The use of combination therapy for the improvement of colistin activity against bacterial biofilm

Abstract

Colistin is used as a last resort for the management of infections caused by multi-drug resistant (MDR) bacteria. However, the use of this antibiotic could lead to different side effects, such as nephrotoxicity, in most patients, and the high prevalence of colistin-resistant strains restricts the use of colistin in the clinical setting. Additionally, colistin could induce resistance through the increased formation of biofilm; biofilm-embedded cells are highly resistant to antibiotics, and as with other antibiotics, colistin is impaired by bacteria in the biofilm community. In this regard, the researchers used combination therapy for the enhancement of colistin activity against bacterial biofilm, especially MDR bacteria. Different antibacterial agents, such as antimicrobial peptides, bacteriophages, natural compounds, antibiotics from different families, N-acetylcysteine, and quorum-sensing inhibitors, showed promising results when combined with colistin. Additionally, the use of different drug platforms could also boost the efficacy of this antibiotic against biofilm. The mentioned colistin-based combination therapy not only could suppress the formation of biofilm but also could destroy the established biofilm. These kinds of treatments also avoided the emergence of colistin-resistant subpopulations, reduced the required dosage of colistin for inhibition of biofilm, and finally enhanced the dosage of this antibiotic at the site of infection. However, the exact interaction of colistin with other antibacterial agents has not been elucidated yet; therefore, further studies are required to identify the precise mechanism underlying the efficient removal of biofilms by colistin-based combination therapy.

Introduction

Colistin is an antimicrobial agent that is extracted from Paenibacillus polymyxa, which belongs to class E of the polymixin group [1]. Two forms of colistin sulfate and colistin methane sulfonate are used in oral-topical and injectable forms for humans, respectively [2]. The general mechanism and goals of colistin are binding to the negative charge of lipopolysaccharides (LPS) in the outer membrane of Gram-negative bacteria, increasing membrane permeability and bacterial lysis, producing reactive oxygen species, and inhibiting respiratory enzymes [3]. In addition to the fact that some bacteria, such as Proteus species, Morganella morganii, Serratia species, Providencia species, Burkholderia pseudomallei, Neisseria species, and Edwardsiella tarda, are intrinsically resistant to colistin, resistance can be seen through chromosomal mutations of genes involved in LPS biosynthesis or glucose transport pathways and the transfer of the mcr-1 gene through a plasmid [4].

Colistin is often used as a last line of defense in critical clinical conditions such as bacteremia/sepsis and ventilator-associated pneumonia in the intensive care unit and against Gram-negative bacteria such as Pseudomonas spp., Acinetobacter spp., and Enterobacteriales [5]. According to the studies, the prevalence of resistance to colistin is increasing, especially in multi-drugs resistant (MDR) bacteria, and the scientific community has demanded to reduce the use of colistin [1]. In addition, one of the limitations of the widespread use of colistin is the high incidence of poisoning, such as renal and neurotoxicity, neuromuscular blockade, and, in some cases, fatal manifestations [6].

The point to consider is that one of the biggest problems in the treatment of bacterial infections is the formation of biofilm by the most important pathogens, and the activity of colistin is often impaired when faced with biofilm [7]. Biofilm formation is a bacterial community in which microorganisms are enclosed in an exopolysaccharide matrix, which leads to extensive phenotypic changes in the bacterial population and makes them 1–1000 times more resistant than their planktonic form [7, 8].

Noteworthy, a recently published study reported that a minimum inhibitory concentration (MIC)-dependent concentration of colistin could not suppress Escherichia coli growth. On the other hand, this antibiotic could lead to bacterial regrowth, with the possibility of resulting in colistin resistance. Moreover, increased expression of the phoQ gene could lead to increased biofilm formation in colistin-induced resistance bacteria, which prevents colistin from reaching the site of action, finally inducing antibiotic resistance [9]. Therefore, in addition to the increased prevalence of colistin-resistant strains in recent years, the biofilm community could also decrease the inhibitory activity of this antibiotic.

To this end, researchers have used new therapeutic options, including combination therapy, to increase the activity of colistin against bacterial biofilm, especially MDR isolates [10]. Antimicrobial peptides, bacteriophages, natural compounds and natural products, antibiotics, N-acetylcysteine, and quorum-sensing inhibitors are among the agents that have shown promising results when used with colistin. The purpose of combined treatment is to overcome resistance to colistin and other antimicrobial agents, increase the effectiveness of the combination compared to monotherapy, and reduce the toxicity associated with colistin [6, 11]. It should be noted that previous studies have shown the existence of synergistic interactions when combining colistin with antimicrobial drugs against Gram-negative clinical isolates and reducing hospital mortality [12]. Considering the therapeutic and practical role of colistin in the clinic and as the last line of treatment, the purpose of this review study is to examine and discuss the combined use of this drug along with other therapeutic agents and drug carriers for inhibition and destruction of bacterial biofilm in a comprehensive way.

Biofilm

Bacterial organisms have been found to undergo evolutionary processes in response to various obstacles and difficulties encountered in hostile settings. These problems may encompass those that are induced by the existence of host immunity, the presence of an antimicrobial agent, and restrictions in nutritional availability. Biofilm production is a notable survival strategy employed by bacteria [13–15]. A biofilm refers to a complex assemblage of many microbial organisms that are enveloped inside a self-generated polymeric matrix. This matrix serves to anchor the biofilm to either living (biotic) or non-living (abiotic) surfaces. It is worth noting that the production of biofilms is a capability exhibited by nearly all bacteria, given the presence of appropriate environmental conditions [16]. Bacterial aggregation and biofilm maturation encompass both reversible and irreversible stages, which are influenced by numerous conserved and/or species-specific variables. During the initial stage, the microorganism establishes a reversible attachment to a surface by engaging in weak contact, such as van der Waals forces, with either an abiotic or biotic surface. Various surfaces can exist in different forms, encompassing earth and aquatic systems as well as indwelling medical equipment. Additionally, surfaces can be found in living tissues, such as heart valves, tooth enamel, the lung, and the middle ear [17]. The second stage involves the process of irreversible attachment, which is facilitated by flagella, pili, and other surface appendages, as well as specific receptors [18].

