Identification And Characteristics Of Sample A: Potential Pathogen Causing Diarrhoea

Sample Preparation

Sample Preparation 

Sample A has been isolated from cheese and the collection method has been done in an “FDA Approved manner” (fda.gov 2022). This describes that the samples have been collected via aseptic technique by using sterile forceps or needles and have been stored in a “sterile”, “leakage proof” and “sealed’ container (Suo et al. 2018). After that, the sample has been effectively incubated for 24 to 48 hours at 37°C. After that, different biochemical tests were performed to identify the organism and its viability. It was observed that sample A is capable of causing severe diarrhoea in patients. It can be stated that the strain might be a strain of “Staphylococcus aureus” as the bacterium has been isolated from cheese and it has been observed that the bacterium causes diarrhoea in patients. Further tests have been performed to confirm the strain and to determine its contamination and pathogenesis.

The first sample that has been isolated from cheese might be “Staphylococcus aureus” which is a “gram-positive” organism and has a specific “round shape” (Ramesh et al. 2019). The organism is responsible for causing “gastrointestinal abnormalities” in humans. Food poisoning is one of the most common adverse effects that is caused by the Staphylococcal toxin (Feng et al. 2021). The major source of staphylococcal infections is food sources such as “milk”, “cheese” and “mucosal secretions”. The major symptoms for “staphylococcal infections” are “excessive abdominal cramp”, “severe nausea”, “vomiting’, “stomach pain”. The symptoms may last up to 8 hours depending on the disease severity (Maktabi et al. 2021).

The present report aims to identify the potential sample organism collected from cheese that is responsible for causing diarrhoea. The report significantly explains the phenotypic characteristics of the microorganism with appropriate “biochemical testing”, “gram staining” and “colony counting”. Serial dilution has been performed to dilute the sample and up to 10^-8 concentrations have been taken. The samples of concentrations “10^-5,10^-6,10^-7 and 10^-8” have been taken. The dilution has been effectively plated on specific “Tryptic Soy Agar or TSA agar” and the total sample has been incubated overnight at 37°C. The phenotypic characteristics have been identified which helps to understand the potential pathogenesis of the organism and also helps to understand the role of this organism in toxin production.

Results

CFU calculation

CFU calculations are effective as this helps in understanding the number of bacteria present in the sample (Saber and Kandala 2018). CFU can be calculated by effectively dividing the “total number of colonies” by the total “dilution factor”. This helps in understanding the bacterial population (Almwafy 2020).

 

Figure 1: Serial Dilution

(Source: Learning Materials)

In cheese sample,

Total number of bacteria in dilution 10^-5 is: 150

Therefore, CFU/ml (150/10⁵) x (1000/100)

= 0.015

Total number of bacteria in dilution 10^-6  is: 70

Therefore, CFU/ml= (70/10⁶) x (1000/100)

= 0.0007

Total number of bacteria in dilution 10^-7  is: 30

Therefore, CFU/ml= (30/10⁷) x (1000/100)

= 0.000003

Total number of bacteria in dilution 10^-8 is: 5

Therefore, CFU/ml= (5/10⁸) x (1000/100)

= 0.00000005

Therefore the average CFU/ml is= (0.015+0.0007+0.000003+0.00000005)/4= 0.0039257625

Sample A	Isolated from cheese
CFU/ml	0.0039257625
Culture characteristics	Large colonies are seen which are “facultatively anaerobic”. The colours of the colonies are “yellow” and “white”.
Biochemical identification
Oxidase	Negative
Catalase	Positive
Coagulase	Positive
Gram staining	Gram-positive in nature. These are small chain cocci that can be seen in a “clustered” format”. The bacterium usually grows in pairs and has a diameter of 0.5 μm to 1.0 μm
Pigmentation	Yellow and white colonies
Toxin production	“Hemolysin”, “enterotoxin”, “exfoliative toxin”, “toxic shock syndrome toxin”
Confirmation	Staphylococcus aureus

Table 1: Confirmation test 1 for sample A

(Source: Created by the author)

According to the above results the bacteria has shown positive results in catalase and coagulase and have shown negative results in oxidase. Different studies have shown that S. aureus is facultative anaerobes that significantly yield “lactic acid” through anaerobic fermentation (Maktabi et al. 2021). However, sometimes aerobic respiration can also be seen in these bacteria. Further analysis has shown that the positive coagulase result can only be seen in Staphylococcus aureus and Staphylococcus intermedius (Saber and Kandala 2018). However, the colony morphology has shown that large yellow and white colonies have appeared which again proves that the sample species is S. aureus.

