**2. Control of microbial virulence and resistance**

### **2.1 Quorum sensing inhibition**

Quorum sensing (QS) is a cellular signaling system, which is developed in response to population cell density [3, 4]. QS cascade relays on the release of signaling molecules called QS autoinducers/signals. The QS signals are produced at low levels with the start of microbial growth and accumulate upon increase in the cell density. Quorum sensing signals coordinate the microbial virulence behaviors such as secretion of toxins, secretion of exoenzymes, microbial motility, adhesion and biofilm assembly [11]. Furthermore, microbial communication systems have been assigned in fungi [12] and viruses [13]. Studies of QS provide significant insights into different mechanisms that control the interactions in microbial communities and how these interactions affect microbial pathogenesis. Several QS systems are well understood including Gram-negative bacteria that produce acyl-homoserine lactone (AHL) signals, including *Pseudomonas aeruginosa*, *Vibrio* sp., *Acinetobacter baumannii* and *Serratia marcescens* [5, 14, 15]. Alternatively, Gram-positive species such as *Staphylococcus aureus* utilize autoinducer peptide (AIP)-based QS systems [16].

Various strategies for quorum sensing inhibition have been explored. The quorum sensing inhibition approaches could be accomplished via interference with the synthesis of QS signals, elimination of the signal accumulation and disruption of signal-receptor interaction [17–19].

#### *2.1.1 Interference with the synthesis of the autoinducing signals*

One of the main quorum sensing inhibiting approaches is the interference with the synthesis of the autoinducing signals [20]. AI-2 compounds are considered as "universal" signal molecules of Gram-negative and Gram-positive bacteria [14, 21]. Moreover, they are encountered in species communications. The biosynthesis of AI-2 requires two main enzymes: methylthioadenosine/S-adenosylhomocysteine nucleosidase (MTA/SAH nucleosidase) and LuxS. AI-2 molecules contribute in various virulence behaviors, biofilm formation and host-pathogen interaction. Therefore, targeting AI-2 elaborates broad spectrum quorum sensing inhibition [22, 23]. In this instance, Gutierrez group have identified the transition analogs, 5′-methylthio- (MT-), 5′-ethylthio-(EtT-) and 5′-butylthio- (BuT) DADMeimmucillin, which specifically bind and inhibit MTA enzymes in *Escherichia coli* O157:H7. Also, 4,5-dihydroxy-2,3-pentanedioneS-ribosyl-homocysteine analogs have been developed as competitive inhibitor of LuxS [24–26].

On other instance, inhibiting AHL-synthesis has been extensively studied, for instance, triclosan inhibited both N-3-oxo-dodecanoyl-l-homoserine lactone and N-butyryl-l-homoserine lactone [27, 28], anthranilate derivatives are a *Pseudomonas* quinolone signal inhibitors [28], and proanthocyanidins have been approved as inhibitor of LasI/RhlI AHL synthases expression [29]. Furthermore, precursors of *Pseudomonas* quinolone signals (PQS) such anthranilatic acid derivatives reduced the pathogenicity of *P. aeruginosa* in lung-infected mice [15].

#### *2.1.2 Elimination of the QS signals accumulation*

Other common strategy is eliminating the accumulation of the QS signals, which have been attained by degrading the QS signal using enzymes or through sequestering the signal by synthetic polymers [30, 31] or utilizing antibodies that bind with the signals. Synthesized monoclonal antibodies (AP4-24H11) by Park group provoke high binding affinity for sequestering AIP-IV and decrease α-hemolysin

**195**

*New Approaches for Competing Microbial Resistance and Virulence*

production in *S. aureus* with relief of abscess formation in the infected murine model [32]. Kaufmann and coauthors inhibited the *P. aeruginosa* QS cascade via development of AHL-specific monoclonal antibodies. Synthetic polymers such as itaconic acid sequester the signaling molecules AHL and attenuate QS in *V. fischeri*

