**4.1 Chaperones**

Bacterial chaperones like DnaK, belonging to the heat shock proteins (Hsp)70 family, are produced by cells in response to exposure to stressful conditions [65]. The interaction between the two domains of such Hsp70, namely ATPase and the substrate-binding domain, triggers the chaperone-based activity of DnaK which are also enhanced by the co-chaperone such as DnaJ (Hsp40 family) and chaperone GroE (Hsp60 family) [66]. DnaK acts on unfolded and partially folded protein chains by binding and controlling their configuration [67].

Besides stress response, DnaK plays a significant housekeeping role in maintaining normal bacterial cellular growth and homeostasis [68]. Thus, any

*Strategic Role Players of Important Antimicrobial-Resistant Pathogens DOI: http://dx.doi.org/10.5772/intechopen.92742*

alterations in the *dnaK* gene reduce the growth of bacteria within the host [69]. In fact, during infection, bacteria activate their heat shock genes like DnaK to protect their cellular machinery from the consequently activated host immune system for defense mechanisms and thereby strengthen their virulence strategy [69]. This phenomenon, thus, provides an insight into structural mechanism of DnaK, leading to misfolding and its role in controlling protein activity contributing to the pathogenicity of multidrug-resistant bacteria, such as the opportunistic human pathogen *A. baumannii* [70]. In fact, DnaK mutants showed decreased viability and improved susceptibility under strained circumstances during systemic infection as reported for *dnaK* mutants of *S. aureus* with increased sensitivity to oxacillin and methicillin [71] and *dnaK/dnaJ* mutants of *E. coli* having increased sensitivity to fluoroquinolones [72].

#### **4.2 Efflux pumps**

benzalkonium chloride concentrations [55]. In fact, the EP protein, SugE, in *Enterobacter cloacae,* which is a member of small multidrug resistance (SMR) protein family, has been found to be responsible for resistance against toxic compounds such as cetylpyridinium chloride and benzalkonium chloride [56]. Another EP protein of the resistance nodulation cell division (RND) category, AcrB, has also been found to be very essential in the pathogenicity and antibiotics resistance of

*Antimicrobial Resistance - A One Health Perspective*

Several health interventions have been proposed as alternatives to current antibiotic therapy and prevent the resistant mechanisms of which, the development of new drug classes, use of vaccines or other therapeutic strategies are noteworthy (**Figure 4**) [59]. In fact, using computational approaches, certain proteins and/or phenotypes, having plausible involvement in antibiotic resistance, are proposed

Bacterial chaperones like DnaK, belonging to the heat shock proteins (Hsp)70 family, are produced by cells in response to exposure to stressful conditions [65]. The interaction between the two domains of such Hsp70, namely ATPase and the substrate-binding domain, triggers the chaperone-based activity of DnaK which are also enhanced by the co-chaperone such as DnaJ (Hsp40 family) and chaperone GroE (Hsp60 family) [66]. DnaK acts on unfolded and partially folded protein

Besides stress response, DnaK plays a significant housekeeping role in maintaining normal bacterial cellular growth and homeostasis [68]. Thus, any

chains by binding and controlling their configuration [67].

*Alternative strategies to combat antimicrobial resistance and their mechanisms of action.*

*E. cloacae* [57, 58].

**4. The role players**

[60–64] as discussed below.

**4.1 Chaperones**

**Figure 4.**

**10**

Antibiotic resistance can be triggered, in MDR bacteria, by four discrete mechanisms viz. target modification, reduced permeability and improved efflux, drug inactivation and drug extrusion by the multidrug efflux pumps (EP) [73]. Due to their poly-substrate specificity, besides having the potential to expel a broad variety of antibiotics, these EP also manage the development of other resistance mechanisms by decreasing intracellular antibiotics concentration and stimulating mutation accumulation [73]. Consequently, over-expression of multidrug EP is involved with clinically related antibiotic resistance. Thus, there has been increasing evidence of EP having biochemical functions in bacteria along with their appearance under strict regulations in response to some physiological and environmental signals [73]. Hence, a systematic knowledge of EP is important for the development of EP inhibitors as promising AMR intervention strategies.

