FOOD POISONING, PATIENT 4

POSTED BY: HUANG JIAFENG

Patient 4: Ng Ming En

Sex: Male Age: 33

CLINICAL PRESENTATION

Complaints: severe vomiting, diarrhea, abdominal cramps

Diagnosis: Food poisoning

No antibiotic treatment

Chemicals, heavy metals, parasites, fungi, viruses and bacteria are possible causes of food poisoning. However, bacteria related food poisoning is the most common.

SPECIMEN COLLECTION

Stool sample

PRELIMINARY INVESTIGATIONS

Since the patient does not show any clinical symptoms of fever, a number of microorganisms and viruses are eliminated. The suspected microorganisms include Staphylococcus aureus, Bacillus cereus, Clostridium perfringens, Escherichia coli and Vibrio cholerae.

Below shows the characteristics and the expected test results for each suspected microbe I have tabulated.

References

1. Brooks, G. F., Butel, J. S. & Ornston, L. N.; “Jawetz, Melnick & Adeberg’s Medical Microbiology”, 23rd edition, Appleton & Lange, 2004.

2. Kansas State University. (2002). IDENTIFICATION, QUANTIFICATION, AND CHARACTERIZATION OF WASTES/RESIDUES. Retrieved on December 5, 2006, from http://www.oznet.ksu.edu/swr/Home/welcome.htm>modules>module2>microbiological characteristics.

3. Timothy Paustian. (2006). Typical results for biochemical tests. Retrieved on December 5, 2006, from http://www.bact.wisc.edu>Microbiology web textbook>Virtual microbiology>7-4 Typical results for biochemical tests.

The reason why for some of the test NA was stated is because that particular test is not necessary to be carried out. However, some of the microorganisms since I have found the expected results of the tests, although not necessary to carry out, but is stated FYI.

Actually the stool specimen should be first cultured on Blood agar, Xylose-Lysine-Deoxycholate (XLD) agar and MacConkey agar to isolate and obtain pure cultures, followed by gram staining. For Gram negative bacilli, oxidase test was first carried out. A negative oxidase result allows us to proceed on to doing the Triple sugar iron test, indole test, methyl red test, simmons citrate test, urease test and Voges Proskauer test. For positive oxidase test, API 20NE can be done.

For Gram positive cocci, a catalase test was done, followed by a coagulase test. For Gram positive bacilli, a Malachite Green stain was carried out to check for spores. Afterwhich, the specific confirmatory tests could be done to further conclude the microorganism.

Blood Agar: Contains mammalian blood (usually sheep), typically at a concentration of 5–10%. BAP are an enriched, differential media used to isolate fastidious organisms and detect hemolytic activity. β-hemolytic activity will show complete lysis of red blood cells surrounding colony, while α-hemolysis will only partially lyse hemoglobin and will appear green. γ-hemolysis is the term referring to a lack of hemolytic activity.

MacConkey Agar: A selective and differential media used to differentiate between Gram negative bacteria while inhibiting the growth of Gram positive bacteria. The addition of bile salts and crystal violet to the agar inhibits the growth of most Gram positive bacteria, making MacConkey agar selective. Lactose and neutral red are added to differentiate the lactose fermenters, which form pink colonies, from lactose nonfermenters that form clear colonies.

Xylose-Lysine-Deoxycholate (XLD) agar: XLD is used for the culture of stool samples, and contains two indicators. It is formulated with deoxycholoate which is a selective agent that inhibits Gram-positive bacteria, while the growth of Gram-negative bacilli is encouraged. The colonies of lactose fermenters appear yellow. Xylose is fermented by almost all of the enteric bacteria except for a few. Thiosulphate and ferric ammonium citrate are the H2S indicators in the medium. Phenol red is the pH indicator.

Malachite green stain: It is a basic dye. Basic dyes are salts of the colored organic bases containing amino and imino groups and also combined with a colorless acid, such as hydrochloric or sulfuric. They are brilliant and most fluorescent among all synthetic dyes. Basic dyes are cationic which has positive electrical charge and are used for anionic fabrics which are negative-charge-bearing, such as wool, silk, nylon, and acrylics where bright dying is the prime consideration. Malachite green does not contain the mineral malachite; the name comes from the similarity of color. This chemical dye is primarily designed to be used as a dye for silk, leather, and paper. Malachite green in dilute solution is widely used medicinally as a local antiseptic. It is effective against parasites, fungal infections and gram-positive bacteria.

Since the principles of the common biochemical test e.g oxidase, coagulase, triple sugar iron test, etc had been covered by the other group members, please refer to the above entries for more information.

