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MECHANISM
OF RESISTANCE

The
bacterial cells produce resistance against antibiotics by three
mechanisms:

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Preventing
the antibiotic attack to the target site by mechanism of altered
permeability(via efflux pumps)

By
transforming the target site

By
the inactivation of antibiotics (by enzymes)1

1.
ENZYMATIC ACTION:

The
?-lactamses classes of enzymes are responsible for this activity.
They are also called as pencillinase or cephalosporinase.
Penicillinase is mostly active against penicillin whereas
cephalosporinase are active against cephalosporins. Sometimes both
types are present in the same organism. It is one of the defense
mechanism against antibiotics in gram negative bacteria. ?-
lactamases cleaves the ?-lactam ring of pencillin and other
antibiotics1. They are classified into four classes (A to D). They
all have mechanism that involves the nucleopilic attack of ?- lactam
antibiotics on the active site serine which results in acyl-enzyme
intermediate 3. This is a type nucleophilic substitution reaction
in which ?-lactam ring act as a nucleophile. This intermediate is
hydrolyzed by base activated water molecule. Generally ?-lactamases
inhibitors are given in combination with ?-lactam antibiotics to
counteract this resistance.

Extended
spectrum beta lactamases (ESBLs) intercede protection from all
pencillins, third generation cephalosporins. They are for the most
part distinguished in E.coli yet additionally have been found in
enterobacteriaceae.

2.
TARGET ALTERATION:

The
second mechanism includes target modification so that the antibiotic
can’t bind legitimately. Due to the imperative cell elements of the
objective locales, creatures can’t sidestep antimicrobial activity
by abstaining from them completely. Generally the target for ?-lactam
antibiotics is to inhibit cell wall synthesis. This is achieved by
their capacity to bind covalently with radiolabelled penicillin.
These proteins to which antibiotics (?-lactam antibiotics) bind are
called Pencillin Binding Protein (PBP). A given life form contains
four to eight PBPs with sub-atomic sizes of 35 to 120 kDa. There is
no fundamental connection between identically numbered PBPs of two
random organisms, although taxonomically related organisms have
comparable PBPs. There are two types of PBPs in an organism based on
their molecular weight- Low molecular weight and high molecular
weight PBP. Most of the microorganisms are resistant to penicillins
because of the alteration of PBPs. This alteration mostly occurs due
to procured mutation or due to any gene transfer. Resistance to
macrolides is due to the attainment of one of 21 erm genes. These
genes codes for rRNA methylases which are the enzymes for the
methylation of adenine residues in 23s rRNA. These inhibits the
binding of macrolides to 50s ribosomal subunit 4. PBP2a belong to
the class of high molecular weight PBPs. Most of the PBPs have high
transpeptidase activity which is required for the crosslinking of
membranes proteins. This transpeptidase activity of PBPS has been
inhibited by methicillin but they were not able to inhibit PBP2a
because they largely depend on trans glycosylase activity.

PBP2a
is encoded by a mecA gene which is present at the 30kb DNA segment.
These are present in the chromosome of MRSA. This is an additional
gene present in MRSA which are found to be absent in MSSA
(methicillin susceptible staphylococcus aureus). Vancomycin was one
of the important drugs which were used for the treatment against MRSA
infections, but unfortunately in the last few years MRSA has acquired
resistance against vancomycin. This is due to presence of van A which
caused alteration in the vancomycin binding site. It is accompanied
with three fold increase in the production PBP2a and PBP2′.
Similarly most of the drugs including oxacillin became ineffective
due to the presence of PBP2a 5. Their major fuction is to provide
the transpeptidase and transglycosylase activity which are important
for the cross-linking of cell wall. These are inhibited by ?-lactam
antibiotics in susceptible species.

The
other target alterations include alteration in protein synthesis,
alteration DNA synthesis. The mechanism of action of macrolides and
lincosamide is different from that of ?-lactam antibiotics. They
inhibit the protein synthesis by binding with 50s ribosomal subunit.
But MRSA shows resistance toward macrolides and lincosamide by the
alteration in 23s ribosomal component of 50s ribosomal subunit. These
occurs during the post transcriptional period where the 23s rRNA
component get altered and therefore the macrolides cannot bind to the
50s subunit. The drugs which act by inhibiting the dna replication
and transcription are the quinolines especially fluoroquiolines. They
mainly inhibit the dna gyrase and topoisomerase enzymes. The
resisitance to quinolines in mrsa occurs by mutation in the
structural gene which alters both the enzymes. This reduces the
affinity of enzymes to quinolines 1.