Moreover, disturbing enzymes responsible for biosynthesis of QS signals is a chief method, which affects both production and accumulation of different signals and perturb quorum sensing circuit [30]. Acylases, lactonases and oxidoreductases are the widely identified enzymes that target AHLs. AHL lactonases are broad AHL degrading enzymes, which produce its effect via hydrolyzing the ester bond of the AHL ring [34]. Lactonases have been isolated from various *Bacillus* sp., which harbor *aiiA* (autoinducer inactivation gene) [35, 36]. Ulrich study showed that, the heterologous expression of *aiiA* in *Burkholderia thailandensis* and *P. aeruginosa* lowered the levels of AHL and QS-related virulence factors [37]. Other important AHL lactonases are AttM and AiiB, which have been isolated from *Agrobacterium* sp. [38], AhlD from *Arthrobacterium*, AhlK from *Klebsiella* [39] and AidC from *Chryseobacterium* [40], QsdA from *Rhodococcus erythropolis* strain W2 [41], AiiM of *Microbacterium testaceum* [42], AidH of *Ochrobactrum* sp. T63 [43] and QsdH of *Pseudoalteromonas yunnanensis* [44]. Furthermore, paraoxonases 1, 2 and 3 (PON1 to −3) are mammalian lactonases were identified in the airway epithelia and mam-

AHL acylases enzymes (*aiiD)* and homologs were found in *Ralstonia* [46], *Actinoplanes utahensis* and *Pseudomonas* sp. The purified AiiD protein has the ability to degrade 3OC10HSL into HSL and 3-oxodecanoic acid. In addition, PvdQ, QuiP and HacB are specific AHL acylases of *P. aeruginosa*, in addition, HacA and HacB acylases of *Pseudomonas syringae* [47, 48]. Furthermore, the broader substrate specificity of AHL acylase (AhlM) was detected in *Streptomyces* sp. strain M664

Oxidoreductases from *Rhodococcus erythropolis* inactivates AHLs (oxidation or reduction) with subsequent elimination of bacterial virulence *in vivo. Rhizobium* strain NGR234 possess diverse AHL-inactivation loci: *dhlR*, *qsdR1* and *qsdR2*, with lactonases activity, *aldR*, and *hydR-hitR* [50]. Enzymatic degradation of other QS autoinducers have been described: *carA* and *carB* from *Bacillus*, *E. coli* DH10B, *Staphylococcus* and *Pseudomonas* as the genes responsible for inhibition of DSF signaling [51]. Hod (3-hydroxy-2-methyl-4(1*H*)-quinolone 2,4-dioxygenase) stimulates the cleavage of PQS and attenuates PQS-regulated virulence factors. Roy and coauthors elicit the AI-2 activation activity of endogenous LsrK in *E. coli*, however, exogenously phosphorylation of AI-2 by LsrK eliminates its intracellular transport

Interference with signal detection through eliminating the QS signal-receptor binding represents a successful approach [53, 54]. Various synthetic and natural AHL analogs have been reported to block the binding of the signal with specific receptors in *P. aeruginosa* and *Vibrio* sp. The prototype signal inhibitors, halogenated furanones, which are produced from *Delisea pulchra* represent a good example [55, 56]. Natural analogs have been also isolated with signal-receptor interference including ajoene [57], eugenol [58], flavonoids [59], iberin [60], furocoumarins [61], ellagic acid, penicillanic acid and patulin [62], phenethyl amide [63] and

The synthetic furanone derivative C-30 interferes and hinders the interaction of AHLs with the receptors [65]. Other furanone analogs have been developed

with activity towards medium- and long-chain AHLs [49].

and hinders subsequent activation of AI-2 [52].

1H-pyrrole-2-carboxylic acid [64].

*2.1.3 Elimination of the QS signal-receptor interaction*

*DOI: http://dx.doi.org/10.5772/intechopen.90388*

[31, 33].

malian sera [45].

*Microorganisms*

**2.1 Quorum sensing inhibition**

of signal-receptor interaction [17–19].

*2.1.1 Interference with the synthesis of the autoinducing signals*

have been developed as competitive inhibitor of LuxS [24–26].

the pathogenicity of *P. aeruginosa* in lung-infected mice [15].