The accumulation of multilayered cells occurs through cellular division; after which they initiate the synthesis of their own extracellular polymeric substance (EPS) matrix. This matrix mostly consists of polysaccharides, proteins, and extracellular DNA, which play a crucial role in promoting the formation of the biofilm [19]. The detection of EPSs can be achieved through microscopic examination as well as chemical analysis. EPSs serve as the foundational matrix or structural framework for the formation and maintenance of biofilms. EPS has a high level of hydration, with approximately 98% of its composition consisting of water. Additionally, EPS displays a strong adherence to the underlying surface. Furthermore, the EPS biofilm matrix serves as a protective barrier for microbes, shielding them from the effects of antimicrobial medications and the host immune system. Hence, the microorganisms implicated in co-infections have the ability to form polymicrobial biofilms, which exhibit not only inherent genotypic resistance but also phenotypic resistance or tolerance to antimicrobial medicines that are linked to the biofilm matrix [20]. Water channels are present in a fully developed biofilm, facilitating the efficient distribution of nutrients and signaling chemicals throughout the biofilm structure. Biofilm cells exhibit detachment either individually or in aggregates due to both internal and extrinsic stimuli. Ultimately, these cells become dispersed, leading to their colonization of various ecological niches [13].

Biofilm-associated organisms have a reduced growth rate compared to planktonic organisms, perhaps because of the constraints imposed by nutrition and/or oxygen deprivation on the cells [21]. Cell detachment from the biofilm occurs due to two primary mechanisms: cellular proliferation and division, or the elimination of biofilm aggregates that encompass substantial quantities of cells. The potential for detached cells to induce a systemic infection is contingent upon various aspects, encompassing the host immune system’s reaction [22]. Biofilms hold considerable importance in the field of public health due to the fact that microorganisms associated with biofilms demonstrate a significant reduction in their susceptibility to antimicrobial treatments. The biofilm represents a suitable niche for the exchange of resistance genes [17, 23]. The impact on susceptibility can be categorized as either intrinsic, which refers to characteristics inherent in the biofilm mode of growth, or acquired, which is associated with the acquisition of resistance plasmids. There exist a minimum of three rationales accounting for the inherent antimicrobial resistance exhibited by biofilms. Initially, it is imperative for antimicrobial drugs to effectively permeate the EPS matrix in order to establish contact with and neutralize the microorganisms residing within the biofilm [24]. EPSs inhibit the diffusion process through two mechanisms: chemical reactions with antimicrobial compounds and restriction of their transport rate. Additionally, organisms linked with biofilms exhibit diminished growth rates, resulting in a decreased uptake of antimicrobial drugs into the cell. Consequently, this phenomenon impacts the kinetics of inactivation [25]. Furthermore, the local environment encompassing the cells inside a biofilm may offer favorable conditions that serve to enhance the organism’s protection [25]. In relation to acquired resistance, studies have demonstrated that the exchange of plasmids within biofilms can occur under many circumstances. Numerous bacterial species have demonstrated the ability to transfer plasmids to diverse bacterial counterparts. The potential factors contributing to increased plasmid transfer within biofilms encompass the heightened likelihood of cellular interaction and the little impact of shear pressures on both the disruption of cell-to-cell contact and the integrity of the pili essential for conjugation [26].

Finally, it is noteworthy to mention that a crucial aspect of addressing biofilm is the recognition of polymicrobial biofilm communities that engage in mutual interactions, resulting in a synergistic impact. These interactions frequently result in an elevated level of resistance to both host and antimicrobial drugs across all participating species [27, 28]. This phenomenon can arise from either heightened tolerance or adaptive resistance, which stem from the synergistic interactions between different species, or from the presence of antibiotic-resistant organisms inside a polymicrobial biofilm, which confers protection to other species within the biofilm against antibiotic interventions. Species inside polymicrobial biofilms frequently exhibit heightened pathogenicity, an enhanced capacity to digest and exploit organic molecules within their surroundings, and provide a conducive milieu for the dissemination of adaptive characteristics and genes associated with antimicrobial resistance, both within and across species [29, 30]. Due to this feature, the management of diseases associated with microbial biofilms has emerged as a highly complex issue within the healthcare system. The cumulative impact of infections linked to biofilms can be highly debilitating for patients, as these illnesses have the ability to persist for extended periods, leading to a loss of hope for patients regarding their recovery. In particular, biofilm has been identified in various types of wounds, including chronic leg ulcers, diabetic foot ulcers, pressure ulcers, burns, malignant wounds, and surgical wounds [31–33].

Combination use of colistin with other antibiotics

Researchers are considering combining colistin with other antibiotics to improve biofilm eradication. Noteworthy, antibiotics from different families inhibit bacteria using various mechanisms, and synergy assessment has clarified the interaction of two drugs in combination against bacterial isolates. Therefore, combination therapy could suppress bacterial communities more efficiently in comparison to monotherapy.

To this end, researchers considered colistin-carbapenems combination therapy to inhibit the biofilm community of Gram-negative bacteria. In a recently published study, different concentrations of colistin-meropenem showed a synergistic effect against the biofilm structures of A. baumannii and P. aeruginosa strains, while an indifference effect was found against K. pneumoniae [34]. The results of another investigation also showed that colistin-meropenem combination therapy decreases the biofilm formation of Myroides odoratimimus strains by 92.4%. Noteworthy, colistin showed a low inhibitory effect against biofilm when used alone, while the level of inhibition improved approximately threefold when used in combination with meropenem or ciprofloxacin [35]. In line with these findings, other studies also reported the synergistic effect of colistin-carbapenems combination therapy against a biofilm community of different bacteria [36–38]. In this regard, Tamayo et al. reported that the colistin-doripenem combination not only showed synergistic effects against biofilm-embedded P. aeruginosa cells but also reduced the emergence of colistin resistance strains. The authors proposed that colistin’s ability to destroy the outer membrane of Gram-negative bacteria enhances the permeability of these bacteria and could allow greater access of doripenem to the critical penicillin-binding proteins located on the cytoplasmic membrane, where the carbapenems act [38].

