Bacteriocins produced from different sources and exhibit antimicrobial and antibiofilm activity.
The spread of multidrug-resistant pathogens together with the development of fatal cases of infectious microorganisms is on the rise. Therefore, there must be new approaches for combating pathogenic microorganisms, either by overcoming antibiotic resistance or via inhibiting their virulence factors. Several virulence factors extremely increase the antimicrobial resistance of various species of pathogens; as a result, the screening of antivirulence agents has gained more and more attention recently. In this aspect, non-traditional strategies that are considered promising in overcoming virulence and pathogenicity of microorganisms will be discussed including; quorum sensing inhibition, antibiofilm, control of the global regulators, bacteriocins and bacteriophages. Applying these methods could provide innovative approaches for competing microbial resistance and virulence.
- bacterial virulence
- quorum sensing inhibition
- global regulators
- phage therapy
- inhibition of biofilm formation
The high incidence of microbial resistance and the spread of multidrug-resistant and pan drug-resistant pathogens have been developed to threaten human mankind. Fortunately, there are upcoming alternative therapeutic approach for eliminating bacterial virulence and host-pathogen interaction [1, 2]. Quorum sensing signals [3, 4] and global regulators represent the main players to control virulence circuits and coordinate host-pathogen interaction . Thus, targeting these regulators provide a promising trend to overcome microbial pathogenicity. Bacterial cells have the ability to grow in matrices of polysaccharides, proteins and DNA forming biofilm . The cell communities inside the biofilm matrices are highly resistant to antibiotics . In this chapter, we will focus on the agents that are known to exhibit antibiofilm assembly including bacteriocins.
Moreover, bacteriophages have specific ability to infect and lyse bacteria . Hence, phage therapy has many potential applications in the treatment of infectious diseases, with high therapeutic index and diminished adverse effects [9, 10]. Inhibitors of quorum sensing signaling, control of the global regulators, and the development of antibiofilm agents will be discussed in detail in this chapter. Additionally, the use of bacteriophages either for eradication of bacterial infections or as an efficient delivery system for antimicrobial agents will be described in this part.
2. Control of microbial virulence and resistance
2.1 Quorum sensing inhibition
Quorum sensing (QS) is a cellular signaling system, which is developed in response to population cell density [3, 4]. QS cascade relays on the release of signaling molecules called QS autoinducers/signals. The QS signals are produced at low levels with the start of microbial growth and accumulate upon increase in the cell density. Quorum sensing signals coordinate the microbial virulence behaviors such as secretion of toxins, secretion of exoenzymes, microbial motility, adhesion and biofilm assembly . Furthermore, microbial communication systems have been assigned in fungi  and viruses . Studies of QS provide significant insights into different mechanisms that control the interactions in microbial communities and how these interactions affect microbial pathogenesis. Several QS systems are well understood including Gram-negative bacteria that produce acyl-homoserine lactone (AHL) signals, including
Various strategies for quorum sensing inhibition have been explored. The quorum sensing inhibition approaches could be accomplished via interference with the synthesis of QS signals, elimination of the signal accumulation and disruption of signal-receptor interaction [17, 18, 19].
2.1.1 Interference with the synthesis of the autoinducing signals
One of the main quorum sensing inhibiting approaches is the interference with the synthesis of the autoinducing signals . AI-2 compounds are considered as “universal” signal molecules of Gram-negative and Gram-positive bacteria [14, 21]. Moreover, they are encountered in species communications. The biosynthesis of AI-2 requires two main enzymes: methylthioadenosine/S-adenosylhomocysteine nucleosidase (MTA/SAH nucleosidase) and LuxS. AI-2 molecules contribute in various virulence behaviors, biofilm formation and host-pathogen interaction. Therefore, targeting AI-2 elaborates broad spectrum quorum sensing inhibition [22, 23]. In this instance, Gutierrez group have identified the transition analogs, 5′-methylthio- (MT-), 5′-ethylthio-(EtT-) and 5′-butylthio- (BuT) DADMe-immucillin, which specifically bind and inhibit MTA enzymes in
On other instance, inhibiting AHL-synthesis has been extensively studied, for instance, triclosan inhibited both N-3-oxo-dodecanoyl-l-homoserine lactone and N-butyryl-l-homoserine lactone [27, 28], anthranilate derivatives are a
2.1.2 Elimination of the QS signals accumulation
Other common strategy is eliminating the accumulation of the QS signals, which have been attained by degrading the QS signal using enzymes or through sequestering the signal by synthetic polymers [30, 31] or utilizing antibodies that bind with the signals. Synthesized monoclonal antibodies (AP4-24H11) by Park group provoke high binding affinity for sequestering AIP-IV and decrease α-hemolysin production in
