Purification and characterization of an antimicrobial peptide … · 2017-10-11 · Purification and characterization of an antimicrobial peptide produced by Bacillus sp. strain P7

Purification and characterization of an antimicrobial peptide

produced by Bacillus sp. strain P7

A dissertation submitted to the University of Manchester for the degree of Master of

Science in Medical Microbiology in the Faculty of Medical and Human Science

2014

Paulina Fernández Soto

School of Medicine

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Table of Contents

List of Figures .............................................................................................................. 3

List of Tables ............................................................................................................... 3

Abstract ........................................................................................................................ 4

Declaration .................................................................................................................. 5

Intellectual Property Statement ................................................................................ 6

Acknowledgments ....................................................................................................... 8

Preface .......................................................................................................................... 9

Dedication .................................................................................................................... 9

1. Introduction ....................................................................................................... 10

2. Materials and Methods ..................................................................................... 15

2.1. Bacterial strains ............................................................................................ 15

2.2. Indicator strains ............................................................................................ 15

2.3. Screening for antimicrobial peptide production ........................................... 15

2.4. Broth optimization for antimicrobial peptide production ............................ 16

2.5. Assays to test antimicrobial peptide activity ................................................ 16

2.6. Purification of the antimicrobial peptide ...................................................... 17

2.7. Characterization of the antimicrobial peptide .............................................. 19

2.8. Strain identification by sequence analysis of the 16S rRNA gene ............... 21

3. Results ................................................................................................................. 24

3.1. Screening of bacteriocin production using simultaneous antagonism ......... 24

3.2. Broth Optimization ....................................................................................... 25

3.3. Antimicrobial peptide production and purification ...................................... 26

3.4. Characterization of the peptide .................................................................... 28

3.5. 16S rRNA sequence analysis ....................................................................... 30

4. Discussion ........................................................................................................... 31

5. References .......................................................................................................... 36

Word Count ( 6,181 )

3

List of Figures

Figure 1: Well diffusion results of NB optimization ..................................................... 25

Figure 2: Well diffusion assay: AMP-P7 produced by P7 from NB with 5% yeast

extract. ............................................................................................................................ 26

Figure 3: Range of activity of purified peptide from P7 ............................................... 28

List of Tables

Table 1: Antimicrobial peptide activity of the environmental bacteria isolates. ........... 24

Table 2: Nutrient Broth optimization for antimicrobial peptide production. ................ 25

Table 3: Oasis fractions and zones of inhibition ........................................................... 27

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Abstract

Background: Antimicrobial peptides are considered to be an alternative to combat

antibiotic-resistant bacteria due to their broad-spectrum activity and lack of interaction

with specific protein binding sites. This project aimed to screen a library of

environmental samples for antimicrobial-peptide-producing bacteria, and attempted to

purify and characterize potential novel agents produced by these bacteria.

Methods: A library of environmental strains were screened for antimicrobial peptide

production against several indicator strains, using the simultaneous antagonism method.

The best producer strain was designated as P7 and its antimicrobial peptide as AMP-P7.

Antimicrobial peptide production from P7 was achieved in Nutrient Broth with 5%

yeast extract, and AMP-P7 inhibitory activity was monitored by either the well

diffusion or spot-on-lawn methods. Partial purification of AMP-P7 was achieved by

using Oasis HLB column chromatography.

Results: A potential novel antimicrobial peptide (AMP-P7) targeting S. aureus NCTC

7447 was identified from a library of environmental strains. A BLAST search revealed

that the 16S rRNA gene sequence of P7 strain was 99.9% similar to Bacillus tequilensis.

Conclusion: This study reports a potential novel antimicrobial peptide with narrow

spectrum activity against S. aureus NCTC 7447, produced by Bacillus tequilensis.

Further studies to improve AMP-P7 purification and characterization are required to

establish its potential used in medicine and industry.

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Declaration

I, Paulina Fernandez Soto, hereby declare that no portion of the work referred to in the

dissertation has been submitted in support of an application for another degree or

qualification of this or any other university or other institute of learning.

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Intellectual Property Statement

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dissertation) owns certain copyright or related rights in it (the “Copyright”) and s/he has

given The University of Manchester certain rights to use such Copyright, including for

administrative purposes.

ii. Copies of this dissertation, either in full or in extracts and whether in hard or

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must form part of any such copies made.

iii. The ownership of certain Copyright, patents, designs, trade marks and other

intellectual property (the “Intellectual Property”) and any reproductions of copyright

works in the dissertation, for example graphs and tables (“Reproductions”), which may

be described in this dissertation, may not be owned by the author and may be owned by

third parties. Such Intellectual Property and Reproductions cannot and must not be

made available for use without the prior written permission of the owner(s) of the

relevant Intellectual Property and/or Reproductions.

iv. Further information on the conditions under which disclosure, publication and

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Policy (see http://documents.manchester.ac.uk/display.aspx?DocID=487), in any

relevant Dissertation restriction declarations deposited in the University Library, The

University Library’s regulations

(see http://www.manchester.ac.uk/library/aboutus/regulations) and in The University’s

Guidance for the Presentation of Dissertations.

http://documents.manchester.ac.uk/display.aspx?DocID=487

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8

Acknowledgments

I would like to thank my supervisor Dr. Guoqing for his corrections and comments

about this project. I would also want to express my sincere gratitude and recognition to

John Moat for all the time he spent with us in the laboratory, for his advice and

comments during this project. Likewise, my sincere recognition to Dr. Issam for his

help in the development of this project. Finally, my sincere gratitude to all the staff of

the Medical Microbiology Department.

