CHAPTER 1.................................................................................................................................. 1
2.1 Introduction
While in the past antimicrobial agents may have been a vital component of modern medicine, this is no longer the case (Grandclément et al., 2016). The World Health Organization published a report in 2014 entitled “Antimicrobial resistance: Global report on surveillance”, highlighting the urgency of antimicrobial resistance, and the threat it poses on health care worldwide (Grandclément et al., 2016). Antimicrobial resistance often occurs due to the use
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of antimicrobial agents as growth enhancers in agriculture (Ramalingam and Amutha, 2013), non-compliance by patients failing to complete a course of antimicrobial agents (Cruz et al., 2014) or the presence of sub-lethal doses of antimicrobial agents in food and water supplies (Dorotkiewcz-Jach et al., 2015). As antimicrobial resistance contributes to as many as 700 000 deaths per year, a figure which could jump to 10 million by 2050 (Indranigrat et al., 2016), alternative treatments are urgently needed.
Quorum sensing inhibition (QSI) has gained popularity as such an alternative therapeutic option (García-Contreras, 2016), with the Gram-negative acyl homoserine lactone QSI being the most widely studied (Rekha et al., 2016). Many bacteria employ quorum sensing (QS) to communicate and regulate virulence and other community behaviours such as biofilm formation (Müller et al., 2015; Banerjee and Ray, 2016; Zhang and Li, 2016).
Quorum sensing inhibition could, therefore, be used to inhibit these virulence traits to treat infections by important pathogens such as Pseudomonas aeruginosa (García-Contreras, 2016;
Hassan et al., 2016), one of the primary organisms recovered from hospital sewage and associated with a large number of hospital infections (Otton et al., 2017).
Marine microorganisms have gained interest, as they are gaining impetus as a promising source of biologically active compounds (Abbamondi et al., 2014). Relatively little research has, however, been undertaken on these marine natural products, as they are difficult to cultivate under standard conditions (Abbamondi et al., 2014). It has been estimated that only 0.01 - 0.1% of marine microbial species are known to scientists, highlighting how much work still needs to be done (Moghadamtousi et al., 2015). Marine species have developed QSI enzymes and mechanisms as a result of competition for limited habitats and nutrients (Grandclément et al., 2016). Marine sponges and their associated microorganisms are considered the topmost producers of natural products within their environment (Kurian and Elumalai, 2017).
The diversity of sponge and sponge-associated bacterial-derived natural products is vast, and includes fatty acids, terpenes, nucleosides and halogenated amino acids (Kurian and Elumalai, 2017). Bacillus subtilis strains, associated with sponge Aplysina aerophoba, produce a multitude of antimicrobial compounds effective against multi-drug resistant Staphylococcus aureus (Bramhachari et al., 2016) while Bacillus spp. from the sponge Tedania anhelans possess potent antioxidant potential (Kurian and Elumalai, 2017). From the South China Sea, the sponge-associated Bacillus atrophaeus from Dysidea avara was shown to produce two
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different antibiotics, Bacillamide C and Neobacillamide A and Bacillus cereus QN03323, a sponge-associated bacterium from the marine sponge Halichondria japonica, was shown to produce two novel antibiotics, YM-266183 and YM-266184 (Bramhachari et al., 2016).
Furthermore, coral-associated Bacillus horikoshii and marine sediment-associated Bacillus pumilus S8-07 have also shown QSI potential (Musthafa et al., 2011). Evidently, with more than 3% of the Bacillus genome being dedicated to the production of secondary metabolites (Bastos et al., 2013), these organisms are one of the top candidates for antimicrobial production. Isolates of the genus Bacillus can be broken up into a number of groups based on highly similar 16S and 23S rRNA sequences, including the Bacillus subtilis and the more common Bacillus cereus groups (Patiño-Navarrete and Sanchis, 2017). This latter group consists of 8 species, namely Bacillus toyonensis, Bacillus weihenstephanensis, Bacillus mycoides, Bacillus cytotoxicus, Bacillus pseudomycoides, Bacillus thuringiensis and Bacillus cereus (Ceuppens et al., 2013; Patiño-Navarrete and Sanchis, 2017).
Bacillus spp. isolates also possess great potential as quorum sensing inhibitors.
Halobacillus salinus, from the same family, produces two powerful QSI compounds, N-(2- phenylethyl)-isobutyramide and 3-methyl-N-(2-phenylethyl)-butyramide, capable of inhibiting violacein production in Chromobacterium violaceum CV026 (Tang and Zhang, 2014;
Saurav et al., 2016). Similarly, B. cereus D28 produced cyclo-L-proline-L-tyrosine, capable of inhibiting QS in C. violaceum ATCC 12472 (Tang and Zhang, 2014; Saurav et al., 2016). Bacillus species are also known producers of AHL-degrading lactonase enzymes, which hydrolyze the AHL lactone ring (Zhang and Li, 2016; Guendouze et al., 2017). Lactonases can be divided into two clusters, with all lactonases belonging to the more extensive AiiA cluster being exclusively produced by these Gram-positive bacilli (Castillo-Juarez et al., 2017). Producers of these lactonases include B. thuringiensis, B. anthracis, and B. cereus (Banerjee and Ray, 2016;
Castillo-Juarez et al., 2017). As a result, Bacillus species have shown strong QSI potential, such as the inhibition of biofilm formation in E. coli (Durai et al., 2013).
Even more important is the ability of Bacillus species to inhibit QS in P. aeruginosa.
Bacillus spp. SS4 inhibited QS in P. aeruginosa PAO1, with B. pumilus inhibiting biofilm formation by the same organism (Kumar et al., 2013; Chang et al., 2017). Furthermore, when the aiiA gene from B. cereus A24 was expressed in P. aeruginosa, a reduction in both C4- homoserine lactone (HSL) and 3-oxo-C12-HSL was observed, resulting in reduced virulence factor production (Fetzner, 2015).
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Overall, this indicates that sponge-associated bacteria, especially those belonging to the genus Bacillus, have the potential to lessen the virulence of P. aeruginosa through QSI strategies (Chang et al., 2017). This chapter serves then as an investigation into the QSI potential of extracts from five sponge-associated Bacillus species against P. aeruginosa, using C. violaceum as a primary screening organism.