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Review Article Bacterial Resistance Mechanism against Oxidative Stress Artee Mishra* and Kaushala Prasad Mishra Division of life Science, Research Cen...

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Review Article Bacterial Resistance Mechanism against Oxidative Stress Artee Mishra* and Kaushala Prasad Mishra Division of life Science, Research Center, Nehru Gram Bharti University, Allahabad- 211002

ABSTRACT From the last decades, oxidative stress has been one of the revolutionary subjects among the biological researcher in whole world. It is an imbalance mechanism between peroxidants and antioxidants in the body. Several reason can be assigns for its importance; knowledge about reproduction and metabolism of the reactive oxygen and nitrogen species. Several bacteria mutant have allowed the demonstrations of variety of critical gene for enzymatic defense and DNA repair as well as an oxyR regulon system. At a certain steady state level of reactive oxygen species are continuously produced and eliminated by microorganism normally maintaining ROS. Regulation of membrane permeability and antioxidant potential are two principle mechanism used by bacteria to protect against oxidative stress. Microorganism continuously deal with stress situation such stress may include change environment pH, temperature, humidity etc., a wide variety of environment stimulate including nutritional stress, chemicals stress and radiation. Surviving in adverse condition bacteria have developed sensory system that facilitate to change in the environment condition. Keywords: Oxidative stress, ROS, Microorganism, Resistance

Address for Correspondence: Artee Mishra Division of life Science, Research Center, Nehru Gram Bharti University, Allahabad- 211002 E mail: [email protected] Conflict of Interest: None Declared!

QR Code for Mobile Users (Received 6 February 2015; Accepted 7 March 2015; Published 30 April 2015) ISSN: 2347-8136 ©2014 JMPI

INTRODUCTION The bacterial stress response enables bacteria to survive adverse and fluctuating conditions in their immediate surroundings. Various bacterial mechanisms recognize different environmental changes and mount an appropriate response (2). A bacterial cell can react simultaneously to a wide variety of stresses and the various stress response systems interact with each other by a complex of global regulatory networks. Bacteria can survive under diverse environmental conditions and in order to overcome these adverse and changing conditions, bacteria must sense the changes and mount appropriate responses in gene expression and protein activity. The stress response in bacteria involves a complex network of elements that acts against the external stimulus. Bacteria can react simultaneously to a variety of stresses and the

various stress response systems interact (crosstalk) with each other (5). A complex network of global regulatory systems leads to a coordinated and effective response. These regulatory systems govern the expression of more effectors that maintain stability of the cellular equilibrium under the various conditions. Stress response systems can play an important role in the virulence of pathogenic organisms (24). In bacteria some of the most important stress response systems are (i) Heat shock response, controlled by the sigma factor sigma 32. (ii) Envelope stress response, controlled mainly by the sigma factor sigma E and the Cpx twocomponent system.(iii)Cold shock response, which governs expression of RNA chaperones and ribosomal factors. (iv)General stress response, which depends on the sigma factor

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sigma S. (v) (p)ppGpp-dependent stringent response which reduces the cellular protein synthesis capacity and controls further global responses upon nutritional downshift (34). Sigma factor (σ factor) is a protein needed only for initiation of RNA synthesis. It is a bacterial transcription initiation factor that enables specific binding of RNA polymerase to gene promoters. The specific sigma factor used to initiate transcription of a given gene will vary, depending on the gene and on the environmental signals needed to initiate transcription of that gene. Every molecule of RNA polymerase holoenzyme contains exactly one sigma factor subunit, which in the model bacterium Escherichia coli is one of those listed below (30). The number of sigma factors varies between bacterial species. Bacteria and especially those capable of persisting in diverse environments, such as Escherichia coli provide particularly valuable models for exploring how single-celled organisms respond to environmental stresses. For example, most bacteria associated with foodborne infections (e.g., some E. coli serotypes, Salmonella enterica serovar Typhimurium, Listeria monocytogenes) can survive under diverse conditions, both inside and outside of the host. To survive extreme and rapidly changing conditions, bacteria must sense the changes and then respond with appropriate alterations in gene expression and protein activity (50). Therefore, one important scientific challenge is to identify mechanisms that control the switch or switches that allow free-living bacteria to adjust to and invade a host organism. These „stresses‟ typically elicit protective and/or adaptive responses that serve to enhance bacterial survivability. Because they impact upon many of the same cellular components and processes that are targeted by antimicrobials, adaptive stress responses can influence antimicrobial susceptibility. In targeting and interfering with key cellular processes, antimicrobials themselves are „stressors‟ to which protective stress responses have also evolved. Oxidative stress: It reflects an imbalance between the systemic manifestation of reactive oxygen species and a biological system's ability to readily detoxify the reactive intermediates or to repair the resulting damage. Disturbances in the normal redox state of cells can cause toxic effects through the production of peroxides and free radicals that damage all components of the cell, including proteins, lipids, and DNA (41). Further, some reactive oxidative

