MMIC PBL Case 2 (2nd Post)

Name: Kwan Siew Yan (outpatient)
Sex: Female
Age: 29
Complaints: Diarrhea
Diagnosis: Enterocolitis
Antibiotic treatment (if any): Nil
Specimen: Stool

Possible Organisms
1) Salmonella enteritidis
2) Shigella species
3) Campylobacter jejuni
4) Staphylococcus aureus
5) Clostridium difficile
6) Bacillus cereus
7) Enteroinvasive E coli (EIEC)

Investigation required:
1) Culture
2) Microscopy
3) Biochemical tests
4) Serology; ELISA and cytotoxicity tests
5) Antibiotic susceptibility tests

Possible Microorganisms	Culture	Microscopy	Biochemical Tests
Salmonella enteritidis	-MacConkey’s agar: non-lactose fermenting (colourless) colonies -Salmonella-shigella agar: colonies with black centre due to H2S production	-gram-negative rods - motile	-TSI agar test: alkaline slant and acid butt with gas and H2S -IMViC tests: gives test result of - + - + respectively.
Shigella species	-MacConkey’s agar: same as Salmonella enteritidi-Salmonella-shigella agar:colonies without black centre as no H2S is produced	-gram-negative rods - non-motile	-TSI agar test: alkaline slant and acid butt with no gas, no H2S
Campylobacter jejuni	-campylobacter selective media (e.g. Skirrow medium or Campy BAP): grow in scanty amounts at 42 degrees celsius	-curved, gram-negative rods that appear either comma or S-shaped -motile with a single polar flagellum	-positive oxidase test
Staphylococcus aureus	-Blood agar: yellow or gold colonies	-gram-positive cocci in clusters	-catalase-positive -coagulase-positive
Clostridium difficile	- Blood agar	- gram-positive -spore-forming rods	Not done
Bacillus cereus	- Blood agar	- gram-negative -spore-forming rod	Not done
Enteroinvasive E coli (EIEC)	-MacConkey’s agar: lactose-fermenting colonies	-gram-negative rods	-TSI agar test: alkaline slant and acid butt with gas, no H2S - IMViC tests: gives test result of + + - - respectively

Possible Microorganisms	Serology; ELISA and cytotoxicity tests	Antibiotic susceptibility tests	Treatment
Salmonella enteritidis	Not done	-resistant to ampicillin and chloramphenicol	-enterocolitis is self limiting in 2-3 days, thus antibiotic treatment is not required.
Shigella species	Not done	-susceptible to ciprofloxacin, ampicillin, doxycycline and trimethoprim-sulfamethoxazole	-fluid and electrolyte replacement without antibiotics or - one of the following antibiotics: ciprofloxacin, ampicillin, doxycycline and trimethoprim-sulfamethoxazole
Campylobacter jejuni	Not done	-susceptible to erythromycin	- usually symptomatic treatment only; erythromycin for severe disease
Staphylococcus aureus	Not done	- susceptible to penicillin G, nafcillin and vancomycin	-penicillin G for sensitive isolates -nafcillin is used to treat beta-lactamase resistant strains -vancomycin is used to treat isolates resistant to nafcillin
Clostridium difficile	-Exotoxin in stool detected by cytopathic effect on cultured cells. Identified by neutralization of cytopathic effect with antibody -Assessment of the A and B toxins in stool can be detected by using ELISA	-susceptible to metronidazole	-metronidazole. Vancomycin, although effective, should not be used as it may select for vancomycin-resistant enterococci
Bacillus cereus	Not done	-susceptible to vancomycin, chloramphenicol, gentamicin, tetracyclines or sulfonamides	-only symptomatic treatment is given antibiotics such as vancomycin, chloramphenicol, gentamicin, tetracyclines or sulfonamides
Enteroinvasive E coli (EIEC)	Not done	- susceptible to trimethoprim-sulfamethoxazole, aminoglycoside, ampicillin, cephalosporin and sulfonamides	- treatment can be difficult because of multiple drug resistance as mediated by ``R’’ plasmid

MacConkey’s agar is 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. [2]

Salmonella-shigella agar are designated as moderately selective media based upon the degree of inhibition of gram-positive microorganisms that they inhibit due to their content of bile salts, brilliant green and citrates. [3] This media is used to find out whether the suspected microorganism is either Salmonella enteritidis or Shigella sp.

Skirrow’s agar is peptone and soy protein base agar supplemented with lysed horse blood and vancomycin (inhibits Gram-positives), polymyxin B (antifungal), and trimethoprim (broad spectrum). Campy BAP is brucella agar supplemented with sheep blood and vancomycin, trimethoprim and cephalothin (inhibits streptococci) [4]

Blood agar is 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 (or non-hemolytic) is the term referring to a lack of hemolytic activity. [2].

After the microorganisms had been isolated, microscopy is used to observe the morphological characteristics so that to identify the organism. Gram stain is a method of differentiating bacterial species into two large groups (Gram-positive and Gram-negative) based on the chemical and physical properties of their cell walls. Gram-positive bacteria have cell wall made of peptidoglycan which stains purple and Gram-negative bacteria which stain pink. [5]. Spore stain will be required to identify spores. Wet mount microscopy is used initially before isolation of organisms to identify their motility.

Antibiotic Susceptibility Test, also known as Kirby-Bauer test, is performed by swabbing the bacterium on the agar and the antibiotic discs are placed on top. The antibiotic diffuses from the disc into the agar in decreasing amounts the further it is away from the disc. If the organism is killed or inhibited by the concentration of the antibiotic, there will be no growth in the immediate area around the disc. This is called the zone of inhibition. The zone sizes are looked up on a standardized chart to give a result of sensititive, resistant, or intermediate. [6] This can help to provide the necessary treatments for the patient.

Pictures
Salmonella enteritidis on Salmonella-shigella agar
[7]


[8]


Staphylococcus aureus (gram stain)
[9]

Campylobacter jejuni wet mount microscopy[10]


Clostridium difficile (gram stain)[11]


Bacillus cereus on blood agar[12]


Enteroinvasive E coli (EIEC)on MacConkey agar[13]













References
1) Levinson, W. (2006). Review Of Medical Microbiology and Immunology. (9th ed.). US: The McGraw-Hill Company
2) http://en.wikipedia.org/ >Agar_plate
3) http://www.bd.com/ >SS_Agar
4) http://www.life.umd.edu/ >Media
5) http://en.wikipedia.org/ >Gram_stain
6) http://www.rlc.dcccd.edu/ >antibiotics
7) http://www.rci.rutgers.edu/ >salmonellashigella
8) http://textbookofbacteriology.net/ >Shigella
9) http://en.wikipedia.org/ >Staphylococcus_aureus
10) http://www.umm.edu/ >fecal culture
11) http://en.wikipedia.org/ >Bacillus_cereus
12) http://www.spiceisle.com/ > Enteric demonstration

Soong Ci Liang
TG01
0503333G