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Biofilms are essential virulence constituents of mycobacteria and thus there exists a need
to develop a greater information base regarding biofilms and Mtb., as well as how biofilm
formation and maturation can be inhibited using plant-based bioactives and natural
products.
The process of quorum sensing, as illustrated in
Fig. 9.3, relies on the presence and
cooperation of three individual mechanisms; the production of autoinducer molecules,
the subsequent recognition of autoinducer molecules via a m embrane-bound or cyto-
plasmic receptor, as well as the effecti ve gene response to autoinducer stimuli (
Antonioli
et al., 2019
). Cell density thresholds are indicated by the concentration of the autoinducer
molecule within the extrace llular environment. Once the autoinducer molecule reaches a
particular threshold concentration, coordinated behaviors are activated, resulting in
population-dependent changes in gene expression, including biofilm formation and viru-
lence factor production. Mai ntenance of the dynamic equilibrium in homogenous or het-
erogeneous bacterial populations relies on these intricate cell-to-cell communication
strategies that ensure peaceful cohabitation of the ecological niche in question (
Antonioli
et al., 2019
).
The petroleum ether fraction showed the best activity and was further fractio nated and
purified by using bioassay-guided isolation to yield the very potent compound 1 biofilm
inhibitor (E)-2-(methyl (phenyl) amino) ethyl 2-(2-hydroxyundecanamido)-7, 11-dimethyl-
3-oxotetradec-4-enoate, with an IC
50
of 4-32 μg/mL (Jiang et al., 2019). Various biofilm
parameters were investigated; namely, the ability of the extract and compound 1 to inhibit
biofilm formation, disrupt mature biofilms, and disperse preformed mycobacterial bio-
films, all of which are essential components of the dynamic nature of biofilms. At a con-
centration of 4 μg/mL, compound 1 was able to successfully inhibit the various
mechanisms by which mycobacteria form biofilms thus decreasing the overall biofilm
biomass (
Jiang et al., 2019).
FIGURE 9.3 A schematic representation of quorum sensing in both Gram-negative (A) and Gram-positive
bacteria (B). Autoinducer molecules are synthesized intracellularly and transported to the extracellular environ-
ment. When high enough concentrations of the autoinducer molecule are present in the extracellular environment,
receptor binding and subsequent signal transduction occur. This results in the induction of gene expression cas-
cades. Source: From Paul, D., Gopal, J., Kumar, M., & Manikandan, M. (2018). Nature to the natural rescue: Silencing
microbial chats. Chemico-Biological Interactions, 280,8698.
https://doi.org/10.1016/j.cbi.2017.12.018.
314 9. Mycobacterial quorum quenching and biofilm inhibition potential of medicinal plants
Medicinal Plants as Anti-infectives
Quorum sensing versus quorum quenching
Bacterial cells are frequently known to recognize the cell density of their population
and to regulate their gene expression accordingly. This process, known as quorum sens-
ing, encapsulates the notion that when it comes to coordinated cellular strategies, it will be
more fruitful to activate such mechanisms when the population is of a particular size or
density threshold, resulting in a greater and more effective response (
Antonioli, Blandizzi,
Pacher, Guilliams, & Hasko
´
, 2019
). The process of quorum sensing, as illustrated in
Figure 9.3, and relies on the presence and cooperation of three individual mechanisms; the
production of autoinducer molecules, the subsequent recognition of autoinducer molecules
via a membrane-bound or cytoplasmic receptor as well as the effective gene response to
autoinducer stimuli (Antonioli et al., 2019). Cell density thresholds are indicated by the
concentration of the autoinducer molecule within the extracellular environment. Once the
autoinducer molecule reaches a particul ar threshold concentration, co-ordinated beha-
viours are activated, resulting in population-dependent changes in gene expression includ-
ing biofilm formation and virulence factor production. Maintenance of the dynamic
equilibrium in homogenous or heterogeneous bacterial populations relies on these intricate
cell-to-cell communication strategies that ensure peaceful cohabitation of the ecological
niche in question (
Antonioli et al., 2019).