species act as cellular messengers in redox signaling. Thus, oxidative stress can cause disruptions in normal mechanisms of cellular signaling. Production of reactive oxygen species is a particularly destructive aspect of oxidative stress. Such species include free radicals and peroxides. Some of the less reactive of these species (such as superoxide) can be converted by oxidoreduction reactions with transition metals or other redox cycling compounds (including quinones) into more aggressive radical species that can cause extensive cellular damage(30). Most long term effects are caused by damage to DNA. It is the inevitable consequence of living in an oxygenrich environment, occurs when the cellular redox balance is upset by increased doses of reactive oxygen species (ROS). Microorganisms living in aerobic environments are constantly exposed to ROS, which are generated by the aerobic metabolism and environmental agents. ROS, including superoxide radical (O2), hydrogen peroxide (H2O2) and hydroxyl radical (NOH), are highly reactive molecules that can damage key cellular components, including DNA, proteins, carbohydrates and lipids (23). Reactive oxygen species (ROS): it is chemically reactive molecule containing oxygen and peroxides. ROS formed as a natural byproduct of the normal metabolism of oxygen and have a important role in cell signaling and homeostasis (3). To defend themselves against ROS injuries, microorganisms have developed different constitutive and inducible mechanisms, including scavenging systems, like superoxide dismutases (SOD) and catalases/peroxidases, export of redox-cycling substances, like the AcrAB-TolC efflux pump, and DNA repair systems, like DNA glycosilases. One possibility is that multiple redox-active flavoproteins all contribute a small portion to the overall production of oxidants under normal conditions. Other enzymes capable of producing superoxide are xanthine oxidase, NADPH oxidases and cytochromes P450. Hydrogen peroxide is produced by a wide variety of enzymes including several oxidases. Reactive oxygen species play important roles in cell signalling, a process termed redox signaling (4). Thus, to maintain proper cellular homeostasis, a balance must be struck between reactive oxygen production and consumption. The best studied cellular antioxidants are the enzymes superoxide dismutase (SOD), catalase, and glutathione peroxidase. Less well studied (but probably just as important) enzymatic antioxidants are the peroxiredoxins and the recently

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discovered sulfiredoxin. Other enzymes that have antioxidant properties (though this is not their primary role) include paraoxonase, glutathione-S transferases, and aldehyde dehydrogenases. The amino acid methionine is prone to oxidation, but oxidized methionine can be reversible (45). Oxidation of methionine is shown to inhibit the phosphorylation of adjacent Ser/Thr/Tyr sites in proteins. This gives a plausible mechanism for cells to couple oxidative stress signals with cellular mainstream signaling such as phosphorylation(47). Free radicals are highly reactive and are capable of damaging almost all types of biomolecules (Proteins, lipids, carbohydrates & nucleic acid). The fact is that free radicals beget free radicals i.e. generate free radicals from normal compounds which continues as a chain reaction. Oxidative stress can arise when cell cannot adequately destroy the excess free radicals formed. These free radicals can damage cell membranes and lipoproteins by a process called as lipid peroxidation(10). Proteins may also be damaged by ROS/NOS, leading to structural changes and loss of enzyme activity. Free radicals may cause DNAstrand breaks which can cause cell mutation. The body has several mechanisms to counteract these attacks by using DNA repair enzymes and orantioxidants (22).

present at high concentrations, maintain a strong reducing environment in the cell, and its reduced form is maintained by glutathione reductase using NADPH as a source of reducing power. Cells posses number of enzyme and repair activity in response to elevated level of antioxidants provide bulk protection against deleterious reaction involving active oxygen in bacteria such as: Oxidant •O2 , superoxideanion

