Pharmacology Science Research Project.docx

BHS034-3 Pharmacology Science Research Project
Answer:
Objectives
The main objective of the research was to determine measurements of MIC value for
various bacterial which are isolated and plot the data collected for the
development(growth) of the bacterial to varying concentration of antibiotic; in determining
the MIC of individual antibiotic from the maximum concentration to minimum at which no
development was observed.
Specific Objectives
To identify PPK inhibitor activity compounds, knowing that Gallien has PPK inhibitor
activity.
To determine the PPK activity that will be able to make bacterial more susceptible, that is:
“the modified Kirby-Bauer disk diffusion susceptibility test” using the chosen assay.
Use the indirect measurements of compound ability of organism inhabitation to establish
the possible evidence of bacterial development (growth) around the disks to assure its
functionality at a specific concentration.
To use our Kwon assay to identify the type of Gallien present in disc susceptibility.
To use the assays that have been performed to identify Gallien as PPK inhibitors.
Research Question
How to perform disc susceptibility tests in the antibiotic drug discovery process?
The Basic Anatomies Of Bacterial Cell
Gram-negative bacterial have tinny cell’s walls enclosed with another lipid’s membranes
called; outer membrane (O.M.). on the other hand, Gram-positive bacterial have a
membrane which is cytoplasmic enclosed by cell wall which is rigid and tough. The outer
membrane of Gram-negative bacterial acts as the supplementary layer of protection by
preventing substances from entering the bacterium. Periplasm is the gap that separate
cytoplasmic membrane from the outer membrane. These membranes have porins channels
that allow drug molecules to pass through. The cell walls have a rigid layer that provides the
shape of bacterium and prevent bacterial from mechanical stresses and osmosis.

Cytoplasmic membranes contain flow to and FRO the cell, thus, maintaining bacterial
components and cytoplasmic in defined space(Baijal and Downey, 2021, p. 21).
Classifications Of Antibiotic By Mechanism Action
Antibiotic Targeting Cell Wall
Cell walls of bacterial are enclosed by peptidoglycan consisting huge sugar polymer.
Peptidoglycan confines crosslinking with glycan strands by peptide chains action and trans
glycosidases that extends from sugars in the polymer to form crosslinks of one peptide to
another. Glycine residues undergoes crosslink the D alanyl alanine portion of the peptide
chain in the presence of BPs (penicillin-binding proteins). Thus, strengthening the cell’s
walls of glycopeptides and Beta?lactam by inhibiting cell wall synthesis.
Β-Lactams (Beta?Llactam) Antibiotics
PBPs are the primary targets of the Beta?lactam agents. It is assumed that the Beta?lactam
ring mimic the portion of D alanyl D alanine in peptide chain which PBP normally binds.
The PBP interacts with the Beta?lactam ring and is not available to synthesize new
peptidoglycan. The disruption of the peptidoglycan layer leads to the lysis of the bacterium.
Glycopeptide
Glycopeptide binds on the portion of D alanyl D alanine into peptide side chain of the
precursor peptidoglycan subunit. The giant drug molecules of vancomycin inhibit cell’s
walls synthesis by preventing the binding between PBP and D alanyl subunit.
Protein Biosynthesis Inhibitors
The RNA (mRNA) transcription process uses data of bacterial DNA in the synthesize of RNA
molecules. On the other hand, the translation process uses a macromolecular ribosome
structure to synthesize proteins available in mRNA. Factors of cytoplasmic and ribosomes
catalyze protein biosynthesis. The 70S bacterial ribosomes comprise two ribonucleoprotein
subunits which are; 30S and 50S. The biosynthesis of protein is inhibited by antibacterial
when it targets the 30S or 50S subunit of the bacterial ribosome inside the bacterium. They
mostly target the bacterial ribosome; therefore, they must be energy-dependent for active
bacterial transport mechanism when entering the bacterial cell, which needs air(oxygen)
and a dynamic motive force of proton when passing the cytoplasmic membrane. Hence, AG
has poor activity against anaerobic bacterial since they work in aerobic conditions. The AG
synergize with antibiotic which inhibits cell’s walls synthesis like; glycopeptides and
Beta?lactam since they permit the passage of A.G. through the cell at a low dosage. A.G.
interacts with the 16S rRNA of the 30S subunit close to the A site by a hydrogen bond. Thus,
resulting premature termination hence, misreading of mRNA translation(Bozhüyük,
Micklefield, and Wilkinson, 2019, p.88).

