Medicinal plants with anti-biofilm activity.
Abstract
Biofilms are structured aggregates of bacterial cells that are embedded in self-produced extracellular polymeric substances. Various pathogens initiate a disease process by creating organized biofilms that enhance their ability to adhere, replicate to accumulate, and express their virulence potential. Quorum sensing, which refers to the bacterial cell-to-cell communication resulting from production and response to N-acyl homoserine lactone signal molecules, also plays an important role in virulence and biofilm formation. Attenuation of microorganisms’ virulence such that they fail to adapt to the hosts’ environment could be a new strategic fight against pathogens. Thus, agents or products that possess anti-biofilm formation and/or anti-quorum sensing activities could go a long way to manage microbial infections. The incidence of microbial resistance can be reduced by the use of anti-biofilm formation and anti-quorum sensing agents.
Keywords
- biofilm
- quorum sensing
- bacteria
- acyl homoserine lactone
1. Introduction
Biofilm is a population of cells growing on a surface and enclosed in an exopolysaccharide matrix [1]. The physiology, structure and chemistry of the biofilm vary with the nature of its resident microbes and local environment [2].
Most important feature among biofilms is that their structural integrity critically depends upon the extracellular matrix produced by their constituent cells. They are notoriously difficult to eradicate and are a source of many recalcitrant infections [2]. Biofilms are associated with serious health issues stemming from persistent infections due to the contamination of medical devices (intravenous and urinary catheters), artificial implants and drinking water pollution among others [3].
Intercellular signaling, often referred to as quorum sensing (QS), has been shown to be involved in biofilm development [4]. Quorum sensing relies on small, secreted signaling molecules; much like hormones in higher organisms, to initiate coordinated responses across a population and it contributes to behaviors that enable microbes to resist antimicrobial compounds [5]. Quorum sensing signaling activation can lead to antimicrobial resistance of the pathogens, thus increasing the therapy difficulty of diseases [4].
The key concern about biofilms is their contribution to the development of resistance against antimicrobial agents, and with the on-going emergence of antibiotic-resistant pathogens, there is a current need for development of alternative therapeutic strategies [6].
An anti-virulence approach by which quorum sensing is impeded could be a viable means to manipulate bacterial processes, especially pathogenic traits that are harmful to human and animal health and agricultural productivity [7]. Further research into the identification and development of chemical compounds and enzymes that facilitate quorum-sensing inhibition (QSI) by targeting signaling molecules, signal biogenesis, or signal detection are required [7]. Anti-QS agents can abolish the QS signaling and prevent the biofilm formation, therefore reducing bacterial virulence without causing drug-resistant to the pathogens, suggesting that anti-QS agents could be potential alternatives for antibiotics [8]. An effective clinical strategy for treating bacterial diseases in the near future will be to combine anti-QS agents with conventional antibiotics since this can significantly improve the efficacy of therapeutic drugs and decrease the cost of human healthcare [9].
2. Microbial biodiversity in biofilm systems
Biofilms are mixed microbial cultures normally consisting predominantly of prokaryotes with some eukaryotes. Thus, in addition to microbial cells, the surrounding environment contains a range of macromolecular products in which exopolysaccharide secreted by the cells is the dominant macromolecular component, while the water content is probably about 90–97% [10, 11]. Secreted products also include enzymes and other proteins, bacteriocins, and low mass solutes and nucleic acid released through cell lysis. The lysis may occur either naturally with cell aging or through the action of phage and bacteriocins.
Opportunistic pathogens, viruses, parasitic protozoa, toxin releasing algae and fungi and enteric bacteria e.g.
Biofilms present complex assemblies of microorganisms attached to surfaces. They are dynamic structures in which various metabolic activities and interactions between the component cells occur [10]. Studies on microorganisms and biofilm formation have revealed diverse complex social behavior including cooperation in foraging, building, reproduction, dispersion and communication among microorganisms [14]. The organisms within a biofilm setup may include a single or diverse species of microorganisms. In the biofilm, bacteria can share nutrients and are sheltered from harmful factors in the environment, such as desiccation, antibiotics, and a host body’s immune system.
