Anti-Quorum Sensing Compounds from Rare Actinobacteria

Abstract

Actinobacteria have exceptional metabolic diversity and are a rich source of several useful bioactive natural products. Most of these have been derived from Streptomyces, the dominant genus of Actinobacteria. Hence, it is necessary to explore rare actinobacteria for the production of novel bioactive compounds. Amongst the novel metabolites, anti-quorum-sensing agents, which can curb infection without killing pathogens, are gaining importance. Not many studies are targeting anti-quorum-sensing agents from rare actinobacteria and this research area is still in its infancy. This field may lead to novel bioactive compounds that can act against bacterial quorum-sensing systems. These agents can attenuate the virulence of the pathogens without challenging their growth, thereby preventing the emergence of resistant strains and facilitating the elimination of pathogens by the host’s immune system. Therefore, this chapter describes the general characteristics and habitats of rare actinobacteria, isolation and cultivation methods, the methods of screening rare actinobacteria for anti-quorum sensing compounds, methods of evaluation of their properties, and future prospects in drug discovery.

1. Introduction

Actinobacteria are Gram-positive and high G + C containing bacteria with exceptional metabolic diversity. They are a rich source of several useful bioactive natural products many of which have been reported for their potential roles as antimicrobial, antibacterial, antiviral, anticancer, and antifungal compounds. More than 22,000 bioactive secondary metabolites from microorganisms have been identified and published in the scientific and patent literature, and about half of these compounds are produced by actinobacteria [1]. Currently, approximately 160 antibiotics have been used in human therapy and agriculture, and 100–120 of these compounds, including streptomycin, erythromycin, gentamicin, vancomycin, avermectin, etc., are produced by actinobacteria [2].

Most of these antibiotics in clinical use today have been developed from compounds isolated from Streptomyces, the dominant genus [3]. However, the recent search for the novel compounds from Streptomyces species has often led to the rediscovery of known compounds. Hence, the focus of screening programs has shifted to bioactive compounds from non-Streptomyces genera; also referred to as rare actinobacteria [4].

Recent evidence has demonstrated that rare actinobacteria, might represent a unique source of novel biologically active compounds, and methods designed to isolate and identify a wide variety of such actinobacteria have been developed. These methods include a variety of pre-treatment techniques in combination with appropriately supplementing selective agar media with specific antimicrobial agents.

At present, not more than 50 rare actinobacterial taxa are reported to produce 2500 bioactive compounds [5]. Thus, it is crucial that new groups of rare actinobacteria be pursued as sources of novel pharmaceutically active metabolites. Amongst the novel metabolites, anti-quorum sensing (AQS) agents, which can curb infection without a killing action, are gaining importance. Bacterial cell–cell communication, dubbed quorum sensing, is intricately related to virulence. An associated phenomenon is bacterial swarming which allows the spread of disease and virulence.

The discovery that many pathogenic bacteria employ quorum sensing (QS) to regulate their pathogenicity and virulence factor production makes the QS system an attractive target for antimicrobial therapy. Targeting the pathogenesis instead of killing the organism may provide less selective pressure for the development of resistance. Therefore, it has been suggested that inactivating the QS system in bacteria using QS inhibitors holds great promise for the treatment of infectious diseases. These compounds can attenuate the virulence of the pathogens without challenging their growth, thereby preventing the emergence of resistant strains and facilitating the elimination of pathogens by the host’s immune system. Therefore, a search for anti-quorum-sensing agents as attractive alternatives to treat infection has become logical and gathered momentum [6].

Although antimicrobial properties of actinobacteria have been extensively studied, less is known about AQS activities of rare actinobacteria which may be a rich source of active compounds that can act against bacterial quorum-sensing systems.

