**3. Effects of microbial secondary metabolites on antibiotic tolerance and resistance**

Secondary metabolites can bring profound variation in microbial physiology, metabolism, and stress responses [29]. Several evidence suggest that these molecules can modulate microbial susceptibility to commonly used antibiotics. This section explores which types of secondary metabolites alter antimicrobial susceptibility, and how and why this phenomenon occurs.

In a given environment, microorganisms are rarely found in isolation. Due to such reasons, the presence of secondary metabolites in a microbial community exerts evolutionary pressure on both secondary metabolite-producing and non-producing members to develop means to withstand them. Generally, secondary metabolites alter the state of metabolism which directly has an impact on an organism's ability to withstand antibiotics assault by either increasing its tolerance or making itself more resistant.

Although antibiotics tolerance and resistance are often treated in a similar manner, they offer different abilities to the microorganism. The phenomenon of antibiotic

tolerance is the ability of organism to survive transient antibiotic exposure while resistance is the ability of organism to grow in the presence of antibiotics at a given concentration [30–32]. Another generic term commonly used is antibiotic resilience which refers to the ability of a bacterial population to be refractory to antibiotic treatment, which can arise from an increase in tolerance and/or resistance.

There are common modes of action through which secondary metabolites molecules can alter antibiotic efficacy in both single-species and polymicrobial communities. Specifically, secondary metabolites can regulate multidrug resistance efflux systems, can modulate the toxicity of antibiotics through interactions with reactive oxygen species (ROS) and can induce antibiotic tolerance to provide an overlooked route for the evolution of antibiotic resistance.

The knowledge of such interactions between secondary metabolites and antibiotic efficacy is beneficial as this could be applied to optimize the use of existing antimicrobial drugs and generate targets for novel therapeutic strategies.

#### **3.1 Induction of efflux systems**

One common mechanism bacteria use to tolerate clinical antibiotic treatment is by activation of efflux pumps that export toxins out of the cell [33, 34]. However, efflux pumps exist long before human use of synthetic antibiotics and therefore are presumed to have originally evolved to transport other, naturally occurring, substrates, such as secondary metabolites [35, 36]. Importantly, efflux pumps vary in their specificity, with regard to both their regulation and their substrate affinity [37]. Therefore, it is essential to understand how the secondary metabolite interacts with the transcriptional regulation of the efflux system, as well as which classes of drug the efflux system can transport before one predict whether a secondary metabolite will increase antibiotic resilience in its producer through the induction of a particular efflux system or not. A well-known example of a secondary metabolite that affects the efflux system is indole. Indole is a signaling molecule self-produced by *E. coli* that triggers the expression of certain multidrug efflux pumps in enteric bacteria [38, 39]. It is also found that subpopulation of mutants in *E. coli* which is more resilient towards norfloxacin and gentamicin antibiotics treatment produces high levels of indole which give population-level resistance [40]. This behavior is characterized as a "charity" as the subpopulation responds in favor to support the rest of the community. It was also found that indole achieve such a role by upregulating the MdtEF-TolC efflux system [40].

As mentioned earlier, efflux pumps are evolved to transport in general various molecules including secondary metabolites, efflux pumps can also provide protection against secondary metabolites that are toxic to their producers. For instance, phenazines are redox-active and toxic secondary metabolites produced by the *Pseudomonas aeruginosa*, which activates expression of the MexGHI-OpmD efflux system via the redox-sensing transcription factor SoxR [41–43]. In addition, phenazines also serve other important roles both for the microbes and other species as it can increase *P. aeruginosa* virulence in the lungs of patients with cystic fibrosis, or help in protecting plants against fungal pathogens [44, 45].

A related phenomenon has been observed in the Gram-positive bacterium *Streptomyces coelicolor*. This bacterium produces a natural antibiotic, actinorhodin, that stimulates the expression of a transporter similar to those that export tetracycline [46, 47].