Moreover, disturbing enzymes responsible for biosynthesis of QS signals is a chief method, which affects both production and accumulation of different signals and perturb quorum sensing circuit . Acylases, lactonases and oxidoreductases are the widely identified enzymes that target AHLs. AHL lactonases are broad AHL degrading enzymes, which produce its effect via hydrolyzing the ester bond of the AHL ring . Lactonases have been isolated from various
AHL acylases enzymes (
2.1.3 Elimination of the QS signal-receptor interaction
Interference with signal detection through eliminating the QS signal-receptor binding represents a successful approach [53, 54]. Various synthetic and natural AHL analogs have been reported to block the binding of the signal with specific receptors in
The synthetic furanone derivative C-30 interferes and hinders the interaction of AHLs with the receptors . Other furanone analogs have been developed including S-phenyl-l-cysteine sulfoxide and diphenyl disulfide  and tetrazole derivatives . Furthermore, synthetic LasR derivatives have been developed such as indole derivatives, non-AHL-like antagonists , the synthesized azines derivatives, 4-(alkyloxy)-6-methyl-2H-pyran-2-one  and aspirin . Triphenyl hybridγ-butyrolactones and cyclopentanones derivatives are potent inhibitors of LuxR . Putative LasI inhibitors have been identified using molecular docking methods including the trans-cinnamaldehyde , (z)-5-octylidenethiazolidine-2, 4-dione  and fatty acyl purified from marine
2.2 Control of the global regulators
Beside the QS regulons, other global regulators exhibit crucial functions in dominating the expression of various genes in assortment style as a response to environmental stimuli and changes, most notably the temperature change . These so-called global regulators enable the bacterial communities to survive different environmental stresses including starvations, pH changes and temperature fluctuations, through the quick conformation of bacterial physiology and structure .
Among many regulators that coordinate gene expression in bacteria, in Gram-negative bacteria, the global regulator termed histone-like nucleoid-structuring (H-NS) protein is relatively significant and of paramount importance . H-NS has been considered as the main model of studying how global regulators can affect bacterial structure and physiology. The H-NS protein is incorporated in the regulation of many genes responsible for controlling the physiological functions of Gram-negative bacterial cells involving cellular functions, survival under different environmental conditions and production of various virulence factors [81, 82]. Moreover, in Gram-positive bacteria, there are several global regulatory loci . Among them in the
This would open up novel approaches for the treatment and eradication of pathogenic bacteria utilizing inhibitors or modulators of these global loci to vanquish the global concerns of antimicrobial resistance and immune evasion of microbial pathogens. Among these approaches, the interesting inhibitor of SarA (SarABI), 4-[(2,4-diflurobenzyl)amino] cyclohexanol, was confirmed as SarA-based new curative medicament against
2.3 Biofilm inhibition and eradication
Biofilm is a sessile community of microbial cells that is found to be attached to animate or inanimate surface, and usually surrounded by a matrix of polysaccharides, proteins and DNA . The cells in these sessile communities differs phenotypically form those present in planktonic communities . Bacterial cells in planktonic forms are almost one thousand times more sensitive to antibiotics than their biofilm counterparts . Additionally, biofilms act as a defense mechanism against different stress conditions or immune cells attack .
In this part, we will focus on the agents that are known to exhibit antibiofilm activity.
2.3.1 Antimicrobial peptides
Antimicrobial peptides (AMPs) that are crucial players of innate immunity are reported to prevent biofilm formation in different pathogens. AMPs with antibiofilm activity are either natural or synthetic. The human cathelicidin peptide, LL-37, has been demonstrated to have antibiofilm activity in case of
Another synthetic peptide, S4(1–16) M4Ka, has been found to inhibit biofilm formation and detach bacterial cells in
The natural AMP piscidin-3, obtained from fish, exhibits nucleosidase activity and can degrade extracellular DNA of
The anionic surfactant, sodium dodecyl sulfate, has been reported to destruct biofilm via enhancing the formation of central cavity within biofilm . Cetyltrimethylammonium bromide (Catanionic surfactant), together with application of high shear stress, increased the detachment of
2.3.3 Free fatty acids
Free fatty acids obtained via hydrolysis of lipids by enzymes . Certain members of free fatty acids are reported to exhibit antibiofilm activity . For example, cis-2-decenoic acid from
2.3.4 Metal chelators
Removal of metals from the microbial environment via metal chelators renders bacteria more susceptible to antimicrobial agents, as metals are essential for different cellular processes . Ethylenediaminetetraacetic acid (EDTA), the most-known metal chelator, has been reported to exhibit antibiofilm activity against
Based on their target, the antibiofilm enzymes are classified into three types: polysaccharide-degrading enzymes, nucleases and proteases.