The coagulase test is an effective biochemical test that helps in determining the strain S. aureus from other strains of S. epidermis. S. aureus is an effective bacterium that produces coagulase (Feng et al. 2021). Due to this reason, S. aureus gives positive results in coagulase tests.

 

Figure 2: Catalase Test

(Source: Learning Materials)

On the other side, an oxidase test is performed to differentiate S. aureus from other micrococcal strains. Positive results in the catalase test describe that the bacterium produces catalase as a virulence factor. The bacterium can resist hydrogen peroxide killing by specifically converting H2O2 into H2O and O2 with the help of this virulence factor (Maktabi et al. 2021). The large yellow colonies of the bacterium appear due to the presence of carotenoids.

Confirmation

One of the major toxins that are produced by S. aureus is hemolysin, enterotoxins, exfoliative toxins etcetera. These protein toxins have high-temperature tolerance and do not destroy after cooking the food. These are the major reasons for food poisoning caused by bacteria. Milk, cheese and other dairy products are some major food items that can have S. aureus contamination. The bacterium has a specifically high tolerance to salt due to coagulase produced by the bacteria. S. aureus, for this reason, can remain active and can release toxins in cooked food items (Feng et al. 2021). Food poisoning caused by S. aureus can have symptoms such as “diarrhoea”, nausea, vomiting, abdominal cramps etcetera.

Different preventive measures are available that can help in reducing the effectiveness of S. aureus food poisoning. This includes “effective temperature measuring”. Cooked foods should be stored either at a temperature higher than 140 degrees Fahrenheit or at a temperature less than 40 degrees Fahrenheit (Saber and Kandala 2018). This inhibits bacterial growth. After cooking, foods should be stored effectively. Stored foods should be kept in an airtight container that is “shallow and wide”. Cooked foods should be effectively stored in the refrigerator after 2 or more hours. This prevents the growth of S. aureus bacteria however does not inhibit the growth.

Sample Preparation 

The second sample has been prepared from a “beef meat sample”. The sample has been collected aseptically with the help of a sterile needle and has been stored at 37 degrees centigrade for 48 hours (Safdar and Malik 2020). After storing the sample has been diluted by using serial dilution and four different dilutions have been prepared from 10^-5 to 10^-8. After that 100μl sample has been taken from each diluted sample and they were plated in a specific agar known as “TSA agar”. After that, the samples were effectively stored at 37 degrees centigrade in an incubator for 2 hours. The colony morphology of the plated samples has been observed after 24 hours. The phenotypic analysis has been performed through biochemical tests, gram staining, toxin production and analyzing the nature of colonies. It can be stated that the sample might be a strain of Bacillus cereus (Majumdar and Gupta 2020).

Discussion-Findings

The organism Bacillus cereus is an effective organism that is “gram-positive” and is “facultatively anaerobic”. This motile organism is rod-shaped and can form spores (KUPRADIT et al. 2020). The organism is a “beta-hemolytic organism” that produces different types of toxins that are effective in causing food poisoning. The toxins produced by this organism are primarily enterotoxin. Generally, two types of toxins are produced by this organism; “emetic toxins” and “diarrheal toxic” (Jha 2020). Both of these are effective in causing food poisoning. The main source through which the organism infects is “potatoes”, “fried rice”, “raw meat” and vegetables. This is one of the most common organisms that affect more than 64000 people each year. The major clinical symptoms of the disease are excessive “Watery diarrhoea”, “abdominal cramps”, “fatigue” etcetera.

The present experiment aims to identify the sample that has been collected from beef meat through effective phenotypic characteristics and aims to determine the sample organism from different biochemical tests and gram staining tests.