My gratefulness also to my sponsor the Ecuadorian Government who granted me with a

scholarship to study at The University of Manchester with the program "Universidades

de Excelencia, 2013".

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Preface

I obtained my undergraduate degree in Clinical Biochemistry in 2011, and worked for

almost 2 years as microbiologist in charge of the diagnostic of zoonotic diseases such as

Tuberculosis and Brucellosis at the International Center of Zoonosis in Ecuador. After

that, to improve my knowledge and research abilities, I decided to come to England and

study at this University. My aim is to become a researcher in the antibiotic resistance

area.

Dedication

I dedicate all this year of study and MSc project to my parents and siblings who have

supported me during this time. Besides, to all my friends, and church family who have

helped me to face difficult times in England. Finally, all my effort is dedicated to God

who is the provider of all wisdom and intelligence.

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1. Introduction

Microbial resistance to antibiotics is a natural phenomenon that is accelerated by

selective pressure due to overuse and misuse of antibiotics in medical and agricultural

areas (WHO 2014). More than 50% of all the antibiotics prescribed for people are

neither needed nor effective (CDC 2013). Likewise, every year an estimated 10,000-

20,0000 tonnes of antibiotics are manufactured and most of them are used for

agricultural purposes (Laxminarayan et al. 2013). The reduction of antibiotic efficacy to

treat serious infections is a worldwide problem, at least 23,000 people die each year as a

result of these infections in the USA (CDC 2013).

Core actions to combat this overwhelming health problem include: appropriate

use of antibiotics, a global surveillance system of antibiotic use and resistance,

prevention of infections and spread of resistance, improvement of medical diagnostics

to assure correct antibiotic therapy, and development of new antibiotics (CDC 2013;

Laxminarayan et al. 2013; Metz & Shlaes 2014). Numerous investigations are being

conducted into membrane-permeabilizing antimicrobial peptides (AMPs) as an

alternative antimicrobial strategy (Wimley & Hristova 2011; Cotter et al. 2013). AMPs

present broad-spectrum activity against drug-resistant bacteria and fungi along with

showing no specific affinity for a particular protein binding site. Both properties

decrease the risk of induced bacterial resistance to antimicrobial peptides (Wimley &

Hristova 2011).

AMPs can be classified into either ribosomal or non-ribosomal peptides

according to their biosynthetic pathways (Hancock 1997). Ribosomally synthesized

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AMPs are known as bacteriocins (Abriouel et al. 2011), and they are the most studied

subgroup of AMPs (Cotter et al. 2013). Bacteriocin activity was first demonstrated by

Gratia (1925) with Colicin produced by Escherichia coli, followed by the description

of food-grade lactic acid bacteria (LAB) in 1928 (Cotter et al. 2005; Yang et al. 2014).

LABs are used as food preservatives or antimicrobial peptides (Nishie et al. 2012).

Nonribosomal peptides (NRPs) are peptides produced by bacteria, fungi, and

streptomycetes synthesized on multienzyme complexes instead of being synthesized on

ribosomes (Hancock 1997). NRPs present broad spectrum activity and

immunomodulator, or antitumor activities (Caboche et al. 2010). NRPs are

distinguished from ribosomally synthesized peptides by two structural characteristics.

NRPs present often a cyclic primary structure rather than lineal, and they are made of a

vast biodiversity of monomers apart from the known 20 amino acids residues (Caboche

et al. 2010).

Bacteriocins characteristics make them ideal as alternatives to antibiotics.

Bacteriocins can inhibit the growth of bacteria of the same species (narrow spectrum) or

other genera (broad spectrum) and their spectrum of activity often depends on the

mechanisms of action of each bacteriocin (Cotter et al. 2005; Snyder & Worobo 2014).

They are small, heat-stable peptides made of short chains of around 20-60 amino acid

residues, however longer chains can also be found (Snyder & Worobo 2014). Most

bacteriocins are products of Gram-positive bacteria, as reported in BACTIBASE

dataset. A few bacteriocins from Gram-negative bacteria have been described and even

fewer from Archaea domain (Hammami et al. 2013).

