Introduction where 90% of them found improvements. However,

IntroductionAn increase of multi-drug resistant bacteria and thedecreasing effects of antibacterial agents poses as a threat to the well beingof humans. New antimicrobial agents have not been developed since 1960 due to thecost 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 bedeveloped was discovered in 1987 by Zasloff from and African frog skin. It was demonstratedto act against 3109 bacterial clinical isolates and tested on individuals withfoot ulcers where 90% of them found improvements. However, from easy over-useof antibiotics, FDA approval was denied (Y. Jerold Gordon and Eric G. Romanowski 2006).

Antimicrobial peptides have a broadeffect towards gram-negative and gram-positive bacteria, by decreasing the rateof resistance being developed by target pathogens due to their mode of actionon the cytoplasmic membrane. Bacteria compete by upregulating genes that codefor these AMPs to fight off bacteria in the environment. Many eukaryotic cellslike lymphs, are involved in the production of AMPs. They have been found toinfluence the inflammatory response during infection.

Lipopolysaccharides thathave been released from bacteria, can induce AMP production in humans and blockcytokine release by macrophages (Ali Adem Bahar and Dacheng Ren, 2013).  The rumen in eukaryotes is a chamber where fibre is brokendown into components by bacteria that are able to be digested. Therefore, it isunable to function without microbial populations present. This environment ishighly competitive microbiome, which allows for the investigation into AMPs. Bysequencing three fosmid metagenomic libraries from the rumen biome, three AMPscan be identified and used to used to test their role in common bacteria suchas E.coli and S.epidermidis.   Materials and Methods Three fosmid metagenomic sequences were obtained from therumen biome and identified using BLAST database.

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These were then passed throughthe AMPA database to identify the propensity and probability of each AMP.  For the experiment the antimicrobial potential of AMPs andciprofloxacin using MIC assays against E.coliand S.epidermidis was tested. Theplates were labelled correctly and 500µL of E.coli was added into a 50mL Greiner tubes that contained 24.

5mLstrength LB broth using a pipette, mixed by shaking lightly and labelledappropriately. 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 apipette 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 otherwells. A serial dilution was then carried out from column 1 row A-F, and intocolumn 2 using a pipette at 100µl andmixing by pipetting up and down three times. The same technique was carried outfor 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.

epidermidisLB 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 ciprofloxacinand 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 threesequences for AMPs From the data of the predicted AMPs, sequence 3 identifiesto 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 treatedwith AMP3.            Figure 2: MIC Assay of E.

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

               Figure 3: MIC Assay of E.colion wells containing AMP2 and ciprofloxacin.In rows A,B and C it contains AMP2, in rows D, E and Fcontains ciprofloxacin antibiotics. There is visibly no growth in rows containingciprofloxacin antibiotics and in columns 1- 4 in AMP2 containing wells. TheAMP2 MIC result shows for column 1 is 64mg/mL wherecolumn 2 and 3 shows an MIC of 132mg/mL.

  Therefore, the MIC for AMP1 is 64 mg/mL.              Figure 4: MIC Assay of E.colion wells containing AMP3 and ciprofloxacin.In rows A, B and C it contains AMP3, in rows D, E and Fcontains ciprofloxacin antibiotics. There is visibly no growth in rowscontaining 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.epidermidison wells containing AMP1 and ciprofloxacin. In rows A, B and C it contains AMP1. There is visibly nogrowth in rows 1- 10 containing ciprofloxacin antibiotics and in columns 1 and2 in AMP1 containing wells. Therefore, the MIC for AMP1 is 264 mg/mL.

The MIC for ciprofloxacin is1mg/mL              Figure 6: MIC Assay of S.epidermidison wells containing AMP2 and ciprofloxacin. In rows A, B and C it contains AMP2. There is visibly nogrowth in rows 1- 10 containing ciprofloxacin antibiotics and in columns 1,2and 3 in AMP2 containing wells. Therefore, the MIC for AMP1 is 132 mg/mL. The MIC for ciprofloxacin is1mg/mL              Figure 7: MIC Assay of S.epidermidison wells containing AMP3 and ciprofloxacin.In rows A, B and C it contains AMP3.

There is visibly nogrowth in rows 1- 10 containing ciprofloxacin antibiotics and in columns 1 in AMP3containing wells. Therefore, the MIC for AMP3 is 512 mg/mL. The MIC for ciprofloxacin is1mg/mL   Figure 8: A table of gathered results of MICs from the AMPassays 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   DiscussionFrom the results it is clear to see that each bacterium wassomewhat limited in terms of growth by AMPs used. Indicating that the antimicrobialpeptides used have successfully permeabilised the membrane of both the Grampositive (S.epidermidis) and Gramnegative (E.coli) bacteria. From thedata of the predicted AMPs (figure 1), sequence 3 identifies to having the lowest propensity at0.

18. Therefore, by using this AMP, the bacteria should have little to nogrowth when treated with AMP3. However, in both bacteria plates the AMP2 (Figure 3and 6) was most effective in terms of preventing growth of bacteria with MIC of64mg/mL for Ecoli and 132mg/mL for S.epidermidis. The lower the MIC the more effective, as only a lowdosage of AMP is required to eradicate the microbes.

Therefore, the Ecoli had the lowest MIC for AMP2 comparedto the S.epidermidis. In the Ecoli plates, there is no growth at allfor ciprofloxacin, but a slight growth in S.

epidermidiswells containing ciprofloxacin at 1mg/mL. ‘Ciprofloxacin may be considered as first-line treatment for a number ofinfections 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 throughoutthe experiment slightly weaker, therefore a stronger concentration of ciprofloxacinshould be used in the future.

In a research paper by Linda B Oyama et al, the effectof Lynronne-1, Lynronne-2 and Lynronne-3 is analysed its effects againstseveral pathogens like S.aureus. Withinthis 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, containedwater as a control, there is growth in the majority of these wells from theassay. This indicates contamination in these wells, within the workingenvironment there are some factors that may have contributed to this result. Thelab at which the work was carried out was above room temperature, some large windowswere open which could have contributed to microbes getting in and contaminationof some wells.

A multi-channel pipette was not available also, therefore singlepipette 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 goodaseptic environment and resources are available and used.  Overall, the use of AMPs is astep forward in overcoming the challenges in antibiotic resistance. The resourcescan be obtained from natural resources at low cost due to the technologiesdeveloped and use of computational approaches. From this it is shown that therumen microbiome is one of many resources from development of AMPs for future antibacterialtreatment.

The three peptides used in this experiment, Lynronne-1, 2 and 3, areidentified as having antimicrobial properties and can be used to fight againstbacteria. Another factor that could’ve been analysed when carrying out this experimentis if AMP-resistant bacteria could have occurred, this could have been analysedby continuing to expose the bacteria to AMPs for a longer duration andcontinuously comparing MICs.