Tetracycline
Tetracycline, like chlortetracycline, tetracycline, minocycline, or doxycycline, prevents tRNA
from binding with A site by acting within the preserved sequence of 16S rRNA of the 30S
ribosomal subunit.
50S Subunit Inhibitors
Macrolides
The early stage of synthesizing protein is affected by translocation which targets preserved
sequence of the center of peptidyl transferase in the 23S rRNA of the 50S ribosomal subunit
that are caused by the premature detachment of incomplete peptide chain. The lincosamide,
streptogramin B, and macrolides have same mechanisms of action.
Chloramphenicol
Protein synthesis are inhibited by chloramphenicol therefore preventing the binding of
tRNA in A site of the ribosomes since they interact with the conserved sequence of peptidyl
transferase cavity of the 23S rRNA of the 50S subunit.
Oxazolidinone
Linezolid is the newest member that has been approved of this entirely synthetic group's
novel class of antibiotics. Oxazolidinone interferes with the synthesis of protein in various
phases as follows:
binds to 23SrRNA which belongs to 50S subunit to inhibit synthesis of protein, and
interact with peptidyl-tRNA and suppress 70S inhibition.
DNA Replicates Inhibitors
Quinolone
The DNA gyrase of bacterial enzyme is inhibited by fluoroquinolones that nick the DNA of
double-stranded by introducing negative supercoil, which seals the nicked end. This is
useful in preventing excess positive supercoils in the strand when they separate to allow
transcription and replication. About two of both A and B subunits are contained in DNA
gyrase. The subunits carry out the nicking of DNA. B subunits introduce negative supercoils,
whereas A subunits reseal the strand. Fluoroquinolones interfere with strand cutting and
resealing function A subunit by binding with it at high affinity. The topoisomerase (IV) is the
original action target of gram-positive bacterial, which nick and separate daughter DNA
strands after replication. This enzyme has a more significant relationship that confers
highest potential to gram-positive bacterial. The mammal cell have topoisomerase (II), that
have lowest affinity to fluoroquinolones thus, lowest toxicity to cell in place of

topoisomerase IV and DNA gyrase(Cai, and Zhang, 2018, p.32).
Inhibitors Of Folic Acid Metabolism
Trimethoprim And Sulfonamides
Trimethoprim and Sulfonamides drugs inhibit distinct stages in metabolism of folic acid.
when combining trimethoprim and sulpha drugs acts on different stages on similar
biosynthetic pathways, indicating reduced mutation rate and synergy resistances. The
dihydropteroate synthase is wholly inhibited with a higher affinity for the enzyme by
Sulfonamides than the natural substrate, P aminobenzoic acid. Trimethoprim’s agent
inhibits the enzyme dihydrofolate reductase by acting late in folic acid synthesis.
Accumulation of antibacterial s and mechanisms of antibacterial resistance prevention from
the decrease of the intake or increase the efflux of the antibacterial from the cell. Porins
facilitate the outer membrane permeability to drug molecules into the cell, and its bilayer
enhances self-uptake and diffusion. In gram-negative bacterial, the porin channels are
located in the outer membrane. At the same time, tinny hydrophilic molecules of quinolones
and Beta?lactam are located through porins and cross the outer membrane. The decreased
rates of Beta?lactam antibiotics and fluoroquinolones' entrance into the cell are due to the
reduced number of porin channels. The lower permeability of all antibiotic classes in
Pseudomonas aeruginosa increases the bacteria’s resistance to antibiotics(Chakraborty,
2021).
Efflux Pumps
The efflux pumps are protein membranes that maintain low intracellular concentration and
export antibiotics from the cell. The efflux mechanisms pump the antibiotics out of the cell
and prevent them from reaching the target at the same speed that they used when entering
the cell. Efflux pumps are housed in a cytoplasmic membrane. All classes of antibiotics
except polymyxin are prone to activating efflux systems. Hence, they are specific to
antibiotics. Many efflux pumps are multidrug-resistant organisms since they have the
potential of pumping and transporting a wide variety of antibiotics such as
fluoroquinolones, macrolides, and tetracyclines(Eustáquio and Ziemert, 2018.).
Target Molecule Modification
The resistance mechanism of antibacterial to drug binding to reach the target area is caused
by acquired changes and natural variations. The spontaneous mutation of a bacterial gene
on the chromosome changes the target site. Minor alteration of the target molecule
significantly affects antibiotic binding since antibiotic interaction with the target molecule is
specific(Fazle and Baek, 2020, p.4973).