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Bacteriocins produced by Gram-positive bacteria are grouped in four classes

based on their biochemical, structural and genetic properties (Klaenhammer 1993;

Cintas et al. 2001; Hammami et al. 2013). Class I bacteriocins or lantibiotics are small

(<5kDa), thermostable peptides containing atypical amino acids (lanthionine and

methyllanthionine) that undergo post-translational modification. Class I is subdivided

into type A and type B. Class II non-lantibiotic bacteriocins are small (<10kDa),

thermostable, unmodified peptides which differ in their structure and give rise to four

subclasses: IIa (pediocin-like bacteriocins), IIb (dipeptide bacteriocins), IIc (non-

pediocin-like single-chain) and IId (differ from IIa-IIc e.g. enterocines). Class III are

large (˃30kDa) thermolabile bacteriocins and class IV are complex peptides containing

lipid or carbohydrate groups. In addition, there are new high molecular weight

bacteriocins which are phage-tail-like molecules. Bacteriocins produced by Gram-

negative bacteria are narrow spectrum antimicrobial peptides and present two groups

named colicins and microcins (Chen & Hoover 2003; Nishie et al. 2012; Hammami et

al. 2013; Karpiński & Szkaradkiewicz 2013).

Bacteriocins can present several modes of action. In general, bacteriocins act

either at the cell envelope or within the cell (Cotter et al. 2013). Bacterial cell death may

arise by different means such as leakage of cell contents, cell lysis (by peptidoglycan

inhibition), protein synthesis inhibition (by cleavage of 16S rRNA), removal of critical

ion gradients and DNA degradation (Vriezen et al. 2009; Hammami et al. 2013). Thus,

mode of action between bacteriocins and antibiotics differs significantly, and makes

bacteriocins a suitable alternative to antibiotics (Hammami et al. 2013).

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The benefits of bacteriocins are seen not only in the food industry to improve

quality and safety, but also many of the bacteriocin properties suggest their potential

value in a clinical setting, including treatment for malignant cancers (Lancaster et al.

2007; Nishie et al. 2012). In food industry, bacteriocins are used as natural food

preservatives to replace chemical preservatives. In addition, bacteriocins present

sensitivity to proteases of the gastrointestinal tract, making them safe food additives that

can be easily digested in the gastrointestinal tract (Cleveland et al. 2001;Yang et al.

2014). Moreover, studies showed growth inhibition of important drug-resistant bacteria

such as methicillin-resistant S. aureus (MRSA) and vancomycin-resistant enterococci

(VRE) when lantibiotics such as nisin, mersacidin, mutacin 1140 and lacticin 3147 were

used (Nishie et al. 2012). Bacteriocin-producing Bacillus strains present antifungal

activity and may also be used as probiotics since they posses inhibitory activity against

C. perfringens and C. difficile (Abriouel et al. 2011). Bacteriocins are considered as

potential treatment against tumor cells. For example, nisin was used to treat head and

neck squamous cell carcinoma reducing cell proliferation (Joo et al. 2012). The fact that

bacteriocins are ribosomally synthesized allows them to be manipulated by genetic

engineering which may increase their antibacterial activity or structural stability (Nishie

et al. 2012).

The simplest way of screening bacteriocin activity is by direct simultaneous

antagonism in agar media contained in petri dishes (Chen & Hoover 2003). The

principle is based on Antonie van Leeuwenhoek´s studies in which the product from

one microorganism inhibit the growth of another (Chen & Hoover 2003). Although

known as a laborious method with some limitations, screening for inhibitory activity of

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organisms has led to the discovery of novel antimicrobials such as penicillin (Snyder &

Worobo 2014).

This project aimed to screen a library of environmental samples for

antimicrobial-peptide-producing bacteria, and attempted to purify and characterize

potential novel agents produced by these bacteria.

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2. Materials and Methods

2.1. Bacterial strains

A library of environmental strains was provided by the Department of Medical

Microbiology at the University of Manchester to be tested for bacteriocin production.

The strains were cultivated on Columbia Blood Agar media (CBA)(Oxoid Ltd.,

England) and incubated at 37 ºC aerobically and with CO2 for 24 hours.

2.2. Indicator strains

The environmental bacteria were assessed for activity against Staphylococcus

aureus NCTC 7447, Methicillin-Resistant S. aureus (MRSA) University strain 393,

Streptococcus pyogenes NCTC 8330, Enterobacter cloacae NCTC 5920, Klebsiella

pneumoniae 169, Bacillus subtilis NCTC 831, and Escherichia coli DH5 isolates

provided by the Department of Medical Microbiology.

2.3. Screening for antimicrobial peptide production

Screening for antimicrobial peptide production was achieved using the

simultaneous antagonism method (Burton et al. 2013). Indicator strains were suspended

in 3 ml of distilled water to gain a turbidity equivalent to a 0.5 McFarland Standard,

then diluted according to BSAC methods (British Society for Antimicrobial

Chemotherapy, May 2013) and plated onto Columbia Agar (CA) (Oxoid Ltd., England)

to produce a lawn of growth. Pure-producer strains were picked from the plates and

stabbed into the previous plated CA and incubated at 37 ºC aerobically and with CO2

(according to bacteria requirements), for 24 hours. The plates were then checked for

16

antimicrobial activity by observing inhibition zone around the stabbed bacterial

colonies. The zone of inhibition against indicator strains were assigned with arbitrary

score ranging from +1 as minimum inhibition to +4 as maximum inhibition.