2. Defining rare Actinobacteria

Rare Actinobacteria are defined as certain types of Actinobacteria which are abundant in various habitats but are difficult to isolate [7]. These include all non-Streptomyces actinobacterial genera like Actinomadura, Actinoplanes, Amycolatopsis, Dactylosporangium, Planomonospora, Planobispora, Salinispora, Streptosporangium, Verrucosispora and Microbiospora [8]. Even though a major percentage of antibiotics are secreted by bacteria from the genus Streptomyces [9] rare actinobacteria account for 25–30% of these. The importance of rare actinobacteria is further demonstrated by the fact that many of the successful bioactive products in the market like rifamycin, erythromycin, teicoplanin, vancomycin, and gentamycin are produced by such rare actinobacteria [10]. Consequently, rare actinobacteria are being unveiled as highly prospective sources of bioactive compounds.

Members of the genus Actinomadura have been reported to secrete about 350 different types of bioactive compounds exhibiting a wide range of mechanisms of action [11, 12]. Some strains of the genus Actinoplanes are known for the production of acarbose, a secondary metabolite that is an α-glucosidase enzyme inhibitor [13] and is currently being used in treating type 2 diabetes [14]. While numerous bioactive compounds are isolated from the strains of Amycolatopsis, vancomycin and rifamycin are the most familiar ones [15]. Research also led to the discovery of the first naturally occurring tetracycline C2 amides called dactylocyclines which are produced by members of the genus Dactylosporangium [16]. Metabolomic studies conducted on the genus Planomonospora led to the discovery of multiple compounds with biosynthetic activities such as Ureylene-containing oligopeptide antipain, the thiopeptide siomycin and sphaericin and lantibiotic 97,518 [17]. Planobispora rosea is found to secrete a novel antibiotic called GE 2770A and thiazolyl peptides which are protein synthesis inhibitors [18]. Arenimycin is another antibiotic produced by the marine actinobacterium Salinispora arenicola and is found to be effective against drug resistant S. aureus and a few other Gram-positive bacteria [19]. Molecular studies done on compounds isolated from Streptosporangium oxazolinicum, resulted in the discovery of three novel alkaloids having anti-trypanosomal activity [20]. Studies conducted using liquid chromatography mass spectrometry (LC–MS) and one strain-many compounds strategy led to the detection of another bioactive compound abyssomicin from the marine actinobacterium Verrucosispora [21]. Members of the genus Micromonospora have been a major source of Gentamicin, apart from which about 740 different antibiotics are found from other strains [22]. All the above mentioned actinobacteria and few more are a subject of intense investigations and prove to be a rich source of many novel antibiotics when modern approaches such as transcriptomics and metabolomics are employed.

3. Habitats of rare Actinobacteria

Actinobacteria inhabit a wide range of habitats with diverse climatic conditions, including those of extreme temperatures and pH as well as marine waters, deserts and soil [23]. However, they are mainly found in soil. It is also observed that even though actinobacteria are found in all layers of soil, their density decreases with increasing depth [24]. Environmental factors such as pH of the soil, humus content and soil type directly influence the population density and type of rare actinobacteria present [7]. Table 1 summarizes a few rare actinobacteria isolated from various habitats.

Habitat	Genus	Reference
Forest Soil	Conexibacter, Actinospica,Catenulispora, Sinomonas, Longispora etc.	[25, 26, 27, 28, 29]
Desert Soil	Yuhushiella, Dietzia, Kineococcus, Microbacterium etc	[30, 31, 32, 33]
Garden Soil	Ornithinimicrobium	[34]
Alkaline soil	Myceligenerans, Yonghaparkia	[35, 36]
Soil sample from Oil springs	Smaragdicoccus	[37]
Sandy Soil	Krasilnikovia, Sphaerosporangium, Micromonospora	[38, 39, 40]
Farmland Soil	Ruania, Agromyces	[41, 42]
Saline Soil	Zhihengliuella, Nocardiopsis, Nesterenkonia, Brevibacterium, Kocuria	[43, 44, 45, 46, 47]
Soil from tropical rainforest	Dactylosporangium, Saccharopolyspora, Pseudonocardia Planotetraspora, Sphaerisporangium	[48, 49, 50, 51, 52]
Soil sample from Paddy fields	Humihabitans, Humibacillus, Arthrobacter	[53, 54, 55]
Soil near wastewater Treatment facility/Activated Sludge	Flexivirga, Micrococcus, Gordonia, Nocardioides	[56, 57, 58, 59]
Dried Seaweed	Phycicoccus, Labedella, Phycicola, Aeromicrobium	[60, 61, 62, 63]
Marine Water	Tessaracoccus, Paraoerskovia, Marinactinospora, Demequina, Verrucosispora	[64, 65, 66, 67, 68]
Caves	Knoellia, Hoyosella, Jiangella	[69, 70, 71]
Roots of Plants	Plantactinospora, Phytohabitans, Actinophytocola, Flindersiella, Phytomonospora	[71, 72, 73, 74, 75, 76]
Leaves of Plants	Frondicola, Rhodococcus, Nonomuraea	[77, 78, 79]
Stems of Plants	Glycomyces, Saccharopolyspora, Kineosporia, Nocardia	[80, 81, 82, 83]
Glaciers	Cryobacterium, Leifsonia, Arthrobacter	[84, 85, 86]
Volcanic rocks	Allocatelliglobosispora, Thermoactinospora,	[87, 88]