Alterations in either 30S subunits or 50S subunits of the ribosomes lead to drug resistance,
affecting proteins synthesis, that is, A.G., macrolides, chloramphenicol, and tetracycline. A.G.

binds with 30S ribosomal subunits, and on the other hand, macrolides, chloramphenicol,
streptogramin B, and lincosamides bind with 50S ribosomal subunits in suppressing the
synthesis of proteins.
Modification of the PBP causes alteration in PBP thus, favoring resistance mechanism in
Gram-positive bacterial. And the developing of resistance to Gram-negative bacterial results
to production of Beta?lactamases. The low affinity of Beta?lactam antibiotic is caused by
mutation in penicillin-binding proteins. The resistance of Enterococcus faecium to
Streptococcus and ampicillin pneumoniae to penicillin undergoes similar mechanism. On
the other hand, staphylococcus aureus, have same resistivity to oxacillin and methicillin by
genetic integration of free elements; staphylococcal cassette chromosome mec into the
chromosome of S. aureus which have gene resistivity of mec A which have, PBP2A protein
that is responsible in changing staphylococcal PBP. PBP2A have higher resistivity to
Beta?lactam antibiotic. S. aureus have cross-resistant to streptomycin, erythromycin,
tetracycline, all Beta?lactam antibiotic.
Alteration of cell wall precursor: glycopeptides inhibits cell’s walls synthesis by binding to D
alanyl D alanine residues of peptidoglycan precursors in Gram-positive bacterial, for
example, in teicoplanin or vancomycin. Glycopeptides do not crosslink with D alanyl
alanine, changing it to D alanyl lactate. Thus, developing high resistance(Van A?type
resistance) to teicoplanin and vancomycin caused by Enterococcus faecalis and E. faecium
and strains. The resistances are less sensitive to vancomycin but more sensitive to
teicoplanin on type Van C and B.
Fluoroquinolone’s resistivity is caused by topoisomerase and mutated?DNA gyrase:
Quinolone binds to DNA gyrases of subunit A. The mechanisms of resistance involve the
modification of two enzymes; that is, topoisomerase of part C and part E gene and DNA
gyrase of gyr A and gyr B gene. the mutation in gene par C and gyr A results in failure in
replication(binding) which is caused by fluoroquinolones.
Ribosomal protection mechanisms cause tetracyclines resistance.
Resistance to rifampicin is established by mutation of RNA polymerase.
Inactivation Of Antibiotic
The antibiotic are inactivated by three major enzymes: chloramphenicol acetyltransferases
(AACs), aminoglycoside modifying enzymes, and Beta?lactamases.
Aminoglycoside Modifying Enzymes (AGE)
A.G. has poor activity against anaerobic bacterial since they work in aerobic conditions. The
AG synergize with antibiotic which inhibits cell’s walls synthesis like; glycopeptides and
Beta?lactam since they permit the passage of A.G. through the cell at a low dosage. A.G.
interacts with the 16S rRNA of the 30S subunit close to the A site by a hydrogen bond. Thus,
resulting premature termination hence, misreading of mRNA translation Protein synthesis
are inhibited by chloramphenicol therefore preventing the binding of tRNA in A site of the
ribosomes since they interact with the conserved sequence of peptidyl transferase cavity of
the 23S rRNA of the 50S subunit(Greco, Keller, and Rokas, 2019, p.22).

Beta?Lactamases
PBPs are the major targets of the Beta?lactam agents. It is assumed that the Beta?lactam
ring mimic the portion of D alanyl D alanine in peptide chain which PBP normally binds.
The PBP interacts with the Beta?lactam ring and is not available to synthesize new
peptidoglycan. The disruption of the peptidoglycan layer leads to the lysis of the bacterium.
The Beta?lactamases are preventive enzymes and are grouped using two major grouping
systems: functional and structural. The classification by structural(Ambler)
The class A Beta?lactamases: are also called clavulanic acid susceptible or penicillinase.
