SOLIDOR^TM) was performed according to a protocol described in the previous study [7], in which catheter fragments were used, approximately 1 cm. These fragments were immersed in PA chloroform for 1 hour, for dilation of the material. Then, the fragments were removed and left for another 30 minutes in a solution containing 200, 400 or 600 µg/mL of CPZ. The entire process was performed aseptically and, to ensure the sterility of the process, some fragments were placed in tubes containing BHI broth and incubated at 37°C for 24 hours. Fragments submitted to chloroform immersion, but not to CPZ impregnation, were used as substrates to control the growth of microbial biofilms [7].

2.6. Effect of chlorpromazine, impregnated in catheter fragments, on biofilm formation by E. coli, K. pneumoniae, P. aeruginosa and P. mirabilis isolates

In experiments to evaluate the effect of CPZ impregnated in Foley catheter fragments on biofilm formation by uropathogens, 2 P. aeruginosa isolates (P. aeruginosa ATCC 29,213 and PAM1) were selected and three concentrations of CPZ were used in the process of impregnation, 200, 400 and 600 µg/mL. Whereas the impregnation concentration of 400 µg/mL was evaluated in a previous study [7] with E. coli strains (E. coli ATCC 25,922 and enteroaggregative E. coli - EAEC), K. pneumoniae (K. pneumoniae ATCC 600,703, KL1S and KL2M) and P. mirabilis (P. mirabilis ATCC 29,906 and PRMR1), only CPZ concentrations of 200 and 600 µg/mL were used in the staining assay.

After impregnating the fragments with CPZ, an in vitro assay was carried out to assess the effect of CPZ on biofilm formation by uropathogens on catheter fragments. Biofilms were prepared as previously described, using a 24-well polystyrene plate, each well adjusted to a final volume of 1000 µL, consisting of 875 µL of 1% BHI-glucose broth and 125 µL of bacterial inoculum. Following the addition of the broth to the wells, CPZ-impregnated and non-impregnated catheter fragments were placed into the wells, and the bacterial inoculum was then added. The plates were incubated at 37°C for 48 hours.

At the end of the incubation period, the culture medium was removed, and the wells were washed twice with 1 mL of sterile saline solution to eliminate bacterial cells that had not adhered to the biofilm. The catheter fragments were subsequently transferred to 15 mL Falcon tubes containing 2 mL of sterile saline solution and vortexed for 1.5 minutes to dislodge the adhered bacterial cells.

The resulting bacterial suspension was serially diluted at 1:1000 and 1:10000. Then, 10 µL of each dilution were plated, using a Drigalski loop, on BHI agar for P. aeruginosa, E. coli, and K. pneumoniae, and on CLED agar for P. mirabilis, in duplicate, to determine colony-forming units after incubation at 37°C for 24 hours [7]. All assays were performed in duplicate at two separate time points. Catheter fragments treated with chloroform but not impregnated with CPZ served as the biofilm growth control.

In addition, biofilms grown on CPZ-impregnated and non-impregnated catheter fragments were analyzed by confocal laser scanning microscopy (CLSM), using the methodology described in a previous study [7,19]. The COMSTAT software was used to analyze the images produced by CLSM and the evaluated parameters obtained were maximum thickness, biomass, average thickness of biomass, roughness coefficient and surface–volume ratio, while the thresholding was calculated automatically by the Otsu method.

2.7. Effect of the antimicrobials ciprofloxacin and meropenem on mature biofilms of E. coli, K. pneumoniae, P. aeruginosa and P. mirabilis formed in catheter fragments impregnated with chlorpromazine

To evaluate the effect of antimicrobials on mature biofilms formed on fragments of catheters, the same protocol as previously described was performed. Ciprofloxacin or meropenem, in concentrations referring to MIC against each isolate (Table 1), were added to the wells after 48 hours of biofilm formation, that is, with mature biofilms formed on catheter fragments, impregnated with CPZ or not. In a simplified way, the experiment was set up in duplicate, the first two wells being the experiment’s growth control, with only BHI-1% glucose broth plus the bacterial inoculum and the unimpregnated catheter fragment, two wells with BHI-1% glucose broth plus the bacterial inoculum and the impregnated fragment, two wells with BHI-1% glucose broth plus the MIC of the antimicrobials for each bacterial isolate, the bacterial inoculum and the fragment impregnated with chlorpromazine and, for finally, two wells with BHI-1% glucose broth plus the MIC of the antimicrobials for each strain, the bacterial inoculum and the non-impregnated catheter fragment.

