**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.

### **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**.

#### **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

*Anti-Quorum Sensing Compounds from Rare Actinobacteria DOI: http://dx.doi.org/10.5772/intechopen.106526*


#### **Table 2.**

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

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 Marin et 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.
