Antibiotics Are A Natural Substance Biology Essay

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In 1980, FQ antibiotic was first synthesised from Nalidixic acid. It is the first men made antibiotic which attacks both the gram-positive and gram-negative bacteria. FQ works in two ways, DNA Gyrase and Topoisomerase IV. DNA Gyrase targets the gram-negative bacteria and Topoisomerase IV attacks the gram-positive bacteria. CCCP is a weak acid and toxic. It is used with FQ, where it acts as a substance of bacteria efflux pumps. Efflux pump decreases the bacteria's inhibition rate or MIC of bacteria. Bacterial antibiotic has efflux pumps of five superfamilies. ABC is one of the superfamily which encodes pumps components induced by FQ. ABC is ATP binding cassetle which are primarily active transporters energized by ATP hydrolysis.

Used samples from CVL (Veterinary Laboratories Agency: VLA Weybridge). Samples are resistance and their resistance profile was confirmed. Looked for mechanisms of FQ in isolated bacteria. The results did not gave any clear idea about it workings. More works needed to be done and further experiments are being performed in order to understand this phenomenon.

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1.3 Key world Fluoroquinolones resistance, veterinary isolated bacteria, Nalidixic acid, ciprofloxacin, carbonyl cyanide 3- Chlorophenylhydrazone(CCCP), multidrug, efflux pump, p-glycoprotein, CVL.

2. Introduction

Antimicrobial agents have been used for the treatment of human and animal disease for more than 50 years. In 1928, Sir Alexander Fleming first discovered antibiotic Penicillin in St. Mary's hospital. On an average every year 4 to 6 new antibiotics are introduced in medical practice, of which most is not used. The word antibiotics come from Greek, where Anti means 'against' and bio means 'life', so antibiotics means against life.

Antibiotics are a natural substance derived from natural substance or synthetic compounds. Antibiotics are selective sensitive toxins which are toxic to microorganisms but not to human. Usually antibiotic resistance is harmful for other microorganisms, viruses, fungi and parasites. Best antibiotics have a wide range of therapeutic properties and the level of toxicity that is harmful to human is relatively high (Schwarz S et al 2001). So small doses of antibiotic are used to kill or prevent the growth of microorganisms.

Early periods of antimicrobial discovery were tempered by the emergence of bacteria stains with resistance to this therapeutics. All type of quinine family including Fluoroquinolones are synthesis derived from Nalidixic acid. Nalidixic acid was first introduced in 1962 and in 1970 the first generation of quinine family was introduced. In the 1980s new potent of drugs were discovered from nalidixic acid. This drug has a fluorine atom on six carbon and piperazine ring at seven carbons. It is called Fluoroquinolones. Ciprofloxacin, norfloxacin and levofloxacin are different type of Fluoroquinolones drug. In 1994, University of Alabama transferred low level quinolone resistance along with several other antibiotics to E. coli and other gram negative organisms, during this test E. coli plasmid caused an 8 to 32 fold decrease in susceptibility for Nalidixic acid and other fluoroquoione test also increased minimum inhibitor concentration.

Antibiotic resistance to inhibit their bacterial targets, antibiotics needs to cross the cellular envelopes to be activated by bacterial enzymes before they can gain access to the targets (MARTINEZ J.L et al 2000). Bacteria have protection determinants against the antibiotic effect. These include antibiotic-inactivating enzymes and MDR efflux pumps (MARTINEZ J.L et al 2000).

Three main types of intrinsic are relevant for the emergence of antibiotic-resistant mutants:

The synthesis and cell positioning of the antibiotic target Genes are involved in; mutations in these genes can be denominated target-structural mutations; (MARTINEZ J.L et al 2000).

The antibiotic to the target access are genes involved which are needed for the biochemical access of the antibiotic; mutations in these genes are named target-access mutations (MARTINEZ J.L et al 2000).

Genes involved in the protection of the target from the drug, including detoxification by antibiotic-modifying enzymes or efflux of the antibacterial compounds; mutations which activate the expression of those are named target-protection mutations (MARTINEZ J.L et al 2000).