CONFIRMATORY TEST for each microorganism is stated below:

Staphylococcus aureus

Specimens planted on blood agar plates give rise to typical colonies in 18 hours at 37 degrees Celsius, but hemolysis and pigment production may not occur until several days later and are optimal at room temperature. S. aureus ferment mannitol. Specimens contaminated with a mixed flora can be cultured on media containing 7.5% NaCl as the salt inhibits most other normal flora but not S. aureus. S. aureus produces catalase and coagulase so by doing these tests may show. A susceptibility testing would show resistance to penicillin G due to the production of Beta-lactamase if the organism is S. aureus as approximately 90% of S. aureus produces Beta-lactamase. Resistance to nafcillin, oxacillin and methicillin occurs in about 20% of S. aureus. Serological tests for diagnosis of S. aureus infections have little practical value. [1]

Clostridium perfringens

Specimens are inoculated into chopped meat-glucose medim and thioglycolate medium and onto blood agar plates and they are incubated anaeroically. The growth from any one of the media is transferred into milk. A clot torn by gas in 24 hours is suggestive of C. perfringens. Once pure cultures are obtained they are identified by biochemical reactions such as various sugars in thioglycolate, actions on milk, hemolysis, and colony form lecithinase activity is evaluated by the precipitate formed around colonies on egg yolk media. Final identification rests on toxin production and neutralization by specific antitoxin. C. perfringens rarely produces spores when cultured on agar in the laboratory. [1] See Nagler test below

Vibrio cholerae

Growth is rapid in peptone agar, on blood agar with a pH near 9.0 or on TCBS agar and typical colonies can be picked up in 18 hours. For enrichment, a few drops of stool can be incubated for 6 – 8 hours in taurocholate-peptone broth (pH8 – 9). Organisms from this culture can be stained or subcultured. Vibrio cholerae are further identified by slide agglutination tests using anti-O group 1 antisera and by biochemical reaction patterns. [1]

Bacillus cereus

Mannitol-Egg yolk-Polymyxine-Agar or Polymyxine-Egg yolk-Mannitol-Bacillus Agar are highly specific for Bacillus cereus as it is polymyxin resistant. Bacillus cereus colonies can be differentiated from colonies of the other polymyxin resistant organims growing on the plate by their inability to ferment mannitol and the presence of lecithinase. B. cereus colonies appear pink (cannot ferment mannitol, pH increases - phenol red turns reddish) and have a halo (insoluable lipids are released by the action of lecithinase, an enzyme found in B. cereus). Clearing zones visible on the blood agar are due to Beta-hemolysis of the red blood cells in the agar. The ability to lyse red blood cells closely parallels toxin production in B. cereus strains. B. cereus can also be confirmed using immunological detection. [1]

Escherichia coli

MacConkey Sorbitol Agar is suitable for the isolation of E. coli. The medium contains sorbitol instead of lactose and it is recommended for the detection of enteropathogenic strains of Escherichia coli 0157:H7 which ferments lactose but does not ferment sorbitol and hence produce colorless colonies. Sorbitol fermenting strains of Escherichia coli produce pink-red colonies. The red color is due to production of acid from sorbitol, absorption of neutral red and a subsequent color change of the dye when pH of the medium falls below 6.8. [1]

Mannitol Salt Agar (MSA): MSA is also a selective and differential media. Mannitol is the differential part, it indicates organisms that ferment mannitol. If mannitol fermentation is occurring, lactic acid will be produced, and the pH will drop causing the MSA plate to turn yellow. The salt portion is selective for halophiles; organisms that cannot withstand a high salt content will be unable to grow.

Nagler test: The Nagler test is a biochemical test that is used to identify organisms which liberate phospholipases (lecithinases) e.g. Clostridium perfringens. The alpha toxin of C. perfringens has phospholipase activity and hence, when grown on a medium containing egg yolk phospholipid, the organism can break down this insoluble triglyceride. Phosphoryl choline release is seen as an area of opacity around the bacterial colonies. This is described as being Nagler positive. Antitoxin can inhibit a positive response.

Kirby-Bauer test (for determining antibiotic resistance spectrum): The kirby-bauer method for this purpose is based on the diffusion of antibiotics from antibiotic-impregnated paper discs into an inoculated culture medium in a petri plate. The diameter of the area of growth inhibition around the antibiotic disc will give information that is useful for the effective treatment of the patient.

MacConkey Sorbitol Agar: is a selective agar for the direct isolation and differentiation of enterohemorrhagic (EHEC) E. coli O157:H7-strains.

Thiosulfate citrate bile salts sucrose (TCBS) agar: TCBS Agar is highly selective for the isolation of V. cholerae and V. parahaemolyticus as well as other vibrios. Inhibition of gram-positive bacteria is achieved by the incorporation of oxgall, which is a naturally occurring substance containing a mixture of bile salts, and sodium cholate, a pure bile salt. Sodium thiosulfate serves as a sulfur source and, in combination with ferric citrate, detects hydrogen sulfide production. Sucrose is included as a fermentable carbohydrate for the metabolism of vibrios. The alkaline pH of the medium enhances the recovery of V. cholerae. Thymol blue and bromthymol blue are included as indicators of pH changes.

References

1. Brooks, G. F., Butel, J. S. & Ornston, L. N.; “Jawetz, Melnick & Adeberg’s Medical Microbiology”, 23rd edition, Appleton & Lange, 2004.

Some of the possible causes of food poisoning but are eliminated as their distinct symptoms includes fever are: Listeria, Salmonella,, Shigella, Vibrio parahaemolyticus, Yersinia enterocolitica, Campylobacter jejuni, etc.