Bacteria, fungi, viruses, protozoa and cyanobacteria that are common pathogens are all involved in biofilm formation [15].
2.1 Bacterial biofilms
About 99.9% of all bacteria live in biofilm communities [16]. A biofilm usually begins to form when a free-swimming bacterium attaches to a surface. Pathogenic organisms are found on most food items including seafoods and biofilm forming pathogens are found on such seafoods as crabs [17], pacific oysters [18], shrimps [19] etc. Public health and clinical microbiologists recognize that biofilms are present everywhere in nature and are responsible for a number of human infections. Infectious caused by microbial communities include urinary tract infections, middle-ear infections, dental plaque, gingivitis, endocarditis, cystic fibrosis. Biofilms on persistent indwelling devices such as catheter, contact lenses, heart valves and joint prostheses are also responsible for many recurrent infections [20, 21]. Biofilms on indwelling medical devices may be composed of Gram-positive or Gram-negative bacteria. Bacteria commonly isolated from these devices include the Gram-positive
The organisms that form biofilms on medical devices originate from patient’s skin microflora, exogenous microflora from health-care personnel, or contaminated infusates. Biofilms associated with catheters may initially be composed of single species, but with the passage of time they become multi-specie communities. Some urinary tract and bloodstream infections are also caused by biofilm-associated indwelling medical devices with 50–70% of infections related to catheter [12]. Chronic infections, inflammation and tissue damage caused by many strains of single species are often found in polymicrobial communities [24].
Bacteria that reside in a biofilm community usually will not grow when cultured, a situation normally referred to as “viable, but not culturable”. The reason is that to change to the planktonic state from a biofilm-producing phenotype, bacteria require complex and specific environmental and signaling factors that are not available in a culture plate [25]. This therefore suggests that analyzing biofilm samples for bacterial infective agents during infections may show negative results and the real cause of the infections may not be detected if culturing is the only investigative procedure.
2.2 Fungal biofilms
Many medically important fungi produce biofilms and they include
2.3 Protozoan biofilms
Free-living protozoans are single celled eukaryotic organisms and are divided into amoebae, flagellates and ciliates. All the three protozoan groups have been found in fresh water biofilms. Although many different species are found in association with biofilms, their level of association differs. The protozoans
2.4 Virus involvement in biofilms
Viruses are obligatory intracellular parasites and are found in communities where cells in which they live are found. Viruses are, thus, found in biofilms communities associated with the bacteria, fungi and protozoa they infect.
Many phages may produce polysaccharases or polysaccharide lyases. Some phages are also known to produce enzymes that degrade the poly-Q-glutamic acid capsule of
Many biofilms possess an open architecture with water-filled channels, which would allow the phage access to the biofilm interior [36]. As biofilms age and cells die and slough off, potential new viral receptor sites may become available. As bacteria excel at adapting to differing nutrient conditions, changes to the host cell surface could be expected with either loss or gain of possible phage receptors. A further factor which might influence phage retention within biofilms lies in the role of hydrophobic and electrostatic interactions. In the interaction of a coliphage with both hydrophobic and hydrophilic membranes, a critical factor in the retention of the phage was its iso-electric point [37].
In complex biofilms in natural environments, eukaryotic algae may also be present [38]. Under these circumstances algal cell lysis through viral action is also possible as many viruses for algal species have now been isolated and identified [39].
3. Biofilms in respiratory tract infections
It is becoming progressively more accepted that biofilm formation is an important cause of morbidity in respiratory tract infections [40]. Biofilms may be involved in some respiratory infections, including ventilator-associated pneumonia, bronchiectasis, bronchitis, cystic fibrosis and upper respiratory airway infections [41].