Host or the environment using antibiotic rapidly appeared to be resistant bacteria but slowly lost the resistance, or even was absent in some antibiotic. This reflects the minimal survival cost to the emerging resistant strains. In addition, resistance genes are often linked with genes specifying resistance to other antimicrobials or toxic substances on the same plasmids (Levy S B& Marshall B 2004).

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Like human medicine antibiotic is also used in food production, prevention of animal disease and control of bacterial infection and growth promotion. Using antibiotics in food and agriculture always raises questions, specially about the relationship between antimicrobial use in animals and the resistance problem in humans. In 2005, US FDA banned using prophylactic Fluoroquinolones in poultry farms. In early 1990, US used Fluoroquinolones in boiler poultry. It was concluded earlier in the decade that this practice was ill-advised as it might result in extensive resistance to these agents who could result in food poisoning in consumers that would be difficult to treat with the fluoroquinolones used clinically.

The resistance of Fluoroquinolones in different mechanisms is questionable. It is not clear how it works in DNA dynamaies. Fluoroquinolones were the first entirely men made antibiotics which are entirely synthetic and have no naturally occurring structural counterpart. Therefore any resistance that has occurred is a selective response to the presence of these agents. Still there is continued interest in the development of FQ, some of which have much greater in vitro activity and broader spectra than Nalidixic acid. The emergence of resistance can significantly shorten the useful life time of antimicrobial agents and resistance to Nalidixic acid and the FQ is the result of chromosomal mutation (Watanabe, M. et al 1990).

2.1 Mechanisms of Fluoroquinolones action

Fluoroquinolones are different among antimicrobial agents in clinical use because they directly inhibit DNA synthesis. Inhibition appears to occur by interaction of the drug with complexes composed of DNA and either of the two target enzymes, DNA gyrase and topoisomerase IV (David C. Hooper 2001). These enzymes are structurally related to each other, both being tetrameric with pairs of two different subunits. The GyrA and GyrB subunits of DNA gyrase are respectively homologous with the ParC and ParE subunits of topoisomerase IV. Both enzymes are type 2 topoisomerases, which act by breaking both strands of a segment of DNA, passing another segment through the break, and then resealing the break. For DNA gyrase, this topoisomerization reaction results in introduction of DNA supercoils, thus affecting the negative supercoiling of DNA necessary to initiate DNA replication and remove positive supercoils that accumulate before an advancing replication fork ( Willmott CJ et al 1994 ). For topoisomerase IV, the topoisomerization reaction results in separation of the interlocking of daughter DNA strands that develop during replication; this facilitates the segregation of daughter DNA molecules into daughter cells. Fluoroquinolones appear to trap the enzyme on DNA during the topoisomerization reaction, forming a physical barrier to the movement of the replication fork, RNA polymerase, and DNA helicase (Shea M E et 1990). The collision of the replication fork with these trapped complexes triggers other poorly defined events within the cell that ultimately result in cell death (David C. Hooper 2001).

2.2 Mechanisms of Fluoroquinolones resistance

Fluoroquinolone resistance mechanisms include one or two of the three main mechanistic categories, in the drug target or in the permeation of the drug to reach its target (David C. Hooper 2001). No specific quinolone modifying or degrading enzymes have been found as a mechanism of bacterial resistance to fluoroquinolones, although some fungi can degrade quinolones by metabolic pathways (David C. Hooper 2001).