H2O2, hydrogen peroxide

•OH, hydroxyl radical

ROOH, organic hydroperoxide RO•, alkoxy and ROO•, peroxy radicals

HOCl,hypochlorous acid

Figure1: Pathway of ROS production and clearance

Response of the cells: Bacterial cell involved to distinct stress response peroxides stimulon and superoxide stimulon responds to oxidative stress condition (1). Anticipation from oxidative damage bacteria have enzymatic component which directly scavenges active oxygen species or may act by producing non enzymatic antioxidants. Some molecules are constitutively present and help to maintain an intracellular reducing environment or to scavenge chemically reactive oxygen. Among these molecules are nonenzymatic antioxidants such as NADPH and NADH pools, β-carotene, ascorbic acid, βtocopherol, and glutathione (GSH) (26). GSH,


Description One-electron reduction state of O2, formed in many autoxidation reactions and by the electron transport chain. Rather unreactive but can release Fe2+ from iron-sulfur proteinsand ferritin. Undergoes dismutation to form H2O2 spontaneously or by enzymatic catalysis and is a precursor for metal-catalyzed •OH formation. Two-electron reduction state, formed by dismutation of •O−2 or by direct reduction of O2. Lipid soluble and thus able to diffuse across membranes. Three-electron reduction state, formed by Fenton reaction and decomposition of peroxynitrite. Extremely reactive, will attack most cellular components Formed by radical reactions with cellular components such as lipids and nucleobases. Oxygen centred organic radicals. Lipid forms participate in lipid peroxidation reactions. Produced in the presence of oxygen by radical addition to double bonds or hydrogen abstraction. Formed from H2O2by myeloperoxidase. Lipid soluble and highly reactive. Will readily oxidize protein constituents, including thiol groups, amino groups andmethionine. Formed in a rapid reaction between •O−2 and NO•. Lipid soluble and similar in reactivity to hypochlorous acid. Protonation forms peroxynitrous acid, which can undergo homolytic cleavage to form hydroxyl radical and nitrogen dioxide.

Catalase and Superoxidase dismutase (SOD): Oxygen is sometimes toxic. Small amounts of superoxide free radicals are formed during the normal respiration of organisms that use oxygen as the final electron acceptor. Obligate anaerobes from some oxygen free radicals that are toxic to the cell (49). Hence, if bacteria want to grow in

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oxygen environment, enzymes like catalase and superoxidase dismutase must be present for neutralization of the toxic form of oxygen (oxygen radical). During normal aerobic respiration, hydrogen ions are produced and have to be removed by bacterial cell. The electron transport system (ETS) in cellular respiration (a part of glycolysis) involves these H+ ions and combines them with oxygen to form water (12). Water is harmless. Energy is given off and stored in the form of Adenosine Triphosphate. Hydrogen Peroxide is formed by the cytochromes in ETS (13). Water being harmless is not required to be removed by the bacteria. So, H2O2 is harmful to bacteria cell that requires it to be removed instantly. Functions of catalase to protected bacteria from toxic hydrogen peroxide (H2O2) accumulation, which can occur during aerobic metabolism (14). Glutathione reductase: Glutathione plays a key role in maintaining proper function and preventing oxidative stress in human cells. It can act as a scavenger for hydroxyl radicals, singlet oxygen, and various electrophiles. Reduced glutathione reduces the oxidized form of the enzyme glutathione peroxidase, which in turn reduces hydrogen peroxide (H2O2), a dangerously reactive species within the cell (25). In addition, it plays a key role in the metabolism and clearance of xenobiotics, acts as a cofactor in ceratin detoxifying enzymes, participates in transport, and regenerates antioxidants such and Vitamins E and C to their reactive forms. The ratio of GSSH/GSH present in the cell is a key factor in properly maintaining the oxidative balance of the cell, that is, it is critical that the cell maintains high levels of the reduced glutathione and a low level of the oxidized Glutathione disulfide. This narrow balance is maintained by glutathione reductase, which catalyzes the reduction of GSSG to GSH. Glutathione reductase (GR) also known as glutathione-disulfide reductase (GSR) is an enzyme that in humans is encoded by the GSR gene (7). Glutathione reductase catalyzes the reduction of glutathione disulfide (GSSG) to the sulfhydryl form glutathione (GSH), which is a critical molecule in resisting oxidative stress and maintaining the reducing environment of the cell. Glutathione reductase functions as dimeric disulfide oxidoreductase and utilizes an FAD prosthetic group and NADPH to reduce one mole of GSSG to two moles of GSH (20).