On the other hand, the results of a study published in 2019 indicated that the addition of colistin to meropenem produced no relevant benefits against extended-spectrum-β-lactamase (ESBL)-producing K. pneumoniae; however, this combination therapy protected against the emergence of colistin-resistant subpopulations [39]. Other beta-lactams, such as ceftazidime, also showed a synergistic effect in combination with colistin against bacterial biofilm. In this regard, a recently published study reported that colistin monotherapy could significantly destroy the biofilm structure of P. aeruginosa, but monotherapy leads to the emergence of colistin-resistant strains. Nonetheless, colistin-ceftazidime combination therapy prevented resistance emergence to both antibiotics and improved killing in comparison to each monotherapy [40]. This supports the finding by Gómez-Junyent et al., who reported colistin plus meropenem and ceftolozane/tazobactam as the most applicable therapeutic approaches for the treatment of MDR and extensively drug-resistant (XDR) P. aeruginosa biofilm-associated infections, respectively [41]. Although the interaction of colistin and beta-lactams against biofilm structure is not well known, the findings of previous studies have shown that bacteria with low metabolic profiles within the biofilms are more susceptible to colistin, while bacteria that are more metabolically active and present in the outer layers of the biofilm are more susceptible to beta-lactams [40, 42–45]. Therefore, colistin/beta-lactam combination therapy can efficiently kill both groups of bacteria and reduce the emergence of antibiotic resistance. Additionally, as mentioned, colistin destroys the outer structure of biofilm and facilitates the penetration of beta-lactams to subpopulations within biofilm layers. Altogether, colistin and beta-lactam’s functions on various biofilm layers and cellular pathways could explain the synergy observed with the combination. Furthermore, colistin/beta-lactam combination therapy could effectively destroy the biofilm community of MDR bacteria and reduce the emergence of colistin-resistant strains. However, it seems this combination therapy has variable effects against various Gram-negative bacteria; thus, further studies are needed to evaluate whether the benefits of the colistin/beta-lactam combination therapy are obtained over longer periods or against different carbapenem-resistant or ESBL-producing bacteria.

In addition to beta-lactams, rifampicin, also in combination with colistin, showed promising results for the inhibition of bacterial biofilm [37, 46, 47]. Geladari et al. reported that colistin plus rifampicin interacted in synergy to decrease the viability of carbapenem-resistant K. pneumoniae biofilm cells at low rifampicin concentrations. Notably, the synergies observed with this combination therapy were higher than those observed with other combinations such as colistin/meropenem and colistin/tigecycline [37]. Of note, the authors supposed that colistin leads to the disruption of the outer bacterial membrane and improves the penetration and intracellular concentration of rifampicin to suppress DNA transcription or the mutual killing of resistant subpopulations by each drug [37, 48, 49]. Additionally, rifampicin’s ability to penetrate biofilm cells is supported by clinical combination studies using rifampicin to treat prosthetic material infections [49]. The result of another study demonstrated that polymixin-rifampicin combination therapy, after one hour, reduced the gene expression of quorum-sensing (QS)-regulated virulence factors, such as biofilm formation and secretion systems of P. aeruginosa. On the other hand, this combination therapy, after four hours, enhanced the expression of peptidoglycan biosynthesis genes. Notably, the combination therapy caused a substantial accumulation of nucleotides and amino acids that lasted at least 4 h, indicating that bacterial cells were in a state of metabolic arrest [46]. Therefore, the colistin-rifampicin combination therapy had a synergistic effect against the biofilm community of different bacteria at relatively low concentrations, and this combination therapy should be considered for the management of biofilm-associated infection and warrant further assessment in appropriate in vivo models.

In the end, the double combination of colistin and tigecycline also showed promising inhibitory effects against bacterial biofilm. Tigecycline, a member of the glycylcycline class of semisynthetic antimicrobial agents, has the potential for disruption of biofilm structure; however, the findings of the in vitro catheter model study showed that tigecycline monotherapy could be related to the regrowth of bacteria [50]. To this end, the combination of this antibiotic with colistin was considered by researchers for better inhibition of biofilm cells. Sato et al. reported that the biofilm community of MDR-A. baumannii was eradicated with colistin but not tigecycline. Noteworthy, the combination usage of colistin with a high concentration of tigecycline effectively eradicated biofilm, while attenuation happened with the combination of colistin and low concentrations of tigecycline [51]. In line with these results, another study also reported that just high concentrations of these drugs could lead to synergistic effects in the double combination therapy against K. pneumonia biofilm, whereas at low concentrations, the combination treatment indicated indifferent results [37]. It is noteworthy to mention that efflux pumps are considered important factors for virulence and antibiotic resistance in Gram-negative bacteria [52]. Previous studies reported that different efflux pumps such as EmrAB have an important role in resistance to colistin, and low concentrations of tigecycline lead to the upregulation of this efflux pump [53, 54]. Therefore, the authors suggested that one possible explanation for this observation might be that a low concentration of tigecycline increases the expression of efflux pumps and consequently attenuates the bactericidal activity of colistin in combination therapy [51].

Collectively, in combination therapy, the second antibiotic targeting distinct bacterial subpopulations with different antimicrobial susceptibilities could complete the function of colistin against bacterial biofilm. In addition to this so-called subpopulation synergy (where different drugs target cells with different susceptibilities), mechanistic synergy has also been proposed for combinations involving colistin, whereby each drug acts on different metabolic pathways or otherwise enhances killing by the second drug [38]. However, the mechanism involved in the synergistic efficacy of the combination of colistin and other antibiotics is not well known; therefore, further studies using validated in vitro and animal biofilm models are needed.

Finally, it is worth noting that other antibiotics such as clarithromycin, levofloxacin, azithromycin, fosfomycin, minocycline, mefloquine, and aminoglycosides were also used in combination with colistin for the inhibition of bacterial biofilms (Table 1).

Table 1.

Other studies used colistin-based combination therapy for the inhibition of bacterial biofilm