A general pattern for most quinolones has emerged: DNA gyrase is the primary drug target in gram-negative bacteria, and topoisomerase IV is the primary target in gram-positive bacteria. These differences correlate with relative drug sensitivities in several cases, the more sensitive of the two enzymes being the primary target defined by genetic tests (Berger J M 1996). The first step in mutational resistance in the drug target usually occurs by an amino acid change in the primary enzyme target, with a rise in MIC of the cell predicted to be determined by the effect of the mutation itself or by the level of intrinsic sensitivity of the secondary drug target (David C. Hooper 2001). Higher levels of resistance may then occur by second mutational steps, in which amino acid changes are selected in the secondary target enzyme. Further mutations result in additional amino acid changes in either enzyme, depending on which was least resistant in the cell under selection. On mechanistic grounds, this pattern of stepwise mutations in alternating target enzymes implies that both high intrinsic potency against the primary target and the similarity of potency against both targets will affect the likelihood of selection of first-step resistant mutants. Thus, fluoroquinolones with a high therapeutic index (defined as the concentration of drug at the site of infection divided by the MIC of the drug for the target bacterium), in which drug concentration exceeds the MIC of a first-step mutant, are unlikely to select spontaneous first-step mutants present in the infecting bacterial population; such mutants are inhibited or killed by these concentrations. Furthermore, the greater the extent to which a fluoroquinolone has similar potency against both enzyme targets, the lower the MIC increment for a first-step drug target mutant (David C. Hooper 2001).Thus, for drugs with low increments in resistance for first-step mutants because of similar activities against both target enzymes, the extent to which drug concentrations can exceed the MIC of first-step mutants may be enhanced (David C. Hooper 2001). These principles would predict that selection of fluoroquinolone resistance could occur readily with ciprofloxacin against species such as Staphylococcus aureus and Pseudomonas aeruginosa, organisms in which single mutations cause MICs of ciprofloxacin that approach or exceed the dominant mechanisms of fluoroquinolone resistance identified are chromosomal mutations causing reduced affinity of DNA gyrase and topoisomerase IV for fluoroquinolones and overexpression of endogenous MDR pumpsachievable serum concentrations (David C. Hooper 2001).

3. Methods

Sample collection

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Methods material from CVL (Veterinary Laboratories Agency: VLA Weybridge).

ID

Date

Sample ID

Ceph or quin

MIC CIP

For GyrA

169

20/02/2008

289

E

64

Yes

s193

05/11/2009

45

E

64

Yes

212

10/08/2009

4

E

64

No

219

02/09/2009

3

E

64

No

Antibiotics used: ciprofloxacin and Nalidixic acid

Toxic used: carbonyl cyanide 3- Chlorophenylhydrazone (CCCP). Dimethyl sulfoxide (DMSO)

Other bacteria used: E.coli

Commonly used media were: Nutrient agar (OXOLD), Nutrient broth

3.1 Disk diffusion test: Used Ciprofloxacin and Nalidixic acid (different dilution) in E.coli to get standard kill zone

Using spread plate of E.coli and serial dilution of CIP and NX. Dilution rate 10-1 to 10 -05.

Stock solution of 100 mg of CIP or NX and 5 ml of distil water.

Incubate at 37o c for approximately 20 to 24 hours, using filter paper in spread plate and putting different dilution solution.

3.2 Use FQ (Ciprofloxacin and Nalidixic acid) in infected eggs

Stock solution of CIP and Nalidixic acid (0.032 g of FQ add in 5 ml of nutrition broth).

Incubate at 37o c for approximately 24 hours.

Mixture made by 1ml of Stock solution FQ , 1ml of culture of infected eggs and 8ml nutrition broth.

Incubate at 37o c overnight.

Sanicate used to break down the cell of mixture.

Used in spread plate of infected eggs.

3.3 Toxic test

Stock solution of CCCP (0.004 mg/ ml of CCCP + 0.20 ml DMSO + 20 ml of nutrition broth).

Stock solution of FQ (CIP) and CCCP (1ml of Stock solution of CIP+ 1ml of stock solution of CCCP+1ml of sample culture +7ml of nutrition broth).

Stock solution of sample and stock solution of FQ (CIP).

Incubation at 37Ö¯ c for 15 min.

Sanicate used to break down the cell of mixture.

Used in spread plate of infected eggs.

Stock solution CIP and toxic CCCP in spread plate of infected eggs which disrupts the proton motive force and inhibits active accumulation of the drug. Accumulation assays was performed by using cell cultures were grown until the late log phase. After harvesting by centrifugation and resuspension in the inhibitor CCCP was added if required at time zero. To separate the cells from extra cellular ciprofloxacin, the samples was centrifuged and frozen until required.