SHV?1 and TEM?1 are Class A Beta?lactamases of Enterobacteriaceae members. They are
penicillinase having fewer activities against cephalosporin. They are progenitors called
ESBL (extended spectrum Beta?lactamases). These enzymes have altered their substrate
profile due to the substitution of amino acids permitting the hydrolysis of most
cephalosporins. The extended-spectrum Beta?lactamases are inhibited by Beta?lactamases
inhibitors like; tazobactam, clavulanic acid, or sulbactam, sensitive to methoxy
cephalosporins(carbapenems and cephamycin) and they resist third-generation
cephalosporins(cefotaxime, ceftriaxone, ceftazidime); penicillin; aztreonam; cefoperazone;
and cefamandole.
The class B Beta?lactamases are metallo Beta?lactamases; they require catalysis enzymes
like heavy metals or zinc and chelating agents to inhibit their activities. These enzymes
resist carbapenems, clavulanate, aztreonam, and sulbactam inactivation’s like, New Delhi
Metallo Beta?lactamase.
The class C Beta?lactamases are called cephalosporinases. All Gram-negative bacterial
produce them apart from, Klebsiella and Salmonella. They hydrolyze cephalosporins when
compared to class A Beta?lactamases. They can bind the bulky extended-spectrum penicillin
since of their huge cavities. Amp C Beta?lactamases are example of class C Beta?lactamases.
They are not inhibited by clavulanate and are resistant to all Beta?lactam and not to
carbapenems.
The class D Beta?lactamases are oxacillin hydrolyzing enzymes that exists in aeruginosa and
Enterobacteriaceae. clavulanic acid have weak inhabitation on them but are inhibited by
sodium chloride and resistant to cloxacillin, penicillin, methicillin, and
oxacillin(Hemmerling, and Piel, 2022, p.1).
Literature Review
In the past twenty years, the effectiveness of polyphosphate for P. aeruginosa virulence was
discovered from the pioneer task of late Nobel laureate Arthur Kornberg. The discovery of
an effective antibacterial drug was crucial in better treating bacterial infections. In this case,
polyphosphate is critical since its antibiotic tolerance, stress response, and P. aeruginosa
virulence, thus, indicating better results when used in antibiotic to target microbial. For the
first time using antibiotic treatments, small molecules of Gallein distracts polyP
(polyphosphate) in agents; they inhibit PPK1 and PPK2 (PPKs families) which contains P.

aeruginosa by showing dual susceptibility when inhibiting PPK. During inhibitor treatment,
the phenocopies of PPKs are deleted, resulting in a reduction of cellular accumulation of
polyphosphate by forming attenuated biofilm, pyoverdine, motility, and pyocyanin. The
Gallein synergized with antibiotic and caused Caenorhabditis elegans infection to attenuate
P. aeruginosa virulence by exhibiting negligible toxicity on HEK293T cells and nematodes,
hence, indicating that PPK inhibitors are the reference point to drug discovery(Hug, Panter,
Krug, and Müller, 2019, p.319).
Both PPK1 and PPK2 enzymes are encoded by P. aeruginosa in bacterial pathogens to
maintain polyphosphate homeostasis. The PPKs have distinct catalytic mechanisms and
structures, but they can consume and synthesize polyphosphate. In this case, PPK2 enzymes
can compensate for losses in PPK1. The research established that small molecules of Gallein
distract polyP (polyphosphate) in agents; they inhibit PPK1 and PPK2 (PPKs families),
which contain P. aeruginosa by showing dual susceptibility when inhibiting PPKs. During
inhibitor treatment, the phenocopies of PPK are deleted and therefore, resulting in the
reduction of cellular accumulation of polyphosphate in DPPK1, DPPK2, and wild type (W.T.)
strains to levels of DPPK1, DPPK2A, DPPK2B and DPPK2C in knockout control, forming
attenuated biofilm, pyoverdine, motility, and pyocyanin. The Gallein synergized with
antibiotic and caused Caenorhabditis elegans infection to attenuate P. aeruginosa virulence
by exhibiting negligible toxicity on HEK293T cells and nematodes, hence, indicating that
PPK inhibitors are the reference point for treatments(Kang, and Wang, 2020, p.1562.).