Novel treatment modalities that aim to interfere with the quorum sensing process have
attained much attention as of late. The inhibitory process is known as quorum quenching;
an attractive target for the inhibition of bacterial virulence (
Paul et al., 2018). Many patho-
genic organisms that infect humans, animals, and plants have been shown to regulate their
virulence via effective quorum sensing strategies (
Paul et al., 2018). Innovative quorum
quenching agents should thus be able to selectively inhibit the process of quorum sensing,
preventing the appearance of undesirable bacterial phenotypes. With further research and
development, quorum quenching has the ability to hold great promise as a new avenue in
treating bacterial and fungal infections and the various population-dependent responses
that these cells exhibit (
Tegos & Hamblin, 2013).
Biofilms
Background on biofilms
Although planktonic cells can move about freely, this level of exposure makes the bacil-
li susceptibl e to the environmental conditions in which they reside. To increase the likeli-
hood of survival, bacteria form biofilms that reduce the level of exposure by creating a
highly organized three-dimensional matrix consisting of sessile cells that are encapsulated
within an extracellular polysaccharide structure. The formation of a biofilm relies on the
random collision of planktonic cells and the subsequent attachment to a surface (biotic or
abiotic) or to one another via a host of cellular appendages such as flagella and pili
(
Shirtliff, Mader, & Camper, 2002). Following attachment, the quorum sensing abilities of
the bacteria are activated in an attempt to accelerate the maturation of the biofilm and to
enhance cell to cell communication for coordinated responses (
Shirtliff et al., 2002).
315Biofilms
Medicinal Plants as Anti-infectives
The maturation of the biofilm involves the secretion of greater volumes of the extracel-
lular polymeric substance (EPS) and the generation of nutrient channels within the matrix.
This complex configuration allows bacterial populations to maximize nutrient uptake and
the subsequent retention of such nutrients within the biofilm (
Shirtliff et al., 2002).
Furthermore, biofilm formation often occurs in nutrient-dense locations thus making it a
highly efficient and capable dynamic structure concerning nutrient acquisition. Another
advantage of biofilm formation is that of resistance to physical detachment by shear stress.
Due to the extensive adhesive interactions between bacteria and the matrix, bacterial cells
are in essence anchored within the biofilm. Thirdly and most medically relevant is the
inherent ability of bacterial cells in biofilms to resist penetration by antimicrobial sub-
stances as well as the ability to avoid consumption via phagocytic processes (
Karami et al.,
2020
). This subsequently enables bacterial persistence and the generation of chronic infec-
tions (
Zhang, 2014). A diagrammatic illustration of the process of biofilm formation and
maturation is illustrated in
Fig. 9.4.
The mechanisms by which bacterial cells within a biofilm acquire antibiotic resistance
can be classified into two main categories, namely, the limited accessibility of cells in a
biofilm to the environment and secondly the acquisition of genetic material conferring
antibiotic resistance. Planktonic cells are naturally more exposed and are thus more sus-
ceptible to clearance via antimicrobials and immunological mechanisms. Due to their
diminished exposure to the environment, bacterial cells within a biofilm resist penetration
by antibiotics via the protective ability of the extracellular matrix and the presence of
efflux pumps which increase the minimum inhibitory concentration (MIC) value and pre-
vent drug accumulation within the biofilm and individual cells (
Khaledi et al., 2016).
Furthermore, in the case of TB, alveolar macrophages have limited access to bacterial cells
within a biofilm thus hindering this immunological defense strategy. Although polysac-
charides have been shown to form the core component of the extracellular matrix, proteins
and exogenous DNA are also present in biologically significant concentrations (
Khan,
Jeong, Park, Kim, & Kim, 2019
). This exogeno us DNA exists in many forms such as trans-
posons and plasmids; more importantly, however, is the potential presence of antibiotic
FIGURE 9.4 Schematic representation of the process of biofilm formation. Source: From da Silva, D.P.,
Schofield, M.C., Parsek, M.R., & Tseng B.S. (2017). An Update on the Sociomicrobiology of Quorum Sensing in Gram-
Negative Biofilm Development. Pathogens 2017, 6, 51. (CC BY-SA 4.0). Accessed January 2022.
https://doi.org/10.3390/
pathogens6040051
.