Table 1.

Habitats of rare actinobacteria.

4. Isolation of rare Actinobacteria

4.1 Pretreatment methods

Isolation of rare actinobacteria is a strenuous task, mainly because they are slow growing. After collection of samples, the type of pretreatment method used decides the viability and isolation of the species under study. While several types of pretreatment methods are available, the requirement of each organism is different. A few common methods include suspending samples in distilled water followed by incubation in a rotary shaker, air drying the samples, or heat treatment in oven at 45֯C–65֯C [89]. Alferova and Terekhova have reported a modified procedure for treatment of soil samples with calcium carbonate under humid conditions [90]. This gave efficient increase in the number of isolates as well as representatives of rare genera. Chemical treatment methods such as treating the sample with 1.5% phenol or physical methods combined with other conventional or sucrose gradient centrifugation methods also proved to be highly effective [91]. Isolation of rare actinobacteria is still an area of active research and hence a particular pretreatment method does not guarantee the isolation of a specific actinobacteria. However, other prescribed pretreatment methods available in literature can be tried to achieve exclusive isolation. These methods are summarized in Table 2.

Pretreatment method	Type of Actinobacteria	Antibiotic for selective isolation	Suggested isolation media	Reference
Physical methods Fresh samples w/o pretreatment Air dried in room temperature for 2 weeks Samples kept in oven at 40֯C-65֯C Samples heated in oven at 110֯C for 1 h Pre-treatment followed by calcium carbonate Chemical methods SDS 0.05% and Yeast extract 5% Phenol 1.5% Chloramine-T	Thermophilic Actinobacteria	Cycloheximide, Kanamycin	Czapeck medium, glycerol asparagine medium, Oatmeal medium	[92, 93]
	Halophilic and Alkalophilic actinobacteria	Nalidixic acid	Starch-casein agar, glycerol asparagine medium, T3 medium,	[30, 94]
	Acidophilic; Actinobacteria	Cycloheximide, Nystatin	ISP4, ISP2, Acidified oatmeal agar and modified Bennett’s agar	[95, 96]
	Actinobacteria from Plant origins	Pimaricin, penicillin G and polymyxin B	Sodium Propionate medium, Yeast extract medium, ISP2, ISP3, Potato dextrose agar	[97, 98]
	Actinobacteria from Soil	Actidione, Nystatin	HV Agar, Glycerol-arginine medium, Medium supplied with superoxide dismutase, PDA	[30, 99, 100]
	Marine Actinobacteria	Cycloheximide, Nystatin	Glycerol-asparagine, Glycerol-glycine, Chitin, Starch-Casein	[101, 102, 103]
	Rare Actinobacteria	Nystatin	HV, HP, Trehalose-Proline agar, ISP5, B4,	[89, 104]

Table 2.

Pretreatment method, antibiotic and isolation media used for selective isolation of rare actinobacteria.

4.2 Use of antibiotics for selective isolation

Pretreatments do reduce a fraction of unwanted predominant fast-growing organisms. However, use of antibiotics in isolation media along with pretreatment substantially increases the chances of selective isolation as it effectively eliminates fast growing and competitive bacteria. By virtue of this property, antibiotics like gentamicin and novobiocin were successfully used to isolate members of the genus Micromonospora [24]. Similarly, members of genus Microtetraspora were isolated using nalidixic acid and trimethoprim [104]. Currently available antibiotics for selection of actinobacteria along with the type of actinobacteria isolated are summarized in Table 2.