Antibacterial agents	Class	Bacterium	Methods	Outcome and comments	Reference
OligoG CF-5/20 7	Alginate oligomer	P. aeruginosa	High-molecular-weight alginate polymer bead and mouse lung infection model	The combination therapy remarkably decreased the MBEC for colistin.	[55]
Low-molecular-weight alginate oligosaccharide	Alginate oligosaccharide	P. aeruginosa	CLSM and SEM evaluation	Combining therapy effectively destroyed both intercolony branching/bridging and microcolony structures in both non-mucoid and mucoid models.	[56]
Alginate oligosaccharide	Alginate oligosaccharide	P. aeruginosa	Greiner glass-bottomed optical 96-well plate, CLSM	This compound significantly induced bacterial death and reduced the formation of biofilm.	[57]
D-amino acids	Amino acid	P. aeruginosa	CLSM, 96-well plates and biofilm dispersal assays	The addition of D-amino acids improved the colistin function and reduced the count of viable bacteria and MBIC.	[58]
Clarithromycin	Antibiotic	A. baumannii	In vitro antibiotic lock model	Colistin-clarithromycin combination therapy showed bactericidal activity against bacteria embedded in the biofilm community.	[59]
Amikacin and levofloxacin	Antibiotic	P. aeruginosa (CR-isolates)	MBECs were examined by counting the live bacteria in the biofilm, CLSM, and animal biofilm infection model.	Combined use of colistin with levofloxacin or amikacin should be considered for inhibition of biofilm-associated infections.	[60]
Azithromycin	Antibiotic	K. pneumonia	Biofilm formation in vials and polystyrene 96-well plates	Azithromycin can improve the effectiveness of colistin.2	[61]
Fosfomycin	Antibiotic	Different GNB	Biofilm chequerboard and quantitative antibiofilm assays	Colistin-fosfomycin combination therapy showed a synergistic effect against the biofilm community of the majority of tested strains.	[62]
Fosfomycin	Antibiotic	CR P. aeruginosa	Biofilm formation in polystyrene 96-well plates	Colistin-fosfomycin combination therapy showed an inhibitory effect against biofilm.	[36]
Ceftazidime-avibactam	Antibiotic	XDR P. aeruginosa	MTT Methythiazolyl tetrazolium assay	The combination therapy can suppress the biofilm formation and decrease the production of drug-resistant bacteria.	[63]
Clarithromycin or esomeprazole	Antibiotic	K. pneumonia	In vitro catheter biofilm model	The combination therapy showed a synergistic effect.	[50]
Rifampicin	Antibiotic	CA A. baumannii	Polystyrene microtiter plate biofilm assay	Monotherapy was not effective at reducing bacteria in biofilm, while colistin-rifampicin combination therapy significantly decreased the bacteria.	[47]
Clarithromycin	Antibiotic	P. aeruginosa	In vitro catheter biofilm model	The combination therapy was most effective at reducing bacterial count in biofilm in comparison to the monotherapy.	[64]
Tobramycin	Antibiotic	P. aeruginosa	Static and dynamic biofilm experiments. CLSM analysis and lung infection in the animal model	The antibiotic combination significantly decreased the bacterial count and was more effective in managing infection in the animal model than monotherapy.	[65]
Inhaled combination dry powder formulation of colistin and rifapentine	Antibiotic and drug delivery	P. aeruginosa	Polystyrene microtiter plate biofilm assay	Combination dry powder increased the antibacterial against the biofilm community.	[66]
Mefloquine	Antimalarial medicine	P. aeruginosa	Biofilm eradication test and biofilm formation inhibition test	The combination therapy decreased biofilm formation and removed pre-formed established biofilms.	[67]
Nisin	Lantibiotics; bacteriocins	P. aeruginosa	Static microtiter plate assays	Nisin could significantly reduce the concentration of colistin for inhibition of biofilm.	[68]
Enterocin DD14 and nisin	Bacteriocins	Colistin-resistant E. coli	Microtiter plate biofilm assay	The combination therapy eradicated bacterial biofilm	[69]
Chitosan-coated human albumin nanoparticles	Drug delivery	A. baumannii, K. pneumonia	Biofilm formation in polystyrene 96-well plates	This compound inhibited the formation of biofilm 4- and 60-fold higher than free colistin.	[3]
Cephalosporin nitric oxide-donor prodrug	Drug delivery	P. aeruginosa	A crystal violet staining technique and CLSM	This compound leads to the near-complete eradication of the biofilm community.	[70]
Polymeric derivative with glyco-polypeptide architecture	Drug delivery	P. aeruginosa, S. aureus	Crystal violet method, static chamber system	This compound leads to the eradication of established clinically relevant biofilms.	[71]
Clomiphene citrate and Auranofin5	FDA-approved drugs	P. aeruginosa	Formation of biofilms on glass discs and CLSM	The combination therapy showed antibiofilm activity	[72]
Exopolysaccharide biosynthetic glycoside hydrolases	Glycoside hydrolases	P. aeruginosa	CLSM, microtiter dish biofilm assay	The addition of enzyme to colistin decreased bacterial count.	[73]
HBED	Iron chelators	P. aeruginosa	Microtiter plate and flow cell biofilm assay	Combination therapy significantly increased the effect of colistin microcolony killing and leads to the almost complete removal of the biofilm.	[74]
EDTA	Iron chelators	Colistin-resistant K. pneumonia	Crystal violet assay and catheter-related biofilm infection mouse model	Combination therapy remarkably reversed colistin resistance in both planktonic and mature biofilms of colistin-resistant and eradicated colistin-resistant bacteria from catheter-related biofilm infections.	[75]
CHIR-090 6	LpxC inhibitors	P. aeruginosa	Bead biofilm assay, biofilms in flow cell chambers, and mouse biofilm implant model of infection	The combination therapy, at sub-inhibitory concentrations, indicated synergistic activity and suppressed the formation of biofilm of colistin-tolerant bacteria.	[76]
Maipomycin 1	Natural compound	A. baumannii	MTT assay and CLSM analysis	Maipomycin increased the antibiofilm activity of colistin.	[77]
Nutrient dispersion compounds	Nutrient dispersion	P. aeruginosa	CLSM	The combination therapy leads to the remarkably significant decrease in the live bacterial population.	[78]
PFK-158 4	PFKFB3 inhibitor	Colistin-resistant GNB	Biofilm formation assay and SEM	The combination therapy significantly reduced biofilm formation and decreased the cell arrangement density of biofilm.	[79]
Furanone C-30 3	QS inhibitors	Colistin-resistant GNB	Biofilm formation inhibition assays and standardized in vitro biofilm mode	The combination therapy suppressed the formation of bacterial biofilm and showed a better eradication effect on established biofilm than monotherapy.	[80]
N-(2-pyrimidyl) butanamide 8	QS inhibitors	P. aeruginosa	CLSM, 24 well microtiter plates containing BBM and AHL analogs	Synergistic antibiofilm activity was detected under both anaerobic and aerobic conditions.	[81]
Ultrasound patches	Ultrasound	P. aeruginosa	Filter-biofilms	Significantly enhanced the bacterial killing of the biofilm community.	[82]
Low-frequency ultrasound	Ultrasound	Pan-resistant biofilms of A. baumannii	24 well microtiter plates	Colistin/vancomycin low-frequency ultrasound combination therapy decreased the count of bacteria in biofilms after 8 h and a continuing decline until 24 h.	[83]