316 9. Mycobacterial quorum quenching and biofilm inhibition potential of medicinal plants
Medicinal Plants as Anti-infectives
resistance genes contained within these mobile genetic elements. Uptake of this exogenous
DNA is a possibility. However, bacteria rapidly regain their susceptibility to antibiotic
treatment after exiting the biofilm and thus the transfer of antimicrobial resistance genes is
less of a contributor to overall resistance than what was previously thought (
Stewart &
Costerton, 2001).
Furthermore, bacteria possess the ability to physiologically dissociate from the matrix in
an attempt to develop additional colonies and biofilms within the host. This ability is criti-
cal in ensuring the sustainability of the species as biofilms are sessile structures that cannot
evade changes in environmental conditions. Disassembly and dispersal of the biofilm are
imperative in the innate ability of bacterial cells within a biofilm to relocate when local
conditions deteriorate (
Boles & Horswill, 2011).
Biofilms and Mycobacterium tuberculosis
The genus Mycobacterium includes both pathogenic species belonging to the Mtb complex,
and environmental nontuberculous species. The genus Mycobacterium (family
Mycobacteriaceae), currently includes more than 170 recognized species (
Forbes, 2017).
Mycobacteria have a common pathogenic factor being the formation of extracellular, poly-
meric matrices called biofilms. Biofilm formation is mainly influenced and regulated by the
availability of nutrients, ions, and carbon sources. The first report of the concept of biofilms
dates back to 1978 when initial observations were published (
Costerton, Geesey, & Cheng,
1978; Esteban & Garcı
´
a-Coca, 2018
). Koch (1982) described the appearance of “cells which
are pressed together and arranged in bundles.” More scientific papers on the topic began to
surface about a decade later, while mycobacterial cells forming aggregates or pellicles were
described in earlier days of mycobacteriology (
Calmette, 1936; Loo
¨
wenstein, 1920).
Mycobacterial biofilms are defined in the same way as any other biofilms. Some mycobac-
teria, however, have the ability to form biofilm structures on liquidair media interfaces and
exhibit sliding motility on agar surfaces (
Ojha et al., 2008). The fast-growing nonpathogenic
M. smegmatis is a model organism that has been extensively studied for mycobacterial biofilm
formation (
Chakraborty & Kumar, 2019; Danese, Pratt, & Kolter, 2000). Biofilm development
starts with the bacterial adhesion and progresses through the different stages of surface attach-
ment, sessile growth, biofilm maturation, and dispersal. Adhesins from the bacterial cell wall
mediate the initial attachment of bacteria to the surfaces which is an important virulence trait
of microbial pathogenesis. Following attachment to the surface, sessile bacteria initiate the syn-
thesis of extracellular matrix which is composed of several glycopeptides, DNA, and other
molecules. Mycobacteria lack surface fimbriae or pili and do not produce the usual exopoly-
saccharide components of extracellular matrix but can attach to different surfaces and form
developed biofilms (
Esteban & Garcı
´
a-Coca, 2018).
Research studies have been conducted on biofilm formation of several mycobacterial
species to better understand key components needed for its formation and how it is used
for bacterial survival. A study conducted on different species of rapidly growing mycobac-
teria revealed that biofilm development follows a sigmoid growth kinetic. This study’s
findings were later confirmed in clinical strains. Nutrients, carbon sources (such as glucose
and peptone), and ions (Ca
21
,Mg
21
,Zn
21
) are known to play an influential and
317Biofilms and Mycobacterium tuberculosis
Medicinal Plants as Anti-infectives
regulatory role in bacterial behavior and biofilm formation. The study also indicated that
tap water, as the nutrient source, can allow biofilms to form. This provides an explanation
as to why mycobacterial biofilms can be found in water sources (
Esteban & Garcı
´
a-Coca,
2018; Esteban et al., 2008
).