4.3 Use of specific isolation media

It is observed that the growth of rare actinobacteria is highly sensitive to contamination by some known fast-growing organisms such as fungus, other bacteria and a few common streptomyces. Hence conventional methods of isolation are ineffective in isolating rare actinobacteria and there is a need to find more advanced and highly selective isolation methods [24]. Studies have shown that in abundance of nutrients, Actinobacteria prefer exponential growth over the production of secondary metabolite [105]. It is also reported that enzymes for secondary metabolite production are inhibited in presence of glucose [106]. Factors such as the type of chemical or physical pre-treatment used [24], pH, temperature and duration of incubation as well as sources of essential nutrients used greatly affect the rate of growth of rare actinomycetes as well as metabolite production [107]. Effect of each of these factors should be considered while designing a culture medium.

A carbon source can thus either be suitable for growth or for metabolite production but not both. Generally, monosaccharides or sugars that are metabolized rapidly are found to be most suitable for growth while polysaccharides or sugars that metabolize slowly are more suitable for antibiotic production [107]. A comparison between inorganic and organic nitrogen sources used revealed that use of organic nitrogen source resulted in maximum growth as well as metabolite production [104]. Evidences have shown that antibiotic accumulation increases as soon as the nitrogen source used in medium is entirely utilized by the organism [108]. Addition of excess inorganic phosphates resulted in rapid growth since it aids the consumption of Carbon and Nitrogen sources as well as accelerates the rate of cellular respiration but it lowers the production of secondary metabolites [109].

Antibiotic production begins extensively in mid log and late log phase and is continuous in the stationary phase of bacterial growth curve [110]. Multiple studies done on various species of actinobacteria suggest that, antibiotic production is maximum at neutral pH [111] and temperature of 30°C [112, 113]. Further, requirement of other important constituents of growth medium such as trace metals and minerals vary with species and culture conditions.

Apart from all the above-mentioned common isolation media, the requirement of each bacterium is different and hence the selective media should be designed keeping in mind the nutritional requirements of target organism. In order to facilitate the process of designing selective isolation media, information from various taxonomic, phenotypic and antibiotic sensitivity databases can be used [97].

5. Anti-quorum sensing

Quorum sensing is a mechanism of cell-to-cell communication seen in bacteria which occurs by the means of certain autoinducer or chemical signal molecules. The concentration of these autoinducer molecules increases with increase in cell density. Once a certain threshold concentration is reached, these autoinducer molecules lead to alteration of gene expression in the population [114]. It is now known that quorum sensing is the underlying mechanism of a wide spectrum of bacterial physiological processes such as virulence [115, 116], bioluminescence [117], motility [118], sporulation [119], conjugation [120], development of genetic competence [121] as well as synthesis of antibiotics [122].

Considering the implications of quorum sensing in various aspects of bacterial life processes, it is evident that inhibiting quorum sensing could have potential therapeutic applications [123, 124]. There are various strategies in which the quorum sensing pathways can be inhibited. Together, they are called as quorum-quenching and the compounds or molecules used to do so are called as AQS compounds. Strategies used in quorum quenching include: Inhibition of synthesis of autoinducer molecule, designing analogues of autoinducer molecule or receptor analogues [125] and antibody or enzyme catalyzed hydrolysis of autoinducer molecule [126].

N-acyl homoserine lactones (AHLs) are an important class of signaling molecules produced by Gram negative bacteria which are known to govern the population density [127]. Lactonases are the enzymes which hydrolyse either the amide linkage between lactone and acyl side chain or affects the ester bond thereby inhibiting the signaling molecule [128]. Further, acylases [129] and oxidoreductases [130] are also found to have quorum quenching activities. Degradation of signaling molecules is also possible via antibody mediated catalysis. Lamo Marinet al. in their studies reported that hydrolysis of AHL is efficiently achieved by original squaric monoester monoamide hapten [131]. Kristina MSmith and YigongBu reported two novel compounds: N-(2-oxocyclohexyl)-3-oxododecanamide which was a moderate antagonist and N-(trans-2-hydroxycyclopentyl)-3-oxododecanamide which was a strong antagonist of autoinducer molecules of P. aeruginosa [125].