2.4. Broth optimization for antimicrobial peptide production

Among the four broths tested for antimicrobial peptide production: Nutrient

Broth (NB), Tryptic Soy Broth (TSB), de Man, Rogosa and Sharpe (MRS) Broth, and

Brain Heart Infusion (BHI); Nutrient Broth presented the greatest yield of peptide after

the well diffusion assay was performed. Further optimisation of this broth was carried

out by addition of sugars such as lactose, sucrose, glucose and yeast extract. All media

were purchased from Oxoid Ltd., England. The sugars and yeast extract were added

individually at a concentration of 5% to each liquid media, then 1 ml (5%) of a 0.5

McFarland suspension of the producer strain was added to a baffled Erlenmeyer flask

containing 20 ml of NB with the different additives. The samples were incubated on a

shaker at 37 ºC for 18 hours. After incubation, 1 ml of the samples were transferred to a

1.5 ml Eppendorf tube and centrifuged at high speed (13,300 RPM) for 5 minutes to

remove cellular debris, then the cell-free supernatants were assayed for antimicrobial

activity using the well diffusion method.

2.5. Assays to test antimicrobial peptide activity

2.5.1. Well diffusion assay

The antimicrobial peptide activity was monitored during the extraction and

purification procedures by well diffusion assay with the indicator strain Staphylococcus

17

aureus NCTC 7447 (Sharma et al. 2009). On the CBA plate, circular wells of 1 cm in

diameter were cut using a sterile cork borer and then partially sealed with low melting

temperature agarose (Sigma-Aldrich Company Ltd.). The wells were filled with 50 µl of

either cell-free supernatant or purified extracts. After complete diffusion of the

supernatant into the agar was observed, the plate was sterilized with chloroform vapour

for 20 minutes to kill any viable bacteria, and then removed and allowed to dry. The

plate was then inoculated with an indicator strain and incubated at 37 ºC for 24 hours.

Inhibitory activity of the peptide was indicated by the zone of inhibition produced after

incubation.

2.5.2. Spot-on-lawn method

Spot-on-lawn method was also used to check antimicrobial peptide activity. It

comprised in spotted 20 µl of sample into a CBA plate, then allowing it to be absorbed

by the agar. After that, the plate was sterilized with chloroform for 20 minutes, and then

proceeded in the same way as in the well diffusion method (Sandiford & Upton 2012).

2.6. Purification of the antimicrobial peptide

2.6.1. Concentration of the antimicrobial-peptide-containing supernatant

To extract and purify the antimicrobial peptide, a baffled Erlenmeyer flask

containing 150 ml of Nutrient Broth with 5% yeast extract was inoculated with 15 ml

(10%) of a 0.5 McFarland suspension containing the producer strain. The sample was

incubated on a shaking incubator at 37 ºC for 18 hours. After incubation, the sample

was divided in 50 ml Falcon tubes and centrifuged at high speed (3,000 RPM) for 20

18

minutes. The sample was pooled and assayed using well diffusion method to confirm

the presence and inhibitory activity of the antimicrobial peptide.

The extraction and purification of the antimicrobial peptide were performed by

Solid Phase Extraction methods (SPE) (Pingitore & Salvucci 2007). The SPE methods

used in this study were Strata-XL-C followed by Oasis HLB columns.

2.6.2. Strata-XL-C chromatography

A Strata-XL-C column was first conditioned using 40 ml of 90% methanol

(pH2) and 80 ml of 99.9% pure methanol (Fisher Scientific UK Ltd., UK). Then, 80 ml

of ultra-pure water was used to wash out the methanol. After that, 150 ml of the

supernatant adjusted to a pH 6.1 with 1 molar HCl (pH range from 5.8 to 6.2) was

loaded through the column using a sterile syringe. The obtained fractions were collected

in 50 ml Falcon tubes and labelled as "flow through". Next, the column was washed

with 40 ml of ultra-pure water, followed by a second wash with 40 ml of 50% methanol

(pH2). The active peptide was eluted with 60 ml of 90% methanol (pH2). All fractions

were collected and assessed by the well diffusion assay. The characteristics of the

Strata-XL-C column used was 100 µl Polymeric Strong Cation 5g/60ml Giga tube

(Phenomenex Ltd., UK).

2.6.3. Oasis HLB column chromatography

The previous active fractions (flows through) were pooled and centrifuged at

high speed (3,000 RPM) for 20 minutes. The supernatant was collected and acidified to

pH 2 with trifluoroacetic acid (TFA) (Sigma Aldrich, USA) before loading on the Oasis

19

HLB With LP Extraction Cartridge (Waters Corporation, USA) column. The column

was equilibrated first with 2 ml of pure acetonitrile (ACN) (Fisher Chemical, UK Ltd.)

followed by 2 ml of Buffer A (ultra-pure water with 0.1% TFA). After loading 20 ml of

the supernatant, the column was washed with 2 ml of Buffer A, and air was pushed

through the column to dry it. The antimicrobial peptide was eluted with 2 ml of

increasing concentrations (from 10% to 90%) of acetonitrile with 0.1% TFA. All the

fractions were collected at each concentration and assessed by spot-on-lawn assay.

