Siphophages Have Noncontractile Tails

Published Date: 02 Nov 2017

Abstract

With the advancement of technology, challenges have arise with the maintainance of probiotics in the dairy products. The phase contamination in the dairy products is one of the major problem in the dairy industry. For this reason it is necessary to develop effective steps to prevent phase contamination from dairy products. Different techniques can be developed to isolate this phase from dairy samples and also to eradicate them. These techniques are also helpful for those microbial threats which is new to dairy industry.

Introduction

Eradicating contamination is one of the devastating problem in the industrial biotechnological processes. If it occurs, it can result in complete loss of facility productivity. Among them the most devastating are those caused by bacteriophages. These are viruses that infect bacteria ultimately lysing them. Phages are present in the ecosystem as well as in air, in the soil and in the water. They are likely the most abundant biological entities on Earth.

The International Committee on Taxonomy

Of Viruses (ICTV) states that all known phages infecting LAB are tailed

phages and members of the Caudovirales order.

Tailed bacteriophages phages are,

classified into three families:

Podoviridae members have short and noncontractile tails.

myophages have tails with a contractile sheath and a central tube.

siphophages have noncontractile tails.

Microbiology based industry is greatly affected by phage due to bacterial infection. As the infection increases, results can be completely loss of the whole batches. During the fermentation insufficient lactic

acid production , high pH and increased lactose production can be occurred for phage infection. Several unwanted consequences may arise for this infection. Such as residual lactose can affect the quality of the product. Not only that these components may give rise the growth of pathogens which ultimately make devastating results for consumer health.

Lactic acid bacteria belong to a large group of beneficial bacteria with similar properties. All are the metabolic end product of lactic acid. They are widespread in nature and are also present in the human digestive tract. Although best known is the role of lactic acid bacteria in the production of fermented dairy products, they are also for the preservation of vegetables, used in baking, wine making and the salting of fish, meat or sausages.

When the yogurt fermentation work two cultures, Streptococcus thermophilus and Lactobacillus bulgaricus together, and stimulate each other in growth. The result is shorter fermentation times, and a product with different characteristics than the one in which only a culture is used.

With yogurt and other fermented milk products, there is considerable potential to use effective probiotic cultures, which have a positive impact on our gut flora. The global market for such products is constantly growing due to the demand by increasingly health-conscious consumers.

The aim of this practical is to enumerate the effects of bacteriophages in dairy products and how to eradicate them.

Isolation and enumeration of phage

There are two principal methods of growing virulent phage, both based on the ability of phage to lyse and therefore kill the host bacterial cell.

One method introduces phage into a fluid bacterial culture medium. After a period of incubation, the phage lyse the bacteria in the broth culture resulting in a clearing of the fluid medium.

The other method, known as the "plaque assay", introduces phage into a soft agar overlay along with some bacterial host cells.

Identification of bacteria

Once an organism has been fully characterized, it may be identified by comparing it with the information held in reference sources such as Bergey’s Manual of Systematic Bacteriology

Cellular Morphological Characteristics

1. Shape

2.Size

3.Arrangement of cells

Cultural characteristcs

1. Colony Characteristics (surface colonies on solid media).colony shape, size, colour, surface character, odour.

2.Liquid culture. The amount of

growth, surface growth, pellicle,

uniform, flocculent, deposits.

Biochemical Classification

1.Obtained Energy: Energy obtained

from chemical compounds

(by fermentative or

oxidative mechanisms)

2.Utilized carbon compounds: Simple

(e.g. organic acids, sugars, hydrocarbons)

or complex (e.g. starch, chitin, cellulose,

fats) carbon compounds (e.g. citrate as sole

carbon source. Ability to degrade complex

carbon compounds, but requiring other carbon

sources to support growth.

3.Nitrogen source:

Inorganic (nitrate, ammonia etc.) or organic

(urea, amino acids etc.) sources or nitrogen

fixation

Materials and Method

Method

Week 1

every group brought to the laboratory a sample of a diary foodstuff of their choice. The yoghurt which was used in the lab was organic Greek style coconut yoghurt which contented lactobacillus acidophilus and bifidobacterium.

