Vibrio cholera
Vibrio cholera affects the intestinal tract. It also causes mild to severe diarrhea, vomiting, and dehydration.Since the patient does not complain of dehydration, food poisoning caused by Vibrio cholera is ruled out.
With the elimination of above possible casaultive agents, the most likely cause of food poisoning in this patient is :
1. Staphylococcus aureus
Staphylococcal food poisoning is the most common form of food poisoning.
“some foodborne diseases are caused by the presence of a toxin in the food that was produced by a microbe in the food. For example, the bacterium Staphylococcus aureus can grow in some foods and produce a toxin that causes intense vomiting.”
Besides causing severe vomiting, Staphylococcus aureus also causes diarrhea and abdominal cramps. These are the exact complaints of the patient.
Laboratory Diagnosis (S.Aureus)
Microscopy
On gram staining, gram positive and cocci-shaped colonies are found, which are arranged in irregular clusters.
Culture
The purpose of this test is to isolate bacteria or other organisms that might be causing the symptoms so they can be identified.
Blood agar supports the growth of most bacteria including Staphylococcus aureus, Listeria monocytogenes, and yeast, which are infrequently implicated in food poisoning or gastrointestinal infections, but do not grow on the other media.
Upon culturing on blood sheep agar, Staphylococcus aureus, if present, shows beta haemolytic characteristics, and yield yellow or gold colonies.
Typical test characteristics of S. aureus:
Catalase activity- Positive
Coagulase production- Positive
Thermonuclease production- Positive
Lysostaphin sensitivity- Positive
Anaerobic utilization of mannitol- Positive
Anaerobic utilization of glucose- Positive
Biochemical Tests
1. Catalase Test
Principle
Hydrogen peroxide (H2O2) is decomposed by catalase enzyme to water and oxygen. Bacteria are unable to protect themselves from the lethal effect of hydrogen peroxide, which is accumulated as a product of carbohydrate metabolism. Catalase is a hemoprotein. Catalytic decomposition of hydrogen peroxide leads to the reduction of ferric iron (Fe3+) to ferrous iron (Fe2+) and the re-oxidation of the latter by oxygen. This reaction can be summarized by the following equation:
Catalase
H2O2 --------------------------> 2H2O + O2
Procedure
• A clean microscope slide is labeled for the organism to be tested.
• One drop of 3% H2O2 is added to the slide.
• Several isolated colonies of the organism is transferred to the H2O2 on the microscope slide using a sterile inoculating loop.
Caution
Avoid touching the surface of the media plates containing sheep or human blood, as red blood cells have catalase activity and will give a false-positive result.
Interpretation of positive test: Evolution of gas bubbles
Negative test: No gas bubbles
Should Staphylococcus aureus be the suspected bacteria, the catalase test would be positive.
2. Coagulase Test
Principle
The coagulase test serves to differentiate Staphylococcus aureus from Staphylococcus epidermidis. Should coagulase be present, a clot will be formed in a tube of citrated platelet-rich plasma (~ >150 x 106 platelets/cc plasma). The citrate, which is an anti-coagulant, is added to avoid auto-clotting.
Procedure
• A generous loopful of stool is added to a tube of citrated rabbit plasma.
• Using the loop, the inoculum is thoroughly homogenized
• The tube is then incubated at 37o C for 1 to 4 hours.
• The tube is observed at 30 minute to hourly intervals for the first 2 hours to detect the presence of a clot by tipping the tube gently on its side.
• Should clotting be seen within 24 hours, the coagulase test is considered positive.
Antibiotic Susceptibility Test
1. Agar Disk Diffusion Method• With a sterile loop, the tops of four to five colonies of S. aureus from pure culture are picked up.
• The colonies were suspended in 5 ml of sterile physiologic saline.
• The inoculum turbidity is standardized to equivalent of a 0.5 McFarland standard.
• The entire surface of a Mueller-Hinton agar plate is inoculated using a sterile swab.
• Disks containing 10 µg of penicillin, 10 µg of ampicillin, 30 µg of cephalexine, 1 µg of oxacillin, and 30 µg of kanamycin are then placed using a sterile forcep onto the agar surface and gently pressed down to ensure contact.
• Plates are incubated at 35°C for 20 h.
• Subsequently, the diameter of the zone of inhibition around each disk is measured.