Table 1.

Selected bacterial isolates and minimum inhibitory concentrations (MICs) and minimum biofilm elimination concentrations (MBECs) for chlorpromazine (CPZ), ciprofloxacin (CIP) and meropenem (MER) against Pseudomonas aeruginosa (n = 6) Escherichia coli (n = 2), Klebsiella pneumoniae (n = 3) and proteus mirabilis (n = 2).

			MIC (µg/mL)			MBEC (µg/mL)
Isolate	Species	Origin	CPZ	CIP	MER	CPZ	CIP	MER	Reference
PATCC	P. aeruginosa	ATCC 29,213	312,5	0,5	0,25	1250	–	–
PAS1	P. aeruginosa	Urine	312,5	32	0,125	625	–	–
PAR1	P. aeruginosa	Urine	312,5	16	0,5	625	–	–
PAR4	P. aeruginosa	Urine	312,5	> 32	0,25	625	–	–
PAR5	P. aeruginosa	Urine	625	1	16	1250	–	–
PAM1	P. aeruginosa	Urine	312,5	> 32	4	625	–	–
ECAT	E. coli	ATCC 25,922	39.06	0.008	0.0625	625	78.12	64	Sidrim et al. 2019
EAEC	E. coli	Reference strain	39.06	0.0625	0.125	625	312.5	> 128	Sidrim et al. 2019
KLAT	K. pneumoniae	ATCC 600,703	156.25	0.25	0.125	625	> 625	> 128	Sidrim et al. 2019
KL1S	K. pneumoniae	Urine	78.12	0,125	0.125	625	> 625	32	Sidrim et al. 2019
KL2M	K. pneumoniae	Urine	39.06	0.0312	0.25	312.5	> 625	> 128	Sidrim et al. 2019
PRAT	P. mirabilis	ATCC 29,906	625	0.0312	0.5	1,250	> 625	> 128	Sidrim et al. 2019
PRMR1	P. mirabilis	Urine	625	16	1	1,250	> 625	> 128	Sidrim et al. 2019

− Test not performed.

2.8. Statistical analyses

The data obtained in the study were first analyzed for symmetry, revealing an asymmetric distribution. After that, in order to evaluate the effect of CPZ on the formation of biofilms of the strains included in the study, the Mann–Whitney test was applied for unpaired analyses and the Wilcoxon test for paired analyses. The overall effect of impregnating the catheters with CPZ and antimicrobials on the bacterial CFU counts was also analyzed, for which analysis of variance (ANOVA) was used, with the Kruskal–Wallis posthoc test. In addition, Dunn’s analysis was used to evaluate the effect of the combinations (impregnated catheter, antimicrobial and impregnated catheter + antimicrobial) on each strain individually. For all tests applied, p values  < 0.05 allowed significant conclusions to be drawn.

3. Results

3.1. MIC and MBEC of chlorpromazine, ciprofloxacin and meropenem against the study strains

CPZ showed MICs ranging from 312.5 to 625 µg/mL against P. aeruginosa. Almost all P. aeruginosa isolates have the same MIC for CPZ, 312.5 µg/mL, with the exception of PAR5 strain, which has a MIC of 625 µg/mL, as demonstrated by Table 1. The MBEC’s were statistically (p < 0.0001) higher than the MICs, being at least two times higher than the MICs for the P. aeruginosa isolates tested, with the exception of PATCC, which presented a MBEC four times higher than the MIC. As for the antimicrobials included in the study, there was a great variability of MICs. For CIP, MICs ranged from 0.5 to > 32 µg/mL, while for MER, MICs ranged from 0.125 to 16 µg/mL.