EP are present in almost all bacterial species involved in AMR. They can be located on plasmids or chromosomes that encode this class of proteins. The five families of bacterial EP, found to be involved in MDR, are the major facilitator superfamily (MFS), the multidrug and toxic compound extrusion (MATE) family, the ATP-binding cassette (ABC) superfamily, the small multidrug resistance (SMR) family, and the resistance-nodulation-division (RND) family, based on their composition, energy sources and substrates used [73]. Importantly, only RND superfamily is found in Gram-negative bacteria due to its structure containing tripartite complex and the efflux systems of the other four families are widely distributed in both Gram-positive and -negative bacteria. These EP can be either single or multiple-component transporters depending on their specific classes. They comprise both an inner and an outer membrane transporter, like the RND type. It has been found that RND family pumps are frequently associated with therapeutically important bacterial resistance such as AcrB in *S.* Typhimurium and *E. coli* and MexB in *P. aeruginosa* owing to their tripartite complex, enabling various drugs to be immediately extruded from cytoplasm to outside the bacterial cells [74].

In fact, antibiotics such as fluoroquinolone, tetracycline, rifampin, novobiocin, chloramphenicol and B-lactams were used to analyze the substrate profile of housekeeping efflux system AcrAB-TolC in *E. coli* [74]. Similarly, the *S.* Typhimurium AcrAB-TolC efflux system was also capable of expelling various antibacterial agents such as tetracycline, quinolones and chloramphenicol [75, 76]. The two RND efflux pumps, MexAB-OprM and MexXY-OprM, homolog to AcrAB-TolC system in *E. coli*, are also expressed in *P. aeruginosa*. Thus, these systems can actively export chloramphenicol, tetracycline and fluoroquinolones. In addition to these substrates, MexAB-OprM export B-lactams and novobiocin whereas MexXY system exports aminoglycosides (**Table 1**) [77].


triggered by ArcAB-TCS system in the M9 glucose medium [84, 85]. Similar to *E. coli*, MdtABC and AcrD are also stimulated by the *Salmonella* BaeSR TCS in

**Bacteria Inhibitors TCS Mechanisms Reference**

complex

DHp domain of HK

Algr1-2 Inhibition of phosphorylation/

signal transduction

Bis-phenol VanR-S — [90]

Inhibition of key interacting residues of

dephosphorylation of Algr2 Inhibition of DNA-binding activity of Algr1

VanR-S Inhibition of autophosphorylation [90]

Affects membrane fluidity, disturbing

WalK-R Binds to the HK cytoplasmic domain for the inhibition of autophosphorylation

PhoP-Q Inhibition of formation of the PhoP-DNA

[89]

[90]

[90]

[90]

[90]

XR770 BaeSR

*Strategic Role Players of Important Antimicrobial-Resistant Pathogens*

NSC9608 (8 compounds, NCI library)

*DOI: http://dx.doi.org/10.5772/intechopen.92742*

Thiazole derivatives

Thiazole derivatives

*Representative TCS targets with their known inhibitors.*

Walkmycin B and Waldiomycin

Salicylanilide KinA/

Spo0F

OmpR/ EnvZ

Again, PhoPQ TCS, the core virulence regulator in *Salmonella*, controls the activation of the RND type MacAB pump [87, 88]. TCS was also revealed to be involved in regulating efflux pumps in other species. In *A. baumannii*, the expression of RND type efflux pump AdeABC has been reported to be regulated by AdeRS-TCS (**Table 2**). The AdeRS-TCS regulatory system is encoded by *adeRS* genes, being positioned in the upstream region of *adeABC* genes [91]. Inactivation of AdeR or AdeS resulted in *A. baumannii* being susceptible to aminoglycosides which are the substrates of this pump, indicating the vital role of AdeRS in *adeABC* activity. The nature of these inducing signals and the AdeRS activation mechanism,

TCS can play an important role in drug discovery. There are several ways to target TCS proteins. Of these, the structure-based virtual screening (SBVS) analysis is carried out using compound databases containing a broad range of prospective

Biofilms, in both single and multi-species groups, communicate and cooperate to perform complex processes with each other and their environment [94]. With the scientists aiming to understand the intercellular interactions that encourage the development of biofilms, they are presently a serious health issue, playing a major role in abiotic device-related diseases such as catheters, prosthetic valves and con-

Biofilm formation can be explored in different stages comprising (a) distinctive adhesion of the planktonic bacteria (PB) to a solid surface [96], (b) micro-colonies

inhibitors, including structures known to be antibacterial [93].

response to metal ions [86].