*2.1.2 Elimination of the QS signals accumulation*

One of the main quorum sensing inhibiting approaches is the interference with the synthesis of the autoinducing signals [20]. AI-2 compounds are considered as "universal" signal molecules of Gram-negative and Gram-positive bacteria [14, 21]. Moreover, they are encountered in species communications. The biosynthesis of AI-2 requires two main enzymes: methylthioadenosine/S-adenosylhomocysteine nucleosidase (MTA/SAH nucleosidase) and LuxS. AI-2 molecules contribute in various virulence behaviors, biofilm formation and host-pathogen interaction. Therefore, targeting AI-2 elaborates broad spectrum quorum sensing inhibition [22, 23]. In this instance, Gutierrez group have identified the transition analogs, 5′-methylthio- (MT-), 5′-ethylthio-(EtT-) and 5′-butylthio- (BuT) DADMeimmucillin, which specifically bind and inhibit MTA enzymes in *Escherichia coli* O157:H7. Also, 4,5-dihydroxy-2,3-pentanedioneS-ribosyl-homocysteine analogs

On other instance, inhibiting AHL-synthesis has been extensively studied, for instance, triclosan inhibited both N-3-oxo-dodecanoyl-l-homoserine lactone and N-butyryl-l-homoserine lactone [27, 28], anthranilate derivatives are a *Pseudomonas* quinolone signal inhibitors [28], and proanthocyanidins have been approved as inhibitor of LasI/RhlI AHL synthases expression [29]. Furthermore, precursors of *Pseudomonas* quinolone signals (PQS) such anthranilatic acid derivatives reduced

Other common strategy is eliminating the accumulation of the QS signals, which have been attained by degrading the QS signal using enzymes or through sequestering the signal by synthetic polymers [30, 31] or utilizing antibodies that bind with the signals. Synthesized monoclonal antibodies (AP4-24H11) by Park group provoke high binding affinity for sequestering AIP-IV and decrease α-hemolysin

**2. Control of microbial virulence and resistance**

Quorum sensing (QS) is a cellular signaling system, which is developed in response to population cell density [3, 4]. QS cascade relays on the release of signaling molecules called QS autoinducers/signals. The QS signals are produced at low levels with the start of microbial growth and accumulate upon increase in the cell density. Quorum sensing signals coordinate the microbial virulence behaviors such as secretion of toxins, secretion of exoenzymes, microbial motility, adhesion and biofilm assembly [11]. Furthermore, microbial communication systems have been assigned in fungi [12] and viruses [13]. Studies of QS provide significant insights into different mechanisms that control the interactions in microbial communities and how these interactions affect microbial pathogenesis. Several QS systems are well understood including Gram-negative bacteria that produce acyl-homoserine lactone (AHL) signals, including *Pseudomonas aeruginosa*, *Vibrio* sp., *Acinetobacter baumannii* and *Serratia marcescens* [5, 14, 15]. Alternatively, Gram-positive species such as *Staphylococcus aureus* utilize autoinducer peptide (AIP)-based QS systems [16]. Various strategies for quorum sensing inhibition have been explored. The quorum sensing inhibition approaches could be accomplished via interference with the synthesis of QS signals, elimination of the signal accumulation and disruption

**194**

production in *S. aureus* with relief of abscess formation in the infected murine model [32]. Kaufmann and coauthors inhibited the *P. aeruginosa* QS cascade via development of AHL-specific monoclonal antibodies. Synthetic polymers such as itaconic acid sequester the signaling molecules AHL and attenuate QS in *V. fischeri* [31, 33].

Moreover, disturbing enzymes responsible for biosynthesis of QS signals is a chief method, which affects both production and accumulation of different signals and perturb quorum sensing circuit [30]. Acylases, lactonases and oxidoreductases are the widely identified enzymes that target AHLs. AHL lactonases are broad AHL degrading enzymes, which produce its effect via hydrolyzing the ester bond of the AHL ring [34]. Lactonases have been isolated from various *Bacillus* sp., which harbor *aiiA* (autoinducer inactivation gene) [35, 36]. Ulrich study showed that, the heterologous expression of *aiiA* in *Burkholderia thailandensis* and *P. aeruginosa* lowered the levels of AHL and QS-related virulence factors [37]. Other important AHL lactonases are AttM and AiiB, which have been isolated from *Agrobacterium* sp. [38], AhlD from *Arthrobacterium*, AhlK from *Klebsiella* [39] and AidC from *Chryseobacterium* [40], QsdA from *Rhodococcus erythropolis* strain W2 [41], AiiM of *Microbacterium testaceum* [42], AidH of *Ochrobactrum* sp. T63 [43] and QsdH of *Pseudoalteromonas yunnanensis* [44]. Furthermore, paraoxonases 1, 2 and 3 (PON1 to −3) are mammalian lactonases were identified in the airway epithelia and mammalian sera [45].