Treatment Therapy
The larger the zone diameter, the more sensitive the bacteria is to the antibiotic. Through this test, it is observed that the largest zone diameter is seen around Penicillin G disk. Hence, Ng Ming En can be treated using Penicillin G antibiotic.
Case study 2
Patient Name: Kwan Siew Lan
Complaints: Diarrhea
Diagnosis: Enterocolitis
Reasons why the microorganisms were excluded:
•Staphylococcus aureus and Bacillus cereus
These organisms produce enterotoxins in food, causing nausea and vomitting – and to a much lesser extent diarrhea. Since these 2 organisms are more likely to cause nausea and vomitting instead of diarrhea, they can be ruled out since the patient’s symptom is only diarrhea.
• Clostridium difficile
Clostridium difficile is a common infection that is acquired while in the hospital that causes diarrhea. However, it is ruled out because it usually causes Pseudomembranous colitis and not enterocolitis.
Escherichia coli
E. coli is a common cause of diarrhea. Of particular interest is the E. coli O157:H7, a strain of E. coli that produces a toxin that causes hemorrhagic enterocolitis (enterocolitis with bleeding). However, patient is diagnosed with only enterocolitis, but not hemorrhagic enterocolitis, and therefore this organism is ruled out.
• Shigella Flexneri
Shigella sp. are the principal agents of dysentery. This disease differs from profuse watery diarrhea, as the dysenteric stool is scant and contains blood, mucus, and inflammatory cells. The patient is only reported of having diarrhea and not dysenetery as a symptom, therefore this microorganism was ruled out.
http://gsbs.utmb.edu>/microbook>/ch022.htm
Other microorganisms
The other organisms such as vibrio cholerae, entamoeba hitolytica, rotavirus, and Norwalk virus and are found to only cause diarrhea and other gastrointestinal symptoms such as nausea, vomiting and abdominal cramps. These organisms do not have any relations in causing enterocolitis.
Reasons why the organisms were included:
• Salmonella sp.
Salmonella sp. are known to cause enterocolitis with symptoms nausea, vomiting, diarrhea. Since this organism matches the patient’s complaints and diagnosis, the infection is very likely to be due to Salmonella sp.
• Campylobacter jejuni
This organism is known to cause enterocolitis with fever, abdominal cramps, watery to bloody diarrhea. The above symptoms are caused by toxin (endotoxin & exotoxin) produced by Campylobacter sp. Since this organism matches the patient’s complaints and diagnosis, the infection is very likely to be due to Campylobacter jejuni.
Laboratory diagnosis for Salmonella sp.
• They are facultative anaerobes
• They are Gram-negative rods
• They are non lactose fermentors
• They produce H2S
Culture
Differential medium cultures – The stool is layed onto an EMB or MacConkey agar as it permits the rapid detection of lactose nonfermenters (organisms that would grow on the plate would be salmonellae, shigellae, proteus etc). Gram positive organisms are also inhibited when these plates are used.
Selective medium cultures – The specimen would also be plated onto an XLD plate, which favours the growth of salmonellae and shigellae over other Enterobacteriaceae.
Enrichment cultures – The specimen is also placed in selenite F or tetrathionate broth which inhibit replication of normal intestinal flora and permit multiplication of salmonellae. After incubation of 1-2 days, the sample from the broth is plated onto a differential and selective media.
Salmonella sp. on XLD plate
Microscopy
Gram negative bacilli of Salmonella sp
Biochemical reactions
• Indole test – negative
• Motility-positive
• Glucose(TSI) – positive
• Lysine decarboxylase(LIA) – positive
• H2S(TSI and LIA) – positive(blackening)
Note: Enterocolitis is self limiting in 2-3 days. Thus antibiotic treatment not required. (no antibiotic susceptibility tests)
Laboratory diagnosis for campylobacter jejuni
o Gram negative, “S” or “gull wing” shaped
o Motile with a single polar flagellum
o micro-aerophilic (5% O2 with 10% CO2)
Culture
The media that can be used are:
• Campylobacter selective media at 42º C, 10% carbon dioxide, 3-4 days incubation. The incubation atmosphere is made by placing the plates in an anaerobe incubation jar without the catalyst and to produce the gas with a commercially available gas generating gas pack.