3.2. Effect of chlorpromazine on in vitro biofilm formation by the study isolates

The concentrations used for the 6 P. aeruginosa isolates were the MIC found in the CPZ susceptibility tests (Table 1), plus two subinhibitory concentrations (MIC/2 and MIC/4). CPZ was able to significantly reduce (p < 0.05) the biomass of P. aeruginosa biofilms at the 3 tested concentrations. The concentration referring to the MIC reduced the biomass of biofilms in formation by 60.6%, when compared to the growth control. The concentrations of MIC/2 and MIC/4 reduced the biomass of biofilms in formation by 40.3% and 38.3%, respectively, when compared to the growth control (Figure 1(a)).

Figure 1.

Effect of the efflux pump inhibitor chlorpromazine (CPZ) on biofilm formation.

(a) Pseudomonas aeruginosa (n = 6). Data expressed as mean and standard deviation of biomass absorbance, measured by crystal violet staining. (b) E. coli, K. pneumoniae, P. mirabilis and P. aeruginosa. Data expressed as mean and standard deviation of log CFU count/catheter fragment. (c) Effect of impregnation of Foley catheters with chlorpromazine (CPZ at 200 µg/mL, 400 µg/mL and 600 µg/mL) on biofilm formation by P. aeruginosa. Data expressed as mean and standard deviation of log CFU/catheter count. (d) Enterobacteriaceae. Data expressed as mean and standard deviation of log CFU/catheter count.

3.3. Effect of chlorpromazine, impregnated in catheter fragments, on the formation of biofilms by P. aeruginosa, E. coli, K. pneumoniae and P. mirabilis isolates

Chlorpromazine, at all concentrations tested (200, 400 and 600 µg/mL), impregnated in Foley catheter fragments, was able to significantly reduce (p < 0.05) the formation of biofilms by all isolates included in the research. The concentration of 200 µg/mL reduced, by up to 30.4%, the formation of biofilms. The concentration of 400 µg/mL reduced the formation of biofilms by uropathogens by 43.9%, representing the greatest inhibition observed, combining the data from the present study obtained against P. aeruginosa with those obtained in the previous study using the same concentration of CPZ against E. coli, K. pneumoniae and P. mirabilis [7]. Finally, the highest concentration used, 600 µg/mL, significantly reduced (p < 0.05), by up to 27.2%, the formation of biofilms on the catheter fragments when compared to the growth control, with the lowest inhibition observed (Figure 1(b)).

Analyzing the action of CPZ on the biofilms formed by P. aeruginosa isolates, alone, it is possible to observe a statistically significant reduction (p < 0.05) only at the concentration of 400 µg/mL, when compared to the growth control (Figure 1(c)).

For isolates of the Enterobacteriaceae family, CPZ was able to reduce the formation of biofilms by all isolates, both at the lowest concentration (200 µg/mL) and at the highest (600 µg/mL). At the concentration of 200 µg/mL, there was a 38.2% reduction in the CFU/catheter fragment count, and at the 600 µg/catheter fragment count, there was a smaller reduction in the CFU/catheter fragment count, being 32% when compared to growth control (Figure 1(d)).

Separating the enterobacteria included in the research by species, it is possible to observe that all concentrations impregnated in the catheter fragments were able to reduce (p < 0.05) the counts of CFU/catheter fragment for E. coli. The lowest concentration tested (200 µg/mL) was the one that showed the greatest reduction, of 81.2%, while the concentration of 600 µg/mL reduced the CFU count/catheter fragment by 61.9%, when compared to the growth control (Figure 2).

Figure 2.

Effect of impregnating Foley catheters with chlorpromazine on biofilm formation by Enterobacteriaceae. (a) E. coli with CPZ at 200 and 600 µg/mL; (b) K. pneumoniae with CPZ at 200 and 600 µg/mL; (c) P. mirabilis with CPZ at 200 and 600 µg/mL. Data expressed as mean and standard deviation of log of CFU/catheter count.

As for the isolates of K. pneumoniae and P. mirabilis, only at the concentration of 600 µg/mL it was possible to observe a statistically significant reduction (p < 0.05) in the counts of CFU/catheter fragment, being 8.2% and 39.6%, respectively. Although there is no statistical difference, it is possible to observe a tendency toward a reduction in CFU/catheter fragment counts at a concentration of 200 µg/mL, for K. pneumoniae isolates, but very prominent for P. mirabilis isolates (Figure 2).

