Antibiotics are one of the major discoveries of the 20th century, but their excessive use has generated a rapid development of resistance to this class of drugs (López-Jácome et al. 2019). Currently, resistance to antimicrobials is a global public health problem, hence, the WHO has urged to develop new effective therapies against antimicrobial resistant bacteria (Ferri et al. 2017). Identification of new antimicrobial action mechanisms that do not induce resistance is an option that has been raised in recent years (Tillotson & Theriault 2013). One of them is anti-virulence therapies, which seek to interfere in the production of virulence factors that bacteria use to establish themselves and cause harm (Muñoz-Cazares et al. 2018). The novelty of these therapies is that they do not directly affect the viability of bacteria (as do antibiotics) because their target is a system or metabolic pathway considered non-vital for the bacterial cell (Mühlen & Dersch 2016). To date, different targets that reduce virulence and damage to the host when they are blocked have been identified (Castillo-Juárez et al. 2017).
Of the most studied targets are quorum sensing (QS), biofilm formation, type 3 secretion systems (T3SS) and swarming (Muñoz-Cazares et al. 2018). QS is a complex phenomenon designed to promote multicellular behavior of unicellular organisms, for which population-level coordination in time and space is required for the expression of virulence factors (Muñoz-Cazares et al. 2017). Biofilms are microbial aggregates that allow bacteria to protect themselves from environmental changes, which include tolerance to high doses of antimicrobials (Muñoz-Cazares et al. 2018). Similarly, swarming is a social phenomenon that involves rapid coordinated movement by flagella and type IV pili of bacteria on a semisolid surface (Köhler et al. 2000).
Chromobacterium violaceum and Pseudomonas aeruginosa are the main bacterial models that have been used to identify anti-virulence activity (Castillo-Juárez et al. 2013). C. violaceum is an opportunistic pathogen of animals that regulates the production of violacein by a QS system (Montes de Oca-Mejía et al. 2015). The facility with which QS inhibition is determined through observation of pigment production has made this bacterium one the main biosensors for quorum quenching (Castillo-Juárez et al. 2013). P. aeruginosa is an opportunistic pathogen of animals and plants and one of the main causes of nosocomial infections. This bacterium regulates the production of virulence factors such as pigments, toxins, enzymes, biofilm formation and swarming through QS. It possesses at least three hierarchically organized QS systems that coordinate production of these virulence factors; hence their inhibition is more complex (Castillo-Juárez et al. 2017).
Natural products derived from plants are so far the main source of the largest number of metabolites with anti-virulence properties (Silva et al. 2016, Chandra et al. 2017). However, the number of species investigated remains minimal in contrast to the enormous diversity of existing plants.
The Bromeliaceae family is composed of 58 genera and 3,408 species native to America distributed from Argentina to the southern United States (Benzing 2000, Luther 2014). Some species have important pharmacological activities, such as anthelmintic (Stepek et al. 2005), antinociceptive (de Lima-Saraiva et al. 2014), gastroprotective (Machado et al. 2013), photoprotective (de Oliveira-Júnior et al. 2017), anticancer (Lowe et al. 2017), hypoglycemic (Witherup et al. 1995) and antibacterial (Faller et al. 2017) activity.
In Mexico, there are19 genera and 422 species of bromeliads, of which 230 species correspond to the Tillandsia genus, one of the most diverse genera in our country (Espejo-Serna & López-Ferrari 2018). This genus includes species that are mainly ornamental and medicinal (Mondragón-Chaparro et al. 2011). In traditional medicine, they are used to treat infections, coughs, bronchitis, burns and gastritis (Sandoval-Bucio et al. 2004). Moreover, their bactericidal activity, mainly against Gram-positive bacteria, has been documented (Castillo-Juárez et al. 2009, Vite-Posadas et al. 2011, Silva et al. 2013). Interestingly, their anti-virulence properties have not been investigated, although the chemical composition of this family is characterized by the presence of compounds identified in other species as possessing the ability to reduce virulence, such as flavonoids, sterols, cinnamic acid derivatives and lignans (Manetti et al. 2009, Silva et al. 2016).
Therefore, the objective of this research was to analyze the violacein inhibition and anti-virulence potential of three species of the Tillandsia genus (T. recurvata (L.) L., T. schiedeana Steud. and T. fasciculata Sw.) distributed in Mexico and their effect on the inhibition of virulence factors in P. aeruginosa.
Materials and methods
Plant material. Plants were collected in December 2016 in the west-central area of the State of Mexico. T. recurvata and T. schiedeana were collected in "La Pedrera", Ixtlahuaca (19° 10. 662' N; 100° 15.303' W), altitude 1,368 m asl, and T. fasciculata was collected in "El Pedregal", Santo Tomás de los Plátanos (19° 11.167' N; 100° 15.912' W), altitude 1,216 m asl. The specimens were identified by Dr. María Flores Cruz (Figure 1 A-C) and deposited in the Collection of Living Bromeliads at the University Center for Conservation and Research of Mexican Bromeliads (CUCIBROM).