#### **3.2 Modulation of oxidative stress**

The idea of oxidative stress become well known when it was proposed that regardless of the specific cellular targets of various antibiotic classes, bactericidal antibiotics exert their lethal effects in part by inducing oxidative stress [48]. Although this hypothesis resulted in major controversies [49, 50], evidence reviewed elsewhere [51] suggests that bactericidal antibiotics do impact cellular redox states and that the resulting increase in ROS and oxidative stress can contribute to cell death. Such phenomena relate well with secondary metabolites as they also interface with cellular redox homeostasis and oxidative stress responses. Below three different modes of action are discussed by which secondary metabolites can potentially antagonize or potentiate the toxicity of antibiotics: upregulation of oxidative stress response genes; direct detoxification of ROS; and increased endogenous ROS generation.

#### *3.2.1 Upregulation of defenses against oxidative stress*

Secondary metabolites that upregulate the expression of oxidative stress responses can prime bacterial cells for tolerance and/or resistance to antibiotics, which is similar to the protective effects of exposure to sublethal concentrations of oxidants such as H2O2 [52]. Among this class of metabolites, indole is a non-toxic molecule produced by *E. coli*, which activates various genes such as thioredoxin reductase, DNA-binding protein (Dps), and alkyl hydroperoxide reductases that activate only during oxidative stress response [53]. Furthermore, the frequency of *E. coli* persisters can increase by at least an order of magnitude if it is exposed to indole to three different classes (fluoroquinolones, aminoglycosides, and β-lactams) antibiotics. In addition, deletion of oxyR substantially diminishes this effect, which demonstrates that upregulation of oxidative stress responses by a secondary metabolite can contribute to bacterial persistence [53].

Another example of the secondary metabolite is pyocyanin (PYO) produced by *P. aeruginosa*. PYO increases superoxide dismutase activity [54] and upregulates the transcription of several other oxidative stress response genes, including those encoding alkyl hydroperoxide reductases, thioredoxin reductase, catalase, and ironsulfur cluster biogenesis machinery [43]. Interestingly, PYO has been shown to increase the frequency of gentamicin-resistant mutants in *P. aeruginosa* cultures that is independent of drug efflux, as PYO does not upregulate aminoglycoside-transporting efflux pumps [55]. A plausible explanation for this phenomenon is that PYO-induced oxidative stress responses counteract ROS-related gentamicin toxicity. The reasoning behind the explanation is as follows: (1) Gentamicin is known to promote increased intracellular ROS levels [56, 57]. (2) Pretreating cells with oxidants can prime them to tolerate antibiotics [52]. This consequently could decrease the rate at which spontaneous mutants are lost from the population [58], thus the frequency of resistant mutants increases as observed experimentally. PYO has been demonstrated to promote the growth of *P. aeruginosa* in the presence of other antibiotics such a β-lactam antibiotic carbenicillin and other aminoglycosides such as kanamycin, streptomycin, and tobramycin [59]. These antibiotics have been shown to perturb cellular redox states [60, 61] rather than as substrates for PYO-regulated efflux systems [55, 62]. This suggests that the observed decreases in antibiotic efficacy could be related to PYO-induced oxidative stress responses.

#### *3.2.2 Detoxification of ROS*

Another way to protect against antibiotic assaults of oxidative stress is by directly detoxifying antibiotic-induced ROS by upregulating the antioxidant activity. Ergothioneine, is one of two major sulfur-containing redox buffers in mycobacteria, along with mycothiol which help in detoxifying ROS during the stress response. This molecule is so important for the *Mycobacterium tuberculosis* that loss of ergothioneine biosynthesis genes decreases minimum inhibitory concentrations (MICs) for various clinical antibiotics such as rifampicin, bedaquiline, clofazimine, and isoniazid. Additionally, loss of gene also decreases the survival rate by at least 30–60% under treatment at the MICs compared to wild type [63].