• Skirrow medium or Campy BAP medium
Vibrio cholera affects the intestinal tract. It also causes mild to severe diarrhea, vomiting, and dehydration.Since the patient does not complain of dehydration, food poisoning caused by Vibrio cholera is ruled out.
With the elimination of above possible casaultive agents, the most likely cause of food poisoning in this patient is :
1. Staphylococcus aureus
Staphylococcal food poisoning is the most common form of food poisoning.
“some foodborne diseases are caused by the presence of a toxin in the food that was produced by a microbe in the food. For example, the bacterium Staphylococcus aureus can grow in some foods and produce a toxin that causes intense vomiting.”
Besides causing severe vomiting, Staphylococcus aureus also causes diarrhea and abdominal cramps. These are the exact complaints of the patient.
Laboratory Diagnosis (S.Aureus)
Microscopy
On gram staining, gram positive and cocci-shaped colonies are found, which are arranged in irregular clusters.
Culture
The purpose of this test is to isolate bacteria or other organisms that might be causing the symptoms so they can be identified.
Blood agar supports the growth of most bacteria including Staphylococcus aureus, Listeria monocytogenes, and yeast, which are infrequently implicated in food poisoning or gastrointestinal infections, but do not grow on the other media.
Upon culturing on blood sheep agar, Staphylococcus aureus, if present, shows beta haemolytic characteristics, and yield yellow or gold colonies.
Typical test characteristics of S. aureus:
Catalase activity- Positive
Coagulase production- Positive
Thermonuclease production- Positive
Lysostaphin sensitivity- Positive
Anaerobic utilization of mannitol- Positive
Anaerobic utilization of glucose- Positive
Biochemical Tests
1. Catalase Test
Principle
Hydrogen peroxide (H2O2) is decomposed by catalase enzyme to water and oxygen. Bacteria are unable to protect themselves from the lethal effect of hydrogen peroxide, which is accumulated as a product of carbohydrate metabolism. Catalase is a hemoprotein. Catalytic decomposition of hydrogen peroxide leads to the reduction of ferric iron (Fe3+) to ferrous iron (Fe2+) and the re-oxidation of the latter by oxygen. This reaction can be summarized by the following equation:
Catalase
H2O2 --------------------------> 2H2O + O2
Procedure
• A clean microscope slide is labeled for the organism to be tested.
• One drop of 3% H2O2 is added to the slide.
• Several isolated colonies of the organism is transferred to the H2O2 on the microscope slide using a sterile inoculating loop.
Caution
Avoid touching the surface of the media plates containing sheep or human blood, as red blood cells have catalase activity and will give a false-positive result.
Interpretation of positive test: Evolution of gas bubbles
Negative test: No gas bubbles
Should Staphylococcus aureus be the suspected bacteria, the catalase test would be positive.
2. Coagulase Test
Principle
The coagulase test serves to differentiate Staphylococcus aureus from Staphylococcus epidermidis. Should coagulase be present, a clot will be formed in a tube of citrated platelet-rich plasma (~ >150 x 106 platelets/cc plasma). The citrate, which is an anti-coagulant, is added to avoid auto-clotting.
Procedure
• A generous loopful of stool is added to a tube of citrated rabbit plasma.
• Using the loop, the inoculum is thoroughly homogenized
• The tube is then incubated at 37o C for 1 to 4 hours.
• The tube is observed at 30 minute to hourly intervals for the first 2 hours to detect the presence of a clot by tipping the tube gently on its side.
• Should clotting be seen within 24 hours, the coagulase test is considered positive.
Antibiotic Susceptibility Test
1. Agar Disk Diffusion Method• With a sterile loop, the tops of four to five colonies of S. aureus from pure culture are picked up.
• The colonies were suspended in 5 ml of sterile physiologic saline.
• The inoculum turbidity is standardized to equivalent of a 0.5 McFarland standard.
• The entire surface of a Mueller-Hinton agar plate is inoculated using a sterile swab.
• Disks containing 10 µg of penicillin, 10 µg of ampicillin, 30 µg of cephalexine, 1 µg of oxacillin, and 30 µg of kanamycin are then placed using a sterile forcep onto the agar surface and gently pressed down to ensure contact.
• Plates are incubated at 35°C for 20 h.
• Subsequently, the diameter of the zone of inhibition around each disk is measured.