CFU calculation

CFU calculation helps in understanding the total bacterial population level in the diluted samples. CFU is usually calculated through the effective division of a total number of colonies and dilution factor.

In beef meat sample,

Total number of bacteria in dilution 10^-7 is: 50

Therefore, CFU/ml= (50/10⁷) x (1000/100)

= 0.0000005

Total number of bacteria in dilution 10^-8 is: 5

Therefore, CFU/ml= (5/10⁸) x (1000/100)

= 0.00000005

Therefore the average CFU/ml is= (0.0000005+ 0.00000005)/2= 2.75 x10^-7 

Sample B	Isolated from Beef meat
CFU/ml	2.75 x10-7
Culture characteristics	Large colonies are seen which are “dull”, “grey”, “spreading” colonies that have “irregular perimeters” and have a comparatively “rough surface”.
Biochemical identification
Oxidase	Negative
Catalase	Positive
Coagulase	Negative
Gram staining	Gram-positive in nature. However, the organism shows effective gram variability and can act as a gram-negative organism also.
Pigmentation	white colonies in a green background
Toxin production	Enterotoxin
Confirmation	Bacillus cereus

Table 2: Confirmation test 2 for sample B

(Source: Created by the author)

The above table shows the gram staining and the biochemical test results of Bacillus cereus. The organism shows negative results on the oxidase test which indicates that the organism does not possess “cytochrome C oxidase”. The organism has shown a positive catalase test which describes its ability to restrict the bactericidal activities of H2O2 (Fasolato et al. 2018). This shows that B. cereus can show effective pathogenesis against hydrogen peroxide due to the presence of catalase enzyme which is a virulence factor. It has been proved from the gram staining that the organism is a gram-positive organism that is “facultatively anaerobic” and also produces toxins. This indicates that this organism is a strain of Bacillus cereus (Jha 2020).

Virulence Factor

The first test that has been performed for biochemical identification is the oxidase test which is primarily done to determine the presence of cytochrome C oxidase which is required for electron donation in microbial “electron transport chain”. The negative result indicates that the organism is a “facultative anaerobe” which produces “lactic acid” through effective fermentation or uses “aerobic respiration” (Fasolato et al. 2018). The second biochemical test that has been performed is the catalase test. This is done to detect the presence of “catalase enzymes”. The positive result indicates that the organism has a specific virulence factor which is catalase. This enzyme is required as this protects the bacterium from the “toxic by-products of O2 ” such as hydrogen peroxide which can be produced during aerobic respiration. This proves that the bacterium can be aerobic also (Enyidi and Onyenakazi 2019). The third test is the coagulase test. This test is usually performed to effectively differentiate cocci bacteria from pathogenic staphylococci.

 

Figure 3: Coagulase test

(Source: Learning Materials)

The negative result indicates that the pathogen is not staphylococci. Therefore, it can be stated that this oxidase negative, catalase-positive, a coagulase-negative bacterium that is gram-positive and also produces an enterotoxin and firm spores is a strain of “Bacillus cereus”.

The major toxin produced by this bacterium is “emetic toxin” which is a heat-stable toxin such as cereulide. Another toxin produced by this bacterium is enterotoxin which is another heat-stable toxin. Bacillus cereus is usually present in raw meat and processed meat and other fried foods (Fasolato et al. 2018). The heat-stable toxins produced by this bacterium can remain stable at cooking temperature. The toxins can effectively withstand a temperature of 120 degrees Celsius for approximately 1.5 hours (Jha 2020). Moreover, the bacterium can withstand the “reheating” of food”. This toxin causes severe diarrhoea in individuals and causes dehydration, nausea, and vomiting. The infection is usually diagnosed through watery diarrhoea, abdominal pain, fatigue and dehydration.

The infection and toxin release from B. cereus should be managed effectively by ensuring that foods are being cooked at a temperature higher than 62 degrees centigrade and the cooked foods are being stored effectively at a temperature below 4-5 degrees centigrade. The cooking temperature for foods should be higher than 78 degrees centigrade to prevent infection (Fasolato et al. 2018).