Six bottle was prepared which had sterile saline and labelled them from 10-1 to 10-6 then 10-1 bottle was weighed and added 1 gram of yogurt on the bottle then it was mixed thoroughly and series diluted was carried out 10-1 to 10-6

Above, the medium is diluted to 6 times each, that is, in calculating the count, the number of dilution must then be multiplied again. The greater the dilution, the less bacteria to grow on the agar. After that 10-5 bottle solution was prepared for further experiment and two MRS agar dishes was libelled them as a 1- (MRS 10-5 25C SEK bioyoghurt ) 2-(Caco3 10-5 23C SEK).

10-5 = MRSA and CaCo3

After examine the two spread plates of isolation lactic acid bacteria which were MRS agar and CaCo3 there was no growth.

The reason of no growth on the plates could be the way the preparation of the dilution been handled. Nevertheless, it has probably possible impact on the result also It could be that something else has killed the bacteria.

Establishment of pure cultures (week 2)

There was no growth the spread plates of isolation of lactic acid bacteria for week 1. So to examine the colony those producing an acid product ( lactic acid in this case it was not possible to do that because of there was no any growth onto the plates so new colonies were given in the lab to carry on the week 2 experiment.

There was no any clear zone around them so to examine the colony shape and its characteristics and to count the colonies with haloes and those without and to express these in terms of colony forming units per ml of any acid positive and acid negative and the total colonies, so to do this we were given two agar spread plates which was spread the bacteria over the surface of the agar plates using a sterile rod spreader. (MRSA and Yoghurt)

Representative colonies need to be subculture to give pure cultures.

Phage induction (week 3)

Lysogenic phage can be induced by the antibiotic Mitomycin C.

The MRS broth liquid cultures are divided equally into two sterile test tubes

Control bottle went cloudy

Mitomycin C added to a final concentration of 0.2 mg/ml

After couple hours the test tube that was mitomycin in it was checked and the solution should remained clear lysis and filtered the solution through 0.2 nanometer filter to remove unlysed cells then added a drop of chloroform until next week.

After that five agar dishes was prepared which was contain STA nutrient agar soluble starch, CA skim milk with nutrient, GA nutrient agar supplement, NA nutrient agar, and MRS is name of the inventor which has complete supplement and nutrient for the bacteria to growth. Then was inserted each plate with unknown culture and streaked on the plates.

Gram Staining of Bacteria

Individual bacterial cells are hard to see, partly because they are small, but also because they are almost transparent in addition to magnification under a microscope, optical tricks must be used to be able to see them.

A slide was placed with a bacterial smear on a staining rack, then applied crystal violet to just cover the smear then left I minute the dye on then it poured off the stain with water and shake the slide to remove excess water.

The slide been flooded the smears with grams iodine solution to allow stand for one minute then it poured off the iodine with water. The slide been decolourised by washing with alcohol with contact time was 30 seconds by which time most of the blue colouration was removed and washed with water, Then slide was stain with safranin for 30 seconds washed with water and shake off excess water and allow to air dry. after staining completed smears been examined microscopically using the procedure outlined and recorded cell morphology and gram reaction.

Cellular morphology: gram stain slides were examined using the oil immersion high power lens on the microscope in order to determine the cellular morphology.

Enumeration of recovery phage (week 4)

To prepare the subculture for phage recovery used last week’s solution to dilute 4 times. The solution was prepared and diluted into four bottles which have right nutrients on them and labelled them from the 10-1, 10-2, 10-3, 10-4 then 0.3ml was taken from last week solution and added 0.1ml lysate on 10-1 bottle and shake it then flame it and closed the lid after was taken 0.1ml from 10-1 bottle and added to the 10-2 bottle and the series diluted continued up to 10-4 same procedure.

Examination of biochemical tests

Starch in STA plate: iodine solution was put into the STA palate and waited for 2 minutes then checked the result if the result is positive the colonies turn blue.

Casein hydrolysis: casein hydrolysis was put on to the CA plate and checked afterwards for the result if it become positive the agar in the dish will start of opaque and added casein hydrolysis in the plate.

Test for gelatine hydrolysis: to test gelatine hydrolysis a drop of ammonia sulphate was put the GA plate and waited to see if there is no white participated around the colonies so the results become positive.

Test for catalase: to hydrolysis the catalase in the NA plate use drops of H2O2 and covered with a lid fir few minutes if the result is positive there will be a small bubble on the agar gel.