Treatment Therapy
The larger the zone diameter, the more sensitive the bacteria is to the antibiotic. Through this test, it is observed that the largest zone diameter is seen around Penicillin G disk. Hence, Ng Ming En can be treated using Penicillin G antibiotic.
Case study 2
Patient Name: Kwan Siew Lan
Complaints: Diarrhea
Diagnosis: Enterocolitis
Reasons why the microorganisms were excluded:
•Staphylococcus aureus and Bacillus cereus
These organisms produce enterotoxins in food, causing nausea and vomitting – and to a much lesser extent diarrhea. Since these 2 organisms are more likely to cause nausea and vomitting instead of diarrhea, they can be ruled out since the patient’s symptom is only diarrhea.
• Clostridium difficile
Clostridium difficile is a common infection that is acquired while in the hospital that causes diarrhea. However, it is ruled out because it usually causes Pseudomembranous colitis and not enterocolitis.
Escherichia coli
E. coli is a common cause of diarrhea. Of particular interest is the E. coli O157:H7, a strain of E. coli that produces a toxin that causes hemorrhagic enterocolitis (enterocolitis with bleeding). However, patient is diagnosed with only enterocolitis, but not hemorrhagic enterocolitis, and therefore this organism is ruled out.
• Shigella Flexneri
Shigella sp. are the principal agents of dysentery. This disease differs from profuse watery diarrhea, as the dysenteric stool is scant and contains blood, mucus, and inflammatory cells. The patient is only reported of having diarrhea and not dysenetery as a symptom, therefore this microorganism was ruled out.
http://gsbs.utmb.edu>/microbook>/ch022.htm
Other microorganisms
The other organisms such as vibrio cholerae, entamoeba hitolytica, rotavirus, and Norwalk virus and are found to only cause diarrhea and other gastrointestinal symptoms such as nausea, vomiting and abdominal cramps. These organisms do not have any relations in causing enterocolitis.
Reasons why the organisms were included:
• Salmonella sp.
Salmonella sp. are known to cause enterocolitis with symptoms nausea, vomiting, diarrhea. Since this organism matches the patient’s complaints and diagnosis, the infection is very likely to be due to Salmonella sp.
• Campylobacter jejuni
This organism is known to cause enterocolitis with fever, abdominal cramps, watery to bloody diarrhea. The above symptoms are caused by toxin (endotoxin & exotoxin) produced by Campylobacter sp. Since this organism matches the patient’s complaints and diagnosis, the infection is very likely to be due to Campylobacter jejuni.
Laboratory diagnosis for Salmonella sp.
• They are facultative anaerobes
• They are Gram-negative rods
• They are non lactose fermentors
• They produce H2S
Culture
Differential medium cultures – The stool is layed onto an EMB or MacConkey agar as it permits the rapid detection of lactose nonfermenters (organisms that would grow on the plate would be salmonellae, shigellae, proteus etc). Gram positive organisms are also inhibited when these plates are used.
Selective medium cultures – The specimen would also be plated onto an XLD plate, which favours the growth of salmonellae and shigellae over other Enterobacteriaceae.
Enrichment cultures – The specimen is also placed in selenite F or tetrathionate broth which inhibit replication of normal intestinal flora and permit multiplication of salmonellae. After incubation of 1-2 days, the sample from the broth is plated onto a differential and selective media.
Salmonella sp. on XLD plate
Microscopy
Gram negative bacilli of Salmonella sp
Biochemical reactions
• Indole test – negative
• Motility-positive
• Glucose(TSI) – positive
• Lysine decarboxylase(LIA) – positive
• H2S(TSI and LIA) – positive(blackening)
Note: Enterocolitis is self limiting in 2-3 days. Thus antibiotic treatment not required. (no antibiotic susceptibility tests)
Laboratory diagnosis for campylobacter jejuni
o Gram negative, “S” or “gull wing” shaped
o Motile with a single polar flagellum
o micro-aerophilic (5% O2 with 10% CO2)
Culture
The media that can be used are:
• Campylobacter selective media at 42º C, 10% carbon dioxide, 3-4 days incubation. The incubation atmosphere is made by placing the plates in an anaerobe incubation jar without the catalyst and to produce the gas with a commercially available gas generating gas pack.
• Skirrow medium or Campy BAP medium