QS, quorum-sensing inhibitor; CR, carbapenem-resistant; MBECs, minimal biofilm eradication concentrations; CLSM, confocal laser-scanning microscopy; DD, disc diffusion; GNB, Gram-negative bacilli; XDR, extensively drug-resistant; MTT, methythiazolyl tetrazolium assay; HBED, N, N'-bis (2-hydroxybenzyl) ethylenediamine-N, N'-diacetic acid (iron chelators); SEM, scanning electron microscopy; BBM, basic medium for biofilm formation; AHL, N-acyl homoserine lactone

¹This compound was isolated from rare actinomycetes strain Kibdelosporangium phytohabitans XY-R10

²The authors reported that the DD method and broth growth-based assays may not be good predictors of antibiotic susceptibility in biofilms

³QS inhibitors

⁴6-phosphofructo2-kinase/fructose-2, 6-bisphosphatase 3 (PFKFB3) inhibitor

⁵Compounds with antibacterial and antibiofilm activities that are commercially available

⁶LpxC inhibitors

⁷Alginate oligomer

⁸QS inhibitors

Drug delivery platforms

Colistin, as mentioned, is considered a promising therapeutic approach for the management of MDR Gram-negative bacteria. However, the increased prevalence of colistin-resistant strains in recent years has restricted the clinical usage of this antibiotic [84]. Noteworthy, recently published studies reported that different nanoparticles and drug platforms could be used for different clinical purposes [85–87]. To this end, the use of various drug platforms is suggested by researchers to deliver intact colistin at the site of infection and shield its interactions with bacterial biofilm and airway mucus, thereby enhancing the interaction of this antibiotic with bacteria.

Sans-Serramitjana et al. proposed that nanoencapsulation could improve the efficacy of colistin against MDR infections by overcoming the limitations of conventional pharmaceutical forms. In this concept, these authors evaluated the antibiofilm activity of nanostructured lipid carriers (NLC)—colistin and free colistin against P. aeruginosa. The findings indicated the more rapid killing of P. aeruginosa bacterial biofilms by NLC-colistin than by free colistin [88].

In line with these results, another study that was published in 2016 also reported that NLC-colistin had the same antibacterial function as free colistin against the planktonic community of P. aeruginosa; nonetheless, nanoencapsulated colistin was much more efficient in the eradication of biofilms than free colistin [89]. Furthermore, nano-embedded microparticles (NEMs) for sustained delivery of colistin in the lung were used in another investigation. To this purpose, the emulsion/solvent diffusion technique was used for the production of poly(lactide-co-glycolide) (PLGA) containing colistin. Noteworthy, poly (vinyl alcohol) (PVA) and chitosan (CS) were used to modulate surface properties and enhance the transport of synthesized nanoparticles through artificial cystic fibrosis (CF) mucus. Additionally, these nanoparticles were spray-dried in various carriers for the production of MEM. Colistin-loaded NEM significantly removed the biofilm of P. aeruginosa and showed a prolonged efficacy in biofilm eradication compared to the free drug. The results of the confocal analysis confirmed that the antibiofilm activity of nanoparticles could be associated with their ability to penetrate into bacterial biofilms and to sustain the release of colistin inside the biofilm community [90].

Notably, the coating of the nanoparticle surfaces with chitosan could facilitate the transport of nanoparticles through mucus, probably as a consequence of mucus fiber collapse, and produce large channels that can paradoxically enhance the penetration of cationic chitosan-modified nanoparticles. Therefore, the engineering of PLGA nanoparticles containing colistin should be considered for killing bacterial biofilm, especially in patients with CF [91]. Taken together, the possible entrapment and slow penetration of colistin within the biofilm due to electrostatic interactions with the negatively charged alginate matrix can significantly decrease the availability of colistin in the bottom layers of the biofilm. In this regard, the use of different nanoplatforms such as NLC and PLGA could easily penetrate the biofilm structure; thus, the colistin-nanoplatform can reach the bacteria located in the deeper layers of the biofilm faster and more easily than free colistin [88, 92, 93].

Osteomyelitis, one of the most important local infections, is mostly caused by Methicillin-resistant Staphylococcus aureus (MRSA); nevertheless, the prevalence of Gram-negative associated bone infections has significantly increased over the last few years [94]. To this end, different bacteria such as E. coli, P. aeruginosa, and A. baumannii have attracted much attention owing to their ability to reach antibiotic multi-resistance. Additionally, bacterial biofilm leads to antibiotic resistance and is responsible for prolonged antibiotic treatment and an aggressive surgical approach in osteomyelitis [95, 96]. As mentioned in the previous parts, the reports of colistin-resistant bacteria were increased in recent years, and this drug is not available in bone void fillers for local high-dose delivery [97]. In this regard, the use of different drug platforms was considered by researchers for the enhancement of colistin’s efficacy against bacterial biofilm and the improvement of the concentration of this antibiotic at the site of infection.

Aguilera-Correa et al. used bone-targeted mesoporous silica nanoparticles that were functionalized with gelatin/colistin coating. The nanoparticles significantly reduced the number of MRSA in the bone just 24 h after only one dose. It is noteworthy to mention that S. aureus by secretion of different enzymes, such as cysteine proteases, serine proteases, and metalloproteases, could degrade gelatin coating and accelerate the delivery of antibiotics from drug platforms on the infected bone [98]. In another study, the authors used mesoporous silica nanoparticles that have been loaded with moxifloxacin and further functionalized with colistin and Arabic gum. This nanosystem showed high affinity toward the biofilm community of E. coli because of Arabic gum coating and antibacterial activity because of the colistin disaggregating effect and moxifloxacin bactericidal effect. The nanosystem, in a short time, could release large amounts of moxifloxacin; therefore, it could lead to the preparation of a high concentration of antibiotics nearby bacteria and in the site of infection from a low quantity of nanoparticles that might decrease the potential side effects associated with other administration routes. Additionally, colistin can directly kill bacteria because this antibiotic is easily absorbed on the surface of nanoparticles. Interestingly, Arabic gum improves the adsorption capacity of nanoparticles, potentially diminishing the final nanoparticle dose that would be required during the treatment if colistin alone was to be used [96].

Finally, in a recently published study, microcontainers, reservoir-based microdevices, were co-loaded with colistin and ciprofloxacin for inhibition of P. aeruginosa biofilm. The results of this study showed that co-loaded microcontainers are superior to monotherapy and completely killed all of the bacteria in the planktonic community. Furthermore, antibiotics in microcontainers work significantly faster (just five hours) than simple perfusion of antibiotics in biofilm. The authors proposed that this effect is caused by the burst release of the antibiotics from the microcontainers, which creates an immediate high concentration of antibiotics at the local site of infection and ultimately more destroyed biomass [99].