AHL acylases enzymes (*aiiD)* and homologs were found in *Ralstonia* [46], *Actinoplanes utahensis* and *Pseudomonas* sp. The purified AiiD protein has the ability to degrade 3OC10HSL into HSL and 3-oxodecanoic acid. In addition, PvdQ, QuiP and HacB are specific AHL acylases of *P. aeruginosa*, in addition, HacA and HacB acylases of *Pseudomonas syringae* [47, 48]. Furthermore, the broader substrate specificity of AHL acylase (AhlM) was detected in *Streptomyces* sp. strain M664 with activity towards medium- and long-chain AHLs [49].

Oxidoreductases from *Rhodococcus erythropolis* inactivates AHLs (oxidation or reduction) with subsequent elimination of bacterial virulence *in vivo. Rhizobium* strain NGR234 possess diverse AHL-inactivation loci: *dhlR*, *qsdR1* and *qsdR2*, with lactonases activity, *aldR*, and *hydR-hitR* [50]. Enzymatic degradation of other QS autoinducers have been described: *carA* and *carB* from *Bacillus*, *E. coli* DH10B, *Staphylococcus* and *Pseudomonas* as the genes responsible for inhibition of DSF signaling [51]. Hod (3-hydroxy-2-methyl-4(1*H*)-quinolone 2,4-dioxygenase) stimulates the cleavage of PQS and attenuates PQS-regulated virulence factors. Roy and coauthors elicit the AI-2 activation activity of endogenous LsrK in *E. coli*, however, exogenously phosphorylation of AI-2 by LsrK eliminates its intracellular transport and hinders subsequent activation of AI-2 [52].

#### *2.1.3 Elimination of the QS signal-receptor interaction*

Interference with signal detection through eliminating the QS signal-receptor binding represents a successful approach [53, 54]. Various synthetic and natural AHL analogs have been reported to block the binding of the signal with specific receptors in *P. aeruginosa* and *Vibrio* sp. The prototype signal inhibitors, halogenated furanones, which are produced from *Delisea pulchra* represent a good example [55, 56]. Natural analogs have been also isolated with signal-receptor interference including ajoene [57], eugenol [58], flavonoids [59], iberin [60], furocoumarins [61], ellagic acid, penicillanic acid and patulin [62], phenethyl amide [63] and 1H-pyrrole-2-carboxylic acid [64].

The synthetic furanone derivative C-30 interferes and hinders the interaction of AHLs with the receptors [65]. Other furanone analogs have been developed

including S-phenyl-l-cysteine sulfoxide and diphenyl disulfide [66] and tetrazole derivatives [67]. Furthermore, synthetic LasR derivatives have been developed such as indole derivatives, non-AHL-like antagonists [68], the synthesized azines derivatives, 4-(alkyloxy)-6-methyl-2H-pyran-2-one [69] and aspirin [70]. Triphenyl hybridγ-butyrolactones and cyclopentanones derivatives are potent inhibitors of LuxR [71]. Putative LasI inhibitors have been identified using molecular docking methods including the trans-cinnamaldehyde [72], (z)-5-octylidenethiazolidine-2, 4-dione [73] and fatty acyl purified from marine *Streptomyces* sp. [74]. Additionally, meta-bromo-thiolactone is a potent inhibitor of RhiI and subsequent PQS cascade [11].

In *S. aureus*, the interference with agr system has been accomplished using solonamide A and B that are cyclodepsipeptides derivatives, which purified from marine *Photobacterium* and reduced the expression of *hla* and RNAIII. Solonamide can act through competitive inhibition of agr system such as *S. aureus* agr system via structure similarity to the AIPs [75]. Other *S. aureus* quorum-sensing inhibitors have been identified including linear peptidomimetics as competitive inhibitors to AgrC [76], savirin as potent inhibitor of AgrA [77] and the polyhydroxy anthraquinone ω-hydroxyemodin as inhibitor of AgrA [78].