PREVENTING
THE ANTIBIOTIC ATTACK TO THE TARGET SITE (Efflux Pumps)

A
drug has to reach its specific target to produce its therapeutic
action. Drug resistance can be acquired by preventing the antibiotic
from reaching its target site. This can be achieved by means of
efflux pumps. Efflux pumps are membrane proteins that export
antibiotics out of the cell. A drug concentration inside the cell
depends on its permeability through the cell membrane. Resistance to
penicillin in gram negative bacteria occurs mainly by this mechanism.
The outer membrane of bacteria contains aqueous channels formed by
proteins called porins which allows the rapid entry of most of the
antibiotics. Mutations in the gene coding for porins can prevent the
entry antibiotics into the cell. Efflux pumps are one of the
important systems which are involved in the expulsion of tetracycline
from the cell 6. It is one of the most identified and studied
mechanism by which tetracycline resistance is caused. The
tetracycline accumulation in bacteria is related with energy supplied
in the cell. The studies have shown that tetracycline expelled out of
the cell in presence of energy and its accumulation increases when
energy is not supplied. This was carried out by specific proteins
present in the outer membrane called tet protein. The efflux system
by the tet protein is indeed a antiport system which export one
molecule of tetracycline out of the cell for one proton which is
entering into the cell. Efflux pumps may be of single or multi
component pumps. The presence of multi drug efflux pumps is the
reason for multi-drug resistance in most of the bacteria. Efflux
pumps are regulated by specific genes which can themselves be a
target for the antibacterial agents. They are mostly regulated by
global receptors which include marA, soxS, rob. They are also
regulated by two component system. Any mutations in these genes can
reduce the resistance and therefore it increases the susceptibility
of organisms to antibiotics. Efflux inhibitors can be used for this
purpose.

ANTIBACTERIAL
EFFECT OF CURCUMIN AGAINST METHICILLIN RESSISTANT STAPHYLOCOCCUS
AUREUS

MRSA
(METHICILLIN RESISTANT Staphylococcus aureus) is a rapidly spreading
infectious disease and their ability of high resistance to
antibiotics lead to some serious provocations regarding the treatment
of this disease 8.Curcuma longa a herbaceous rhizome have many
actions in human body. It is native to India and south Asian
countries. It has less advers effects in pregnancy and lactation
period23. The major constituent curcumin have exhibited a wide
range of pharmacological action at low doses 9.At higher doses
curcumin shows genotoxicity24. curcumin is extracted from turmeric
by solvent extraction with the help of various methods such as
Soxhlet, microwave etc. 9. Curcumins antibacterial activity was
tested against the strains of MRSA by combining with other
antibiotics such as oxacillin,ampicillin and norfloxacin showed
positive results by reducing the minimum inhibitory concentration of
the drug thus reducing the bacterial count to a high extent8.A
standard broth microdilution method was used here.curcumin were
prepared in MHB in sterile conditions8.a cheker bord test was done
to evaluate 2 or more drug combinations and respective assays were
performed with OXI,AMP,CIP and NOR1.Interactions between the in
vitro drugs were determined by the fractional inhibitory
concentration or(FIC)index8
FIC
index = FICa+FICb=A/MICa + B/MICb where,
A=CONCENTRATION
OF DRUG A
B=CONCENTRATION
OF DRUG B
FIC
and MIC are the respected minimum inhibitory concentratiom values for
drugs A and B 8.
The
cheker board broth method provided more precise results and suitable
for studies with multiple concentration of compounds.The main
mechanism behind curcumin is able to reverse the resistance when
used in combination with other drugs9.
DISADVANTAGE
OF CURCUMINS
There
are also certain things which challenge the use of curcumin as an
antibiotic which mainly include evidences of cytotoxcicity. Another
one includes the ability to promote lung cancer. It is linked to the
induction of reactive oxygen species(ROS) such as hydrogen
peroxide9.nausea,diarrhea and increased serum alkaline phosphatase
are one of the major side effects.in additiom to this curcumin has
poor solubility and low bio availability. In presence of detoxifying
enzymes curcumin will get degraded rapidly. But studies have also
shown that the products obtained from degradation of curcumin is
mainly responsible for the pharmacological actions9.curcumin
treatment may cause growths despite the anticancer activity of
curcumin is very much archived.As curcumin is an effective iron
chelator,it might conceivably influence fundamental iron digestion
particularly the individuals who have problematic iron
status.Moreover ,curcumin has been accounted for to hinder the
compounds that use medications, such as cytochrome p450s. this may
prompt the aggregation of non metabolized medicates in blood and ends
up causing toxicity9. Notwithstanding potential danger, poor
solvency, and low boo availability, curcumin experiences several
difficulties when it is regulated either through oral or intra venous
course because of the idea of body frame work.a lot of curcumin may
get corrupted within the sight of detoxifying and metabolic
compounds, or it might tie to the circulatory proteins,for example,
egg white which may possibly diminish its action.9
DESISTANCE
Curcumin
has demonstrated powerfull antibacterial action and other
pharmacological activites in the past few years8. Curcumin has been
showcased all around as a well being supplement essential for its
cell reinforcements and calming properties.likewise, it additionally
can possibly be created into a anti-toxin against Methicillin
Ressistant Staphylococcus Aureus and other bacterial strains later
on.