3.1 Upper respiratory tract infections
Infectious diseases that affect the upper respiratory tract include otitis media, sinusitis, tonsillitis, adenoiditis, pharyngotonsillitis, adenoiditis and chronic rhinosinusitis [42]. In otitis media, infections may be as a result of both respiratory viruses and bacteria such as non-capsulated
The most cited reason for childhood visits to physicians is otitis media with effusion (OME) and is again one of the most reasons for antibiotic therapy in children. Even though OME is regarded as a sterile inflammatory process, current data using a chinchilla model suggest that viable bacteria are present in intricate communities referred to as mucosal biofilms [44]. It is interesting to know that intracellular
Biofilms were seen in the sinus tissues of 72% of patients affected by chronic rhinosinusitis and the cultured organisms identified included
3.2 Tissue-related infections
3.2.1 Cystic fibrosis (CF)
Cystic fibrosis (CF) is a protracted disease of the lower respiratory tract. The most frequent serious clinical complication in CF today is chronic endobronchial infection with
3.2.2 Cystic fibrosis with chronic lung infections
A major difficulty in this type of infection is contamination of lower respiratory secretions with the normal oropharyngeal flora, particularly as members of the normal flora (e.g.
3.2.3 Chronic obstructive pulmonary disease (COPD)
The role of biofilms in patients with COPD has not been directly validated but has been hypothesized considering the evidence showing that the respiratory tracts of these patients are frequently colonized by pathogens. Murphy and Kirkham [50] have recently confirmed that biofilms do play a role in COPD where they identified major outer membrane proteins of Non-typeable
3.2.4 Non-cystic fibrosis bronchiectasis
In bronchiectasis not due to cystic fibrosis, infections result in changes in the muscular and elastic components of the bronchial wall, which become distorted and expanded. Airways gradually become unable to clear mucus, leading to serious lung infections, which in turn cause more damage to the bronchi [52]. Recently biofilm formation has been demonstrated
3.2.5 Bronchitis
Prolonged bacterial bronchitis may be caused by chronic infections of the respiratory tract. In children especially, the condition appears to be secondary to impaired mucociliary removal that produces an environment favorable for bacteria to become established, usually in the form of biofilms. The most commonly involved bacteria include
3.2.6 Diffuse pan-bronchiolitis
Diffuse pan-bronchiolitis (DPB) is an unusual inflammatory lung disease of unknown etiology found in adult Japanese patients. With this disease, chronic endobronchial infection with
3.3 Device-related infections
In device-related infections such as ventilator-associated pneumonia (VAP), biofilms result in microbial persistence and reduced response to treatment. Biofilm formation within the first 24 h after intubation has been reported in 95% of endotracheal tubes [57]. Pathogens in both endotracheal tube biofilm and secretions accrued within the airways/endotracheal tubes in 56 to 70% of patients with VAP have been reported.
3.4 Biofilm forming organisms associated with respiratory tract infections
This section presents the role of biofilms in respiratory tract infections, with specific emphasis on the biofilms formed by
3.4.1 Biofilms formed by Pseudomonas aeruginosa
3.4.2 Biofilms formed by Staphylococcus species
The adherence of
3.4.3 Biofilms formed by Haemophilus influenzae
Non-typeable
3.4.4 Biofilms formed by other microorganisms
4. Quorum sensing
In the control of microbial infections, two strategies are normally envisaged; killing the organisms or attenuation of the organisms’ virulence such that they fail to adapt to the host environment. The former approach is what is generally favored; the latter lacks specific targets for rational drug design. It has, however, been realized that Gram-negative bacteria use small molecules known as acyl homoserine lactones to regulate the production of secondary metabolites and virulence factors, and this could offer a novel target to address the strategy of attenuating the organisms’ virulence thereby impairing their adaptation to the host system. Recent research has highlighted the importance of cell-to-cell interactions or communications, referred to as Quorum Sensing (QS), in microorganisms. Many bacterial species employ a complex mechanistic communication system to transmit information among themselves. Bacteria can act in response to a variety of chemical signals produced by the same species along with others produced by other species, and this provides a way for intraspecies and interspecies cross-communication and interruption of signals. The ability of bacteria to dispatch, pull together, and process information allow them to act as “multicellular” organisms and enhance their survival in complex environments [82].
Any mechanism capable of disrupting QS signals can be used to reduce survival of the microorganism thereby preventing or reducing virulence in the host environment. Such methods of interruption of the QS include:
Disruption of biosynthesis of signal molecules,
Application of QS antagonists (e.g. use of extracts from higher plants and algae and other chemical compounds),
Chemical inactivation of quorum sensing signals,
Biodegradation of signal molecule.