Test for the carbohydrate: two bottle of solution was provided from for each bench also there was one API was provided for each bench to fill up the solution in each chamber of APi used lops to collect the bacteria from MRS agar gel and put on peptic solution then filled the API chamber to let the bacteria to grow in 4 -5 hours if the bacteria used carbohydrates as an energy source it will change from white to blue.

Result:

Dilution

MRSA Agar

CuCO3 Agar

10-3

0

0

10-4

0

2

10-5

0

1

 

After transferring the provided organism onto MRS agar, the organism produced white/cream colored medium sized colonies.  Their edges were smooth with a convex rise.

Gram staining the provided organism showed it to be Gram +ve bacilli diploid or chain linked.

The organism in MRS broth was too little n volume to accurate describe how it dispersed and grew in a liquid state.  Did show signs of white, cloudy turbidity however.

Table 2.

Illustrates the hydrolysis results on media growth and biochemical test exposure to the unknown organism.

Catalase

Casein Agar

Starch

Gelatin Agar

-ve

-ve

-ve

-ve

 

The Agar stab plate produced white growth mostly throughout the medium and a small amount at the surface.  This would suggest it to be a facultative aerobe or aero tolerant and positve motility.  

The PFU plates produced no growth while the API strip confirmed the unknown organism to be Lactobacillus paracasei ssp paracasei with an ID of 95%.

DOUBTFUL PROFILE

Strip

API 50 CHL V5.1

Profile

- - - - - + - -+ -+ + + + - - - - + - - - + - - + + + + +- -+ - - - - - - -+ -+ - - - - - - -

Note

Significant taxa

%ID

T

Tests against

Lactobacillus curvatusssp curvatus

98.8

0.38

ADO 0%

MAN 12%

TRE 25%

TUR 12%

Next taxon

% ID

T

Tests against

Lactobacillus paracaseisspparacasei 1

0.5

0.1

ADO 13%

SAC 93%

SOR 36%

MLZ 93%

AMY 98%

GEN 80%

ARB 100%

GNT 93%

Discussion

During the work different problems was encountered that there has been diluent because there was not much growth. Probably the bacteria was killed by residual detergent in the bottles, to explain why this is happened is that irrespective sample and irrespective of the agar because the agar was three different types of agar

Exoenzymes are proteins that are secreted by bacteria in large quantities into the environment.

The catalase tests bacteria did not show any evolution oxygen from hydrogen peroxide

Agar stab: there was a little bit grown at the bottom which means that it doesn’t like oxygen.

The reason for API test run was to determine which carbohydrate sources that the bacteria can utilise.

Some of the strips can be read straight away. After added all the reagents provided API colour chart which contain negative results and positive results were examined .The positive results score certain number point and the test sets 3. If the first test is positive then the score is 1, so if the second test is positive then it scores 2 and if the third is positive it scores 4. So in sets three the maximum score is 7.

There were some technical problems which were encountered such as (1) insofar as it was not possible to recover lactic acid bacteria from the majority of the samples. This was despite the range of samples brought to the lab and the fact that three different agar types were used (two in-house and one from a commercial supplier). Therefore these cannot be the cause of the lack of growth.

The only common component of the procedure is therefore the diluent used to dilute the samples. It is likely that the bottles containing the diluent had not all been rinsed thoroughly during the glass washer cycle. Any traces of detergent would have affected viability of the bacteria, lactic acid bacteria are sensitive to strong oxidizers (e.g. hydrogen peroxide) as they rely on fermentative metabolism. For this reason the bacteria that were recovered successfully had to be used by the entire class. This affects the range of bacteria recovered which is unfortunate, but does not affect the ability to complete the work with the available bacteria.

As the bacteria were recovered from a food sample the species and strains recovered will not be identical. Additionally it is not known that at the outset what bacteria are going to be present in the samples selected. Similarly it is not known to what extent, if at all, the recovered strain harbour a latent bacteriophage infection.

Conclusion

The yogurt manufacturers have in recent years increasingly recognized that sell their products even better when the ingredients are mixed into yogurt, the health-conscious consumer has known from the pharmacist. Yogurt with aloe vera supplement, a few mg of omega-3 fatty acids, bifidus acidophilus supplements or with other helpful substances come to the consumer better. A great merit of the yogurt maker is therefore that in the meantime already large segments of the population, the meaning is conscious of how important a healthy intestinal flora for health.