6. Methods of screening and evaluation of anti-quorum sensing compounds isolated from rare Actinobacteria

While the method of preparation of bacterial extracts remains the same, various conventional and virtual screening methods are used to screen compounds for their AQS activity.

6.1 Pigment inhibition assays

Formation of violacein, a purple pigment in the reporter strain Chromobacterium violaceum is controlled by quorum sensing [132]. Quantifying the inhibition of violacein synthesis, by paper disc diffusion as well as flask incubation assay is thus used as a method for screening AQS activity of a given compound. Presence of a zone of inhibition in paper disk diffusion plates and significant reduction in violacein content in flasks, are considered as positive results. This method was efficiently used to test the AQS activity of Vanilla extract [133], pigments extracted from Auricularia auricular [134], essential oils [135], fruit extracts of Passiflora edulis [136], and a few compounds isolated from plant endophytic bacteria [137]. Similarly, inhibition of pigment production in Serratia marcescens is also a coherent test to determine the quorum quenching activity of a compound [138].

6.2 Swarming motility and biofilm formation assays

Swarming and biofilm formation are other quorum sensing controlled processes seen in bacteria such as P. aeruginosa [139], S. marcescens [140], P. mirabilis [6] and are known to play a major role in pathogenesis. Reduction in diameter of the swarm zone, alteration in swarming patterns and significant reduction in biofilm formation [141] indicate that given compound has AQS activity. While reduction in swarm zone diameter and alteration of swarming patterns can be quantified directly, inhibition in biofilm formation can be quantified using microtiter plate method and observing topographical changes in biofilm by scanning electron microscopy [125] as well as confocal laser scanning microscopy. Wijaya et al., have reported that crude extracts from marine actinobacteria isolated from Indonesia, show significant reduction of biofilm formation by inhibiting quorum sensing in Gram positive and Gram negative pathogenic bacteria [142]. Similar activity is reported in extracts from Nocardiopsis sp [143].

6.3 Molecular and physiochemical methods

Genes encoding polyketide synthase I and II (PKS-I and PKS-II), and nonribosomal peptide synthetases (NRPS), are responsible for synthesis of novel AQS compounds in rare actinobacteria [138, 144]. PCR Screening of a given bacterium for these genes is helpful in detecting the presence of quorum quenching activity [145]. Purified compounds isolated from the broth cultures of such bacteria can be further subjected to Physiochemical screening methods such as UV–Vis spectrophotometry, TLC to determine the nature of compound which exhibits AQS activity [138]. Some strains of Nocardia [146] and Micromonospora [147] are subjected to such studies and novel AQS compounds were found.

6.4 In silico screening methods

Biosynthetic gene clusters in a given bacteria can be identified using genome mining studies. 20 biosynthetic gene clusters are reported in a single strain of Pseudoalteromonads most of which were NPRS and PKS gene clusters [148]. CLUSEAN (CLUster SEquence ANalyzer) [149] and antiSMASH [150] are tools used for analysis of bacterial secondary metabolite biosynthetic gene clusters and can be used to predict AQS activity in a given bacterium. Using structures available in various databases, performing molecular docking [151] studies on compounds having AQS activities is possible and can provide remarkable information on molecular interactions involved in inhibition of quorum sensing.

7. Conclusion

Though work on Rare actinobacteria and bioactive compounds from them is gathering momentum, not many studies are targeted towards isolation and identification of AQS compounds from rare actinobacteria and this research area is still in its infancy. These studies may lead to novel bioactive compounds that can act against bacterial quorum sensing systems. These agents can attenuate the virulence of the pathogens without challenging their growth, thereby preventing the emergence of resistant strains.

Such potent anti-quorum -sensing compounds may lead to the development of alternative therapies to address the glaring problem of antibiotic resistance.

This would be of immense medical and commercial benefit.