*Salmonella enterica* Typhi and/or Typhimurium

*Pseudomonas aeruginosa*

*Enterococcus faecium*

*Staphylococcus aureus*

*Methicillin-resistant Staphylococcus aureus*

**Table 2.**

however, remain unclear [92].

**4.4 Biofilms**

tact lenses [95].

**13**

*means unknown.*

#### **Table 1.**

*Selected multidrug resistance efflux pump regulators.*

#### **4.3 Two-component systems**

Two component systems (TCS) are commonly found in bacteria, allowing them to respond to various fluctuations in the environment. Canonically, TCS are composed of a response regulator (RR) and a histidine kinase (HK) [78]. The membrane associated HKs can detect and transform various environmental sensations by autophosphorylation. The HKs can then transphosphorylate their cognate partners, the RRs, which then influence the expression of downstream genes to affect the concerned phenotype [78].

A thorough investigation of the correlation between efflux pumps and TCSs in *E. coli*, revealed the involvement of several RRs in drug resistance [79]. Among them, *mdtABC* and *acrD* expression was triggered by the BaeSR and CpxAR TCS in response to indole [80, 81] and envelope stress [79], respectively, while no signals were detected when the EvgSA TCS triggered the activation of EmrKY and MdtEF [82, 83]. Moreover, the expression of the MdtEF efflux pump was


*Strategic Role Players of Important Antimicrobial-Resistant Pathogens DOI: http://dx.doi.org/10.5772/intechopen.92742*

#### **Table 2.**

*aureus*

*Representative TCS targets with their known inhibitors.*

triggered by ArcAB-TCS system in the M9 glucose medium [84, 85]. Similar to *E. coli*, MdtABC and AcrD are also stimulated by the *Salmonella* BaeSR TCS in response to metal ions [86].

Again, PhoPQ TCS, the core virulence regulator in *Salmonella*, controls the activation of the RND type MacAB pump [87, 88]. TCS was also revealed to be involved in regulating efflux pumps in other species. In *A. baumannii*, the expression of RND type efflux pump AdeABC has been reported to be regulated by AdeRS-TCS (**Table 2**). The AdeRS-TCS regulatory system is encoded by *adeRS* genes, being positioned in the upstream region of *adeABC* genes [91]. Inactivation of AdeR or AdeS resulted in *A. baumannii* being susceptible to aminoglycosides which are the substrates of this pump, indicating the vital role of AdeRS in *adeABC* activity. The nature of these inducing signals and the AdeRS activation mechanism, however, remain unclear [92].

TCS can play an important role in drug discovery. There are several ways to target TCS proteins. Of these, the structure-based virtual screening (SBVS) analysis is carried out using compound databases containing a broad range of prospective inhibitors, including structures known to be antibacterial [93].

#### **4.4 Biofilms**

**4.3 Two-component systems**

*Selected multidrug resistance efflux pump regulators.*

*Staphylococcus aureus*

*Adapted from [73]. means unknown.*

**Table 1.**

**12**

concerned phenotype [78].

Two component systems (TCS) are commonly found in bacteria, allowing them to respond to various fluctuations in the environment. Canonically, TCS are composed of a response regulator (RR) and a histidine kinase (HK) [78]. The membrane associated HKs can detect and transform various environmental sensations by autophosphorylation. The HKs can then transphosphorylate their cognate partners, the RRs, which then influence the expression of downstream genes to affect the

QacA MATE MepR Chlorhexidine, cetrimide, dequalinium

**Efflux pump Pump type Regulator Regulator family Inducible signal**

MexAB RND MexR MarR Superoxide stress

MexCD RND NfxB LacI/GalR Biocide chlorhexidine MexEF RND MetT LysR Chloramphenicol, GSNO