References

1. Carminati D, Giraffa G, Quiberoni A, Binetti A,

Suárez V, Reinheimer J. Advances and trends in starter

cultures for dairy fermentations. In: Mozzi F, Raya R,

Vignolo G, eds. Biotechnology of lactic acid bacteria:

Novel applications. Iowa, USA: Wiley-Blackwell 2010;

177-192.

2. Emond E, Moineau S. Bacteriophages and food fermentations.

In: McGrath S, van Sinderen D, eds.

Bacteriophage: Genetics and Molecular Biology.

Horizon Scientific Press/Caister Academic Press 2007;

93-124.

3. Moineau S. Applications of phage resistance in lactic

acid bacteria. Antonie Van Leeuwenhoek 1999;

76:377-82; PMID:10532393; http://dx.doi.

org/10.1023/A:1002045701064.

4. Madera C, Monjardín C, Suárez JE. Milk contamination

and resistance to processing conditions determine

the fate of Lactococcus lactis bacteriophages

in dairies. Appl Environ Microbiol 2004; 70:7365-

71; PMID:15574937; http://dx.doi.org/10.1128/

AEM.70.12.7365-7371.2004.

5. Suárez VB, Capra ML, Rivera M, Reinheimer JA.

Inactivation of calcium-dependent lactic acid bacteria

phages by phosphates. J Food Prot 2007; 70:1518-22;

PMID:17612087.

6. Surono S, Hosono A. Starter cultures. In: Fuquay J, Fox

P, McSweeney P, eds. Encyclopedia of dairy science 2°

Edition (Volume 2), Academic Press, Elsevier Science,

USA 2011; 477-482.

7. Suárez V, Reinheimer J, Quiberoni A. Bacteriophages

in dairy plants. In: Quiberoni A, Reinheimer J, eds.

Bacteriophages in dairy processing, Nova Science

Publishers, 2012; pp. 53-78.

8. Mäyrä-Mäkinen A, Bigret M. Industrial use and production

of lactic acid bacteria. In: Salminen S, von

Wright A, Ouwehand A, eds. Lactic acid bacteria:

microbiological and functional aspects, 3° Edition,

New York, Marcel Dekker, Inc. 2004; 175-198.

9. Nath KR, Wagner BJ. Stimulation of lactic acid bacteria

by a micrococcus isolate: evidence for multiple effects.

Appl Microbiol 1973; 26:49-55; PMID:4199337.

10. Ringø E, Bendiksen HR, Gausen SJ, Sundsfjord A,

Olsen RE. The effect of dietary fatty acids on lactic acid

bacteria associated with the epithelial mucosa and from

faecalia of Arctic charr, Salvelinus alpinus (L.). J Appl

Microbiol 1998; 85:855-64; PMID:9830121; http://

dx.doi.org/10.1046/j.1365-2672.1998.00595.x.

11. Quiberoni A, Suárez VB, Binetti AG, Reinheimer JA.

Bacteriophage. In: Fuquay J, Fox P, McSweeney P, eds.

Encyclopedia of dairy science 2° Edition (Volume 1),

Academic Press, Elsevier Science, USA 2011; 430-438.

12. Quiberoni A, Moineau S, Rousseau GM, Reinheimer

J, Ackermann H-W. Streptococcus thermophilus bacteriophages.

[Review]. Int Dairy J 2010; 20:657-64; http://

dx.doi.org/10.1016/j.idairyj.2010.03.012.

13. Brüssow H, Suarez JE. Lactobacillus phages. In:

Calendar R, Abedon ST, eds. The bacteriophages, 2°

Edition, Oxford University Press, New York 2006.

14. Villion M, Moineau S. Bacteriophages of lactobacillus.

Front Biosci 2009; 14:1661-83; PMID:19273154;

http://dx.doi.org/10.2741/3332.

15. Capra ML, Binetti AG, Mercanti DJ, Quiberoni A,

Reinheimer JA. Diversity among Lactobacillus paracasei

phages isolated from a probiotic dairy product plant. J

Appl Microbiol 2009a; 107:1350-7; PMID:19486389;

http://dx.doi.org/10.1111/j.1365-2672.2009.04313.x.

16. Quiberoni A, Suárez VB, Reinheimer JA. Inactivation

of Lactobacillus helveticus bacteriophages by thermal

and chemical treatments. J Food Prot 1999; 62:894-8;

PMID:10456743.