Therefore, as mentioned in this section, different drug platforms, such as liposomes, PLGA, and microcontainers, have an acceptable capacity as delivery systems for inhibition of bacterial biofilm by reaching immediate high local colistin concentrations at the site of infection, especially in patients with CF and osteomyelitis. Additionally, the use of drug platforms decreases the desired dose of colistin, therefore reducing the side effects of this antibiotic. Drug platforms could be functionalized by different antibacterial agents such as natural compounds, mucin-degrading enzymes, metal nanoparticles, and antibiotics; thus, the use of drug platforms can enhance the antibiofilm activity of colistin. Altogether, drug platforms containing colistin may be an interesting strategy for the inhibition and removal of bacterial biofilms; however, further studies are required to identify the precise mechanism underlying the efficient removal of biofilms by these platforms.

In the end, other drug platforms, such as colistin-loaded human albumin nanoparticles, bi-functional alginate oligosaccharide–polymixin conjugates, and colistin-conditioned surfaces, showed promising results for inhibition of bacterial biofilms (Table 1) [3, 57, 100].

N-Acetylcysteine

N-Acetylcysteine (NAC) is commonly administered as an antioxidant due to the ability of the free thiol group to react with nitrogen species and reactive oxygen by constituting a precursor of intracellular glutathione. Additionally, NAC is known as a mucolytic agent; therefore, this compound clears thick mucus from the lungs and is used in combination therapy with antibiotics for the treatment of lower respiratory tract infections [101, 102]. The results of the recently published studies also showed that NAC could destroy the matrix architecture of bacterial biofilm and enhance biofilm breakdown. As mentioned, NAC is a nebulized mucolytic agent and is used for the management of patients with CF. These patients are mostly infected with MDR Gram-negative bacteria such as P. aeruginosa and Stenotrophomonas maltophilia, and the biofilm community of these bacteria is a main problem in CF patients. Altogether, colistin is used as a last resort in these patients, and combination therapy with this antibiotic and NAC for removing MDR bacterial biofilm could be an applicable therapeutic approach (Fig. 1).

Fig. 1.

The use of combination therapy for enhancement of colistin activity against bacterial biofilm

In this regard, Aksoy et al. reported that NAC reduced the minimum biofilm inhibitory concentration (MBIC) of different antibiotics such as colistin against E. coli, Proteus mirabilis, and Pseudomonas putida [103]. In another experiment, 18 S. maltophilia were collected, and the effect of the colistin-NAC combination therapy was evaluated against these bacteria. The combination therapy indicated synergism against the colistin-resistant strains, suggesting that NAC could antagonize the mechanisms involved in colistin resistance. Additionally, a dose-dependent potentiation of colistin activity at sub-MIC concentrations by NAC was also clearly observed against S. maltophilia biofilms [104]. In line with these results, Polline and colleagues reported a significant synergistic antibiofilm function of N-acetylcysteine (8000 mg/L) plus colistin (8 mg/L) against colistin-susceptible and colistin-resistant strains [101].

Noteworthy, an artificial sputum medium (ASM) model was used in two studies in order to mimic the bacterial biofilm environmental conditions experienced in CF mucus. In one of these studies, monotherapy with NAC (8000 mg/L) indicated strain-dependent and limited antibiofilm activity against P. aeruginosa. However, the use of colistin (2 to 32 mg/L) plus NAC (8000 mg/L) demonstrated a relevant antibiofilm synergism against all strains. Furthermore, this combination therapy also demonstrated a clear synergism against bacterial biofilms grown in ASM. Noteworthy, the colistin concentration that allowed observation of synergism was much higher (i.e., 32 × the MIC) in the ASM model in comparison to the Nunc-TSP lid system. In this regard, the authors proposed that the requirement for a higher dosage of colistin could be due to the strong ionic interactions of this antibiotic with ASM components such as mucin and extracellular DNA. In addition, the results showed that treatment of bacterial planktonic cultures with NAC could reduce the virulence of P. aeruginosa, such as anaerobic respiration, zinc starvation response, and flagellum-mediated motility. Hence, pretreatment with NAC could prevent biofilm formation and lung infection [105].

In line with these results, Aiyer et al. evaluated the impact of synergistic and additive colistin-NAC combination therapy in an ASM model using lung macrophages and bronchial cells to model Achromobacter xylosoxidans infection. Noteworthy, this bacterium is a Gram-negative bacillus that has been related to chronic colonization in CF and antibiotic resistance. The findings of this study showed that combination therapy is well tolerated by both cells and could lead to the synergistic and remarkable decrease in bacterial counts [106]. These data indicated that the antibiofilm synergism of colistin-NAC combinations against P. aeruginosa strains is also preserved under environmental conditions mimicking the CF mucus, which is promising for clinical applications.

Noteworthy, NAC itself can suppress bacterial proliferation and growth by preventing cysteine utilization by bacterial cells and reducing the formation of EPS and polysaccharides in many bacterial species [107]. NAC can destabilize the biofilm structure by interacting with the main components in the biofilm matrix or by chelating magnesium and calcium [108]. As mentioned in previous parts, NAC, at concentrations achievable by topical administration, could suppress the biofilm community of CF-associated Gram-negative bacteria and revert the colistin-resistant phenotype. In this setting, NAC might revert the colistin resistance phenotype and remarkably improve the efficacy of this antibiotic against bacterial biofilm. Nevertheless, the report of exact interactions between colistin and NAC that lead to synergism in combination therapy is not easy to hypothesize due to the relevant knowledge gaps on the mechanisms of action of both compounds. In this regard, further studies with a focus on NAC and colistin interactions are encouraged because understanding the mechanisms of such a synergism would be relevant to the discovery of new antibacterial agents for inhibition of MDR bacterial biofilm in different infections such as CF and catheter-associated infections.

Natural compounds

Recently published studies have reported that different natural products or natural compounds (NCs) not only could reduce the formation of biofilm but also could eradicate an established biofilm structure [109–111]. Furthermore, probably a combination treatment of NCs with conventional antibiotics, because of their increasing effectiveness and potency while minimizing the toxicity and dosage of antibiotics and reducing the likelihood of developing resistant strains, should be considered as an applicable therapeutic approach to manage MDR and biofilm-associated infection [112]. In this regard, the results of recently published studies indicated that different NCs such as resveratrol, chrysin, kaempferol, plumbagin, naringenin, cinnamaldehyde, thymol, and capsaicin could enhance the efficacy of colistin against biofilms of Gram-negative bacteria, even colistin-resistant strains [113–120].