This method is used from L. J. V. Piddock et al (1997) 'Non-gyrA-mediated ciprofloxacin resistance in laboratory mutants of Streptococcus pneumoniae',. Journal of Antimicrobial Chemotherapy page 39, 609-615.

4. Results

4.1 Disk diffusion test: Use of Ciprofloxacin and Nalidixic acid (different dilution) in E.coli to get standard kill zone

1. Different dilution rate of ciprofloxacin in E.coli spread plate:

Dilution rate

Kill zone(1)

Kill zone(2)

Average kill zone

10-1

1.4

1.2

1.3

10-2

1.7

1.8

1.75

10-3

1.7

1.9

1.8

10-4

1.9

1.6

1.75

10-5

1.9

1.9

1.9

2. Different dilution rate of Naldioxie acid in E.coli spread plate:

Dilution rate

Kill zone(1)

Kill zone(2)

Average kill zone

10-1

1.3

1.3

1.3

10-2

1.6

1.5

1.55

10-3

1.8

1.7

1.75

10-4

1.9

1.7

1.8

10-5

2.1

1.6

1.85

First generations of FQ are more active in gram negative bacteria. Gram negative bacteria are attacked by FQ in topoisomearse. It was proposed that this antagonism was due to the inhibition of the "self-promoted" uptake pathway by cations. This pathway involves an interaction between the drug and the lipopolysaccharide (LPS) of the gram-negative outer membrane (Levy S B& Marshall B 2004).

Disk diffusion test is a most common test for antibiotic susceptibility. In this test the bacterial isolate is inoculated uniformly onto the surface of an agar plate. A filter disk impregnated with a standard amount of an antibiotic is applied to the surface of the plate and the antibiotic is allowed to diffuse into the adjacent medium. The result is a gradient of antibiotic surrounding the disk. Following incubation, a bacterial lawn appears on the plate. Zones of inhibition of bacterial growth may be present around the antibiotic disk. The size of the zone of inhibition is dependent on the diffusion rate of the antibiotic, the degree of sensitivity of the microorganism, and the growth rate of the bacterium. The zone of inhibition in the disk diffusion test is inversely related to the MIC (Mayer, 2010).

E.coli is a gram negative rod shape bacteria. E.coli is an ideal and standard gram negative bacteria, which is used to find out how FQ works in gram negative bacteria. The test used 5 different dilution rate of FQ in E.coli spread plate. The decrease in drug dilution rate increases the kill/ inhibition zone. Smaller amount of drug resulted in better inhibition zone in E.coli or gram negative bacteria. Inhibition kill zone in the E.coli bacteria spread plate means that E. coli is resistance to FQ (Ciprofloxacin and Nalidixic acid). It helps to know that both the antibiotics are working. Ciprofloxacin has a broad spectrum of activity against gram-positive and gram-negative bacteria. Ciprofloxacin mutation occurs spontaneously at a low frequency in susceptible populations of bacteria (Mahamoud Aet al 2007). Nalidixic acid is the first synthetic quinolon antibiotic. Nalidixic is a rapid, specific and reversible inhibitor of bacterial DNA replication (J.James et al 1974). Nalidixic acid affected both the gram negative and gram positive bacteria.

4.2 Using FQ (Ciprofloxacin and Nalidixic acid) in infected eggs

The egg samples used were infected eggs from CVL (Veterinary Laboratories Agency: VLA Weybridge). White eggs are the best host in in-vitro test. Samples were 36 strains and mixed with Ceph or quinolones. There Minimal Inhibitory Concentration rate (MIC) of Ciprofloxacin were different. MIC 8 was most sensitive, 16 and 32 were less sensitive and 64 were resistance. Some were GyrA mutant. The experiment used four different samples which were resistance (MIC 64), two samples of GyrA mutant and two other which were not mutants. FQ resistance in bacteria are mutations in FQ targets, topoisomearse IV and DNA gyrase. Low level of FQ resistance is related to amino acid substitutions in the quinolone resistance-determining region of topoisomerase IV subunit ParC or DNA subunit of GyrA. Topoisomearse IV and DNA Gyrase contribute together to high- level resistance (OIS GUERIN F et al 2000). The basic quantitative measures of the in-vitro activity of antibiotics are the minimum inhibitory concentration (MIC) and the minimum bactericidal concentration (MBC). The MIC is the lowest concentration of the antibiotic that results in inhibition of visible growth (Mayer 2010).