Prevention measures

Sample Preparation 

The sample has been effectively collected from “chicken meat” and the sample has been collected after following the guidelines of the FDA for aseptic collection of microorganisms (fda.gov 2022). The sample has been collected by using an aseptic needle and the sample has been stored in a sealed and airtight container to prevent any environmental contamination. After that, the samples were prepared effectively after diluting the sample through serial dilution. The serial dilution has been performed in four different concentrations of 10^-5, 10^-6, 10^-7, and 10^-8. After incubating the diluted samples for approximately 48 hours at 37 degrees celsius the sample tubes have been plated on TSA agar after taking 100 microlitre samples from each dilution. These plates have further incubated at a temperature of 37 degrees centigrade for 24 hours. After that, the colonies have been counted and the organism has been identified through its colony morphology, phenotypic characteristics and different biochemical tests. It has been assumed that sample C might be a strain of Escherichia coli.

The organism Escherichia coli is a gram-negative organism and has a specific rod-shaped morphology with “rounded ends” (Hassani et al. 2022). The organism also possesses “filamentous” or “elongated rods” which makes it different from “staphylococcal colonies”. The organism is “facultatively anaerobic” and also a “non-spore-forming” organism. E. coli is a faecal coliform bacteria that produces a specific toxin known as “Shiga toxin”. Although the bacteria is a “harmless” organism, the Shiga toxin produced by the organism can cause severe diarrhoea and sometimes causes excessive damage to the “intestinal epithelial lining” (Majumdar and Gupta 2020). The major infection sources are “raw and improperly cooked meat”. E. coli infection also can occur through “unpasteurized milk”, “sprouts”, “salami” etcetera. The symptoms include “bloody diarrhea”, “severe abdominal cramps”, “nausea”, “excessive vomiting” etcetera. Sometimes fever arises, however, the temperature ranges between 100-101 degrees Fahrenheit. The toxin produced by E. coli can produce other significant diseases such as “Hemolytic Uremic Syndrome or HUS” (Menge 2020). HUS is a specific kidney disorder that occurred due to a Shiga toxin infection. In this disease, the blood vessels of the kidneys get damaged effectively and this creates “blood clots” in the “kidney blood vessels”. It has been observed that the toxin causes severe “inflammation” and “swelling” in the blood vessels. The blood clot restricts the normal kidney function and sometimes the disease can be “life-threatening” (Abdelkarim et al. 2020).

The present report aims to identify sample C from chicken meat with appropriate phenotypic analysis, different biochemical analyses and colony morphology analyses have been performed. The report also aims to determine the toxin produced by this organism and its effect on humans.

CFU calculation

CFU or “Colony Forming Unit” calculations are effective in determining the total number of bacterial colonies that are present in the sample. CFU has usually calculated in “per ml” calculation which helps in determining the number of bacteria that are present per ml of the solution. CFU calculation is performed by dividing the total number of colonies that have been counted from the TSA plates by the dilution factor.

In chicken meat sample,

Total number of bacteria in dilution 10^-5 is: 74

Therefore, CFU= (74/10⁵) x (1000/100)

= 0.0074

Total number of bacteria in dilution 10^-6  is: 32

Therefore, CFU= (32/10⁶) x (1000/100)

= 0.00032

Total number of bacteria in dilution 10^-7  is: 14

Therefore, CFU= (14/10⁷) x (1000/100)

=0.000014

Total number of bacteria in dilution 10^-8 is: 4

Therefore, CFU= (4/10⁸) x (1000/100)

= 0.00000004

Therefore the average CFU is= (0.0074+0.00032+0.000014+0.00000004)/4= 0.00193351

Sample C	Isolated from Chicken meat
CFU	0.00193351
Culture characteristics	Large colonies can be seen which can be distinguished from other colonies through their “greyish-white colour”. Significant characteristics of E. coli colonies are “moist”, “opaque”, effective “translucent discs” etcetera.
Biochemical identification
Oxidase	Negative
Catalase	Positive
Coagulase	Negative
Gram staining	Gram-negative in nature. The organism is a facultatively anaerobic and rod-shaped bacterium. The organism is a “faecal coliform” organism that is non-motile.
Pigmentation	No -pigment
Toxin production	Shiga toxin (type of endotoxin)
Confirmation	Escherichia coli