Different molecules in the formation of biofilms and their composition have been exam-
ined. In the well-studied mycobacterial model, Mycobacterium smegmatis, glycopeptidoli-
pids are important for the initial surface attachment (
Recht & Kolter, 2001). The same
molecules have been shown to play a role in sliding motility, a property that several myco-
bacterial strains have that may be, although not always related, to biofilm spreading on
surfaces. Shorter chain mycolic acids play a role in the structure of biofilms and are pro-
posed to form a hydrophobic extracellular matrix. Mycolic acids are associated with higher
resistance to disinfectants and antibiotics associated with these microorganisms. The
mycolic acids found in the cell wall provide a permeability barrier. Other macromolecules
such as GroEL1 chaperones play a role in biofilm development in M. smegmatis. The com-
plexities of mycobacterial biofilm structure and development are continuously being inves-
tigated for a better understanding on its clinical impact and how to deal with its
occurrence (
Sharma, Misba, & Khan, 2019).
Mycobacterial biofilms harbor an extensive, drug-tolerant population of cells. The EPSs pro-
duced by mycobacteria disrupt the diffusion of antimicrobials throughout the matrix, thus
protecting individual bacterial cells from exposure (
Solokhina, Bonkat, Kulchavenya, &
Braissant, 2018
). This makes mycobacterial biofilms incredibly difficult to treat and enables
chronic persistence within the human body (
Ojha et al., 2008). Drug-tolerant bacteria are bac-
teria that exhibit a diminished response to the presence of high concentrations of antimicrobial
agents and transiently survive in such environments (
Crabbe
´
, Jensen, Bjarnsholt, & Coenye,
2019
). Drug-resistant bacteria are bacteria that possess the ability to significantly withstand the
effects of a drug which is usually effective against them and generally increase the MIC value
of the drug in question (
Crabbe
´
et al., 2019). The location in which mycobacterial biofilms
form is of the utmost clinical relevance. Studies by
Solokhina et al. (2018) have shown that
necrotic granulomas and caseous foci are saturated with bacterial cells, and cavities are lined
with mycobacterial biofilms despite their avascular nature. The presence of vasculature in
infectious processes is important for the hematogenous dissemination of the infectious organ-
ism. Tuberculosis bacilli have been shown to possess the ability to overcome low oxygen ten-
sion environments as seen in avascular structures despite being a strict aerobe. Not only does
this ensure their persistence, but it also ensures their inherent tolerance to antimicrobial com-
pounds that are distributed via the vasculature. Remote lesions have also been shown to
sequester an immunologically, chemically, and physically resistant population of cells which
ensure recalcitrant infection processes (
Solokhina et al., 2018).
During in vitro studies, disruption of mycobacterial biofilm formation is often achieved
with the addition of a chemical detergent which prevents the aggregation and clumping of
bacterial cells; a step critical in the formation of biofilms. Detergent-free media, on the other
hand, enables the formation of mycobacterial biofilms on the airmedia interface which is
commonly known as a pellicle (
Ojha et al., 2008).Thequestionthenarises,whynotusedeter-
gents as biofilm disrupting agents or as adjuvants in TB treatments? The answer lies in both
theeffectsthatithasonthehostaswellastheeffectithasonthebacterium.Detergentsare
generally classified as toxic to the human bodyandthustheirusageandefficacyinvivo
318 9. Mycobacterial quorum quenching and biofilm inhibition potential of medicinal plants
Medicinal Plants as Anti-infectives
would be ethically questioned. Secondly, in terms of the bacterium, detergents have been
shown to significantly alter several properties of the bacterial envelope, lipid structure, and
function as well as the permeability of bacterial cell membranes. Therefore, the reliability of
detergents to produce scientifically ethical and effective adjuvants is limited (
Ojha et al., 2008).