The experiment did not produce the expected results. Most of the plates were contaminated with other bacteria. Possible reasons of this unexpected result could be:

1 Proteins broke down when used in the centrifuge.

2. The drugs washed away while washing.

Most of the sample but not to all FQ

4.3 Toxic test result

Toxic test is a continuous and rapid process of using FQ (Ciprofloxacin) in infected eggs. This experiment used toxic CCCP and DMSO. In this method samples were incubated in four different conditions.

CCCP is a weak acid which is highly noxious and cytotoxic also acts as substrates of bacterial efflux pumps. It is also commonly used as a disconnecting agent and inhibitor of photosynthesis. CCCP is a proton ionophore, which transport proteins across lipid bi-layer protein.

DMSO is a reserpine. This solvent is widely used in organic chemistry, cell biology and chemical technology. It is also used for protected biology structure. DMSO increases permeability across membranes and changing prangoperties of protein in the cell DMSO found into phospholipids bi-layers, it can produce new phases and change their stability. It is also effect on the repeat spacing distance and modifies hydration forces (M Alexander et al 1999).

Sample 212's kill zone

Sample

Kill zone(1)

Kill zone (2)

Kill zone (average)

No drug

(-FQ and -CCCP)

1.0

0.7

0.8

0.8

0.9

0.75

With drug

(+FQ and +CCCP)

1.1

0.8

1.3

0.7

1.2

0.75

With FQ

1.3

1.2

1.4

1.1

1.35

1.15

With CCCP

1.0

1.0

1.1

0.9

1.05

0.95

Sample 212's kill zone resulting from different incubation condition, MIC is 64 but it is not GyrA mutant. The sample is a resistant bacteria. This sample is working well in with FQ incubation condition.

Sample without FQ and CCCP also gave a kill zone, which is interesting. The samples might have been contaminated with air bacteria. This bacteria gave a nice and small kill zone.

The CCCP stock amount was taken incorrectly. Though all sample used stock measurement of 10 ml but in this sample 20 ml of CCCP stock was used. Also the amount of CCCP in stock was wrong as it should have been 0.082 mg of CCCP and 0.12 ml of DMSO, but instead the amount used was 0.004 ml of CCCP and 0.19 mg of DMSO. Sample with CCCP rate inhibition zone for the 20 ml CCCP stock solution.

FQ and CCCP solution with sample created sample kill zone the FQ. The CCCP amount was too small an amount to make much affect in the reaction.

Sample 219's kill zone

Sample

Kill zone(1)

Kill zone (2)

Kill zone (average)

No drug

(-FQ and -CCCP)

0.9

0.9

0.8

1.0

0.85

0.95

With drug

(+FQ and +CCCP)

0.8

1.0

0.9

1.1

0.85

1.05

With FQ

0.8

1.1

1.0

1.1

0.9

1.1

With CCCP

0.9

1.1

1.1

1.0

1.0

1.05

In sample 219's the kill zone resulted from different incubation conditions. Sample MIC is 64 but is not GyrA mutant. It is a resistant bacteria. The kill zone of this sample is nearly the same as the sample 212.

Sample 193's kill zone

Sample

Kill zone(1)

Kill zone (2)

Kill zone (average)

No drug

(-FQ and -CCCP)

0

1.3

0

1.0

0

1.15

With drug

(+FQ and +CCCP)

0

0

0

0

0

0

With FQ

1.1

1.3

0.9

1.1

1.1

1.2

With CCCP

0

1.0

0

1.1

0

1.05

In sample 193's the kill zone resulted from different incubation conditions. Sample MIC is 64 and GyrA mutant. It is a resistance bacteria. This sample is working well in with FQ incubation condition in both duplicate samples.