Table 3: Confirmation test 3 for sample C

(Source: Created by the author)

The above table shows that the expected bacterium has shown positive results on catalase tests and negative results on coagulase and oxidase tests. The positive results in catalase prove that the organism is “facultatively anaerobic” which describes that the sample microorganism has a catalase enzyme. This can be confusing as Bacillus cereus also shows similar results on biochemical tests. However, there are significant characteristics differences between the sample C bacterium and Bacillus cereus (Lee et al. 2021). The gram staining proves that the sample organism is gram-negative, unlike other sampled organisms. Moreover, the colony morphology has shown that the organism does not have significant pigment and the colonies that have appeared are “greyish white” in colour and have a round appearance which cannot be seen in other colonies. This proves that this gram-negative, non-motile, non-spore-forming, rod-shaped, oxidase negative, coagulase-negative and catalase-positive bacteria is Escherichia coli.

The biochemical tests have explained that the organism has shown positive results in catalase tests which describes that this organism can restrict killing by H2O2 and can survive itself by producing water and oxygen from hydrogen peroxide. It has been shown that E. coli has specific catalase enzymes known as “hydroperoxides I” and “hydroperoxides II”. These two catalase enzymes can effectively break hydrogen peroxidase into “H2O and O2” (Menge 2020). This helps in protecting the bacterium from the harsh environment. The bacterium has shown negative results in coagulase which indicates that the bacterium is not a staphylococcal strain. Negative results on oxidase tests have shown that this bacterium does not produce “cytochrome C oxidase” as a part of their “microbial electron transport chain”. Gram staining has proved that the organism is gram-negative. The biochemical straining has proved that the organism is “facultatively anaerobic”. Moreover, the colony morphology identification has proved that this bacterium has a clustered round-shaped colony that has a greyish white colour. Again, the phenotypic morphology has proved the organism is a rod-shaped organism that is “non-spore-forming” and is “non-motile”. This evidence also suggests that the sample is a strain of Escherichia coli which produces Shiga toxin (Lee et al. 2021). This is a type of endotoxin that causes gastrointestinal infection in humans. This virulence factor of E.coli is responsible for causing severe kidney infection also.

The major toxin of Escherichia coli is Shiga toxin which is a type of endotoxin and is responsible for causing severe diarrhoea in humans. The level of diarrhoea depends on the severity of the infection and the major symptoms are “blood in stools”, “vomiting”, “nausea”, and fever.  In some cases, the Shiga toxin causes severe kidney damage which can lead to life-threatening situations for individuals (HUS). The infection occurs from “raw and uncooked meat”, “unpasteurized milk” etcetera. The presence of toxins increases the pathogenesis of this pathogen. Different studies have shown that only specific strains of E. coli are toxin-producing bacterium. These are known as “Shiga Toxin-Producing E. coli” or STEC (Abdelkarim et al. 2020). The major virulence factor of STEC is Shiga toxins which have been proven to be one of the major “Cytotoxic and Class II ribosome-inactivating proteins’ ‘. This toxin is one of the most potent virulence factors of E. coli which not only causes foodborne illnesses but causes HUS disease which can impair effective kidney functions.

Different types of preventive measures are available for reducing E.coli contamination; this includes effective hygiene maintenance practices such as “handwashing”, “avoidance of consumption of raw meat and uncooked foods” etcetera. Proper hygiene practice can reduce the effectiveness of the disease. Foods should be cooked properly at a temperature higher than 120 degrees centigrade. Meat should be cooked thoroughly and individuals should properly wash their hands and “cutting utensils” after handling raw meat (Majumdar and Gupta 2020). Proper storage of meat, milk, juices and other raw and fresh food items can help in preventing the contamination of Escherichia coli. Effective storage, hygiene maintenance and proper treatment can help in preventing the disease.

Reference

Abdelkarim, E.A., Hafez, A.E.E., Hussein, M.A., Ali, S.S. and Elsamahy, T.S., 2020. Prevalence of escherichia coli in marked poultry carcasses in Egypt. Adv. Anim. Vet. Sci, 8(s1), pp.55-61.