Mycobacterial biofilms are unique concerning the content of the EPS they produce (Ben-
Kahla & AL-Hajoj, 2016
). Research has shown that novel lipids derived from mycolic acid
as well as free mycolic acids are present in abundance in mycobacterial biofilms (
Ojha
et al., 2008
). This has important implications regarding how Mtb form biofilms as well as
how they ensure their subsequent maturation. During the late stages of biofilm matura-
tion, mycolic acids in the EPS have been shown to increase in concentration with mutants
defective in maturation potential such as the ΔgroEL1 mutant, lacking the ability to pro-
duce these lipids (
Ojha et al., 2008). Not only do Mtb biofilms differ from other bacteria
concerning the content of the EPS but they also exhibit extensive EPS variations between
Mtb bacilli themselves (
Ben-Kahla & AL-Hajoj, 2016). These variations are presumed to be
due to the various evolutionary pressures that Mtb bacilli face when occupying a host of
different ecological niches (
Ojha et al., 2008). Despite playing a critical role in the cellular
integrity of mycobacterial cells and the way in which Mtb interacts with the immune sys-
tem, mycolic acid concentration is imperative in the phenotype of the biofilm produced
with respect to its integrity and thickness (
Ben-Kahla & AL-Hajoj, 2016). Mycolic acid syn-
thesis is thus an important therapeutic target for the disruption of mycobacterial biofilms.
Conventional TB treatment is inefficient in the inhibition of mycobacterial quorum sens-
ing and the subsequent clearan ce of biofilms, prompting the development of novel thera-
pies aimed at combatting mycobacterial biofilm formation, maturation, and disassembly.
Virulence factors
Background on virulence factors
The pathogenicity of a bacterium, or the ability to cause disease within host cells, is
dependent on an array of parameters that involve the complex interactions between patho-
gen and host. One of these essential parameters is the quantitative characteristic—virulence,
which is defined as the ability to overcome several host defenses that may be initiated upon
pathogen challenge and induce varying degrees of host-cell damage (
Ufimtseva et al., 2018).
The microbial virulence of a pathogen is a complex characteristic that involves the produc-
tion and secretion of a host of virulence factors which are important regulators of pathoge-
nicity. Virulence factor secretion is yet another trait regulated by quorum sensing, as the
feasibility and success of infection is enhanced when bacteria achieve a certain threshold
population density (
Paul et al., 2018). Microorganisms produce virulence factors in varying
quantities and in response to a myriad of different environmental stimuli.
Virulence factors and Mycobacterium tuberculosis
The way in which Mtb regulates its virulence is not via the secretion of a single viru-
lence factor but rather via an extensive and complex combination of virulence responses
319Virulence factors and Mycobacterium tuberculosis
Medicinal Plants as Anti-infectives
that ensure its dynamic adaptation to host immunity and defenses (Madacki, Mas Fiol, &
Brosch, 2019
). Over the years, studies have shown that an increasing number of virulence
genes are required for survival and replication of M. tuberculosis bacilli in vivo. An initial
study by
Sassetti and Rubin (2003) showed that over 196 genes associated with virulence
were required for the survival of Mtb. Subsequent deep sequencing molecular advances
have isolated an additional 400 genes required for the effective infection of host cells by
Mtb (
Zhang et al., 2013). Although a large proportion of these virulence genes encode
functions in basic metabolism, the vast remainder of such gene products is on the front-
line combatting host immune defenses. Examples of these virulence gene products include
a host of ESX/type VII secretion systems as well as complex lipids of the cell envelope
which ensure survival as well as a high degree of virulence in the host cell (
Madacki et al.,
2019
). To examine the effects of these individual gene products, one needs to examine the
host cell and its properties and the way in which host and pathogen interact on a physico-
chemical and biological level.