With FQ and CCCP incubation sample didn't get any kill zone. When washing the sample, it might have lost the antibiotics and toxic.

Sample 169's kill zone

Sample

Kill zone(1)

Kill zone (2)

Kill zone (average)

No drug

(-FQ and -CCCP)

0

1.1

0

1.3

0

1.2

With drug

(+FQ and +CCCP)

0

0

0

0

0

0

With FQ

0.9

1.1

0.8

1.2

0.85

1.05

With CCCP

1.1

1.1

1.2

1.3

1.15

1.2

In sample 169's kill zone resulted from different incubation condition. Sample MIC is 64 and GyrA mutant. It is a resistance bacteria. This sample is working well in without FQ and CCCP incubation condition. All are three sample are giving better result in FQ incubation condition. But this sample is giving a kill zone of FQ smaller then incubation with CCCP toxic.

The effects of reserpine and CCCP, inhibitors of the NorA (multidrug efflux protein) efflux system, on the MIC of a selected number of antibiotics were determined. Sample 219 had a reduced inhibition zone and also sample 212 duplicated sample were reduced. There is one sample in 212's increase its inhibition zone. It may be CCCP is a substrate for the putative transporter is insensitive to CCCP, like ABC transporter (Piddock L J.V et al 2002).

Efflux pump could have happened in the bacteria. An efflux mechanism is a complex protein structure in bacterial cell. It can pump out the antibiotics before it can work in the cell. In some cases efflux pumps can act to decrease intracellular quinolone concentration. CCCP use affects the energy level of the bacterial membrane. It reduces the viability of the bacterium and cell death via the distraction of the proton-motive force of the membrane (Li XZ et al 2004). They could effect on the efflux pump, it is one of the causes of an increase in the penetration of the FQ. Inhibitors of efflux pumps in molecules were initially documented as inhibitors of vesicular monoamine transporters and blockers of transmembrane calcium entry. CCCP is an inhibitor of MDR pumps of cancer cells and parasites and also improves the activity of tobramycin. Reserpine inhibits the activity of Bmr and NorA, two Gram-positive efflux pumps (Li XZ et al 2004). They alter the generation of the membrane proton-motive force required for the function of MDR efflux pumps. Although these molecules are able to inhibit the ABC transporters involved in the extrusion of antibiotics (Mahamoud A et al 2007).

Discussion

Thanks to Dr Nick Coldham and Prof Martin Woodward and all the staffs of VLA Weybridge, who worked on the sample, making it highly MIC and Gyrse A mutant. They also screened it for FQ resistance to cheek the VLA results and tried to look for susceptible strains, also looking for steady state in FQ.

Novel assay is a selected key station using samples from VLC. Those samples had high MIC. Some of the samples were Gyrase A mutation and some were not. Mutation with Gyrase A work with Topoisomearse IV, they are highly resistance. With out mutation Gyrase A sample only works with Topoisomearse IV.

In 1980, FQ was first developed as an anti malaria drugs, though It was not much useful as an anti malaria drug. Later it was introduced as a class of borad- spectrum agents applicable to a range of gram negative infections including urinary tract infection, sexually transmitted diseases, bone and join infection, etc (Poole K 2000). Broad spectrum of activity, it is unfortunate that resistance to FQs has increased in a number of gram-negative organisms, where FQs have been employed ( Acar, J. F. 1997). Resistance is due usually to mutations in the genes for the bacterial targets of the FQs (DNA gyrase [GyrA] and topoisomerase IV [ParC]) or to active efflux of the agents via antibiotic efflux pumps (Poole K 2000). FQ is also used in the USA for animal feed and water especially for chicken. In chicken body, they made FQ resistance bacteria, which human eats and thus being infected with FA resistance bacteria.