2.7. Characterization of the antimicrobial peptide

The active fractions were pooled and rotary evaporated for 2 hours using a

Savant SpeedVac Concentrator (Thermo Scientific, Germany) to eliminate the ACN

and concentrate the antimicrobial peptide.

Methanol evaporation of active fractions obtained from Strata-XL-C column

was performed in a water bath at 70 ºC for 38 hours.

The samples were neutralized to pH 7 with 1 molar NaOH to perform tests such

as: range of activity, MALDI-TOF, Minimum Inhibitory Concentration (MIC) and

Haemolytic Assay. Due to the small quantity of the sample pH variation was measured

with strips (SIGMA-pH Test Strips 6.0-7.7).

2.7.1. Testing range of activity

The range of activity of the partially purified antimicrobial peptide was

performed by well diffusion assay on CBA plate with five indicator strains

Staphylococcus aureus NCTC 7447, MRSA University strain 393, Escherichia coli

20

DH5, Streptococcus pyogenes NCTC 8330 and Enterobacter cloacae NCTC 5920. The

indicators were streaked in a line (Figure 3).

2.7.2. MALDI-TOF Mass spectrometry

The molecular mass of the partially purified antimicrobial peptide was

determined by matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF)

mass spectrometry . MALDI-TOF was performed by the Core Protein Facility at the

Michael Smith Building of the University of Manchester.

2.7.3. Minimum Inhibitory Concentration

The MIC was determined according to the protocol recommended by the

Clinical Laboratory Standards Institute (formerly National Committee for Clinical

Laboratory Standards) (CLSI 2008). To test the MIC of the partially purified

antimicrobial peptide, suspensions of Staphylococcus aureus NCTC 7447, MRSA

University strain 393, and Escherichia coli DH5 were prepared based on CLSI

methods. The test was performed in a sterile 96-well U-shaped microplate (Thermo,

Scientific, England). First, using sterile pipettes, 100 µl of Müller-Hinton Broth (Oxoid

Ltd., England) was added in each row from well 1 to 12 for each strain to be tested.

Then, 100 µl of purify peptide was added to the first well, mixed and 100 µl were taken

from the first well to do the 2-fold dilutions of the peptide until well number 11, well 12

was used as a growth control. After that, 5 µl of each bacterial suspension previously

prepared was added to each well. The microplate was incubated at 37 ºC for 24 hours.

The lowest concentration that inhibited the growth of the organisms was defined as the

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MIC. In this study, the MIC was measured by comparing the results obtained with the

positive controls. The purify peptide was inoculated in a CBA plate to test its sterility.

2.7.4. Haemolytic Assay

To determine the haemolytic activity of the partially purified antimicrobial

peptide a haemolytic assay was performed (Fernandez-Lopez et al. 2001). First, horse

blood (Thermo Scientific, Oxoid, England) was centrifuged for 10 minutes (3,000

RPM) and the supernatant was removed. Next, the erythrocytes were washed 3 times

with 0.9% saline solution and then re-suspended to a concentration of 5% in 0.9% saline

solution. Subsequently, 100 µl of saline solution was added until well number 10 of a

U-shape microplate. An amount of 100 µl of peptide was added to the first well and 2-

fold dilutions were performed until well number 10, well 11 containing a 100 µl saline

only (Negative control-0% haemolysis) and well 12 containing a 100 µl of 1% triton in

saline (Positive control-100% haemolysis). Finally, 100 µl of 5% horse blood was

added until well number 12 and the plate was incubated at 37 ºC for 30 minutes. The

haemolysis results were measured by comparing the positive and negative controls.

2.8. Strain identification by sequence analysis of the 16S rRNA gene

To identify the producer strain, the region coding for 16S rRNA was sequenced.

First, a Gram stain of the producer was performed to select the correct chromosomal

DNA extraction method, which in this case was focused on Aerobic Spore-bearers

(ASB). In an Eppendorf tube a loopful of cells from a fresh culture were suspended in 1

ml of lysis buffer pH 7.6 with 50 µl of lysozyme (20mg/ml) (SIGMA-ALDRICH,

England) and heated at 37 ºC for 30 minutes. Next, proteinase K from Tritirachium

22

(35mg/ml) (SIGMA-ALDRICH, England) was added to the mixture and heated at 57 ºC

for 30 minutes. As soon as the turbidity of the sample was reduced, it was taken out

from the heater. The DNA extraction was confirmed by running the sample on a E-gel

(Invitrogen E-gel 2% agarose, Life Technologies, Paisley, UK).