Noteworthy, inhibition of QS in Gram-negative bacteria, especially P. aeruginosa, was considered by researchers as a promising therapeutic approach for inhibition of bacterial biofilm and infections [121]. In this regard, different NCs, such as cinnamaldehyde, could suppress the expression of Las-, Rhl-, and PQS in P. aeruginosa. The P. aeruginosa QS system is comprised of three hierarchically integrated QS systems, Las, Rhl, and PQS, to manage the expression of different virulence factors and biofilm-related genes of this bacterium [118, 119, 122]. Therefore, NCs, by suppressing the QS system in bacteria, could inhibit the formation of bacterial biofilm and make them more susceptible to colistin.

In addition to QS inhibition, NCs could reduce the biofilm formation by inhibition of bacterial attachment in the first step of the switch of planktonic bacteria to a biofilm phenotype. In this manner, the results of a study showed that chrysin, a component of honey, could downregulate the expression of csuA/B and katE [120]. Noteworthy, the chaperoneusher pili (Csu) assembly system, including transport proteins (CsuC and CsuD) and pilin subunits (CsuA/B, CsuA, CsuB, and CsuE), is a main player in A. baumannii adhesion to the medical appliances surfaces [120, 123]. Moreover, RpoS has an association with the formation of established biofilm by inducing motility-related genes and by suppressing EPS synthesis, and katE is a reporter gene of RpoS [120, 124, 125]. To this end, when the expression of the mentioned genes is downregulated by colistin-chrysin combination therapy, the formation of biofilm is also inhibited.

Furthermore, NCs showed interaction with the outer membrane and altered the potential of the cell membrane. For example, the findings of a study showed that thymol can enhance the permeability of membranes to overcome colistin resistance. Capsaicin can also destroy the permeability of the outer membrane, which could enable hydrophobic capsaicin molecules to pass through the LPS in the outer layer of bacteria and reach their target, thus playing a synergistic role [113, 114]. Therefore, NCs are permeabilizers and should be considered a promising adjuvant to colistin against Gram-negative bacteria. Finally, it should be noted that NCs could suppress the expression of efflux pumps, and this possible mechanism should be evaluated in future studies as a possible synergistic effect in the colistin-NCs combination therapy [77]. Hence, NCs can improve the efficacy of colistin against bacterial biofilm and overcome colistin resistance; therefore, the synergy between colistin and NCs warrants further experimental evaluation and confirmation using different animal models of infection and different dosage combinations. However, some disadvantages, such as poor stability and solubility, hinder the use of NCs in clinical settings. To this end, the use of drug delivery platforms can overcome these physicochemical barriers [119]. Additionally, some of the NCs are used as antioxidant agents, while the production of reactive oxygen species (ROS) is one of the important antibacterial mechanisms mediated by colistin [126]. Therefore, co-administration of NCs that remove ROS could increase the persister cells, and this issue should be evaluated in further studies.

Antimicrobial peptides

Antimicrobial peptides (AMPs), host defense peptides that mainly consist of protein fragments, are produced by both prokaryotes and eukaryotes and act as a first line of defense for the immune system [127]. AMPs are broad-spectrum antibacterial agents that kill bacteria rapidly with low cytotoxicity for eukaryotic cells [127, 128]. Additionally, these agents have an antibiofilm capacity using different mechanisms, such as disruption of membrane and EPS, downregulation of biofilm-associated genes, interference with cell signaling, and inhibition of stringent response [129].

In this regard, researchers considered colistin-AMP combination therapy a promising antibiofilm strategy. Morroni et al. reported synergic activity for colistin-LL37 combination therapy against ESBL- and carbapenemase-producing and mcr-1 carrying E. coli. Noteworthy, AMP LL-37 is a 37-amino acid peptide that is proteolytically released from the human cathelicidin hCAP-18. AMP LL-37 significantly reduced the E. coli biofilm at 1 × MIC concentration, and the expression of mcr1, a colistin-resistant-associated gene, did not affect the function of this AMP [130]. The results of another study also showed that a combination of colistin and different AMPs such as LL-37 could decrease minimum biofilm eradication concentration (MBEC) by eightfold [131]. Other authors also reported that a combination of the sub-MIC concentration of CRAMP (a cathelicidin-associated AMP) with colistin indicated interesting synergistic activity against the P. aeruginosa biofilm community and reduced the expression of QS-regulated genes, resulting in inhibitory effects on QS-regulated virulence phenotypes (pyocyanin and rhamnolipid) [132].

Furthermore, the findings of a recently published study demonstrated that colistin, in combination with AMPs, including citropin 1.1, MP temporin A, and tachyplesin I linear analogue, have an additive or synergic function in the treatment of pre-established S. aureus and P. aeruginosa single- and double-species biofilm [133]. Finally, the combination of colistin with melittin, an alpha-helical hydrophobic AMP, completely inhibited the biofilm formation of MDR-strong biofilm producer A. baumannii. Furthermore, the results of this study showed that melittin decreased the expression of the bap gene. This phenomenon decreases the accumulation of extracellular matrix in the periphery of bacteria, which consequently enhances the penetration of antibiotics into the cytoplasm. Besides, it is noteworthy to mention that melittin can bind to the resistant gene and inhibit its expression by its DNA binding activity [134].

It seems colistin-AMP combination therapy leads to synergistic effects due to the diversity of mechanisms of action found in colistin and AMPs. For instance, colistin, after binding to the LPS of Gram-negative bacteria, leads to the disruption of the outer membrane, while AMPs interact with the cytoplasmic membrane. Therefore, the combination of these two antibacterial agents causes a perturbation of both the outer and cytoplasmic membranes and could explain the synergistic relation between colistin and different AMPs such as LL-37 [6, 130, 135]. Additionally, as mentioned, colistin-AMP combination therapy could suppress the biofilm formation in P. aeruginosa by downregulation of the rhl system gene, resulting in decreased expression of pyocyanin and rhamnolipid. It should be noted that pyocyanin and rhamnolipid are important virulence factors in P. aeruginosa that are mainly regulated by the rhl system and manage the secretion of the extracellular DNA in bacteria (eDNA) and play a vital role in the formation and diffusion of biofilm [132, 136, 137]. Therefore, AMPs could destroy the structure of biofilm by downregulating the QS-associated gene and consequently improve the interactions of colistin with biofilm. However, the studies in this field are very limited, and further in vivo studies are needed to evaluate the synergistic activity of colistin and AMPs and determine the possible clinical dose through pharmacokinetic experiments.