Different Mechanisms of FQ resistance are:

Altered permeability may be due to the inability of the antimicrobial agent to enter the bacterial cell or alternatively to the active export of the agent from the cell ( Mayer 2010)

Resistance is often the result of the production of an enzyme that is capable of inactivating the antimicrobial agent ( Mayer 2010)

Resistance can arise due to alteration of the target site for the antimicrobial agent ( Mayer 2010)

Replacement of a sensitive pathway. new enzyme replace by the sensitive one ( Mayer 2010)

FQ used on infected eggs did not create any kill zone first time. But those eggs were FQ resistance. VLC had already cheeked there resistance. It should not have happened. But when the same sample and same stock was used in the second method it created a kill zone (incubation with FQ). This means that the sample was resistance. Some how this did not worked the first time.

Incubation with CCCP also created kill zone. The kill zone size was smaller than the incubation except in one sample. This means that the CCCP toxic killed the bacteria. So when FQ and CCCP worked together it should have given a bigger kill zone but it did not. CCCP working with multidrugs resistance helps efflux pumps. Sub lethal level of antibiotics can lead to multidrug resistance. This occurs via bacterial antibiotic mediated radical formation of mutations, some of which confer antibiotic resistance. Low- level resistance likely provides a first step towards clinically significant resistance (Goldstein, 2007) (Kohanski M A et al 2010).

There are a number of mechanisms that the bacteria can develop by antibiotic resistance with horizontal transfer of resistance genes, drug specific of natural occurring resistance etc. quinolones is DNA damaging antibiotics, can stimulate the emergence of drug resistance by SOS -independent recombination and induction of RecA-mediated processes SOS -regulated error-prone polymerases and homologous recombination (Kohanski M A et al 2010).

5.1 Efflux affected in bacteria in inhibitor stage

Drug efflux pumps play a key role in drug resistance and also serve other functions in bacteria. There has been a growing list of multi-drug and drug-specific efflux pumps characterised from. These pumps are mostly encoded on the chromosome, although they can also be plasmid-encoded (Li XZ et al 2004).

Efflux is a general mechanical response of bacteria resistance to antibiotics. Active of drug transport is involves in low natural susceptibility, selection or acquisition of additional resistance mechanisms and cross- resistance to chemically unrelated class of molecules (???). Efflux mechanisms of inhibition bacteria target are:

Increased the intercellular concentration of antibiotics.

Restore the drug susceptibility of resistance clinical stains.

Reduced the capability for acquired additional resistance (Mahamoud A et al 2007).

Multi drug resistance efflux can be done in gram-negative and gram-positive bacteria. Generally gram-negative bacteria is more resistance to antibiotics owing to the sophisticated architecture of their cell envelope, in outer and inner membrane which describe the periplasmic space. Can toxic compound pocked up in the periplasm and expelled in to externals medium, strongly reducing the number of molecules reaching their eytopasmic targets (Mahamoud A et al 2007).

Bacterial antibiotic has efflux pumps of five super-families. ABC is one of the superfamily which encodes pumps components included by FQ. ABC is ATP binding cassetle which are primary active transporters energized by ATP hydrolysis.

p-glycoprotein is a multidrug resistance genes. It is a one of the most well-recognised efflux transports in tissues, including kidney, brain and intestine. P-glycoprotein inhibition show different drug-drug interaction.

Cancer treatment is the most important improvement in chemotherapy in case of multidrug resistance. Normal way from multidrug resistance is attributable to the membrane transport protein. P-glycoprotein is increasesed drug efflux. Using long term culture increasing drug concentrations, a procedure used to select cells with efflux- mediated resistance to anticancer agents. FQ act as antibiotics by impairing bacterial topoisomerases at low concentrations, inhibit the eukaryotic topoisomearse II and kill mammalian cells at large concentration. Reduced Ciprofloxacin, which brought almost to control by ATP depletion t (Marquez B et al 2009).

6. Conclusion

FQ are antimicrobials bind to the A subunit of DNA gyrase in topoisomerase and prevent supercoiling of DNA, thereby inhibiting DNA synthesis. The result is not clear resistance and mechanism of FQ. It need more identification and more testing to get significant statement. As my project was a part of bigger project, it needs more lab work and more work to reach a viable conclusion.