Introduction where 90% of them found improvements. However,

Introduction

An increase of multi-drug resistant bacteria and the
decreasing effects of antibacterial agents poses as a threat to the well being
of humans. New antimicrobial agents have not been developed since 1960 due to the
cost of development. It has been since recognised that antimicrobial peptides
(AMPs) are catering to an alternative way to provide antibiotics. The first antimicrobial peptide to be
developed was discovered in 1987 by Zasloff from and African frog skin. It was demonstrated
to act against 3109 bacterial clinical isolates and tested on individuals with
foot ulcers where 90% of them found improvements. However, from easy over-use
of antibiotics, FDA approval was denied (Y. Jerold Gordon and Eric G. Romanowski 2006). Antimicrobial peptides have a broad
effect towards gram-negative and gram-positive bacteria, by decreasing the rate
of resistance being developed by target pathogens due to their mode of action
on the cytoplasmic membrane. Bacteria compete by upregulating genes that code
for these AMPs to fight off bacteria in the environment. Many eukaryotic cells
like lymphs, are involved in the production of AMPs. They have been found to
influence the inflammatory response during infection. Lipopolysaccharides that
have been released from bacteria, can induce AMP production in humans and block
cytokine release by macrophages (Ali Adem Bahar and Dacheng Ren, 2013).

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The rumen in eukaryotes is a chamber where fibre is broken
down into components by bacteria that are able to be digested. Therefore, it is
unable to function without microbial populations present. This environment is
highly competitive microbiome, which allows for the investigation into AMPs. By
sequencing three fosmid metagenomic libraries from the rumen biome, three AMPs
can be identified and used to used to test their role in common bacteria such
as E.coli and S.epidermidis.

 

 

Materials and Methods

Three fosmid metagenomic sequences were obtained from the
rumen biome and identified using BLAST database. These were then passed through
the AMPA database to identify the propensity and probability of each AMP.

 

For the experiment the antimicrobial potential of AMPs and
ciprofloxacin using MIC assays against E.coli
and S.epidermidis was tested. The
plates were labelled correctly and 500µL of E.coli was added into a 50mL Greiner tubes that contained 24.5mL
strength LB broth using a pipette, mixed by shaking lightly and labelled
appropriately. The same steps were then carried out using the S.epidermidis bacteria and labelled.
From the plates, in column 1 row A, B and C, 180 µl of broth and 20µl of AMP 1 was added using a
pipette and mixed. Into column 1 row D, E and F 180µ of E.coli LB broth was added along with 20µl of ciprofloxacin. 100µl of E.coli LB broth was added into column 1 row G and H and 100µl of E.coli LB broth in the 4 other
wells. A serial dilution was then carried out from column 1 row A-F, and into
column 2 using a pipette at 100µl and
mixing by pipetting up and down three times. The same technique was carried out
for columns 2-3, 3-4 etc until the twelfth column where 100µl of the contents was discarded.
This technique was then repeated into two more wells containing AMP2 and AMP3.
It was again repeated using the S.epidermidis
LB broth into three more assay plates containing AMP1, AMP2 and AMP3.

 

Each assay then contained diluted AMPs from 512µg/mL in column 1 down to 0.25 µg/mL in column 12 and 32 µg/mL in column 1 of ciprofloxacin
and downwards. The plates were then sealed and incubated for one week at 4oC .

 

Results

Figure 1: A table to show the AMPA results of the three
sequences for AMPs

From the data of the predicted AMPs, sequence 3 identifies
to be antimicrobial due to it having the lowest propensity at 0.18. Therefore,
by using this AMP, the bacteria should have little to no growth when treated
with AMP3.

 

 

 

 

 

 

 

 

 

 

 

Figure 2: MIC Assay of E.coli
on wells containing AMP1 and ciprofloxacin.

In rows A,B and C it contains AMP1, in rows D, E and F
contains ciprofloxacin antibiotics. There is visibly no growth in rows
containing ciprofloxacin antibiotics and in columns 1 and 2 in AMP1 containing
wells. Therefore, the MIC for AMP1 is 264 mg/mL. 

 

 

 

 

 

 

 

 

 

 

 

 

 

Figure 3: MIC Assay of E.coli
on wells containing AMP2 and ciprofloxacin.

In rows A,B and C it contains AMP2, in rows D, E and F
contains ciprofloxacin antibiotics. There is visibly no growth in rows containing
ciprofloxacin antibiotics and in columns 1- 4 in AMP2 containing wells. The
AMP2 MIC result shows for column 1 is 64mg/mL where
column 2 and 3 shows an MIC of 132mg/mL.  Therefore, the MIC for AMP1 is 64 mg/mL. 

 

 

 

 

 

 

 

 

 

 

 

 

Figure 4: MIC Assay of E.coli
on wells containing AMP3 and ciprofloxacin.

