RESEARCH PROPOSAL

Title: Screening for bacteriocin producers against mastitis-causing pathogens: Staphylococcus

aureus, Escherichia coli, and Streptococcus uberis in cow milk followed by purification and

characterization of collected bacteriocins.

NAME OF APPLICANT:

MS. NGUYEN LE THANH

BSc in Biotechnology, School of Biotechnology

International University, Vietnam National University in HCMC, Vietnam

Contact detail: lethanh.iuetv@gmail.com

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Brief description of the project:

Due to both economic losses and animal health impact, mastitis infections should be tackled in no

time. Furthermore, as frequent failure of antibiotic therapy and the lack of effective vaccine

measures, the need to find an antimicrobial alternative to fight against major mastitis pathogens

such as Staphylococcus aureus, Escherichia coli, and Streptococcus uberis is necessary.

Bacteriocins, ribosomally synthesized peptides with antimicrobial characteristics, from Lactic acid

bacteria (LAB) and Staphylococci appear to be able to eliminate such pathogens. This study is

designed to explore stable bacteriocins to solve the problem of mastitis infection in cows.

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1. Background and rationale for the project:

Mastitis is believed to be the disease that costs the most in the dairy industry (Sordillo & Streicher,

2002). In addition, the consequences of mastitis do not only include economic losses but also affect

animal well-being; the disease is a primal reason for culling or death of dairy cattle (Grohn, Eicker,

Ducrocq, & Hertl, 1998). There are three main species of pathogens that resposible for mastitis,

namely Staphylococcus aureus, Escherichia coli, and Streptococcus uberis (Chaneton, Tirante,

Maito, Chaves, & Bussmann, 2008). From which S. aureus show an increasing resistance rate to

antibiotics and failure of bacteriological treatment for mastitis (Barkema, Schukken, & Zadoks,

2006). As antibiotic treatments often fail and there is also a lack of effective vaccine measures,

other measures to fight against mastitis should be employed. Bacteriocins with their antimicrobial

characteristics and a narrow spectrum activity range are the candidate of interest.

Bacteriocins are peptides that ribosomally synthesized, naturally secrected by bacteria to kill

similar or closely related species (Klaenhammer, 1993). Accoding to Cotter, Ross, and Hill (2013),

they can be categorised into two classes, i.e. Class I includes proteins that significantly modified

through post-translational process, and Class II with peptides that are unmodified. In which, class

I bacteriocins are proven to be effective in eliminating Gram-positive pathogens (e.g. Methicillin-

resistant Staphylococcus aureus (MRSA), Clostridium difficile, Streptococcus pneumoniae, etc.)

(Brumfitt, Salton, & Hamilton-Miller, 2002; Cotter et al., 2013; Goldstein, Wei, Greenberg, &

Novick, 1998; Severina, Severin, & Tomasz, 1998).

Lysostaphin is a staphylolytic enzyme secreted by Staphylococcus simulans, and was demonstrated

by Wall et al. (2005) as a transgene to protect the mammary gland against a mastitis-causing

pathogen in cows. However, E.coli and S.uberis were not inhibited by lysostaphin in this study. As

a result, other types of bacteriocins need exploring for their abilities to supress all three pathogens.

Most bacteriocins are found in plants but some do originate from bacteria. From bovine milk, a

number of bacteriocins produced from lactic acid bacteria (LAB) and Staphylococci were

identified (Brito, Somkuti, & Renye, 2011; Todorov & Dicks, 2006).This study means to

investigate the population of bacteriocin producers in cow milk, both LAB and Staphylococci, as

a way to ensure the stability of antimicrobial peptides against mastitis in cows.

2. Suggested methodological background:

2.1. Suggested flow chart of the study:

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2.2. Steps in experiment design:

2.2.1. Collection of raw milk for screening and Isolation of bacteriocin

producers:

Raw cow milk (unpasteurized) samples would be collected from local dairy farms, in a sterile

capped tubes, and serially diluted (10-1 – 10-6) in sterile water. The diluted samples are to be plated

(0.1 ml suspension) onto de Man Rogosa Sharpe (MRS) agar and blood agar, then incubated at

37°C for 48 h (Brito et al., 2011; Tagg & McGiven, 1971; Todorov & Dicks, 2006).

Screening of bacteriocin-producing isolates will be achieved by well-diffusion method (Tagg &

McGiven, 1971) against the indicator bacteria i.e., Staphylococcus aureus, Escherichia coli, and

Streptococcus uberis. Bacteriocin producing isolated would then be sub cultured before preserved

in 20% glycerol at -20°C. Bacteriocin producing strains can then be Gram stained and examined

microscopically for cellular morphology and Gram-stain phenotype. Catalase activity can be tested

by spotting colonies with 3 % hydrogen peroxide as described by Pal, Jamuna, and Jeevaratnam

(2005).

2.2.2. Production of crude bacteriocin:

Collection and Isolation of

samples

ction of Produ crude ba cteriocins

Purification of collected

bacteriocins

Determination of Protein

Molecular Weight Determination

Characterisation of collected bacteriocins

Effect of pH

Effect of Temperature

Effect of Proteolytic

Enzyme

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The isolated strains should be transfer to broth cultures, 10% inoculum: 10 CFU/ml in 1000 ml.

Incubate overnight, 48 h at 37°C. After incubation, the whole broth was centrifuged at 10,000 rpm

for 20 minutes, and the cell-free supernatant can be used as crude bacteriocin (Ogunbanwo, Sanni,

& Onilude, 2003).

2.2.3. Purification of bacteriocin:

Crude bacteriocins collected then will be saturated with 70% ammonium sulfate and stored at

4°C to precipitate the proteins. The pellet will be collected after centrifugation at 10,000 rpm, 4°C,

30 minutes. Then it put for dissolve in potassium phosphate buffer (25ml, 0.05M, pH 7.0) and be

dialyzed against the same buffer at 4°C overnight. Assay of the bacteriocin activity should be

carried out and titer coud be used to determine whether in the precipitate or supernatant containing

bacteriocin (Ogunbanwo et al., 2003).

2.2.4. Determination of Protein

Protein concentration of the bacteriocin will be measured by applying the method of Lowry,

Rosebrough, Farr, and Randall (1951), using bovine serum albumin (BSA) as the standard.

2.2.5. Molecular Weight Determination

According to Laemmli (1970), molecular weight of the purified bacteriocin can be identified using

12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). 2.2.6.

Characterisation of collected bacteriocins

Effect of pH:

0.5 ml of purified bacteriocin should be added into 4.5 ml of nutrient broth at different pH values

and incubated for 30 minutes at 37°C (Motta & Brandelli, 2002).

Effect of Temperature:

0.5 ml of purified bacteriocin should be added into 4.5 ml of nutrient broth, overlaid with paraffin

oild to avoid evaporation, and heated at different temperatures (30, 40, 50, 60, 70, 80, 90 and

100°C) for 10 min (Sharma, Kapoor, & Neopaney, 2006).

Effect of Proteolytic Enzyme:

1mg/ml of purified bacteriocin will be treated for 1h with various enzymes. All enzymes used

should be dissolved in phosphate buffer (0.5M, pH 7.0). For control samples, untreated bacteriocin

plus buffers, buffers alone, and enzyme solutions alone can be employed (Paik, Bae, Park, & Pan,

1997; Sankar et al., 2012)

BIBLIOGRAPHY

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