22.214.171.124 Polysaccharide-degrading enzymes
Alpha amylase enzyme was found to inhibit biofilm formation by
126.96.36.199 Nucleases enzymes
Deoxyribonuclease I (DNase I) degrades DNA in biofilm matrix . Moreover, it was shown to have antibiofilm activity and to detach the biofilms produced by different bacterial species . Such nuclease can prevent the initial adherence of microbial cells to surfaces via the degradation of cell surface-associated nucleic acids that act as surface adhesins . Furthermore, DNase I has been found to increase the sensitivity of bacterial cells in biofilm matrix to antibiotics, resulting in reduction of biofilm mass .
Proteases act as antibiofilm agents because they are able to inhibit cell-cell communication, in biofilms, via hydrolysis of extracellular protein fibers and surface adhesins . Subtilisins, a class of serine proteases produced by Bacillus species, were reported to prevent the adherence of microorganisms to surfaces . The coating of silicone surfaces with multiple layers of amylase or acylase has been found to inhibit biofilm formation in case of
2.3.6 Amino acids
d-Amino acids have been shown to inhibit biofilm formation in
2.3.7 Nitric oxide generators
Exogenous generation of nitric oxide (NO) by agents, for example, sodium nitroprusside has been shown to trigger the bacterial growth from the biofilm form to the planktonic form via the reduction of the level of cyclic di-GMP inside the bacterial cells . Further NO-generators, for example, S-nitroso-N-acetyl penicillamine and S-nitroso-l-glutathione were found also to induce the dispersion of
2.3.8 Natural agents
Alkaloids are a group of natural organic compounds that contain a nitrogen atom and are present in different species of plants. The alkaloid berberine has been reported to inhibit biofilm formation in
Guaijaverin, a flavonoid obtained from the leaves of
Terpenes are a large class of natural hydrocarbons that are synthesized in microorganisms, plants and animals. Bakuchiol, isolated from the seeds of
Bacteriocins are proteins or peptides that are produced by bacteria or archaea, and are usually active against strains of bacteria that are related or unrelated to the producer strain . Several bacteriocins are reported to exhibit antibiofilm activity and/or antimicrobial activity. The results of some these reports are summarized in Table 1.
|Bacteriocin||Source||Antimicrobial activity||Antibiofilm activity|
|Mutacin 1140||Oral biofilm-associated with |
|Epidermicin NI0||MRSA, |
2.5 Phage therapy
Phage therapy, which is also termed viral phage therapy, is the utilization of bacteriophages as medicaments for controlling and treating diseases brought by pathogenic bacterial infections . Bacteriophages, like other viruses, are obligate intracellular parasites that utilize the enzymatic machinery of their hosts for establishing their physiological functions and replication . The hosts for bacteriophages are bacteria, and phages have unique ability to specifically infect bacterial hosts resulting in their lysis .
There are many conceivable usages for phage therapy in the treatment of crucial diseases in plants, animals as well as human [8, 145]. An outstanding advantage of utilizing bacteriophages over commonly used antibiotics, during treating infectious diseases, is their selectivity and specificity to infect and lyse infectious bacteria only without harming the host . Besides, bacteriophages cause no harm to other organisms that live in a commensalism within hosts, such as the normal flora in human, which decreases significantly the incidence of superinfections or other opportunistic infections . Moreover, due to their mode of action that phages replicate
Historically, the first trials for the utilization of bacteriophages as medicaments for treating bacterial pathogens was reported in the Eastern world before the discovery of marvelous medicaments so-called antibiotics; however, there was any report of their usage in the Western world [150, 151]. The ability of bacteriophages to infect and lyse pathogenic bacteria was discovered by the scientists Frederick Twort and Felix D’Hérelle, who worked on
It has been estimated that there are more than 100 different phage species and at least 10 phages for each bacterium. The International Committee for the Taxonomy of Viruses (ICTV) was affirmed at 1971 with the objective to always bring to date the taxonomic guidelines of viruses. The ICTV classified tailed bacteriophages (bacterial infecting phages) under the order of viruses which is termed
Compared with antibiotics and other therapeutic regimens, the steps and cost of production of bacteriophages are much easier and cheaper, respectively . The easiest process for capturing of bacteriophages is done through collecting samples that seem to involve high titers of phages like sewage water samples. The collected samples are inoculated with the host bacterium, which seems to be infected by phages, on suitable growth medium. The successful isolation of certain lytic phage is depicted by the presence of clear inhibition zones in which bacteria cannot grow termed plaques; which indicates the lytic power of the isolated phage. Thereafter, the titer of isolated phage is increased by passing the phage in its specific bacterial strain several times to increase its concentration. Then, the pure supernatants containing phages are gained by centrifugation of bacterial/phage mixture, filtered through bacterial filters to remove any bacterial debris and pure phages are participated using special solutions containing NaCl and polyethylene glycol 8000 (PEG8000) .