Furthermore, the CLSM analysis demonstrated, in general, in addition to a smaller biofilm thickness, biofilms with non-viable cells in the impregnated fragments when compared to the non-impregnated controls, evidencing what was already mentioned in the previous sections. It is important to emphasize that P. aeruginosa (PATCC) presented lower cell viability in biofilms formed in all concentrations tested and a greater reduction (p < 0.5) in biomass at the concentration of 400 µg/mL (Figure 3).

Figure 3.

Impregnation of Foley catheters with chlorpromazine (CPZ) and its effects on biofilm formation by Pseudomonas aeruginosa (PAATCC), Escherichia coli (EAEC), proteus mirabilis (PRAT) and Klebsiella pneumoniae (KLAT). (A) Representative images, obtained by scanning electron microscopy, of non-impregnated catheter fragments exposed to chloroform (growth control) and catheter fragments exposed to chloroform impregnated with CPZ at 200, 400, and 600 mg/L. (B) Effect of impregnation of catheters with CPZ on biofilm formation by uropathogens; data expressed as mean and standard deviation of colony-forming units (CFU) per catheter fragment, normalized with the growth control. *indicates statistically significant differences between catheters impregnated with CPZ and non-impregnated control (p < 0.05).

3.4. Effect of the antimicrobial ciprofloxacin and meropenem on mature biofilms isolated from chlorpromazine-impregnated catheter fragments

To evaluate a possible positive interaction between the antimicrobials chosen for the study and the CPZ, the experiment was performed combining the impregnated fragments, at the three concentrations (200 µg/mL, 400 µg/mL and 600 µg/mL) with or without the presence of the antimicrobials.

At all concentrations used in the study, CPZ impregnated in the catheter fragments, as well as MER reduced the CFU count/catheter fragment, when compared to that of the growth control. There was no reduction in biofilms formed on non-impregnated catheter fragments in the presence of CIP, but there was a reduction (p < 0.05) in biofilms formed on CPZ-impregnated catheter fragments in the presence of CIP. The highest concentration of CPZ used (600 µg/mL), combined with CIP, was able to cause a significant reduction (p < 0.05) in biofilms formed in impregnated fragments, when compared to non-impregnated fragments in the presence of CIP and to growth control (Figure 4).

Figure 4.

Effect of the antimicrobials ciprofloxacin (CIP) and meropenem (MER) on mature biofilms of E. coli (n = 2), K. pneumoniae (n = 3), P. mirabilis (n = 2) and Pseudomonas aeruginosa (n = 2) grown on catheter fragments impregnated with chlorpromazine (CPZ) at (a) 200 µg/mL, (b) 400 µg/mL and (c) 600 µg/mL. Control: non-impregnated catheter fragments. Data expressed as mean and standard deviation of log CFU/catheter count.

4. Discussion

According to the Centers for Disease Control and Prevention (CDC), approximately 75% of hospital-acquired urinary tract infections (UTIs) are associated with the use of urinary catheters, and 15–25% of hospitalized patients use urinary catheters [21]. This increases the risk of healthcare-associated infections (HAIs), creating selective pressure, and promoting the spread of resistant pathogens within the hospital environment [22].

Microbial colonization of catheters is promoted by the formation of biofilms. Biofilms are responsible for the failure of medical devices and for the persistence and spread of numerous diseases in hospital settings [22]. A new strategy that has gained attention in preventing biofilm formation in catheters is coating them with compounds that have antimicrobial activity [23].

The present study aimed to analyze the coating of urinary catheters with CPZ. CPZ is a phenothiazine widely used as an antipsychotic, but it also has antimicrobial activity, like other phenothiazines, such as promethazine, thioridazine and trifluoperazine [24], having proved to be an effective strategy in combating microbial biofilms, because in addition to its antimicrobial effect, it also acts as an efflux pump inhibitor [7]. Efflux pumps are important transmembrane proteins that act as transporters of important metabolites for microbial physiology, in addition to secreting self-inducing molecules responsible for microbial quorum sensing and excreting antimicrobials, making these microorganisms resistant to various antimicrobials [16,25].