It is established that PPK2 and PPK1 are valuable drug targets in P. aeruginosa,
hence, indicating that PPK inhibitors are the reference point for drug discovery. The linear
polymer of inorganic phosphate residues ranging from about one thousand monomers in
length forms Inorganic polyphosphate. PPK1 and PPK2 (PPKs families) enzymes synthesize
and consume polyphosphate in the bacterial. PPK1 enzyme catalyzes polyphosphate
synthesis by ATP, a phosphor donor. At the same time, the PPK2 enzyme destroys
polyphosphate to phosphorylate nucleotides; established by Nobel Arthur Kornberg. PPK1
is an essential determinant for virulence in P. Aeruginosa. Since, during inhibitor treatment,
the phenocopies of PPK are deleted, resulting in the reduction of cellular accumulation of
polyphosphate by forming attenuated biofilm, pyoverdine, motility, and pyocyanin in
relevant pathogens, comprising Campylobacter jejune, Escherichia coli, Francisella
tularensis, Proteus mirabilis and Mycobacterium tuberculosis. PPKs families’ enzymes have
potential targets for antibacterial therapeutics by establishing their various inhibitors, but
in compounds, low nanomolar activity or micromolar against PKKs classes have not been
found (Khabthani, Rolain, and Merhej, 2021, p.2297).
The inhibitors targeting PPK1 and PPK2 (PPKs families) enzymes can provide the best
therapeutic options due to high bacterial pathogens priorities like Klebsiella, P.
aeruginosa, pneumoniae, Acinetobacter Baumann, C. jejune, and F. tuberculosis consists a
minimum of one of PPKs. The P. aeruginosa genome is encoded by PPK1, PPK2A, three PPK2
enzymes, PPK2C and PPK2B. The PPK2B and PPK2A of P. aeruginosa generate sufficient

polyphosphate to form granules as a supplementary of PPK1 inactivation. At the same time,
PPK2A facilitates exit for the near-normal cell cycle. During inhibitor treatment, the
phenocopies of PPK are deleted, resulting in the reduction of cellular accumulation of
polyphosphate by forming attenuated biofilm, pyoverdine, motility, and pyocyanin. The
hence, indicating that PPK inhibitors are the reference point to drug discovery as directed
by World Health Organization(Larsen, Pearson, and Neilan, 2021, p.56).
Pyoverdine in P. aeruginosa is the principal of siderophore, which serves to import and
chelate iron into the bacterial cell. It leads to the formation of distinctive green color during
the culture of P. aeruginosa. The phenocopies of PPK are deleted, resulting in a reduction of
cellular accumulation of polyphosphate by forming attenuated biofilm, pyoverdine, motility,
and pyocyanin. The Gallein synergized with antibiotic and caused Caenorhabditis elegans
infection to attenuate P. aeruginosa virulence by exhibiting negligible toxicity on HEK293T
cells and nematodes. Genetic and phenotype analysis are used in the determination of
mutation and bacterial resistance to antibiotic respectively. This is essential to health
practitioners when establishing the best antibiotic to give patients(Milke, and Marienhagen,
2020, p.6057).
In this study, P. aeruginosa, for PPKs enzymes, is an ant virulence target when purified and
expressed in PPK1, PPK2A, PPK2B, and PPK2C in biochemical research. Using analog
synthesis and compound screening new family of polyhydroxylated is identified as small
molecules of Gallein distract polyP (polyphosphate) in agents; they inhibit PPK1 and PPK2
(PPKs families), which contains P. aeruginosa by showing dual susceptibility when
inhibiting PPKs. During inhibitor treatment, the phenocopies of PPK are deleted, resulting
in a reduction of cellular accumulation of polyphosphate by forming attenuated biofilm,
pyoverdine, motility, and pyocyanin(Scott, and Piel, 2019, p.404).
When conducting Gallein distracts polyP (polyphosphate) in agents, they inhibit PPK1 and
PPK2 (PPKs families), which contain P. aeruginosa by showing dual susceptibility when
inhibiting PPK. During inhibitor treatment, the phenocopies of PPKs are deleted and
therefore, resulting in the reeducation of cellular accumulation of polyphosphate by forming
attenuated biofilm, pyoverdine, motility, and pyocyanin. The Gallein synergized with
antibiotic and caused Caenorhabditis elegans infection to attenuate P. aeruginosa virulence
by exhibiting negligible toxicity on HEK293T cells and nematodes, hence, indicating that
PPKs inhibitors provide a foundation for the future design discovery and development of
new antibiotic drug(Silva, Blom, Keller?Costa and Costa, 2019, p.4002).
