Mechanism Of Fluoroquinolones Resistance In Veterinary Isolated Bacteria Biology Essay

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Aim: Isolates of Fluoroquinolones resistance bacteria from CVL (VLA weybridge) their resistance profile confirmed.

Summary:

1.3 Key world: Fluoroquinolones resistance, veterinary isolated bacteria, Nalidixic acid, ciprofloxacin, carbonyl cyanide 3- Chlorophenylhydrazone

2. Introduction:

Antimicrobial agents have been used for the treatment of human and animal 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 is 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. So small doses of antibiotic are used to kill or prevent the growth of microorganisms.

Early periods of antimicrobial discovery was tempered by the emergence of bacteria stains with resistance to this therapeutics. All type of quinine family including Fluoroquinolones is syntheses 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 drug were discovered from nalidixic acid. This drug have 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.

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. [1]

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 human. 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.

In the resistance of Fluoroquinolones different mechanisms is questionable. Fluoroquinolones is first man made entirely antibiotics. Fluoroquinolones 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 shorter 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).

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. 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. 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. The collision of the replication fork with these trapped complexes triggers other poorly defined events within the cell that ultimately result in cell death. (http://www.cdc.gov/ncidod/eid/vol7no2/hooper.htm)(26/03/2010).

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. 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. 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. 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. 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. 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. 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.

(http://www.cdc.gov/ncidod/eid/vol7no2/hooper.htm)(26/03/2010)

3. Methods:

Sample collection:

Methods material from CVL (Central veterinary laboratory).

ID

Date

Sample ID

Ceph or quin

MIC CIP

For GyrA

169

20/02/2008

289

E

64

Yes

193

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 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 distill 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 (Cirprofloxacin 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 borth.

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

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

4. Results:

4.1Use Ciprofloxacin and Nalidixic acid (different dilution) in E.coli 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

E.coli is a gram negative rod shape bacteria. First generations of FQ are more active in gram negative bacteria. Gram negative bacteria are attacked by FQ in topoisomerase. 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.

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.

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.

Nalidixic acid is the first synthetic quinolon antibiotic. Nalidixic is a rapid, specific and reversible inhibitor of bacterial DNA replication. Nalidixic acid affected both the gram negative and gram positive bacteria.

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

Sample used on infected eggs from CVL. White eggs are the best host in in-vitro test. Samples are 36strains and mix with Ceph or quinolones. There minimal inhibitory Concentration rate (MIC) of Ciprofloxacin is different. MIC 8 is most sensitive, 16 and 32 are less sensitive and 64 are resistance. Some are mutant with GyrA.

Used four different samples which are resistance (MIC 64), two samples of GyrA mutant and two other not mutant.

This experiment did not produce the expected results. Most of the plate was contaminated with other bacteria. Possible reasons of contamination could be:

1. Maybe efflux pumps done when washing bacteria and drugs.

2. Proteins broken down when used in the centrifuge.

3.

4.3 Toxic test result:

Toxic test is a continues and rapid process of using FQ in infected eggs. These methods used toxic CCCP and DMSO.

Sample 212

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 219

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

Sample 193

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

Sample 169

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

Efflux pumps can act to decrease intracellular quinolone concentration.

Conclusion:

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