CPZ MICs against P. aeruginosa isolates were 312.5 µg/ml for 5/6 strains tested. Only one isolate included in the study had a different MIC than the others, with a MIC of 625 µg/mL. These results corroborate those of the study by Nehme et al. [26], in which CPZ MICs against P. aeruginosa ranging from 256 to 1024 µg/mL were observed. When compared to previous studies with other bacterial species, it can be observed that the MICs of CPZ against P. aeruginosa were higher than the MICs against isolates of E. coli and K. pneumoniae, however, lower than those of P. mirabilis [7]. In addition, CPZ MBEC against P. aeruginosa isolates were 2x higher than the MIC for the same tested isolates. The greater tolerance to CPZ by P. aeruginosa biofilms may be associated with several factors, such as the presence of persistent cells, low penetrability caused by the presence of an extracellular matrix and the expression of efflux pumps, one of the important factors for the increase of resistance of microorganisms in planktonic and sessile state [18]. All these factors can make biofilms more resistant to external actions and may present resistance up to 1000x greater when they detect planktonics [18].

Baugh et al. [25], demonstrated in his study that CPZ was able to inhibit, by more than 50%, the biofilms formation of P. aeruginosa, in vitro, at a concentration of 512 µg/mL. Even using a lower concentration, in our study, CPZ also inhibited, in vitro, the formation of biofilms at all tested concentrations, with the greatest reduction observed when a concentration of 312.5 µg/mL was used, reducing 60% of the biomass of the biofilms tested. The anti-biofilm action of CPZ can be attested, because at concentrations lower than the MIC of CPZ for each isolate, it was possible to observe a reduction in biofilm formation, corroborating the study previously carried out with E. coli, K. pneumoniae and P. mirabilis, in which MIC and subinhibitory concentrations were able to inhibit biofilm formation in vitro [7].

The evaluation and testing of impregnation of materials used in clinical medicine is already a well-researched reality in an attempt to combat HAI’s, whether in the impregnation of dressings or even medical devices, such as central venous catheters or urinary catheters [27–29]. In our study, at the different concentrations used, CPZ impregnated in catheter fragments reduced the biofilms formed by all uropathogens included in the study, with the smallest reduction observed for P. aeruginosa isolates. Such behavior was also observed in another study that tested the impregnation of catheters with antibiofilm compounds (BPA – benzalkonium chloride, polyacrylic acid and glutaraldehyde) to inhibit the formation of biofilms by different uropathogens, in which there was a total reduction of E. coli biofilms, but there was no reduction in biofilms by P. aeruginosa [2]. We can suggest that, among other factors associated with biofilms, in our study, the greater expression of efflux pumps in P. aeruginosa isolates may be involved in this greater resistance to the compound. This hypothesis can be reinforced with the study by Vikram, Bomberger and Bibby [30], in which, they evaluated the action of glutaraldehyde against species of Pseudomonas spp. without success but highlighted that by chemically inhibiting the expression of efflux pumps, there was a potentiation of the action of glutaraldehyde in the inhibition of biofilm formation in these species.

For enterobacteria, in the study by Sidrim et al. [7], the concentration of 400 µg/mL, impregnated in catheter fragments, inhibited the formation of biofilms in these medical devices by more than 50%. This finding corroborates the demonstration that the best concentration for impregnation of these catheter fragments is 400 µg/mL, since, in our current study, the concentration of 200 µg/mL inhibited the formation of biofilms of these enteropathogens by 38%, while the concentration of 600 µg/mL inhibited it by 32%. To reinforce what was previously written, when analyzing by species, E. coli showed a reduction in both concentrations tested, while the isolates of K. pneumoniae and P. mirabilis only showed a reduction in the highest concentration tested. For P. aeruginosa isolates, in our current study, the concentration of 400 µg/mL was the only one capable of inhibiting (P < 0.05) the formation of biofilms, reinforcing our hypothesis that this is the ideal concentration for impregnation of catheters as a strategy to combat biofilms by the microorganisms under study. The use of efflux pump inhibitors appears to be an effective strategy to combat biofilms, either by having antimicrobial action or by inhibiting the formation of biofilms [26,31].