Phage

Bacteriophages (phages), viruses that infect bacteria, are considered by scientists as an applicable approach for the management of MDR bacteria. Additionally, recent studies have reported phages as antibacterial agents with inhibitory effects against bacterial biofilm. However, some disadvantages, such as the narrow host range and the development of phage resistance, restrict the clinical usage of phages [138]. To this end, the combination of phages and antibiotics was considered by scientists to inhibit bacterial infection. Colistin is one of these antibiotics that showed promising inhibitory effects against MDR bacterial biofilm when used in combination with phages and phage-derived enzymes. Vashisth et al. evaluated the synergistic effects of Myophage φAB182 in combination with different antibiotics such as colistin against the biofilm community of MDR-A. baumannii. The results of this study showed that φAB182 has the highest synergy with colistin in comparison to other antibiotics such as ceftazidime, polymixin B, and cefotaxime. Furthermore, colistin-phage combination therapy also eradicated the biofilm of A. baumannii [139].

In line with these results, another investigation also reported that colistin-phage T1245 combination therapy not only could reduce bacterial density up to approximately 80% but also could reduce biomass and bacterial viability in 3-day established biofilms. After the combination therapy, scanning microscopic evaluation showed visible alterations in cell morphology, with membrane poration and cell lysis as indicated by the presence of cell debris [140]. vWU2001 was another phage that, in combination with colistin, remarkably inhibited carbapenem-resistant A. baumannii in comparison to monotherapy. This combination therapy also leads to a significantly greater enhancement in G. mellonella survival and in bacterial clearance, as compared with that of phage or colistin alone [141]. Therefore, the use of colistin in combination with phages could be used for the inhibition of MDR-A. baumannii biofilm. The exact interaction of phages and colistin is not yet elucidated, but some possible mechanisms have been reported by recent studies. For instance, colistin interaction with the outer membrane of Gram-negative bacteria could increase both phage adsorption and phage DNA injection [142]. Phage-antibiotic combination therapy may lead to changes in bacterial morphology, rapid cell lysis, and phage maturation [143]. Additionally, colistin could cause cell clustering, and this phenomenon enhanced the phage’s ability to travel on the adjoined cell surface, increasing phage infection efficiency [144].

It is noteworthy to mention that phages produce the endolysins during the final stages of the replication cycle to cleave the bacterial cell wall and produce progeny virions. Endolysins showed good inhibitory functions against Gram-positive bacteria; however, the presence of the outer membrane restricted their function against Gram-negative bacteria [145, 146]. In this regard, colistin could be employed to assist the endolysins in overcoming the impenetrability of the outer membrane [146]. Finally, depolymerases that are encoded by phages are responsible for destroying EPS, LPS, and capsular polysaccharides (CPS) of the host bacteria during phage invasion. Therefore, phages can destroy biofilm structure and improve the penetration of the antibiotic to the deeper layers of biofilm by inducing the synthesis of enzymes such as polysaccharide depolymerase [147]. Hen et al. used depolymerase Dpo71, derived from a baumannii phage, in combination with colistin, for inhibition of the biofilm community of MDR-A. baumannii. Dpo71 improved the inhibitory effect of host immune cell activity against bacteria and also acts as an adjuvant to assist or improve the function of colistin. An exact evaluation indicated that the enhanced bactericidal effect of colistin is attributed to the improved outer membrane destabilization capacity and binding rate to bacteria after stripping off the bacterial capsule by Dpo71. Moreover, the combination of Dpo71 could remarkably improve the colistin activity against biofilm and enhance the survival rate of A. baumannii-infected Galleria mellonella. The results of the microscopy evaluation showed that Dpo71 could significantly destroy the biofilms but was not able to decrease the count of viable cells in the biofilms. Colistin-Dpo71 combination therapy significantly reduced the count of viable bacterial cells and residual biomass of biofilm compared with monotherapy. It seems that the antibiofilm activity of his combination therapy is associated with the improved colistin penetration within the biofilm matrix after the EPS depolymerization by the Dpo71 [148].

Therefore, the combined use of colistin and phage depolymerase should be considered for the inhibition of the biofilm of MDR bacteria. However, knowledge of the exact interaction of extracellular structures of Gram-negative bacteria cleavage by phage depolymerases is still largely missing. Additionally, the biofilm’s susceptibility to phage depolymerase treatments varied, depending on the bacterial strains, the activity of the depolymerase enzyme, and the type of bacteria. Therefore, although colistin-phage combination therapy may represent promising treatment strategies for managing MDR bacteria, more confirmatory studies in this field are required.

Conclusion

As mentioned in the previous sections, the use of combination therapy could boost the inhibitory effects of colistin against the MDR bacterial biofilm community. To this end, the use of different antibacterial agents, antibiotics, and drug platforms in combination with colistin could suppress biofilm formation and remove established biofilm. Additionally, combination therapy reduces the chance of acquiring colistin resistance and decreases the required dose of this antibiotic at the site of infection. Therefore, colistin-based combination therapy could open new frontiers in the treatment of biofilm-related infections; however, the exact mechanism underlying the efficient removal of biofilms by combination therapy has not yet been reported. To this end, further investigation of preclinical and clinical studies as well as bio-toxicity evaluation for the human body is needed before the clinical usage of colistin-based combination therapy for the management of biofilm-associated infections.

Abbreviations

MDR

Multi-drug resistant

LPS

Lipopolysaccharides

MIC

Minimum inhibitory concentration

ESBL

Extended-spectrum-β-lactamase

XDR

Extensively drug-resistant

QS

Quorum sensing

MRSA

Methicillin-resistant Staphylococcus aureus

NAC

N-acetylcysteine

CF

Cystic fibrosis

MBIC

Minimum biofilm inhibitory concentration

ASM

Artificial sputum medium

NCs

Natural compounds

MBEC

Minimum biofilm eradication concentration

Author contribution

AJ conceived and designed the study. RA, MA MS, MM, MA, and RL contributed to comprehensive research and wrote the paper. Notably, all authors have read and approved the manuscript.

Data availability

The authors confirm that the data supporting the findings of this study is available within the article.

Declarations

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Conflict of interest

The authors declare no competing interests.