The intracellular environment of host macrophages is a critical area of TB research as
mycobacterial cells are ingested by these immunological cells upon entry into the pulmo-
nary alveoli (
Madacki et al., 2019). As part of the generic immunological response, macro-
phages form a phagosome post-bacilli ingestion. The phagosome fuses with a lyso some to
produce a phagolysosome; a hostile acidic environment that is induced to clear the
infected cell of infection. Tuberculosis bacilli have developed an array of microbial viru-
lence strategies to prevent consumption by phagolysosomes and to ensure subsequent sur-
vival and replication success by delaying the formation or differentiation of such
immunological structures. Studies have shown that escape from phagosomal confinement
and the prevention of phagosomal acidification are regulated by the ESX-1 type VII secre-
tion system as mutants deficient in this system were unable to escape from the phagosome
via phagosomal rupture (
Madacki et al., 2019). This secretion system is, therefore, a critical
virulence factor during TB infection, as the ability to overcome phagosomal immune
defenses is imperative for the survival of the species within the host.
Covalently linked to the outer mycobacterial cell wall layer, phenolic glycolipids are
another example of a well-known mycobacterial virulence factor. A study by
Reed et al.
(2004
) showed that the presence of these phenolic glycolipids resulted in a hypervirulent
phenotype in a murine TB model and that all known pathogenic Mycobacterium strains
contain these essential virulence factors (
Reed et al., 2004). Furthermore, mutants deficient
in this virulence factor were found to be attenuated in guinea pig TB mo dels (
Reed et al.,
2004
). From a host immunity perspective, phenolic glycolipids were also shown to inhibit
the formation of pro-inflammatory cytokines which are critical in the formation of the
inflammatory environment (
Reed et al., 2004). If such an environment cannot be generated,
TB bacilli have a head start in their ability to overcome host responses and ultimately
secure a more efficient and long-lasting infection (
Madacki et al., 2019).
Although the protein constituents of the mycomembrane are a vastly underexplored
topic, recent studies have identified and characterized several integral proteins of the
mycomembrane that contribute substantially to their virulence (
Madacki et al., 2019). The
channel-forming protein, CpnT, contains an N-terminal exotoxin domain which has been
determined to be responsible for the ability of TB bacilli to induce necrosis in host cells
(
Danilchanka et al., 2014). Caseous necrosis is a debilitating complication of pulmonary
320 9. Mycobacterial quorum quenching and biofilm inhibition potential of medicinal plants
Medicinal Plants as Anti-infectives
mycobacterial infection and significantly alters the structural and functional integrity of
the lung tissue. Following necrosis, CpnT limits cytokine production which allows for a
silent escape and dissemination to both surrounding and distant tissues (
Madacki et al.,
2019
). Due to the severity of disease that cooperating virulence factors can induce, it is nec-
essary to develop novel pharmaceuticals that can be used to attenuate their toxic effe cts.
As a quorum sensing-regulated process, virulence factor production can be addressed by
innovative quorum quenching agents that aim to redu ce their synthesis or inhibit their
secretion. Using medicinal plants which are selected upon the basis of their ethnobotanical
and ethnopharmacological uses, effective and ethically sound medicines can be discovered
which target mycobacterial quorum sensing and subsequent virulence factor production.
Medicinal plants as quorum quenching agents
Nosocomial infections are infections that originate within the confines of the hospital
environment and are those which were not incubating prior to hospital admission.
Pseudomonas aeruginosa is an important cause of nosocomial respiratory infections espe-
cially in patients with preexisting pulmonary deficiencies (
Vandeputte et al., 2010). The
key determinant in its virulence lies in its ability to regulate communication and gene
expression via quorum sensing. A study (
Vandeputte et al., 2010) showed how leaf and
bark extracts of Combretum albiflorum (Tul.) Jongkind were able to interfere with the tran-
scription and regulation of quorum sensing genes in P. aeruginosa thereby inhibiting bio-
film formation and maturation (
Vandeputte et al., 2010). The inhibition of biofilm
formation and maturation is critical in eradicating the chronicity of such respiratory condi-
tions caused by P. aeruginosa. The bioactive compounds of C. albiflorum extract were frac-
tionated and characterized, revealing primarily flavonoids with the particular abundance
of the catechin, a flavan-3-ol derivative, which was found to be the active quorum sensing
inhibitor (
Vandeputte et al., 2010).