Agents capable of inhibiting the growth of microorganisms or disrupting the quorum sensing mechanisms of the microorganisms or interrupting the biofilm formation may be useful in the fight against microbial pathogenicity.
4.1 Anti-quorum sensing activity
It has now become apparent that different types of microorganisms have evolved the ability to recognize and act in response to the presence of other microorganisms in their neighborhood. Most Gram-negative bacteria produce and respond to
The unpleasant side effects of antibiotics (such as ototoxicity and nephrotoxicity associated with the aminoglycosides) have led to preference for preventive rather than curative approach towards fighting infectious diseases. Inhibition of quorum sensing activity has been hypothesized as one approach that can be useful in preventing bacterial infection. It could provide an additional approach to antibiotic mediated bactericidal or bacteriostatic activity thereby reducing the risk of successful establishment of infections or resistance development in the bacteria. This is supported by the protective effect of QS inhibition demonstrated in animal infection models. A simple animal infection model on QS was launched in
Many bacteria produce AHL molecules in response to QS and so could be used as biomonitor organisms in screening of compounds for anti-QS activity. Such bacteria include
5. Medicinal plants with biofilm inhibition activity
Natural products have been identified to inhibit biofilm formation in microorganisms. The exact mechanism for most of the agents is yet to be elucidated. Medicinal plants have been identified as rich source of bioactive compounds that have the capability of interfering with biofilm formation but most of these studies are still in the early stages of drug development. The anti-biofilm effects of medicinal plants have been proposed to be due to the inhibition of formation of polymer matrix, suppression of cell adhesion and attachment, interruption of extracellular matrix formation and reduction in virulence factors production and activation, thereby blocking QS network and biofilm development [85].
Medicinal plants belonging to various plant families reported to have biofilm inhibitory activity are listed in Table 1; the part of the plant (leaves, fruits, stem bark, rhizome) used, the various solvents used for extraction and their ability to inhibit cell adhesion or to eradicate biofilm formed by different pathogens have been mentioned.
Plant name | Family | Part used | Solvent | Biofilm inhibition activity | Reference |
---|---|---|---|---|---|
|
Lythraceae | Fruit | Methanol | Inhibit biofilm formation in |
[86] |
|
Lamiaceae | Aerial parts | Ethanol | Inhibit biofilm formation by 60.9% at 0.78 mg/mL | [87] |
|
Ericaceae | Fruit | Decoction | Reducing 47% MRSA biofilm viable counts. 12.5 mg/mL | [88] |
|
Commelinaceae | Whole plant | Distilled water | Inhibited the biofilm formation at 250 μg/mL | [89] |
|
Zingiberaceae | Rhizome | Aqueous | Removed 30 to 40% of biofilm at 5–0.63 μg/mL | |
|
Euphorbiaceae | Aerial parts | Methanol | Biofilm inhibition and eradication activity against |
[90] |
|
Combretaceae | Dried fruit | Ethanol | Inhibition biofilm formation by 89.8 and 92.2% at 125 and 250 μg/mL, respectively | [91] |
|
Meliaceae | Leaf | Distilled water | Reduced biofilm completely by 35% at 5% w/v | [92] |
|
Burseraceae | Stem bark | Distilled water | Inhibition of cell adhesion above 80% at 4.0 mg/mL | [93] |
|
Fabaceae | Fruit | Distilled water | Inhibition of biofilm formation was determined to be 77.8 ± 5.0% at 4.0 mg/mL | |
|
Theaceae | Leaves | Ethanol | Inhibited the cell adhesion by 78.7% 0.5%w/v | [94] |
6. Conclusion
Combatting biofilm and quorum sensing is a good strategy to reduce microbial pathogenicity and thus fight infections. This can be achieved by finding effective agents that can inhibit biofilm formation and disrupt quorum sensing mechanisms. Natural products particularly medicinal plants are a rich source of bioactive compounds that have served as useful leads in the development of drugs. Rigorous evaluation of medicinal plants can therefore lead to novel anti-biofilm and anti-quorum sensing agents.
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