Past research has shown that PPKs families' enzymes have potential targets for
antibacterial therapeutics by establishing their various inhibitors. Still, compounds with low
nanomolar activity or micromolar against PKKs classes have not been shown. There,
creating room for developing disc susceptibility inhibitors that act on all types of enzymes

using an attractive methodology. This is made possible by using RT1 and ellagic acid as a
reference point to synthesize compounds that can hit Gallein compounds that inhibit PPK1,
PPK2B, and PPK2A at low micromolar concentration. The PKK's enzymes are catalyzed
under the same chemical reactions since they have similar structural homology and
sequence. PPK2 enzymes are minor thirty-five kDa. It shares mechanistic and structural
similarities with thymidylate kinases; here, walkers A and B fill the position where the
nucleotide substrate of Mg21 is catalyzed to perform a nucleophilic attack on the terminal
phosphate of polyphosphate. Whereas PPK1 enzymes are large, about seventy-five kDa, it
shares mechanistic and structural similarities with the phospholipase D family of proteins
when catalyzed by phosphohistidine. Still, the research suggests that Gallein acts as a
nucleotide mimetic in static binding sites for PKKs enzymes classes. The weakness of
inhibition of PPK2C by Gallein is due to catalyzing of class II PPK2 enzymes to change
nucleoside of monophosphates into nucleoside diphosphates. PPK2A and PPK2B belong to
class I enzymes that catalyze phosphorylate nucleosides into diphosphates. The structural
comparisons of PPK2 (class I) and Meiothermus rubber PPK2 (class III) of F. tuberculosis
phosphorylate into diphosphates or nucleoside mono. Its distinct nucleotide-binding
orientations identify this. Adenosine moiety is mainly shifted to M. rubber protein. Thus,
PPK2C has less impact on static binding sites of Gallein. Therefore, the structural similarity
of NSC 9037 and Gallein suggests the best inhibitor results of M. tuberculosis PPK2, which is
a Galleon-based inhibitor that can be used in combating PKK2 bacterial pathogens carriers
(Zhang, Sun, Wang, Chen, Xue, Zhang, Zhu, Duan, and Yan, 2021, p.1).
Materials And Methods
Materials
List of compounds for testing against Gallein
Rupesh
EMC1059 B3
EMC1059 D5
Mohammed
BU10016 (EMC1059D3)
EMC1048
Ahsan
BU10078

EMC1041
Dozen
BU10009 (EMC1058C1)
EMC1058C3
Aruna
BU10004
EMC1085M3
Manor
BU10007 (EMC1058B2)
EMC1058L1
Lama
BU10077 (EMC1092NI)
EMC1051
Viviene
BU10067 (EMC1085MI)
EMC1085L3
Said
BU10003 EMC1058A2
EMC 1080
Method
The plates were set up by making stocks of each three bacterial at an absorbance at 600 nm
of 0.05. fill strips of 8 wells with 180 ul of bacterial either singly or in duplicate or triplicate;
for each row, this means 8 x 180 ul = 1440 ul (so effectively 5 MLS for three rows). Provide

for a control row having no addition of antibiotic to check for growth, although typically,
this should not be needed for the lowest dilution of antibiotic still allows development.
Make a thick stock of each bacterium by scraping colonies from the plate or growing stock
in the media for a few hours. In obtaining the desired absorbance of about 0.05, dilute and
fill the test plates. Begin by measuring the absorbance with bacterial stock using a
spectrometer set to 600 nm using a blank of L.B. media.
The computation of the amount of stock required is given as:
The volume of dense stock =
Then, add the media to the final total volume obtained(this was done for each bacterial
test).
How Serial Dilutions Of Antibiotic Were Made
The setup of the serial dilutions of the antibiotic to be tested was done as follows. The test
required the setup of 4 sets, each having 8 Eppendorf tubes (1.5ml tubes) and one set for
each of the antibiotic. A 2-fold serial dilution was then made from a 4-fold series to give a
more considerable range of dilutions by changing the volumes of water in each tube to 450
ul, and the volume transferred to 150 ul by making up enough diluted antibiotic for several
plates (300 ul of diluted antibiotic in each tube). More samples were done by adjusting the
volumes as 3 x 20 ul per bacterial for each measurement in triplicate. The four sets of eight
tubes were placed in a rack; each group was labeled from one to eight, as demonstrated
below:
Fill all of the tubes apart from the first one of each set with 300 ul of water.
Makeup to 600 ul of antibiotic concentration to begin within the test for the first tube of
each set.
Transfer about 300 ul into the second tube from the first tube, then mix it. Then, transfer
300 ul to the third from the second tube, and mix it. Repeat the above, moving 300 ul from
each tube to the next until the last tube. To obtain sets of serial dilutions to test than
required to complete this for all four antibiotic tests.