#### *3.2.3 Synergistic interactions between secondary metabolites and antibiotics*

So far secondary metabolites have been demonstrated to decrease antibiotic efficacy by attenuating oxidative stress. However, they can also amplify the toxicity of antibiotics by increasing ROS generation. 2-heptyl-3-hydroxy-4-quinolone is one example, also known as the *Pseudomonas* quinolone signal (PQS) produced by *P. aeruginosa*. PQS is a redox-active molecule that possesses both antioxidant properties and pro-oxidant activity as it can reduce not only free radicals but also metal ions. Reduction in metal ions concentration such as iron is lethal for the cell as it facilitates ROS formation through the Fenton reaction [64].

#### **3.3 Interspecies antibiotic resilience**

So far, we have discussed examples of how secondary metabolites affect their producers susceptibility to antibiotics. However, secondary metabolites can also modulate interspecies antibiotic resilience. Such study is beneficial as it will potentially show how interactions among members of a polymicrobial infection might affect antibiotic treatment outcomes [65, 66]. For example, one study has demonstrated that indole, which is produced by *E. coli*, can increase antibiotic tolerance of *Salmonella enterica subsp. enterica serovar Typhimurium* [38, 67]. *S. Typhimurium* does not produce indole, yet it becomes more than threefold tolerant against ciprofloxacin in the presence of exogenously added indole, as well as in the case where it is cocultured with indole-producing *E. coli* [67]. Indole induces the same OxyR-regulated oxidative stress responses in *S. Typhimurium*, as in *E. coli*, and the deletion of oxyR decreases tolerance against ciprofloxacin in *S. Typhimurium* mediated by it [67]. Thus, indole acts like an interspecies modulators of antibiotic resilience. Besides indole, putrescine is another secondary metabolite that acts similar to indole. For example, *Burkholderia cenocepacia* produces putrescine to protect itself from polymyxin B but it also protects its neighboring species in the co-cultures, including *E. coli* and *P. aeruginosa* [68]. There are definitely more such secondary metabolites that exist which have the potential of being interspecies modulators of antibiotic resilience.

Importantly, although the above examples suggest that certain secreted secondary metabolites have the potential to raise the community-wide level of antibiotic resilience in polymicrobial communities, it may not be this case always. As mentioned earlier that secondary metabolites can be toxic or non-toxic and can increase or decrease resilience against antibiotics, it is important to consider whether the stress caused by the secondary metabolite is tolerable to the non-producing species. If the molecule-caused toxicity outweighs the benefits it provides against antibiotics, the

non-producing species would not gain a benefit. In such a case, the secondary metabolite might even act synergistically with the clinical antibiotic [69].

#### **4. The regulation of the secondary metabolism of** *Streptomyces*

So far, we have discussed what different kinds of secondary metabolites exist, how they affect cellular metabolism and help in survival in competitive and stressful environments. This last section focuses on the factors on which the biosynthesis of secondary metabolites is regulated. This will show how different conditions lead to the secretion of secondary metabolites which leads to phenomena discussed in the previous two sections. As the exploration of regulation of secondary metabolites among various species is still in its early stages except for Streptomycetes, this section will focus on its regulatory mechanisms behind biosynthesis of secondary metabolites.

Streptomycetes and other actinobacteria are renowned as a rich source of natural products of clinical, agricultural, and biotechnological value. Sequencing genomes of numerous streptomycetes has revealed that they all possess the capacity to produce multiple secondary metabolites [70, 71] implies that it can repel a large number of competitors using either individual or combination of molecules using their synergistic characteristics [72]. The genes of enzymes responsible for the production of individual secondary metabolites are found clustered. Furthermore, these clustered genes are commonly associated with regulatory gene/s that regulate their transcription or resistance genes.

This section discusses some of the factors which regulate the production of secondary metabolites in *Streptomyces*.