In another study by
Vadakkan et al. (2018), violacein which is a purple pigment pro-
duced when bacterial populations reach a certain cell density was used as a reporter mole-
cule for the visualization of quorum quenching activities of Tribulus terrestris L. root
extract. Chromobacterium violaceum produces violacein during a natural quorum sensing
response and is commonly used as a reporter strain due to its ability to visually illustrate
the results of potential quorum quenching compounds. This study found that a three times
daily exposure to 300 μg/mL of root extract was sufficient to significantly interfere with
quorum sensing gene regulation by inhibiting the production of the reporter molecule,
violacein (
Vadakkan et al., 2018).
Although the ultimate goal of research and development into medicinal plants and their
therapeutic actions is often to produce a pharmaceutical formulation, the role of the die-
tary ingestion of medicinal plants and their phytochemical constituents in quorum
quenching strategies should not be set aside. Curcumin, the major phytochemical present
in turmeric (Curcuma longa L.), has long been used in indigenous knowledge systems as a
source of antimicrobial and antiinflammatory therapeutics (
Packiavathy, Priya, Pandian, &
Ravi, 2014
). Scientific confirmation of anecdotal evidence was investigated in a study by
Packiavathy et al. (2014) whereby curcumin was investigated for its quorum quenching
321Medicinal plants as quorum quenching agents
Medicinal Plants as Anti-infectives
potential in vitro against Pseudomonas aeruginosa, using a host of various quorum sensing
processes as targets (
Packiavathy et al., 2014). These targets included biofilm formation,
swarming motility, and the production of various EPSs such as alginate, which are all reg-
ulated via quorum sensing. The study sho wed that curcumin had a substantial quorum
quenching effect on biofilms via the disruption of biofilm architecture and the dislodging
of biofilm biomass. At a concentration of 100 μg/mL, curcumin reduced biofilm biomass
by 89% in P. aeruginosa (
Packiavathy et al., 2014). Furthermore, alginate, a key component
of the EPS in Pseudomonas biofilms, was inhibited by 63%. Finally, both swimming and
swarming motility were inhibited at increasing concentrations of curcumin in a dose-
dependent manner (
Packiavathy et al., 2014). As swarming motility is a key regulator of
biofilm formation, the inhibition of such a strategy would form a key component of novel
quorum quenching agents . Even more promising was the fact that curcumin showed ther-
apeutic synergism when combined with various antimicrobial drugs such as azithromycin,
which holds great promise for its use in complementary medicine (
Packiavathy et al.,
2014
).
Medicinal plants and mycobacterial quorum quenching
The chronicity of mycobacterial infections has long been associated with the presence of
several quorum sensing strategies that mycobacteria employ, in an attempt to enhance the
efficiency of infection, biofilm formation, and virulence factor production (
Solokhina et al.,
2018
). Mycobacterial biofilms have been well described concerning their ability to harbor
an extensive drug-tolerant population of cells which is known to complicate and extend
the length of conventional treatment strategies (
Jiang, Gan, An, & Yang, 2019). Novel
agents that aim to induce a supportive, adjuvant, or synergistic effect have been vastly
understudied and only a few known studies are currently in existence. Research has sub-
sequently turned to the use of natural products in combatting the prolific rate of antimi-
crobial resistance and assisting in producing a therapeutic intervention that is efficient in
mycobacterial quorum quenching and which is safe for human consumption.
Phytochemistry and ethnopharmacology research for new antimicrobial sources with
novel modes of action has been of interest for many years. Plant extracts may be repre-
sented as potential superior sources of antimicrobial compounds than synthetic drugs
(
Romero et al., 2016). Aqueous extracts of Vaccinium oxycoccos L. (fresh fruit), Azadirachta
indica A. Juss. (fresh leaves), Hippophae rhamnoides L. (dried fruit), Juglans regia L. (dried
bark), and ground spices were evaluated for their efficacy for antibiofilm activity.