In rows A, B and C it contains AMP3, in rows D, E and F
contains ciprofloxacin antibiotics. There is visibly no growth in rows
containing ciprofloxacin antibiotics and in column 1 in AMP3 containing wells.
Therefore, the MIC for AMP3 is 512 mg/mL. 

 

 

 

 

 

 

 

 

 

 

 

 

Figure 5: MIC Assay of S.epidermidis
on wells containing AMP1 and ciprofloxacin.

 

In rows A, B and C it contains AMP1. There is visibly no
growth in rows 1- 10 containing ciprofloxacin antibiotics and in columns 1 and
2 in AMP1 containing wells. Therefore, the MIC for AMP1 is 264 mg/mL. The MIC for ciprofloxacin is
1mg/mL 

 

 

 

 

 

 

 

 

 

 

 

 

Figure 6: MIC Assay of S.epidermidis
on wells containing AMP2 and ciprofloxacin.

 

In rows A, B and C it contains AMP2. There is visibly no
growth in rows 1- 10 containing ciprofloxacin antibiotics and in columns 1,2
and 3 in AMP2 containing wells. Therefore, the MIC for AMP1 is 132 mg/mL. The MIC for ciprofloxacin is
1mg/mL 

 

 

 

 

 

 

 

 

 

 

 

 

Figure 7: MIC Assay of S.epidermidis
on wells containing AMP3 and ciprofloxacin.

In rows A, B and C it contains AMP3. There is visibly no
growth in rows 1- 10 containing ciprofloxacin antibiotics and in columns 1 in AMP3
containing wells. Therefore, the MIC for AMP3 is 512 mg/mL. The MIC for ciprofloxacin is
1mg/mL 

 

Figure 8: A table of gathered results of MICs from the AMP
assays

Ecoli

AMP

MICmg/mL 

Ciprofloxacin
MICmg/mL 

Lynronne-1

264

 

Lynronne-2

64

 

Lynronne-3

512

 

S.epidermidis

Lynronne-1

264

1

Lynronne-2

132

1

Lynronne-3

512

1

 

 

Discussion

From the results it is clear to see that each bacterium was
somewhat limited in terms of growth by AMPs used. Indicating that the antimicrobial
peptides used have successfully permeabilised the membrane of both the Gram
positive (S.epidermidis) and Gram
negative (E.coli) bacteria. From the
data of the predicted AMPs (figure 1), sequence 3 identifies to having the lowest propensity at
0.18. Therefore, by using this AMP, the bacteria should have little to no
growth when treated with AMP3. However, in both bacteria plates the AMP2 (Figure 3
and 6) was most effective in terms of preventing growth of bacteria with MIC of
64mg/mL for Ecoli and 132mg/mL for S.epidermidis. The lower the MIC the more effective, as only a low
dosage of AMP is required to eradicate the microbes. Therefore, the Ecoli had the lowest MIC for AMP2 compared
to the S.epidermidis. In the Ecoli plates, there is no growth at all
for ciprofloxacin, but a slight growth in S.epidermidis
wells containing ciprofloxacin at 1mg/mL. ‘Ciprofloxacin may be considered as first-line treatment for a number of
infections in which gram-negative pathogens are proven or strongly suspected,
including complicated urinary tract infections, bacterial prostatitis,
bacterial diarrhea’ (T J Louie, 1994) all of which is more common with Ecoli than S.epidermidis. However, the strength of ciprofloxacin used throughout
the experiment slightly weaker, therefore a stronger concentration of ciprofloxacin
should be used in the future. In a research paper by Linda B Oyama et al, the effect
of Lynronne-1, Lynronne-2 and Lynronne-3 is analysed its effects against
several pathogens like S.aureus. Within
this project, all three said AMPs were effective against bacterial pathogens,
proving that the rumen microbiome may be used for therapeutics in the future.

 

In well rows G and H, contained
water as a control, there is growth in the majority of these wells from the
assay. This indicates contamination in these wells, within the working
environment there are some factors that may have contributed to this result. The
lab at which the work was carried out was above room temperature, some large windows
were open which could have contributed to microbes getting in and contamination
of some wells. A multi-channel pipette was not available also, therefore single
pipette was used. These factors may have contributed to unreliable results; therefore,
the experiment would need to be carried out again, in the future by ensuring a good
aseptic environment and resources are available and used.

 

Overall, the use of AMPs is a
step forward in overcoming the challenges in antibiotic resistance. The resources
can be obtained from natural resources at low cost due to the technologies
developed and use of computational approaches. From this it is shown that the
rumen microbiome is one of many resources from development of AMPs for future antibacterial
treatment. The three peptides used in this experiment, Lynronne-1, 2 and 3, are
identified as having antimicrobial properties and can be used to fight against
bacteria. Another factor that could’ve been analysed when carrying out this experiment
is if AMP-resistant bacteria could have occurred, this could have been analysed
by continuing to expose the bacteria to AMPs for a longer duration and
continuously comparing MICs. 

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