#### **4.1 Cluster-situated regulators (CSRs)**

Generally, a single regulatory gene regulates several gene clusters associated with secondary metabolites production. This way multiple chemical signals which can trigger activation of the regulatory gene can activate specific or multiple genes clustered corresponding to secondary metabolite production. Some of the gene clustered include the clusters for streptomycin in *S. griseus* and actinorhodin (ACT) in *S. coelicolor*. Their corresponding transcription factors are called StrR and ActII-ORF430 respectively [73]. Both StrR and ActII-ORF430 transcription factors directly activate the transcription of genes of the corresponding clusters that encode biosynthetic enzymes. Moreover, evidence suggests that the cellular level of a CSR is the principal factor that determines the level of transcription of the biosynthetic genes it targets. This correlates closely with the level of secondary metabolite produced [74, 75]. Thus, factors that control the production of ActII-ORF4 and StrR will ultimately regulate the production of ACT and streptomycin respectively. Both these transcription factors are under regulation with many activators, repressors, and inducers which are explained well in detail here [76].

#### **4.2 The stringent response and nutrient deprivation**

Stringent response is an stress response of bacteria in reaction to amino-acid starvation, fatty acid limitation, iron limitation, heat shock, and other stress conditions. During stringent response, accumulation of (p)ppGpp enables bacteria to survive sustained periods of nutrient deprivation. For several *Streptomyces* species,

#### *Diverse Survival Functions of Secondary Metabolites in Nature DOI: http://dx.doi.org/10.5772/intechopen.101977*

mutations that block the synthesis of (p)ppGpp (guanosine tetra- and pentaphosphate) have been found to alter antibiotic production and hinder morphological development [77]. In general, the stringent response enhances transcription of numerous genes associated with the stationary phase of batch culture and stress responses. Stimulation of (p)ppGpp synthesis, either by subjecting growing cultures to amino acid starvation [78] or inducing expression of a truncated version of relA that confers ribosome independent (p)ppGpp synthetase activity [79, 80] increases the level of actII-ORF4 transcription and production of the corresponding antibiotics.

#### *4.2.1 Regulation of secondary metabolism by carbon*

The availability and source of carbon have a substantial effect on the production of antibiotics and morphological development [81]; for example, glucose blocks production of ACT by *S. coelicolor* [82]. Lack of carbon source triggers a stringent response which as explained above leads to accumulation of (p)ppGpp which increases the level of actII-ORF4 transcription and production of the corresponding antibiotics.

#### *4.2.2 Regulation of secondary metabolism by nitrogen*

Numerous studies have shown that the source of nitrogen can influence the production of antibiotics. In the presence of sources of nitrogen that are favorable for growth, production of many, but not all, secondary metabolites is reduced [83–85]. One interpretation of this tendency is that by supplying a good source of nitrogen, the available carbon can be used for growth and generating biomass. Thus cell does not experience or experience of lesser extent of the stringent condition in the presence of suitable nitrogen source.

#### **4.3 Upsetting of zinc and iron homeostasis**

Zinc is an essential trace element and cofactor required for the structure and function of many proteins. Being important, it is under tight regulation by Znr, a zinc-responsive transcriptional repressor that regulates genes encoding a high-affinity uptake system for zinc, as well as zinc-free paralogues of ribosomal proteins in many bacteria, including streptomycetes [86, 87]. Znr also directly represses a promoter within the cluster of coelibactin, a non-ribosomally synthesized peptide predicted to have siderophore (metal-chelating) activity in *S. coelicolor* [88, 89]. AbsC, a pleiotropic regulator which is required for the production of ACT and RED (chromosomallyencoded antibiotics, the prodiginine complex RED, which is red in color), represses promoters of the coelibactin cluster under the specific condition of low zinc [88]. Although the underlying regulatory mechanism is still unknown, under low zinc concentration if upregulation of genes encoding a high-affinity uptake system for zinc via Znr does not work, AbsC potentially becomes active which increases the production of ACT and RED for antibiotics production.

Iron is another essential metal that is under tight regulation [90]. Members of the DmdR (divalent metal-dependent) family i.e., DmdR1 and DmdR2 are the key regulatory components of iron homeostasis in *S. coelicolor* [91, 92]. The dmdR1 gene overlaps with adm gene on the opposite strand and disruption of the overlapping gene increases the production of RED and ACT which leads to antibiotics production [91]. Although the details of the DmdR1/Adm system remain to be uncovered, it is likely that physiological cellular stress indirectly affects antibiotic production.