Azadirachta indica was found to be the most effective in the study and had substantial bio-
film reduction potential of M. smegmatis, followed by Vaccinium oxycoccos and spices with
28% and 26% biofilm reduction potential, respectively (
Abidi et al., 2014). Leaves from
Parinari curatellifolia Planch. ex Benth. have been studied for their efficacy against biofilm
formation and the growth of M. smegmatis. The water, dichloromethanolic, and ethanolic
leaf extracts were shown to inhibit biofilm formation. Phytochemical analysis of the plant
revealed the presence of saponins, steroids, alkaloids, flavonoids, tannins, and cardiac
glycosides (
Bhunu, Mautsa, & Mukanganyama, 2017).
322 9. Mycobacterial quorum quenching and biofilm inhibition potential of medicinal plants
Medicinal Plants as Anti-infectives
In addition to testing several plants, nature-inspired synthetic molecules have also
been considered and heavily investigated. These naturally derived compounds have
unlocked the development of a plethora of synthetic therapeutic agents and the discovery
of many novel antibiofilm agents. Sesamol is an orga nic compound isolated from sesame
seeds.
Hans, Sharma, Hameed, and Fatima (2017) demonstrated that sesamol was a
potent M. smegmatis biofilm inhibitor that reduced the metabo lic a ctivity and dry weight
of biofilm biomass by 70% and 58%, respectively. Carvacrol [2-Methyl-5-(1-methylethyl)
phenol] is a volatile monoterpene that is a major constituent of several essential oils of
the Labi atae family. This compound has been approved as safe for usage in food pro-
ducts. Research has demonstrated its antioxidant, antit umor, antihepatotoxic, antibacte-
rial, antiinflammatory, analgesic, and insecticidal biological activity (
Hyldgaard, Mygind,
& Meyer, 2012; Langeveld, Veldhuizen, & Burt, 2014; Magi, Marini, & Facinelli, 2015;
Nostro & Papalia, 2012
). Carvacrol has been reported to be effective against biofilm for-
mations of rapidly growing mycobacteria, namely, M. phlei, M. smegmatis,andM. fortui-
tum (with antimycobacterial MIC values of 80100 μg/mL). The study observed that
concentrations above the MIC were able to cause a significant disaggregation effect on
biofilm biom ass and the metabolic activity of the cells protected by the biofilm matrix at a
maturation phase. At concentrations below the MIC, the compound was able to disrupt
biofilm formations (
Marini et al., 2019). Arisa ema sinii K. Krause, a Chinese medicinal
herb, extract (80% ethanol in water) exhibited antibiofilm activity against M. tuberculosis.
Historically speaking, this medicinal herb has be en empl oyed by local populations to
treat pulmonary and lymphatic TB and thus its ethnobotanical and ethnopharmacological
properties have directed further research into novel lead compounds isolated from A.
sinii. The petroleum ether fraction showed the best activity and was further fractionated
and purified by using bioassay-guided isolation to yield the very potent compound 1
(
Fig. 9.5) biofilm inhibitor (E)-2-(methyl (phenyl) amino) ethyl 2-(2-hydroxyundecanami-
do)-7, 11-dimethyl-3-oxotetradec-4-enoate, with an IC
50
of 4-32 μg/mL (Jiang et al., 2019).
Various biofilm parameters were investigated; namely the ability of the extract and com-
pound 1 to inhibit biofilm formation, disrupt mature biofilms and disperse pre-formed
mycobacterial biofilms; all of which are essential components of the dynamic natur e of
biofilms. At a concentration of 4 μg/mL, compound 1 was able to successfully inhibit the
FIGURE 9.5 The chemical structure of (E)-2-(methyl
(phenyl) amino) ethyl 2-(2-hydroxyundecanamido)-7,11-
dimethyl-3-oxotetradec-4-enoate (compound 1). Source:From
Jiang, C.-H., Gan, M.-L., An, T.-T., & Yang, Z.-C. (2019).
Bioassay-guided isolation of a Mycobacterium tubercu losis bioflim
inhibitor from Arisaema sinii Krause. Microbial Pathogenesis,
126,351356.
https://doi.org/10.1016/j.micpath.2018.11.022.
323Medicinal plants and mycobacterial quorum quenching
Medicinal Plants as Anti-infectives