Figure 2: Diagrammatic presentation of Reduced glutathione reductase, glutathione peroxidase and glutathione interact to reduce hydrogen peroxide to water, in order to protect the cell from oxidative damage(7)

Reduced glutathione reductase, glutathione peroxidase and glutathione interact to reduce hydrogen peroxide to water, in order to protect the cell from oxidative damage. Prokaryotic cells contain catalysts able to repair directly some covalent modifications to the primary structure of proteins. One of the most frequent modifications is the reduction of oxidized disulfide bonds: (i) thioredoxin reductase transfers electrons from NADPH to thioredoxin via a flavin carrier, (ii) glutaredoxin is also able to reduce disulfide bonds, but using GSH as an electron donor and, (iii) protein disulfide isomerase facilitates disulfide exchange reactions with large inactive protein substrates, besides having chaperone activity. Oxidation of methionine to methionine sulfoxide can be repaired by methionine sulfoxide reductase(25). Efflux pumps: are proteinaceous transporters localized in the cytoplasmic membrane of all kinds of cells. Some are primary active transporters utilizing Adenosine triphosphate hydrolysis as a source of energy, whereas others are secondary active transporters (uniporters, symporters, or antiporters) in which transport is coupled to an electrochemical potential difference created by pumping out hydrogen or sodium ions outside the cell(9). In bacterial efflux systems, certain substances that need to be extruded from the cell include surface components of the bacterial cell (e.g. capsular polysaccharides, lipopolysaccharides, and teichoic acid), proteins involved in bacterial pathogenesis (e.g. hemolysis, heme-binding protein, and alkaline protease), heme, hydrolytic enzymes, S-layer proteins, competence factors, toxins, antibiotics, bacteriocins, peptide antibiotics, drugs and siderophores. Bacterial efflux transporters are classified into five major superfamilies, based on the amino acid sequence and the energy source used to export their substrates (40).

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The major facilitator superfamily (MFS) 1. The ATP-binding cassette superfamily (ABC) 2. The small multidrug resistance family (SMR) 3. The resistance-nodulation-cell division superfamily (RND) 4. The Multi antimicrobial extrusion protein family (MATE).

Figure 3: The different families of metal exporters include the Resistance-Nodulation-Cell division (RND) type transporters, the P-type ATPase family (forming a covalent phosphorylated intermediate), The small multidrug resistance family (SMR), Major facilitator superfamily (MFS), and the Multi antimicrobial extrusion protein family (MATE) (25).

ATP-binding cassette superfamily(ABC): In bacteria these ABC permeases arrange into 6 transmembrane α-helical segments that associate as covalently linked homo-dimers or heterodimers, accompanied by two nucleotide binding domains for ATP hydrolysis to carry out certain biological processes including translocation of various substrates across membranes and nontransport-related processes such as translation of RNA and DNA repair(15). They transport a wide variety of substrates across extra- and intracellular membranes, including metabolic products, lipids and sterols, and drugs. ABC transporters are classified as proteins based on the sequence and organization of their ATPbinding cassette (ABC) domain(s). Bacterial ABC transporters are essential in cell viability, virulence, and pathogenicity. They are divided into three main functional categories. In prokaryotes, importers mediate the uptake of nutrients into the cell. The substrates that can be transported include ions, amino acids, peptides, sugars, and other molecules that are mostly hydrophilic. The membrane-spanning region of the ABC transporter protects hydrophilic substrates from the lipids of the membrane bilayer thus providing a pathway across the cell membrane. Exporters or effluxers, which are both present in prokaryotes and eukaryotes, function as pumps that extrude toxins and drugs out of the cell. In gram-negative bacteria, exporters transport lipids and some polysaccharides from the cytoplasm to the periplasm(17). The third subgroup of ABC proteins do not function as transporters, but are