PCR (model GeneAmp PCR system 9700 Applied Biosystems) reaction was

performed using 2X BioMix Red (Bioline Ltd., London, UK) ready to use reaction

mixture that includes an ultra-stable Taq DNA polymerase with 1μl of template DNA

extract. PCR amplification was performed with both forward and reverse universal PCR

primers at a concentration of 10 µmol/µl. These were 63f (5'-CAG GCC TAA CAC

ATG CAA GTC-3') and 1387r (5'-GGG CGG WGT GTA CAA GGC-3') (Eurofins,

Germany). The PCR cycle was run at 94 ºC for 2 minutes, followed by 35 cycles at 94

ºC for 1 minute, primer-annealing step at 60 ºC for 1 minute and an extension step at

72ºC for 1 minute. The reaction was completed with an extension step at 72 ºC for 5

minutes, and the product was kept at 4 ºC.

The PCR product was confirmed by running it into an E-gel. For sequencing, the

PCR product was cleaning up using the Illustra ExoProStar 1-step method by adding 2

µl of it to 5 µl of PCR product in a 0.2 ml PCR tubes. The sample was incubated at 37

ºC for 15 minutes to activate the enzymes followed by incubation at 80 ºC for 15

minutes to inactivate the enzymes. Sequencing was performed by staff at the University

of Manchester, department of DNA Sequencing facility at Stopford Building using

3730 48-capillary Applied Biosystems Genetic Analyzer.

23

Finally, the DNA sequence data was analyzed by BioEdit and searched using the

Basic Local Alignment Search Tool (BLAST) of NCBI (http://blast.ncbi.nlm.nih.gov),

and compared against the 16S ribosomal RNA sequences in the database.

24

3. Results

3.1. Screening of bacteriocin production using simultaneous antagonism

Among the 14 strains tested, strain P7 was chosen for peptide extraction and

purification since it presented the best inhibitory activity against S. aureus NCTC 7447,

MRSA University strain 393, Strep. pyogenes NCTC 8330 and E. coli DH5 as observed

in Table 1.

Table 1: Antimicrobial peptide activity of the environmental bacteria isolates.

Indicator strains

Producer Strain and

Zone of Inhibition

P7 P9 P14

S. aureus NCTC 7447 4+ 3+ 2+

MRSA University strain 393 4+ - -

Streptococcus pyogenes NCTC 8330 4+ - -

Enterobacter cloacae NCTC 5920 - - -

Klebsiella pneumoniae 169 - - -

Bacillus subtilis NCTC 831 - - -

Escherichia coli DH5 1+ - -

25

3.2. Broth Optimization

P7 strain grew and produced the potential antimicrobial peptide named AMP-P7

in Nutrient Broth (NB) with a zone of inhibition of 10 mm in diameter. Incubation in

other broths showed no antimicrobial peptide activity when supernatants were assessed

by the well diffusion method. Once additives were incorporated to NB the zones of

inhibition slightly increased (Table 2 & Figure 1).

Table 2: Nutrient Broth optimization for antimicrobial peptide production.

Producer

Strain

Nutrient Broth and Zone of inhibition

(Well diffusion method)

NB+

lactose 5%

NB+

glucose 5%

NB+

sucrose 5%

NB+

yeast extract 5%

P7 strain 14 mm 13 mm 11 mm 15 mm

Figure 1: Well diffusion results of NB optimization

Nutrient broth with: lactose (1), sucrose (2), yeast extract (3), glucose (4)

Indicator strain: S. aureus NCTC 7447

1

2 3

4

26

3.3. Antimicrobial peptide production and purification

NB with 5% yeast extract was selected for AMP-P7 production. After 18 hours

of incubation, the supernatant was tested by the well diffusion assay, and a zone of

inhibition of 20 mm in diameter was observed (Figure 2). This result was considered as

the positive control for AMP-P7 activity presence. Following this results, AMP-P7

purification was initiated with Strata-XL-C chromatography

Figure 2: Well diffusion assay: AMP-P7 produced by P7 from NB with 5% yeast

extract. Indicator strain: S. aureus NCTC 7447

3.3.1. Strata-XL-C chromatography

A bigger zone of inhibition was found in the "flow through" fractions than in the

eluted fractions after assessment of the supernatants by the well diffusion method. This

result suggests that AMP-P7 bound weakly to the column, as zone of inhibition was

also found when the sample was eluted with methanol 90%. Therefore, "flows through"

27

fractions were processed to be purified through Oasis column. Moreover, the fractions

eluted at methanol 90% containing AMP-P7 peptide were assessed by the well diffusion

method after methanol evaporation.

3.3.2. Oasis HLB column chromatography

After adjusting the previous "flows through" to pH 2, the supernatant was

assessed to verify AMP-P7 presence. A zone of inhibition was observed (10 mm)

showing its presence but in small amounts. No zone of inhibition was observed in the

loading and washing fractions which indicates that the AMP-P7 bound to the column.