Then add about 20ul of each serial dilution to set up the antibiotic test from the most dilute
to highest concentration to the 8th to first columns on the microplate. Do not change tips as
filled in each row by filling this order. Fill one row at a time for each of the four antibiotic.
Add 180 ul of stock across four rows of wells and fill the set of wells to test with bacterial
from the stocks made., using a new tip for each row. The figure below shows the summary
of how to make serial dilution.
Measurements
After setting up the plates, the measurement was done from the initial absorbance in the
microplate reader was obtained by ensuring the reader is set to 600 nm and readings of the

absorbance for the plate made. The uniformity in absorbance across all wells was observed,
implying that the setup of plates was correct. The plate was then incubated at 37 C for at
least 3 hours to let the cells grow to repeat the measurements to determine if the cells had
grown. Plot results of absorbance versus dilution to determine the lowest concentration
needed to inhibit the cells and MIC(Minimum Inhibitory Concentration).
How Disc Susceptibility Test Was Performed
Preparation Of Mueller-Hinton Plate
Inoculum Preparation
use a sterile to inoculate, needle or loop, and touch four or five isolated colonies of the
organism to be tested.
Using sterile falcon tubes, suspend the organism containing 5 mL of L.B. Broth.
Incubate bacterial overnight in a shaker incubator at 37°C and record the OD600nm should
be 1.0 x 109 cells ml-1
Inoculation Of The M.H. Plate
Dilute overnight culture broths 1 in 10 using sterile Eppendorf tubes and read the O.D. at
600 nm, expected to have 1.0 x 108 cells ml-1.
Using a sterile spreader, from the 107 concentrations of O/N broth with different strains,
take 100 µl and spread onto M.H. agar plates.
The same procedure can be used for each broth strain onto M.H. agar plates.
Allow inoculated plates to dry for 30 minutes at room temperature.
Results
Discussion
The disc susceptibility of Gallein distracts polyP (polyphosphate) in agents; they inhibit
PPK1 and PPK2 (PPKs families), which contain P. aeruginosa by showing dual susceptibility
when inhibiting PPK. During inhibitor treatment, the phenocopies of PPKs are deleted and
therefore, resulting in a reduction of cellular accumulation of polyphosphate in DPPK1,
DPPK2, and wild type (W.T.) strains to levels of DPPK1, DPPK2A, DPPK2B and DPPK2C in
knockout control, forming attenuated biofilm, pyoverdine, motility, and pyocyanin. The
hence, indicating that PPKs inhibitors are the reference point for treatments. It is
established that PPK2 and PPK1 are valuable drug targets in P. aeruginosa, therefore,
implying that PPK inhibitors are the reference point for drug discovery. The linear polymer
of inorganic phosphate residues ranging from about one thousand monomers in length
forms Inorganic polyphosphate. PPK1 and PPK2 (PPKs families) enzymes synthesize and
consume polyphosphate in the bacterial. PPK1 enzyme catalyzes polyphosphate synthesis
by ATP, a phosphor donor. Glycopeptides inhibit cell wall synthesis by binding to D alanyl D
alanine residues of peptidoglycan precursors in Gram-positive bacterial, for example, in
teicoplanin or vancomycin. Glycopeptides do not crosslink with D alanyl alanine, changing it
to D alanyl lactate. Thus, developing high resistance(Van A?type resistance) to teicoplanin
and vancomycin caused by Enterococcus faecalis and E. faecium and strains. The resistances

are less sensitive to vancomycin but more sensitive to teicoplanin on type Van B and C
resistance.
Pyoverdine in P. aeruginosa is the principal of siderophore, which serves to import and
chelate iron by reaction inhibitor in E. coli into the bacterial cell. The phenocopies
of PPK are deleted, resulting in a reduction of cellular accumulation of polyphosphate by
forming attenuated biofilm, pyoverdine, motility, and pyocyanin. The Gallein synergized
with antibiotic and caused Caenorhabditis elegans infection to attenuate P. aeruginosa
virulence by exhibiting negligible toxicity on nematodes. The determination of bacterial
resistance to antibiotic of all classes; phenotypes and mutations responsible for bacterial
resistance to antibiotic.