Almwafy, A., 2020. Preliminary Characterization And Identification Of Gram Positive Hemolysis Bacteria. Al-Azhar Journal Of Pharmaceutical Sciences, 62(2), Pp.96-109.

Enyidi, U. and Onyenakazi, G., 2019. Effects of Substitution of Fishmeal with Bambaranut Meal on Growth and Intestinal Microbiota of African Catfish (Clarias gariepinus). Aquaculture Studies, 19(1), pp.09-23.

Fasolato, L., Cardazzo, B., Carraro, L., Fontana, F., Novelli, E. and Balzan, S., 2018. Edible processed insects from e-commerce: Food safety with a focus on the Bacillus cereus group. Food microbiology, 76, pp.296-303.

fda.gov, 2022. BAM Chapter 1: Food Sampling/Preparation of Sample Homogenate. [online] U.S. Food and Drug Administration. Available at: <https://www.fda.gov/food/laboratory-methods-food/bam-chapter-1-food-samplingpreparation-sample-homogenate> [Accessed 19 April 2022].

Feng, T., Leptihn, S., Dong, K., Loh, B., Zhang, Y., Stefan, M.I., Li, M., Guo, X. and Cui, Z., 2021. JD419, a Staphylococcus aureus Phage With a Unique Morphology and Broad Host Range. Frontiers in microbiology, 12, p.840.

Hassani, S., Moosavy, M.H., Gharajalar, S.N., Khatibi, S.A., Hajibemani, A. and Barabadi, Z., 2022. High prevalence of antibiotic resistance in pathogenic foodborne bacteria isolated from bovine milk. Scientific Reports, 12(1), pp.1-10.

Jha, S.K., 2020. Isolation and characterization of bacteria with biochemical importance from soil samples of Ranchi city, India. Asian J. Microbiol., Biotechnol. Env. Sci, 22(4), pp.648-653.

KUPRADIT, C., INNOK, S., WORARATPHOKA, J. and KETUDAT-CAIRNS, M., 2020. Prevalence and characterization of pathogenic bacteria in bulk tank raw milk, Thailand. Walailak Journal of Science and Technology (WJST), 17(6), pp.588-599.

Lee, K.S., Jeong, Y.J. and Lee, M.S., 2021. Escherichia coli Shiga toxins and gut microbiota interactions. Toxins, 13(6), p.416.

Majumdar, R.K. and Gupta, S., 2020. Isolation, identification and characterization of Staphylococcus sp. from Indian ethnic fermented fish product. Letters in Applied Microbiology, 71(4), pp.359-368.

Majumdar, R.K. and Gupta, S., 2020. Isolation, identification and characterization of Staphylococcus sp. from Indian ethnic fermented fish product. Letters in Applied Microbiology, 71(4), pp.359-368.

Maktabi, S., Zarei, M., Ghafuri, Z. and Ataee, R.A., 2021. Biotyping and enterotoxigenicity of coagulase-positive and coagulase-negative staphylococci isolated from different raw meat. Iranian Journal of Veterinary Research, 22(2), p.113.

Menge, C., 2020. Molecular biology of Escherichia coli Shiga toxins’ effects on mammalian cells. Toxins, 12(5), p.345.

Ramesh, N., Archana, L., Royam, M.M., Manohar, P. and Eniyan, K., 2019. Effect of various bacteriological media on the plaque morphology of Staphylococcus and Vibrio phages. Access Microbiology, 1(4).

Saber, N. and Kandala, N.J., 2018. The inhibitory effect of fluphenazinedecanoate and caffeine on Staphylococcus aureus efflux pumps. Cur. Res. Micro. Biotech, 2108(6), p.2.

Safdar, Y. and Malik, T., 2020. Antibacterial activity of the rose extract. Open Acc J Comp & Alt Med, 2(4), pp.194-201.

Suo, B., Yang, H., Wang, Y., Lv, H., Li, Z., Xu, C. and Ai, Z., 2018. Comparative proteomic and morphological change analyses of Staphylococcus aureus during resuscitation from prolonged freezing. Frontiers in microbiology, 9, p.866.