Table 3: Oasis fractions and zones of inhibition

Fraction

(%)

Increasing concentrations of acetonitrile with 0.1% TFA

10 20 30 40 50 60 70 80 90 100

Zone of

Inhibition

(mm)

-

-

-

-

-

-

11

11

11

8

AMP-P7 (Table 3) was eluted at 70%, 80%, 90% and 100% acetonitrile. Similar

clear inhibition zones were obtained for 70%, 80% and 90% fractions; therefore, these

three fractions were combined and processed again with Oasis column to concentrate

the AMP-P7 peptide. After rotary evaporation of the acetonitrile from the fractions and

adjustment of pH (pH 7.7), fraction 90% was used for MALDI-TOF mass spectrometry

analysis, the 80% fraction was used for range of activity, MIC and haemolytic assays.

28

3.4. Characterization of the peptide

3.4.1. Testing range of activity

The inhibitory activity of AMP-P7 was tested against the indicator strains that

showed sensitivity in the initial simultaneous antagonism assay. However, inhibitory

activity was detected only against Staphylococcus aureus NCTC 7447 which since the

beginning showed to be greatly inhibited by AMP-P7 (Figure 3) suggesting that higher

concentration of AMP-P7 is required to confirmed its inhibitory activity.

Figure 3: Range of activity of purified peptide from P7

a) S. aureus NCTC 7447, b) Enterobacter cloacae NCTC 5920, c) MRSA University

strain 393, d) Strep. pyogenes NCTC 8330 and e) E. coli DH5

In addition, no zone of inhibition was observed when the methanol 90% fraction

was tested for presence of AMP-P7 after water-bath evaporation, suggesting that either

Zone of

inhibition a

e

a

d

a

b

c

a

29

the amount of AMP-P7 in the sample was low or that AMP-P7 is not heat stable at 70

ºC. Besides, all the active fractions containing the AMP-P7 also underwent pH

adjustment from 2 to 8 which could inactivate its antimicrobial activity.

3.4.2. Minimum Inhibitory Concentration

MICs were examined against S. aureus NCTC 7447, MRSA University strain

393, and E. coli DH5. MICs for all the indicator strains were ˃1/2 neat concentration

since no growth inhibition of the indicator strains was observed. These results suggest

that the amount of AMP-P7 was insufficient to determine the MIC necessary to inhibit

bacterial growth especially when serial dilution of AMP-P7 was required.

3.4.3. Haemolytic Assay

AMP-P7 showed haemolytic activity only at the first dilution (1:2 the Neat),

after this dilution no haemolysis was detected.

3.4.4. MALDI-TOF Mass spectrometry

The molecular weight of AMP-P7 eluted at 90% acetonitrile was measured by

MALDI-TOF mass spectrometry. The analysis gave 5 molecular masses: 565.598 Da,

615.943 Da, 889.426 Da, 928.159 Da. It was not possible to obtain a chromatogram of

the results due to technical problems with the mass spectrometry machine.

30

3.5. 16S rRNA sequence analysis

After a BLAST search was performed, it revealed that the 16S rRNA gene

sequence of P7 strain was 99% similar to Bacillus tequilensis strain 10b. No data of

any antimicrobial peptide produced by Bacillus tequilensis strain was found in

BACTIBASE dataset.

31

4. Discussion

A potential narrow spectrum antimicrobial peptide targeting S. aureus produced

by Bacillus tequilensis was obtained. Narrow spectrum bacteriocins are considered as

'designer drugs' and may be used to combat specific microorganism (Riley & Wertz

2002). As known S. aureus is part of the normal human microbiota, especially found in

the nose of 30% healthy people (Humphreys 2007). Infections cause by S. aureus

comprises from minor skin infections to severe pneumonia (Den Heijer et al. 2013). It is

speculated that resistance genes are acquired first by the normal commensal microbiota,

and this may increase the acquirement of resistance in pathogens (De Lastours et al.

2010).

DNA data result of P7 strain was similar to Bacillus tequilensis. In 16S rRNA

sequence analysis, it is likely to obtain sequences that share a great level of similarities

due to sequencing errors, amplification errors, and the possibility of microheterogeneity

(Fox et al. 1992). In addition, there is no a universal primer of sufficient length that is

100% match to all bacteria (Baker et al. 2003). Due to time constraint, the designing of

Bacillus specific primers to confirm the identification of the P7 strain as Bacillus

tequilensis could not be performed. Further identification of P7 strain should combine

confirmation of specie by specific primer design, and comparison of its phenotypic

characteristics described by Gatson et al. (2006).

Bacillus tequilensis was first describe in 2006, after isolation from an

approximately 2000-year-old shaft-tomb at a site called Huitzilapa, near the city of

Tequila in the Mexican state of Jalisco (Gatson et al. 2006). Up to now the only

32

reported clinical application was described by Pradhan et al. (2013), who found an

halophilic biosurfactant produce by B. tequilensis CH able to inhibit biofilm formation

of E. coli and S. mutans on both hydrophilic and hydrophobic surfaces. The rest of

applications found involve biotechnological or industrial focus. (Amulya et al. 2014;

Wang et al. 2014; Sondhi et al. 2014; Chiliveri & Linga 2014). No report of any

antimicrobial peptide produced by B. tequilensis has been documented; thus, the

described AMP-P7 peptide might be considered as a potential novel antimicrobial

peptide produced by this bacterium.

