Etnobotánica

  

DOI: 10.17129/botsci.2380

Anti-virulence activities of some Tillandsia species (Bromeliaceae)

Actividades antivirulencia de algunas especies de Tillandsia (Bromeliaceae)


Abstract

Background:

Using molecules that inhibit bacterial virulence is a potential strategy to fight infections, with the advantage that, in contrast to bactericidal compounds, they do not induce resistance. Several compounds with anti-virulence properties have been identified in plants, however, they represent a small portion of the total diversity, and in Mexico there are still few studies on this matter.

Hypothesis:

Extracts of species of the Tillandsia genus inhibit the expression of diverse virulence factors without affecting the bacterial growth.

Studied species:

Tillandsia recurvata (L.) L., T. schiedeana Steud. and T. fasciculata Sw.

Study site and date:

These species were collected in December 2016 in the municipalities of Ixtlahuaca and Santo Tomás de los Plátanos, State of Mexico.

Methods:

The ability of dichloromethane (CH2Cl2) and methanol (CH3OH) extracts to inhibit production of violacein in Chromobacterium violaceum was evaluated, as well as the virulence factors regulated by quorum sensing, motility and biofilm in Pseudomonas aeruginosa. In addition, the bioactive fractions obtained were partially identified by 1H NMR.

Results:

CH2Cl2 and CH3OH extracts reduced violacein production from 43 to 85 %, but only those from CH2Cl2 reduced protease activity, biofilm formation and swarming. Interestingly, CH3OH extracts stimulated the formation of biofilms by up to 37 %. Presence of terpenes and phenolic compounds in these species was confirmed. In T. schiedeana glycosylated compounds and cycloartane-type triterpenes were identified.

Conclusion:

The species of Tillandsia show anti-virulence activity, mainly on factors related to adhesion and dispersion in Pseudomonas aeruginosa.

 

Key words: 

Anti-biofilm; anti-virulence; bromeliads; quorum quenching; Tillandsia.

Resumen

Antecedentes:

Usar moléculas que inhiben la virulencia bacteriana es una estrategia potencial para combatir infecciones, con la ventaja que, a diferencia de los compuestos bactericidas, no inducen resistencia. Se han identificado en plantas diversos compuestos con propiedades antivirulencia, no obstante, representan una pequeña porción de la diversidad total, además en México son aún escasos los estudios al respecto.

Hipótesis:

Los extractos de especies Tillandsia inhiben la expresión de factores de virulencia sin afectar el crecimiento bacteriano.

Especies de estudio:

Tillandsia recurvata (L.) L., T. schiedeana Steud. y T. fasciculata Sw.

Sitio y año de estudio:

Las especies se colectaron en diciembre de 2016 en los municipios de Ixtlahuaca y Santo Tomás de los Plátanos, Estado de México.

Métodos:

Se evaluó la capacidad de extractos de diclorometano (CH2Cl2) y metanol (CH3OH) para inhibir la producción de violaceína en Chromobacterium violaceum, así como factores de virulencia regulados por percepción de quórum, motilidad y biopelícula en Pseudomonas aeruginosa. Las fracciones bioactivas obtenidas se identificaron parcialmente por RMN de 1H.

Resultados:

Ambos extractos redujeron la producción de violaceína del 43 a 85 %, pero solo los de CH2Cl2 redujeron la actividad proteolítica, formación de biopelícula y motilidad. De modo interesante los extractos de CH3OH estimularon la formación de biopelícula hasta un 37 %. Se confirmó la presencia de terpenos y compuestos fenólicos en estas especies. En T. schiedeana se identificaron compuestos glicosilados y triterpenos tipo cicloartano.

Conclusión:

Las especies de Tillandsia muestran actividad antivirulencia, principalmente sobre factores relacionados con la adhesión y dispersión en Pseudomonas aeruginosa.

 

Palabras clave: 

Antibiopelícula; antivirulencia; bromelias; apagado de quórum; Tillandsia.


 

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).