Caution must be given during isolation of phages as a type called lysogenic bacteriophage may be isolated rather than the required bacterial pathogen killing type, which is called lytic bacteriophage. Lysogenic bacteriophages do not lyse bacterial cells, but they perform as tools for transfer of genetic elements of the nucleic acid between bacteria; including the genes responsible for antibiotic resistance. Fortunately, the most abundant phages are of the lytic type not the lysogenic [8, 145, 150].
Practically, bacteriophages can be dispensed and used through many routes including; less commonly oral or systemic route and most commonly topical route as sprays, liquid solutions or their application on surgical dressings for the treatment of wound infections . The possibility of their clearance during the presence in blood stream by immune system or presence of any trace hazards of chemicals or parts of the bacterial host used during their production, made bacteriophage usage as intravenous injections uncommon and very rare [148, 149]. Lyophilization of bacteriophages and their production as solid dosage forms as pills or tablets do not decrease their potency and increase their shelf life as oral dosage forms [155, 156]. The supplementation of oral forms of phages, either solid or liquid, with antacid increases its stability, as it protect them from the high acidity during their bypassing in the stomach [155, 156].
The application of bacteriophages as therapeutic medicaments has been extensively reported. For example, in the field of human health promotion and food protection, different bacteriophages have been employed to eradicate common bacterial pathogens that may cause food spoilage as
A more recent policy, termed enzybiotic, for using phages as therapeutic agents is the utilization of their enzymes only, which are produced by recombinant technology, combined with other antibacterial agents or as a separate antibacterial agents .
As other therapeutic regimens for controlling bacterial pathogens, the patients may develop extensive fever and shock, when the bacteria are lysed due to the release of what is called pyrogens or endotoxins within the patient . This problem can be coped during phage therapy through the utilization of genetically modified phages that harbor enzymes having the ability to lyse these endotoxins and the other bacterial structures into harmless products .
Examples of therapeutic approaches of bacteriophages and their enzymes are illustrated in Table 2.
|Infection/disease||Model||Causative agent||Route of administration of phages/enzymes||Treatment outcomes||Reference|
|Chronic otitis||Human||Oral administration of phages||Successful treatment|||
|Typhoid||Human||Oral administration of phages||Successful treatment|||
|Diabetic foot ulcer||Human||Topical application of phages||Successful treatment|||
|Sepsis||Murine||Intraperitoneal and oral administration of phages||Successful treatment|||
|Pneumonia||Murine||Intranasal administration of phages||Successful treatment|||
|Ulcers and wounds||Human||Topical application of phages||Successful treatment|||
|Meningitis||Murine||Intraperitoneal or subcutaneous administration of phages.||Successful treatment|||
|Sepsis||Murine||Intraperitoneal administration of phages||Successful treatment|||
|Bacteremia||Murine||Intraperitoneal administration of phages||Successful treatment|||
|Ileocecitis||Hamster||Oral administration of phages||Successful treatment|||
|Dysentery||Human||Oral administration of phages||Successful treatment|||
|Cholera||Human||Oral administration of phages||Successful treatment|||
|Pneumonia||Murine||Intraperitoneal administration of Cpl-1 lysin enzyme||Successful treatment|||
|Bacteremia||Murine||Intraperitoneal administration of PlySs2 lysin enzyme||Successful treatment|||
|Application of PlyG lysin enzyme||Significant reduction in bacterial density|||
|Endophthalmitis||Murine||Application of Ply187 lysin as eye drops||Successful treatment|||
|Bacteremia||Murine||Administration of PlyF307 lysin enzyme||Successful treatment|||
|Application of ABgp46 lysin enzyme||Significant reduction in bacterial density|||
Various approaches have been developed for competing microbial virulence and resistance. Quorum sensing signals and global regulators play an essential role in controlling the gene expression of virulence factors, and the expression of proteins required for adaptation to environmental and stress condition. Therefore, control of these regulators will stop the microbial pathogenicity. In addition, biofilms act as a defense mechanism against host immunity and antimicrobial therapy. Natural and synthetic compounds have approved activities in eradication of biofilm formation. Besides, phage therapy, which is currently successful in destruction of bacterial pathogens that do not respond to conventional antimicrobials. These methods would open up new perspectives for management the up growing problem of microbial resistance. Further,