ANTIBIOTIC
COMBINATIONS AGAINST MRSA
Methicillin-safe
Staphylococcus aureus (MRSA) is a considerable general medical issue
around the world, causing huge dreariness and mortality and raised
medicinal services costs. There were an expected 94 360 obtrusive
MRSA contaminations in the United States in 2005, causing more than
18 000 passings for every year. Methicillin-safe S aureus
pervasiveness has expanded in the course of the most recent 10 years;
MRSA-related doctor’s facility releases have multiplied more than 10
years, with doctor’s facility releases for MRSA skin and delicate
tissue contamination tripling since 2004.Infections caused by MRSA
are related with longer healing center stays and an expanded
budgetary weight on society, costing an expected US $14.5 billion for
all inpatient days in 2003.An case of the expanded grimness and
mortality related with MRSA can be seen when contrasting the yearly
disease rates and death rates in the United States for MRSA, AIDS,
viral hepatitis, and tuberculosis. Methicillin-safe S aureus is
evaluated to cause a larger number of contaminations than alternate
maladies consolidated and a bigger number of passings every year than
AIDS1.
?-
lactam binding action, A detailed study
A
vast assortment of anti-microbial blends are of now being used
against MRSA contaminations the vast majority of the anti-microbial
mixes act synergically and is bactericidal against MRSA. Drugs, for
example, vancomycin and linezolid are the most broadly utilized.
Twofold blends, for example, vancomycin with imipenem and fosfomycin
with cetazolin were utilized however wound up as disappointments
blog. Most
antimicrobial operators utilized for the treatment of bacterial
contaminations might be sorted concurring their important system of
activity. There are 4 major modes of activity: (1) impedance with
cell divider synthesis,(2) restraint of protein blend, (3)
interferencewith nucleic corrosive union, and (4) hindrance of a
metabolic pathway3. Antibacterial
medications that work by repressing microscopic organisms lcell
divider blend incorporate the b-lactams, for example, the
penicillins, cephalosporins, carbapenems, and monobactams,and the
glycopeptides, including vancomycin and teicoplanin3
PBPs
are characterized as those bacterial proteins that predicament
penicillins and otherfJ-lactam anti-infection agents covalently. PBPs
are promptly distinguished and their relative sums quantitated by
brooding of bacterial films with 14C penicillin G, trailed by
sodium dodecylsulfate (SDS) gel electrophoresis and ftuorography4.
The liking of a PBP for guaranteed (nonradiolabeled) fJ-lactam is
normally communicated as the grouping of anti-microbial required to
lessen 14Cpenicillin G authoritative to the PBP by half and is
resolved after preincubation with the unlabeled fJ-lactam under given
states of time,temperature4. Studies went for reasoning which PBPs
are deadly focuses for f3-lactam anti-microbials have used two key
methodologies: examination of mutants with adjusted PBP examples and
relationships between’s in vivo and in vitro impacts of fJ-lactams.
In this manner, the physiological results of inactivation of specific
PBPs, either by change or by treatment with exceptionally particular
fJlactams, have been inspected and the in vivo elements of the PBPs
inferred4. ?-Lactam specialists restrain synthesisof the bacterial
cell divider by meddling with the compounds required for the blend of
the peptidoglycanlayer. Vancomycin and teicoplanin additionally
meddle with cell divider amalgamation, yet do as such by official to
theterminal D-alanine buildups of the incipient peptidoglycan chain,
along these lines keeping the cross-connecting steps required for
stable cell divider synthesis 3. Notwithstanding penicillins and
cephalosporins, the vancomycin group of glycopeptide anti-microbials
additionally focuses on the peptidoglycan layer in the cell-divider
gathering. But instead than focusing on the catalysts engaged with
peptide crosslinking, vancomycin
ties up the peptide substrate6 and in this manner keeps it from
responding with either the transpeptidases or the transglycosylases.
The net impact is the same: inability to make peptidoglycan
crosslinks prompts a weaker divider that inclines the treated
microbes to a murdering lysis of the cell wall layer. The container
molded undersurface of the vancomycin anti-infection makes five
hydrogen bonds to the D-Ala-D-Ala dipeptide end of each uncrosslinked
peptidoglycan pentapeptide side chain, which represents the high
fondness of the anti-microbial for its objective, both in halfway
crosslinked dividers and in the lipid II middle of the road. Since
b-lactams and vancomycin chip away at contiguous advances – substrate
and catalyst , they indicate cooperative energy when utilized as a
part of combination5. For anti-microbials to be compelling they
should achieve their particular bacterial targets and collect at
fixations that can demonstration in some sensible time period. For
instance, the protein-amalgamation hardware is situated in the
cytoplasm so antibacterials that are inhibitors of protein union must
go through the cell films (external andinner porousness obstructions
for Gram-negative microscopic organisms; internal layer hindrances
for Gram-positive microbes) and after that collect to a sufficiently
high focus to hinder the specific vulnerability advance of protein
get together. Both Gram-positive and Gram-negative microscopic
organisms that wind up plainly impervious to antibiotic medications
generally overproduce related film proteins (with relative sub-atomic
masses of 42,000) that go about as a fare or efflux pump for the
drug5. A structural representation of penicillin binding protein
from staphylococcus aureus is shown below. It shows penicillin like
antibiotic binding site, non active sites and transpeptidase region
(domain which is responsible for the synthesis of cell wall in
bacteria).

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