Bacillus species produce both types of AMPs: ribosomal (e.g., subtilin) and

nonribosomal (e.g., daptomycin) peptides (Lee 2011). It is important to distinguish

whether or not an AMP is a true bacteriocin, due to the emergence of resistance to some

nonribosomal peptides by certain bacteria (Abriouel et al. 2011; ). Further studies, of

AMP-P7 peptide, will involve its classification in any those two types of AMPs. There

are around 1164 NRPs (Caradec et al. 2014), however the study of them are beyond the

scope of this project.

Abriouel et al. (2011), proposed a new classification for bacteriocins produced

by Bacillus species that includes three classes: Class I contains bacteriocins that

undergo post-translational modifications (e.g., subtilin, subtilosin A, mersacidin). Class

II bacteriocins are small (0.77-10kDa), heat and pH stable, non-modified and linear

peptides (e.g., coagulin, lichenin). Class III contains large bacteriocins (˃30kDa) (e.g.,

megacin) with phospholipase activity. Bacteriocins that have not being able to be

grouped are called bacteriocins-like inhibitory substances (BLIS). (Abriouel et al. 2011;

33

Mongkolthanaruk 2012). It was suggested that AMP-P7 may belong to the Class II

small bacteriocins as its molecular mass ranged between 0.56kDa to 0.92kDa.

Previous studies showed that the production of bacteriocin may be affected by:

amount of carbon, nitrogen and phosphate, cations, surfactants, inhibitors and pH

(Parente & Ricciardi 1999). In addition, bacteriocin operon is commonly regulated by

specific environmental conditions and stress; therefore, bacteria may not express the

same range of activity under laboratory conditions (Snyder & Worobo 2014). Although

some studies suggest MRS, BHI and TSB mediums for peptide production from

Bacillus species (Marrec 1998; Aunpad & Na-Bangchang 2007; Xie et al. 2009), the

greatest yield of peptide production compare with the other broths was obtained using

NB with 5% yeast extract.

Production of high amounts of bacteriocins by some producer strains is not

always possible (Pingitore & Salvucci 2007). In this study, although NB with 5% yeast

extract allowed the greatest peptide production, few amount of AMP-P7 was obtained

according with the zone of inhibition observed when the well diffusion method was

performed. Therefore, to concentrate antimicrobial peptides prior purification,

ammonium sulfate concentration, absortion-desortion, organic solvent extraction, or

lyophilization can be performed (Aunpad & Na-Bangchang 2007; Pingitore & Salvucci

2007).

The best purification method will depend on the previous knowledge of the

peptide (Parente & Ricciardi 1999; Pingitore & Salvucci 2007). Solid-Phase Extraction

(SPE) methods were used to purify the AMP-P7 peptide. Oasis HLB column allowed

34

partial purification of AMP-P7. However, although Oasis HLB column allows the

extraction of low-concentration analytes, generally the analyte after elution steps ends

up in a large solution volume making difficult its detection (Kitt & Harris 2014).

Reverse-phase high-performance liquid chromatography (RP-HPLC) has shown great

contribution to obtain high purification of bacteriocins, combination of SPE and RP-

HPLC methodology are performed in numerous studies (Chaney 2009; Aunpad & Na-

Bangchang 2007; Pingitore & Salvucci 2007; Sandiford & Upton 2012).

After successful purification is performed, further studies will involve

quantification of AMP-P7 peptide by gas chromatography, mass spectrometry or high-

performance liquid chromatography (HPLC) (Kitt & Harris 2014). In addition, to

elucidate primary structure of peptides, Tandem mass spectrometry techniques are

commonly used; however, because some AMPs undergo post-translational modification

it is difficult to identify a complete sequence of the peptide by using only mass

spectrometry. Hence, to identify uncommon acids that are part of AMPs, Nuclear

Magnetic Resonance spectroscopy (NMR) is recommended (Kim et al. 2010).

As stated previously, production of high amounts of bacteriocins by some

producer strains is not always possible although optimization of extraction and

purification methodology is achieved. The application of the methodology here

described may be beneficial to obtain previous characterization of numerous potential

novel antimicrobial peptides. However, once characterization and potential importance

of the antimicrobial peptide is found, studies may be focus on obtaining bacteriocins by

genetic engineering (Nishie et al. 2012) using either mutating bacteriocin-encoding

genes or by fusing genes from different bacterial species (Gillor et al. 2005).

35

In conclusion, the present study described a partially purified antimicrobial

peptide produced by Bacillus tequilensis. AMP-P7 initially exhibited an antimicrobial

activity against S. aureus NCTC 7447, MRSA University strain 393, Strep. pyogenes

NCTC 8330 and E. coli DH5. However, after extraction and purification processes,

AMP-P7 inhibitory activity was found only against S. aureus. Numerous reports about

B. tequilensis applications has been conducted but none of them describe antimicrobial

peptide production by B. tequilensis. Further studies should focus on optimising

extraction and purification methods used to obtain more reliable characterisation of the

potential antimicrobial peptide isolated during this project.

36

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