Motility caused by flagellar requires the critical establishment of its acute infections, which
is compromised by Gallein treatments in a pink-dependent fashion, with previous
corroborating reports linking polyphosphate to this phenotype in P. aeruginosa. Once again,
our results show that deletion of all four pink genes or treatment with Both PPK1 and PPK2
enzymes are encoded by P. aeruginosa in bacterial pathogens to maintain polyphosphate
homeostasis. The PPKs have distinct catalytic mechanisms and structures, but they can
consume and synthesize polyphosphate. In this case, PPK2 enzymes can compensate for
losses in PPK1. The research established that small molecules of Gallein distract polyP
(polyphosphate) in agents; they inhibit PPK1 and PPK2 (PPKs families), which contain P.
aeruginosa by showing dual susceptibility when inhibiting PPK. During inhibitor treatment,
the phenocopies of PPK are deleted and therefore, resulting in a reduction of cellular
accumulation of polyphosphate in DPPK1, DPPK2, and wild type (W.T.) strains to levels of
DPPK1, DPPK2A, DPPK2B and DPPK2C in knockout control, forming attenuated biofilm,
pyoverdine, motility, and pyocyanin. The Gallein synergized with antibiotic and caused
Caenorhabditis elegans infection to attenuate P. aeruginosa virulence by exhibiting
negligible toxicity in the cell. Inhibiting PPK1 and PPK2 enzymes values are supported in C.
elegans models of disease for S.K. and L.K. assays in Gallein treatment of wild type. P.
aeruginosa phenocopied attenuates the virulence of the D polyphosphate strain. P.
aeruginosa pathogenesis is used in L.K. to mediate and reduce pyoverdine levels in D
polyphosphate cells and galleon treatment when Gallein is combined with ciprofloxacin and
antibiotic tetracycline, it synergetic protection of C. elegans in L.K.
Gallein shows inhibitors in mammalian C. elegans and G protein-coupled receptors (GPCRs),
referred to as OCTR1. They are used in innate immunity in targeting Gallein. But in the
absence of additives, the P. aeruginosa strain is effective in treating infections in C. elegans
with D polyphosphate compared to the wild-type strain due to off-target OCTR1 inhibitors.
PPK2 and PPK1 are valuable drug targets in P. aeruginosa, indicating that PPK inhibitors
are the reference point for drug discovery. The linear polymer of inorganic phosphate
residues ranging from about one thousand monomers in length forms Inorganic
polyphosphate. PPK1 and PPK2 (PPKs families) enzymes synthesize and consume

polyphosphate in the bacterial. PPK1 enzyme catalyzes polyphosphate synthesis by ATP, a
phosphor donor. In contrast, the PPK2 enzyme destroys polyphosphate to phosphorylate
nucleotides; established by Nobel Arthur Kornberg. PPK1 is an essential determinant for
virulence in P. Aeruginosa. Since, during inhibitor treatment, the phenocopies of PPK are
deleted, resulting in the reduction of cellular accumulation of polyphosphate by forming
attenuated biofilm, pyoverdine, motility, and pyocyanin in relevant pathogens, comprising
Campylobacter jejune, Escherichia coli, Francisella tuberculosis, Proteus mirabilis and
Mycobacterium tuberculosis. PPKs families' enzymes have potential targets for antibacterial
therapeutics by establishing their various inhibitors.
Conclusion
Drug discovery for antibiotic is a great relief; the prolonged use of antibiotic causes the
emergence of new infections. The use of antibiotic drugs to treat bacterial over a long time
makes some bacterial more resistant and competitive to resist antibacterial drugs. The
resistance of bacterial to antibacterial is a critical problem when it comes to treating
microbial infections. These biochemicals resistance mechanisms to antibacterial are; target
modification, antibiotic inactivation, relief of metabolic pathway, and altered permeability.
The bacterial phenotypes and mutations are the leading cause of bacterial resistance to
antibiotic. Therefore, research on establishing bacterial phenotypes and genetic analysis are
essential in determining bacteria's antibiotic resistance. Genetic and phenotype analysis are
used in the determination of mutation and bacterial resistance to antibiotic respectively.
This is essential to health practitioners when establishing the best antibiotic to give
patients.
Future Work
PPKs families’ enzymes have potential targets for antibacterial therapeutics by establishing
their various inhibitors, but in compounds, low nanomolar activity or micromolar against
PKKs classes have not been established. Therefore, future research will be based on
discovering the inhabitancy of compounds with low nanomolar activities and micromolar.
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