AcrD RND BaeSR TCS Indole, zinc, copper

MdtABC RND BaeSR TCS Indole, zinc, copper

MepA MFS QacR TetR Rhodamine 6G, TPP

MacAB ABC PhoQP TCS Magnesium MdsABC RND GolS MerR Gold

MexXY RND MexZ TetR Tetracycline, erythromycin, gentamicin

RamA AraC Indole, bile salts

CpxAR TCS Indole, zinc, copper

CpxAR TCS Indole, zinc, copper

NalD TetR

RamR TetR SoxS AraC AcrR TetR

AdeABC RND AdeRS TCS

*Antimicrobial Resistance - A One Health Perspective*

AcrAB RND MarA AraC

AcrEF RND AcrS TetR

*Acinetobacter baumannii*

*Pseudomonas aeruginosa*

*Salmonella Typhimurium*

A thorough investigation of the correlation between efflux pumps and TCSs in *E. coli*, revealed the involvement of several RRs in drug resistance [79]. Among them, *mdtABC* and *acrD* expression was triggered by the BaeSR and CpxAR TCS in response to indole [80, 81] and envelope stress [79], respectively, while no signals were detected when the EvgSA TCS triggered the activation of EmrKY and

MdtEF [82, 83]. Moreover, the expression of the MdtEF efflux pump was

Biofilms, in both single and multi-species groups, communicate and cooperate to perform complex processes with each other and their environment [94]. With the scientists aiming to understand the intercellular interactions that encourage the development of biofilms, they are presently a serious health issue, playing a major role in abiotic device-related diseases such as catheters, prosthetic valves and contact lenses [95].

Biofilm formation can be explored in different stages comprising (a) distinctive adhesion of the planktonic bacteria (PB) to a solid surface [96], (b) micro-colonies

(MC) formation surrounded by protective secreted molecules known as the matrix of extra polymeric substances (EPS) having up to 97% water as the main component [97] and (c) dispersal including shedding of PB or MC from the mature biofilm [97]. The last phase can encourage further biofilm colonization of the host which can eventually benefit the bacteria with a limited supply of nutrients and waste accumulation [97]. Importantly, the transition from planktonic growth to surface life is triggered by several environmental signals known as various stresses for the bacteria based on their ecological niche [98]. These include UV radiation, pH changes, oxygen tension, osmolarity, iron availability, temperature, nutrient supply and desiccation [98], which may disrupt their fundamental functions such as growth and survival capability. The environmental indications, however, vary significantly between organisms. Thus, *P. aeruginosa* will form biofilms under most circumstances [99, 100] while *E. coli* O157:H7 produce a biofilm under low-nutrient conditions only [101].

Recent advances in biofilm research have proven its connection to various pathways and proteins [61]. For instance, defects in MDR EP activity reduced the biofilm formation and thus, EP inhibitors have been employed as a promising biofilm inhibition approach for strains of *E. coli* and *Klebsiella* [102], *Salmonella* [103], *P. aeruginosa* and *S. aureus* [104]. However, certain other reports show that despite the elimination of planktonic cells through pharmacological intervention, the sessile forms are resistant and continue to proliferate within the biofilm [105]. This is more of prominence on abiotic surfaces [95], such as catheters [106], contact lenses [107] and prosthetic cardiac valves [108]. Thus, alginate mucoids, with EPS overexpression, from *P. aeruginosa* species isolated from cystic fibrosis patients, were found to improve AMR by promoting the biofilm formation [109].

#### **5. Conclusion**

The constant increase in AMR is a significant public health concern that needs to be addressed now. This review starts with an introduction to AMR followed by the threats from the clinically important MDR pathogens and their rise. With the existing management strategies for MDR by the scientists still ongoing, we have taken up this study to propose an integrated approach to deal with MDR threats. Thus, the review ends with new connections of important bacterial components with MDR.

**Author details**

Malaysia

**15**

Shama Mujawar1†, Bahaa Abdella1,2† and Chandrajit Lahiri<sup>1</sup>

*Strategic Role Players of Important Antimicrobial-Resistant Pathogens*

*DOI: http://dx.doi.org/10.5772/intechopen.92742*

\*Address all correspondence to: chandrajitl@sunway.edu.my

† These authors have contributed equally to the work.

provided the original work is properly cited.

1 Department of Biological Sciences, Sunway University, Petaling Jaya, Selangor,

© 2020 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium,

2 Faculty of Aquatic and Fisheries Sciences, Kafrelsheikh University, Egypt

\*

#### **Acknowledgements**

The authors acknowledge the support of Sunway University, Selangor, Malaysia for providing the computational facilities and wishes to thank Hend Salah for the valuable contribution in developing the artwork for the concept provided.

#### **Conflict of interest**

The authors declare no conflict of interest.

*Strategic Role Players of Important Antimicrobial-Resistant Pathogens DOI: http://dx.doi.org/10.5772/intechopen.92742*