rather involved in translation and DNA repair processes. These cells were shown to express elevated levels of multidrug-resistance (MDR) transport protein which was originally called Pglycoprotein (P-gp), but it is also referred to as multidrug resistance protein 1 (MDR1) or ABCB1(6). In multidrug-resistant cells, the MDR1 gene is frequently amplified in multidrugresistant cells. This results in a large overproduction of the MDR1 protein. Drugs such as colchicine and vinblastine, which block assembly of microtubules, freely cross the membrane into the cytosol, but the export of these drugs by ABCB1 reduces their concentration in the cell. Therefore, it takes a higher concentration of the drugs is required to kill the cells that express ABCB1 than those that do not express the gene (11). Resistance nodulation cell division superfamily (RND): The resistance-nodulationcell-division (RND) superfamily multidrug exporter of bacteria are stress inducible. Their role in the corresponding stresses response and identities of the inducing signal or factor molecule are remaining uncertain (38). A solute transporter consists of a large periplasmic domain that assembles with periplasmic fusion proteins and an outer membrane pore to form a complete tripartite channel from the inner cytoplasm past the outer membrane in Gramnegative bacteria. This family of membrane proteins is an efficient mechanism for membrane transport via proton antiport because this system completely extrudes harmful substances from the bacterial cell. The most well known RND efflux pump is the AcrAB-TolC system of E.coli. RND type multidrug system in Pseudomonas aeroginosa are MexAB-oprM, MexCD-oprJ MexEF-oprN and MexXY-oprM(42). Small multi-drug resistance superfamily (SMR): The small multi-drug resistance (SMR) groups of drug or proton antiporters are composed of 4 transmembrane spanning αhelical segments that function arguably as a tetramer or dimer(29). Members of this family are capable of transporting substrates such as aminoglycosides, and lipophillic cations. The well characterized SMR pumps include Smr of S. aureus and EmrE of E.coli (21). Major facilitator superfamily (MFS): The major facilitator superfamily (MFS) is a class of membrane transport proteins that facilitate movement of small solutes across cell membranes in response to chemiosmotic gradients. Transporters of the MFS consist of 12 or 14 α-helical segments that typically function

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as a monomer within the inner-membrane (37). The MFS group of drug transporters consists of two domains that center around a central pore with two domains that switch conformations from the cytoplasmic side of the membrane to the periplasmic as a result of a gradient of Na+ or H+ ion. MFS proteins constitute a major portion of the drug efflux pumps in bacteria (36). For example, nearly half of the 37 efflux proteins in the E. coli genome belong to the MFS of transporters. The MFS family was originally believed to function primarily in the uptake of sugars but subsequent studies revealed that drugs, metabolites, oligosaccharides, amino acids and oxyanions were all transported by MFS family members. These proteins energetically drive transport utilizing the electrochemical gradient of the target substrate (uniporter), or act as a cotransporter where transport is coupled to the movement of a second substrate (28). For example, in the best studied MFS transporter, LacY, lactose and protons typically bind from the periplasm to specific sites within the aqueous cleft. This drives closure of the extracellular face, and opening of the cytoplasmic side, allowing substrate into the cell. Upon substrate release, the transporter recycles to the periplasmic facing orientation (39). Exporters and antiporters of the MFS family follow a similar reaction cycle, though exporters bind substrate in the cytoplasm and extrude it to the extracellular or periplasmic space, while antiporters bind substrate in both states to drive each conformational change. Multi-antimicrobial extrusion protein (MATE): The MATE family of multi-drug efflux pumps also known as multidrug and toxin extrusion or multidrug and toxic compound extrusion is a family of proteins which function as drug/sodium or proton antiporters. The MATE proteins in bacteria, archaea and eukaryotes function as fundamental transporters of metabolic and xenobiotic organic cations (36). The first multi-drug efflux pump of this family, NorM, was discovered in 1998 in the laboratory of Tsuchiya, who cloned the norM gene from the Gram-negative bacterium V. parahaemolyticus and found the pump to be energized by Na + instead of H+. AcrAB efflux pump system and contained a restriction endonuclease cloning system. Later, the gene encoding YdhE, another MATE efflux pump, was removed from the E. coli KAM3, and the resulting strain was called KAM32(18). These strains were later used to characterize the efflux pump activities of not only MATE transporters, but of many others. Using these new E. coli strains the range of

substrates for NorM was found to include agents such as ciprofloxacin, norfloxacin, kanamycin, streptomycin, trimethoprim, daunomycin, doxorubicin, DAPI, acriflavine, and ethidium bromide. This new efflux pump represents an emerging and promising new avenue as a molecular target for putative efflux pump inhibitors for the treatment of infectious disease caused by multi-drug resistant E. cloacae(31). Mechanism Bacterial efflux pumps: Several mechanisms have evolved in bacteria which confer them with antibiotic resistance. These mechanisms can chemically modify the antibiotic, render it inactive through physical removal from the cell, or modify target site so that it is not recognized by the antibiotic (27). The most common mode is enzymatic inactivation of the antibiotic. An existing cellular enzyme is modified to react with the antibiotic in such a way that it no longer affects the microorganism. An alternative strategy utilized by many bacteria is the alteration of the antibiotic target site (34).