Gupta et al. [32] observed that P. aeruginosa strains exposed to subinhibitory concentrations of antibiotics exhibited attenuated virulence, including a reduced ability to form biofilms. The authors suggest that this effect is related to the inhibition of the quorum-sensing mechanism under subinhibitory conditions. It can thus be assumed that, at the concentration of 400 µg/mL, there was considerable inhibition of efflux pumps, hindering the release of quorum-sensing molecules and thereby preventing the maintenance of the biofilm – although without eliminating it, as observed at the MBEC concentration.

In addition to the antimicrobial and antibiofilm activity of CPZ, other studies also report CPZ as a potentiating agent for classical antimicrobials, acting synergistically with these antibiotics [7,33,34]. In our study, it was possible to observe a greater reduction in biofilms when there was a combination of antimicrobials with CPZ-impregnated catheter fragments, such as ciprofloxacin, which, combined with CPZ, potentiated the reduction in biofilm formation. This reduction was compared both with the non-impregnated fragment in the presence of CPZ and with the impregnated fragment without the addition of antimicrobials, demonstrating a possible potentiating action of CPZ, even at subinhibitory concentrations.

Thus, this study demonstrates that CPZ at a concentration of 400 µg/mL was the most effective in combating biofilms associated with urinary tract infections. In addition, CPZ acted by enhancing the action of antimicrobials. Therefore, our findings reinforce the idea of CPZ, impregnated in Foley catheters, as an effective strategy in the prevention of catheter-associated urinary tract infections. More studies need to be carried out to corroborate our findings.

5. Conclusion

The new findings of this study reinforce the potential use of chlorpromazine, impregnated in indwelling catheters, as an example of drug repositioning, as an effective strategy to prevent catheter-associated urinary tract infections. Our study demonstrates the strong antimicrobial and anti-biofilm potential of chlorpromazine, which is effective against several clinically relevant species in the hospital setting and contributes to a better understanding of the role of efflux pumps in the dynamics of microbial biofilm formation, maturation, and maintenance. Its use could serve as an adjuvant strategy, in combination with another antibiofilm compound or antimicrobial agent, enhancing their activity (as observed with ciprofloxacin). Further studies are needed to confirm the clinical applicability of this therapeutic approach. Thus, we hope our work paves the way for continued exploration of this field, allowing the study to advance and helping to close the remaining knowledge gaps.

Funding Statement

This work was supported by grants from the National Council for Scientific and Technological Development (CNPq; Brazil) process numbers [405312/2021–0, 403795/2021–4, and 316340/2023–5] and from the Coordination for the Improvement of Higher Education Personnel (CAPES; Brazil).

Article highlights

• Chlorpromazine has antimicrobial and antibiofilm action against uropathogen isolates.

• Chlorpromazine inhibits biofilm formation by P. aeruginosa and enteropathogens.

• Chlorpromazine, impregnated in Foley catheters, inhibits bacterial biofilm formation.

• Chlorpromazine, impregnated at a concentration of 400 µg/mL in Foley catheters, was the one that most inhibited the formation of bacterial biofilm.

• Chlorpromazine, impregnated in Foley catheters, potentiates the antibiofilm activity of antimicrobials.

Author contributions

BR Amando and JJC Sidrim contributed to the data acquisition, drafting, revising and final approval. FIF Gomes, CL Melgarejo, PRH Souza, VM Dantas Junior, FVM Portela and RFM Guedes contributed to the data acquisition and analyses, drafting, and final approval. GMM Guedes, CL Costa Sousa, PCP and Riello, GB contributed to the data acquisition and final approval. RA Cordeiro contributed to drafting and revising, and final approval. MFG Rocha contributed to data analyses, revising and final approval. DSCM Castelo-Branco contributed to the work conception, data acquisition, drafting and revising, and final approval.

Disclosure statement

The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties

Writing assistance

No writing assistance was utilized in the production of this manuscript

Reviewer disclosures

Peer reviewers on this manuscript have no relevant financial or other relationships to disclose.