Figure 4: Bacterial efflux mechanisms(21)

Three mechanisms of antibiotic resistance found in bacteria. Most, but not all, resistance mechanisms are encoded by plasmids, which are potentially transmissible to other bacteria. Clockwise 12 o'clock: Efflux pumps are highaffinity reverse transport systems located in the membrane that transport the antibiotic out of the cell. This is the mechanism of resistance to tetracycline. 4 o'clock: A specific enzyme modifies the antibiotic in a way that it loses its activity. In the case of streptomycin, the antibiotic is chemically modified so that it will no longer bind to the ribosome to block protein synthesis. 9 o'clock: An enzyme is produced that degrades the antibiotic, thereby inactivating it. For example, the penicillinases are a group of beta-lactamase enzymes that cleave the beta lactam ring of the penicillin molecule (33). Gene repair or SOS mechanism: The SOS response is a global response to DNA damage in which the cell cycle is arrested and DNA repair

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and mutagenesis are induced. it is the essential pathway of bacterial acquisition of bacterial mutation which leads to resistance of oxidative stress condition. The increase rate of miutation during SOS response is caused by their low fidelity of DNA polymerase, Pol III, Pol IV and Pol V. DNA repair enzyme include endonuclease IV, which is induced by oxidative stress, and exonuclease III, which is induced in the stationary phase and in starving cells. Both enzymes act on duplex DNA cleaning up DNA 3' termini (19). Mechanism:

Figure 5: Gene repair mechanism (12)

During normal growth, the SOS genes are negatively regulated by LexA repressor protein dimers. Under normal conditions, LexA binds to a 20-bp consensus sequence (the SOS box) in the operator region for those genes. Some of these SOS genes are expressed at certain levels even in the repressed state, according to the affinity of LexA for their SOS box. Activation of the SOS genes occurs after DNA damage by the accumulation of single stranded (ssDNA) regions generated at replication forks, where DNA polymerase is blocked. RecA forms a filament around these ssDNA regions in an ATPdependent fashion, and becomes activated (44). The activated form of RecA interacts with the LexA repressor to facilitate the LexA repressor's self-cleavage from the operator. Once the pool of LexA decreases, repression of the SOS genes goes down according to the level of LexA affinity for the SOS boxes. Operators that bind LexA weakly are the first to be fully expressed. In this way LexA can sequentially activate different mechanisms of repair. Genes having a weak SOS box (such as lexA, recA, uvrA, uvrB, and uvrD) are fully induced in response to even weak SOS-inducing treatments. Thus the first SOS repair mechanism to be induced is nucleotide excision repair (NER), whose aim is to fix DNA damage without commitment to a

full-fledged SOS response (19). If, however, NER does not suffice to fix the damage, the LexA concentration is further reduced, so the expression of genes with stronger LexA boxes (such as sulA, umuD, umuC - these are expressed late) is induced. SulA stops cell division by binding to FtsZ, the initiating protein in this process. This causes filamentation, and the induction of UmuDC-dependent mutagenic repair. As a result of these properties, some genes may be partially induced in response to even endogenous levels of DNA damage, while other genes appear to be induced only when high or persistent DNA damage is present in the cell (43). Efflux pump inhibitors: Several trials are currently being conducted to develop drugs that can be co-administered with antibiotics to act as inhibitors for the efflux-mediated extrusion of antibiotics. However, some of them are used to determine the efflux prevalence in clinical isolates (46). It‟s shown that Verapamil can inhibit P-glycoprotein mediated efflux which can increase oral absorption of some compounds. Some chemicals found in plants have potential as reflex pump inhibitors (32). Chemicals such as Capsanthin and capsorubin, carotenoids isolated from paprika;theflavonoids, rotenone, chrysin, ph loretin and sakuranetin(48).

Figure 6: Diagrammatic drawn of efflux pump inhibitors (38)

CONCLUSION Oxidative stress is imbalance between oxidants and antioxidants in favor of the oxidants which are formed as a normal product of aerobic metabolism. Bacteria have different efflux system possible playing a role in ameliorating the effect of oxidative stress. Removal of their detoxification and repair system has been shown to make bacteria more susceptible to oxidants and immune attach manipulation of endogenous bacteria ROS production remain largely explored. This possibility, understanding the precise molecular mechanism of drug translocation across the biological membrane in terms of kinetics, biochemistry and of molecular structure changes during drug and cation

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